Genetics Update for the Next Gene ration Clinician

(A Compilation of Articles from Genetic Clinics)

Editor Dr Prajnya Ranganath Associate Editors Dr Dhanya Lakshmi N Dr Shagun Aggarwal Foreword & Guidance Dr Shubha R Phadke

Published by The Society for Indian Academy of (SIAMG)

Genetics Update for the Next Generation Clinician

Editor

Dr Prajnya Ranganath Associate Professor & Head, Department of Medical Genetics, Nizam's Institute of Medical Sciences, Hyderabad

Associate Editors

Dr Dhanya Lakshmi N Assistant Professor, Department of Medical Genetics, Nizam's Institute of Medical Sciences, Hyderabad

Dr Shagun Aggarwal Associate Professor, Department of Medical Genetics, Nizam's Institute of Medical Sciences, Hyderabad

Foreword & Guidance

Dr Shubha R Phadke Professor & Head, Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow President, Society for Indian Academy of Medical Genetics, Editor, Genetic Clinics

Preface

his book is essentially a compilation of happening on an almost daily basis, an attempt interesting and clinically relevant review has been made to update them to the extent Tarticles from past issues of Genetic possible. Clinics, which have been put together to serve as a quick reference guide for medical genetics- I sincerely thank the Associate Editors Dr related information. 'Genetic Clinics', the Dhanya Lakshmi and Dr Shagun for their help quarterly journal of the Society for Indian with the compilation and editing of this book. I Academy of Medical Genetics (SIAMG), is being am also extremely grateful to Dr Shubha regularly published since July 2008 and is now Phadke, Editor of Genetic Clinics, for her in its tenth year of publication. It was started guidance and inputs, and my colleagues in the with the objective of providing comprehensive Editorial Board of Genetic Clinics (Dr Ashwin and updated information about various aspects Dalal and Dr Girisha KM) for their help. I would of medical genetics to clinicians and basic also like to express my sincere thanks to Mr scientists in an easily readable and Girish MP and Dr Niranjana from Fastbucks understandable format, and over the years has Services, Bangalore, for the technical support covered many topics of practical interest. and Genzyme, India for the funding support, without which this book would not have seen This selection of articles from Genetic Clinics the light of day. covers diverse aspects of medical genetics such as principles of genetic tests, approach to This book is the brainchild of Professor Shubha common genetic conditions, prenatal and Phadke (President - SIAMG) and other reproductive genetics etc., that are of practical Executive Committee members of SIAMG. We, importance and clinical relevance, and can be the members of the editorial team, have tried used as a desk-guide by clinicians. Some of our best to bring it to fruition and we sincerely these articles were originally written a few hope that our readers would benefit from this years back and as medical genetics is a rapidly endeavour. evolving specialty with technological advances

Dr Prajnya Ranganath Editor

Foreword

'Paralearning' through the Landscape of Medical Genetics

am glad to introduce this bouquet of articles on different aspects of medical genetics to the audience of medical doctors and basic scientists interested in the latest Idevelopments in genomic . The tremendous developments in deciphering the genome and identifying phenotypic effects of genomic variations have brought genetics to the clinic. The diagnosis and management of genetic disorders namely monogenic disorders and chromosomal disorders have got a big impetus due to genomic techniques like next generation sequencing and microarray. The same techniques have revolutionized research and patient care of cancers, multifactorial disorders and infectious disorders leading to a paradigm shift in medical practice.

We are glad that these exciting developments in technology and the scientific and clinical expertise to use it are now available in India. The medical practitioners therefore have to update themselves to this new platform of clinically applicable knowledge. The pace of developments is too fast to keep oneself updated even for medical geneticists. 'Genetic Clinics', a three-monthly publication, has been a continued and regular effort of the Society for Indian Academy of Medical Genetics (SIAMG) to disseminate Dr Shubha R Phadke knowledge of the latest developments in medical genetics to interested doctors, students and scientists. The articles on Professor and Head, clinically relevant aspects of diagnosis and management of Department of Medical Genetics, genetic disorders as well as scientific marvels are presented in an easy-to-read fashion. We have selected some articles relevant to Sanjay Gandhi Postgraduate the present time from the previously published issues of Genetic Institute of Medical Sciences, Clinics and have compiled them in the form of a book. This Lucknow, India. compilation of articles will give an overview of clinical genetics and President, Society for Indian genomic diagnosis to make one interested and excited about the Academy of Medical Genetics field. Most of the articles chosen are on clinical aspects and will make one comfortable in approaching patients and families with Editor, Genetic Clinics genetic disorders. The articles on use of latest technology for diagnostics are meant to clarify the principles of the tests and hence provide the clinician the power to order the appropriate test in each situation, and interpret the results with a good understanding of the limitations. Pre-test and post-test counseling are extremely important in the pre and post-natal evaluation of genetic disorders. Addition of practical scenarios, practical questions, photo quiz and crosswords has been done to improve understanding and add fun to the learning process.

I am sure the book will provide a good overview of genomic medicine and give clinicians an opportunity to update themselves on clinically relevant genetics-related topics. The objective is to enthuse the medical fraternity in India about genetics, so that they continue to learn with 'Genetic Clinics' in the future. I feel that reading of the book can be compared with paragliding over the beautiful landscape of genomic medicine and I am sure everyone will enjoy this 'paralearning' experience. Shubha Phadke 8th December 2017

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1. Principles and Applications of Genetic Laboratory Techniques

Chapter 01 Karyotyping in Clinical Practice 01 Girisha KM Chapter 02 Multiplex Ligation-Dependent Probe Amplification: A Versatile Technique in Molecular 05 Diagnostics Kausik Mandal, Shubha R Phadke Chapter 03 Cytogenetic Microarray: A Revolution in Cytogenetics 09 Meenal Agarwal Chapter 04 Clinical Cytogenetics in the Diagnosis and Prognosis of 15 Krishna Reddy CH, Ashwin Dalal Chapter 05 Triplet Primed PCR (TP-PCR) – A Versatile Method for Molecular Diagnosis of Triplet 22 Repeat Disorders Padma Priya T, Ashwin Dalal Chapter 06 Mendelian Disease Gene Identification and Diagnostics using Targeted Next 26 Generation Sequencing Daniel Trujillano, Rami Abou Jamra, Arndt Rolfs Chapter 07 Diagnostic Yield of Exome Sequencing in Neurodevelopmental Disorders 31 Moirangthem Amita, Shubha R Phadke

2. Genetic Approach to Common Clinical Scenarios

Chapter 08 Approach to a Child with Dysmorphism/ Congenital Malformation 37 Prajnya Ranganath Chapter 09 Approach to Intellectual Disability 43 Prajnya Ranganath Chapter 10 Approach to Disorders of Sex Development 51 Prajnya Ranganath Chapter 11 Inborn Errors of Metabolism Presenting in the Newborn Period: Representative Phenotypes and 57 Diagnostic Approach Ratna D Puri, Shubha R Phadke Chapter 12 An Approach to Genetic Disorders Affecting the White Matter 70 Vijayalakshmi SR, Prajnya Ranganath Chapter 13 Approach to a Child with Macrocephaly: the Dysmorphologist's View 85 Sumita Danda Chapter 14 Radial Ray Defects: Genetics and Syndromic Etiologies 91 Sankar VH

3. Prenatal, Perinatal and Reproductive Genetics

Chapter 15 Recurrent Loss: From Chromosomes to Genes 97 Meenal Agarwal, Shubha R Phadke Chapter 16 Rapid Detection (RAD) Techniques for Prenatal Diagnosis 102 Pranita Pai, Shagun Aggarwal, Anju Shukla, Girisha K M Chapter 17 Fetal Dysmorphology: An Indispensable Tool for Synthesis of Perinatal Diagnosis 107 Shagun Aggarwal Chapter 18 Non-immune Fetal Hydrops: An Update 116 Gayatri N, Ashwani Tandon, Prajnya Ranganath Chapter 19 Fetal Therapy- Current Approaches and Future Possibilities 124 Shagun Aggarwal Chapter 20 Newborn with Down : Care and Counseling 131 Shubha R Phadke, Rekha Gupta 4. Mendelian Disorders

Chapter 21 Spinal Muscular Atrophy 135 Dhanya Lakshmi N, Girisha KM Chapter 22 Duchenne Muscular Dystrophy 140 Udhaya H Kotecha Chapter 23 Fragile X Revisited - Novel Testing, Screening and Treatment Strategies 145 Priyanka Srivastava, Shagun Aggarwal Chapter 24 MECP2 Gene-Related Disorders 150 Meenakshi Lallar, Shubha R Phadke Chapter 25 : Recent Advances in Diagnosis and Management 155 Prajnya Ranganath, Gaurav Kumar Rai Chapter 26 Disorders of the RAS-MAPK Pathway: Prototype of Pathway Disorders with Overlapping 161 Phenotypes Prajnya Ranganath, Dhanya Lakshmi N, Shubha R Phadke Chapter 27 Inherited Disorders of DNA Repair 169 Lekshmi Nair, Surya Prabha B, Prajnya Ranganath Chapter 28 Distal Arthrogryposis 177 Siddram J Patil Chapter 29 Osteogenesis Imperfecta: An Update 181 Vartika Giri, Priyanka Srivastava, Meenakshi Lallar, Shubha R Phadke Chapter 30 Six Lethal Skeletal Dysplasias Which a Pediatrician Should Never Miss 190 Sheela Nampoothiri

5. Recent Advances in Medical Genetics

Chapter 31 Management of Lysosomal Storage Disorders: The Current Scenario 195 Prajnya Ranganath, Dhanya Lakshmi N Chapter 32 Genome Editing: The 'CRISPR' Way 202 Annapurna Gupta, Meenal Agarwal Chapter 33 Next Generation Sequencing in New Born Screening - Current Insights 207 Deepika Delsa Dean, Sarita Agarwal

6. Heart-to-Heart Talk

I. The Burden of Diagnosis 213 Shubha R Phadke ii. Looking Beyond… The Vision of a Blind Man 215 Shagun Aggarwal, Shubha R Phadke iii. Amniotic Cavity Full of LOVE ! 216 Shubha R Phadke iv. Short – on sensitivity ! 218 Divya Aggarwal, Shubha R Phadke v. Unacceptable Truth or Acceptable Lie !! 220 Ashwin Dalal

7. Genetic Exercises

I. Genetic Counseling Exercises 221 Dhanya Lakshmi N, Shubha R Phadke ii. Crosswords 227 Dhanya Lakshmi N iii. Answers to Photoquizes 231 Prajnya Ranganath Section 1 Principles and Applications of Genetic Laboratory Techniques

Chapter 1

Karyotyping in Clinical Practice

Girisha KM Department of Medical Genetics, Kasturba Medical College, Manipal University, Manipal, India

Correspondence to: Dr Girisha KM Email: [email protected]

As early as in 1882, Walther Flemming, an Structure of a Chromosome Austrian cytologist and professor of Anatomy had published the first illustration of human The word ‘chromosome’ is derived from the Greek chromosomes. It then took several decades words for ‘colored body’. Human cells contain 46 before Tjio and Levan in 1956 could prove that the chromosomes comprising 22 pairs of total number of chromosomes in a human cell is numbered from 1 to 22 and a pair of sex 46. In 1959, Lejeune studied the chromosomes chromosomes (two X chromosomes in females from patients with and described and one X and one Y in males). The numbers are an extra chromosome in each cell. Since assigned in descending order of length, size and then, several chromosomal abnormalities are well centromere position of each chromosome pair. characterized. The indications for chromosomal Each chromosome is made up of a shorter ‘p’ arm analysis are briefly discussed here. and the longer ‘q’ arm joined at the centromere. The ends of chromosomes are referred to as telomeres. Metacentric chromosomes have centromere at the middle position of the chromosome. Submetacentric chromosomes have centromere between the middle and the tip and in acrocentric chromosomes, the centromere is present near the tip. Acrocentric chromosomes may have satellites beyond the centromeres. Karyotype

Karyotype refers to the ordered display a) of chromosomes starting from the largest to followed by sex chromosomes. A karyotype is prepared either by using computer software or manually arranging the images of chromosomes (cut from a photograph). Chromosomes can be analyzed from any actively dividing cells. Usually, peripheral blood lymphocytes are stimulated to undergo multiplication by phytohemagglutinin in a culture bottle. Other common sources are marrow, amniocytes, chorionic villi and skin fibroblasts. The dividing cells are arrested in metaphase by colchicine, a mitotic inhibitor. These cells are then b) treated with hypotonic solution to destroy the cell membranes and then fixed with fixative made up Figure 1 a) A metaphase spread as seen under of methanol and acetic acid. The cell pellet of the light microscope. b) A normal male appropriate quantity is dropped on to glass karyotype. slides to get ‘metaphases’ (chromosomes from a

1 Chapter 1 single cell are usually found in groups). The at two places and rejoined in the reverse chromosomes are then ‘banded’ using trypsin and orientation. Inversions may be pericentric when stained by Giemsa to give G-bands, with alternate the breaks are on either side of the centromere dark and light bands of various sizes along the or paracentric when they are on one side. length of chromosomes. Several other banding Translocation involves transfer of a segment techniques are available, but are used in specific of a chromosome to another chromosome. indications. Modifications of the technique permit Reciprocal translocations represent one of the high resolution banding. Metaphases are then most common structural rearrangements in man. seen under a microscope (Figure 1a), imaged, It results when two different chromosomes individual chromosomes identified based on their exchange segments (Figure 2). Robertsonian size and band pattern, and then arranged to get translocations are non-reciprocal and occur the ‘karyotype’ (Figure 1b). when the long arms of any two acrocentric chromosomes join to produce a single metacentric Chromosomal Abnormalities or submetacentric chromosome. Other structural abnormalities are isochromosome (both the Chromosomal abnormalities are either numerical arms are similar), ring, dicentric or acentric or structural. Polyploidy refers to the cell that chromosomes. Microdeletions are small contains a multiple of 23 chromosomes. A triploid deletions that usually escape detection during cell contains 69 chromosomes and tetraploidy routine karyotyping because of the small size refers to presence of 92 chromosomes. Most of the deletions. A specific test like FISH commonly seen abnormalities are (Fluorescent in situ hybridization) or MLPA (not a multiple of 23 chromosomes). These include (Multiplex ligation-dependent probe amplification) (only one copy of a chromosome in is necessary to detect these abnormalities. otherwise diploid cell) or (3 copies of Chromosomal microarray, a molecular cytogenetic a chromosome) and (4 copies of technique, can detect copy number variations a chromosome). Common examples include (CNVs) anywhere in the genome. trisomy 21 (Down syndrome), trisomy 18 (Edward syndrome), trisomy 13 (), When to order a Karyotype? monosomy X () and (47,XXY). A contains two or more Karyotype is one of the most commonly used cell lines with different chromosomal constitution, ‘genetic’ tests. By asking for a karyotype, which are derived from a single zygote. the clinician intends to look for structurally visible chromosomal abnormalities under the microscope. These include numerical and large structural chromosomal abnormalities. Such situations are common in clinical practice.

Figure 2 Partial karyotype showing translocation between chromosomes 7 and 11.

Structural chromosomal abnormalities are identified by the band pattern. Balanced rearrangements have no loss/gain of chromosome material whereas unbalanced rearrangements have either loss or gain of chromosomal segments. Deletions refer to loss of a part/segment of a chromosome. In duplication, an extra copy of a genomic segment results in a partial trisomy. In Figure 3 Child with Down syndrome. inversion, a segment of chromosome is broken

2 Chapter 1

Suspected known Chromosomal : The overall yield of karyotype is reported to be in Down• syndrome is by far the commonest the range of 4-28% (selection criteria varies in chromosomal abnormality encountered by a different studies; the reported diagnostic yield is pediatrician in the clinics. In most situations, the higher especially in studies that include Down diagnosis is obvious by clinical examination alone syndrome). As cognition is likely to be determined (Figure 3). However, newborn babies may have by several genes spread throughout the genome, only few identifiable features at birth posing even a small aberration is expected to have diagnostic challenge to even an experienced significant effect on mental function. However, the pediatrician. About 4% of cases of Down syndrome Cytogenetic microarray technique, which has a result from Robertsonian translocations. In these much higher resolution of up to 10-100 kb as situations, one of the parents may be a normal compared to karyotyping which has a resolution of carrier of this translocation, which increases only around 5 Mb, is now recommended as the the risk of recurrence of Down syndrome to first line genetic investigation in patients with IDD. 5-15% (100% if the translocation involves two 21 chromosomes). Here the risk of recurrence thus Disorders of Sex Development: Individuals increases from 1% in the family with Down with• ambiguous genitalia, delayed or incomplete syndrome due to trisomy 21. To give a definite risk pubertal development, oligo / azoospermia and of recurrence, karyotype of affected child is primary amenorrhea need a karyotype. Often essential in all cases of Down syndrome. Turner syndrome and Klinefelter syndrome are The definitive diagnoses of trisomy 18, trisomy diagnosed in this manner. Many a times they aid 13, Turner syndrome (Figure 4) and Klinefelter further diagnostic evaluation as in a case of syndrome are established by karyotyping. ambiguous genitalia. Short stature in a pre-pubertal female: Turner• syndrome should be ruled out as short stature can be the only manifestation of this condition in pre-pubertal females.

Pregnancy loss and infertility: Chromosomal• structural rearrangements can often lead to recurrent spontaneous abortions and infertility. The chance of karyotypic abnormality is higher (about 5.5%) if the first trimester losses are 3 or more. Some of these families may also have abnormal offspring with malformations and mental retardation due to unbalanced chromosomal abnormalities. Hence, karyotype not only identifies the cause of poor reproductive Figure 4 Webbing of neck and pedal edema outcome, but also serves to offer counseling and with hypoplastic nails in a child with for future . Monosomy X. Parents of a child with structural Special Chromosomal Studies: In some chromosomal• abnormality: Some of the situations,• specific studies on chromosomes aid structural chromosomal abnormalities arise from the diagnosis. (fragile site balanced rearrangements in parents who run on ) and chromosomal breakage the risk of future offspring with chromosomal syndromes like Fanconi anemia, , abnormalities and birth defects. Parents of a child ataxia telangiectasia etc., illustrate conditions with translocation-associated Down syndrome where modified cytogenetic studies confirm the need karyotyping. clinical diagnosis. Malignancies: Some hematological malig- Unexplained Intellectual Disability: All nancies• are known to be associated with children• with idiopathic/ unexplained global chromosomal abnormalities. Chronic myeloid developmental delay/ intellectual disability are , for instance, requires karyotype for conventionally evaluated through karyotyping. diagnosis (to look for , a

3 Chapter 1 balanced translocation involving chromosomes 9 What is the limitation of karyotype as a and 22) as well as assessing the response to therapy. diagnostic test?

Child with monogenic disorder: Usually Karyotype detects numerical chromosomal children• with known monogenic condition do not require karyotyping and require molecular genetic abnormalities and large rearrangements of tests such as gene sequencing for confirmation of chromosomes. Small submicroscopic alterations the diagnosis. Karyotyping may be considered if below 4-5Mb size are usually not picked up by the child has additional unexplained mental routine karyotype. It is important to remember retardation or if the child has more than two that most Mendelian disorders have mutations monogenic disorders. involving only one or very few nucleotides, and are not diagnosed by karyotype. It is also worthwhile Prenatal diagnosis: In pregnancies at risk of to note that even though karyotype is a commonly a• chromosomal abnormality, karyotype may used genetic test with a fair diagnostic yield, it has be done from chorionic villi or amniocytes. limited resolution, which can be explained by Commonly, couple with previous child with the large size and complexity of the human chromosomal abnormality, advanced maternal genome. Identification of structural abnormalities age, carriers of balanced chromosomal has provided clues to the location of genes of rearrangements, fetal malformation detected by several single gene disorders like Duchenne sonography, positive , fetal markers of muscular dystrophy and contributed immensely to aneuploidy are offered prenatal fetal karyotyping. research in identifying disease genes. GeNeVerse An Ode to a Chromosome - by a Cytogenetics Trainee Black and white little banded bar, How I wonder what you are Down through the microscope I gaze, Trying to focus through the haze How do I identify each one in this mix, Amongst the chromosomes forty and six There you lie inconspicuously on the slide, Looking insignificant but encompassing the genome wide Within your recesses, in each fold, The entire code of life you hold A little less here, a little more there, Can make the whole system go haywire Please oblige, help me correctly identify, Stop at the right length in metaphase and comply Show me clearly your centromere and each band, Don’t bend over, as straight as possible please stand If only you’d stay true to the textbook prototype, I wouldn’t have to struggle so, to get a good karyotype. (Based on real life experiences!) - By Dr. Prajnya Ranganath

4 Chapter 2

Multiplex Ligation-Dependent Probe Amplification (MLPA): A Versatile Technique in Molecular Diagnostics

Kausik Mandal, Shubha R Phadke Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow

Correspondence to: Dr Kausik Mandal Email: [email protected]

Introduction Each probe mix is designed either for a particular disease or conditions with similar phenotype. Each In the history of medical genetics, identification of probe mix can contain up to 40 to 50 probes correct number of chromosomes in the mid-1950s (which can be called probe pairs) for different was a major breakthrough. In the past three target regions of the genome. All the probe pairs decades, DNA technology has revolutionized diag- in a probe mix have a pair of common sequences nostics in medical genetics. Various cytogenetic as (say F and R, one at each end) (Figure1). These well as molecular genetic techniques like fluores- common sequences F and R, act as templates for cent in-situ hybridization (FISH) and its variations, common primers during the PCR reaction. Each polymerase chain reaction (PCR) and its modi- probe pair for a target sequence has a stuffer fications and comparative genomic hybridization sequence (attached to one of the probes in the (CGH) and its advanced version array-CGH, have probe pair). The length of the stuffer sequence been used to screen the genome or part of the is different for different probe pairs, thus making genome for identification of mutations and loss the amplification product of each probe pair ofa or gain of DNA (copy number variations). Most unique length. After the probes hybridize with of the known genetic disorders are known to the complementary sequences in the sample DNA, be associated with hundreds of mutations and the two probes in a probe pair are ligated by a some with mutations involving more than one thermostable ligase enzyme. If the target DNA gene. Scientists are always in search of techniques does not have the complementary sequence, the which are fast, technically less demanding, and probe will not hybridize and hence, will not get cost effective yet sensitive enough to detect the ligated. The ligation reaction is very specific; even a minutest variations in the genome. Multiplex single base pair mismatch may hamper the ligation Ligation-dependent Probe Amplification (MLPA) is step. In the next step, all the ligated probe sets are a multiplex PCR method detecting abnormal copy simultaneously amplified by PCR, using a universal numbers of up to 40 to 50 different genomic primer pair for the above mentioned F and R DNA or RNA sequences in a single reaction. For regions. In case of defect in ligation, further PCR most hereditary conditions, gene (partial or whole) amplification does not take place. The resulting deletions or duplications account for less than 10% amplification products of a commercially available of all disease-causing mutations, for some other MLPA kit range between 130 and 480 nucleotides disorders this is 10-30% or even higher (Aretz et in length. There is a difference of 7 to 8 base pairs al., 2007; Redeker et al., 2008). The inclusion between two neighbouring PCR products. Hence, of MLPA in clinical settings can therefore signifi- the PCR products can be separated by capillary cantly increase the detection rate of many genetic electrophoresis. The peak height or peak area for disorders. each probe reflects the relative abundance of the target sequence in the DNA. Principle of MLPA Technique of MLPA

There are more than 300 commercially available The basic steps include DNA denaturation, addition probe mixes (also called probe sets) for MLPA. of probe mix for overnight hybridization, ligation

5 Chapter 2 by a thermostable ligase, PCR of ligated products using a universal PCR primer pair and detection of products by capillary electrophoresis (Figure 1). It is recommended that during each experiment of MLPA, control samples or standards are run along with test samples; but in most clinical situations, other samples in the same set act as controls. In- terpretation of result is done by statistical analysis using software. In step 4 as depicted in Figure 1, the peaks representing a specific area of genome are demarcated by arrows. The peak in the up- per panel (patient) is smaller as compared to the corresponding peak in the lower panel (control). The heights of other peaks in the upper and lower panel are comparable. This suggests a of that part of genome in the patient. The dosage quotient for each probe is calculated by dividing the normalized peak height in the patient by the mean of normalized peak heights of the controls. The result is depicted as mean ratio. If the value of ratio of the normalized peak height in the patient to the mean of normalized peak heights of the controls is 1.35 or more, it is taken as duplication and if it is 0.65 or less, it is taken as deletion. The representative data and its interpretation are given in step 5 of Figure 1.

Figure 1 Principle and steps of MLPA.

Step 1: Denaturation and hybridization – the probe will hybridize with complementary se- quences in the sample DNA. Step 2: Hybridized pairs of the probes will ligate with each other. Step 3: PCR of the ligated products- only ligated probes will be amplified. Step 4: Capillary electrophoresis- the amplified products are separated on a sequencer. Step 5: Analysis.

6 Chapter 2

Strengths of the Technique It is not sensitive enough to differentiate be- • tween large and small deletions as whatever It requires a thermocycler (PCR machine) be the length of the deleted segment, the • and a sequence type capillary electrophoresis final analysis will detect it as a deletion of (Sequencer), which are routinely available in the segment for which the probe set was most molecular biology laboratories. designed.

It is a multiplex technique, screening multiple Only known mutations can be detected. It is unlikely to precisely detect unknown muta- • areas of the genome simultaneously. • tions. Large number of samples can be handled It is not a direct method like FISH, where the simultaneously, with results being available • result can be visually interpreted. It requires within 24 to 48 hours. • statistical tools for interpretation of result.

It is sensitive and requires very small amount It cannot directly detect mosaicism; in re- of DNA (20 ng DNA). • • cent studies, scientists are trying to make it possible. It does not require living cells as for karyotype. • As this technique detects relative abundance It identifies the frequent, single gene aberra- • of a specified segment of a genome as com- • tions which are too small to be detected by pared to other parts of the same genome, FISH. It can be used to distinguish sequences it does not detect triploidy or tetraploidy, differing in only one nucleotide by designing where the DNA in the whole genome is probes. (Schouten et al., 2002) proportionally increased.

The technique is versatile and can be de- Some copy number variations are polymor- signed for certain disorders and group of • • phisms and it may be difficult to decide disorders with near similar phenotype. whether a particular deletion is a variation seen in normal people or a disease-causing It is flexible. One can use few extra probes in mutation. • addition to what is available commercially. Sequence variations near the target site may It can be used to study RNA and epigenetic • influence the peak height. • alterations (methylation) in the genome. A detected copy number variation sometimes It is reproducible (coefficient of variability of • needs to be confirmed by another probe • each probe is 3% to 8% when human DNA mix or other methods like FISH or CMA samples of 40-100 ng are analysed). (cytogenetic microarray).

It is cost effective when compared to other Scope of MLPA • contemporary techniques (Palomares et al., 2002). This technique is widely used for various disorders. The whole is extensive. Each Weaknesses of MLPA probe mix is a mixture of probe sets designed to detect copy number variations for diseases with It is a screening method, and is not as sen- similar manifestations or single gene disorders • sitive as sequencing or ARMS- PCR to detect with known deletions and duplications. point mutations. Thus, as of now, this It is being extensively used for mental retarda- technique can detect only 10 to 15% of all tion and other neurogenetic disorders. Identifica- mutations in single gene disorders in most tion of etiology of cases with mental retardation cases (except in diseases like DMD where without an obvious etiology or clinical clue to eti- around 70% mutations are due to full exon ology is a challenge to clinicians and researchers. deletions or duplications). Recently, new Small deletions and duplications of various parts probe sets are being developed by scientists of the genome have been found to cause mental for some known point mutations. retardation. These deletions / duplications may

7 Chapter 2 be too small to be detected in the traditional tect methylation defects in disorders like Beckwith- method of chromosomal analysis. The deletions / Wiedemann Syndrome, Russell-Silver Syndrome, duplications of the ends of chromosomes (telom- Prader-Willi syndrome and . It eres and subtelomeric regions) are found in about is also used for mRNA analysis, pharmacogenetics, 5 to 7% cases of mental retardation without an basic research (e.g. cytochrome P-450, repair obvious cause. MLPA has been found to be a genes) and animal experiments. very useful technique to search for deletions/ du- It is evident from the above list that the scope plications of subtelomeric regions. For screening of this technique is immense and researchers are of subtelomeric regions of chromosomes in cases developing new probe sets for research into newer of mental retardation, more than one probe mix disorders. It is not far when this technique will are available commercially. Probe mixes are also be incorporated into routine diagnostics for most available for common microdeletion syndromes genetic disorders. However, it is to be emphasized and X-linked mental retardation. This makes MLPA that at present, MLPA is not useful to detect point an important modality of investigation in cases mutations and for most single gene disorders this with mental retardation (Mandal et al., 2009). technique will detect only deletions and duplica- In addition to microdeletion syndromes, MLPA tions, which is not more than 10 to 15% of all is also useful in identification of mutations for mutations. some monogenic conditions. Duchenne muscular dystrophy (DMD), Spinal Muscular Atrophy (SMA) References and Limb-girdle muscular dystrophy (LGMD) are neuromuscular disorders, which can be screened 1. Aretz S, et al. High proportion of large genomic by this technique. Deletions and duplications in deletions and a genotype phenotype update the DMD gene account for more than 70% cases in 80 unrelated families with juvenile polyposis of DMD. DMD is a very big gene and deletions are syndrome. J Med Genet 2007; 44: 702-709. found in all parts of the gene. MLPA being able to 2. Mandal K, et al. Use of multiplex ligation- perform 40 PCRs (multiplex) in one test, can test dependent probe amplification (MLPA) in for all areas of the gene in one test. Probe mixes screening of subtelomeric regions in children are also being designed for diseases like retinitis with idiopathic mental retardation. Indian J pigmentosa, neurofibromatosis, Parkinsonism etc. Pediatr 2009; 76:1027-1031. The other important use of the technique is 3. Palomares M, et al. MLPA vs multiprobe FISH: for prenatal diagnosis of aneuploidy. Probe mix comparison of two methods for the screening is commercially available for chromosomes 21, 18, of subtelomeric rearrangements in 50 patients 13, X and Y. Probes for subtelomeres and common with idiopathic mental retardation. Clin Genet microdeletion syndromes can also be used for 2006; 69: 228-233. rapid screening in prenatal samples. However, it is 4. Redeker EJ, et al. Multiplex ligation-dependent always advisable to use another method like FISH probe amplification (MLPA) enhances the molec- along with this technique for confirmation. ular diagnosis of aniridia and related disorders. MLPA is also being used for cancers and Mol Vis 2008; 14: 836-840. hereditary cancer syndromes like neuroblastoma, 5. Schouten JP et al. Relative quantification of 40 retinoblastoma, breast cancer, Li-Fraumeni syn- nucleic acid sequences by multiplex ligation- drome (LFS), ataxia-telangiectasia, etc. dependent probe amplification. Nucleic Acids The technique of MLPA is being modified to de- Res 2002; 30(12): e57.

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8 Chapter 3

Cytogenetic Microarray: A Revolution in Cytogenetics

Meenal Agarwal Clinical Genetics Division and Medical Genetics Laboratory, GenePath Dx, Causeway Healthcare Private Ltd., Pune (Formerly: Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow)

Correspondence to: Dr Meenal Agarwal Email: [email protected]

Introduction gions are spotted on a slide. There is extreme miniaturization as the small chip has about 3 mil- Intellectual disability (ID) is the most common clin- lion probes corresponding to various regions of the ical situation encountered in medical genetics for whole genome on it. The probes are fluorescently counseling. Confirmation of diagnosis is the first labelled. Test DNA is hybridized to the probes on step to provide prognosis, genetic counseling and the chip. The excess of un-hybridized probes is option of prenatal diagnosis to the family. Despite washed and the slide is scanned by the scanner the exhaustive investigations, etiology remains which quantifies the fluorescence signal intensity unidentified in 30-50% of the cases (Moeschler et at each of the millions of spots. Scanning and al., 2006). With the advent of molecular cytogenetic interpretation is done by computerized software. techniques i.e. fluorescence in situ hybridization The signal intensity of each probe is compared with (FISH), multiplex ligation probe dependent ampli- reference data and analysis is done by platform fication (MLPA) and now cytogenetic microarray specific software. The results are provided inthe (CMA) in the evaluation of ID, the diagnostic yield form of copy number losses i.e. deletions or copy has been increasing. CMA is a technique to study number gains i.e. duplications. The results not chromosomes at a very high level of resolution. only give the exact size of the area deleted or It can detect gains or losses of sizes as small duplicated but also the exact location in the form as 10 kb. FISH and MLPA techniques provide of nucleotide numbers of the ends of the region higher resolution than traditional karyotype but and hence one knows the gene content of the they only analyze the few targeted regions (up to region. Some CMA platforms use comparative hy- 45 regions in MLPA) in the genome. In contrast bridization technique in which patient and control to this, CMA provides whole genome coverage like DNA samples tagged with differentially coloured a karyotype but at an extremely high resolution. fluorochromes are co-hybridized with the probes That is the reason for the higher diagnostic yield of on the chip. The signals of different fluorochromes this technique in the evaluation of developmental at a spot are evaluated by the relative comparison disabilities, multiple congenital anomalies (MCA) of strength of the signals. and/or autistic spectrum disorders (ASDs). More- There are various CMA platforms available over, do not need to have a clinical (Affymetrix, Illumina, etc) and recently a few guide- diagnosis before ordering CMA. With an experi- lines have also been published regarding the use ence of CMA in a very large number of cases of array platforms, their probe density and overall with developmental disabilities it is clear that CMA genome coverage (Kearney et al., 2011). Almost all detects abnormalities in about 20% of cases with of these platforms use combination of probes for intellectual disability (Manning et al., 2010). Now, copy number variants (CNV) and single nucleotide CMA is recommended as the first-tier test in the polymorphisms (SNP). Usually the probe density is investigation of ID/MCA/ASDs (Miller et al., 2010) higher in the areas for known abnormalities but in addition the whole genome is covered by a Principle of CMA and platforms backbone of probes. Because of use of SNP probes in the plat- CMA is based on the principle of complementary forms, in addition to copy number variations, these hybridization of nucleotides. The probes for re- microarray platforms also detect areas of homozy-

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Figure 1 Flow chart showing classification and interpretation of each variant (followed at the Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow). gosity which are important to identify uniparental ter of Unbalanced Chromosome Aberra- disomy and help to locate autosomal recessive loci tions (http://umcecaruca01.extern.umcn.nl: in consanguineous families. 8080/ecaruca/ecaruca.jsp, ECARUCA). On the basis of this analysis each CNV is classified into benign, pathogenic or variants of unknown signif- Analysis of variations identified in CMA icance (VUS), as presented in Figure 1. These VUS are further divided into probably benign, probably Each variant identified by the software is anal- pathogenic or of unknown significance on the basis ysed on the basis of its size (no guidelines are of available information (Figure 1). (Kearney et al., available about size cut off, but most cytogenetic 2011; de Leeuw et al., 2012) laboratories use 200 kb as a size limit for reporting a variant), gene content, published case reports/ Illustrative cases series and database search. The various databases which are used for such bioinformatic analysis A few cases which illustrate the utility of the are PubMed (ncbi.nlm.nih.gov/pubmed), On- chromosomal microarray technique are described line Mendelian Inheritance in Man (omim.org, here. OMIM), DECIPHER (http://www.sanger.ac.uk/ PostGenomics/decip), Database of Genomic Vari- Case 1: ation (DGV), UC Santa Cruz Database (http: 8 months old female child with developmen- //www.genome.uscs.edu, UCSC), ISCA (https:// • tal delay, facial dysmorphism (hypertelorism, isca.genetics.emory.edu/iscaBrowser/) and straight eye brows & deep-set eyes) and post European Cytogeneticists’ Association Regis- axial polydactyly in the left foot (Figure 2a).

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CMA showed presence of 3 Mb deletion in • the 22q11.2 region (Figure 3b). Interpretation: Pathogenic • Comments: Though 22q11.2 microdeletion • is a known syndrome, the facial phenotype was not characteristic and congenital heart disease which is a common feature of 22q deletion syndrome was not present. The postaxial polydactyly which was present in this case is seen only in 15% of cases of 22q11 microdeletion. Therefore, in this case clinical suspicion of this syndrome was not possible (Figure 3).

a)

b)

Figure 2 a) Photograph of patient showing facial dysmorphism (hypertelorism, deep set eyes, straight eyebrows and deep-set a) eyes). b) Cytogenetic microarray re- sults (Affymetrix 2.7 M array) showing deletion at 1p36 region.

CMA identified 3 Mb deletion in the 1p36 • region (Figure 2b) Interpretation: Pathogenic • Comments – 1p36 microdeletion is a known • and clinical features of this patient were consistent with the char- acteristic features of this syndrome described b) in literature. Figure 3 a) Photograph of a 2-years-old girl with Case 2: subtle facial dysmorphism. b) Cyto- 3 years old female child with global develop- genetic microarray (Illumina Cytochip) • mental delay, subtle facial dysmorphism and result showing 3 Mb deletion in 22q11.2 postaxial polydactyly (Figure 3a). region.

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Case 3: Case 6: 17 years old boy with intellectual disability, Child with global developmental delay and • subtle facial dysmorphism and post axial • polydactyly polydactyly. CMA showed 1.1 Mb loss at 6q12.3 region. CMA identified 3.4 Mb deletion in the region • This pericentromeric region variant has been of 7q12 (Figure 4). • • reported in DGV and no pathogenic pheno- Interpretation: Pathogenic types were reported to be associated with it in DECIPHER/OMIM/PUBMED. • Comments: On bioinformatic analysis, GLI3 Interpretation and comment: CMA report did • gene which is known to be associated with polydactyly was found to be situated in this • not show any probable pathogenic gain/loss. region. Hence there was a genotype pheno- However, this does not rule out single gene type correlation. (Figure 4) disorders or imbalances of regions not repre- sented on the microarray platform. Case 4: Case 7: 14 years old boy with intellectual disability, facial dysmorphism and . 10 months old boy with global developmental • delay. CMA showed the presence of 14 Mb deletion • CMA showed 424 Kb copy number gain on • in 10q21.11. • 3p26.3, and 518 kb copy number gain on Interpretation: Pathogenic 18p23. • Comments: This region has not been not Bioinformatic analysis: Both these regions known to be associated with ID previously. • • were partially reported in DGV and were not Because of the presence of a de novo large mi- associated with any pathogenic phenotype crodeletion (CMA of the parents was normal), in DECIPHER/OMIM/PubMed. CHL1 gene, this variant was interpreted as pathogenic. which is a member of the L1 gene family of neural cell adhesion molecules, is situ- Case 5: ated in the 3p26.3 region. It is a neural 4 months old female child with failure to recognition molecule that may be involved in • thrive and microretrognathia signal transduction pathways. The deletion of one copy of this gene may be responsible CMA showed 9 Mb deletion in 7q36.1 and 13 for mental defects in patients with the 3p- Mb duplication in 11q24.1. • syndrome. Thus, this variant was interpreted Interpretation: Pathogenic as a variant of unknown significance probably • Comments: These double segment imbal- pathogenic. The other copy number gain • ances may be due to separate chromosomal at 18p23 was not very well reported in DGV events or an inherited/ de novo chromosomal and had the associated OMIM phenotype of rearrangement. Extended pedigree analysis the 18q deletion syndrome. This region had showed that 2 relatives on the paternal side no genes and was interpreted as variant of were having varying degrees of ID. Her pater- unknown significance. nal first cousin, who was 4 years old with GDD, facial dysmorphism and congenital heart dis- ease, was found to have 9 Mb duplication at Application of CMA as an adjunct to 7q36.1 and 13 Mb deletion on 11q24.1. In this traditional karyotyping family, familial chromosomal rearrangement is the most likely diagnosis. Fluorescent in Traditional karyotypes detect abnormalities but in situ hybridisation (FISH) for the involved chro- some cases the exact delineation of the cytogenetic mosomal segments will be the investigation abnormality may not be possible due to the low of choice to diagnose the presence of bal- level of resolution of the technique. In such a anced chromosomal rearrangements in this situation, chromosomal microarray may be able family. to provide additional information which can be

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LOC646999; RALA; LOC349114; CDC2L5; C7orf11; C7orf10; INHBA; LOC285954; LOC285954; GLI3; C7orf25; PSMA2; MRPL32

Genes in the deleted region

Figure 4 Cytogenetic microarray (Illumina Cytochip) results in case 3, showing 3.4 Mb deletion in the 7p14.1 region, harbouring the GLI3 gene. interpreted in the context of the cytogenetic ab- 3. Difficulty in interpretation of variants ofun- normality observed and thus give a clear picture. known significance. (De Gregori et al., 2007; Gribble et al., 2005). In the 4. Incomplete penetrance/variable expressivity/ following situations CMA can be a useful adjunct to interaction between many variants. the karyotype: 5. Rapid addition of new data implies that inter- To identify cryptic imbalances at the break- pretation of variants is likely to change in the • points in cases with cytogenetically apparent future. This needs continued interpretation balanced translocations. of a CMA report and puts a huge burden on To identify extra material on a chromosome. clinical cytogeneticists and the family as well. • To identify an extra chromosome of unknown 6. Cost of CMA is a special issue especially in the • origin (a ). Indian scenario where the family has to pay for the test. Many a times, parental CMA is Limitations of CMA also required for the analysis of variants. This further adds to the cost. MLPA using probes Like any other diagnostic technique, CMA has its of subtelomeric and common microdeletion own limitations. These are as follows: regions, still can be considered as the first-tier test in Indian patients with ID. 1. Availability of various CMA platforms which 7. Does not identify origin of marker chromo- differ in their probe density and overall some in some cases / low level of mosaicism/ coverage. balanced chromosomal rearrangements. 2. No stringent guidelines regarding interpreta- 8. Does not identify monogenic causes of intel- tion of copy number gains/ losses. lectual disability.

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CMA in prenatal diagnosis complex chromosome rearrangements: a study of 59 patients. J Med Genet 2007; 44: 750-762. CMA is now being increasingly recommended in 2. de Leeuw N, et al. Diagnostic interpretation of prenatal samples with or without fetal malforma- array data using public databases and internet tions. In various studies the overall yield of CMA sources. Hum Mutat 2012; 33: 930-940. in prenatal diagnosis is said to be in the range of 3. Gribble SM, et al. The complex nature of con- 2-3%. The yield can be as high as 8-10% in prenatal stitutional de novo apparently balanced translo- samples where antenatal ultrasonography has de- cations in patients presenting with abnormal tected one or more malformations (Yatsenko et al., phenotypes. J Med Genet 2005; 42:8-16. 2012). 4. Kearney HM, et al. American College of Medical Genetics recommendations for the design and Conclusions performance expectations for clinicalgenomic copy number microarrays intended for use in CMA has proven to be of significant utility in the the postnatal setting for detection of consti- evaluation of patients with ID/MCA/ASDs. The tutional abnormalities. Genet Med 2011; 13: American College of Medical Genetics has ad- 676-679. vocated CMA as a first-tier investigation in the 5. Kearney HM, et al. American College of Medical evaluation of children with intellectual disability, Genetics standards and guidelines for inter- autism and malformation syndromes. Now with pretation and reporting of postnatal constitu- the use in prenatal diagnosis, it is likely to rev- tional copy number variants. Genet Med 2011; olutionize the diagnostic yield in as well. 13:680-685. However, physicians and concerned families need 6. Manning M, Hudgins L and the Professional to understand its limitations and the need for Practice and Guidelines Committee. Array- genetic counseling both before and after the test based technology and recommendations for results. As per the recommendations of the Amer- utilization in medical genetics practice for detec- ican College of and Gynecology (ACOG) tion of chromosomal abnormalities. Genet Med and the Society for Maternal-Fetal Medicine (De- 2010;12: 742-745. cember 2013; reaffirmed in 2015), chromosomal 7. Miller DT, et al. Consensus statement: chromo- microarray analysis is recommended (instead of somal microarray is a first-tier clinical diagnostic karyotyping) when the has one or more test for individuals with developmental disabil- major structural abnormalities in USG and in a ities or congenital anomalies. Am J Hum Genet structurally normal fetus in USG, undergoing inva- 2010; 86:749-764. sive prenatal diagnostic testing, either CMA or fetal 8. Moeschler JB, Shevell M and the Committee karyotyping can be done. on Genetics. Clinical Genetic Evaluation of the Child with Mental Retardation or Developmental References Delays. 2006; 117; 230416. 9. Yatsenko S, et al. Application of chromoso- 1. De Gregori M, et al. Cryptic deletions are a mal microarray in the evaluation of abnormal common finding in “balanced” reciprocal and prenatal findings. Clin Genet 2013; 84: 47-54.

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14 Chapter 4

Clinical Cytogenetics in the Diagnosis and Prognosis of Leukemias Krishna Reddy CH, Ashwin Dalal Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics, Hyderabad

Correspondence to: Dr Ashwin Dalal Email: [email protected]

Introduction traditionally have been designated as acute or chronic, based on the percentage of blast cells and Leukemias are a group of disorders characterized the course of the disease. According to the recently by accumulation of malignant white cells in the proposed classification system the blast cell count bone marrow and blood. They are neoplastic, required to classify as acute leukemia is more than clonal disorders of the hematopoietic stem cells. 20% either in bone marrow or peripheral blood, They can be broadly classified as acute and chronic and does not require any minimum blast cell per- leukemias. The term acute, historically referring centage when certain morphologic and cytogenetic to a rapid onset and usually fatal outcome, indi- features are present. cates the relatively undifferentiated nature of the There have been several changes in the classifi- leukemic cells. Acute leukemias usually present cation of the leukemias over the last few decades. with bleeding manifestations, severe anemia and Two widely used classification systems include: one severe infections, where as many patients with devised by a group of French, American, British chronic leukemias are asymptomatic or present (FAB) hematologists and the other by the World with splenomegaly, fever, weight loss, malaise, Health Organization (WHO). The traditional classifi- frequent infections, bleeding, thrombosis, or lym- cation of acute leukemia used criteria proposed by phadenopathy. Based on the cell of origin they are the French– American–British Cooperative Group classified as myeloid and lymphoid leukemias. In (FAB), using the 30% bone marrow blast cell the WHO classification, the term “Myeloid” includes cutoff. This classification system originally distin- all cells belonging to the granulocytic (neutrophil, guished different leukemia types by morphologic eosinophil, basophil), monocytic/macrophage, ery- features and cytochemical studies (Hamid et al., throid, megakaryocytic and mast cell lineages 2011). The recent WHO 2008 classification takes (Vardiman et al., 2009). 20% bone marrow blast cell cutoff and includes The overall annual incidence of these disorders all the features like morphology, cytochemistry, in the general population is about 4 per 100,000 immunophenotype and genetics (Vardiman et al., with approximately 70% of them being acute 2009; Yin et al., 2010) (Table-1 & Table-2). myeloid leukemia (AML). AML accounts for about 15% of childhood leukemias and for approximately Genetic Abnormalities in leukemias 80% to 90% of acute leukemias in adults, with the median age at diagnosis of about 70 years. Acute Cytogenetic abnormalities seen in leukemias are lymphoblastic leukemia (ALL) is primarily a child- mainly translocations, deletions, ploidy changes hood disease, with the peak incidence between the and mutations in specific genes. They differ ages of 2 to 3 years. Chronic leukemias predom- based on the type of leukemia and are useful in inantly occur in adults, the common ones being classification and prognosis of the leukemia. chronic myeloid leukemia comprising of 40% of all Acute Myeloid Leukemia: Acute myeloid leukemias followed by chronic lymphoid leukemia. leukemia• (AML) is a tumor of hematopoietic pro- genitors caused by acquired oncogenic mutations Classification that impede differentiation, leading to the accumu- lation of immature myeloid blasts in the marrow. Leukemias are broadly classified into myeloid or It is the common form of acute leukemia in adults lymphoid, based on their cell of origin. Leukemias and accounts for 25% of all leukemias diagnosed

15 Chapter 4 in adults, and is increasingly common with age. Gain or loss of whole or part of chromosome Cytogenetic analyses and molecular analyses are • – -7/del(7q), -5/del(5q), +8, +9, +11, del currently used to risk-stratify AML. Cytogenetic (11q), del(12p), -18, +19, del(20q) +21, abnormalities can be detected in approximately 50% to 60% of newly diagnosed AML (Kumar et al., Mutations involving FLT3, KIT, NPM1, CEPBA 2011). • genes. The t(8;21) is more fre- Table 1 WHO 2008 classification of Acute Myeloid Leukemia and related quent in the young and is neoplasms. rare beyond the age of 50 years. The translocation Acute myeloid leukemia with recurrent genetic abnormalities results in the generation on the derivative chromo- • – AML with t(8;21)(q22;q22); RUNX1-RUNX1T1 some 8 of a consistent – AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11 hybrid gene, ETOAML- 1 – APL with t(15;17)(q22;q12); PML-RARA that encodes a novel mes- sage for haematopoietic – AML with t(9;11)(p22;q23); MLLT3-MLL cell proliferation (Rueda et – AML with t(6;9)(p23;q34); DEK-NUP214 al., 2004). – AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1 Most common chro- – AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1 mosomal abnormalities t(8;21) and inv(16), dis- – Provisional entity: AML with mutated NPM1 rupt the CBF1α and – Provisional entity: AML with mutated CEBPA CBF1β genes, respectively. Acute myeloid leukemia with myelodysplasia-related changes These two genes en- code polypeptides that Therapy-related myeloid neoplasms • bind one another to form • Acute myeloid leukemia, not otherwise specified a CBF1α/CBF1β transcrip- • – AML with minimal differentiation tion factor that is required – AML without maturation for normal hematopoiesis. The t(8;21) and the inv(16) – AML with maturation create chimeric genes en- – Acute myelomonocytic leukemia coding fusion proteins that – Acute monoblastic/monocytic leukemia interfere with the function of CBF1α/CBF1β and block – Acute erythroid leukemia the maturation of myeloid – Acute megakaryoblastic leukemia cells. – Acute basophilic leukemia The t(15;17) transloca- tion is found in approx- – Acute panmyelosis with myelofibrosis imately 95% of (Acute Myeloid sarcoma Promyelocytic leukemia) • Myeloid proliferations related to Down syndrome APLs, a specific subtype • Blastic plasmacytoid dendritic cell neoplasm of AML (Figure 1). The t(15;17) translocation is al- • ways associated with APL and leads to the expres- sion of PML-RARα onco-fusion gene in hematopoi- Genetic abnormalities seen in AML are: etic myeloid cells. The PML-RARα onco-fusion Structural rearrangements protein acts as a transcriptional repressor that interferes with gene expression programs involved • – Balanced chromosomal translocations, in differentiation, apoptosis, and self-renewal. particularly t(8;21), inv(16), and t(15;17). Generally, patients with APL t(15;17) phenotype – Translocations involving 3q21 and 3q26 represent a unique group characterized by distinct and t(1;7) biological features and good prognosis, particularly

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Table 2 WHO 2008 classification of Acute Lymphoid Leukemia.

B lymphoblastic leukemia/ • – B lymphoblastic leukemia/lymphoma, NOS – B lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities * B lymphoblastic leukemia/lymphoma with t(9;22)(q34;q11.2);BCR-ABL 1 * B lymphoblastic leukemia/lymphoma with t(v;11q23);MLL rearranged * B lymphoblastic leukemia/lymphoma with t(12;21)(p13;q22) TEL-AML1 (ETV6-RUNX1) * B lymphoblastic leukemia/lymphoma with hyperdiploidy * B lymphoblastic leukemia/lymphoma with hypodiploidy * B lymphoblastic leukemia/lymphoma with t(5;14)(q31;q32) IL3-IGH * B lymphoblastic leukemia/lymphoma with t(1;19)(q23;p13.3);TCF3-PBX1 T lymphoblastic leukemia/lymphoma • when all-trans retinoic acid (ATRA) is used as part et al., 2008). NPM1 mutations often coincide with of remission induction (Kumar et al., 2011). mutations in FLT3, particularly with the ITD-type mutations. The NPM1 mutations in AML are as- sociated frequently with normal karyotypes. The CEBPA gene provides instructions for making a pro- tein called CCAAT/enhancer-binding protein alpha. This protein is a transcription factor, which means that it binds to specific regions of DNA and helps control the activity of certain genes. It is believed to act as a tumor suppressor, helping to prevent cells from growing and dividing too rapidly or in an uncontrolled way. CEBPA are detected in 15% of patients and associated with favorable response to therapy (Verhaak et al., 2005). Other molec- ular markers, such as IDH1, IDH2, and DNMT3A have been suggested to be predictive of risk and response to treatment. Acute Lymphoblastic Leukemia (ALL): ALLs are neoplasms composed of the immature T or Fluorescent In Situ Hybridization show- • Figure 1 B cell, called lymphoblasts. ALL is a malignant ing the translocation in a patient with clonal proliferation of lymphoid progenitor cells, Acute Promyelocytic leukaemia. The cell most commonly of the B-cell lineage (B-ALL). In shows 2 fusion signals and 1 indepen- the pediatric population, ALL accounts for 81% of dent red and 1 independent green signal childhood leukemias; leukemia overall accounts for (Vysis Dual color dual fusion probe). one third of cancers diagnosed in children between Among the mutated genes FLT3 (ITD) is the most ages 0–14 years. Overall, T-ALL has a bad prognosis common, seen in one third of AML cases. FLT3 when compared to B-ALL. Approximately 90% of is a receptor tyrosine kinase that is expressed ALLs have numerical or structural chromosomal on hematopoietic progenitor cells and indicative changes. of poor prognosis. Other mutated genes are Genetic abnormalities seen in B-ALL are: NPM1, c-KIT, CEBPA. NPM mutations seen in 30% Hyperdiploidy of cases, it is a protein which normally acts as a • Hypodiploidy nucleocytoplasmic shuttling protein and regulates • Balanced chromosomal translocations the p53 pathway (Verhaak et al., 2005; Stirewalt • t(12;21), t(1;19), t(9;22), t(4;11).

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respectively. The t(9;22)(q32;q11), or Philadelphia (Ph) chromosome, is present in 20 to 30% of adults and 1 to 5% of children with ALL at the cytogenetic level and is associated with older age, higher leukocyte count, and more frequent CNS involvement at the time of diagnosis (Woo et al., 2014; Ruth et al., 2013). MLL (mixed-lineage-leukemia) gene rearrange- ments at 11q23 are present in 80% of all infant B-ALL cases and 10% of all childhood B-ALL. The most common gene rearrangements include t(4;11)(q21;q23) encoding MLL-AF4, t(9;11)(p22;q23) encoding MLL-AF9, t(11;19)(q23;p13.3) encoding MLL-ENL, t(10;11)(p13-14;q14-21) encoding MLL- AF10 and t(6;11)(q27;q23) encoding MLL-AF6 (Woo et al., 2014; Ruth et al., 2013). The t(4;11)(q21;q23) is also associated with high-risk features, most Figure 2 Karyotype of a patient with Acute notably a high WBC count and age of onset lymphoblastic leukaemia showing hy- year (Hamerschlak et al., 2008). < 1 podiploidy for chromosome 10 and 18 Conventional karyotyping identifies structural and a marker chromosome. chromosomal aberrations in 50% of T-ALL. Numer- ical changes are rare, except for tetraploidy which High hyperdiploidy (51–65 chromosomes) is one is seen in approximately 5% of cases. The com- of the most common cytogenetic abnormalities ob- mon abnormalities involve the rearrangements served in childhood B-ALL. It is seen in 25-30% involving T-cell receptor genes like deregulation of of total childhood B-ALL cases, with the highest homeobox genes TLX1 (HOX11) t(10;14)(q24;q11), frequency in the 1 to 4 year age range. High hyper- TLX3 (HOX11L2) t(5;14)(q35;q32), and TAL1 diploidy is characterized by a nonrandom gain of (SCL,TCL5) t(1;14)(p32;q11), t(1;14)(p34;q11) and chromosomes, including +X, +4, +6, +10, +14, +17, t(1;7)(p32;q34); deregulation of MYB gene- +18, and +21. This diagnosis confers a good prog- duplication t(6;7)(q23;q34); and fusion gene re- nosis in childhood B-ALL (Woo et al., 2014; Ruth et arrangements like PICALM-MLLT10 (CALM-AF10) al., 2013). Approximately 20% of hyperdiploid ALL t(10;11)(p13;q14) and MLL-fusions. Favorable prog- have activating mutations in the receptor tyrosine nosis is associated with subtypes HOX11 or MLL-ENL kinase FLT3. Hypodiploidy is characterized by fewer (Ruth et al., 2013; Hamerschlak et al., 2008). than 46 chromosomes and is seen in 5-8% of total Up to 70% of T-ALLs have gain-of-function mu- B-ALL cases (Figure 2). The majority of hypodiploid tations in NOTCH1, a gene that is essential for B-ALL contain 45 chromosomes. The remainder T-cell development. A high fraction of B-ALLs of hypodiploid cases is much rarer and includes have loss-of-function mutations in genes that are high-hypodiploid (40–44 chromosomes). required for B-cell such as PAX5, E2A and EBF. The t(12;21) is the most common chromosomal rearrangement in ALL. It occurs in 25% of children Chronic Myeloid Leukemia (CML): It is a with B-ALL and confers an excellent prognosis. clonal• stem cell disorder characterized by the ac- This translocation joins the TEL (or ETV6) gene on quisition of an oncogenic BCR/ABL fusion protein, chromosome 12p with the AML1 (or CBFA2) gene usually the result of a reciprocal translocation on and is associated with an t(9;22)(q34;q11) and by proliferation of granulo- early pre-B immunophenotype, a younger child- cytic elements at all stages of differentiation. The hood population, and non hyperdiploidy. The majority of CML cases are in adults. The frequency ETV6-RUNX1 fusion protein is thought to disrupt of this type of leukemia is 1 per 1 million chil- the normal expression of RUNX1-regulated genes dren up to the age of 10 years. Among adults, by converting RUNX1 to a transcriptional repressor. the frequency is around 1 in 100,000 individuals The t(1;19)(q23;p13) translocation is found in (Hamerschlak et al., 2008). CML can be divided into one fourth of patients with the pre-B immunophe- three clinically distinct phases: an initial chronic notype. This translocation represents fusion of the phase followed by an accelerated phase which E2A and PBX1 genes on chromosomes 1 and 19, subsequently leads to blast crisis.

18 Chapter 4

and/or molecular RT-PCR testing for further evalu- ation and documentation of a BCR-ABL transcript (Hsiao et al., 2011). Based on the breakpoint region in BCR gene three transcripts are formed first, the major breakpoint cluster region (M-) and chimeric protein derived from this mRNA is 210-kd. This is the most common breakpoint region. Sec- ond, the minor breakpoint cluster region (m-bcr) and resultant mRNA is translated into a 190-kd protein. Recently, a third breakpoint cluster region (μ-bcr) was identified, giving rise to a 230-kd fusion protein (Deininger et al., 2000). Identification of fusion transcript helps in targeted treatment by tyrosine kinase inhibitors and in prognosis by identifying minimal residual disease. The p53 and Rb1 genes are most often im- Figure 3 Karyotype of a patient with Chronic plicated in the transformation. Alterations in myeloid leukaemia showing t(9;22) the p53 and Rb1 genes occur in about 30% and (Philadelphia translocation). 20% of blast crisis cases, respectively. Additional clonal cytogenetic abnormalities, seen are , isochromosome 17q, or duplication of the Ph The t(9;22) translocation leads to derivative chromosome. Recently, it has been observed that chromosome 22 also referred to as the Philadel- in greater than 85% of cases, CML is associated phia chromosome (Ph) (Figures 3 and 4) seen in with the appearance of mutations that interfere 95% of AML as well as in some acute lymphoblas- with the activity of Ikaros, a transcription factor tic leukemias (ALL) (Pokharel, 2012). The ABL that regulates the differentiation of hematopoietic gene located on chromosome 9 is the human progenitor. homologue of the v- oncogene carried by the Abelson murine leukemia virus and it encodes a nonreceptor tyrosine kinase. Human Abl is a ubiq- uitously expressed 145-kd protein with 2 isoforms arising from alternative splicing of the first exon. The activity of the c-Abl tyrosine kinase domain is regulated by the SH3 and SH2 regions. The SH3 domain is a negative regulator of c-Abl activity, as a deletion mutant of this domain activates the kinase, whereas the SH2 domain is a positive reg- ulator. The BCR gene (Breakpoint cluster region) located on chromosome 22 encodes a 160-kd Bcr protein which, like Abl, is ubiquitously expressed. Figure 4 Fluorescent In Situ Hybridization show- The first N-terminal exon encodes a serine thre- ing the t(9;22) translocation in a patient onine kinase. As a result of the t(9;22)(q34;q11) with chronic myeloid leukaemia. The Bcr-Abl fusion, the N-terminus of Bcr binds to the left cell shows two green and two SH2 domain of Abl in a phosphotyrosine indepen- red independent signals suggestive of dent manner, constitutively activating the tyrosine normal FISH for t(9;22)(bcr-abl) whereas kinase. This oncoprotein is associated with en- the right side cell shows 2 Fusion signals hanced expression of several major downstream (suggestive of presence of t(9;22)(bcr- effectors such as Ras, phosphoinositide 3-kinase abl)) and 1 independent red and 1 (PI-3K) and protein kinase B (Deininger et al., 2000; independent green signal each. Di Bacco et al., 2000). Approximately 5% of cases appear to be “Ph- negative” by conventional cytogenetics because Chronic Lymphocytic Leukemia: Chronic of the presence of a cryptic or submicroscopic lymphocytic• leukemia (CLL) is characterized by the BCR-ABL rearrangement. These cases require FISH accumulation of mature appearing lymphocytes

19 Chapter 4 in the blood, marrow, lymph nodes, and spleen. also to know the bad and good prognostic features, They are characterized by the absolute lymphocyte which help in the treatment of the disease. Cytoge- count 4000/mm . Genetic abnormalities com- netic studies include karyotyping and Fluorescence 3 monly> encountered are deletions of 13q14.3, 11q, In-Situ Hybridization (FISH) for detection of the and 17p, and trisomy 12q; translocations are rare. deletions and translocations. Molecular analysis The most frequent structural abnormality in includes PCR, Real Time PCR and the mutational CLL is a deletion at 13q14. Approximately one analysis for detection of the fusion transcript and half of the abnormalities involves an interstitial mutation in specific genes. The major applications deletion and almost invariably is associated with of each technique are: loss of the Rb gene. Translocations involving 13q are found in the remaining one half of cases, and Applications of Immunophenotyping most of these involve a breakpoint at 13q14, which • is the site of the Rb gene, with translocations from – Diagnosis and classification a variety of other chromosomes. A deletion of – Assessment of prognosis 11q22–q23 which occurs in 20% of cases, is seen in younger patients, and is associated with marked – Monitoring of minimum residual disease lymphadenopathy, rapid disease progression, and – To monitor effectiveness of treatment poor survival. Trisomy 12q13 occurs as a result of duplication of one homolog and is seen in 10 Applications of Cytogenetic analysis to 20% of CLL patients. Trisomy 12 is frequently associated with “atypical” CLL and CLL/PLL. A dele- • – Confirmation of diagnosis of specific tion of 17p13 is usually associated with a mutation subtype of p53, and these patients usually have aggressive and drug-resistant disease (Puiggros et al., 2014). – Assessment of prognosis Diagnostic Workup – Assessment of clonality – Detection of minimum residual disease Laboratory work up in diagnosis of leukemias includes: Applications of Molecular genetic studies Morphological examination of peripheral • – Diagnosis of specific subtype • blood and bone marrow. Cytochemistry – Early detection of minimum residual dis- • Immunophenotyping ease Cytogenetic analysis • – Assessment of prognosis • Molecular genetic studies Assessment of clonality •The diagnosis of a hematological neoplasm – usually starts from a clinical suspicion, although for chronic leukemias the diagnosis is sometimes an Prognostic Markers incidental one. A blood count and is an essential first step whenever leukemia, lymphoma Prognostic markers help in predicting the survival or other hematological neoplasm is suspected. of the patients and for planning the appropriate Diagnosis is based on peripheral smear and confir- treatment for the disease. The presence of favor- mation is done by bone marrow examination. The able markers in a case means that the survival rate next step in the diagnostic process depends on the of the patient for that particular disease is more clinical features and the specific condition that is and unfavorable markers have less survival rates. suspected. With advances in immunophenotyping Some of the markers are classified as intermediate and other techniques, the role of cytochemistry in markers based on their survival rate, which have hematological diagnosis has declined considerably. less clinical implication when compared to bad and Further typing of leukemia into myeloid or lym- good markers and further evaluation is required phoid either B cell or T cell, requires the analysis of (Table-3). Genetic prognostic markers for the antigen expression by immunophenotyping. chronic leukemias are not well established as in Cytogenetic and molecular genetic studies are the case of acute leukemias. The major prognostic done for molecular classification of leukemia and markers are:

20 Chapter 4

Table 3 Prognostic genetic markers in tist’s Perspective and Challenge. Mariastefania Antica (Ed.), 2011. ISBN: 978-953-307-553-2. Leukemias. 2. Deininger MW, et al. The molecular biology of chronic myeloid leukemia. Blood 2000; 96: Type of Favorable Unfavorable 3343-3356. Leukemia 3. Di Bacco A, et al. Molecular abnormalities in ALL Hyperdiploidy Hypodiploidy chronic myeloid leukemia: deregulation of cell growth and apoptosis. Oncologist 2000; 5: ( 50) t(12;21) t(9;22) 405-415. > t(4;11) 4. Forero RM, et al. Genetics of Acute Lym- t(1;19) phoblastic Leukemia, Prof. Margarita Guenova AML t(8;21) -7/del(7q) (Ed.), InTech, 2013. DOI: 10.5772/55504. 5. Hamerschlak N. Leukemia: genetics and prog- t(15;17) -5/del(5q) nostic factors. J Pediatr (Rio J) 2008; 84: S52-57. inv(16) +8 6. Hsiao HH, et al. Additional chromosome ab- +9 normalities in chronic myeloid leukemia. Kaoh- CLL del 13q del 17p siung J Med Sci 2011; 27: 49-54. 7. Kumar CC. Genetic abnormalities and chal- Trisomy 12 del 11q lenges in the treatment of acute myeloid CML Ph chromosome + Trisomy 8 leukemia. Genes Cancer 2011; 2: 95-107. del 22q 8. Lidiane R, et al. Translocation t(8;21)(q22;q22) in Acute Myeloid Leukaemia. Rev Bras Hematol Hemoter 2004; 26: 66-67. 9. Pokharel M. Leukemia: A Review Article. IJARPB. Conclusions 2012; 2: 397-407. 10. Puiggros A, et al. Genetic abnormalities in Leukemias are among the commonest cancers chronic lymphocytic leukemia: where we are worldwide. Their diagnosis depends mainly on and where we go. Biomed Res Int 2014; the morphology, cytochemistry and immunophe- 435983. notyping, but cytogenetic and molecular studies 11. Stirewalt DL, et al. Identification of genes with are essential for the molecular classification and abnormal expression changes in acute myeloid predicting the prognosis of the disease. Common leukemia. Genes Chromosomes Cancer 2008; cytogenetic studies include karyotyping and FISH 47: 8-20. for finding the translocations and deletions, and 12. Vardiman JW, et al. The 2008 revision of the molecular studies include PCR, Real Time PCR and World Health Organization (WHO) classification mutation analysis of the relevant genes. Hence, for of myeloid neoplasms and acute leukemia: ra- accurate early diagnosis and treatment, a stepwise tionale and important changes. Blood 2009; evaluation of the case with the suitable diagnostic 114: 937-951. tests is employed for the favorable outcome. 13. Verhaak RG, et al. Mutations in nucleophosmin (NPM1) in acute myeloid leukemia (AML): asso- ciation with other gene abnormalities and pre- Acknowledgements viously established gene expression signatures and their favorable prognostic significance. We would like to thank Dr Ashwani Tandon, Nizam’s Blood 2005; 106: 3747-3754. Institute of Medical Sciences, Hyderabad for kindly 14. Woo JS, et al. Childhood B-acute lymphoblas- providing the karyotype and FISH images. tic leukemia: a genetic update. Exp Hematol Oncol 2014; 3: 16. References 15. Yin CC, et al. Recent advances in the diagno- sis and classification of myeloid neoplasms– 1. Abdul-Hamid G. Classification of Acute comments on the 2008 WHO classification. Int Leukemia. In: Acute Leukemia -The Scien- J Lab Hematol 2010; 32: 461-476.

21 Chapter 5

Triplet Primed PCR (TP-PCR) – A Versatile Method for Molecular Diagnosis of Triplet Repeat Disorders

Padma Priya T, Ashwin Dalal Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, Telangana

Correspondence to: Dr Ashwin Dalal Email: [email protected]

Introduction successive generations. A repeat length of greater than 50 to 1000 results in the manifestation of Trinucleotide repeats are highly polymorphic re- DM1. The severity of the disorder and age of onset gions in the human genome which act as key of symptoms correlates well with the number of players in a number of diseases known as triplet repeats. Another triplet repeat disorder is FRAX repeat disorders. Of these disorders, Friedreich which is the most common cause of inherited ataxia (FA), Myotonic dystrophy type 1 (DM1) and mental retardation. It is caused by large CGG Fragile X syndrome (FRAX) are caused by expansion expansions in the 5’ untranslated region of FMR1 of the triplet repeats in the non-coding regions of gene, consequently resulting in hypermethylation corresponding genes. The other diseases caused of the CpG island present in the promoter of FMR1 due to triplet repeat expansion include Huntington further leading to the gene silencing. Normal disease, Spinocerebellar ataxias, etc. Here we de- individuals carry the repeats in the range of 5-54 scribe application of a modified PCR assay for the while the affected individuals have 200 CGG diagnosis of Friedreich ataxia, Myotonic dystrophy repeats. Premutation individuals have> repeats in and Fragile X syndrome. the range 55-200. There is an inverse correlation between repeat size and age of onset of the disease in FA and DM1. Overview of the diseases The triplet repeats tend to result in both somatic and germline instability and have a strong ten- FA is an autosomal recessive disease caused by dency to further expand in successive generations. the expansion of GAA repeats present in the first This increasing number of repeats in subsequent intron of frataxin or FRDA gene resulting in defi- generations explains the phenomenon known as cient frataxin mRNA and protein levels. Increased anticipation. Owing to the broad spectrum of accumulation of mitochondrial iron due to loss of clinical presentations, handicapping nature of the frataxin function and subsequent increased free disease phenotypes and unavailability of curative radical generation and oxidative stress underlies treatments, effective preventive measure is the the pathophysiology of FA. In normal individu- only available help to the families with these dis- als the GAA repeats range from 7-22 and in orders. Molecular diagnostic tests are imperative affected cases these can be more than 66to for the early clinical diagnosis and timely prenatal 1000 triplet repeats. Myotonic dystrophies, also diagnosis. The molecular diagnostic tests are also known as dystrophia myotonica, are a group of helpful in carrier detection and newborn screen- autosomal dominant disorders with highly variable ing especially in case of FRAX. Routine molecular phenotypes of which DM1 is caused by unstable diagnostics involves a traditional PCR, amplifying expansion of CTG repeats in the 3’ untranslated the repeat region followed by agarose gel elec- region of the myotonic dystrophy protein kinase trophoresis whereas the gold standard is southern (DMPK) gene located on chromosome 19. In nor- blotting to determine the size of the repeats for mal population, the number of CTG repeats in the these disorders. However, the larger expanded DMPK gene is between 4 and 37. The intermediate alleles get frequently missed by these traditional alleles with 35-49 triplets are not disease-causing PCR approaches and southern blotting is a very but show instability while getting transmitted to cumbersome and expensive method for routine

22 Chapter 5 diagnostics. Southern blot also need radioactiv- high sample throughput. (Warner et al.,1996) ity which most laboratories prefer to avoid. In The traditional PCR based approach is based on this review we describe a new modified triplet use of two primers (P1 and P2) flanking the repeats primed PCR (TP-PCR) approach for the identifica- followed by agarose gel electrophoresis and sizing tion and characterization of expanded alleles and of number of repeats (Figure 1A). a complete molecular diagnosis of FA, DM1 and The basic principle behind TP-PCR method is FRAX. that this method involves two rounds of PCR re- actions using 3 primers, P1, P3 and P4 (Figure 1B). The primer P1 is site specific and primer P4 consists of trinucleotide repeats and tail of primer P3. The properties of primer P3 are such that (a) it should contain little or no self complementarity A) (b) no homology to known human sequences. In the first round a locus specific primer P1anda fluorescently labelled primer P4 which contains the specific repeat sequences is used. This pair of primers produces products of varying sizes based on site of binding of primer P4 (Figure 1C). However, since a 10:1 molar ratio of P3 to P4 is B) maintained, primer P4 is exhausted in the early amplification cycles. In the latter cycles, primer P3 along with P4 undergoes the PCR reaction which results in PCR products of varying sizes and the extent of variation will depend on num- ber of repeats present (Figure 1D). The products are then resolved by capillary electrophoresis on C) an automatic sequencer to interpret the results. In presence of expansion of triplet repeats be- yond a threshold, the electropherogram shows a characteristic pattern of multiple peaks. Friedreich Ataxia

D) TP-PCR being a highly sensitive technique, can be used as a first step in the molecular diagnosis of FA as it can accurately detect the expansion of Figure 1 A) Illustration of binding of locus specific large expanded alleles which can be seen as a primers P1 and P2 during short PCR. characteristic ladder like pattern of the repeats in B) Illustration of binding of primers P1, the electropherogram (Ciotti et al., 2006). After P3 and P4 during TP-PCR. C) Amplifica- TP-PCR if there is an expansion of the repeats in tion during early PCR cycles by P1 and the pathogenic range ( 250 bp) then to confirm, P4. D) Amplication of the products of whether it is an affected> case or a heterozygous previous cycle by P3 and P4. carrier, a short PCR using locus specific primers P1 and P2 has to be performed, which will detect the normal alleles (Figure 2). Triplet primed PCR (TP-PCR) Although normal PCR with locus specific primers P1 and P2 detects the larger expanded allele, the TP PCR method has many advantages and suits results are not always consistent leading to a false well especially for rapid handling and testing of few negative diagnosis which can result in adverse samples as required in routine laboratory practice repercussions in the concerned families. Hence, by since the method is PCR-based, rapid, and not TP-PCR in combination with short PCR, genotype of labor intensive. The analysis can be done using a the affected patient, carrier and normal individuals small amount of DNA. It is a closed tube system can be accurately determined which will in turn that does not require post-PCR handling, and has a help in the accurate diagnosis and prenatal diag-

23 Chapter 5 nosis in the affected FA families. (Ciotti et al.,2006; which have a common 5’ sequence (tail) for the Cossee et al., 2001) TP-PCR amplification. The paired primer P4 has the sequence (TGC) at its 3’ terminus, specific to the triplet repeat to5 be amplified (Ciotti et al., 2006) The TP-PCR electropherogram of an affected DM1 individual shows a typical ladder of 3 base pair periodicity with the peak height diminishing with increasing product size (Figure 3). Role of TP-PCR is hence crucial in autosomal dominant dis- eases like DM1 where routine PCR most often fails to amplify the larger expanded alleles resulting in false negative results.

Figure 2 Upper panel A: Products of short PCR (2% agarose gel) L (1Kb ladder), lanes A) 1, 2 (normal cases showing single PCR product of approximately 200 bp), lane 3 (affected case showing absence of PCR product since the short PCR cannot amplify the region with large number of repeats) and electrophoregram of a normal case (TP-PCR) showing few peaks within 100 bp size. B) Lower panel B: Short PCR of affected cases showing absence of PCR products due to large expansion (2% agarose gel) Figure 3 A) Electrophoregram of a normal indi- L (1Kb ladder), Lane 1(Homozygous vidual (negative for DM1) showing few expansion), Lanes 2 and 3 (Heterozy- peaks of less than 100 bp. B) Elec- gotes showing absence of PCR product trophoregram of an affected DM1 case from normal allele) and right panel – showing peaks of varying sizes beyond electrophoregram of an affected case threshold. (TP-PCR) showing peaks due to PCR products of varying sizes well beyond the threshold of 250 bp. Fragile X syndrome

Southern blotting has been considered as a gold Myotonic dystrophy Type 1 standard when it comes to determine the size of large expanded alleles in FRAX. However, it is a The CTG expansions in a DM1 affected individual very time consuming, expensive and highly labor can be very large i.e., even greater than 4-5 kb. intensive procedure. Hence recently a fluorescent So usual short PCR fails to amplify such large ms-PCR assay for FRAX has been described that alleles and detects only the shorter allele. As classifies normal, premutation, and full mutation DM1 is an autosomal dominant disorder failure of affected males and females according to their amplification of the expanded allele in a short PCR unique electrophoregram patterns. (Zhou et al., poses quite a difficult situation. 2006) TP-PCR efficiently detects the larger CTG expan- Due to the difficulty in amplification of theGC sions even though it cannot size them. A locus rich CGG repeats in FMR1 the genomic DNA is specific primer P1 is used in combination witha modified according to published protocols. The pair of primers P3 and fluorescently labelled P4 bisulfite modified DNA is used in the mTP-PCR

24 Chapter 5 assay which employs three primers, a Ned-labelled overcomes the shortcomings of a traditional PCR forward primer upstream of the repeat (P1), an by detecting the very large expanded alleles of even unlabeled tailed reverse primer annealing within 5 kb accurately. Southern blot is a very tedious, the modified methylated repeat (P4) and a second >time consuming and expensive procedure which unlabelled reverse primer specific to the tailed (P3) requires very large amount of DNA to detect the segment of the first reverse primer. PCR prod- pathogenic expanded alleles. However, very small ucts following capillary electrophoresis detect all and poor-quality DNA also gives results accurately methylated alleles and produce a specific pattern in TP-PCR and it is a very rapid and fast process in the presence of a premutation or full mutation when compared with southern blotting. TP-PCR (Figure 4). (Zhou et al., 2006; Nygren et al., 2008) cannot size the expanded alleles for which one has to proceed for southern blotting. However, in rou- tine diagnostics for patient diagnosis and prenatal diagnosis, TP-PCR can act as a versatile technique to detect the expanded alleles in patients with triplet repeat diseases. References

1. Ciotti P, et al. Triplet repeat primed PCR (TP PCR) in molecular diagnostic testing for Friedreich ataxia. J Mol Diag 2004; 6: 285-289. 2. Cossee M, et al. Analysis of FRDA patients with interrupted GAA expansions in the frataxin gene by fluorescent triplet primed PCR. Eur JHum Genet 2001; 9: 403. 3. Nygren AO, et al. Methylation-Specific Multiplex Ligation-Dependent Probe Amplification En- ables a Rapid and Reliable Distinction between Male FMR1 Premutation and Full-Mutation Alle- Figure 4 Representation of results of mTP-PCR les J Mol Diag 2008; 10: 496-501. assay of normal, premutation and full 4. Warner JP, et al. A general method for the mutation males (Left panel) and fe- detection of large CAG repeat expansions by flu- males (right panel). orescent PCR. J Med Genet 1996; 33: 1022-1026. 5. Zhou Y, et al. Simplified molecular diagnosis of fragile X syndrome by fluorescent methylation- In conclusion, TP-PCR qualifies as a highly specific PCR and GeneScan analysis. Clin Chem reliable, sensitive and robust technique which 2006; 52: 1492-1500.

GeNeImage

Contributed by: Dr. Usha Dutta, Dr. Ashwin Dalal COLORS have Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics, revolutionized not Hyderabad just films but Email: [email protected] diagnostics too !!!!

25 Chapter 6

Mendelian Disease Gene Identification and Diagnostics using Targeted Next Generation Sequencing

Daniel Trujillano1, Rami Abou Jamra1, Arndt Rolfs1, 2 1Centogene AG, Rostock, Germany 2Albrecht-Kossel-Institute for Neuroregeneration, Medical University Rostock, Rostock, Germany

Correspondence to: Dr Daniel Trujillano Email: [email protected]

During the last few months we have observed for most of them, that disease gene associations for the first time since the introduction of the for most of the common disorders would have first massive parallel sequencers in 2007, that been established, and that personalized preven- the cost of sequencing a human genome has tive medicine would be a reality. Although some not changed significantly (Figure 1) [Wetterstrand., of his claims have come true, most of the post- 2015]. These numbers challenge a trend that genomic era promises are yet to be accomplished has been maintained during years beating Moore’s [Collins., 2010]. law, and indicates that in the medium term we should not expect sequencing to be significantly cheaper until the next technological revolution arrives. This can have profound consequences in the genomics field, which has heavily relied during the last years on the fact that each sequencing run was cheaper than the last. Most of the recent big genomic achievements have been based on brute force experiments, made possible by the rapid technological advances. The present change of cycle would require more ingenious and elegant ideas to keep publishing interesting findings after the genomics boom of the last decade. So, it is time to fully exploit the potential of the current sequencing technologies, which are likely to be static for a while. Moreover, it is also time to tone down the rhetoric around the advent of $1000 human genomes and to start working seriously on the clinical translation of our research based on Figure 1 Evolution of the cost of sequencing a the technology that we currently have, not what we human genome. From [Wetterstrand, expect to have tomorrow [Hall, 2013]. 2015]. Current next generation sequencing technolo- Genomics as an enhanced approach to health- gies have the potential to address the mismatch care has the potential to transform the quality between promises and achievements in medical of life worldwide, allowing the widespread imple- genetics, still present more than ten years since mentation of more tailored medical care based on the human genome project was drafted. Shortly individual risk. It seems quite likely that whole before the completion of the first human genome, human genome sequencing (and eventually, pro- Francis Collins, one of the leaders of the Human teome, metabolome, microbiome, etc.) would be Genome Project predicted that by 2010 the genetic a routine component of everyone’s health record causes of most Mendelian diseases would have available to both patients and physicians for pre- been unveiled and therapies would be available dictive and purposes. This

26 Chapter 6 is poised to have a transforming effect in clinical the reference genome and to align de novo se- practice, including diagnosis and decision-making quences that are missing in the reference genome for appropriate therapeutic procedures. sequence. Since DNA sequence variants may vary from single nucleotides up to several kb, specific Identification of Mendelian disease algorithms have been developed for single base substitutions, insertions/deletions and structural genes by exome sequencing variants.

During the last decades, positional cloning strate- gies have led to the discovery of several new insights in human genetics. However, when this approach is used for rare Mendelian disorders, the results often are not conclusive due to the lack of complete pedigrees, the unavailability of large family collections and marked locus heterogeneity. Thus, in small and non-consanguineous families, neither linkage analysis nor homozygosity mapping are likely to succeed in identifying the responsi- ble gene of a given rare Mendelian disease. As a consequence of these limitations, the underly- ing genetic cause of more than three thousand disorders of Mendelian inheritance still remain to be determined (http://www.ncbi.nlm.nih. gov/Omim/mimstats.html). In that respect, advances in next generation se- quencing technologies, especially exome sequenc- ing, represent an important milestone in genomics, Figure 2 Overview of the bioinformatics analy- providing an effective alternative for the discovery ses for NGS data. From the mapping of candidate genes and mutations that underlie of the raw sequencing reads to the Mendelian disorders that have been resistant to annotation of the detected variants. conventional approaches, thanks to an unprece- dented ability to identify rare variants. Next Once the variant calling process of a given sam- generation sequencing technologies have been a ple or project is finished, the next task is the an- much awaited step forward from linkage mapping notation of each detected sequence variant (Figure for Mendelian disease gene discovery, since it has 2). During this process information regarding the enabled mapping of genes for monogenic traits in alignment of the variant to a specific base position families with small pedigrees and even in as few in a gene, the in silico assessment of the variant’s samples as two unrelated individuals [Lalonde et potential to disrupt gene function (“pathogenicity”) al., 2010]. [Kumar et al., 2009; Schwarz et al., 2010], and the presence of the variant in databases such as db- Next generation sequencing bioinformatics: SNP, 1000 Genome project, Exome Variant Server Due• to the sheer magnitude of the genomic etc are gathered and recorded. Several annotation information produced by the current next gen- tools are available, such as the Genome Analysis eration sequencing equipment (several hundred Toolkit (GATK) [McKenna et al., 2010], SeattleSeq Gigabases can now be generated in just one se- (www://gvs.gs.washington.edu/), or ANNOVAR quencing run), the experimental bottleneck has [Wang et al., 2010], among many other. shifted from data acquisition towards its correct storage and processing. Efficient methods to align Disease gene/variant filtering strategies: millions of short-read sequences to the human Next• generation sequencing technologies produce genome (matching the short reads to a preexisting sheer numbers of genotype calls on the order of reference genome) and the calling of variants (de- per exome, for the combined exomes of a termination of the best guess for the genotype, or small4 family, and 5 per genome. Thus, after data 10 10 6 other sequence feature, at each aligned position) acquisition and variant10 calling the main challenge have been developed, allowing to access most of in the downstream analysis of next generation se-

27 Chapter 6 quencing data is to “winnow” the list of variants to downstream analyses are focused on the iden- be able to differentiate known and potential novel tification of very rare or novel loss-of-function disease-causing mutations (the “wheat”) from both mutations that introduce truncations to the en- technical artifacts and benign genetic variation (the coded protein, i.e. nonsense and non-synonymous “chaff”). variants, splice acceptor and donor site mutations and coding InDels, anticipating that synonymous variants are far less likely to be pathogenic. This filtering strategy, which has been successfully ap- plied in several studies, substantially reducing the list of candidate variants, making feasible their individual confirmation prior to expression and functional testing (Figure 3). Another parameter that has to be taken in to account during variant/gene prioritization is the inheritance pattern, since for autosomal dominant disorders each gene must show at least one po- tentially causative variant per individual, whereas in autosomal recessive disorders, candidate genes must have either homozygous or compound het- erozygous mutations (Figure 4) [Robinson et al., 2011].

Figure 3 Variant prioritization process for Mendelian disorders.

Depending on the capture design and the depth of coverage, an average exome contains between five to ten thousand variant calls representing Figure 4 Strategies for Mendelian disease gene either non-synonymous substitutions in protein identification. coding sequences, small InDels, or alterations of the canonical splice-site dinucleotides (NS/SS/I), being between 100 and 200 homozygous protein All in all, the variant filtering strategy must be truncating or stop loss variants. Thus, the mere flexible enough to allow adjustment of all analytic identification of an apparently causative variant parameters. But even more importantly, those cannot be taken as a proof that it is relevant to the performing the analysis must understand the ra- disease being investigated, and additional variant tionale, procedures, and assumptions inherent in filtering and functional analyses are required to each step. assign causativeness. When exome sequencing is applied to Exome sequencing example in a diagnostics Mendelian disorders, the filtering strategy is de- setup:• Exome sequencing is a powerful tool and signed to highlight rare or de novo, high penetrance is often the only possibility to clarify the cause(s) of protein-modifying mutations responsible for a disorders. As an example, we recently performed large phenotypic effect, as well as all variants exome sequencing in an 18 years old male index previously associated with the disease. Thus, the patient and his consanguineous parents. The index

28 Chapter 6 patient presented with short stature, bilateral nys- variants because it involves a drastic reduction tagmus, cerebellar ataxia, and intellectual disability in the sequencing required. In fact, as opposed (Figure 5). Bioinformatic analysis and medical eval- to whole genome sequencing, exome sequencing uation revealed a candidate mutation in the gene requires about 20-fold less ( 5%) sequencing to KIF1C. The variant is a missense, is rare in public achieve the same depth of∼ coverage, which is databases and is predicted to be pathogenic by sev- translated into considerably less raw sequence and eral pathogenicity prediction programs. Mutations lower costs. Despite the inherent costs of genomic in KIF1C lead to the autosomal recessive spastic capture in addition to sequencing, according to ataxia 2 with horizontal nystagmus, distal muscle the list prices, the all-in cost of exome sequenc- atrophy, cerebellar gait ataxia, tremor, spasticity of ing is roughly 10- to 20-fold less than for the the lower limbs, cerebellar atrophy (in some pa- whole genome. Also, exome sequencing requires tients) and it has the onset in teenage years. Given less complicated analyses than whole genome se- the results of the exome sequencing, we concluded quencing, and the number of variants detected that the phenotypic spectrum of the patient can be is up to two orders of magnitude lower as a clarified to a large extent via this variant; it clarifies consequence of only retrieving variants affecting the nystagmus and the cerebellar ataxia, as well the coding regions of the genome. This reduces as the intellectual disability. The etiology of short data fatigue and simplifies the analyses for the stature is still not clarified. If no exome sequencing identification of disease-causative variants. was performed, the would follow the In addition to the technical limitations inherent medical rule of finding one cause of a phenotypic to genomic enrichment, such as selection bias spectrum, and would thus go for other differential and uneven capture efficiency, the main limita- diagnoses that include short stature. This would tion of the targeted resequencing approach is the be, however, misleading, and only through analysis impossibility to efficiently capture and sequence of the whole exome we were able to find that a the repetitive and low-complexity, and GC-rich ge- symptom of the patient is not correlated to the nomic sequences that are refractory to enrichment. rest of the phenotype. It may be possible that the However, the constant optimization of the capture phenotypic spectrum of mutations in KIF1C may and next generation sequencing chemistries will get extended to short stature too. gradually close the capture gaps (mainly due to uniqueness constraints, homopolymer runs, am- biguous bases or other factors that are known to cause issues in either oligonucleotide synthesis or hybridization), and reduce enrichment variability between samples and targets. Exome sequencing has proven its reliability for the identification of genetic variability underlying relatively simple, single-gene disorders. How- ever, the step from rare monogenic and simple Mendelian disorders to more-complex multigenic disorders is going to be a challenging move. Exome sequencing studies done so far have to be consid- ered as a starting point in the effort to apply these technologies to multigenic diseases. The extent of heterogeneity associated with common complex disorders will have to be mitigated with larger sample sizes and more sophisticated weighting of non-synonymous variants by predicted functional impact. Figure 5 Pedigree of the studied family. Concluding remarks

Exome sequencing advantages and disadvan- Currently, our ability to discover genetic variation tages:• Exome sequencing represents the most in a patient genome is running far ahead of our cost-effective alternative to whole genome se- ability to interpret that variation. The success of quencing for the discovery of highly penetrant rare next generation sequencing for medical genetics

29 Chapter 6 hinges on the accuracy in distinguishing causal other area of biomedical research. Especially, from benign alleles, which is the key challenge for the advances in the field of next generation se- interpreting DNA sequence data for diagnostics. quencing development and applications, make this Over the last three decades, PCR amplification of an exciting time for the study of how genetic target regions followed by Sanger sequencing has variation affects health and disease. The ulti- been the gold standard for the identification of mate game changer in clinical genetics will be clinically relevant mutations in the terms of routine the routine sequencing of individual genomes, but diagnostics. It offers great accuracy, at the expense until this becomes feasible, targeted approaches of being laborious and costly, especially when it are the more convenient interim solution. The comes to the analysis of disorders of heteroge- standardization and further development of the neous etiology for which multiple targets/genes methods described here will provide powerful and might be tested in a stepwise fashion. Such dis- cost-effective techniques for the identification of orders may require extensive screening of several causative variants of heritable disorders caused by genes, using different molecular approaches for known and unknown genes. every type of sequence variant being tested. However, this rather costly, stepwise, and time- consuming technology will be gradually replaced References by next generation sequencing technologies, which offer higher throughput and scalability and, as 1. Collins F. Has the revolution arrived? Nature a corollary, have reduced costs per sequenced 2010; 464, 674-675. nucleotide and shorter turnaround time. Given the 2. Hall N. After the gold rush. Genome Biol 2013; current cost of targeted next generation sequenc- 14, 115. ing of small genomic regions, it is inciting to use 3. Kumar P, et al. Predicting the effects of coding next generation sequencing approaches to screen non-synonymous variants on protein function these genes for diagnostics purposes. using the SIFT algorithm. Nat Protoc 2009; 4, The transition over the next years of next gener- 1073-1081. ation sequencing technologies from basic research 4. Lalonde E, et al. Unexpected allelic hetero- to the routine detection of mutations in genetic geneity and spectrum of mutations in Fowler loci with well documented diagnostic value will syndrome revealed by next-generation exome take advantage not only of the new benchtop next sequencing. Hum Mutat 2010; 31, 918-923. generation sequencing platforms which can be 5. McKenna A, et al. The Genome Analysis Toolkit: much more easily incorporated in the daily clinical a MapReduce framework for analyzing next- practice, but also of automated workflows and generation DNA sequencing data. Genome Res simplified bioinformatics analyses able to gener- 2010; 20, 1297-1303. ate medical report-like outputs adapted to clinical 6. Pop M, et al. Bioinformatics challenges of new laboratories. However, the correct interpretation, sequencing technology. Trends Genet 2008; storage, and dissemination of the large amount of 24, 142-149. the datasets generated remain a major challenge 7. Robinson PN, et al. Strategies for exome and on the path of next generation sequencing to medi- genome sequence data analysis in disease- cal applications [Pop et al., 2008]. These challenges gene discovery projects. Clin Genet 2011; 80, could be addressed with extensive exchange of 127-132. data, information and knowledge between medi- 8. Schwarz JM, et al. MutationTaster evaluates cal scientists, sequencing centers, bioinformatics disease-causing potential of sequence alter- networks and industry. Some genomic centers ations. Nat Methods 2010; 7, 575-576. working in biomedicine have developed collabora- 9. Wang K, et al. ANNOVAR: Functional annota- tive initiatives aiming at bringing everyone together tion of genetic variants from high-throughput to harmonize genomic medical research, set up sequencing data. Nucleic Acids Res 2010; 38, standards in medical sequencing and review the e164. current diagnostic standards according to the new 10. Wetterstrand KA. DNA Sequencing Costs: insights gained from genomic and phenotypic data Data from the NHGRI Large-Scale Genome integration. Sequencing Program. https://www.genome. Genomics is making faster progress than any gov/sequencingcosts/ (accessed April 2015)

30 Chapter 7

Diagnostic Yield of Exome Sequencing in Neurodevelopmental Disorders

Moirangthem Amita, Shubha R Phadke Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow

Correspondence to: Dr Shubha R Phadke Email: [email protected]

Introduction al., 2011; Moeschler et al., 2014). Cytogenetic microarray which is now considered to be the first Neurodevelopmental disorders affect around 3-5% line test in the diagnosis of non-specific intellectual of the population. These are heterogeneous in disability has a yield of 10-20%. This “diagnostic etiology and more befittingly termed “symptom odyssey” lasts many years and more than half of complexes”. The various disorders presenting the patients never receive an etiological diagnosis, with intellectual disability may be congenital or thereby adding to the pain and disappointment acquired, sporadic or familial, syndromic or non- besides the cost incurred by the family. In a survey syndromic. Severe neurodevelopmental disorders of patients with rare diseases it was found that for are mostly genetic in origin and may be due to 25% of participants, the time to diagnosis was ex- a molecular defect at the chromosomal or single tensive, ranging from 5 to 30 years, and during that gene level or due to epigenetic abnormalities. Till time 40% received an incorrect diagnosis (Sawyer date more than 450 genes have been implicated et al., 2016). in intellectual disability. One may wonder as to what is the need for an etiological diagnosis when Whole Exome Sequencing and its Utility majority of these cases cannot be cured. However, diagnosis is a key element in the management of in Neurodevelopmental Disorders the patient, for explaining the course and prog- nosis, and for provision of appropriate care and With the advent of whole exome sequencing (WES), support system. It precludes subjecting the patient there came a paradigm shift in the approach to the to unnecessary and redundant tests and interven- diagnosis of rare diseases, with timely diagnosis tions. It is essential for proper counseling of the of genetic diseases and discovery of new disease- family regarding the recurrence risks and prenatal causing genes. WES uses a high throughput diagnosis, and also for access to research treat- sequencing technique of next generation sequenc- ment protocols. The family’s “need to know” has ing (NGS) to sequence coding regions of all genes an enormous emotional impact and cannot be ig- in the human genome. This helps to identify nored. The elucidation of the underlying molecular the causative gene/ mutation even if the clinical abnormalities is another important step towards evaluation and supportive investigations do not developing treatment strategies (Willemsen et al., provide any clue to the etiology and the causative 2014). genetic defect. This approach is being used for all genetic phenotypes with known or unknown causative genes. The study by Retterer et al. (2016) Diagnostic Approaches on the use of WES on 3040 consecutive cases gave a high diagnostic yield for patients who had Various diagnostic practice guidelines are available disorders involving hearing (55%, N = 11), vision for evaluation of patients with neurodevelopmental (47%, N = 60), the skeletal muscle system (40%, N disorders and consist of clinical evaluation, neu- = 43), the skeletal system (39%, N = 54), multiple roimaging, metabolic profile, cerebrospinal fluid congenital anomalies (36%, N = 729), skin (32%, N examination and specific genetic tests dictated by = 31), the central nervous system (31%, N = 1,082), the clinico-biochemical phenotype (Michelson et and the cardiovascular system (28%, N = 54). Here,

31 Chapter 7 we review the diagnostic yield of WES in neu- clinical expertise of syndrome recognition, causing rodevelopmental disorders. In cases of moderate a shift from a “phenotype first’’ to a “genotype to severe neurodevelopmental disorders, different first’’ approach. Besides, the impact on families in studies have reported a diagnostic yield ranging ending the expensive, often invasive, and stress- from 16-45% using WES (de Ligt et al., 2012; Yang ful diagnostic odyssey cannot be overemphasized. et al, 2013; Yang et al., 2014; Sawyer et al., 2016). However, in both the cases detailed phenotyping WES out-performs conventional approaches in the remains a very important step in the diagnostic diagnosis of disorders with genetic heterogeneity approach to a case with a neurodevelopmental with phenotypic variability and poor specificity. disorder and the clinician has a very important role Besides its utility in the diagnosis of ultra-rare to play in manually judging the candidate sequence conditions and novel gene discovery, it also has an variations identified by mining the exome data. impact on expanding the phenotypic spectrum of already known syndromes. Most importantly, WES allows for re-assessment of data in the light of new Issues Related to WES in Diagnostics knowledge, thus providing additional diagnostic results over years without significant extra costs. In addition to the high cost, the vast amount of WES is an effective approach in the evaluation of data, which is a challenge for analysis and storage, cases with sporadic non-specific ID using family is a major problem being faced. The advantage based strategy (child-parents trios) with yields of of WES is that the need of a clinical differential 35-55% (Willemsen et al., 2014). The use of WES diagnosis is not a must and WES can identify the is not only restricted to identification of these de etiology in a case with a nonspecific or a subtle novo variants but its emerging widespread use phenotype. The same advantage of covering all the in predominantly consanguineous populations has genes poses challenges for analysis analysis as in led to the expansion of the repertoire of genes some cases more than one pathogenic sequence causing autosomal recessive neurodevelopmental variant may be identified or many variations of disorders. Recently, a study in 121 consanguineous unknown significance are identified. These issues families identified pathogenic variants in 68, out will gradually get minimized or resolved as more of which a novel gene was implicated as causative and more parts of the genome get annotated in 30 (Riazuddin et al., 2016). The diagnostic and databases of pathogenic and non-pathogenic yield of WES in cohorts with neurodevelopmental variations become more comprehensive. One of disorders in various studies has been summarized the reservations which many physicians have in in Table 1. While the effectiveness of WES in the implementing this approach is the high cost of diagnosis of intellectual disability has been proven WES. A recent study comparing the cost of WES beyond doubt, the optimal timing of when it should with conventional diagnostic approaches in a co- be done is still debatable. Whether WES should hort of individuals with intellectual disability found be at the first appointment if clinically indicated that the traditional diagnostic trajectory cost was or after the initial tests are normal (e.g. first tier $16,409 per patient while the trio-WES cost was of genes ruled out) or towards the end of the $3,972 only. They concluded that WES resulted diagnostic odyssey (e.g. after extensive, possibly in average cost savings of $3,547 for genetic and invasive tests have occurred), needs to be evalu- metabolic investigations in diagnosed patients and ated. The newer school advocating use of WES $1,727 for genetic investigations in undiagnosed as a first-tier test have undertaken some studies patients (Monroe et al., 2016). Another concern using genomic tools (WES alone or with molecular is the inability of WES to detect copy number karyotyping) in the diagnosis of ID patients. They variations (CNVs), which cause a large proportion have reported diagnostic yields of 32-58% (Anazi et of intellectual disability. But in recent years, there al., 2016; Thevenon et al., 2016). Diagnostic yield as have been many publications evaluating the utility high as 91% has been reported in consanguineous of WES in detecting CNVs by various algorithms. families (Shaheen et al., 2016). Notably, many of These studies have reported a fair rate for de- the molecular defects were not suspected clinically, tection of CNVs, 59 to 89% with the sensitivity highlighting the power of this tool to overcome increasing with CNVs 200kb in size (Tetreault et the limitations of clinical phenotyping. This has al., 2015; Miyatake et> al., 2016). In the current set a trend of “reverse phenotyping”, whereby the situation, WES as a first-tier test in neurodevelop- success of identification of clinical recognizable ID mental disorders appears to be an attractive option syndromes will no longer be highly dependent on especially in families with consanguinity, multiple

32 Chapter 7

Table 1 Summary of the diagnostic yield of WES in intellectual disability (ID) in various studies.

Study Cohort Timing of WES Diagnostic yield de Ligt et al., 100 patients with se- Trios WES Diagnostic yield=16%, 79 de 2012 vere ID All patients with prior ex- novo mutations in 53 /100, tensive genetic diagnostic Potential causative variants work up in 22 novel genes Yang et al., 250 patients (80% All patients had undergone Diagnostic yield – 62/250 2013 with neurological phe- prior genetic testing (mi- (25%), autosomal dominant notype) croarray, metabolic screen- =33, autosomal recessive ing and specific gene se- =16, X-linked disease=9, quencing) Four probands with 2 non- overlapping diagnoses Yang et al., 2000 patients, 87.8% All patients had under- Diagnosis in 504 (25.2%), 2014 with neurological phe- gone some prior diagnos- novel variants in 58%, 6 pa- notype tic workup including specific tients with large deletions, genetic tests 23 had mutations at 2 dif- ferent loci Willemsen et al., 253 individuals from 2 phases- diagnostic (spe- Phase I - diagnosis in 43 2014 234 Dutch families with cific genetic test, microar- (18.4%). unexplained ID ray, metabolic screen) and Phase II - pertinent/plau- research phase (NGS in 30% sible diagnosis in 24 /58 of undiagnosed patients in (41.4%) in NGS cohort. phase I) Total diagnostic potential combining both phases = 59.8%. Sawyer et al., 500 children from All had already received Mutations in known dis- 2015 >362 families with standard of care genetics ease genes for 105 rare genetic diseases, evaluation of 362 families studied mainly neurodevelop- (29%), Neurodevelopmen- mental and dysmor- tal phenotype- yield was phism disorders 31.6% Riyazuddin et al., 121 large consan- All patients had clinical Total yield - 68 families 2016 guineous Pakistani +/- neuroimaging evalua- (56.2%) families with ID tion. No prior genetic test Novel genes - 30 families was done Anazi et al., 337 ID subjects, high Molecular karyotyping, Standard clinical evaluation 2016 prevalence of consan- multigene panels, WES suggested a diagnosis in guinity were used as first-tier tests, 16% (54/337), only 70% compared with standard of these (38/54) were con- clinical evaluation done in firmed. parallel Genomic approach re- vealed a likely diagnosis in 58% (n = 196). These included CNVs in 14% (n = 54, 15% are novel), and point mutations in remain- ing 43%

Note: ID – Intellectual Disability, CNV – Copy Number Variations

33 Chapter 7 affected members, clinical features suggestive of exome sequencing will fill the niche of being the a monogenic phenotype with no obvious cause most versatile, relatively inexpensive and hence and after the cytogenetic microarray is normal. popular application of NGS in the clinic (Tetreault More and more experience and data will provide et al., 2015). Its application early-on in the answers to today’s challenges of technical issues evaluation process of patients with non-specific (like coverage), accurate interpretation of the huge intellectual disability will have a significant impact data generated, incidental findings, functional vali- in ending the “diagnostic odyssey”. dation of novel variants, and need for more robust algorithms for CNV detection. References To get the estimate of clinical utility of WES in the diagnosis of genetic phenotypes, a lot of 1. Anazi S, et al. Clinical genomics expands the work is needed and has already been started. The morbid genome of intellectual disability and important one to be mentioned here is the Clinical offers a high diagnostic yield. Mol Sequencing Exploratory Research (CSER) consor- 2016. doi: 10.1038/mp.2016.113. [Epub ahead tium which includes eighteen projects which not of print] only are exploring clinical utility and clinical validity 2. Bowdin SC, et al. The Sick Kids Genome of clinical genome and exome sequencing, but is Clinic: developing and evaluating a pediatric also looking at the ethical, social and legal impli- model for individualized genomic medicine. cations via multidisciplinary approaches (Green et Clin Genet 2016; 89:10-19. al, 2016). Similar efforts being done are ‘Genome 3. Douzgou S, et al. Dysmorphology services: a Clinic Task Force’ (Fokstuen et al., 2016) and the snapshot of current practices and a vision for ‘SickKids Genome Clinic’ (Bowdin et al., 2016). The the future. Clin Genet 2016; 89: 27-33. analysis of these big studies will provide answers 4. Green RC, et al. CSER Consortium. Clinical to the questions about appropriate use and timing Sequencing Exploratory Research Consortium: of the WES-based diagnosis which is powerful but Accelerating Evidence-Based Practice of Ge- costly and has some issues which need to be nomic Medicine. Am J Hum Genet 2016; 98: sorted. 1051-1066. 5. Fokstuen S, et al. Experience of a multidisci- plinary task force with exome sequencing for Conclusions and Future Perspective Mendelian disorders. Hum Genomics 2016; 10: 24. Diagnosis of genetic disorders is an arduous and 6. de Ligt J, et al. Diagnostic exome sequencing challenging task. The armamentarium of advanced in persons with severe intellectual disability. N genetic testing is improving the etiological diag- Engl J Med 2012; 367: 1921-1929. nosis and thus helping the families. Cytogenetic 7. Michelson D J, et al. Evidence Report: Ge- microarray is considered the first tier test for netic and metabolic testing on children with evaluation of a child with a neurodevelopmental global developmental delay: Report of the disorder. However, it should be noted that de- Quality Standards Subcommittee of the Amer- tailed clinical evaluation, appropriate imaging and ican Academy of and the Practice biochemical investigations constitute the first step Committee of the Child Neurology Society. in the direction of diagnosis. As is the experience Neurology 2011; 77: 1629. of clinical geneticists, a study of different clinical 8. Miyatake S, et al. Detecting copy-number vari- genetics centres has shown that in patients with ations in whole-exome sequencing data using dysmorphism, the diagnosis is achieved in the the eXome Hidden Markov Model: an ‘exome- first visit in 30 to 60% of cases (Douzgou etal., first’ approach. J Hum Genet 2015; 60:175-182. 2016). However, WES offers the hope of diagnosis 9. Moeschler JB & Shevell M. Committee on Ge- in many cases where there was none. In the fu- netics. Comprehensive evaluation of the child ture, whole genome sequencing (WGS) is expected with intellectual disability or global develop- to eventually replace WES and even cytogenetic mental delays. Pediatrics 2014; 134: e903-918. microarray, as it is the single genetic test which 10. Monroe GR, et al. Effectiveness of whole- has the potential to detect the whole spectrum of exome sequencing and costs of the tradi- genetic aberrations ranging from single nucleotide tional diagnostic trajectory in children with variations to complex genomic rearrangements. intellectual disability. Genet Med 2016. doi: However, at present and during the next few years, 10.1038/gim.2015.200. [Epub ahead of print].

34 Chapter 7

11. Retterer K, et al. Clinical application of whole- as a diagnostic tool: current challenges and exome sequencing across clinical indications. future opportunities. Expert Rev Mol Diagn Genet Med. 2016; 18: 696-704. 2015; 15: 749-760. 12. Riazuddin S, et al. Exome sequencing of 16. Thevenon J, et al. Diagnostic odyssey in se- Pakistani consanguineous families identifies vere neurodevelopmental disorders: toward 30 novel candidate genes for recessive in- clinical whole-exome sequencing as a first-line tellectual disability. Mol Psychiatry 2016.doi: diagnostic test. Clin Genet 2016: 89: 700-707. 10.1038/mp.2016.109. [Epub ahead of print]. 17. Willemsen MH, Kleefstra T. Making headway 13. Sawyer SL, et al. Utility of whole-exome se- with genetic diagnostics of intellectual disabili- quencing for those near the end of the diag- ties. Clin Genet 2014; 85: 101-110. nostic odyssey: time to address gaps in care. 18. Yang Y, et al. Clinical whole-exome sequencing Clin Genet 2016; 89: 275-284. for the diagnosis of mendelian disorders. N 14. Shaheen R, et al. Accelerating matchmaking Engl J Med 2013; 369: 1502-1511. of novel dysmorphology syndromes through 19. Yang Y, et al. Molecular findings among patients clinical and genomic characterization of a large referred for clinical whole-exome sequencing. cohort. Genet Med 2016; 18: 686-695. JAMA 2014; 312: 1870-1879. 15. Tetreault M, et al. Whole-exome sequencing

PhotoQuiz - 1 Contributed by: Dr Prajnya Ranganath Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad Email: [email protected]

This 10 months-old male infant presented with global developmental delay, facial dysmorphism, abnormal skull shape and mild hepatomegaly. The skeletal radiographs and metabolic test results are shown. Identify the condition.

Answer on page 231

35 GeNeToon

Contributed by: Dr. Shagun Aggarwal Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad Email: [email protected]

36 Section 2 Genetic Approach to Common Clinical Scenarios

Chapter 8

Approach to a Child with Dysmorphism/ Congenital Malformation Prajnya Ranganath Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad

Correspondence to: Dr Prajnya Ranganath Email: [email protected]

Definition neural tube defect, ventricular septal defect, polydactyly etc. Dysmorphology is a discipline of clinical genetics which deals with the study of abnormal patterns Deformation: Distortion of a normally devel- of human growth and with the recognition and • oped structure caused by mechanical forces study of congenital human structural anomalies usually in the latter half of gestation and most and patterns of birth defects. often involving musculo-skeletal tissues. E.g. Congenital malformations/ birth defects can be club foot, torticollis, plagiocephaly etc. sub-classified as major or minor anomalies. Disruption: Breakdown of an intrinsically nor- • mally developing/ developed tissue due to Major anomalies are those that interfere with some disruptive event such as a mechanical, • the normal functioning of an individual and vascular or infectious insult. E.g. amniotic pose a significant health problem or risk to band sequence. life. E.g. congenital heart defects, neural tube defects, , cleft palate etc. Dysplasia: Abnormal cellular organization within a tissue, almost always of genetic Minor anomalies do not interfere with the • cause. E.g. skeletal dysplasias. • normal functioning of an individual and usu- ally are only of cosmetic significance. E.g. A syndrome is a recognized composite pattern simian crease, accessory nipple, clinodactyly, of 2 or more anomalies with a common specific pre-auricular skin tag etc. aetiology. E.g. Turner syndrome, fetal phenytoin syndrome etc. Major anomalies are present in 2-3% and minor An association is a non-random occurrence of anomalies are present in around 15% of live births. 2 or more anomalies that occur together more Minor anomalies are usually associated with an frequently than expected by chance alone, but increased risk of associated major anomalies and without a known specific aetiology. E.g. VACTERL therefore presence of minor anomalies should (vertebral defects, anal atresia or stenosis, cardiac prompt a thorough search for associated major defects, tracheo-esophageal fistula, radial defects anomalies. and renal anomalies, limb defects) association. A sequence is a pattern of anomalies result- Classification of congenital anomalies ing from a single primary anomaly or factor E.g. Congenital anomalies are classified, on the basis Potter sequence (Primary anomaly - bilateral renal of the developmental stage in which the insult aplasia/ dysplasia decreased fetal urine pro- occurred, the process that caused the change and duction severe oligohydramnios→ compressive the end result, into: effects → flattened facies with→ flattened nose, deformed→ ears, pulmonary hypoplasia & positional Malformation: Primary intrinsic developmen- limb defects). • tal defect usually caused by genetic/ envi- ronmental/ multi-factorial causes (recurrence Approach to a case with dysmorphism risk varies accordingly) which occur during the period of organogenesis which is up to 8 The following step-wise clinical approach should weeks post fertilization for most organs. E.g. be followed in the assessment and management

37 Chapter 8 of an individual with dysmorphism or congenital Neonatal course: malformation(s): • – Feeding and activity 1. Suspicion of a genetic etiology – Any adverse events/ complications 2. Clinical evaluation Post neonatal: history • • physical examination – Physical growth 3. Investigations• – Developmental milestones 4. Analysis and diagnosis – Neurological symptoms especially 5. Confirmation seizures / visual or hearing deficits/ 6. Intervention: behavioural phenotype treatment – Other systemic symptoms • counseling Family history: prenatal diagnosis • • – At least three generation family history / 7. Surveillance• & follow up pedigree – History of recurrent pregnancy losses/ Suspicion of a genetic etiology infertility A genetic aetiology should be suspected in any – Specific information/ medical records of individual with the following: other affected family members – Consanguinity in parents Congenital anomalies: at least 1 major/ – Ethnic background • minor anomalies. > 2 Growth deficit (short stature/ failure to thrive) Physical examination • Developmental delay, intellectual disability or A thorough clinical examination must be done • developmental regression taking the following aspects into consideration: Failure to develop secondary sexual charac- General principles: • teristics • Abnormal genitalia – Thorough head to toe examination to be • Appears ‘different’/ ‘unusual’ done. – Measurements to be taken and com- • History pared with standard tables/ graphs of age and gender norms. A detailed history covering the following aspects – Both parents and other available family should be obtained: members to be examined for similar or Prenatal history: related features. – Clinical photographs to be taken with • – Teratogenic exposures especially in the informed consent of individual/ parent/ first trimester of pregnancy: infections/ guardian: for records, syndrome search, medications/ drugs of abuse/ maternal referral and study of evolution of the illness/ radiation exposure phenotype. – Prenatal complications and antenatal ul- Anthropometric measurements: trasonographic findings • – Height/ length, weight, head circumfer- Perinatal history: ence • – Presentation/ mode/ complications of Assessment of proportionality & symmetry: delivery • – and condition (Apgar – Upper segment/ lower segment ratio score) at birth – Arm span – Birth weight, birth length and head cir- – Individual limb segment measurements cumference; body proportions (in specific cases)

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a) b) c)

Figure 1 Typical gestalt of some syndromes. a) Down syndrome; b) Russell-Silver syndrome; c) .

Head to toe assessment: (for exact descrip- – Forehead - Size: small/ broad/ tall; • tion of each feature refer to Am J Med Genet Shape: sloping/ frontal bossing/ bitem- A 2009 Jan; 149A (1) & Aase JM Diagnostic poral narrowing/ metopic prominence; Dysmorphology textbook). Supraorbital ridges: prominent/ under- developed – Each body part to be examined carefully from head to feet to look for anomalies – Maxilla/ midface – – Cranium – size; fontanelles; sutures; * Cheek bone: prominent/ underde- shape and symmetry veloped/ fullness – Scalp hair - colour and texture; distri- * Malar region: prominence/ flatten- bution; hair whorl patterns; position of ing anterior and posterior hairline * Midface: prominence/ retrusion – Face * Nasolabial folds: prominent/ under- developed * overall impression of facial appear- ance: gestalt e.g. Down syndrome – Mandible - size & shape: micrognathia/ facies, coarse facies, myopathic fa- retrognathia/ prominence cies. See figure 1. – Eyes- eyebrows; palpebral fissure length * overall shape, symmetry and size of (short/long); palpebral fissure slant face: triangular/ broad/ round (up/down); epicanthic folds; eye spac- * face to be divided into sections: ing (use a rough guide of 1:1:1 for ratio forehead, midface and oral region of left palpebral fissure length: inner * face to be viewed from front and canthal distance: right palpebral fissure from side length); palpebral fissure shape; iris * lateral profile better for: depth or colour; pupil shape; cornea/ sclera/ lens; height of structures such as nasal globe position (assessed from lateral bridge, position of mandible relative view: protuberant vs deep set globes). to maxilla and midface development See figure 2. – Facial measurements: – Nose- nasal root; nasal bridge: de- pressed/ prominent/ broad; nasal tip: * Interpupillary distance, inner can- thal distance, outer canthal distance, broad/ flattened; columella (the vertical interalar distance, philtral length, up- ridge separating the nostrils): wide/ over- per lip thickness, lower lip thickness, hanging; nostrils: patency and position intercommisural distance (anteverted); alae nasi. See fig 3. * Measurements to be compared to – Mouth and perioral region - mouth size age and sex norms ( or 2SD and shape; upper and lower lip shape abnormal) < > ⇒ and thickness; gum thickness; philtrum

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a) b) c) d)

Figure 2 Dysmorphic findings in the eyes. a). Upslanting palpebral fissures and epicanthal folds; b). Downslanting palpebral fissures; c). Blepharophimosis and epicanthus inversus; d). Telecanthus.

definition and length; jaw position (prog- * Phallus size and morphology nathia/micrognathia); palate shape * Development, rugosity & pigmenta- – Oral cavity - teeth/ frenulum/ tongue size tion of scrotum and morphology * Size and position of testes – Ears * Development of labia * Position of anus relative to genitalia * Ear position and patency of anus * Ear rotation (normally 15 degrees posterior to the vertical plane of the Systemic Examination: cardiovascular/ per head): anteriorly/ posteriorly rotated • abdomen/ neurological/ respiratory * Ear shape and structure Physical features not found as normal or * Accessory structures: pits/ skin tags • familial traits and which are present in only – Skeleton- a few conditions or are pathognomonic of specific disorders are of more diagnostic Neck: length/ shape (webbed) * help. These are said to be ‘good handles’ for Shape of thoracic cage * diagnosis e.g. white forelock of hair which Sternum: length & shape (pectus * is a good diagnostic clue for Waardenburg carinatum/excavatum) syndrome. * Spine: length/ straight/curved * Limbs: length/shape/ symmetry Radiographs – Joints – contractures; range of joint movement: laxity/ restriction; soft tissue The following radiographic assessment helps in the webbing across joints (pterygium) diagnostic evaluation: – Skin – X ray wrist + hand (anteroposterior (AP) view) in cases with short stature: for bone age Texture: smooth/ coarse/ dry/ • * assessment ichthyotic * Pigmentation: hypo/ hyperpigmen- Genetic skeletal survey for suspected skeletal tation; patchy / generalized • dysplasias/ disproportionate short stature: Naevi/ lentigines * – AP & lateral views of skull Redundancy/ laxity * – AP & lateral views of spine (cervical to Patchy pigmentation may indicate * sacrum) mosaicism – AP view of pelvis with bilateral hip joints – Hands and Feet – AP view of one hand and one foot * Overall shape and size of hand and – AP view of one upper limb (shoulder to foot elbow; elbow to wrist) * Digit number – AP view of one leg (knee to ankle) * Digit shape (e.g. clinodactyly) and length Imaging studies * Webbing between digits * Palmar, plantar and digit creases The following imaging modalities may be used in * Nail morphology the evaluation: – Genitalia and Anus Neuroimaging: • 40 Chapter 8

a) b) c)

Figure 3 Dysmorphic findings in the nose. a). Hypoplastic alae nasi; b). Beaked nose; c). Broadbifidtip of nose.

– MRI brain: in presence of neurologi- e.g. Di George syndrome (22q microdele- cal deficits/ seizures/ microcephaly or tion)/ Angelman syndrome (15q microdele- macrocephaly tion)/ (7q microdeletion) – CT Scan brain: for suspected TORCH in- Metabolic testing: Relevant biochemical in- fections/ cranial contour abnormalities/ • vestigations should be done if a metabolic craniosynostosis (3D CT) etiology is suspected. Metabolic disorders USG abdomen/ 2D Echo: to look for visceral with dysmorphism include: • malformations – Mucopolysaccharidoses, oligosacchari- doses, mucolipidosis, GM1 gangliosido- Analysis sis All clinical and laboratory findings must be – Peroxisomal disorders • analysed together in order to get a diagnosis; – Disorders of cholesterol metabolism (e.g. all features must fit into the diagnosis as far Smith Lemli Opitz syndrome) as possible If the condition cannot be diagnosed based on Single gene mutation analysis: DNA-based molecular genetic tests to be done when a • previous experience or existing knowledge, • one should take the help of resources such as specific monogenic disorder is suspected. dysmorphology databases (e.g. LDDB - Lon- Cytogenetic microarray (CMA) study: don Dysmorphology DataBase and POSSUM • – Pictures of Standard Syndromes and Un- – Can be done in any case with multiple diagnosed Malformations), online resources malformations with or without associ- (OMIM – Online Mendelian Inheritance in ated intellectual disability and without Man) and dysmorphology textbooks. any other identified genetic/ non-genetic cause Genetic Testing – CMA scans the entire genome for copy number variations (microdeletions/ mi- The following genetic tests can help in confirming croduplications) the aetiology in affected cases: Next Generation Sequencing(NGS): Karyotyping: to be done in cases with: • • – congenital malformations – NGS-based multigene panel testing can be used for sequencing large genes as – prenatal onset growth retardation well as genes with similar/ overlapping – disorder of sexual development phenotypes – developmental delay – NGS-based Exome sequencing can be – history of multiple miscarriages in the done in a case with multiple malforma- family tions with or without associated intellec- Fluorescence in situ hybridization (FISH)/ Mul- tual disability, where a specific etiology is not identified and CMA is non-diagnostic. • tiplex ligation - dependent probe amplifica- tion (MLPA): when the phenotype is sug- – Clinical Exome Sequencing can help gestive of a specific microdeletion syndrome detect sequence variants in exons of

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genes with known associated pheno- Follow up types, while Whole Exome Sequencing covers exons of all genes. To assess growth & development • To study course of the disease Intervention • To monitor for known/ anticipated associated Appropriate medical/ surgical management • complications • wherever feasible: eg. surgical correction of cardiac defect, correction of hearing deficit To offer newly available diagnostic tests etc. • To offer newly available therapeutic options Genetic counseling • Sometimes phenotype evolves with age and • Prenatal diagnosis wherever feasible • reassessment at a later age in an undiagnosed case might make diagnosis clear Genetic• Counseling To discuss reproductive risks. Deformations/ disruptions have low risk of • • recurrence (but can recur if the causative intrauterine environmental factor persists or Resources for reference recurs in the next pregnancy). Books: Denovo chromosomal abnormalities and mi- • • crodeletions have a risk of recurrence of – Aase JM. Diagnostic dysmorphology. % 1990. Springer.

42 Chapter 9

Approach to Intellectual Disability

Prajnya Ranganath Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad

Correspondence to: Dr Prajnya Ranganath Email: [email protected]

Definition Etiology

Intellectual disability or mental retardation is de- Intellectual disability can result from a large num- fined as significant sub-average intellectual func- ber of conditions of both genetic and non-genetic tion existing concurrently with deficits in adaptive origin. The broad etiological classification of DD/ behaviour and manifested during the developmen- ID is listed in Table 1. tal period. A precise cause is found only in about 50–70% Intellectual disability/ mental retardation of cases of severe ID; about 35–55% cases of (ID/MR) has 3 components: mild ID remain idiopathic. Amongst the causes identified for severe ID in studies, genetic causes 1. Significantly abnormal intellectual performance have been found to account for up to 25–50% of (determined by a test of intelligence). cases. 2. Impairment of the ability to adapt to the envi- Down syndrome is the most common identified ronment. genetic cause of DD/ ID. It affects 1 in 700–800 3. Onset during development before the age of 18 live born children and accounts for up to 20–30% years. of all cases of MR. Trisomy 21 is found in 95% cases of Down syndrome, translocation involving Developmental delay (DD) is a term usually used chromosome 21 occurs in 3–4% cases and mosaic for young children, especially years of age, trisomy 21 occurs in of cases. in whom formal IQ testing is≤ not 5 possible. It Fragile X syndrome∼ 1%is the most common mono- refers to delay in attainment of milestones and genic disorder associated with ID and is the most implies deficit in learning and adaptation. Global common inherited cause of ID. It accounts for 1–2% developmental delay (GDD) implies impairment of of all cases of ID and has an X-linked inheritance all spheres of development (gross & fine motor, pattern. social & adaptive, and language). DD/ID conditions include: Prevalence

Static non-progressive disorders with onset of The overall prevalence of DD/ ID in the general • cognitive impairment from birth/early child- population world-wide is 2–3%. The prevalence hood. of moderate and severe ID is 0.3–0.5% and of Progressive disorders with onset of cognitive mild ID is 1–3%. Mild intellectual disability is 7–10 • impairment in childhood. times more common than the severe form. Most Hereditary neurodegenerative and metabolic epidemiological studies show affected males to affected females ratio of around 1.3–1.9: 1;to • disorders with neuro-regression beginning some time after a period of normal develop- a large extent the higher prevalence in males is ment. because of X-linked causes of ID. ID/MR is said to be present when the intelli- Diagnostic Approach gence quotient (IQ) is less than 70. Classification of ID based on the IQ is as follows: mild-IQ The approach to a child with DD/ ID includes 50–70, moderate-IQ 35–50, severe-IQ 20–35 and a thorough clinical evaluation (clinical and fam- profound-IQ . ily history, complete dysmorphologic examination, < 20 43 Chapter 9 complete neurologic examination, and systemic for every patient with DD/ ID should include the examination) followed by a judicious use of labora- following: tory tests, imaging studies and appropriate genetic tests. Antenatal period (maternal age at conception, • history suggestive of teratogenic exposures Clinical history: A detailed clinical history [drugs and infections] especially in the first that• should be elicited from the parents/ guardians trimester, maternal illnesses, abnormal ante-

Table 1 Etiological classification of intellectual disability

Genetic Chromosomal anomalies (detectable by conventional cytogenetic techniques) Down syndrome (most common) • Other numerical chromosomal anomalies (e.g. trisomy 13 & trisomy 18) • Structural chromosomal anomalies- deletion/ duplication/ unbalanced translocation/ • Mosaic chromosomal anomalies • Submicroscopic chromosomal anomalies (not detectable by conventional cytogenetic tech- niques and detected by molecular cytogenetic techniques like FISH/ MLPA/ chromosomal microarray) Microdeletion/ microduplication syndromes (e.g. Prader Willi syndrome, Angelman • syndrome, Williams syndrome etc.) Cryptic subtelomeric rearrangements • Cryptic interstitial rearrangements • Single gene disorders Fragile X and other X linked monogenic disorders • Multiple malformation syndromes • Inborn errors of metabolism • Syndromic and non-syndromic monogenic disorders caused by mutations in genes • required for neurogenesis, neuronal migration, neuronal and synaptic functions and transcription signalling cascades

Deregulation of imprinted genes (E.g. Prader-Willi syndrome, Angelman syndrome) of imprinted gene • Deletion of non-imprinted gene • Imprinting centre defect • Multiple malformation syndromes without an identified genetic basis Known sporadic syndromes • New/ private syndromes • Idiopathic (Familial or sporadic cases without a definite identified genetic aetiology)

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Non-genetic CNS dysfunction due to identified prenatal, perinatal & postnatal acquired and environmental causes Teratogenic exposures (e.g. fetal phenytoin syndrome, fetal alcohol syndrome) • Intrauterine fetal infections ( e.g. congenital toxoplasmosis, congenital CMV infection) • Perinatal and postnatal asphyxia • Prematurity • Postnatal intracranial infections • Peri- and postnatal intracranial trauma and vascular accidents (hemorrhage or infarction) • Idiopathic (Cause not identified)

natal ultrasound findings) (failure to thrive, recurrent unexplained acute Perinatal period (prematurity, difficult labour, illness, seizures, ataxia, loss of psychomotor skills, recurrent somnolence/ coma, lethargy, • birth asphyxia, low birth weight, sepsis, hy- perbilirubinemia) abnormal body odour) Postnatal period (seizures or abnormal pos- Family history: • turing, history of intracranial infections/ • trauma/ vascular accidents, spasticity, vi- Detailed minimum 3-generation family his- sual and hearing problems, exposure to lead, • tory with exact medical records wherever abnormal pattern of sleep) possible Detailed developmental history (time of at- Particular emphasis on family members with developmental delay, intellectual dis- • tainment of milestones, any regression of • milestones) ability, seizure disorders, psychiatric disor- ders, congenital malformations, miscarriages, Detailed behavioural history (changes in be- stillbirths, and unexplained early childhood havioural pattern, deterioration in school per- • deaths formance, self-injurious behaviour, repetitive behaviour, autistic behaviour, hyperactivity, Construction of a pedigree of 3 generations or attention deficit); behavioural phenotype can • more to ascertain the pattern of inheritance provide important clues to the diagnosis as listed in Table 2. Physical examination: • History suggestive of metabolic disorders Complete anthropometric assessment • •

a) b) c) d)

Figure 1 Some intellectual disability conditions diagnosable from the typical gestalt. A. Down syndrome, B. , C. Cornelia de Lange syndrome, D. .

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Table 2 Behavioural patterns associated with some intellectual disability conditions

Behavioural pattern Intellectual disability condition Autistic features, perseverative speech, problems in impulse Fragile X syndrome control Repetitive, stereotypic, purposeless hand movements, fits of screaming and inconsolable crying, autistic features, panic-like attacks Self mutilation Lesch Nyhan syndrome Inappropriate happy behaviour and excitability Angelman syndrome Self mutilation, aggression and sleep disturbances Smith Magenis syndrome Overfriendliness, social disinhibition, attention deficit Williams syndrome Autistic features 15q duplication Hyperphagia and sleep disturbances Prader Willi syndrome

(height, weight, head circumference, upper macrocytosis in abnormalities of vitamin B12 segment: lower segment ratio). metabolism etc.) Detailed dysmorphologic evaluation from SGPT for gross liver function assessment • head to toe should be done in every case • of DD/ ID, followed by comparison of find- Serum creatinine for gross renal function ings with available literature, OMIM and • assessment dysmorphology databases (such as London Serum creatine phosphokinase must be Dysmorphology DataBase, POSSUM). There • checked in all young male patients with un- are a large number of dysmorphic and multi- explained developmental delay as Duchenne ple malformation syndromes associated with muscular dystrophy may present in early DD/ ID, which can be diagnosed through thor- childhood with developmental delay ough physical examination. Figure 1 shows some common dysmorphic syndromes asso- Serum T4 & TSH: Hypothyroidism must be ciated with intellectual disability. excluded• in all cases of developmental delay es- Detailed neurological evaluation must be pecially when clinical features of hypothyroidism are seen and in younger children ( years) where • done in every case and must include assess- ment of: no specific cause is identifiable.< 2 Hypothyroidism may be associated with some genetic syndromes – higher mental functions, e.g. Down syndrome, It is very important to not – cranial nerve deficits, miss hypothyroidism as it is a treatable cause of intellectual disability. – motor and sensory system examination, – cerebellar signs, Neuroimaging: – involuntary/ abnormal movements, • Neuroimaging is recommended in the work- – persistence of neonatal reflexes • up of intellectual disability cases particularly Ophthalmological examination should be when at least one of the following is present: • done in all cases of intellectual disability. This can provide important clues to the diag- – abnormal head size: microcephaly/ nosis (Table 3). macrocephaly – seizures Baseline Investigations: – focal motor/ neurological findings on • Complete hemogram including RBC indices neurological examination • (may provide clue to the diagnosis eg. alpha – associated malformations esp. facial thalassemia – mental retardation syndrome, malformations

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Table 3 Ophthalmological findings associated with some intellectual disability conditions

Ophthalmological finding Associated intellectual disability conditions Cataract Galactosemia, congenital rubella syndrome, Lowe syndrome, congenital myotonic dystrophy, Cockayne syndrome Cherry red spot Some lysosomal storage disorders: GM1 gangliosidosis, GM2 gangliosidosis (Tay Sachs and Sandhoff disease), Sialidosis, Galactosialidosis, Mucolipidosis, Niemann Pick disease Corneal clouding Mucopolysaccharidoses, Oligosaccharidoses, Mucolipidoses Chorioretinitis Congenital intrauterine infections Lens dislocation Homocystinuria, Sulfite oxidase deficiency Retinal changes/ pigmentary Norrie disease, Bardet Biedl syndrome, Mitochondrial disor- degeneration ders, Neuronal ceroid lipofuscinosis

In addition, it is preferable to do neuroimag- cause. The label of ‘cerebral palsy due to adverse • ing studies if no other aetiology is obvious; in perinatal events’ should not be given until there is conditions like neuronal migration anomalies definite history and evidence in neuroimaging. and some cases of pre/ perinatal insult there may be no other clinical diagnostic clue. Karyotyping: Neuroimaging helps to identify hypoxic is- To confirm the diagnosis of a clinically sus- • chemic sequelae, vascular insults, neuroec- todermal syndromes, intracranial structural • pected chromosomal disorder such as Down defects and neuronal migration abnormali- syndrome, Trisomy 13 or Trisomy 18. ties. Should be done in all cases with intellectual MRI is generally preferable to CT scan for • disability, where a specific etiology has not been clinically identified. • neuroimaging and is much more informative. However, a CT Scan is a better modality for in Conventional karyotyping (up to 550 band- certain situations such as documentation of • level) yields a diagnosis in 2.5–3% of unex- intracranial calcifications associated with old plained/ idiopathic ID cases. hemorrhage, tuberous sclerosis complex and congenital CMV or toxoplasmosis infection, Fluorescence in situ hybridization (FISH)/ and for craniosynostosis. Multiplex ligation-dependent probe amplification Specific tests based on the clinical features (MLPA) study: in• each individual case: Based on the clinical To confirm the diagnosis of clinically identified findings in each individual case, the following tests • microdeletion syndromes such as Prader-Willi may be additionally required in the work-up. syndrome, Angelman syndrome, Di George Electrophysiological studies: Electroen- syndrome, Williams syndrome etc. • cephalogram (EEG)/ Audiometry/ Electromyo- MLPA panels to check for multiple common gram (EMG) / Nerve conduction study (NCS) • microdeletion syndromes in one test are TORCH serology: for suspected congenital available commercially. • intrauterine infections in infants MLPA panels to test for subtelomeric dele- Ultrasonography to look for visceral malfor- • tions and duplications on all chromosomes in • mations a single test are also available commercially. Echocardiography to look for cardiac malfor- • mations Testing for Fragile X syndrome: Genetic Evaluation: Genetic evaluation Fragile X screening can be done through should• be done in all cases of intellectual dis- • PCR-based tests but confirmation requires ability without a definitely proven environmental Southern Blot testing.

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If fragile X molecular genetic testing is done Whole exome/ whole genome sequencing: • using a phenotypic checklist of 7 features Newer genetic testing modalities such as Whole (proposed by de Vries et al, J Med Genet 1999) Exome and Whole Genome sequencing are now the diagnostic yield is –8% (long jaw, high being used for the evaluation of patients with forehead, large and/or∼ protuberant 7 ears, hy- idiopathic DD/ ID where the first and second tier per extensible joints, soft and velvety palmar genetic investigations are not conclusive for the skin with redundancy on the dorsum of the etiology. hands, testicular enlargement, and behavior The clinically relevant Clinical Exome/ Medical of initial shyness and lack of eye contact Exome/ Mendeliome sequencing, which covers followed by friendliness and verbosity). majority of the genes associated with OMIM-listed phenotypes is now proving to be a cost-effective Younger children have subtle dysmorphism testing modality for the evaluation of idiopathic which may be missed; therefore fragile X test- • DD/ID. ing is recommended in all children especially males with idiopathic ID even in the absence of a family history. Management For most cases of DD/ ID, treatment is mainly Molecular genetic testing: If a specific mono- • symptomatic and supportive. genic disorder is suspected clinically, mutation Mainstay of therapy includes interventions analysis of the relevant gene should be done for • such as early stimulation, physiotherapy, confirmation and for identification of the exact vocational training, special schooling and disease-causing mutations in the family, wherever speech therapy. feasible. Identification of the mutations helps in Institutionalization is to be discouraged and offering accurate carrier screening and prenatal • integration into society is encouraged. diagnosis for family members. A multidisciplinary approach is essential for • appropriate management especially for mul- Metabolic testing: tiple malformation syndromes and multi- systemic involvement. If a specific inborn error of metabolism (IEM) • is suspected clinically, the relevant biochem- Genetic counseling ical assay such as enzyme assay/ plasma amino acid HPLC/ urine GCMS for organic Depends on the aetiology: acids etc. is to be done for confirmation. Chromosomal causes: Most IEMs present with neuroregression and • Usually not familial except when one of the have a progressive course. • parents is a carrier of a balanced chromoso- Routine metabolic evaluation in unexplained mal rearrangement. • ID has an average diagnostic yield of only Risk of recurrence in de novo chromosomal around 1%. • disorders is low (usually ). In translocation Down syndrome,< 1% when one Cytogenetic microarray (CMA): • of the parents is a balanced translocation carrier, the recurrence risk is variable: Chromosomal microarrays have a diagnostic – 2–5% if the father is a carrier yield of around 12–20% in idiopathic ID. • – 10–15% if the mother is a carrier This has been recommended as the first – 100% if either parent is a carrier of the • line diagnostic test for idiopathic ID by the 21; 21 translocation American College of Medical Genetics, but in India the test is still not being done as a Single gene disorders: first-line evaluation because the cost remains • Risk of recurrence depends on the mode of prohibitive. • inheritance: CMA detects genome-wide copy number vari- For autosomal recessive disorders, the risk of • ations (CNVs- deletions/ duplications). • recurrence in siblings is 25%.

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Table 4 Empiric risks of recurrence of intellectual disability with accompanying features, without an identified specific etiology

Accompanying feature with DD/ ID Approximate risk of recurrence Microcephaly alone 1 in 6 to 1 in 8 (10–15%) Microcephaly with other features 1 in 30 (3%) Infantile spasms 1 in 30 to 1 in 100 (1–3%) Non-specific dysmorphic features 1 in 25 to 1 in 30 (3–4%) Malformation 1 in 50 (2%) No specific features; male proband 1 in 13 (8%) No specific features; female proband 1 in 20 (5%) Holoprosencephaly with normal karyotype & no 1 in 20 (5%) forme fruste in parents Lissencephaly type 1 Very low ( ) Lissencephaly type 2 1 in 4 (25%)< 1% Cerebellar hypoplasia 1 in 8 (12.5%) Schizencephaly/ asymmetric porencephaly Very low Cerebral palsy (diplegia/ hemiplegia) 1 in 200 to 1 in 400 Symmetrical spasticity 1 in 8 to 1 in 9 (10–12%) Asymmetric neurological signs 1 in 50 to 1 in 100 (1–2%) Ataxic diplegia 1 in 24 (4%) Congenital ataxia 1 in 8 (12.5%)

For autosomal dominant disorders, the risk Prenatal Diagnosis • of recurrence for the sibling is 50% if one of the parents is affected; in a sporadic case A specific prenatal diagnostic test can be offered (de novo mutation), the risk of recurrence in for future pregnancies in the family only if the exact the sibling is very low but the possibility of aetiology is established in the index case with DD/ gonadal mosaicism in either parent cannot ID. The actual test offered depends on the nature be ruled out. of the genetic problem identified in the proband.

For X-linked recessive disorders, the risk of Fetal karyotyping (preferably in amniotic fluid) is done if the proband has an identified chro- • recurrence for male siblings is 50%, whereas • females usually do not manifest the disorder. mosomal anomaly and/ or there is evidence of a balanced chromosomal rearrangement in either parent. Environmental causes: Recurrence is un- FISH, MLPA or cytogenetic microarray analysis likely but can occur if the causative agent persists • • is done in the fetal sample (chorionic villus antenatally or postnatally in the environment of sample or amniocytes), if proband is identi- the next child also e.g. lead exposure, intrauterine fied to have a chromosomal microdeletion/ exposure to teratogens like alcohol or specific microduplication detected by FISH, MLPA or drugs. microarray. When no etiology is identified: Empiric risks Targeted mutation analysis is done in the fetal of recurrence• can be predicted when no specific eti- • sample (chorionic villus sample or cultured ology is identified, but a specific prenatal diagnostic amniocytes) if the proband has a monogenic test cannot be offered for the next pregnancy. See disorder with identified disease-causing gene Table 4. mutations. In some cases, if the diagnosis

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of the monogenic disorder is confirmed in tect all malformations and some anomalies the proband but the exact disease-causing such as microcephaly/ pachygyria etc. may mutations are not identified, prenatal diag- not become apparent until later gestation. nosis may be done through linkage analysis, provided informative linkage markers are Suggested Reading identified in the family (there is however chance of error because of the possibility∼ 5% of 1. Chelly J, et al. Genetics and pathophysiology recombination). of mental retardation. Eur J Hum Genet 2006; 14: 701-713. Relevant enzyme assay is done in fetal tissue 2. Koolen DA, et al. Genomic microarrays in • (CVS/ cultured amniocytes) if the proband is confirmed to have a metabolic disorder such mental retardation: a practical workflow for as a lysosomal storage disorder. Recent diagnostic applications. Hum Mutat 2009; 30: guidelines suggest that wherever possible, 283-292. targeted mutation analysis must be com- 3. Miller DT, et al. Consensus statement: chro- bined with enzyme assay, for improving the mosomal microarray is a first-tier clinical reliability of the prenatal diagnostic test (to diagnostic test for individuals with develop- close to 100%). Metabolite assays in amniotic mental disabilities or congenital anomalies. fluid, though conventionally done for prenatal Am J Hum Genet 2010; 86: 749-764. diagnosis of inborn errors of metabolism such 4. Morrow EM. Genomic copy number varia- as organic acidurias, are not very reliable. For tion in disorders of cognitive development. IEMs such as organic acidurias, aminoaci- J Am Acad Child Adol Psychiatr 2010; 49: dopathies, fatty acid oxidation disorders and 1091-1104. urea cycle defects also, targeted mutation analysis and/ or the relevant enzyme assay 5. Moeschler JB, et al. Clinical genetic evalua- provide reliable results. tion of the child with mental retardation or developmental delays. Pediatrics 2006; 117: If the proband has a multiple malformation 2304-2316. • syndrome, prenatal diagnosis may be offered for the future pregnancies, through fetal 6. Moeschler JB, Shevell M; Committee on Ge- targeted anomaly scan and fetal echocar- netics. Comprehensive evaluation of the diography at 18 to 20 weeks, followed, if child with intellectual disability or global de- required, by serial antenatal USG monitoring. velopmental delays. Pediatrics. 2014;134: Fetal MRI may also be done in some cases e903-18. such as a CNS malformation in the proband. 7. Rauch A, et al. Range of genetic mutations as- However, the family has to be counselled sociated with severe non-syndromic sporadic that fetal imaging cannot give information intellectual disability: an exome sequencing about intellectual functioning, it cannot de- study. Lancet 2012; 380: 1674-1682.

SIAMG-Genzyme Fellowship in Clinical Genetics

http://iamg.in/images/ GenzymeSIAMG_Fellowship_Curriculum.pdf

50 Chapter 10

Approach to Disorders of Sex Development

Prajnya Ranganath Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad

Correspondence to: Dr Prajnya Ranganath Email: [email protected]

Definition 46, XX DSD: • Disorders of sex development (DSD) are a group Disorders of ovarian development of congenital conditions in which the development • Disorders of androgen excess of chromosomal, gonadal, or anatomical sex is • atypical. – with congenital adrenal hyperplasia – without congenital adrenal hyperplasia DSD is an umbrella term which encom- • passes ambiguous genitalia + primary hypog- Sex Chromosome DSD: onadism. • Numerical sex Ambiguous genitalia refers to a congenital • leading to abnormal gonadal development • defect wherein appearance of the external genitalia is at variance with what is normal for Most have gonadal dysgenesis (poorly formed either male or female sex and includes the fol- • testis: dysgenetic testis; poorly formed ovary: lowing: phallus intermediate in size between streak gonad) normal penis & normal clitoris; aberrantly Klinefelter and Turner syndromes are the located urethral opening; or at least one • most common sex chromosome DSDs – they impalpable gonad. present with primary hypogonadism The terms ‘intersex’, ‘sex reversal’ and Sex chromosome DSDs which present with • ‘hermaphroditism’ are considered controver- • ambiguous genitalia include: 45,X/ 46,XY sial, derogatory and confusing and are no mixed gonadal dysgenesis & 46,XX/ 46,XY longer used. ovotesticular DSD Classification 46, XY DSD: • Normal male karyotype (46,XY) The new classification of DSD, as per the Lawson Wilkins Pediatric Endocrine Society (LWPES) & Eu- • External genitalia: ambiguous or female due ropean Society for Pediatric (ESPE) • to incomplete intrauterine masculinization 2006, is as follows: Male gonad(s) are palpable in majority Sex chromosome DSD: • Formerly called “male pseudohermaphrodite” • 45, X (Turner & variants) • 46, XY DSD includes: • 47, XXY (Klinefelter & variants) • – Disorders of gonadal (testicular) de- • 45,X/46, XY (Mixed gonadal dysgenesis) velopment: • 46,XX/ 46,XY (& variants causing ovotesticular * Complete gonadal dysgenesis (Swyer • DSD) syndrome) 46, XY DSD: * Partial gonadal dysgenesis Testicular regression syndrome • Disorders of testicular development * * Ovotesticular DSD • Disorders of androgen synthesis – Disorders in androgen biosynthesis & • Disorders of androgen action action: • 51 Chapter 10

Table 1 Disorders of Androgen Biosynthesis.

Deficiency Defective synthesis Phenotype (Autosomal recessive) 17 β hydroxysteroid Testosterone deficiency only DSD dehydrogenase 3 β hydroxysteriod Deficiency of testosterone + DSD + congenital adrenal hyperplasia + dehydrogenase glucocorticoid + salt loss Cholesterol mineralocorticoid desmolase (P450scc) 17 α hydroxylase Deficiency of testosterone & DSD + congenital adrenal hyperplasia + (P45017α) excess of glucocorticoid + salt retention + hypertension mineralocorticoid P450 oxidoreductase Deficiency of testosterone + Antley-Bixler syndrome: DSD + (POR) multisystem anomalies craniosynostosis+ skeletal anomalies + renal anomalies + cognitive deficit 7dehydrocholesterol Deficiency of testosterone + Smith Lemli Opitz syndrome: DSD + reductase multisystem anomalies microcephaly + growth retardation + malformations 5 – alpha – reductase Deficiency of DSD - variable degree of under-virilization dihydrotestosterone of male external genitalia

* Androgen biosynthesis defect 46, XX DSD includes: Defect in androgen action * • – Disorders of ovarian development: * Leutinizing hormone receptor defect () * Ovotesticular DSD * Disorders of antimullerian hormone * Testicular DSD (SRY+, dup SOX9) & AMH receptor * Gonadal dysgenesis – Conditions with fetal androgen ex- Syndromic associations with 46, XY DSD cess: include Frasier syndrome, Denys-Drash syn- • – With congenital adrenal hyperplasia drome, and Campomelic dysplasia * 21 hydroxylase deficiency Important disorders of androgen biosynthe- 11 β hydroxylase deficiency sis are listed in Table 1 * • – Without congenital adrenal hyperplasia Disorder of androgen action is the Androgen Aromatase deficiency insensitivity syndrome which is caused by * • P450 oxidoreductase deficiency mutations in the AR gene (which encodes * Maternal androgen intake/ androgen the androgen receptor). It can be partial * secreting tumour (incomplete masculinization of external geni- talia) or complete (female external genitalia), 21-hydroxylase deficiency: depending on residual activity of the andro- • gen receptor. It has an X-linked inheritance Most common form of congenital adrenal pattern. • hyperplasia ( 90%) and the second most DSD after mixed gonadal> dysgenesis 46, XX DSD: Autosomal recessive disorder caused by mu- • tations in the CYP21A2 gene (6p21.3) Have a normal female karyotype (46,XX) • Every newborn with ambiguous genitalia • Have ambiguous genitalia due to masculiniza- • without testes (clinically or on imaging) • tion / virilization of female external genitalia should be evaluated for 21-hydroxylase Formerly called “female pseudo- deficiency, as it is a potential medical • hermaphrodite” emergency

52 Chapter 10

Affected females are either virilized at birth Any abnormal virilized /cushingoid appear- • and have ambiguous genitalia or become • ance of mother virilized postnatally and can present with Medical photography – with sensitivity & menstrual abnormalities and reduced fertility • consent Affected males develop early virilization in • childhood i.e. isosexual pseudoprecocious Clinical indicators of DSD: puberty • Identified in the neonatal period: Salt-losing crisis occurs in 75% of affected individuals. • • ∼ – overt genital ambiguity – apparent female genitalia with enlarged Evaluation clitoris/ posterior labial fusion Evaluation of an individual affected with DSD – apparent male genitalia with bilateral should be done in a step-wise manner. undescended testes/ hypospadias/ mi- cropenis Detailed history: – discordance between genital appearance • Growth, development, associated systemic & a prenatal karyotype • complaints Later presentations in older children/ adoles- Prenatal history: maternal exposure to andro- • cents: • gens/ maternal androgen secreting tumors Detailed family history with at least – previously unrecognized genital ambigu- • 3-generation pedigree: consanguinity and ity family history of genital ambiguity, hirsutism, – inguinal hernia in a girl precocious puberty, amenorrhea, infertility, – delayed or incomplete puberty unexplained sudden infant death (due to salt-losing crisis). – primary amenorrhea or virilization in a girl Androgen insensitivity syndrome (AIS) is an – breast development in a boy • X-linked disorder; males affected with com- plete AIS may have normal appearing ex- – gross or cyclic hematuria in a boy ternal female genitalia and would be reared as females – therefore, it is important to Investigations ask specifically for family history of primary amennorhoea/ infertility in sisters/ maternal First-line tests: aunts/ female cousins on the maternal side. • Cytogenetic analysis Physical examination: • • Thorough examination of genital anatomy – most commonly karyotyping – FISH/ MLPA with X- & Y-specific probes • Groin and scrotal/labial folds to be checked can be done for rapid gender assignment for presence of palpable gonads: a palpable • in a newborn in select cases gonad is almost always a testis (or rarely ovotestis); ovaries & streak gonads do not – differentiates between sex chromosome descend and are therefore not palpable DSD; 46,XY DSD; & 46, XX DSD

Phallic size: width & stretched length mea- Imaging: abdominopelvic ultrasound surements • • Prader scale: objective scoring of degree of – for identification of Mullerian/ Wolffian • virilization of external genitalia structures Complete general & systemic examination – to detect testes in inguino-scrotal region (not useful for intra-abdominal testes) • for syndromic associations: anthropometry, dysmorphology evaluation, systemic exami- – to rule out possible adrenal anomalies/ nation associated renal anomalies

53 Chapter 10

Further evaluation: Measurement of serum concentration of • • 17-OHP (classic form 10,000 ng/ dl; non- i. 46, XY & absent testes & ± Mullerian classic form: ACTH stimulation> may be re- structures: quired to demonstrate increased levels)

Suggestive of complete gonadal dysgenesis Measurement of urinary pregnanetriol (in- creased) • or testicular regression syndrome • FISH/ MLPA – to look for SRY deletion Molecular genetic testing of CYP21A2 for con- • If syndromic: mutation analysis of SOX9 (cam- • firmation & for prenatal diagnosis for future pregnancies of parents • pomelic dysplasia) or WT1 (Frasier/ Denys- Drash syndrome) If non-syndromic gonadal dysgenesis/ testic- Management • ular regression: mutation analysis for SRY, NR5A1, DHH, WNT4 genes Gender assignment: • ii. 46, XY with testes & no Mullerian struc- Complex issue with important psychological tures: Basal testosterone (T) and dihydrotestos- • & social implications terone (DHT) levels to be measured in neonates & post-pubertal individuals. Post hCG stimulation Gender should be assigned after the com- testosterone (T) and dihydrotestosterone (DHT) to • plete diagnostic process be checked in post-neonatal period & childhood: Multidisciplinary approach: geneticist, neona- in normal persons there is a 3 fold elevation of • tologist, endocrinologist, gynaecologist, psy- testosterone over baseline, with hCG stimulation. chiatrist, surgeon & social workers Low T level + high FSH/LH testosterone Factors influencing gender assignment: diag- biosynthesis defect • ⇒ • nosis, genital appearance, fertility potential, Low T level + high FSH/ LH + low anti Mullerian therapeutic/surgical options, familial views, cultural biases • hormone gonadal dysgenesis Normal testosterone⇒ (T), normal or low dihy- Surgical intervention for gender assignment: • drotestosterone (DHT), post hCG stimulation • high T/ DHT 5 alpha reductase deficiency controversy about optimal timing of mutation analysis⇒ of SRD5A2 • American Academy of Pediatrics guidelines →Normal T, normal DHT, normal T/ DHT • recommend genitoplasty between 2 - 6 months of age • androgen insensitivity syndrome mutation⇒ analysis of AR → Feminizing genitoplasty for infants to be iii. 46, XX with normal ovaries & normal • raised as females: internal female genital organs: – removing the corporal bodies/ clitoro- Serum electrolytes – for salt losing CAH plasty • Serum 17-OH-progesterone: elevated in 21 – creating normal-looking introitus and • hydroxylase deficiency CAH labia minora & majora Serum 11-deoxycortisol and deoxycorticos- – vaginoplasty to provide an adequate • terone: elevated in 11β-hydroxylase defi- opening ciency & reduced in 21-hydroxylase deficiency Masculine reconstruction for infants to be iv. For suspected congenital adrenal hyper- reared as males: plasia due to 21-hydroxylase deficiency: •

Serum electrolyte concentrations / plasma – orchiopexy, • renin assay – hypospadias repair Karyotype or FISH for X- and Y-chromosome – removal of retained mullerian duct struc- • detection tures

54 Chapter 10

Gonadectomy: Genetic Counseling • controversy regarding timing but commonly Sex chromosome DSDs: • recommended soon after diagnosis germ cell malignancy occurs only in DSD • risk of recurrence is not significantly elevated in subsequent pregnancies of the parents of • cases with Y-chromosome • an affected child for 46,XY DSDs to be raised as female, it is specific prenatal testing is not required. • best to remove the testes to prevent testicular malignancy & virilization at puberty Autosomal• recessive disorders: for 46, XY DSDs to be raised as male, tes- • e.g. 21-hydroxylase deficiency, 5-alpha reduc- ticular biopsy to be done at puberty for • • tase deficiency, Smith-lemli-Opitz syndrome, e/o premalignant lesions - carcinoma in situ most disorders of the androgen/ estradiol or undifferentiated intra-tubular germ cell biosynthesis pathway etc. neoplasia risk of each subsequent offspring of the Hormone replacement therapy: • affected child’s parents getting both the • to induce & sustain puberty disease-causing mutations is 25%; however, the phenotype may be sex-limited • to induce secondary sexual characteristics & prenatal diagnosis can be done in subsequent • pubertal growth spurt pregnancies through targeted mutation anal- to optimize bone mineral accumulation • ysis in the chorionic villus sample (CVS)/ am- • for psychosocial maturation niocytes, after identifying the disease-causing • Boys with hypogonadism: mutations in the proband • – intramuscular injections of testosterone for congenital adrenal hyperplasia due to for pubertal changes • 21-hydroxylase deficiency, the protocol rec- ommended to minimize the chances of viril- – testosterone gels & patches for viriliza- isation in the female fetus is given in Figure tion of external genitalia 1. Girls with hypogonadism: Autosomal dominant disorders: • – estrogen supplementation to induce sec- • e.g. Frasier syndrome, Denys-Drash syn- ondary sexual changes & for menstrua- • drome, Campomelic dysplasia etc. tions in majority of cases, the parents are normal – progestin added after breakthrough and the disorder occurs because of a de novo bleeding develops or within 1 to 2 years • mutation in the proband; therefore, the risk of continuous estrogen of recurrence in subsequent offspring of the Management of 21 hydroxylase deficiency: parents is not significantly elevated • Glucocorticoid replacement therapy has to be risk of recurrence is 50% if either parent is affected. • given on a regular basis; the dose has to be • increased during periods of stress in some cases there may be gonadal mo- In the salt-wasting form: mineralocorticoid • saicism in either parent leading to a small risk of recurrence (determined empirically for • 9α-fludrohydrocortisone therapy should also be added each disorder) and prenatal testing may be offered for subsequent pregnancies. Monitoring for symptoms & signs of salt- • losing crises to be done & prompt supportive X-linked disorders: therapy to be initiated • e.g. androgen insensitivity syndrome Females virilized at birth may require feminiz- • subsequent male offspring of the parents ing genitoplasty and/or vaginal dilation • • have a 50% chance of recurrence and prena- Primary prevention of symptoms is possible tal diagnosis can be done through targeted • through newborn screening (17-OHP in heel mutation analysis in CVS/ amniocytes after prick -dried blood spot sample) and early identifying the disease-causing mutation in intervention. the proband

55 Chapter 10

* Fetal sex not revealed to the family in accordance with the Pre-Conception and Pre-Natal Diagnostic Techniques (PCPNDT) Act, 1994.

Figure 1 Prenatal evaluation/management of a pregnancy at risk for 21-hydroxylase deficiency. (Adapted from Nimkarn & New, 2006)

Suggested Reading 4. Hughes IA, LWPES Consensus Group, ESPE Consensus Group, et al. Consensus State- 1. Arboleda VA, et al. Disorders of sex devel- ment on Management of Intersex Disorders. opment. In: Genetic Diagnosis of Endocrine Pediatrics 2006; 118: 488-500. Disorders. First edition. Elsevier 2010; pp 227-43. 5. Nimkarn S, New MI. Prenatal diagnosis and treatment of congenital adrenal hyperplasia. 2. Carillo AA, et al. Disorders of sexual dif- Horm Res 2006; 67: 53-60. ferentiation. In: Pediatric Endocrinology. Fifth edition. İnforma Health Care 2007; pp 6. Simpson JL. Disorders of the gonads, genital 365-413. tract, and genitalia. In: Emery and Rimoin’s 3. Houk CP, Lee PA. Consensus statement on Principles and Practice of Medical Genetics. terminology and management: disorders of Fifth edition. Churchill Livingstone Elsevier sex development. Sex Dev 2008; 2:172-80. 2007; pp 2055-92.

56 Chapter 11

Inborn Errors of Metabolism Presenting in the Newborn Period: Representative Phenotypes & Diagnostic Approach

Ratna Dua Puri1, Shubha R Phadke2 1Institute of Medical Genetics and Genomics, Sir Ganga Ram Hospital, New Delhi 2Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow

Correspondence to: Dr Ratna D Puri Email: [email protected]

Introduction immunodeficiency syndromes. The effects ofthe deficient enzyme or product are limited to the Improving healthcare in India and resulting de- system of which the protein is a part. However, cline in infant mortality rate makes contribution some enzymes are involved in basic metabolism of Inborn Errors of Metabolism (IEMs) significant (e.g. Kreb cycle) or metabolic derangements in for neonatal and infant morbidity and mortality. some affect multiple organs in the body. This, Though individually rare, collectively the incidence latter group of disorders are traditionally grouped may be as high as 1 in 800 to 1 in 2500 births. Every as IEMs and the discussion in this chapter is limted neonatologist needs to have a working knowledge to these IEMs. of diagnostic approaches, recent advances and availability of treatments for this group of disor- ders. The clinical of IEMs in neonates mimic those of common illnesses of the neonatal period and thereby a high degree of suspicion is essential to diagnose these disorders. a) The timely diagnosis can improve the outcome for treatable disorders. Some disorders are difficult to treat or are untreatable. However, accurate diagnosis is essential to provide genetic counseling to the family for risk of recurrence and prevention by prenatal diagnosis. Investigations on a very urgent basis and storage of samples are needed, as b) the course in some cases is very fast and fatal. The main aim of this chapter is to present a simplified approach through case studies to enable early recognition and investigations of inborn errors of metabolism so that they do not remain a challenge to suspect and diagnose. Within the purview of this textbook, the disorders discussed here are those with presentations in the neonatal period. Inborn errors of Metabolism (IEMs) are inher- Figure 1 Pathophysiology of IEMs. ited disorders that occur due to deficiency of an a) Normal metabolic pathway. enzyme, transporters or cofactor in a metabolic b) Deficency of enzyme b in the pathway. This leads to deficiency of the final metabolic pathway. product, or accumulation of metabolites proximal ‘a’ - enzyme in metabolic pathway, to the block. (Figure 1) ‘b’ - enzyme in metabolic pathway, Some of the genetic metabolic disorders are ‘x’ - deficiency of enzyme ‘b’. limited to one system like hemophilia or congenital

57 Chapter 11

Table 1 Small Molecule Disease and Organelle Defect.

Feature Small molecule disease Organelle disease Onset Often sudden, acute Gradual Course Remissions & relapses Progressive Physical findings Nonspecific Characteristic Histopathology Nonspecific Characteristic Response to treatment Often quick Response over a period of time Disorders Aminoacidopathy, urea cycle Lysosomal storage disorders, disorders, organic acidemias, peroxisomal disorders, mitochondrial carbohydrate metabolism defects, disorders, synthetic disorders like disorders of nucleotides, glycosylation defects, Smith Lemli porphyrins and metals Opitz syndrome.

Small Molecule vs Organelle Disease iv. Family history – consanguinity, similar illness in a sibling, unexplained neonatal death IEMs can be categorized into small molecule and v. Progressive illness with signs of cerebral dys- large molecule disorders or organelle defects; the function such as poor sucking, limpness, vomit- former have an acute presentation while large ing, irritability, respiratory distress, lip smacking molecule disorders are more insidious in onset movements and hypothermia. and progression. At the bedside, this classification is useful to decide the direction of investigations. vi. Unusual odors like maple syrup or burnt sugar, The small molecule disorders include conditions mousy, cabbage like, sweaty feet. Abnormal where the defect occurs at a single step in the urine odors are best identified on opening a metabolic pathway of a water-soluble metabolite, container with urine that has been closed for a such as an amino acid or monosaccharide. The few minutes at room temperature. diagnosis is by evaluation of the metabolic interme- diaries in physiological fluids, blood, urine and CSF. vii. Difficult to control seizures The organelle disorders are those where the defect viii. Vomiting, chronic hiccups is in an organelle specific enzyme or metabolic process and usually associated with intracellular ix. Persistent metabolic acidosis, ketosis, hypo- storage of metabolic substrates. Table 1 shows glycemia, coagulopathy the differences between the two classes of IEMs. x. Cholestasis with or without hep- Very few of the organelle diseases present during atosplenomegaly neonatal period. xi. Involvement of other systems like cardiomy- When to Suspect an IEM opathy, coarse facies, ascites, cataract, alope- cia, ambiguous genitalia and abnormalities of A high index of suspicion is important as the clinical pigmentation features of many inherited metabolic diseases are non-specific and simulate those of a neonate with IEM in a Neonate: An emergency sepsis The neonate has a limited repertoire of responses i. History of acute deterioration after a period of to illness and therefore a high index of suspicion normalcy after birth. for IEMs is important to make a diagnosis. In the newborn period most IEMs present as life threat- ii. Exaggerated response to an illness ening emergency illnesses that closely resemble a neonate with sepsis. These include: iii. Poor response to treatment of a supposedly i. Acute encephalopathy e.g. urea cycle disor- acquired illness ders, MSUD, organic acidurias

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Table 2 Five Basic Investigations for IEMs Serve as Diagnostic Clues.

Disorder Acidosis Ketosis Lactate Ammonia Glucose Maple syrup urine disease (MSUD) N ++ N N N/ Organic acidemias + ++ N/ N/ ↓ Urea cycle disorders N N N↑ ↑ ↓↓N Lactic acidosis + + ++ ↑↑N N NKH / Sulfite oxidase / Molybedenum N N N N N cofactor deficiency / Phenylketonuria / Peroxisomal disorders / Galactosemia N N N N /N GSD I + + Post-prandial + Fasting N ↓ ↓↓ ii. Liver disease e.g. galactosemia, hereditary suck. The alert resident on rotation elicited a family fructose intolerance, tyrosinemia type I history of parental consanguinity and a previous sibling death on day 8 of life with suspected sepsis. iii. Cardiac failure e.g. Pompe disease, fatty acid He sent off a blood ammonia and lactate and urine oxidation defects, mitochondrial disorders for ketones, suspecting an IEM. There was no lactic acidosis or ketosis. The ammonia was increased iv. Metabolic derangements like hypoglycemia at 1450 µmol/L. A urea cycle disorder was sus- and acidosis pected and the neonate treated and investigated Acute encephalopathy with or without seizures further. Quantitative amino acids by high perfor- is the commonest presentation in the neonatal mance liquid chromatography (HPLC), acylcarnitine period and in infancy. Typically a term, good profile analysis using tandem mass spectrometry weight neonate, who is well for the first few days of (MS/MS), and urine organic acid estimation by gas life develops feeding difficulties, lethargy, vomiting, chromatography - mass spectrometry (GC-MS) was abnormalities of tone and coma. The progression performed. The plasma citrulline levels were high from onset of symptoms is extremely rapid and (1344 nmol/l; ref: 17 5), argininosuccinate and the neonate may be soon in the intensive care arginine low and urine± orotic acid were increased unit with assisted ventilation. A neonate with a which were consistent with citrullinemia type I. He metabolic disorder can also have associated sepsis, was managed with ammonia scavenging agents, and therefore an index of suspicion in a critically dialysis, IV fluids, dextrose, intralipid but contin- sick neonate is important to investigate further for ued to be encephalopathic and died on day 7 of an IEM. Typically five basic investigations are useful life. Molecular analysis for citrullinemia on the as a first step to differentiate the common cate- stored DNA sample confirmed the neonate to be gories of IEMs and facilitate a diagnostic approach homozygous for a nonsense mutation in ASS gene. (Table 2). Analysis and Synthesis: An elevated plasma am- Typical cases illustrating the diagnostic ap- monia level is an indicator of hepatocellular dys- proaches to the above mentioned five metabolic function due to any cause, commonly being inborn derangements are presented below. errors of metabolism, infections or intoxications. Case Scenario 1: Acute encephalopathy with Urea cycle disorders (UCDs) are the commonest hyperammonemia:• A female neonate, born at metabolic disorders with high ammonia levels. A term, appropriate for date, developed respiratory hypotonic neonate with encephalopathy, respira- distress on Day 2 of life. The blood glucose, tory alkalosis and hyperammonemia should alert electrolytes and sepsis screen were normal. There to a diagnosis of a urea cycle disorder. The was progression to lethargy and poor feeding. The investigations and diagnosis of a urea cycle de- arterial blood gases showed alkalosis and blood fect is illustrated in Figure 2. Central to this culture was sent before initiation of antibiotics. diagnostic algorithm is the quantitative analysis Despite treatment she developed multifocal clonic of amino acid levels in the blood and urinary seizures, became limp and had poor gag reflex and orotic acid estimation by GC-MS. An important

59 Chapter 11

CPS – carbamyl phospahte synthetase NAGS – N acetylglutamate synthatase NH3 – ammonia THAN – Transient hyperammonemia of the newborn

Figure 2 Evaluation and diagnosis in a child with high plasma ammonia.

60 Chapter 11 practical point is to collect a free flowing blood the onset of encephalopathy. The information sample for ammonia estimation and transport this about prenatal diagnosis during next pregnancy to the laboratory in ice for immediate estima- of mother was provided and the need to store tion. Transient hyperammonemia of the newborn blood sample of the baby for mutation detection (THAN) unlike UCDs occurs on the first day of was stressed. Prenatal diagnosis can only be done life. Other disorders causing hyperammonemia if the mutation in the affected child is identified due to secondary inhibition of the urea cycle path- before the next pregnancy. way include pyruvate carboxylase deficiency, fatty Most of the IEMs are autosomal recessive and acid oxidation defects, organic acidurias, lysin- there is 25% risk of recurrence in sibs. There can uric protein intolerance and hyperammonemia- be two, three or more consecutive babies affected hyperornithinemia-homocitrullinuria syndrome. with the disorder as happened in this case. Accu- Case 2 a: Acute encephalopathy with rate diagnosis can prevent recurrence by prenatal metabolic• acidosis: diagnosis or provide presymptomatic diagnosis by testing the sib in newborn period and appropriate management to improve the outcome. The case above illustrates the same message.

Case 2b: In contrast to the case discussed above,• case 2b illustrates the utility of timely diag- nosis and appropriate management of propionic academia.A second born to non-consanguineous parents was well till 1 month of age when he suddenly became sick probably after a day with fever. He developed vomiting, lethargy and pro- gressive encephalopathy. Investigations showed normal electrolytes, normoglycemia but persistent metabolic acidosis with an increased anion gap. The family was from Jharkhand and the neonate His ammonia levels were 175 µmol/L. Urine was (5th in birth order) was transferred when he devel- positive for ketones. A possibility of an organic oped lethargy and poor feeding on day 3 of life. academia was maintained and while awaiting the At admission his sensorium was altered, was dehy- results of MS-MS and urine GC-MS analysis he was drated and had metabolic acidosis that persisted started on the emergency protocol management even after he was stabilized. The anion gap was 26, for organic acidemias with protein elimination and blood sugar 50 mg/dl, plasma ammonia was 256 high caloric intake and carnitine supplementation. µmol/L, plasma lactate normal. Sepsis screen was He improved on management and the diagnosis of negative. There was history of 3 sibling deaths, two propionic acidemia was confirmed - elevated levels females and one male, all of who had presented of propionyl carnitine (8.0nmol/ml(ref: 3.5) on similarly. The family history of an autosomal re- MS-MS acylcarnitine profile and increased< methyl- cessive inheritance and presence of high anion gap citrate and 3 hydroxy propionate in urine GC-MS metabolic acidosis strongly suggested an IEM. Tan- testing. After stabilization, he was started on a dem mass spectrometry (MS-MS) showed elevated diet restricted in isoleucine, valine, methionine and propionyl carnitine (C3) and urine GC-MS identified threonine with carnitine and metronidazole sup- elevated 3 - OH propionate, propionyl glycine & plementation. Post diagnosis follow up with the methycitrate. Propionic academia (PA) was diag- above treatment shows normal development. He nosed based on these reports. The neonate was had one minor episode of decompensation after started on emergency protocol for management. a viral illness during infancy that was detected at However in view of a worsening encephalopathy the onset by alert and knowledgeable parents and and guarded prognosis, the parents took the child managed successfully with the emergency regimen home. It is apparent from this family history protocol. The second issue for this couple was to that all the previous siblings had also died of plan their next pregnancy. Propionic academia is propionic acidemia. Unfortunately the possibility an autosomal recessive disorder with a 25% risk of of a metabolic disorder had not been kept and recurrence. Prenatal diagnosis is best performed appropriate investigations not done to prevent at 11 weeks gestation by mutation analysis in recurrence / initiate timely management before a chorionic villi sample. For this purpose the

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FAOD – fatty acid oxidation defects; FBPase – fructose-1,6-bisphosphatase; G6Pase – glucose-6-phosphatase; GSD – glycogen storage disorders; RTA – renal tubular acidosis.

Figure 3 Approach to metabolic acidosis. mutation in the proband in one of the two genes organic acidopathy. The common organic acidurias implicated in PA had to be identified. The mutation prevalent in our country are maple syrup urine dis- was IVS1 5G A and IVS3 2T C in PCCB gene. ease, methyl malonic aciduria, propionic aciduria, Armed with+ this> knowledge,+ prenatal> testing was glutaric aciduria type I and multiple carboxylase possible in the next pregnancy and the fetus was deficiency. not affected. DNA based testing for mutation detection is Analysis and Synthesis: Organic acidurias may very important for providing prenatal diagnosis have vomiting as a prominent phenotype and be during next pregnancy. Two ml blood in EDTA misdiagnosed as pyloric stenosis. The presence vial may be stored for DNA testing later and the of metabolic acidosis should alert to a diagnosis sample can be transported at room temperature. of an organic aciduria as isolated pyloric stenosis has alkalosis. Metabolic acidosis is a common Case 3: Acute encephalopathy with ketosis: complication of almost any illness and is usually A 2500• gms neonate was born at term and dis- secondary to tissue hypoxia. However if there is charged home on day 2 of life. There were no associated ketosis, high anion gap acidosis and adverse perinatal events. On day 5 he was read- normal plasma chloride; persistent acidosis after mitted with history of poor feeding and lethargy. correction of tissue perfusion, with or without He developed seizures, dystonic posturing of limbs previous history of similar complaints, metabolic and progressed to encephalopathy. There was disorders should be investigated for as per the significant respiratory distress for which he re- approach represented in Figure 3. In acidosis due quired ventilation. Investigations for sepsis were to IEM, a urine pH is below 5 and differentiates it negative. Urine ketones were positive and there differentiates from a renal acidosis. Collection of was no metabolic acidosis. It is important to note urine for estimating organic acids by GC-MS and that presence of ketonuria in the neonate is always plasma acylcarnitine levels at the time of acidosis abnormal. The blood sugar was 78 mg/dl and is useful to confirm or exclude the diagnosis ofan electrolytes normal. The dinitro-phenyl-hydrazine

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MSUD – maple syrup urine disease; NKH – non ketotic hyperglycinemia; SO - Sulfite oxidase deficiency; MCO – molybedenum cofactor deficiency; Cbl – cobalamine.

Figure 4 Approach to a neonate in coma.

(DNPH) test for ketones was positive. The quan- disorders. A basic approach presented in Figure titative amino acid analysis showed high levels 4 for inborn errors of metabolism should be fol- of leucine 2900 µmol/l (normal 65-220 µmol/l), lowed alongwith evalation for the acquired causes isoleucine 377 µmol/l (normal 26-100 µmol/l) and of neonatal and infantile encephalopathy. Abnor- valine 384 µmol/l (normal 90-300 µmol/l) and al- malities of ammonia, arterial blood gas analysis, loisoleucine at 655 µmol/l (normal 0-5 µmol/l). This serum electrlytes and urine ketones are important identified the neonate to have Maple Syrup Urine clues to further testing and treatment. disease (MSUD). The neonate was managed with the specific protein restricted food for MSUD and Case 4: A Neonate with Hypoglycemia and the emergency protocol was initiated whenever he Hepatocellular• Disease: A 40 days infant pre- decompensated. The mother was trained to do the sented with failure to gain weight appropriately, DNPH test at home when she felt the child is be abdominal distension and mild jaundice. On ex- unwell and was educated to initiate the emergency amination he had bilateral cataract, conjugated management at home. He performed well with hyperbilirubinemia and mild transaminitis. The good metabolic control and normal developmental blood sugar was low at 40 mg/dl and prothrombin milestones. At 22 months of age, a liver transplant and partial thromboplastin time was normal. The was performed. This was the first ever liver trans- α- fetoprotein levels were normal. Benedict’s test plant in India for MSUD. The boy, now 9 yrs old, for urine reducing substances was positive and the is on no dietary restrictions and doing well. This glucose oxidase stix was negative suggesting the case illustrates that early diagnosis and educating presence of non glucose reducing substances in the family about the disorder and appropriate the urine. Plasma succinylacetone was normal. management of the treatable IEMs can have a Plasma galactose and RBC galactose -1- phosphate good outcome. levels were increased, 6032.9 (N. Range: 56 umol/L) and 4.5 (N. Range: 1mg/dL) respec-< Coma in a neonate or infant is a common tively. Enzyme assay for galatose< 1 phosphate presentation for acwuired as well as metabolic uridylyltransferase enzyme (GALT) confirmed the

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FAOD – fatty acid oxidation defects; GSD – glycogen storage disorders; HFI – hereditary fructose intolerance; IDM – infant of diabetic mother; SGA – small for gestational age.

Figure 5 Approach to hypoglycemia. diagnosis of galactosemia. Molecular analysis in tions should be done at the time of hypoglycemia GALT gene confirmed the presence of a homozy- and it is prudent to store a critical blood and gous mutation, CAT CCT; c. A203C; p.H68P. The urine sample for future use if required. Additional neonate was put on> a galactose free diet. His features to be looked for at the time of assessment cataract regressed spontaneously in 2-3 months. of hypoglycemia include hepatomegaly, timing of The metabolic parameters improved and on fol- hypoglycemia, lactic acidosis, and urine and blood low up the levels of galactose and galactose 1 ketones. Estimate the levels of insulin, cortisol phosphate decreased. For the diagnosis of galac- and growth hormone to evaluate the endocrine tosemia it is imperative to take blood samples causes. Test hepatic function for liver disease, for assay prior to blood transfusion. It is best acquired or inherited, as a cause for hypoglycemia. to combine analysis of the metabolites, galactose Hypoglycemia after a short fast suggests a dis- and galactose 1 phosphate alongwith with GALT order of carbohydrate metabolism and that after assay. This helps to resolve borderline values of a prolonged fast signifies a disorder of fatty acid enzyme that may be obtained as well as diagnose oxidation. Predominant hepatomegaly with short epimerase and galactokinase deficiency. fast hypoglycemia is a feature of gluconeogene- sis disorders and glycogen storage disorder type Analysis & Synthesis: To investigate a neonate III. Hepatocellular disease seen in tyrosinaemia /infant with hypoglycemia for a metabolic etiology, type 1 can be accompanied with hypoglycemia. one can follow the algorithm in Figure 5. Investiga-

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Respiratory chain defects and organic acidurias persistent infections and hyperventilation. Pres- have associated ketoacidosis. Making a timely ence of ketosis with high lactate suggests an IEM diagnosis is imperative as many disorders present- (exceptions include FAOD, PDH, GSD type1), as the ing with hypoglycemia such as glycogen storage ketosis is less likely in acquired causes of hyper- disease I / III, fatty acid oxidation defects, fruc- lactatemia. Among the IEMs, high lactate can be tise 1,6 bisphosphatase deficiency, HMG coA lyase present secondarily in organic acidurias, urea cycle deficiency are easily treatable. Acute hepatocel- disorders (esp. citrullinemia), fatty acid oxidation lular dysfunction can present with hepatomegaly, defects. Primary lactic acidemias include disorders jaundice, hypoglycemia, transaminitis and abnor- of gluconeogenesis, glycolysis, pyruvate carboxy- malities of . Metabolic disorders to be lase deficiency, pyruvate dehydrogenase deficiency considered in the differential include galactosemia, and disorders of mitochondrial respiratory func- tyrosinaemia type 1, fatty acid oxidation defects, tion. Concomitant high levels of CSF lactate and peroxisomal disorders, hereditary fructose intol- blood and CSF alanine levels support a diagnosis erance, mitochondrial respiratory chain defects, of a primary lactic academia. Fasting hypoglycemia congenital disorders of glycosylation, Niemann and hyperlactatemia is seen in glucose 6 phos- Pick C disease, disorders of bile acid metabolism phatase deficiency and fructose 1-6 biphosphatase and neonatal hemochromatosis. deficiency. Postprandial hyperlactatemia suggests GSD type III / VI / glycogen synthase deficiency. In Case 5: Acute encephalopathy with lactic pyruvate dehydrogenase deficiency and disorders acidosis:• Second in birth order to a consan- of respiratory chain function, the lactate levels are guineously married couple, the male neonate highest in the fed state and may be missed if developed poor feeding and lethargy with seizures sampling is performed after an overnight fast. 3 days after birth. The sepsis screen was negative. Lactate pyruvate ratio (L/P ratio) measurements There was hypoglycemia and severe lactic acidosis. aid to differentiate pyruvate carboxylase deficiency The serum ammonia was 78 micromol/litre. Urine and respiratory chain disorders where the ratio is ketones were positive. Blood pyruvate analysis increased ( 30), from normal or low L/P ratio in was not available. There was progressive deterio- PDH deficiency. ration of sensorium with requirement of ventilator > support. The MS/MS analysis for acylcarnitines was Mutation detection confirms the diagnosis and normal. The urine organic acid estimation by gas is essential for preventing recurrence by prena- chromatography- mass spectrometry showed high tal diagnosis. In situations where the metabolic lactate levels. A differential diagnosis of pyruvate profile does not provide a definite diagnosis or carboxylase deficiency, PDH deficiency, disorders a genetically heterogeneous disorder like MSUD of gluconeogenesis like glucose 6 phosphatase is diagnosed, an option of exome sequencing is deficiency, fructose 1-6 biphosphatase deficiency found very useful. Exome sequencing is a test and PEPCK disorder was kept. There was a his- which sequences genes known for all monogenic tory of death of a male sibling in the neonatal disorders in one go using a latest high throughput period with undiagnosed encephalopathy and aci- sequencing technique known as Next Generation dosis. As a specific diagnosis could not be reached Sequencing (NGS). This test has a 40 to 50% diag- by biochemical diagnosis, exome sequencing (se- nostic yield in metabolic disorders and is of great quencing of coding regions of all clinically relevant importance when the clinical / biochemical diagno- genes) was ordered rather than testing one gene sis is not reached or gene for clinically suspected after other. No mutation was identified in the PDH disorder is very large. In such situations, this test gene but a homozygous mutation in the PC gene becomes cost effective also. It can be done later on confirming the diagnosis of pyruvate carboxylase the stored blood sample as little as one millilitre. deficiency. Case 6: Presentation with seizures: At 36 Analysis & Synthesis: Once a high blood lactate hours• of life a term neonate was noted to have is identified, the first step is to exclude false increased irritability and abnormal seizure like elevations due to improper sampling techniques. activity of the face and limbs. There was no A high lactate level from a free flowing blood risk factor for hypoxia or sepsis and the inves- sample can be because of acquired or genetically tigations results were non-contributory. Serum determined IEMs. Acquired conditions associated electrolytes, blood glucose was normal. Video EEG with lactate accumulation included tissue hypoxia, showed a burst suppression pattern. The seizure circulatory collapse, diarrhea, hepatic failure and profile was refractory to antiepileptic therapy.

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Injectable pyridoxine, 100 mg under cardiorespira- good outcomes as with all treatable disorders. tory and video EEG monitoring showed cessation Initiation of treatment at suspicion of an IEM is of the seizure activity. The clinical diagnosis of necessary even before a definitive diagnosis is pyridoxine-dependent epilepsy was confirmed by made. The aims of management include removal molecular diagnosis that confirmed the mutation of the toxic metabolite by reducing substrate avail- in the ALDH7A1 gene. Pyridoxine was continued ability and enhancing toxic metabolite excretion, orally and slowly the antiepileptics were tapered provide adequate calories to prevent catabolism, and stopped. This is another example that illus- co-factor therapy and supportive management. trates the good response to a treatable disorder if Many sick neonates will also require circulatory timely suspected and appropriately treated. and ventilatory support. Analysis & Synthesis: Seizures in the neonate In all sick neonates, proteins are stopped for commonly occur as a result of hypoxic-ischemic 24 – 48 hrs and intaveneous 10% glucose and encephalopahty, infections, cerebral dysgenesis, intralipid infusion initiated to provide calories. If cerebrovascular disorders and transient metabolic hyperglycemia occurs, do not decrease the glucose disturbances. Neonatal seizures with or without infusion but consider insulin. Inadequate calories associated biochemical derangements is also an promote catabolism and aggravation of the en- important manifestation of IEMs. A high index of cephalopathy. Once the neonate improves, small suspicion is important to evaluate for disorders amounts of protein are started orally no later than presenting with isolated neonatal seizures. These 24 – 48 hrs (0.5 mg/kg/day) and gradually increased include nonketotic hyperglycinemia, pyridoxine- to 1 gm / kg/ 24 hours. dependent seizures, glucose transporter defi- The biochemical abnormalities of acidosis, hy- ciency„ sulphite oxidase deficiency, molybdenum poglycemia and hyperammonemia are treated cofactor defect, folinic acid responsive seizures, immediately. Sodium benzoate, sodium phenyl- creatine deficiency syndromes, abnormalities of butyrate / phenylacetate and arginine decrease serine biogenesis and peroxisomal disorders. Peri- ammonia levels and are best given intravenous partum onset of seizures, refractory to treatment depending on availability. (Table) If the neonate or progressive worsening of seizure phenotype, continues to be comatose, dialysis should be ini- electroencephalogram indicative of burst suppres- tiated to decrease the toxic products. Although sion pattern or hypsarrhythmia or magnetic reso- hemodialysis is more efficient, peritoneal dialysis nance imaging of brain showing profound atrophy is more widely available and feasible. or hypoxic- ischemic injury without obvious perina- For neonates with a suspected organic tal insult mandates evaluation for an IEM. Diagnosis acidemia, the acidosis is treated with sodium of the above disorders is by detection of the spe- bicarbonate as per NICU protocol. In addition cific accumulating analyte in the urine, plasma carnitine supplementation and cofactor therapy and CSF. Specific molecular testing is important to with biotin, vitamin B12 (vitamin B12 responsive confirm diagnosis. Non metabolic genetic disor- MMA), and thiamine (thiamine responsive MSUD) ders like pontocerebellar atrophy, various forms is initiated. of infantile epileptiform epilepsy, etc can present Intractable seizures in a setting of a suspected with seizures and neuroimaging, family history and IEM are given a trial of pyridoxine. An initial genetic studies will provide the diagnosis. dose of 100 mg intraveneously of pyridoxine is administered. This can be repeated after 10 mins Miscellaneous Presentations that should in- if there is no response, upto a total dose of 200 clude• evaluation of specific metabolic defects: mg. If there is uncertainity of response / partial Sudden death or stillbirth, ascites or non-immune response, pyridoxine may be continued at 5-15 hydrops fetalis, cardiomyopathy, dysmorphism mg/kg/day upto 7 days to evalaute for response. and malformations, and can be the If the seizures persist, folinic acid at 5 mg/kg/day presenting features of IEMs presenting during orally or IV in three divided doses, daily for 3 days neonatal period. is given. If still no response, pyridoxal phosphate at 30 mg/kg/day for 3 days is tried. In a sus- Treatment pected glucose transporter defect, ketogenic diet is benificial. Many IEMs are treatable and products are now Supportive care for management of hydration, being made available in India as well. Early di- seizures, ventilation and sepsis is similar as for agnosis and initation of therapy is important for any sick neonate. Sodium valproate is to be

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Table 3 Metabolic Emergency Life Saving Medications.

Drug Indication Recommended Dose Route and doses Biotin Biotinidase def. 5-20 mg/day Oral / IV MCD def. Biotin responsive basal ganglia disease Dextromethorphan NKH 5-7 mg/kg/day. Upto 35 Oral; 4 divided doses mg/day have been used Diazoxide Persistent 15 mg/kg/day (newborn) Oral Hyperinsulinemia 10 mg/kg/day (infants) Folinic acid Remethylation defects, 5-15 mg/day Oral / IV DHPR def., Cerebral folate transporter Hydroxocobalamin Disorders of cobalamin 1mg/day IM (Vit B12) metabolism 10mg/day Oral L- Arginine Acute management of 250-500 mg/kg/day Oral / IV hyperammonaemic crisis 4-6 divided doses or urea cycle disorders except arginase def. L- Carnitine Primary or secondary Acute crises - 100 mg/kg/dose IV/PO; 3-4 times daily carnitine deficiency Chronic - 100-200 mg/kg/day PO; 3 divided doses N- NAGS, CPS deficiency 100 mg/kg Oral; 4 times a day Carbamylglutamate NTBC Tyrosinemia type I 1 mg/kg Oral; 1-2 divided doses Pyridoxine Pyridoxine-responsive 100 mg i.v. with EEG IV seizures, Homocystinuria monitoring or 30mg/kg/day Oral / enteral for 7 days Maintenance 5-10 mg per day Oral Pyridoxal Pyridox(am)ine 40 mg/kg per day in 4 divided Oral phosphate 5-phosphate oxidase doses deficiency Sodium benzoate Hyperammonaemia 250-500 mg/kg/day Oral / IV; 4-6 divided doses or continuous i.v. infusion Sodium Hyperammonaemia 250-500 mg/kg/day Oral / IV; 4-6 divided Phenylbutyrate doses Thiamine Thiamine responsive 10-15 mg/day Oral variants of MSUD, PDH def., Complex 1 def. Coenzyme Q10 Primary CoQ10 deficiency 2-15 mg/kg/day Oral

avoided in patients with IEMs. Specific, long term now being made available in India. Table 3 denotes management after stabilization includes protein the important drugs used in management of IEMs restriction and exclusion of amino acids specific to in neonates the diagnosed disorder. These special diets are

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Screening Tests Genetic Diagnosis & Role of New Technology Table 4 lists the investigations to screen a neonate suspected to have an IEM. Special care for handling some samples as discussed above. Further specific Diagnosis of IEM is a challenging task for a neona- investigations to confirm the diagnosis depend on tologist. Clinical suspicion is the first and important the presenting phenotype and screening tests re- step. Working with algorithms and experience with sult. Discussion with a geneticist / metabolic expert the disorders definitely improves the diagnosis. at the time of sampling / results improves making Still many a times, definite etiological diagnosis can a definitive diagnosis and treatment of neonates not be reached even if the possibility of IEM is with IEMs. strong or definite. Lack of easy access to GCMS and MSMS, time lost in sample transport, inappropriate timing of sample collection, metabolic profile mod- Metabolic Autopsy ified by tissue hypoxia, stoppage of feeds, etc. are the reasons for failure of diagnostic efforts. DNA Very often the neonate / child is very sick and based disgnostic test to identify causative mutation metabolic investigations may not be possible im- does not have some of these limitations. DNA is mediately. In this situation blood and urine not affected by timing of sample collection. Tradi- samples must be preserved for diagnostic pur- tionally, sequencing of the gene for the suspected poses. Pretransfusion blood sample in an EDTA disorder is the DNA test done. This is sometime and plain vacutainer as well as on the newborn difficult as the list of differential diagnosis consid- screening filter paper must be stored. Freeze 15 ered is long. New technology of high throughput ml urine at -80C as well as CSF/ tissue biopsy DNA sequencing known as Next Generation Se- samples. Most of the IEMs have a 25 – 50% risk of quencing (NGS) is of great help in such situation. recurrence and prenatal diagnosis in a subsequent This technology is used to sequence many genes pregnancy is not possible in the absence of a (panel) for the disorders with similar phenotype in definitive diagnosis. one go or the whole genome can be sequenced

Table 4 Screening Tests when an Inborn Error of Metabolism is suspected.

Blood Glucose Electrolytes Acid Blood Gas, anion gap (Na+K) – (HCO3+Cl) Ammonia Lactate, Pyruvate Creatinine Calcium SGOT/SGPT, PT & APTT Uric acid Plasma amino acids by HPLC Carnitine and Acylcarnitine profile by MSMS DNA storage (3 ml blood in EDTA or a spot newborn screening filter paper) Urine Odor Ketones, sulfite, MPS screen Reducing substances by Benedict’s reagent and glucose oxidase Organic acids by GC-MS CSF Glucose Lactate Amino acids HPLC – High performance liquid chromatography; GC-MS – Gas chromatography - Mass spectrometry.

68 Chapter 11 in one go and the data is searched to identify the useful investigations in a particular phenotype. the genetic variation which can be the cause of Expected poor outcome may demoralize the clin- the phenotype. NGS technology is commonly ician and may lead to loss of cooperation from used to sequence all the coding regions of genes the family. However, importance of confirmed (Whole Exome Sequencing – WES) or sequencing etiological diagnosis and preferably identification all genes known to be associated with disorders. of disease causing mutations by DNA analysis The diagnostic yield using WES in various clinical should be conveyed to the family. It is essential for presentations may vary from 30 to 50%. It may be genetic counseling and prenatal diagnosis. Storage more in certain situations especially in IEM, where of samples in a serious baby or after death has there are some definite candidate genes. The already been stressed above. In addition, 1 to 2 ml reporting time varies between 2 to 4 weeks and of blood in EDTA IS VERY IMPORTANT. getting a diagnosis of a treatable disorders will be of such great help and WES may cause dramatic Suggested Reading change in the outcome. Mutation detection is also of importance for providing prenatal diagnosis and 1. Bodian DL, et al. Utility of whole-genome should be pursued even if the child is unlikely to sequencing for detection of newborn screen- survive or is not alive. In some situations where ing disorders in a population cohort of 1,696 the baby with the disease is not alive and there neonates. Genet Med 2015; 18:221-230. is no sample for DNA analysis; an attempt can be 2. Clarke JTR. A clinical guide to inherited made to do WES of the parents or one of them metabolic diseases. 3rd Edn. Cambridge: to identify carrier status for an autosomal reces- Cambridge University Press, 2006. sive disorder in both of them or a heterozygous mutation for an X linked disorder in the mother. 3. Christodoulou J, Wilcken B. Perimortem lab- WES also has an advantage that in addition to oratory investigation of genetic metabolic IEMs, other genetic disorders presenting similar to disorders. Semin Neonatol 2000; 9: 275-280. IEM also will be detected; even when the clinician 4. Fazeli W, et al. Mendeliome sequencing has not suspected it. Sometimes the results of enables differential diagnosis and treatment WES are uncertain and care has to be taken while of neonatal lactic acidosis. Mol Cell Pediatr providing genetic counseling. Understanding the 2016; 3: 22. principle of the new technology is essential to 5. Filiano JJ. Neurometabolic Diseases in the learn the strengths and limitations of the powerful Newborn. Clin Perinatol 2006; 33: 411-479. technique. WES is also being evaluated as a first tier test for babies in NICU and also, to be used 6. Leonard JV, Morris AA. Diagnosis and early as for newborn screening of asymptomatic babies. management of inborn errors of metabolism Availability of easy diagnosis for such a complex presenting around the time of birth. Acta disorders will change the approach and outcome Paediatr 2006; 95: 6-14. of these disorders. 7. Saudubray JM, et al. Clinical approach to treatable inborn metabolic diseases: An in- Conclusion troduction. J Inherit Metab Dis 2006; 29: 261-274. Neurometabolic disorders, especially those that present acutely are treatable and delay in diag- 8. Saudubray JM, van den Berghe G, Walter JH. nosis and initiation of treatment increases the Inborn metabolic diseases: diagnosis and morbidity and risk of mortality. A high index of treatment. 5th Edn. Heidelberg; Springer, suspicion is important to diagnose an IEM. The 2012. major hurdle in diagnosing an inborn error is the 9. Tebani A, et al. Clinical Metabolomics: The inability to consider a diagnosis. Once a differential New Metabolic Window for Inborn Errors diagnosis with the help of the basic investigations of Metabolism Investigations in the Post- as detailed above is made, samples for special Genomic Era. Int J Mol Sci 2016;17(7). investigations can be sent to a reference labora- tory as most of these tests are now available in (Reproduced with permission from: Neonatal India. It is helpful to consult a clinician involved Emergencies. Editor Dr Meharban Singh. CBS in diagnosis and management of IEMs to discuss Publishers and Distributors Pvt Ltd, New Delhi)

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An Approach to Genetic Disorders Affecting the White Matter

Vijayalakshmi SR, Prajnya Ranganath Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad

Correspondence to: Dr Prajnya Ranganath Email: [email protected]

Introduction trophy. Hence, all leukodystrophies are genetic encepahlopathies but not all genetic leukoen- The white matter lies beneath the gray matter and cephalopathies are leukodystrophies. consists of millions of nerve fibre bundles (axons) that serve as functional circuits linking different regions of the brain. White matter forms the bulk Myelin and myelination of the deep parts of the brain and the superficial The myelin sheath, a modified plasma membrane parts of the spinal cord. Aggregates of gray matter extension of the oligodendroglial cells, spirals are spread within the cerebral white matter. The around the axons and encases it throughout its gray matter is primarily associated with cognition length except at the nodes of Ranvier. Myelin and processing, while the white matter serves sheaths act as insulation sheaths and serve to to conduct the action potentials and coordinate transmit action potentials by saltatory conduction communication between different brain regions. which is fast and energy efficient. Myelin also Genetic disorders affecting the white matter of provides trophic support and protection for axons. the brain are heterogeneous with variable and Disturbances in myelin therefore result in motor, overlapping phenotypes. There is an ongoing pro- sensory and cognitive impairment. cess for classification of these disorders based on Myelin is made up of an outer layer of glycol- the genetic, histopathology and the neuroimaging ipid (galactocerebroside and sulfatide) and choles- pattern observed in these disorders. Few of the terol, an inner phospholipid layer (phosphoinos- recent consensus definitions are as follows: itol serine, phosphoinositol 4,5,diphosphate and Leukodystrophy: (Greek: leukos (white), dys ethanolamine plasmalogen) and an intervening area of hydrocarbon chains (long chain fatty acids). – (disturbance),• trophê (nutrient)-defective nu- trition) refers to a group of genetic disorders In addition, there are structural proteins namely primarily affecting the white matter with or without the myelin basic protein (MBP) and the proteolipid peripheral nervous system involvement. They are protein (PLP) which maintain myelin structure and characterised by glial cell or myelin sheath abnor- stability, the myelin-associated glycoprotein (MAG) malities with magnetic resonance imaging (MRI) which is essential for initiation of myelination by showing a hyperintense T2 signal in the affected mediating axonal-glial contact, and myelin zero white matter and a variable T1 signal (Vanderver A protein (MZP), myelin oligodendrocyte glycopro- et al., 2015). tein (MOP), oligodendrocyte myelin glycoprotein and 2,3-cyclic nucleotide 3-phosphodiesterase (van Leukoencephalopathy: though often used der Knaap, 2001). interchangeably with leukodystrophy, actually • Myelination begins as early as the 12th week refers to genetic or acquired disorders which of intrauterine life and occurs significantly from have white matter changes comparable to that mid-gestation to the second year of postnatal life. found in leukodystrophies, but primarily have ei- Myelination is a high energy requiring process. Ini- ther neuronal, vascular or systemic involvement tiation of myelin and its maintenance is regulated with myelin involvement occurring as a secondary by the availability of glycolytic and lipid substrates. event (Vanderver A et al., 2015). Abnormalities of myelination can be in the form of Genetic leukoencephalopathies: have been failure of myelin formation (hypomyelination), for- defined• as heritable white matter abnormalities mation of abnormal myelin (dysmyelination) and which do not meet the criteria of being a leukodys- loss of formed myelin (demyelination).

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Classification of myelin disorders Well defined leukoencephalopathies • Undefined leukoencephalopathies There are various classifications of myelin disor- • ders (based on pathological, biochemical, genetic Under this classification, well defined leukoen- and combined clinical/ histopathological/ biochem- cephalopathies have been further subdivided into ical criteria). As per the classification proposed categories as listed in Table 1 (Di Rocco et al., 2004). by van der Knaap for myelin disorders, integrating As per the recent GLIA (Global Leukodystrophy the MRI pattern and pathophysiology (van der Initiative) Consortium consensus statement, more Knaap, 2001), all white matter disorders have been than 30 distinct leukodystrophy conditions have included under the umbrella term of leukoen- been characterized, which are listed below in the cephalopathies and these have been categorized alphabetical order in Table 2 (Vanderver A et al., as: 2015).

Table 1 Types of well defined leukoencephalopathies.

Type of well defined Underlying pathophysiology Examples leukoencephalopathy Hypomyelinating Primary disturbance in the forma- Pelizaeus- Merzbacher (PMD) and disorders tion of myelin • PMD-like diseases Secondary to neuron or astrocyte Cockayne syndrome dysfunction (including abnormal • Tay syndrome interaction between oligodendro- • Salla disease cytes and neurons) • GM1 and GM2 gangliosidoses • Infantile neuronal ceroid lipofusci- • nosis Hypomyelination with atrophy of • the basal ganglia and cerebellum (HABC) syndrome Dysmyelinating disor- Altered sequence of myelination 18q minus syndrome ders (Delayed or dis- • Irregular pattern of myelination • Untreated aminoacidopathies turbed myelination) • Additional component of hy- • Organic acidurias • pomyelination observed • Demyelinating disorders Abnormal myelin composition Metachromatic leukodystrophy (re- (leukodystrophies) • Myelin instability and subse- • duction of cerebroside and accu- • quent loss (demyelination) mulation of sulphatides which is toxic to oligodendroctyes) X-linked Adrenoleukodystrophy • (ALD) (dysfunction of microglia- inflammatory response; incorpo- ration of very long chain fatty acids in myelin leads to membrane insta- bility) Krabbe disease (accumulation of • cerebroside and toxic metabolite psychosine leads to oligodendro- cyte death) Peroxisomal disorders such as Zell- • weger syndrome, neonatal ALD and Refsum disease. (decrease in myelin substrates)

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Disorders related to With myelin loss Canavan disease (accumulation of myelin splitting (cystic • N-acetyl aspartate and precursor degeneration of myelin) N-acetylaspartlyglutamate leads to intramyelinic edema, vacuolization and oligodendrocyte failure) Mitochondrial disorders • 1,2-hydroxyglutaric aciduria Without myelin loss • Megalencephalic leukoencephalo- • pathy with subcortical cysts Disorders secondary to Giant axonal neuropathy axonal damage • Others Alexander disease (primary genetic • disorder of astrocytes) Childhood ataxia with central • nervous system hypomyelination (CACH) Sjogren-Larsson syndrome • Cerebrotendinous xanthomatosis • Leukodystrophy with polyol • metabolism abnormality

Table 2 Different types of leukodystrophies with the causative genes and modes of inheritance.

Disorder Gene(s) Pattern of inheritance Adrenoleukodystrophy X linked (X-ALD) ABCD1 XL Adult onset leukodystrophy with neuroaxonal spheroids and • CSF1R AD pigmented glia (ALSP): • Hereditary diffuse leukoencephalopathy with spheroids • (HDLS) Pigmentary type of orthochromatic leukodystrophy with • pigmented glia (POLD) Aicardi– Goutières Syndrome (AGS) ADAR1 Usually AR • RNASEH2A but maybe AD • RNASEH2B • RNASEH2C • SAMHD1 • TREX1 Alexander disease (AxD) • GFAB AD Autosomal Dominant Leukodystrophy with Autonomic disease • LMNB1 AD (ADLD) • Canavan disease ASPA AR Cerebrotendinous Xanthomatosis (CTX) • CYP27A1 AR Chloride Ion Channel 2 (ClC-2) related leukoencephalopathy • CLCN2 AR with intramyelinic oedema (leukoencephalopathy with ataxia) •

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eIF2B-related disorders (Vanishing White Matter Disease EIF2B1 AR (VWMD) or Childhood ataxia with central nervous system • EIF2B2 hypomyelination (CACH)) • EIF2B3 • EIF2B4 • EIF2B5 Fucosidosis • FUCA1 AR Globoid cell Leukodystrophy (Krabbe) • GALC AR • PSAP Hypomyelination with atrophy of the basal ganglia and • TUBB4A AD cerebellum (H-ABC) • Hypomyelination with brainstem and spinal cord involvement DARS AR and leg spasticity (HBSL) • Hypomyelination with congenital cataract (HCC) FAM126A AR Leukoencephalopathy with brainstem and spinal cord involve- • DARS2 AR ment and lactate elevation (LBSL) • Leukoencephalopathy with thalamus and brainstem involve- EARS2 AR ment and high lactate (LTBL) • Megalencephalic Leukoencephalopathy with subcortical cysts MLC1 AR (MLC) • HEPACAM Metachromatic leukodystrophy (MLD) and its biochemical • ARSA AR variants • PSAP Oculodentodigital dysplasia (ODDD) • GJA1 Usually AD • maybe AR Pelizaeus Merzbacher disease (PMD) PLP1 XL Pelizaeus Merzbacher like-disease (PMLD) • GJC2 AR Peroxisomal Biogenesis disorders (including Zelleweger, • PEX genes AR neonatal Adrenoleukodystrophy and Infantile Refsum) • Pol-III related disorders (4H syndrome - hypomyelination, POLR3A AR hypodontia and hypogonadotropic hypogonadism) • POLR3B Polyglucosan Body Disease (PGBD) • GBE1 AR RNAse T2 deficient leukoencephalopathy • RNASET2 AR Sialic acid storage disorders (Salla disease, Infantile sialic acid • SLC17A5 AR storage disease and Intermediate form) • Single enzyme deficiencies of peroxisomal fatty acid beta AR oxidation: D-Bifunctional Protein Deficiency HSD17B4 • Sterol Carrier Protein X (SCPx) deficiency • SCP2 • Peroxisomal acyl-CoA-Oxidase Deficiency • ACOX1 •Sjögren–Larsson syndrome • ALDH3A2 AR SOX10-associated PCWH - peripheral demyelinating neuropa- • SOX10 AD thy, central dysmyelinating leukodystrophy, Waardenburg • syndrome and Hirschsprung disease 18q minus syndrome Contiguous gene Majority are de •deletion involving novo; deletion the MBP gene can be inherited AD = autosomal dominant; AR = autosomal recessive; XL = X-linked

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Leukoencephalopathies include inherited vas- Clinical features culopathies (eg. Cerebral Autosomal-Dominant Arteriopathy with Subcortical Infarcts and Leukoen- The clinical features are predominantly neurologic cephalopathy (CADASIL) and COLA1 & COLA2- and almost invariably affect the motor system related disorders), inborn errors of metabolism and are progressive in nature. Extra neurologic (e.g. organic acidemias and disorders of aminoacid features provide vital clues to arrive at a specific metabolism), disorders affecting the neurons of diagnosis. the cerebral cortex or other gray matter struc- tures (e.g. infantile variants of GM1 and GM2 Age of onset gangliosidosis and neuronal ceroid lipofuscinosis), those with both white and gray matter involvement The onset of the symptoms is variable ranging from (e.g. mitochondriopathies such as Mitochondrial connatal (at birth) to adulthood (Table 3) and most encephalomyopathy, lactic acidosis, and stroke- of these disorders present with variable severity like episodes (MELAS) and Myoclonic Epilepsy with across all age groups. Ragged-Red Fibers (MERRF) syndromes, POLG- related disorders and familial hemophagocytic Neurologic manifestations lymphohistiocytosis) and acquired myelin disor- ders (eg. multiple sclerosis) which may be of Motor impairment: Motor symptoms are infectious or post infectious etiology or due to the• predominant presenting feature in majority of toxic, hypoxic or non-genetic vascular insults. the white matter disorders, while cognitive decline (personality changes and dementia) and seizures Prevalence are the initial manifestations in neuronal (gray matter) disorders. These caveats may not be appli- There is limited data on the overall prevalence cable in all cases as many disorders with primary of leukodystrophies and the relative frequencies gray matter involvement may also affect the white of different leukodystrophies. Metachromatic matter due the underlying , leading to a leukodystrophy is reported to occur in 1 in 40,000 clinical dilemma. to 170,000 individuals world-wide (von Figura et al., A variable period of normal development fol- 2001). Ethnic preponderance has been reported lowed by regression is the most common presen- in some leukodystrophies. Canavan disease is tation. The pattern of motor regression may vary relatively common with a high carrier frequency in each condition (Table 4). They may also present in the Ashkenazi Jewish population. Two com- with delayed or stagnated development in some mon mutations (E285A and Y231X) accounting for cases. 98% of the disease-causing alleles of the ASPA Hypomyelinating conditions usually present gene (Sistermans et al., 2001). Megalencephalic as developmental delay, while genetic leukoen- leukodystrophy with subcortical cysts (MLC) is a cephalopathies are characterised by a period of common leukodystrophy described in the Agarwal normal development followed by stagnation i.e. community in India and in a study by Gorospe et no further acquisition of skills. Older children and al (2004) all 31 cases tested were found to result adults may report frequent falls, altered gait or from a common mutation (320insC) in the MLC1 difficulty in sporting activities. gene, suggesting a founder effect in this population Spasticity and hypereflexia are characteristic (Gorospe et al. 2004). signs in majority of the white matter disorders. There may be an initial hypotonia which invariably Evaluation of leukodystrophies and progresses to spasticity. Severe hypotonia with genetic leukoencephalopathies head lag is a feature of Canavan disease. A hy- potonic infant with facial dysmorphism, seizures, The clinical diagnosis of leukodystrophies and ge- dolichocephaly and a wide open anterior fontanelle netic leukoencephalopathies is often challenging may suggest . Spasticity with due to considerable overlap in the clinical fea- diminished deep tendon reflexes occurs when tures. Though the advances in recognition of the peripheral nervous system is involved as in the neuroimaging patterns of these disorders has Metachromatic leukodystrophy(MLD), Krabbe and improved the diagnostic yield, more than half of other hypomeylinating conditions such as with these disorders still remain undiagnosed. PLP-null mutations.

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Table 3 Age of onset of common leukodystrophies.

Disorder Infantile (first Late infantile Juvenile Adolescent year) (1-5yrs) (5-12yrs) and adulthood Metachromatic leukodystrophy √ (most common √ √ (MLD) type of MLD) Pelizaeus Merzbacher Disease √ √ (classic form) Krabbe disease √ (classic form) √ √ √ Alexander disease √ (most common √ √ √ variant) Canavan disease √ X-linked adrenoleukodystrophy √ √ Childhood ataxia with central √ √ √ √ nervous system hypomyelina- tion (CACH) Megalencephalic Leukoen- √ √ √ √ cephalopathy with subcortical cysts (MLC) Aicardi–Goutières Syndrome √ (AGS) Giant axonal neuropathy type I √ Hypomyelination with atrophy √ √ of the basal ganglia and cerebel- lum (H-ABC) Leukoencephalopathy with √ √ √ brainstem and spinal cord in- volvement and lactate elevation (LBSL)

Table 4 Pattern of motor regression.

Gradual progressive decline Majority of leukodystrophies • Megalencephalic leukoencephalopathy with subcortical • cysts (MLC) Rapid decline Infantile Krabbe Episodic decline (triggers may be • Childhood ataxia with central nervous system hypomyeli- an event of minor head trauma or • nation (CACH)/Vanishing white matter disease febrile illness) Inborn errors of metabolism • Mitochondrial disorders • Pol III related disorders • X-linked adrenoleukodystrophy • Ataxia: Ataxia, a feature of cerebellar in- and hypogonadotropic hypogonadism) or in asso- volvement,• may occur as an isolated finding, as ciation with predominant spasticity. Sensory ataxia a predominant finding e.g. Childhood ataxia with may be present due to impaired proprioception in central nervous system hypomyelination (CACH) peripheral sensory system involvement. and 4H syndrome (hypomyelination, hypodontia

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Extrapyramidal symptoms: Extrapyramidal acid storage disorders and Sjogren Larsson syn- symptoms• appear with involvement of the deep drome. An acute onset with focal seizures may gray nuclei and are seen in 1,2 hydroxyglutaric be seen in childhood onset X-ALD. Impact seizures aciduria. Choreoathetosis is a feature of the classic (following minor head trauma) are seen in MLC. form of Pelizaeus-Merzbacher disease. Dystonia, Autonomic dysfunction: Autonomic dys- when it occurs, is usually generalised and appears function• causes bladder (retention, incontinence), as repetitive movements, torsion or abnormal pos- bowel (constipation/ incontinence), cardiac (ar- turing, which may be exacerbated by voluntary rhythmias), vascular (postural hypotension) and movements, emotions and physical discomfort. thermoregulatory (decreased sweating) symptoms. Cognitive impairment: Cognitive decline in- It is a prominent feature of adult onset or adoles- evitably• appears in the course of the disease, cent phenotypes of Alexander disease, Autosomal although the nature and severity varies. It presents dominant leukodystrophy with autonomic disease as intellectual disability in childhood, or as de- (ADLD) and MLD, and can affect younger patients mentia and psychiatric features in adult onset late in the disease. leukodystrophies. Cognitive decline can be rapid Others: Progressive loss may appear in in X-ALD and lysosomal storage leukodystrophies. speech,• ability to eat (swallowing, chewing), vision Relative sparing of the mental abilities is seen in and hearing as the disease advances. Deafness MLC and CACH. Irritability is a feature of Krabbe, can occur early in mitochondrial disorders and 18q Canavan and Aicardi–Goutières syndromes. deletion syndrome. Bulbar dysfunction (palatal Seizures: Seizures usually occur in the more myoclonus and dysphonia) is unique to Alexander advanced• stage of the disease, but may be the pre- disease. Macro or microcephaly may be a fea- senting feature of Alexander disease or may occur ture of the different leukodystrophy conditions, as early in the course in Canavan, Krabbe, Cerebro- listed below in Table 5. tendinous Xanthomatosis (CTX), Leukoencephalo- pathy with thalamus and brainstem involvement Extraneurologic manifestations and high lactate (LTBL), Metachromatic leukodys- trophy (MLD), Oculodentodigital dysplasia (ODDD), Associated extraneurologic features provide addi- Megalencephalic leukoencephalopathy with sub- tional clues to the diagnosis. They are summarised cortical cysts (MLC), peroxisomal disorders, sialic in the following table (Table 6).

Table 5 Diagnostic clues from head circumference.

Macrocephaly Alexander disease- head circumference exceeds 98th centile at 6 to 18 months; obstructive • hydrocephalus may be present Canavan disease- occurs by the first year of life; normal head size is described in some variants • Childhood ataxia with central nervous system hypomyelination (CACH) -megalencephaly • Megalencephalic leukoencephalopathy with subcortical cysts - megalencephaly • 1,2-hydroxy glutaric aciduria - relative macrocephaly which is usually an inconsistent feature • GM2 gangliosidosis - infantile Tay-Sachs and Sandhoff • Microcephaly Krabbe syndrome • Cockayne syndrome • Tay () syndrome •

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Table 6 Extraneurologic manifestations of leukodystrophies and genetic leukoencephalopathies.

Clinical feature Disorder Facies Dysmorphism 18q microdeletion Peroxisomal disorders Coarse facies Sialic acid storage disease, Fucosidosis, Multiple sulfatase deficiency, Mucopolysaccharidosis Progeroid appearance Cockayne syndrome Dental anomalies Dental anomalies Oculodentodigital dysplasia (enamel hypoplasia) POL III related disorders (not universal and highly variable- oligodontia, hypodontia, delayed eruption, altered sequence of eruption, abnormal colour /shape) Cockayne syndrome (propensity for cavities (most com- mon), abnormal shape, hypodontia, oligodontia and enamel hypoplasia) Peroxisomal disorders (enamel defects of secondary teeth) Eyes Cataracts At birth Hypomyelination with congenital cataract (HCC), Childhood ataxia with central nervous system hypomyelination (CACH) (only connatal cases), peroxisomal disorders Childhood onset Cerebrotendinous Xanthomatosis Cherry red spot Sialidosis GM1 gangliosidosis GM2 gangliosidosis (helps differentiate from other disorders with infantile onset macrocephaly) Metachromatic leukodystrophy (some cases) Glaucoma Aicardi–Goutières Syndrome, Oculodentodigital dysplasia Optic atrophy Metachromatic leukodystrophy Canavan, Childhood ataxia with central nervous system hypomyelination (CACH), Cerebrotendinous xanthomatosis, peroxisomal disorders (+/-), Pol III related (+/-) Hypomyelinating and mitochondrial disorders Retinitis pigmentosa (night blindness) Refsum disease (adolescent and adults) Peroxisomal disorders Vascular retinal defects Cerebroretinal microangiopathy with calcifications and cysts (Coats plus syndrome) Glistening white dots in the retina Sjogren Larsson syndrome (pathognomonic in a patient with (perifoveal) ichthyosis) Nystagmus Early onset/ PMD & PMLD (prominent feature) congenital SOX10 related disease Later age Oculodentodigital dysplasia, Pol III related 4H syndrome - hypomyelination, hypodontia and hypogonadotropic hypog- onadism),18q del, Alexander, Canavan

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Skin manifestations Angiokeratoma corporis diffusum Fucosidosis Icthyosis Congenital Sjogren Larsson syndrome Childhood Multiple sulfatase deficiency Sialic acid storage disorder Adulthood Refsum disease Hyperpigmentation X-ALD/AMN [Figure 1] Xanthomas Cerebrotendinous xanthomatosis Photosensitivity Cockayne, Tay syndrome Chilblains Aicardi– Goutières Syndrome Endocrinologic manifestations Adrenal insufficiency X-ALD, peroxisome biogenesis disorders Hypothyroidism 4H syndrome - hypomyelination, hypodontia and hypogo- nadotropic hypogonadism), Aicardi– Goutières Syndrome Hypogonadotropic hypogonadism 4H syndrome - hypomyelination, hypodontia and hypogo- nadotropic hypogonadism) Ovarian dysgenesis Ovarioleukodystrophy (CACH), AARS2 mutation-related (Premature ovarian failure) Hepatobiliary manifestations Hepatosplenomegaly Lysosomal storage disorders’s, (multiple sulfatase deficiency, galactosialidosis, sialic acid disorders) Hepatic dysfunction Peroxisomal disorders (isolated hepatomegaly +/- hepatic dysfunction) Aicardi– Goutières Syndrome (congenital period, rarely in infancy) Mitochondriopathies Gall bladder dysfunction MLD (causes gall bladder papillomatosis) Cerbrotendinous xanthomatosis (can present as neonatal jaundice) Skeletal system Chondrodysplasia punctata Peroxisomal disorders Dysostosis multiplex Multiple sulfatase deficiency, sialidosis Short stature Cockayne syndrome, 4H leukodystrophy Hearing deficit Hearing impairment (Commonly Peroxisomal biogenesis disorders (early onset) central origin-sensorineural) SOX 10 associated LD (early onset) RNAseT2 deficiency Refsum disease (adult onset)

Diagnostic workup provide clues to a differential diagnosis. In general, myelinated white matter is hyperintense to the Neuroimaging: MRI pattern recognition is cortex on T1-weighted images and hypointense to an important• step in the further workup towards a the cortex on T2-weighted images. This pattern is specific diagnosis of white matter disorders. Myelin reversed in demeylination with the white matter assessment by MRI with associated features can demonstrating T1 hypointensity and T2 hyperin-

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tensity relative to the cortex. While T1 weighted images are most useful for assessing myelination until the first year of life, T2-weighted images are most useful in later stages of myelination. A high signal on T2-weighted images is abnormal for cerebral white matter after 1.5 years. Furthermore, knowledge of the sequential pattern of myelination is essential for distingushing abnormal and normal patterns of myelination. The minimum sequences recommended are (Parikh et al., 2015):

Sagittal T1 • Axial T1 • T2 weighted Figure 1 Generalised skin hyperpigmentation in • a patient with juvenile onset X -linked Fluid attenuated inversion recovery (FLAIR) (Cys- adrenoleukodystrophy (confirmed by plasma VLCFA analysis). • tic lesions are best detected using FLAIR studies)

Table 7 Hypomyelination on MRI.

With cerebellar Normal corpus callosum involvement Hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) (inconstant) • Oculodentodigital dysplasia • Thin corpus callosum 4H syndrome - hypomyelination, hypodontia and hypogonadotropic hypog- • onadism) Salla disease • Fucosidosis • Cockayne • PMD With basal ganglia • Hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) involvement • (atrophy especially putamen) Oculodentodigital dysplasia • Fucosidosis (globus pallidus) • Mucolipidosis type IV With absence of • 18q del (cerebellar hypoplasia) cerebral atrophy (or • Hypomyelination with congenital cataract (HCC) atrophy in late • HEMS (hypomyelination of early myelinating structure) stages) with normal • PMD basal ganglia • PMLD (hypomyelination of brainstem especially pons) • Salla disease • SOX 10 associated disorders With global atrophy • Infantile sialic acid storage disorders • Aicardi– Goutières Syndrome (calcifications present) •

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Other specific sequences include: Contrast administration- for disorders with an • inflammatory component eg. cerebral X-ALD Susceptibility weighted- for disorders with cal- • cifications eg. Aicardi– Goutières Syndrome, Cockayne syndrome (basal ganglia calcifications), Krabbe (calcifications of thalami, basal ganglia and putamen) MR spectroscopy (MRS)- lactate peak is seen • in mitochondrial disorders and Leukoencephalo- pathy with brainstem and spinal cord involve- ment and lactate elevation (LBSL), while a markedly increased N-acetyl aspartate (NAA) Figure 2 Brain MRI (axial FLAIR image) showing peak is typical of Canavan disease. bilateral deep white matter involve- Diffusion weighted- for AARS2-related leukoen- ment with posterior predominance and • cephalopathy sparing of the subcortical U fibers in Serial imaging with an interval of atleast 6-12 X-linked adrenoleukodystrophy. (Cour- months is pertinent to distinguish between per- tesy: Dr Shubha R Phadke, SGPGIMS, manent hypomyelination and delayed myelination Lucknow) especially in children less than 2 years of age. CT scan is useful for detecting calcifications. A stepwise approach to recognising the MRI pat- tern helps in differentiating various white matter disorders (Schiffmann & van der Knaap, 2009). This involves first recognising whether it is hypomyeli- nation or delayed myelination or demyelination. Hypomyelination: Hypomyelination on MRI is characterised• by mild T2 hyperintensity in com- bination with T1 hyperintensity (=normal signal), T1-isointensity or mild T1-hypointensity relative to Figure 3 MRI brain (axial FLAIR image) showing gray matter structures. Table 7 lists the condi- periventricular hyperintensities in the tions with hypomyelination and additional findings frontal and parieto occipital region in which provide a possible clue to diagnosis. metachromatic leukodystrophy. Demyelination: Demyelination on MRI is characterised• by prominent T2 hyperintensity with prominent T1 hyporintensity relative to gray matter structures. These can be further analysed as to whether they are confluent or isolated and mul- tifocal and further on the area of predominance of the lesion. Confluent and bilateral symmetric lesions are characteristic of genetic white mat- ter disorders while isolated and multifocal are suggestive of acquired causes such as infections and vasculopathies and is also seen in structural chromosomal disorders. Table 8 lists the con- ditions with confluent lesions and their area of predominance. Delayed myelination: Improvement of Figure 4 MRI brain (coronal FLAIR image) show- myelination• on serial MRI is suggestive of de- ing diffuse extensive white matter in- layed myelination and can occur in SOX 10 related, volvement in Canavan disease. MCT8 related and other neuronal disorders.

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Figure 6 MRI brain (axial T2 image) showing Figure 5 MRI brain (axial FLAIR image) showing periventricular deep white matter as diffuse white matter involvement with well as subcortical white matter in- temporal subcortical cysts in a case of volvement in a case of Leigh disease. megalencephalic leukoencephalopathy with subcortical cysts (van der Knaap disease).

Table 8 Disorders with MRI features of demyelination and confluent lesions and their area of predominance.

Confluent lesions Predominant Disorder localization Frontal Alexander disease • Metachromatic leukodystrophy • Aicardi– Goutières Syndrome • X-ALD (frontal variant) • Hereditary diffuse leukoencephalopathy with spheroids (HDLS) Pareito-occipital • X-ALD (involvement of splenium of corpus callosum and sparing of the • occipital arcuate fibers) [Figure 2] Krabbe • Adult Polyglucosan Body Disease (PGBD) • Early onset peroxisomal disorders • Neonatal hypoglycemia Periventricular • MLD (sparing of the arcuate fibers) [Figure 3] • Krabbe (sparing of the arcuate fibers) • Leukoencephalopathy with brainstem and spinal cord involvement and • lactate elevation (LBSL) (sparing of the arcuate fibers) Sjogren Larsson syndrome • Adult Polyglucosan Body Disease (PGBD) • Oculodentodigital dysplasia • Inborn errors of metabolism • Periventricular leukomalacia • HIV related encephalopathy Subcortical • Canavan [Figure 4] • L2 glutaric aciduria • Propionic acidemia • Urea cycle defects • Ribose 5 phosphate isomerase deficiency •

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Kearns Sayre syndrome • Leukoencephalopathy with thalamus and brainstem involvement and high • lactate (LTBL) Diffuse cerebral Childhood ataxia with central nervous system hypomyelination (CACH) / • vanishing white matter disease (cystic degeneration of white matter) Megalencephalic leukoencephalopathy with subcortical cysts (subcortical • sycts in the anterior-temporal regions with sparing of central white matter) [Figure 5] Inborn errors of metabolism • Early onset peroxisomal disorders • Laminin alpha 2 deficiency • Mitochondrial defects [Figure 6] • End stage of progressive white matter disorders Posterior fossa •Cerebellum + middle cerebellar peduncles + Brainstem predominance Peroxisomal disorders • Alexander disease • Autosomal Dominant Leukodystrophy with Autonomic disease (ADLD) • Leukoencephalopathy with brainstem and spinal cord involvement and • lactate elevation (LBSL) Mitochondriopathies •Cerebellum and cerebellar peduncles predominance Histiocytosis • Early onset Mple syrup urine disease • Cerebrotendinous xanthomatosis • FMR1 premutation • FA2Hrelated disorders • Heroin and cocaine toxicity •Brainstem predominance Leukoencephalopathy with thalamus and brainstem involvement and high • lactate (LTBL) Adult Polyglucosan Body Disease (PGBD) • Wilson disease • Leigh syndrome • Dentatorubral-pallidoluysian atrophy (DRPLA) Temporal • • Herpes simplex encephalitis • Aicardi– Goutières Syndrome (AGS) • Congenital Cytomegalovirus infection • RNASET2 deficiency • Metabolic testing diagnosable and those with treatment options may be undertaken. Table 9 lists the biochemical Serial biochemical testing can be done to look for screening tests and the specific target disorders. It the disease etiology. The type of testing, either bio- is also important to rule out nutritional deficiencies chemical testing or direct single gene testing, may which can cause white matter changes such as be dictated by the clinical and neuroimaging find- vitamin B12 deficiency which can be treated. Some ings. If a specific etiology cannot be conclusively laboratory tests useful in diagnosing genetic white established by clinical evaluation and imaging fea- matter disorders are listed below in Table 9. tures then an approach of ruling out the rapidly

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Table 9 Some laboratory tests useful in diagnosing genetic white matter disorders.

Screening test Disorder Enzyme assays Krabbe (galactosyl cerebrosidase) MLD (arylsulphatse A) Multiple sulfatase deficiency (arylsulphatase A,B,C,D) GM1 gangliosidosis (beta galactosidase) GM2 gangliosidosis (hexosaminidase A & B) Sialidosis (neuraminidase) Galactosialidosis (neuraminidase + beta galactosidase) Urinary Analysis Sulfatides MLD Glysoaminoglycans Multiple sulfatase deficiency Organic acids L2 glutaric aciduria ( concentration of L-2-hydroxyglutaric acid and lysine) ↑ Canavan disease ( N-acetylaspartic acid) Mitochondrial disorders↑ (Krebs cycle intermediates) Aminoacids Aminoacidopathies Plasma Very long chain fatty acids ALD (C26:0, ratio of C24:0 to C22:0, ratio of C26:0 to C22:0) Peroxisomal↑ biogenesis disorders ↑ Peroxisomal beta oxidation defects Plasma cholestanol CTX Mitochondrial disorders Blood lactate, pyruvate, aminoacids

Other tests leukodystrophy and genetic leukoencephalopathy genes or whole exome sequencing can be applied Additional specific testing can be done at the to come to a conclusive diagnosis. discretion of the physician and as required, to detect abnormalities which may either provide additional diagnostic clues or may help in the Management overall patient management such as ophthalmo- Treatment options in general at present are largely logic evaluation (including slit lamp examination symptomatic and supportive, while curative ther- and fundoscopy), hearing evaluation, endocrino- apies are limited and inadequate. Supportive logic workup and neurophysiologic studies such therapy is aimed at improving the quality of life as BAER, EMG/NCV,VEP, SSEP) to characterise the and involves various strategies, common as well involvement of cranial and peripheral nerves (AMN, as tailored to individual needs. These include MLD, Krabbe), optic tracts and spinal tracts. management of spasticity (medications, physio- therapy, orthotics), seizure control and prevention Molecular genetic testing (anticonvulsants), surgical release of contractures and scoliosis correction, gastrostomy for severe Molecular diagnostic confirmation can be done by dysphagia, proper wheelchair seating, special ed- sequence analysis of the relevant gene, based on ucation, assistive communication devices, and the recognition of a definitive pattern in MRI or nutritional support. based on the metabolic testing results. However, for many of the leukoencephalopathies, especially Genetic counseling is an important component the rarer types, there is often a significant overlap in of management of these conditions and the af- the clinical and neuroimaging features and reliable fected families can be offered appropriate counsel- metabolic testing may not be available; therefore, ing about the recurrence risks and about prenatal broad spectrum testing in the form of next genera- diagnostic testing options to prevent recurrence. tion sequencing-based multigene panel testing for Specific therapies are available for certain disor-

83 Chapter 12 ders. Early hematopoietic stem cell transplantation cephalic leukodystrophy with cysts is caused by (HSCT), though not curative, attenuates the clinical a common MLC1 mutation. Neurology 2004; 62: course and prolongs survival of infantile Krabbe 878-82. disease and X-ALD. Dietary therapy with oral 3. Parikh S, et al, A clinical approach to the diagno- chenodeoxycholic acid (750 mg/day) corrects the sis of patients with leukodystrophies and genetic biochemical abnormalities and reverses symptoms leukoencephalopathies. Mol Genet Metab 2015; in Cerebrotendinous xanthomatosis. Hormone 114: 501-515. therapy can be life saving by preventing Addiso- 4. Schiffmann R, van der Knaap MS. Invited article: nian crisis in susceptible individuals with X-ALD. an MRI-based approach to the diagnosis of white Early institution (prior to occurrence of MRI ab- matter disorders. Neurology 2009; 72:750-759. normalities) of oral Lorenzo’s oil (erucic acid and 5. Sistermans EA, et al. Mutation detection in oleic acid combination) lowers plasma levels of the aspartoacylase gene in 17 patients with very long chain fatty acids (VLCFA) in patients with Canavan disease: four new mutations in the X-ALD. Early recognition can be beneficial in those non-Jewish population. Europ J Hum Genet conditions with definite treatment options, and on 2000; 8: 557-560. the whole, preventive, symptomatic and support- 6. van der Knaap MS. Magnetic resonance in child- ive care with multidisciplinary involvement, are of hood white matter disorders. Dev Med Child prime importance in the management of patients Neurol 2001; 43:705-712. with genetic white matter disorders. 7. Vanderver A, et al. Case definition and clas- sification of leukodystrophies and leukoen- References cephalopathies. Mol Genet Metab 2015; 114: 494-550. 1. Di Rocco M, et al. Genetic disorders affecting 8. von Figura K, Gieselmann V, Jaeken J. Metachro- white matter in the pediatric age. Am J Med matic leukodystrophy. In: Scriver CR, Beaudet Genet B Neuropsychiatr Genet. 2004; 129B: AL, Sly WS, Valle D, editors. The Metabolic and 85-93. Molecular Bases of Inherited Disease. New York: 2. Gorospe JR, et al. Indian Agarwal megalen- McGraw-Hill; 2001: 3695-3724.

PhotoQuiz - 2 Contributed by: Dr Prajnya Ranganath Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad Email: [email protected]

This 16 years-old male infant presented with short stature, sub-average scholastic performance, history of congenital heart disease operated in childhood, and bilateral undescended testes. Identify the condition.

Answer on page 231

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Approach to a Child with Macrocephaly: The Dysmorphologist’s View

Sumita Danda Department of Clinical Genetics, Christian Medical College and Hospital, Vellore, Tamil Nadu

Correspondence to: Dr Sumita Danda Email: [email protected]

Definition size (including malformations and space occupy- ing lesions), cerebrospinal fluid (CSF) and blood Macrocephaly if present is often the reason for volume, presence of subdural fluid, and overlying referral to a clinical geneticist. It is an integral part tissue scalp. [Box]. Another classification par- of pediatric examination and an important diag- ticularly used by clinical geneticists is syndromic, nostic clue for a dysmorphologist. Macrocephaly non-syndromic and non-genetic (Table 1). is defined as head circumference (occipital-frontal Enlargement of the cranial vault leads to sec- circumference OFC) more than 2 SDs or greater ondary changes in the craniofacial profile leading than 97th centile above the mean for age and sex to a high forehead or dolichocephaly, whereas matched normal standards. This is an objective cranial base enlargement can cause mild hyper- definition. Subjectively it is an apparently increased telorism, down slanting palpebral fissures or a size of the cranium. Many clinicians diagnose mi- triangular face. crocephaly based on an initial impression and confirm it by actual measurements and plotting them on charts. History

Measurement The value of history cannot be ignored and in-depth analysis of prenatal, perinatal, postnatal and family history is required. Macrocephaly may be detected Head circumference is measured from just above by prenatal ultrasonography and lethal conditions the glabella (the most prominent point of the such as Achondrogenesis type 1 and 2 can be frontal bone above the root of the nose) to the detected but this article focuses on approach to most posterior prominent point of the occipital macrocephaly detected after birth. bone using a tape measure. Some standard charts are organised by centiles and others are by standard deviation. Macrocephaly is an absolute Onset term. Ethnic background and overall stature are important and one needs to be familiar with the OFC at birth if available should be recorded. Birth term relative macrocephaly. The term relative weight is important; a high birth weight more than macrocephaly can be used when the head size is th th th 97 centile will indicate an . at the 75 centile with height at the 5 centile for Macrocephaly is present at birth in syndromes age and sex or when OFC is 2 SDs of the mean but such as (SS), Neurofibromatosis is disproportionately above that for the stature. type 1 (NF1) or Megalencephalic Leukoencephalo- Parental OFC measurements are important for pathy with subcortical cysts (MLC). It appears interpretation and approach. later in autism, Canavan and Alexander diseases. Among the metabolic disorders Glutaric aciduria Types and classification type I (GA1), is an exception because unlike other metabolic disorders where macrocephaly mani- The etiological types of macrocephaly depend on fests during infancy or later, in GA1 macrocephaly the factors that determine head size namely brain may present in the neonatal period.

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Table 1 List of causes of macrocephaly.

Hydrocephalus Megalencephaly (Syndromic) Increased skull thickness Achondroplasia Achondroplasia Anemia/ • Basilar impression • Neurocutaneous disorders • Haemoglobinopathies • Benign enlargement • Epidermal nevus syndrome Cleidocranial • Choroid plexus papilloma Neurofibromatosis1 • Craniometaphyseal dysplasia • Meningeal malignancy Tuberous Sclerosis • Epiphyseal dysplasia • Dandy walker malformation Incontinentia pigment • Hyperphosphatasia • Walker - Warburg Hypomelanosis of Ito • Orofaciodigital • X-linked hydrocephalus • syndrome • Porencephaly/ Neuro-cardio-facial Hydranencephaly syndromes • • • Pycnodysostosis Holoprosencephaly Noonan, Costello LEOPARD, • Rickets • Cardiofacio cutaneous (CFC) Overgrowth syndromes • Russell Silver syndrome • Sotos, Weaver • Gorlin MCMTC • Kenny Caffey Simpson-Golabi-Behmel, • Marshall Beckwith- Wiedemann • MPS I, II, VI, VII With mental retardation • Proteus • FRAXA • Lenz- Majewski Megalencephaly • Ruvacalba-Bannayan (Non-Syndromic) • Metabolic • Alexander Canavan Organic aciduria • (Glutaric acid type 1 D2-hydroxy-glutaric aciduria) Galactosemia GM2 Gangliosidosis Globoid leukodystrophy MSUD, MLC, MLD

MSUD- Maple syrup urine disease MLC- Megalencephalic leukoencephalopathy with subcortical cysts MLD- Metachromatic leukodystrophy Non-Genetic causes Hydrocephalus – Haemorrhage, infections & other causes Subdural effusions Post traumatic Arachnoid cysts

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Growth ever, microcephaly instead of macrocephaly could be present in some cases of BWS. Serial values of OFC as well as height and weight should be obtained and plotted on gender and age- Skin examination dependent graphs. Accelerated growth of the OFC is commonly seen in patients with hydrocephalus Examination of the skin can point to a specific which may be identified as part of a syndrome such diagnosis such as presence of cafe au lait spots, de- as Achondroplasia or Walker-Warburg syndrome. pigmented skin lesions or linear pigmentation point to NF1, TS or Linear sebaceous nevus syndrome Development respectively. Lentigines may be present in LEOP- ARD syndrome or the other RAS/ MAPK related It is important to know whether there is im- disorders i.e. Noonan, cardio-facio-cutaneous or provement in development or there is regression. Costello syndrome. Features of lethargy, vomiting, poor feeding, irri- tability, and poor weight gain indicate increased Eye examination intracranial pressure or a metabolic disease. Eye examination could reveal corneal clouding in Family history the mucopolysaccharidoses (MPS types IH or IH/S, VI, VII) cherry red spot in Tay Sachs disease, Lisch Benign familial macrocephaly (BFM), as the name nodules in NF1, or retinal hamartomas in TS. suggests, runs in families. This is an autosomal Additional findings of cardiac murmurs, hep- dominant disorder as are NF1, Tuberous sclerosis atomegaly, and splenomegaly are features that (TS) or MLC. Mendelian inheritance patterns may suggest storage disorders. be recognized from the family history such as two male siblings with hydrocephalus may indicate an Dysmorphology evaluation X-linked hydrocephalus. Additional familial fea- tures, seizures, ataxia, tremors or dementia can Dysmorphism is an important clue for syndrome point to a FRAXA, Autism - macrocephaly-epilepsy, identification e.g. mandibular abnormalities in py- FG syndrome (Opitz and Kaveggia), Proteus syn- cnodysostosis, prognathism in FRAXA, pointed chin drome etc. About 15-35% of autistic children have in Sotos syndrome (Figure 1 a & b) wide mouth in macrocephaly. SGB, adducted thumbs in X-linked hydrocephalus and hemihypertrophy in Klippel-Trenaunay-Weber Physical examination syndrome(KTW).

Examination from head to toe is essential. Differential diagnosis and work up Short stature with limb shortening points to achondroplasia and features for other skele- Careful history and physical examination as stated tal dysplasias should also be looked for care- above will result in a diagnosis in many cases. fully. Macrosomia denotes overgrowth syn- Box 1 lists the causes of macrocephaly. History dromes, more commonly Sotos syndrome, of developmental regression indicates a leukodys- Macrocephaly-cutis marmorata-telangiectasia con- trophy; in Alexander disease the regression is genita (MCMTC), Simpson-Golabi-Behmel syn- rapid typically presenting below 2years of age, in drome (SGBs) and . The shape of Canavan disease it begins between 2-4 months of the head may give additional clues such as dolicho- age with cognitive delay followed by optic atrophy cephaly in Sotos syndrome or frontal bossing in without retinopathy, and in MLC (Figure 1c) there achondroplasia. Increased intracranial pressure is infantile macrocephaly but the rapid course of which accompanies hydrocephalus is indicated neuro-deterioration seen in Alexander or Canavan by tense or bulging anterior fontanel or widely disease is lacking. Skin lesions are helpful to separated sutures. Cranial bruits are diagnos- diagnose neurocutaneous syndromes, but minor tic for a vein of Galen malformation. Coarse manifestations such as ash leaf spots in TS may facies indicate storage disorders such as mu- require Wood’s lamp examination. Overgrowth copolysaccharidoses and macroglossia is evident syndromes present with macrosomia at birth as in Beckwith-Wiedemann syndrome (BWS). How- in SS, BWS, Weaver or SGBs. Mental retardation

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a) b) c)

Figure 1 Patients with a) Sotos syndrome, b) Fragile X syndrome and c) Cystic Megalencephalic leucoencephalopathy. if present in a boy with prominent jaw, long ears phology databases (LMD, POSSUM) or websites or long face may point to FRAXA. Organomegaly (OMIM) on macrocephaly. Additional key words indicates possible storage disorder and the investi- are entered in the search criteria for syndrome gation can be planned to reach a specific diagnosis. identification. The current with Accelerated growth of the cranium points to hy- macrocephaly in OMIM is 277 and will probably drocephalus and eye and fundus examination then keep increasing in the near future. This article help further to rule out raised intracranial tension. focuses on the more common genetic conditions that one encounters in a clinic. The etiological cause of macrocephaly is often determined by brain imaging. In a neonate a neu- rosonogram may be used for screening as it is a simple bedside technique. However, confirmation by MRI brain or CT scan at a later date is often required. MRI of the brain is the most useful test which can help in reaching a diagnosis. MRI brain in In- dividuals with benign familial macrocephaly (BFM) show relative enlargement of the subarachnoid compartments and the interhemispheric fissure Box 1: Causes of macrocephaly. width over the frontal regions so that frontal lobes appear atrophic. Subcortical cysts in MLC and basal ganglia involvement in glutaric acidura Investigations are some other specific findings. Brain scans may demonstrate nonspecific frontal and temporal Clinical diagnosis may be obvious in the first set- lobe atrophy. Hemimegalencephaly is observed in ting as in case of achondroplasia, then targeted TS, KTW, Congenital Proteus and Epidermal nevus mutation detection in the FGFR3 gene will suffice syndromes. for confirmation. Additional investigations may be CT Scan may be done during assessment planned directed to manage complications such as of hydrocephalus and follow up after surgery. brain imaging to rule out hydrocephalus. How- Subdural haemorrhages specifically can occur ever, in other situations the diagnosis may not be in Haemophilia (X linked condition) and glutaric obvious and investigations should be done after acidura. Chronic subdural haematomas could be performing sufficient literature search in dysmor- due to coagulopathies (genetic) or infections.

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When dysmorphic features are present but no Other investigations include complete blood known syndromes are recognized, chromosomal counts and RBC indices to rule out studies are a must particularly if there is associ- , and lead levels. Skull ra- ation with mental retardation. Now, with MLPA diographs may provide clues for diagnosis of (multiplex ligation-dependent probe amplification) craniosynostosis and craniofacial disorders. Fi- and array CGH, a higher yield of detection of nally, exome and whole genome sequencing in chromosomal anomalies is likely. unidentified or new entities can be applied.

Table 2 Genes and patterns of inheritance associated with some common macrocephaly conditions (molecular testing may be carried out to confirm the diagnosis and for appropriate genetic counselling or prenatal diagnosis, as required, for these conditions).

Syndrome/disease Gene Inheritance pattern BRR PTEN AD NF1 NF1 AD KTW VG5Q AD Sotos NSD1 AD Weaver NSD1 AD MCMCT PICK3CA Sporadic (somatic) Simpsom-Golabi-Behmel syndrome GPC5 XL Beckwith-Wiedemann CDKN1 AD Noonan PTPN11, KRAS, AD SOS1, RAF1 Costello HRAS AD LEOPARD PTPN11, RAF1 AD Cardiofaciocutaneous KRAS, BRAF, AD MEK1, MEK2 Fragile X FMR1 XL Glutaric-aciduria type 1 DCDH AR D-2-hydroxyglutaric aciduria D2HGD AR Alexander GFAP AD Canavan ASPA AR MLC MLC1 AD Hunter IDS XL Hurler IDUA AR Tay- Sachs HEXA AR Achondroplasia FGFR3 AD Craniometaphyseal dysplasia ANAH AD, AR Osteopetrosis TGRG1, CLCN7, AR, AD OSTM1, TNFSF11 X-linked aqueductal LICAM XL stenosis/hydrocephalus congenital AR stenosis of aqueduct of Sylvius AD: autosomal dominant, AR: autosomal recessive, XLD: X-linked

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Description of common syndromic Sotos syndrome (SS): SS is the prototype of overgrowth• syndromes and occurs due to muta- conditions with macrocephaly tions in the NSD1 gene. It is an autosomal domi- nant condition and is characterized by overgrowth, Benign Familial Macrocephaly (BFM): Char- macrocephaly and distinctive facial features. It is acteristically• they have a high normal head cir- usually associated with developmental delay. cumference at birth which then increases to more than 2SDs by one year, absence of evidence of syndrome, normal radiographic study of brain and a parent or sibling with macrocephaly or macrocephaly which can be traced through several generations. The criteria for diagnosis have been set forth by DeMyer. Neurofibromatosis type 1 (NF1): Around 20-30%• of NF1 cases have macrocephaly. NF1 occurs due to mutations in the NF1 gene and diag- nosis is easy if café au lait spots, neurofibromas, or family history of NF1 is present. Lisch nodules are characteristically present in the eyes. Relative macrocephaly is more common in NF1. Tuberous Sclerosis (TS): TS has various skin lesions such as ash leaf spots, facial angiofibromas • Figure 2 Adenoma sebaceum and ash leaf mac- (Figure 2), Shagreen patches or ungual fibromas with brain (cortical tubers, seizures) and kidney ules seen in patients with Tuberous (angiolipomas, cysts) involvement. TSC1 and TSC2 sclerosis. gene mutations are found in about 31% and 69% of cases respectively. PTEN Hamartoma-Tumour syndrome (PHTS): Conclusion This• spectrum includes (CS), Bannayan-Riley-Ruvalcaba syndrome (BRRS), PTEN- This write up focuses on macrocephaly as a major related (PS) and Proteus-like dysmorphic feature which when recognized can syndrome All of these macrocephaly-associated point to a syndromic diagnosis and help in the conditions are due to heterozygous mutations in management plan of the individual and genetic the PTEN (Phosphatase and tensin homolog deleted counseling for the family. on chromosome TEN) gene (10q23.31), hence the term PTEN hamartoma - tumor syndromes. All References can be inherited as autosomal dominant disorders with extremely variable expression. 1. Fenichel GM. Disorders of Cranial volume and Macrocephaly-cutis marmorata-telangiectasia shape. In: Clinical Pediatric Neurology: A signs congenita• syndrome (MCMCT): This is charac- and symptoms approach. Sixth edition. Saun- terised by macrocephaly, limb asymmetry, and ders 2009; pp 369-84. vascular stains. Birth weight is increased. There 2. Garg BP, Walsh L. Clinical approach to the is cutis marmorata and a characteristic haeman- child with a large head. Indian J Pediatr 2001; gioma of the philtrum of the upper lip. Cutaneous 68:867-871. features may fade with age. There is macrocephaly 3. Williams CA, et al. Genetic disorders associated with ventricular dilatation and polymicrogyria may with macrocephaly. Am J Med Genet A ;2008: be present. PIK3CA gene mutations are associated. 146A 2023-2037.

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Radial Ray Defects: Genetics and Syndromic Etiologies

Sankar VH Genetic Division, Department of Pediatrics, SAT Hospital, Government Medical College, Thiruvananthapuram, Kerala

Correspondence to: Dr Sankar VH Email: [email protected]

Limb anomalies are a commonly occurring Molecular Embryology group of malformations, deformations and disrup- tions due to the developmental complexity of the Limbs develop from embryonic limb buds. Upper limbs, their extended period of morphogenesis limb buds are first visible in the embryo on Day and their position outside the body wall. Limb 26-30 as an elevation on the anterolateral aspects malformations can be a part of chromosomal aber- of the body wall. Limb development includes rations or an array of single gene disorders or may limb initiation and growth (proximo-distal axis) occur due to environmental teratogens. Radial and its polarization in the antero-posterior and ray defects are a group of limb malformations dorso-ventral axis. It involves several coordinated characterized by unilateral or bilateral absence of processes characterized by a constant equilibrium the radial ray which consists of the radius and between cell mitotic activity and programmed cell thumb (Figure 1). The prevalence of radial ray death. Limb bud formation and growth (proximo- defects is low and varies between 1 in 30,000 to 1 distal axis) are due to rapid cell proliferation in in 100,000 with syndromic causes accounting for the progress zone (PZ) induced by the overlying approximately two-third of cases (Vergult et al., apical ectodermal ridge (AER). The proximo-distal 2013). The common syndromes associated with ra- growth is closely linked to polarization along the dial ray defects are Holt-Oram syndrome, Fanconi antero-posterior axis (under control of the zone of anemia, TAR syndrome and VACTERL association. polarizing activity, ZPA) and the dorso-ventral axis In addition, chromosomal disorders such as tri- (limb patterning) (Grzeschik, 2002). somy 18 can also cause radial ray defects along Limb development involves coordinated func- with significant growth and developmental delay tioning of various interlinked genes which work and other congenital anomalies. In this review by forming a network of signals. Limb bud out- syndromes associated with radial ray defects are growth is promoted by WNT and FGF10. Upper discussed. limb anatomy is specified by TBX5 and lower limb anatomy by TBX4 genes (Isphording et al., 2004). Mutations in T-box genes are associated with syndromes characterized by limb anomalies, the location of which is in agreement with the expres- sion profile of the respective gene i.e. in either the arms only (TBX5-Holt Oram syndrome) or both arms and legs (TBX3-ulnar mammary syndrome). Mutations in the SALL4 gene (SAL-like4), which also encodes a transcription factor, can cause limb anomalies. Mutations in another gene in the same pathway SALL1 (SAL-like1) are known to cause Townes-Brocks syndrome. Proximal-distal growth is controlled by the apical ectodermal ridge (AER) whose formation requires induction by the bone Figure 1 Characteristic hand abnormality in ra- morphogenic protein (BMP) and the homeobox dial ray defects and X-ray showing ab- gene MSX2. The important gene in establishment sent radius and rudimentary thumb. of antero-posterior polarity is the sonic hedgehog (SHH) gene (Villavicencio et al., 2000). Its expres-

91 Chapter 14 sion is confined to the ZPA. A number of molecules ized by bilateral absence of the radii and throm- involved in the SHH pathway are known and in- bocytopenia. Diagnostic criteria by Hall include clude patched-1, smoothened, GL1-1, GLI-2, GLI-3 bilateral absence of the radii in the presence of and TWIST (Biesecker, 2006). WNT7A is a major both thumbs and a thrombocytopenia. The pres- determinant of dorsal development accomplished ence of thumbs distinguishes TAR syndrome from through upregulation of LMX1B and WNT7A is other disorders featuring radial aplasia, which are repressed by Engrailed 1 (En1). usually associated with absent thumbs. Bilateral absence of the radii may be accompanied by ulnar Syndromes with radial ray defects or humeral anomalies and the most severe cases exhibit phocomelia. Lower limb involvement is Holt-Oram Syndrome (OMIM 142900): Holt variable (40-47%) and includes dislocation of the patella and/or of the hips, absent tibio-fibular joint, Oram• syndrome (HOS) is an autosomal dominant disorder occurring in approximately one in every and lower limb phocomelia. Thrombocytopenia, 10,000 live births and is characterized by cardiac which may be transient, is seen in all cases and and upper limb malformations. Affected individ- will be symptomatic in over 90% of cases within uals exhibit limb defects that range from subtle the first four months of life. Other systemic carpal abnormalities, absent digits and tripha- problems reported are cow milk intolerance (60%), langeal thumbs to sloping shoulders and various renal abnormalities (23%), cardiac abnormalities grades of reduction abnormalities of the radius (15%), genital abnormalities (3%) and cleft palate. (Figure 2). Limb defects are usually bilateral but Other associations reported in case series are may be more prominent on the left side. This is fre- facial capillary hemangiomas, deafness, epilepsy quently associated with cardiac defects like ostium and neural tube defects. Differential diagnoses secundum atrial septal defect, ventricular septal include other conditions with radial ray defects; defect or asymptomatic conduction disturbances however, TAR can be differentiated by the presence in most cases. More complex anomalies like tetral- of the thumbs in spite of absent radii and other ogy of Fallot and pulmonary arterial hypoplasia associated malformation. occur rarely. TAR syndrome is autosomal recessive in inher- itance. An inherited or denovo deletion of 1q21.2 is present in a majority of cases. However, in view of the apparent autosomal recessive inheritance an additional causative allele should be there for the development of the disease. A compound inheritance mechanism of a rare null allele and one of two low-frequency SNPs in the regulatory regions of RBM8A, encoding the Y14 subunit of exon-junction complex (EJC) have been found to cause TAR. This is the first disease described to be associated with the deficiency of the exon-junction Figure 2 Holt Oram Syndrome. complex (EJC) (Albers et al., 2013). Fanconi anemia: Fanconi anemia (FA) is Holt-Oram syndrome is caused by mutations in characterized• by physical abnormalities, bone mar- the TBX5 gene and mutations are spread through- row failure and an increased risk of malignancy. out the gene as nonsense, insertion, deletion or Physical abnormalities are present in 65-70% of mis-sense mutations and rearrangements. When cases which include short stature, abnormal skin applying stringent clinical criteria, a detection rate pigmentation, malformations of the skeletal sys- of 74% can be achieved in patients with HOS (Mc- tem and microcephaly. Upper limb malformations Dermott et al., 2005). Nevertheless, not all carriers include anomalies of the thumb (35%) (absent, of the TBX5 mutations have the HOS phenotype, hypoplastic, bifid, duplicated, rudimentary, tripha- indicating phenotypic heterogeneity at this locus. langeal, long), radii (7%) (absent or hypoplastic Thrombocytopenia with absent ra- with abnormal thumbs), hands (5%) (flat thenar dius• (TAR) syndrome (OMIM 274000): The eminence, absent first metacarpal, clinodactyly, thrombocytopenia-absent radius (TAR) syndrome polydactyly) and ulnae (1%) (dysplastic, short) (Fig- is a congenital malformation syndrome character- ure 3). Lower limb anomalies are seen in 5%

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a) b) c)

Figure 3 Thumb abnormalities in Fanconi anemia a) triphalangeal thumb b) rudimentary thumb c) duplication of thumb. of cases which include toe syndactyly, club feet by quantitative real time PCR will detect a further and abnormal toes. Developmental delay can 10-15% cases. Most mutations are private or have occur in 10% of cases. The diagnosis of FA rests been observed in no more than three independent on cytogenetic testing for increased chromosomal families. Inheritance is autosomal dominant with breakages or rearrangements and formation of 95% penetrance. The proportion of cases caused radial figures in the presence of diepoxybutane by denovo mutations is approximately 40-50%. (DEB) or Mitomycin C. Molecular genetic testing is complicated by the genetic heterogeneity with at least 15 genes known to be responsible for the FA complementation groups. Most of these ge- netic abnormalities are inherited in an autosomal recessive pattern except mutations in the FANCB gene, which show X-linked inheritance (Zierhut et al., 2014). SALL 4 related disorders: SALL-4 related disorders• include the Duane-radial ray syndrome (DRRS), Okihiro syndrome and acro-renal-ocular syndrome (AROS), phenotypes previously thought to be distinct entities (Kohlhase, 2015). The Duane-radial ray syndrome (DRRS) and Okihiro syndrome are characterized by radial ray abnor- malities which include hypoplasia/aplasia of radii, hypoplasia/aplasia of thumb, thenar hypoplasia, triphalangeal thumb, duplication of thumb (preax- ial polydactyly) and Duane anomaly (characterized Figure 4 Townes-Brocks syndrome in a father by uni- or bilateral limitation of abduction of the and his son. Hypoplastic radius and eye associated with retraction of the globe and absent thumb are seen. narrowing of the palpebral fissure on adduction). Acro-renal-ocular syndrome (AROS) is clinically Townes-Brocks Syndrome (OMIM 107480): established in individuals with radial ray malfor- Townes-Brocks• syndrome is an autosomal dom- mations, renal abnormalities (renal hypoplasia, inant disorder. Radial ray abnormalities are mild malrotation, ectopia, horseshoe kidney, vesi- reported in 50% of published cases. These consist coureteric reflux, bladder diverticula) and ocular of preaxial polydactyly (bifid thumb), triphalangeal abnormalities (ocular coloboma, Duane anomaly). thumb, hypoplastic thumb, broad thumb, and Rarely, SALL4 mutations may cause clinically typical distal ulnar deviation of the thumb (Figure 4). Holt-Oram syndrome. Direct sequencing of the Anorectal abnormality is characteristic of this con- complete SALL4 coding regions (exons1-4) detects dition. Other abnormalities include auricular, renal mutation in more than 80% of individuals with and cardiac abnormalities (Miller et al., 2012). An DRRS and AROS. Exonic or whole gene deletions important differential diagnosis is the VACTERL

93 Chapter 14 association, where all these abnormalities can oc- If no features typical of SALL-4 related dis- cur. However, the presence of vertebral defects or • orders are found, molecular genetic testing tracheo-oeophageal malformation or both would of the TBX5 gene is suggested as the first strongly favor the diagnosis of VACTERL associa- molecular test. tion. Mutation in the SALL1 gene at 16q12.1 is responsible for this condition. If features typical of SALL-4 related disorders • are present, molecular genetic testing of the VACTERL association: VACTERL association SALL-4 gene is suggested as the first step. comprises• Vertebral defects, Anal atresia, Cardiac defects, Tracheo-Esophageal fistula, Renal malfor- If clinical overlap exists with Townes-Brocks mations, and Limb malformations. There are • syndrome, molecular genetic testing of the some single gene disorders and syndromes which SALL1 gene should be the first test if the radial resemble the VACTERL association which include ray malformations do not include malforma- Fiengold syndrome, 22q11 deletion syndrome, tions of the radius itself. If malformation Townes-Brocks syndrome and Fanconi anemia. of the radius is present, molecular genetic When dysmorphic features, growth abnormalities testing of the SALL4 gene is suggested as the and/or learning disability are present, a syndromic first molecular test. diagnosis or chromosomal abnormality has to be considered (Solomon, 2011). Prenatal Diagnosis

In pregnancies at risk, detailed high-resolution pre- Testing strategy for individuals with natal ultrasound examination may detect upper- typical radial ray abnormalities limb malformations and/or congenital heart mal- formations. A normal ultrasound examination Perform cardiac evaluation, ophthalmologic does not eliminate the possibility of radial ray • evaluation and renal ultrasound examination defects in the fetus. Prenatal testing for the defect in addition to routine physical examination. may be most useful in families with a known

Syndrome Craniofacial Limb anomalies Other anomalies features Nager acrofacial Malar hypoplasia Hypoplasia or aplasia of Conductive deafness dysostosis Micrognathia thumb with or without radius Intelligence normal (OMIM 154400) Preauricular tag Proximal radioulnar synosto- Cleft palate sis with limitation of elbow Rothmund- Frontal bossing Small hands and feet Mental retardation Thomson Small saddle nose Hypoplastic to absent thumbs Cataract syndrome Prognathism Forearm reduction defects Sparse hair (OMIM 268400) Erythema on skin (Figure 5) Poikiloderma Small dystrophic nails Baller Gerold Craniosynostosis Absent/hypoplsatic radii Mental retardation syndrome Micrognathia Curved ulna Congenital heart disease (OMIM 218600) Microstomia Absent/hypoplastic thumbs Renal anomaly Epicanthic fold Fused carpal Imperforate anus Hypertelorism RAPADILINO Long face Absent thumbs Small stature syndrome Narrow palpebral Joint dislocation Hearing defect (OMIM 266280) fissures Stiff interphalangeal joints Infantile diarrhea Long slender nose Pigmentation Cleft palate

Table 1 Other syndromes with radial ray anomalies.

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Figure 5 Bilateral absent thumb in a case of Rothmund-Thomson syndrome. mutation to confirm ultrasound findings. If the approach to the molecular basis of develop- disease-causing mutation has been identified in ment. Int J Dev Biol 2002; 46: 983-991. the family, prenatal diagnosis for pregnancies at 4. Kohlhase J. SALL4-Related Disorders. 2004 increased risk is possible by analysis of DNA ex- Aug 16 [Updated 2015 Jan 15]. In: Adam tracted from fetal cells obtained by MP, Ardinger HH, Pagon RA, et al., editors. (usually performed at 15-18 weeks’ gestation) GeneReviews® [Internet]. Seattle (WA): Univer- or chorionic villus sampling∼ (usually performed at sity of Washington, Seattle; 1993-2017. Avail- 10-12 weeks’ gestation). Because of the significant able from: https://www.ncbi.nlm.nih. ∼variable expressivity observed in most conditions gov/books/NBK1373/ especially with Holt-Oram syndrome both within 5. Isphording D, et al. T-box genes and congenital and among families with the same mutation, the heart/limb malformations. Clin Genet 2004; 66: severity of upper-limb defects and congenital heart 253-264. malformations cannot be accurately predicted by 6. McDermott DA, et al. TBX5 genetic testing molecular genetic testing alone. validates strict clinical criteria for Holt-Oram syndrome. Pediatr Res 2005; 58: 981-986. Acknowledgements 7. Miller EM, et al. Implications for genotype- phenotype predictions in Townes-Brocks syn- Dr S R Phadke, Department of Genetics, Sanjay drome: case report of a novel SALL1 deletion Gandhi Postgraduate Institute of Medical Sciences, and review of the literature. Am J Med Genet A Lucknow and Dr Girisha K M, Department of 2012; 158A: 533-540. Genetics, Kasturba Medical College, Manipal for 8. Solomon BD. VACTERL/VATER Association. Or- providing photographs for publication. phanet J Rare Dis 2011; 6: 56. 9. Vergult S, et al. Complex genetics of radial References ray deficiencies: screening of a cohort of54 patients. Genet Med 2013; 15: 195-202. 1. Albers CA, et al. New insights into the genetic 10. Villavicencio EH, et al. The sonic hedgehog- basis of TAR (thrombocytopenia-absent radii) patched-gli pathway in human development syndrome. Curr Opin Genet Dev 2013; 23: and disease. Am J Hum Genet 2000; 67: 316-323. 1047-1054. 2. Biesecker LG. What you can learn from one 11. Zierhut HA, et al. Genetic counseling for Fan- gene: GLI3. J Med Genet 2006; 43: 465-469. coni anemia: crosslinking disciplines. J Genet 3. Grzeschik KH. Human limb malformations; an Couns 2014; 23: 910-921.

95 GeNeImage

Contributed by: Dr. Shubha R Phadke Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow Email: [email protected]

Beauty in Symmetry

GeNeToon

Contributed by: Dr Prajnya Ranganath Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad Email: [email protected]

96 Section 3 Prenatal, Perinatal and Reproductive Genetics

Chapter 15

Recurrent Pregnancy Loss: From Chromosomes to Genes

Meenal Agarwal1, Shubha R Phadke2 1Clinical Genetics Division and Medical Genetics Laboratory, GenePath Dx, Causeway Healthcare Private Ltd., Pune 1(Formerly: Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow) 2Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow

Correspondence to: Dr Shubha R Phadke Email: [email protected]

Recurrent pregnancy loss (RPL), also known as arm of and inversion Y are seen recurrent spontaneous abortions, habitual abor- in many normal individuals and are not known tions, or recurrent miscarriages are traditionally to be associated with poor reproductive outcome. defined as 3 or more consecutive pregnancy losses Formation of gametes with unbalanced chromo- at less than 20 weeks of gestation. Two to 5 somal constitution is shown in figure 1, in which percent of couples experience RPL. (Kiwi, 2006) The one of the partners is a carrier of a reciprocal cause of RPL is difficult to assess and in fact, no translocation between chromosomes 2 and 9. In cause can be determined in half of the cases in addition to the gametes shown in the figure, 3:1 spite of a battery of investigations. This suggests and 4:0 segregations are possible and will always the presence of unidentified genetic causes. (Kiwi, be unbalanced. 2006) Non genetic causes Chromosomal abnormalities Anatomical anomalies of the uterus such as Mul- In 3-5% of couples with RPL, one partner is found lerian anomalies, uterine fibroids, Asherman syn- to carry a balanced chromosomal rearrangement drome and cervical incompetence are the cause (Warren et al., 2008). Approximately 50% of of RPL in 10-15% of the affected couples. Several these chromosomal rearrangements are balanced endocrinological factors like diabetes mellitus, un- reciprocal translocations, 24% are Robertsonian treated thyroid disease, hyperprolactinemia, luteal translocations, and 12% are sex chromosomal phase defect, high serum androgen and polycystic mosaicism. The remainders are chromosomal ovaries might be associated with RPL but convinc- inversions and other sporadic abnormalities (War- ing evidence for their role in RPL is lacking. Luteal ren et al., 2008). In these couples RPL occurs phase deficiency has been found in 10-28% of due to abnormal segregation of gametes at the couples with RPL and there seems to be evidence time of meiosis. The balanced chromosomal of benefit by progesterone therapy in women with rearrangement carrier is phenotypically normal; a history of RPL. (Potdar, 2005; Haas et al., 2008) however, the separation of chromosomes during meiosis results in an aberrant copy number of Thrombophilia chromosome segments in the conceptus resulting in pregnancy loss. If one of the spouses has Congenital or acquired thrombophilia is another a balanced translocation, the risk of a live born important cause in couples with RPL. Mechanisms child with chromosomal imbalance is about 4% which have been proposed are placental thrombo- and depends upon the particular chromosomal sis, placental infarction, inhibition of thrombolytic segments involved (Kiwi, 2006). Preimplantation system and abnormal prostacyclin metabolism. genetic diagnosis or fetal karyotyping by amnio- Apart from RPL, other obstetric complications centesis is an option for these couples to select that have been associated with thrombophilia are fetuses with normal chromosomal content. Some still birth, early onset of preeclampsia, intrauter- chromosomal variations like pericentric inversion ine growth retardation and placental abruption. of 9, small or large heterochromatin on the q Histopathological examination of placental vascu-

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Figure 1 Gamete formation in an individual with a balanced translocation between chromosomes 2 & 9. lature in pregnancy with thrombophilia also show Autoimmune disorders increased fibrin deposition and thrombus forma- tion. (Adelberg et al., 2002) The most common Many autoantibodies have been studied in asso- inherited thrombophilic disorders are the factor ciation with RPL, but most of the studies have V Leiden mutation and the prothrombin G20210A focused on antiphospholipid antibodies consisting mutation. There is sufficient evidence to rec- of anticardiolipin antibodies (ACA) and lupus anti- ommend factor V Leiden and prothrombin gene coagulant (LAC) directed against negatively charged mutation testing in patients with RPL after exclud- molecules present in the cell membrane. One or ing more common causes. Mutations in genes the other of these is found to be present in 5% - 15% of protein C, protein S and antithrombin have of women with recurrent pregnancy loss and there- also been shown to be associated with RPL but fore testing for these antibodies is recommended as compared to Factor V Leiden mutation and in couples with RPL. Many mechanisms have been prothrombin gene mutation, they are far less com- postulated for the role of these autoantibodies mon and studies are not consistent enough to in the causation of RPL including thrombosis of recommend routine testing for these mutations in placental vessels and infarction, inhibiting the im- all couples with RPL. These tests can be considered plantation process, impaired trophoblast function if there is a strong family history of thrombosis or complement activation. Combined therapy with and tests for other causes of thrombophilia are unfractionated heparin and low dose aspirin may negative. Some mutations in genes of the fib- reduce pregnancy loss by 54% in couples with RPL rinolytic pathway such as the 675 4G/ 5G and showing presence of antiphospholipid antibodies. A844G polymorphisms in the promoter region of Some recent studies also show beneficial effects the plasminogen activator inhibitor-1 (PAI) gene, of this combination therapy in couples with un- have been hypothesized to be associated with RPL. explained RPL with no demonstrable evidence of However, the available data is not adequate to thrombophilia. Other autoantibodies like antinu- provide a convincing association between the PAI clear and antithyroid antibodies can be found in gene and RPL. (Warren et al., 2008; Adelberg et al., increased frequency in couples with RPL but most 2002) studies do not show any significant relationship

98 Chapter 15 with RPL. (Christiansen et al., 2005) Studies from Other possible genetic causes India have reported a significant contribution of genetic and acquired thrombophilias in recurrent It is likely that many other single gene polymor- early and late fetal losses. (Ghosh et al., 2008) phisms may cause or contribute to pregnancy loss but these genetic mechanisms remain poorly un- Immune mechanisms derstood. The likely candidate genes are the genes involved in immune pathways or in the control of Immunological mechanisms have also been pro- placental and fetal development. Carolyn et al posed to be related with RPL due to the break have suggested that multiple mutations in many down of the normal maternal immune system and genes rather than specific gene mutations may be rejection of the fetus. Some studies show that risk factors for RPL, supporting the multifactorial specific immune cells like CD56+ Natural Killer (NK) model. The genes which have been shown to cells may have an abnormal distribution in the have association with RPL in the recent past are peripheral blood and endometrium of women with listed in table 1. Further studies of these polymor- RPL as compared with controls. The total number phisms in larger number of patients from different of T cells is not altered but T cell expression may be populations and search for the pathogenetic mech- altered in the peri-implantational endometrium or anisms involved are necessary before any definite peripheral blood. In some recent studies, multiple conclusions can be drawn. At present, study of cytokine polymorphisms have been reported to be these polymorphisms is not included in routine associated with RSA. However these results have patient care investigations, but it may open up new not been consistent and have not been confirmed strategies for treatment in the future. in other studies. This might be related to differ- ences in the ethnic background. The contribution Recurrent pregnancy loss in clinic of HLA genes to the etiology of RPL is still under discussion. Some prospective studies in the past Recurrent pregnancy loss is a common clinical have shown that sharing of HLA-B and HLA-C problem disturbing equally to the patient and the alleles between the husband and the wife may doctor. Usually investigations are recommended significantly increase fetal loss rates but convincing after three pregnancy losses. If the patient is in her evidence for this hypothesis is still lacking. Hu- thirties, the family may wish to get investigations man Leukocyte Antigen (HLA)-G is a nonclassical done after two pregnancy losses. However after protein that is expressed on the surface of in- a single spontaneous abortion, reassurance to the vading cytotrophoblasts and may play a role in family is needed rather than order expensive list immunoprotection of the developing pregnancy. of investigations. All the investigations including There have been several reports linking HLA-G karyotypes of the husband and wife may be done deficiency and some polymorphisms in this gene in one go rather than doing investigations one after have been associated with increased miscarriage the other. Karyotypes of the couple give more rates in selected populations. Further investigation information regarding the possibility of recurrence is needed to determine the role of HLA-G in normal of chromosomal abnormality in the next pregnancy and abnormal pregnancy. (Warren et al., 2008; and must be done in all cases. Karyotype of the Christiansen et al., 2005) products of conceptions may not be done if there are cost constraints. The chromosomal analysis of Immunotherapy each spontaneous abortion is not indicated. The culture and karyotype of products of conception A number of clinical trials have now been car- obtained after curettage is not only costly, but ried out for inducing immunological tolerance has a high failure rate even in a good laboratory. in the wife for paternal antigens. This in- TORCH group of infections do not cause recurrent cludes administering repeated infusions of the pregnancy losses and serological investigations for husband’s peripheral blood mononuclear cells to TORCH are not indicated in evaluation of a case of the wife. A Cochrane review, concluded that recurrent pregnancy losses. paternal cell immunization, as well as other im- The abnormal test results need to be discussed munologic treatments, such as third party donor with the family regarding the availability of treat- leukocytes, trophoblast membranes, and intra- ment. Detection of thrombophilia, autoimmune venous immunoglobulin provide no significant and endocrine disorders needs appropriate treat- beneficial effect over placebo in preventing further ment. The most important counseling is for miscarriages. (Haas et al., 2008) balanced chromosomal rearrangement. If one

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Table 1 The genes which have been shown to have association with RPL in the recent past.

Author and year No of No of Gene Polymorphism or P value Odds of publication cases controls studied mutation or allele ratio having significant association Karvela et al, 131 - Androgen G1733A polymorphism 0.0004 2.12 2008 receptor Goodman et al, 69 37 Apo E ApoE4 allele 0.036 - 2009 Bolor et al, 2009 26 150 SYCP3 1) c.IVS7 16_19delACTT Mutation found in in intron7 2/26 affected 2) c.657T/C in exon 8 women but in none of the controls Zammiti et al, 372 274 TNFa 1) -238A/G, .019-0.455 0.57-0.85 2009 2) -308G/A .013-0.184 1.52-1.32 Faridi et al, 2009 205 224 KIR BB genotype 0.0001 4.4 Prigoshin et al, 40 53 IFN- +874A?T polymorphism 0.01< - 2004 gamma Coulam et al, 152 65 VEGF -1154AA genotype 0.05 - 2009 < Firouzabadi et al, 97 32 p53 Codon 72 0.041 - 2009 polymorphism Kaare et al, 2009 46 191 p53 C11992A polymorphism 0.0414 2.08

of the partners is found to be a carrier of a pregnancy is around 70% without any treatment balance chromosomal rearrangement then there and it decreases with higher number of losses. is 30 to 50% chance of each pregnancy ending in Possibilities of good outcome should be presented a spontaneous abortion. This may depend on the with caution in families with many spontaneous chromosomes involved and the size of translocated losses. Conveying the truth about inability to segment. Some translocations are likely to lead detect cause and definitive treatment must be to unbalanced gametes in higher proportions and shared with the family. It may help in accepting the risk of spontaneous abortions may be higher. the situation and take appropriate decisions about There is also an increased possibility (5 to 10% taking chances and other options like adoption. and depends on the chromosomal abnormality) of Tests for genes and treatment modalities cur- birth of a child with chromosomal imbalance and rently under research should not be offered unless hence malformations and / or mental retardation. it is a part of research program cleared by ethics In such a family karyotyping from amniotic fluid committee. should be offered if the pregnancy continues be- yond first trimester. In vitro fertilization followed References by preimplantation diagnosis and implanting only chrmosomally balanced embryos is a good option 1. Adelberg AM, et al. Thrombophilias and recur- for the families with chromosomal abnormality. rent miscarriage. Obstet Gynecol Survey2002; If no cause is detected after all the investigative 17: 703-9. work up, the family should be counseled about 2. Bolar H, et al. Mutations of the SYCP3 gene inability to detect the cause of RPL and lack of in women with recurrent pregnancy loss. Am J availability of any definitive treatment. Even after Hum Genet 2009; 84: 14-20. three RPL the chance of a live birth in the next 3. Choi YK, et al. Cytokine gene polymorphisms in

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recurrent spontaneous abortions: a compre- 34-8. hensive review. Am J Reprod Immunol 2008; 10. Haas DM, et al. Progestogen for preventing mis- 60:91-110. carriage. Cochrane Review 2008; 2:CD003511. 4. Christiansen OB, et al. Evidence-based investi- 11. Kaare M, et al. Study of p53 gene mutations gations and treatments of recurrent pregnancy and placental expression in recurrent miscar- loss. Fertil Steril 2005; 83:821-39 riage cases. Reprod Biomed Online 2009;18: 5. Coulam CB, et al. Vascular endothelial growth 430-5 factor gene polymorphisms and recurrent 12. Karvela M, et al. Evidence for association of pregnancy loss. Am J Reprod Immunol 2008;59: the G1733A polymorphism of the androgen 301-5. receptor gene with recurrent spontaneous 6. Faridi RM, et al. Influence of activating and abortions. Fertil Steril 2008; 90:2010e9-12. inhibitory killer immunoglobulin-like receptors 13. Kiwi R. Recurrent pregnancy loss: evalua- on predisposition to recurrent miscarriages. tion and discussion of the causes and their Hum Reprod 2009; 24: 1758-64. management. Clev Clin J Med 2006; 73: 913-21. 7. Firouzabadi RD, et al. Association of p53 poly- 14. Potdar N, et al. The endocrinological basis morphism with ICSI/IVF failure and recurrent of recurrent miscarriages. Curr Opin Obstet pregnancy loss. Aust N Z J Obstet Gynaecol Gynecol 2005; 17:424-8. 2009; 49: 216-9. 15. Warren JE, et al. Genetics of pregnancy loss. 8. Ghosh K, et al. Thrombophilic dimension of Clin Obstet Gynecol 2008; 51:84-95. recurrent fetal loss in Indian patients. Blood 16. Zammiti W et al. Tumor necrosis factor alpha Coagul . 2008;19: 581-4. and lymphotoxin alpha haplotypes in idio- 9. Goodman C, et al. The association of Apopro- pathic recurrent pregnancy loss. Fertil Steril tein E polymorphisms with recurrent preg- 2009; 91: 1903-8. nancy loss. Am J Reprod Immunol 2009;61:

GeNeVerse Think Genetics If you have a patient with muscle weakness or seizure, Send him to a neurologist and a geneticist for good measure.

If you have a patient with an enlarged liver or spleen, He could be having an acquired problem or a defect in some gene.

If you have a patient with a defect in the heart, It may be isolated or of some syndrome it could be a part.

If you have a patient who is not thriving well or short, He may have skeletal dysplasia or a syndrome of some sort.

Keep a genetic etiology always in mind, Especially if a case confounds you, it is very likely to be of a genetic kind.

- By Dr. Prajnya Ranganath

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Rapid Aneuploidy Detection (RAD) Techniques for Prenatal Diagnosis

Pranita Pai1, Shagun Aggarwal2, Anju Shukla1, Girisha K M1 1Department of Medical Genetics, Kasturba Medical College, Manipal University, Manipal 2Department of Medical Genetics, Nizam’s Institute of Medical Sciences and Diagnostics Division, Centre for DNA Fingerprinting & Diagnostics, Hyderabad

Correspondence to: Dr Pranita Pai Email: [email protected]

Introduction testing is performed late in pregnancy, necessitat- ing rapid detection of aneuploidy prior to legal limit Chromosomal abnormalities, both numerical and of termination, which is currently 20 weeks. Rapid structural, are known to occur in approximately 1 diagnosis of aneuploidies in these cases also aids in 200 live births. The diagnosis for chromosomal to relieve anxiety for the family. abnormalities in the antenatal period is usually done by conducting cytogenetic analysis of am- Techniques of Rapid Aneuploidy niotic, chorionic or fetal blood cells obtained by invasive procedures. detection The various indications for fetal cytogenetic Rapid Aneuploidy Detection (RAD) methods are testing include: 1) abnormal ultrasound scan, 2) primarily targeted for the diagnosis of common au- abnormal maternal serum biochemical results, 3) tosomal (13, 18, 21) and sex chromosome advanced maternal age ( 35 years at the expected aneuploidies. Three methods i.e. Fluorescent In time of confinement) ≥4) one of the parent carrier Situ Hybridization (FISH), Quantitative Fluorescent of a chromosomal rearrangement 5) History of Polymerase Chain Reaction (QF-PCR), and Multiplex previous offspring with chromosomal disorder Ligation Dependent Probe Amplification (MLPA) Karyotyping is a well-established cytogenetic have been validated for use in prenatal diagnosis technique which has been extensively used as a (Mann et al., 2004). diagnostic tool for pregnant women undergoing prenatal invasive procedures. The technique is Fluorescent In Situ Hybridization (FISH) tech- considered the gold standard for the detection of nique:• It is usually performed on uncultured autosomal trisomies and sex chromosome aneu- interphase cells with probes designed specifically ploidies, against which all other techniques are for , 18, 21, X and Y. The number compared. Error rates of 0.01-0.02% are reported, of fluorescent signals per cell gives the number of primarily related to maternal contamination, sam- copies of the targeted chromosome (Mann et al., ple mixup and typographical errors, and not the 2004). The technique is known to be almost 100% test perse. In addition to chromosomal aneuploi- sensitive and specific for detection of aneuploidies dies, structural rearrangements upto 5Mb in size, (Grimshaw et al., 2003). Another advantage is the triploidy and mosaicism can also be detected using capacity to detect triploidy where an extra set of karyotype. However, karyotype requires culture of chromosomes is present in the cell. However, the amniotic fluid in order to obtain dividing cells, and FISH technique is non-automated, time consuming this involves a delay of 14-21 days in reporting. and necessitates a skilled technician Mann et al., A large proportion of pregnant women in India 2004; Grimshaw et al., 2003). seek antenatal care during the second trimester. Quantitative Fluorescent Polymerase Chain Additionally, many ultrasound abnormalities be- Reaction• (QF-PCR): This assay has been widely come apparent during the 18-20 weeks anomaly used for the past 20 years for rapid aneuploidy scan. As a consequence, in many cases invasive detection. It is a PCR based molecular method

102 Chapter 16 which uses fluorescent labeled primers to amplify probes are interpreted in an automated manner specific DNA markers which are polymorphic (STRs) to provide information regarding the number of and unique for chromosomes 13, 18, 21, X and copies of that region of the genome. In contrast Y. The amplified products are separated through to the commonly used RAD techniques described capillary electrophoresis. The copy number of a above, chromosomal microarray can detect aneu- specific sequence of each chromosome is deter- ploidies of al 23 chromosomes as well as submi- mined based on the intensity of the fluorescent croscopic copy number abnormalities in a genome signal. The sensitivity and specificity of the assay wide manner. Various studies have shown that is in the range of 95% -100% (Grimshaw et al., CMA can detect additional clinically relevant abnor- 2003; Cirigliano et al., 2009). There are several malities compared to karyotype in 6-7% cases with commercially available QF-PCR kits (Aneufast TM, ultrasound abnormalities and 1-1.7% cases with Chromoquant aneuploidy detection kit) (Allingham- normal ultrasound findings (Oneda et al., 2014, Hawkins et al., 2011). Detection of maternal cell Armengol et al., 2012, Robson et al., 2017). Also, as contamination, triploidy and mosaicism as low as CMA can be done directly on DNA extracted from 15% are important advantages of these techniques chorionic villus sample or amniocytes, culture of (Mann et al., 2004; Cirigliano et al., 2009). However, the cells is not essential, and hence rapid testing this technique is limited by its cost, and rarely with a TAT of 5-7 days is feasible. This approach absence of informative polymorphic markers may provides combined benefits of rapid results as limit its use. well as genome wide coverage. However, it is Multiplex Ligation Dependent Probe Amplifi- limited by cost and in 1-2% cases (Wright et al., 2016, Armengol et al., 2012, Robson et al., 2017), cation• (MLPA): It is also a PCR based method. It is relatively cheaper and less labor intensive than copy number abnormalities of unknown signifi- the FISH technique. The technique involves the cance may be detected, complicating pregnancy use of two fluorescent labeled probes which are counselling and management. To circumvent this hybridized to specific DNA sequences and are then issue, various companies offer low resolution ar- joined by the enzyme DNA ligase. The free ends rays, which look for known and clinically relevant of the ligated probes are complementary to the abnormalities at low cost. Various professional primer which enables the amplification of target bodies recommend that CMA should be first tier sites. The amplified products are separated based test in pregnancies with ultrasound abnormalities, on size using capillary electrophoresis. Each peak and additional diagnostic yields in patients with is considered to be the amplified product of a normal ultrasound indicate that this test should be specific probe. The technique has a capacity to offered to all women undergoing invasive prenatal quantify up to 40-50 different target sequences in testing. one reaction. The commercially available kit MLPA Table 1 summarizes the principles, techniques, P095 kit is useful for the detection of chromosome advantages and limitations of the important rapid 13, 18, 21, X and Y chromosome aneuploidies. aneuploidy detection tests. Prior to ordering any For detection of aneuploidies, a sensitivity and of these tests, appropriate pretest counseling is specificity of 100% is attained by employing this essential. technique (Van Opstal et al., 2009). One of the major drawbacks of this technique is the failure to Conclusion detect triploidies especially in a female fetus. It is a completely automated method, and is being increasingly used as a method for RAD especially There are different rapid prenatal tests and op- where large scale testing of samples is required. tions which can be offered to pregnant women. However, even with different options available, a rapid prenatal aneuploidy test should meet New technology: Chromosomal certain important criteria: (1) vastly accurate microarray with less number of false-negative results; (2) no false-positive results because certain important ir- Chromosomal microarray is based on the principle reversible decisions such as pregnancy termination of hybridisation. Here, many different probes may be taken as a result of an abnormal result; (3) corresponding to different parts of the genome are robust with minimum failure rates and ambiguous made to hybridise with the fluorescently labelled results; (4) rapid with high specimen throughput; test DNA, and the strength of signals from these (5) cost effective, as the rapid test is likely tobe

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Table 1 Comparison of different techniques used for rapid aneuploidy detection.

Description QF-PCR FISH MLPA CMA Principle of Selective Binding of a Two probes of Hybridisation of test the amplification of fluorescently unique length DNA to multiple technique genomic DNA labeled probe hybridized to probes each regions (STRs) by specific for a DNA target DNA representing specific binding of sequence on sequences and genomic regions fluorescently interphase cells and joined by DNA labeled primers to visualized using a ligase. The the target microscope amplified target sequences. The sites separated by products are then size separated by size. False result No false-positive, False-positive rate – No false positive findings minimal false of less than 1 in Minimal false negative negative results 30,000 cases and false-negative rate of less than 1 in 4000 (Tepperberg et al., 2001) Sensitivity 100% for common 100% 100% for 100% for aneuploidies(non- common aneuploidies, 96-98% mosaic), 98.9%* aneuploidies(non- overall (Wright et al., (Allingham-Hawkins mosaic) 2016) et al., 2011) 95.65% (Cirigliano et al., 2009) Specificity 100%* 100% 100% 100% (Allingham-Hawkins et al., 2011) 99.97% (Cirigliano et al., 2009) Mosaicism Can detect as low as Standard practice is Unknown Can detect mosaicism 15% (Mann et al., to score 100 cells to sensitivity for as low as 9-12% 2004) exclude mosaicism mosaicism. (Wright et al., 2016, at a level of greater Further detection Carey et al, 2014) than 10% to 15%, a has to be done by level similar to that employing the of full karyotype FISH technique Maternal cell Results cannot be MCC can rarely Female fetus SNP arrays can detect contamina- obtained in heavily interfere with detection of MCC MCC. aCGH cannot tion blood stained interpretation. not possible. In detect MCC, unless samples (1%) due to Although it is less male fetuses the male fetus (Lamb et the presence of sensitive to MCC, in evidence of MCC al., 2012) MCC (Cirigliano et female fetus MCC is examined from al., 2009) goes undetectable the results of probes located on X chromosome

* Aneufast TM kit used

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Cost Due to automated Comparable to Cheapest Current cost is high methods, this QF-PCR technique Low resolution arrays technique is cost is 3X cost of FISH relatively cheaper than the FISH method. However commercial kits can increase the cost of diagnosis per sample Turnaround Average is 30.5 Reported within 30 hours (Van Mean turnaround Time hours, Median is 24-72 hours Opstal et al., time is 5-7 business 25.1 hours 2009) days (Armengol et al., (Allingham - 2012; Robson et al., Hawkins et al., 2011) 2017) Advantages Reliable, Reliable for Low cost and Detects genome wide automated, detects detection of amplification of abnormalities triploidy and targeted different markers including aneuploidies mosaicism. MCC aneuploidies. in one tube and copy number problems Detects triploidy, defects minimized mosaicism and MCC does not interfere with interpretation Does not require capillary eletrophoresis Disadvan- Commercial kits Non-automated, Cannot detect all Variants of unknown tages may increase the requires skilled cases of triploidy, significance in 1-2% cost. Cannot detect technician, labour and sensitivity to (Wright et al., 2016, structural intensive, intact mosaicism is Armengol et al., 2012,) chromosomal cells, considerable unknown. MCC High cost aberrations time, fails to detect detection not Cannot detect Requires capillary balanced possible in female triploidy unless SNP electrophoresis rearrangements fetus. Cannot array used, balanced equipment and imbalanced detect structural structural aberrations of chromosomal chromosomal chromosomal aberrations rearrangements segments Requires capillary Requires expensive electrophoresis microarray platform equipment and software Kits used Aneufast TM and AneuVysion Assay P095 detection kit Various: Affymetrix, Chromoquant kit (Van Opstal et al., Illumina, Perkin Elmer (Cirigliano et al., 2009) 2009; Allingham - Hawkins et al., 2011)

105 Chapter 16 conducted in addition to full karyotype analysis; (6) abnormalities. Health Tbibechnol Assess 2003; efficient in detection in specimens of low quality 7: 1-146. and quantity and (7) sensitive to detect MCC and 6. Lamb AN, et al. Defining the impact of ma- mosaicism (Mann et al., 2004). Counseling, both ternal cell contamination on the interpretation pre and post test, should accompany the above of prenatal microarray abibnalysis. Genet Med. mentioned testing to facilitate informed decision- 2012;14:914-921. making for the family. Unless CMA is used for 7. Mann K, et al. Strategies for the rapid prena- RAD, a complete karyotype should always follow tal diagnosis of chromosome aneuploidy. Eur J a RAD test, as residual risk of aneuploidies not Hum Genet 2004; 12: 9bib07-915. detected by conventional RAD techniques is upto 1 8. Oneda B, et al. High-resolution chromosomal in 1000. Additionally, as chromosomal microarray microarrays in prenatal diagnosis significantly studies in patients undergoing invasive testing for increase diagnostic pbibower. Prenat Diagn. indications other than ultrasound abnormalities 2014;34:525-353. have shown 1-1.7% yield of clinically significant 9. Robson SC, et al. Evaluation of Array Compara- copy number abnormalities , this should be of- tive genomic Hybridisation in prenatal diagnosis fered to all women undergoing invasive testing as of fetal anomalies: abib multicentre cohort an alternative/adjunct to karyotype. study with cost analysis and assessment of patient, health professional and commissioner References pbibreferences for array comparative genomic hybridisation. Southampton (UK): NIHR Journals 1. Allingham-Hawkins DJ, et al. Prospective vali- Library; 2017 Feb. (Efficacy abibnd Mechanism dation of quantitative fluorescent polymerase Evaluation, No. 4.1.) chain reaction for rapid detection of common 10. Tepperberg J, et al. Prenatal diagnosis us- aneuploidies. Genet Med 2011; 13: 140-147. ing interphase fluorescence in situ hybridiza- 2. Armengol L, et al. Clinical utility of chromo- tion (FISH): 2-year mbibulti-center retrospective somal microarray analysis in invasive prenatal study and review of the literature. Prenat Diagn diagnosis. Hum Genet. 2bib012 ;131:513-523. 2001; 21: 293-301. 3. Carey L, et al. Prenatal diagnosis of chromoso- 11. Van Opstal D, et al. Rapid aneuploidy detec- mal mosaicism in over 1600 cases using array tion with multiplex ligation-dependent probe comparative genomic hbibybridization as a first amplification: a pbibrospective study of 4000 line test. Prenat Diagn. 2014;34:478-486. amniotic fluid samples. Eur J Hum Genet 2009; 4. Cirigliano V, et al. Rapid prenatal diagnosis of 17; 112-121. common chromosome aneuploidies by QF-PCR, 12. Wright D, et al. Validation of a Chromoso- results of 9 years of cbiblinical experience. mal Microarray for Prenatal Diagnosis Using Prenat Diagn 2009; 29: 40-49. a Prospective Cohort of Pregnancies with In- 5. Grimshaw GM, et al. Evaluation of molecular creased Risk for Chromosome Abnormalities. tests for prenatal diagnosis of chromosome Genet Test Mol Biomarkers. 2016;20:791-798.

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106 Chapter 17

Fetal Dysmorphology: An Indispensable Tool for Synthesis of Perinatal Diagnosis

Shagun Aggarwal Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad & Centre for DNA Fingerprinting and Diagnostics, Hyderabad

Correspondence to: Dr Shagun Aggarwal Email: [email protected]

Introduction prognostication and communicating the possibility of intellectual disability and other co-morbidities in Dysmorphology is the science (and art!) of studying such children, helps in decision-making regarding abnormal form, with special emphasis on subtle termination or continuation of pregnancy, facili- findings which provide clue to an underlying diag- tates appropriate postnatal management and also nosis, mostly a genetic syndrome. It has been the provides recurrence risk estimates for subsequent prime tool of the geneticist enabling a syndromic conceptions. In the postnatal scenario, such a diag- diagnosis on basis of patient’s gestalt with findings nosis facilitates emotional closure, recurrence risk like a white forelock, heterochromia iridis, broad counseling and early, definitive prenatal diagnosis thumb, and many other in subsequent pregnancies. subtle features acting as decisive tools in the ge- Any morphological or growth abnormality in the netics clinic. Most individuals with dysmorphism fetal life can be an isolated abnormality of multi- are affected with genetic syndromes, which can factorial origin, the consequence of environmental be due to chromosomal abnormalities, copy num- etiologies like a teratogenic insult, intrauterine ber variations or single gene defects. However, factors, maternal illness, etc. or a component of a various environmental factors can also lead to genetic syndrome. In such a scenario, it is impor- dysmorphism, many times mimicking specific ge- tant to be aware of these possibilities, and perform netic syndromes due to involvement of a common a complete dysmorphological evaluation with an biological pathway. A dysmorphological evaluation aim to distinguish between these different situa- typically involves a head to toe examination look- tions with varied prognosis and recurrence risks. ing for malformations, and minor features showing Figure 1 shows some common fetal abnormalities deviation from the expected norm as per sex, and respective etiologies. age, family background and ethnicity. This often forms the first and most crucial step in establishing Setting of fetal dysmorphology a genetic diagnosis and is subsequently followed by relevant genetic testing for confirmation. In There are two main settings where syndromic the era of next generation sequencing when the diagnosis in the fetus is a possibility and should be rate of gene discovery has surpassed the clini- actively looked for: cal recognition of a new genetic syndrome and reverse phenotyping has become commonplace, a. Abnormal antenatal ultrasound. dysmorphology still remains an important tool in b. Postmortem evaluation of an unexplained fetal the hands of an experienced geneticist. demise or morphologically abnormal fetus. Although as a discipline dysmorphology evolved in the paediatrics setting, it can be extended to Abnormal antenatal ultrasound: Abnormal- the fetal life to enable the diagnosis of a genetic ities• on antenatal ultrasound can be found in syndrome in the fetus. The recognition of a genetic 5-10% of pregnancies. These can vary from growth syndrome in particular, has important implications abnormalities, major or minor malformations, soft for pregnancy management as it aids in accurate markers and liquor or placental abnormalities.

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Fetal abnormality Acquired etiologies Genetic etiologies

Naso-maxillary • Fetal warfarin syndrome • Brachytelephalangic hypoplasia/Binder • Vitamin K deficiency chondrodysplasia punctata facies • • Vitamin K metabolism defects

Talipes- • Oligohydramnios • Chromosomal disorders: sex equinovarus • Uterine malformations chromosome aneuploidy • Multiple pregnancy • Neuromuscular disorders • Skeletal dysplasias Cleft lip • Multifactorial • Isolated Mendelian* • Fetal hydantoin syndrome • Syndromic- chromosomal disorders, copy number abnormalities or Mendelian disorders#

Ventriculomegaly • Fetal infections • Isolated Mendelian* • Intracranial hemorrhage • Syndromic- Chromosomal disorders • Associated with neural • Copy number abnormalities tube defect (multifactorial) • Mendelian disorders#

Hydrops fetalis • Blood group antigen • Chromosomal disorders: Turner isoimmunisation syndrome, Down syndrome • Fetal infections esp. • Mendelian disorders#: Noonan Parvovirus syndrome, primary lymphatic • Hypothyroidism dysplasias, alpha thalassemia, • Congenital heart block, lysosomal storage disorders, others fetal arrhythmias • Cardiac defects Neural tube defect • Maternal diabetes • Isolated Mendelian* • Maternal hyperthermia • Syndromic- Chromosomal, • Folate deficiency Mendelian# eg. Meckel-Gruber syndrome, Spondylocostal dysostosis

*Isolated Mendelian: Isolated defect due to mutation in single gene #Syndromic Mendelian disorder: A spectrum of multiple defects arising due to mutation in single gene

Figure 1 Common fetal abnormalities and their etiologies.

At least 10-30% of prenatally detected malforma- dysmorphic facies, recognize the pattern of abnor- tions are due to a genetic etiology (Beke et al., malities and elicit a detailed family history, which 2005). This figure is much higher for specific ab- would help in synthesis of a syndromic diagnosis. normalities like omphalocele, holoprosencephaly The advent of 3D ultrasound technology provides and cystic hygroma where 50-90% cases can be opportunity for facial dysmorphism recognition, attributed to genetic abnormalities involving the and can be used as an adjunct to the conventional chromosomes. In each of these scenarios, the 2D ultrasonography. antenatal ultrasonography should be performed by a fetal medicine specialist with a dysmorphol- Postmortem evaluation/ Fetal autopsy: ogy or clinical genetics knowledge. Alternatively, Post-mortem• evaluation is an important modal- a clinical geneticist consultation should be sought, ity for establishing the cause of unexplained fetal along with relevant images to enable recognition of deaths as well as a medically terminated mor- phologically abnormal fetus. At least 15-30% of

108 Chapter 17 stillbirths are reported to be due to genetic causes thalidomide, etc. needs to be ascertained as these (Reddy et al., 2012). Besides the histopatholog- are known to be potent fetal teratogens. Some ical examination of the placenta which provides of these can result in fetal malformations which evidence of acquired insults like utero-placental mimic genetic disorders involving defects in the insufficiency and perinatal infections, dysmorpho- common biological pathway. An example is fetal logical evaluation by a geneticist or perinatal warfarin syndrome, arising due to exposure to war- pathologist with expertise in dysmorphology is es- farin in the first half of pregnancy. Warfarin inhibits sential for syndrome recognition. Various studies the activity of Vitamin K, and its fetal effects are indicate that autopsy provides additional findings similar to a brachytelephalangic or modifies the antenatal diagnosis in 20-50% cases chondrodysplasia punctata which is caused by a (Rodriguez et al., 2014). Antenatal series have also mutation in the ARSE gene, important for Vitamin K shown that at least 50% syndromic diagnoses are metabolism in the body. possible only after an autopsy (Stoll et al., 2003). Family history: A three-generation family Hence, all cases with abnormal ultrasound findings pedigree forms the cornerstone of the family his- should undergo a post-mortem evaluation. • tory ascertainment. This can provide important information like consanguinity, which increases Practical approach to fetal the risk of autosomal recessive disorders; pre- dysmorphology vious fetus or child with similar or overlapping phenotype; other family members with pregnancy The evaluation of the fetus in-utero and/or post- losses, infertility or abnormal offspring indicating mortem for syndrome recognition involves the possibility of a chromosomal rearrangement or following steps (depicted in figure 2): single gene etiology; and at times a parent with milder manifestation of the same condition as the Antenatal and medical history fetus. • Family history Ultrasonographic findings: Various ultra- • Ultrasonographic findings sonographic findings may be a manifestation • Reports of serum aneuploidy screen • of an underlying genetic syndrome in the fetus • Postmortem evaluation and a high degree of suspicion as well as careful • Genetic testing search for associated abnormality(ies) is important • Antenatal history: The woman should be to recognise these. asked• about history of potential teratogenic ex- a. Structural/ morphological abnormality: These posure in the form of prescription drugs, high most commonly are malformations i.e. intrinsic grade fever, exposure to environmental toxins and defects in the formation of a structure, but can infection with teratogenic pathogens like rubella, also be deformations due to compressive effects cytomegalovirus, etc. History of decreased fe- on a normally formed structure e.g. varus de- tal movement perception and malpresentations formity in oligohydramnios, or disruptions due to is important in cases with arthrogryposis, poly- sudden insult, traumatic or vascular on a normally hydramnios and small stomach bubble, where formed structure e.g. amputation due to amniotic these finds can provide a clue regarding a primary band. Malformations or intrinsic defects are likely neuromuscular disorder in the fetus. History of to be of genetic etiology. They may be isolated previous pregnancies is also important, as previous or may be associated with other malformations pregnancy losses, pregnancy terminations due to and/or growth problems which indicate an under- similar or overlapping findings, neonatal deaths lying genetic syndrome. A specific spectrum of or previous live abnormal offspring all indicate abnormalities may be characteristic of a specific possible segregation of a genetic disorder in the genetic syndrome e.g. Meckel Gruber syndrome family. presents with encephalocele, polydactyly and mul- Medical history: History of maternal illness ticystic dysplastic kidneys; trisomy 13 presents with like• uncontrolled diabetes, phenylketonuria, thy- holoprosencephaly, midline cleft, polydactyly and roid disorders, etc. needs to be elicited as they can multicystic dysplastic kidneys; and similarly many play an important role in fetal growth and devel- other patterns of malformations indicating a par- opment. Maternal drug use especially antiepileptic ticular diagnosis. As a rule, multiple abnormalities drugs, coumarin derivatives, ACE inhibitors and per se indicate possibility of a genetic syndrome, some rarer drugs like retinoic acid derivatives, whereas a single malformation may or may not be

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Fetus with abnormality Historical details including pedigree

Antenatal Postmortem ultrasound examination

• Soft marker: Single or multiple; • Facial dysmorphism low risk or high risk • External or internal • Malformation: Isolated or malformation: Isolated or multiple multiple • Growth/liquor abnormality: • Growth abnormalities Isolated or associated with soft Organomegaly marker or malformation; • Skeletal dysplasia on radiographs Doppler studies or other • Histopathology of fetal organs indicators of acquired showing specific etiology etiologies • Placental histopathology showing • Adjunct modalities: fetal MRI, evidence of uteroplacental 3 D USG insufficiency

Invasive testing: Fetal tissue Amniocentesis/cordocentesis/ sampling placental biopsy

• Fetal karyotype • Chromosomal microarray • Biochemical testing • Exome sequencing

Diagnosis of fetal syndrome

Dashed arrow reflects that postmortem evaluation may or may not be done depending on ultrasound findings

Figure 2 Practical approach to fetal syndrome diagnosis. genetic in etiology. ploidy risk, which is then used for decision-making regarding invasive testing and fetal karyotyping. b. Soft markers: These are ultrasound findings, Similar to malformations, presence of multiple which may be seen in many normal fetuses, but are markers increases the risk of chromosomal aneu- also indicators of underlying syndromic etiology, ploidy more significantly. primarily chromosomal disorders in some fetuses. Many soft markers have been described and the c. Growth abnormalities: Both fetal growth re- risk of chromosomal disorders associated with striction as well as fetal overgrowth can be due each has been statistically quantified. These risks to maternal and utero-placental factors or due to are integrated with the maternal demographics an intrinsic fetal abnormality. Besides chromoso- and serum screening risks to provide a final aneu- mal disorders, various single gene disorders like

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Fetal abnormality Genetic syndromes reported to be associated

Increased nuchal fold • Trisomy 21 thickness • Other trisomies • Noonan syndrome • Skeletal dysplasias: Achondrogenesis, osteogenesis imperfecta

Absent nasal bone • Trisomy 21 • Skeletal dysplasias: asphyxiating thoracic dystrophy, brachytelephalangic chondrodysplasia punctata Short long bones • Skeletal dysplasias: Various with distinctive features like fractures, bending, macrocephaly, polydactyly • Syndromes with primordial short stature Multicystic dysplastic kidney • Trisomy 13 • Meckel-Gruber syndrome and other ciliopathies

Figure 3 Common ultrasound abnormalities and associated genetic syndromes. microcephalic osteodysplastic dwarfism, Seckel eg in polyhydramnios and auto- syndrome, Smith-Lemli Opitz syndrome (SLOS), somal recessive polycystic kidney disease in oligo- Russel-Silver syndrome, etc. can present with hydramnios. At times, these could be indicators intrauterine growth restriction (IUGR). Another im- of other underlying morphological abnormalities, portant group of disorders presenting with short indicating a syndromic diagnosis. bones and mimicking IUGR is the skeletal dyspla- Figure 3 depicts some common ultrasound sia group, which includes at least 100 different abnormalities and associated genetic syndromes. single gene conditions presenting in the prena- tal period. Fetal overgrowth may also be due Maternal serum screen: These are biomark- to primary overgrowth disorders like Beckwith- ers• in the maternal serum which are assayed Wiedemann syndrome(BWS), Pallister-Killian syn- with the primary aim to screen for common fetal drome and Weaver syndrome, among others. chromosomal abnormalities. Both first and second Hence, it is important to look for additional find- trimester screening protocols are available, which ings like facial dysmorphism and malformations in in combination with ultrasonic soft markers aid all cases of fetal growth abnormalities, where no in screening of low risk women for fetal chro- acquired etiology is apparent. At times, maternal mosomal disorders with high sensitivity and low serum screen results can provide clues to the un- false positive rates. However, these markers are derlying genetic etiology, such as low levels primarily useful for a few specific conditions, and in SLOS and high alfa-fetoprotein in BWS. the detection of most fetal genetic syndromes are d. Liquor abnormalities: Both excess and scanty on basis of ultrasound findings and historical data liquor can be due to underlying genetic etiologies, as detailed above.

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Postmortem evaluation/fetal autopsy: This of the fetus in a post-mortem setting and forms the involves• a comprehensive and step-wise evaluation single most important modality for diagnosis of a

Postmortem external or internal abnormality Genetic syndromes reported to be associated

Radial ray defect • Trisomy 18 • VACTERL association • Fanconi anemia • TAR syndrome • SALL4 mutation • Rothmund-Thomson syndrome

Polydactyly • Trisomy 13 • Meckel-Gruber syndrome and other ciliopathies • Orofaciodigital syndrome • Pallister-Hall syndrome and other GLI3 disorders • At least 400 different disorders

Overlapping fingers • Trisomy 18 • Distal arthrogryposis • Otopalatodigital syndrome

Arthrogryposis multiplex • Trisomy 18 congenita • Multiple pterygium syndrome • Neuromuscular disorders • Skeletal dysplasias • Connective tissue disorders • At least 300 different disorders

Cleft Palate • 22q11.2 deletion • • Smith-Lemli-Opitz syndrome • Otopalatodigital syndrome • Orofaciodigital syndrome Congenital diaphragmatic hernia • • Simpson-Golabi-Behmel syndrome • Pallister-Killian syndrome • Donnai -Barrow syndrome

Truncus arteriosus • 22q11.2 deletion • Trisomies • Holt –Oram syndrome • Townes-Brocks syndrome

Dandy-Walker malformation • Walker-Warburg syndrome • Trisomies • Ritscher-Schinzel syndrome • Meckel-Gruber syndrome • Congenital disorder of glycosylation • Orofaciodigital syndrome

Figure 4 Common postmortem dysmorphic findings and associated genetic syndromes.

112 Chapter 17 genetic syndrome. Typically, autopsy encompasses the smooth brain surface in early gestation among an external examination or dysmorphological eval- others. Gestation-specific photographs should be uation of the fetus, similar to the approach in a used for comparison before concluding a structure clinical genetics clinic; internal dissection to look as abnormal. Figure 5 provides some normal for gross morphological abnormalities of fetal or- gestation-dependent findings. Figure 4 depicts gans and structures; whole body radiogram, both some internal organ abnormalities and respective antero-posterior and lateral views; and histopatho- associated genetic syndromes. logical evaluation of fetal organs and placenta. A standard autopsy proforma helps in maintaining record of the findings. Briefly, the following are the steps, relevant findings and implications during an autopsy: a. Radiographs: A complete fetal radiograph is essential for diagnosis of a skeletal dysplasia and in distinguishing the various types from each other. Cardinal features like platyspondyly, flaring of ends of femur, bent femur, fractures, absent ossification, epiphyseal stippling, etc. all help in providing diagnosis of a specific condition. A radiograph can also provide ancillary information like joint dislocations, spine deformities, missing or supernumerary bones, etc., which may help in diagnosis of a specific genetic syndrome. b. External/Dysmorphological examination: This involves assessment of anthropometric parame- ters like crown rump length, crown heel length, head circumference, chest circumference and foot length. Other parameters like inter-orbital dis- Figure 5 Some gestation-dependent normal tance, hand length, philtrum length, phallus length, morphological findings in fetal life a: and limb segment length may also be assessed as Blake pouch cyst with normal cerebel- required. All the parameters should be compared lum (inset) at 19 weeks; b: Smooth to available centile charts and recorded. This brain at 19 weeks gestation; c: Low is followed by a head to toe examination with set and poorly formed ear placode in a special attention to dysmorphic features. The first trimester fetus; d: Lobulated fetal head, face, neck, spine, chest, abdomen, external kidneys similar in size to fetal adrenals genitalia, extremities, joints and skin are assessed at mid-trimester. for shape, size and appearance, and any deviation from normal searched for and noted. The placenta, d. Gross examination and histopathology of fetal membranes and umbilical cord are also examined organs: All fetal organs are weighed and com- for appearance and any abnormality. The weight pared with gestation-dependent percentiles. A of the placenta and number of cord vessels are detailed histopathological evaluation is performed, recorded. Figure 4 provides some dysmorphic fea- using H&E staining, and if necessary special stains tures and the corresponding genetic syndromes and immunohistochemistry. Many renal and associated with these. brain can be well delineated follow- c. Internal examination: This involves the exam- ing histopathology and in many instances this ination of the intra-abdominal, intra-thoracic and forms the sole basis for diagnosis. An example intra-cranial structures for any abnormalities, in being cystic diseases of kidney, where histopathol- size, shape or morphology. Incision is made on the ogy can distinguish between autosomal recessive, anterior aspect of the trunk and the skull following dominant polycystic kidneys, multicystic dysplastic standard techniques. It is important to be aware kidneys and glomerulocystic kidney disease, all of some normal gestation-dependent findings, like of which can have a similar gross appearance the lobulated appearance of fetal kidney, the rel- and clinical presentation. Similarly, brain patholo- ative large size of adrenals, thymus and liver, the gies like neuronal migration disorders can be developing internal genitalia and lung fissures and well delineated and classified on histopathology.

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Placental histopathology can provide evidence of testing was done by Sanger sequencing of the uteroplacental insufficiency and fetal infections. causative gene in the fetal DNA. However, availabil- This comprehensive approach to an abnormal ity of the Next generation sequencing technology fetus, using historical, imaging and postmortem has made it easier to provide molecular testing, findings provides important diagnostic information as this enables the parallel sequencing of mul- and often leads to the diagnosis of a specific tiple genes enabling interrogation of overlapping dysmorphic syndrome or identification of an ac- phenotypes as well as genetically heterogeneous quired etiology. However, final confirmation ofa conditions. An example would be the skeletal genetic etiology depends on laboratory testing and dysplasias, with at least 100 different single gene identification of the underlying genetic aberration. disorders presenting with short bones on antenatal ultrasound. Exact diagnosis is often not possible Genetic testing for the dysmorphic antenatally, and fetal sampling followed by a NGS- based testing of all skeletal dysplasia genes can fetus be done to arrive at a final diagnosis and provide accurate prognostication to the family. Genetic disorders can broadly be classified into c. Genomic disorders: Another group of genetic three different types, and each of these require a diseases are caused by copy number abnormal- specific laboratory diagnostic approach. ities in the genome i.e. small, submicroscopic a. Chromosomal disorders: These are disorders microdeletions or microduplications involving part arising due to numerical or structural abnormal- of the genome. These conditions require special ities in chromosomes. The common ones with molecular cytogenetic techniques for diagnosis, well described prenatal phenotypes are Down syn- and often in the antenatal period, where a spe- drome (Trisomy 21), Patau syndrome(Trisomy 13), cific diagnosis is not apparent, a chromosomal Edward syndrome (Trisomy 18), Turner syndrome microarray is the most common testing modality and triploidy. A karyotype from the amniotic fluid used. Various antenatal series have found that (following amniocentesis), cord blood (following chromosomal microarray studies from fetal DNA cordocentesis or at birth), intra-cardiac blood (post of a morphologically abnormal fetus indicate a mortem) or skin fibroblasts, is the gold standard copy number abnormality in 6-10% (de Wit et al., for the diagnosis of this group of disorders. These 2014). Presently, microarray studies are recom- on average constitute 10-30% of fetuses with an mended as first tier test in case of morphological antenatal malformation (Beke et al., 2005). Since abnormalities on ultrasound. Postnatal studies karyotyping requires the presence of viable cells, it have also found 2-10% of stillbirths as having copy is essential to obtain suitable samples antenatally number abnormalities, indicating the significant or soon after birth for this investigation. contribution of this group of genetic aberrations to b. Single gene disorders/ Mendelian disorders: fetal abnormalities (Reddy et al.,2012). These are diseases arising due to mutations in in- d. A relatively rarer type of genetic disorders dividual genes. At least 6000 single gene disorders known as imprinting disorders can also present have been described and for 4500 of them the with fetal abnormalities, primarily affecting growth. molecular basis is known. Many of these disorders Examples being Beckwith-Wiedemann syndrome present in the prenatal period with fetal abnormali- presenting with overgrowth, organomegaly, om- ties, some common examples being Meckel-Gruber phalocele and polyhydramnios; and Russel-Silver syndrome, Noonan syndrome, short rib polydactyly syndrome presenting with fetal growth restriction. syndromes, lysosomal storage disorders, etc. The Testing for these conditions requires methylation exact estimate of such disorders in the prenatal studies on fetal DNA. period is not known, however some recent stud- Unlike samples for karyotyping, which require ies employing Next generation sequencing-based viable cells, fetal DNA can be obtained from any novel technologies have found single gene defects fetal sample, including an umbilical cord seg- in 20-30% of fetuses with antenatal malformations ment, either antenatally or post-mortem. The (Drury et al., 2015). The diagnosis of these dis- only prerequisite for suitable DNA sample is that orders is challenging in the laboratory as many the concerned sample should not be exposed to conditions have overlapping features and genetic formalin, which can lead to cross linkage, adduct heterogeneity is common. Conventionally, most formation and fragmentation of DNA, precluding often the diagnosis was made following a post- further molecular studies. Hence, to facilitate mortem evaluation, and then subsequent targeted laboratory testing and confirmation of a genetic

114 Chapter 17 diagnosis, suitable fetal sample should be obtained Once the exact mutation or chromosomal abnor- and stored if immediate testing is not possible. mality or biochemical defect in the index case Storage for purpose of DNA extraction can be done is known, early and definitive prenatal diagnosis at 2-8⁰C for few weeks and at -20⁰C for long term. is possible by chorionic villus sampling at 11-12 For karyotyping, sample can be stored at 2-8⁰C weeks in subsequent conceptions. In absence of and be transferred to the laboratory as soon as a laboratory diagnosis, prenatal diagnosis can be possible, and at least within 48 hours. attempted by ultrasound, however this may not be of utility till later in pregnancy, and milder or Genetic counseling discordant manifestations may not be detected. These issues need to be discussed with the family Appropriate genetic counselling is possible after prior to pregnancy termination, so that appropriate an accurate diagnosis has been made following fetal samples can be obtained. the clinical and laboratory evaluations. Counseling typically addresses the following issues: Conclusion 1. Prognosis: This is relevant in the antenatal setting when a couple is faced with an ultrasound Feta dysmorphology plays an important role in diagnosis of a fetal abnormality. Besides the evaluation of an abnormal fetus with far reach- morbidities of the abnormality and outcome of ing implications for the current as well as future postnatal surgery in structural abnormalities, the pregnancies. A multi-disciplinary approach, with recognition of a syndrome has various implica- clinical geneticist playing a pivotal role is integral tions. Most genetic syndromes are associated with to optimising the care of these special patients and intellectual handicap, which does not have a satis- their families. factory therapy. Additionally, there can be growth issues, presence of other internal malformations References not detectable by imaging, and occasionally pre- mature lethality. This information needs to be 1. Beke A, et al. Trisomies and other chromo- communicated to the couple as it helps in decision some abnormalities detected after positive making regarding pregnancy termination, obstetric sonographic findings. J Reprod Med 2005; 50: management as well as neonatal management. 675-691. 2. Recurrence risk: There is an increased recur- 2. de Wit MC, et al. Additional value of prenatal rence risk associated with genetic etiologies, which genomic array testing in fetuses with isolated is 25% for autosomal recessive disorders, 50% structural ultrasound abnormalities and a nor- for an autosomal dominant disorder with affected mal karyotype: a systematic review of the parent and 50% for male offspring of a carrier literature. Ultrasound Obstet Gynecol 2014; female for X-linked recessive disorders. The risk is 43:139-146. low for chromosomal disorders, unless they arise 3. Drury S, et al. Exome sequencing for prenatal due to a parental chromosomal rearrangement diagnosis of fetuses with sonographic abnor- and for autosomal dominant disorders with nor- malities. Prenat Diagn 2015; 35:1010-1017. mal parents. This risk estimate helps the couple in 4. Reddy UM, et al. Karyotype versus microarray availing prenatal diagnosis services in subsequent testing for genetic abnormalities after stillbirth. pregnancies, and the need and availability of the N Engl J Med 2012; 367: 2185-2193. same should be communicated. 5. Rodriguez MA, et al. Concordance between 3. Prenatal diagnosis: The pre-requisite to defini- prenatal ultrasound and autopsy findings in a tive prenatal diagnosis in subsequent pregnancies tertiary center. Prenat Diagn 2014; 34: 784-789. is identification of the underlying genetic aberra- 6. Stoll C, et al. Prenatal diagnosis of dysmorphic tion in the affected fetus. Hence, laboratory genetic syndromes by routine fetal ultrasound examina- testing and confirmation of the clinical diagnosis tion across Europe. Ultrasound Obstet Gynecol plays an important role in fetal dysmorphology. 2003; 21: 543-551.

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Non-immune Fetal Hydrops: An Update

Gayatri N 1, Ashwani Tandon 2 and Prajnya Ranganath 1 1Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad 2Department of Pathology, Nizam’s Institute of Medical Sciences, Hyderabad

Correspondence to: Dr Prajnya Ranganath Email: [email protected]

Introduction Etiology of Non-immune Fetal Hydrops

‘Hydrops fetalis’ is a Greek term which refers to Non-immune fetal hydrops is a nonspecific finding the pathological accumulation of fluid in fetal soft and can be the manifestation of a wide variety tissues and serous cavities. Non-immune fetal hy- of disorders (Bellini et al., 2009). The cause can drops (NIFH) is defined as fluid accumulation inat be found in nearly 60% of cases prenatally and in least 2 fetal body compartments in the absence of around 85% of cases when postnatal tests are in- red cell isoimmunisation (Moise, 2008). Abnormal cluded. Identification of the exact etiology helps in fluid collection may be ascites, pleural effusion, accurate prognostication of the recurrence risk for pericardial effusion or generalised skin edema subsequent pregnancies of the couple and definite (skin thickness 5mm) (Figure 1). Other frequent prenatal testing can be offered in their future preg- nancies (Moreno et al., 2013). The most common sonographic findings> associated with fetal hydrops include placental thickening and polyhydramnios. etiologies include cardiovascular causes, chromo- The placental thickness (in mm) is normally equal somal anomalies and hematological abnormalities. to the gestational age (in weeks) +/- 10 mm; if the Other conditions associated with NIFH include fetal placental thickness exceeds this range, it is con- infections, fetal malformations, inborn errors of sidered as increased placental thickening (Moise, metabolism, lethal skeletal dysplasias, numerous 2008). With the widespread use of routine antiD other single gene disorders, fetal tumours and prophylaxis in Rh-negative mothers, prevalence placental abnormalities. of RhD alloimmunisation and associated hydrops The important etiological associations of NIFH has dramatically decreased and especially in de- are listed in Table 1 (Moise, 2008). veloped countries, NIFH now accounts for almost 90% of cases of hydrops fetalis. The progressive Pathophysiology of non-immune fetal fall in the incidence of immunologic hydrops fetalis hydrops has fostered growing interest in non-immune fetal hydrops. The world-wide prevalence of NIFH is The basic pathophysiological mechanism of fetal estimated to range from 1 in 1500 to 1 in 3800 hydrops is imbalance in the regulation of fluid be- births (Bellini et al., 2015). tween vascular and interstitial spaces. Fluid move- The identification of fetal hydrops by antena- ments between vascular and interstitial spaces are tal ultrasound requires extensive search for the regulated by filtration of fluid across the capillary etiology which includes a wide range of diseases wall as described by the Starling equation which including several genetic disorders. Even after states that the fluid movement due to filtration undergoing numerous investigations, in a good across the wall of a capillary is dependent on the number of cases the etiology remains unknown. balance between the hydrostatic pressure gradient In addition, the prognosis is usually poor with a and the oncotic pressure gradient across the capil- perinatal loss of 70–90%, except in rare cases of lary. When these pressure gradients are disturbed spontaneous resolution of parvovirus B19 infec- due to various pathophysiological mechanisms, tion. there is an increased fluid accumulation in the

116 Chapter 18 interstitial spaces, which leads to fetal hydrops. librium would therefore be of great importance Increased knowledge and understanding of the in identifying potential therapeutic interventions underlying mechanisms that disturb the fluid equi- (Bellini et al., 2012).

Table 1 Etiological associations of non-immune fetal hydrops.

Etiology Proportion of cases I Cardiac anomalies 17-35%

i. Structural defects - Atrioventricular septal defect isolated - Heterotaxy syndrome - Severe right or left ventricular outflow tract (RVOT/ LVOT) obstruction - Tricuspid dysplasia and Ebstein’s anomaly - Absent pulmonary valve syndrome - Premature closure of foramen ovale - Truncus arteriosus with truncal valve insufficiency ii. Cardiac tumours - Rhabdomyoma - Hamartoma - Hemangioma - Intrapericardial teratoma iii. Cardiomyopathy - Dilated/restrictive - Myocarditis iv. Arrhythmias - Tachyarrhythmias / bradyarrhythmias v. Idiopathic arterial calcification II Chromosomal aberrations 7-16% i. Monosomy X (Turner syndrome) (Figure 2) ii. Trisomy 13/15/16/18/21 iii. Triploidy and Tetraploidy iv. Partial duplications and Partial deletion of chromosomes (Figure 3) III Hematological disorders 4-12% i. Intrinsic - Alpha thalassemia - Erythrocyte enzyme disorders - Erythrocyte membrane disorders ii. Extrinsic hemolysis - Kasabach-Meritt sequence

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iii. Red cell aplasia - Parvovirus B19 infection - Diamond-Blackfan syndrome - Dyserythropoietic anemia - Congenital leukemia iv. Fetomaternal hemorrhage IV Twin–Twin transfusion 3-10% V Infections 5-7% TORCHES CLAP (Toxoplasma, Rubella, Herpes simplex, Echovirus, Syphilis, Cy- tomegalovirus, Coxsackie virus, Leptospirosis, AIDS, Adenovirus, Parvovirus) VI Syndromes 3-4% i. Autosomal dominant disorders - Cornelia de Lange syndrome - Congenital myotonic dystrophy - Noonan syndrome - Tuberous sclerosis ii. Autosomal recessive disorders - Lethal multiple pterygium syndrome (Figure 4) - Neu Laxova syndrome - Cumming syndrome - VII Skeletal dysplasias 3-4% - Asphyxiating thoracic dysplasia - Short rib thoracic dysplasia with/ without polydactyly (Figure 5) - Achondrogenesis - Osteogenesis imperfecta type 2 - Lethal osteopetrosis - Lethal Kneist- like dysplasia - Chondrodysplasia punctate (Conradi-Hunermann variant) - Greenberg chondrodystrophy - Caffey syndrome VIII Gastrointestinal disorders 0.5-4%

i. Intestinal haemorrhage and meconium peritonitis due to bowel perforation ii. Hepatic disorders - Cholestasis /congenital portal hypertension - Hepatitis/hepatic fibrosis - Hepatic cirrhosis with portal hypertension - Polycystic liver disease IX Renal anomalies 2-3% - Congenital nephrosis (Finnish type) - Polycystic kidney disease - Renal vein thrombosis

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X Inborn errors of metabolism 1-2% i. Lysosomal storage disorders (Figure 6) - Mucopolysaccharidosis types 1, 4, 7 - Sphingolipidoses (GM1 gangliosidosis, Galactosialidosis, Farber disease, Gaucher disease, Niemann-Pick disease type A) - Mucolipidosis type 1 (Sialidosis) and type 2 (I cell disease) - Transport defects (Niemann-Pick disease type 3 and Sialic acid storage disease) ii. Non-lysosomal disease - Glycogen storage disease type 2 - Long- chain hydroxyl- acyl CoA dehydrogenase deficiency - Carnitine deficiency - Congenital disorder of glycosylation type I/IX XI Placental causes 1% - Chorioangioma of placenta/ Subchorial placental hematoma - Umbilical cord abnormalities (true knots of cord, umbilical cord torsion, angiomyxoma of umbilical cord, umbilical vein thrombosis) XII Miscellaneous 3-15% XIII Unknown 15-25%

A B C

Figure 1 Antenatal ultrasound findings suggestive of fetal hydrops. A: Fetal scalp edema, B: Fetal ascites, C: Fetal pleural effusion.

A B A B

Figure 2 Fetus with Turner syndrome. A. Au- Figure 3 Fetus with unbalanced chromosomal topsy findings of webbing of neck, translocation. A. Autopsy findings of subcutaneous edema and joint con- facial dysmorphism and generalised tractures. B. Karyotype showing 45,X. subcutaneous edema. B. Karyotype showing 46, SC, der 5, t(5;10) (p15.3; q24.3) mat.

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Detection of Non-immune fetal hydrops

Detailed antenatal ultrasonography (USG) is the initial diagnostic modality for any case with non- immune fetal hydrops, and apart from detecting the hydrops per se also helps to assess the severity of hydrops and to detect associated malformations A B (Figure 1). Sonography can even provide important clues to the underlying cause of the hydrops in many cases. Increased nuchal translucency is often Figure 4 Fetus with autopsy findings suggestive the first sign of NIFH due to chromosomal abnor- of lethal multiple pterygium syndrome. malities. Cases secondary to cardiac abnormality A & B: Cystic hygroma, webbing of usually show significant cardiomegaly (Skoll et al., neck, contractures of joints and ptery- 1991). A fetus with anemia-related hydrops is likely gia across joints. to demonstrate the presence of pleural fluid and skin edema (Skoll et al., 1991). The middle cerebral artery peak systolic velocity (MCA PSV) 1.5 MoM (multiples of median) indicates fetal anemia> in fetuses of more than 16 weeks of gestation. Fetal hydrops associated with metabolic disorders is usually severe with massive ascites and significant thickening of the skin. Additional USG findings in various fetal infections associated with fetal hydrops include intrauterine growth retardation, polyhydramnios/ oligohydramnios, microcephaly, ventriculomegaly, intracranial calcification, cardiac anomalies, liver calcifications and echogenic bowel A B C (SOGC clinical practice guidelines, 2013). However, in most cases, further investigations are required Figure 5 Fetus with short rib thoracic dyspla- to clearly diagnose the etiology. sia. A. Autopsy findings of generalised subcutaneous edema, rhizomelic limb Stepwise evaluation for non-immune shortening and narrow and short tho- fetal hydrops rax. B. Skeletal radiograph showing narrow thorax with short ribs and As NIFH is an etiologically heterogeneous condi- shortening of humerus and femur. tion, each case of NIFH would require stepwise evaluation for all the known causes, in order to ascertain the exact etiological diagnosis. As a sig- C nificant proportion of cases have a genetic etiology, identification of the exact cause in each caseis very important for accurate counseling regarding the recurrence risk and prenatal diagnostic testing for future pregnancies. A B Step 1: Fetal imaging Figure 6 Fetus with Gaucher disease. A & B: i. Detailed obstetrical ultrasound which should Autopsy findings of generalised subcu- include a detailed survey for anomalies of the taneous edema and enlarged liver and fetus, placenta, umbilical cord and amniotic spleen. C: Sequence chromatogram of fluid and assessment of the fetal Doppler (Mid- the GBA gene showing the homozygous dle cerebral artery) and fetal echocardiogram. pathogenic mutation.

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Step 2: Tests in the mother Step 4: Postnatal evaluation

i. VDRL test for syphilis and TORCH serology. i. If the fetal samples have not been procured Maternal TORCH serology should be done in all antenatally or the antenatal samples are inad- cases of NIFH occuring for the first time in the equate, fetal cord blood/ skin biopsy/ umbilical family. cord sample should be collected after delivery for karyotyping/ enzyme assays/ DNA extrac- ii. SS-A and SS-B antibodies to be tested in the tion. If fetal cord blood is being collected, mother in cases of fetal bradyarrhythmia. about 2-3 ml of fetal cord blood should be Step 3: Invasive testing collected in a heparinized vacutainer and about 5ml in an EDTA vacutainer. If fetal autopsy is i. Amniocentesis: For fetal karyotyping or chro- planned, the fetal body and placenta should be mosomal microarray analysis; PCR for Cy- preserved in 10% formalin. tomegalo virus/PCR for parvovirus-B19/toxo- plasmosis in selected cases; DNA extraction ii. Whole body skeletal radiographs of the fetus- for further molecular genetic studies; enzyme anteroposterior and lateral views assay for lysosomal storage disorders. iii. Head to foot external dysmorphology evalua- ii. Fetal blood sampling: Complete blood picture tion with count, white blood cell count and count; TORCH serology/ iv. Internal dissection of fetal organs PCR for viral infections and viral and bacterial v. Histopathology of fetal organs, placenta and cultures; liver function tests including serum umbilical cord (Figure 7) total protein and albumin in some cases. In case of antenatal doppler evidence of fetal vi. Immunohistopathology, as required, for detec- anemia, PCR for Parvovirus B19 and molecular tion of lymphodysplasia; lymphodysplasia may genetic testing for alpha thalassemia should be be the underlying pathophysiological mecha- done in the fetal sample. nism in a number of cases including chromo- somal abnormalities. Immunohistochemical studies with CD31 and CD34 are helpful es- pecially if the lymphodysplasia lesional areas are small and not visible on gross examination (Bellini et al., 2010).

Whole body fetal radiographs, detailed fetal dysmorphology evaluation, internal organ dissec- tion and histopathological examination of the fetal organs and the placenta should done in every case. If a specific etiology is identified with this first-tier evaluation, specific cytogenetic or molecular genetic testing can be done in the fetal sample for confirmation of the same. As per vari- ous literature reports, perinatal autopsy provides important additional information or changes the ultrasonography-based diagnosis in 22-76% cases. Figure 7 Histopathology showing abnormal cal- In cases where autopsy evaluation does not cific deposits in various fetal organs in reveal a specific etiology, karyotype and enzyme a fetus with Idiopathic infantile arterial assays for common NIFH-associated lysosomal calcification. A & B: Haematoxylin-eosin storage disorders can be done. In cases where & Vonkossa stain for pulmonary vessel the above evaluation is inconclusive and the cause calcification, C: Myocardial calcification, remains unknown, higher resolution genetic test- D: Cerebral calcification, E: Renal cortex ing techniques i.e. chromosomal microarray and calcification, F: Placental villi calcifica- exome sequencing can be done in the fetal DNA tion, G: Aortic calcification, H: Renal sample, for copy number variations and single arterial calcification. gene etiologies respectively. Both parents can be

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Table 2 Therapeutic modalities for some causes of non-immune fetal hydrops.

Etiology Therapy Twin to twin transfusion syndrome Laser ablation of placental anastomoses or selective termination Twin-reversal arterial perfusion Percutaneous radiofrequency ablation Cardiac arrhythmias Maternal transplacental administration of antiarrhythmic medications Fetal anemia Fetal blood sampling followed by intrauterine transfusion Fetal hydrothorax/ pleural effusion associated with Placement of thoracoamniotic shunt/ bronchopulmonary sequestration needle drianage of effusion Fetal CPAM - (Congenital pulmonary airway malformation) Macrocystic Needle drainage/ Thoracoamniotic shunt Microcystic Corticosteroid therapy Large bronchopulmonary sequestration NdYAG Laser of the feeding vessel Fetal thyrotoxicosis Antithyroid drugs

tested further, as relevant, for the genetic etiology due to cerebral complications remains high. Fetal identified in the fetus. cytomegalovirus infection has been treated with maternal and direct fetal administration of hyper- Prognosis immune globulin. However there are only a few reported cases where this therapy was attempted Prognosis depends upon the etiology, the gesta- and they did not resolve with this therapy. tional age at onset and whether pleural effusions are present. In general, the earlier the hydrops Genetic counseling occurs, the poorer the prognosis. In particular, pleural effusions and polyhydramnios prior to20 Genetic counseling is an integral component of weeks of gestation are poor prognostic signs be- the management of any family with non-immune cause of increased risks of pulmonary hypoplasia fetal hydrops. If the cause of hydrops is identified, and preterm labour/ premature rupture of mem- the nature of abnormality, pattern of inheritance branes, respectively. On the other hand, absence and recurrence risk in future pregnancies can be of aneuploidy and absence of major structural determined. In cases of hydrops due to cardio- abnormalities confer a better prognosis. Despite vascular anomalies, the recurrence risk depends continued advances in perinatal care NIFH con- on the type of anomaly and varies from 3-50%. tinues to be associated with significant mortality Hydrops due to infections is less likely to recur. (Simpson et al., 2006). Hydrops due to chromosomal abnormalities usu- ally have a recurrence risk of around 1%, unless Therapeutic options they are associated with a familial chromosomal rearrangment, in which case the recurrence risk Fetal treatment for NIHF depends on the etiology would be higher, depending on the nature of the and gestational age. Some of the therapies for chromosomal anomaly. If the fetal hydrops is due selected etiologies are listed in Table 2 (SOGC to autosomal recessive disorders there is 25% risk clinical practice guidelines, 2013). of recurrence in the subsequent pregnancies of the NIFH related to fetal toxoplasmosis treated with couple. If NIFH is due to an autosomal dominant maternal administration of pyrimethamine and sul- condition, most often it would be due to a de novo fadiazine and NIFH related to fetal syphilis treated mutation, but there would be a small but signifi- with penicillin resolves but the overall prognosis cant risk of recurrence in subequent pregnancies

122 Chapter 18 due to the possibility of gonadal mosaicism for the rience in 79 fetuses. Am J Med Genet A 2010; pathogenic mutation in either parent. Idiopathic 152A: 1189-1196. NIFH generally has a low recurrence risk. Prenatal 4. Bellini C, Hennekam RC. Non-immune hydrops testing, as required, can be offered for subsequent fetalis: a short review of etiology and patho- pregnancies of the couple through targeted cy- physiology. Am J Med Genet A 2012; 8A: togenetic/molecular genetic testing, based on the 597-605. etiology identified in the affected fetus. 5. Bellini C, et al. Etiology of non immune fe- tal hydrops: An update. Am J Med Genet A Conclusion 2015;167A: 1082-1088. 6. Moise Jr K. Ultrasound evaluation of hydrops fetalis. In: Ultrasonography in Obstetrics and Non-immune fetal hydrops is a significant cause Gynecology. Fifth edition. Ed. Peter W Callen. of prenatal and perinatal morbidity and mortal- Elsevier Saunders, Pennsylvania, USA; 2008: p ity. With the use of advanced genetic testing 676- 697. technologies such as chromosomal microarray and 7. Moreno CA, et al. Non–immune hydrops fe- whole exome/ whole genome sequencing, we are talis: A prospective study of 53 cases. Am J likely to identify the underlying genetic basis in Med Genet A 2013;161A: 3078-3086. a greater proportion of cases with NIFH. This in 8. Puri RD, et al. Utility of fetal autopsy to corrob- turn would help to provide a greater insight into orate antenatal ultrasound findings. Am J Med the etiopathogenesis of NIFH and help to identify Genet A 2016; 170 A: 2119-2126. potential therapeutic targets for this condition. 9. Skoll MA, et al. Is the ultrasound definition of fluid collections in non- immune hydrops References fetalis helpful in defining the underlying cause or predicting outcome? Ultrasound Obstet 1. Bellini C, et al. Etiology of nonimmune hydrops Gynecol 1991; 1: 309-312. fetalis: a systematic review. Am J Med Genet A 10. Simpson JH, et al. Severity of non-immune 2009; 149A: 844-851. hydrops fetalis at birth continues to predict 2. Bellini C, et al. A diagnostic flow chart for non survival despite advances in perinatal care. immune hydrops fetalis. Am J Med Genet A Fetal Diagn Ther 2006; 21:380-382. 2009; 149A: 852-853. 11. Society of Obstetricians and Gynaecologists of 3. Bellini C, et al. Immunohistochemistry in non Canada (SOGC) clinical practice guidelines. J immune hydrops fetalis: A single center expe- Obstet Gynaecol 2013; 35: e1-e14. PhotoQuiz - 3 Contributed by: Dr Prajnya Ranganath Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad Email: [email protected]

This 10 months-old male child was brought for evaluation of recurrent infections, hepatosplenomegaly, developmental delay and hypopigmented hair. Hair microscopy (HM) showed typical findings. Identify the condition.

Answer on page 231

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Fetal Therapy - Current Approaches and Future Possibilities

Shagun Aggarwal Nizam’s Institute of Medical Sciences and Centre for DNA Fingerprinting and Diagnostics, Hyderabad

Correspondence to: Dr Shagun Aggarwal Email: [email protected]

Introduction or intravenously, which then passes onto the fetus through the uteroplacental circulation. Various Strictly speaking, fetal therapy is defined as a indications and routes of fetal medical therapies therapeutic intervention for the purpose of cor- are mentioned in Table 1. recting or treating a fetal anomaly or condition. Administration of thyroxine by intra-amniotic However, in a broader sense, it encompasses any route in cases of hypothyroid fetal goitre, maternal prenatal treatment administered to the mother administration of dexamethasone for prevention with the primary indication to improve perinatal or of genital ambiguity in female fetuses affected with long-term outcomes for the fetus or newborn. This congenital adrenal hyperplasia and management implies that even conventional practices like folic of polyhydramnios by maternal NSAID therapy are acid supplementation, management of maternal some of the relatively well established therapies thyroid disorders, steroid therapy for lung matura- of documented benefit. The same cannot be tion, management of polyhydramnios and therapy said about newer approaches like administration for fetal infections, all fall under the gamut of fetal of fresh frozen plasma to fetuses with Smith therapy. The need for fetal therapy arises when Lemli Opitz syndrome, where doubtful benefit was a fetus is found to be affected with a condition achieved in a single case report only, and many in-utero which, if left unattended, would lead to other similar rare conditions. In recent years, fetal demise, irreversible injury to the fetus or prenatal steroid therapy for congenital pulmonary obstetric complications. airway malformations (previously called congeni- tal cystic adenomatoid malformations) has shown Guidelines (Ville, 2011) promising results in reducing size of lesions as well as resolution of hydrops. Medical therapy of The International Fetal Medicine and Surgery Soci- the fetus is non-invasive or at the most minimally ety guidelines for fetal therapy are as follows: invasive in nature, hence newer studies and data are bound to emerge at a fast pace and this The natural history of the disease should be field is likely to evolve with promising results in • at least partly understood. the future. Some obvious drawbacks are the non-curative nature requiring continuous and re- The condition should be lethal or could result peated medication during pregnancy followed by • in severe morbidity if not treated in utero. postnatal therapy, which again is likely to be long term and specific adverse effects associated with The fetal intervention should be at least the particular therapy. • partly corrective and the results should com- pare favourably with those obtained following postnatal treatment. Surgical therapies (Deprest et al., 2010; van Mieghem et al., 2012) Medical therapies (Hui et al., 2011) In contrast to medical therapies, fetal surgical The therapeutic approach to the fetus can be medi- interventions are more definitive and may lead cal or surgical. Medical approach is most commonly to almost complete correction of the fetal defect. by the transplacental route, i.e. by administration However, they are invasive and associated with of medication to the mother orally, intramuscularly operative complications like infection, preterm

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Table 1 Fetal medical therapies.

Fetal condition Therapy Route of Evidence of use administration Congenital Dexamethasone Maternal oral Effectively prevents genital Adrenal 20ug/Kg/day ambiguity in female foetuses in Hyperplasia 85% cases Polyhydramnios Indomethacin Maternal oral Efficacy 80-100%; Problems of (300mg/day) premature ductus closure Sulindac (400mg/day) Fetal Levothyroxine Intra-amniotic Oral route not found useful hypothyroid 150-600ug instillation goitre Intra-muscular Fetal Propylthiouracil Maternal oral Fetal teratogenic effects thyrotoxicosis 100-600mg/day due to maternal Grave’s disease Fetal tach- Anti-arrythmic drugs- Maternal oral, Proven efficacy yarrhythmias Digoxin, Sotalol, i.v. amiodarone, Flecainide Fetal i.v, i.m. Congenital heart Immunoglobulin Maternal i.v. Ineffective in 3rd degree block. block Corticosteroids Maternal oral May prevent progression of 2nd degree block Congenital Corticosteroids Maternal i.m. Case series of 43 cases-Lesion pulmonary reduction:82% airway malfor- Hydrops resolve:88% mation(cystic adenomatoid malformation) of lung Fetal biotinidase Biotin Maternal oral Few case reports deficiency Fetal Vit B12 Maternal Few case reports methylmalonic oral/i.m. acidemia Fetal pyridoxine Pyridoxine Maternal oral Few case reports dependent seizures Fetal Smith Lemli Fresh Frozen Plasma Fetal Few case reports Optiz syndrome intravascular, intraperitoneal labour, anaesthetic hazards, etc. As of now, only MRI and fetal karyotyping/chromosomal microar- very limited conditions are amenable to surgical ray are absolute pre-requisites before performing therapy and only few centres in the world have ex- . In addition the following principles perts who can perform these procedures. Before need to be followed: a) Condition should be severe operating on a structural fetal defect, it is essential enough to warrant intervention, b) Prenatal inter- to ensure that the defect is isolated and not part of vention is associated with better prognosis and a multiple malformations syndrome. Hence, fetal c) Condition should not be severe enough to be

125 Chapter 19 irreversible already. The surgical approach can be by slow infusion of the PRBC under continuous through the following routes: ultrasound visualisation. At end of infusion of the 1. Open surgery: This requires a hysterotomy calculated volume, fetal blood is collected and the and general anaesthesia. It has been used for checked to confirm that the transfusion correction of lesions like congenital diaphragmatic has been successful. Subsequent transfusions are hernia, congenital cystic adenomatoid malforma- usually done at intervals of 15-20 days and delivery tion of lung, myelomeningocoele and sacrococ- can be planned after lung maturity is attained. cygeal teratomas. However, the highly invasive Various procedural complications can arise like nature and associated high incidence of complica- fetal demise, bradycardia, cord bleeding, preterm tions limit its utility. labour and infection. The fetal survival rate is 88% and in the absence of hydrops has been reported 2. guided/: This proce- to be as high as 96%. (Moise et al., 2008) dure is done through an endoscopic instrument 2. Myelomeningocele repair: Myelomeningo- which is inserted through a small abdominal & cele gives rise to motor & sensory defects, or- uterine incision. It also requires general/regional thopedic problems involving lower limbs, bowel & anaesthesia and uterine tocolysis. Conditions bladder dysfunction, hydrocephalus secondary to which are amenable to therapy by this approach hindbrain compression (requiring shunt placement are twin-to-twin transfusion syndrome, congenital in 80% of cases), and death due to posterior fossa diaphragmatic hernia, posterior urethral valves, syndrome in 15-30% individuals. The pathophys- amniotic bands and selective fetal termination of iology is believed to be damage to an exposed the anomalous twin. neural tube and cerebrospinal fluid leak leading to 3. Ultrasound guided: This is a minimally inva- Chiari malformation and hydrocephalus. Attempts sive approach requiring local/regional anaesthesia. to prenatally repair the defect have been made on It can be used for radiofrequency ablation, shunt the rationale that closure of the exposed spinal placement in bladder outlet obstruction & cystic cord would help in preventing damage to the neu- thorax lesions, endovascular/cardiac procedures, ral tissues and stop the CSF leak, thereby reversing and fetal blood sampling as well as transfusion. brainstem herniation & hydrocephalus. Various The following is an overview of the commonly animal models of prenatal surgery showed positive used fetal therapeutic procedures: results, following which the first fetoscopic repair 1. Fetal blood transfusion: This was the first was attempted in 1997 and the first open surgery minimally invasive fetal therapy procedure used by in 1998. Presently, the specific pre-requisites for Liley et al in 1963 for management of Rh haemolytic myelomeningocele repair are a) defect between disease. Initial transfusions were given intra- level T1-S1, b) Normal leg movements and bladder peritoneally under fetoscopic guidance. Later, emptying c) Arnold Chiari malformation d) Mild USG-guided intravascular transfusion became the to moderate ventriculomegaly e) 19-26 weeks ges- preferred technique. Besides Rh haemolytic dis- tation. Data from previous procedures at CHOP ease, other causes of fetal anemia like parvovirus (Children’s Hospital of Philadelphia) (n=51) and infection and feto-maternal hemorrhage are also Vanderbilt University (n=178) showed reversal of amenable to this intervention. Presently man- hindbrain herniation in 38%, decreased the need agement of Rh haemolytic disease involves serial for shunting in 43-59%, improved leg and bladder monitoring of middle cerebral artery peak systolic function and improved later cognitive function in velocity for evidence of fetal anemia, followed by selected fetuses. Recently MOMS (Management ultrasound-guided fetal umbilical vein puncture, of Meningomyelocele Study), a multicentric long estimation of fetal hematocrit and subsequent in- duration trial (n=183) in USA, has shown definite fusion of packed RBCs if hematocrit is 30%. The benefit of open meningomyelocele repair in terms RBCs infused have to be O negative,< compatible of survival, shunt requirement and motor function. with maternal blood, washed, filtered, gamma However, there was no cognitive benefit and the irradiated, with a hematocrit 80%. The amount procedure was associated with a significant risk to be transfused is calculated≥ on basis of the fetal of prematurity and scar dehiscence (Adzick et al., weight, fetal hematocrit and the blood unit hemat- 2011; van Bebbington et al., 2011). Following the ocrit. The target hematocrit is usually 40-50%. The promising results of the MOMS trial, open fetal process of transfusion involves ultrasound guided surgery for selective cases of is con- puncture of the umbilical vein, confirmation of fetal sidered the treatment of choice, provided facilities blood, infusion of a fetal paralytic agent followed are available.

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3. Lower Urinary tract obstruction: The promising results in recent times. This involves fe- most common etiology is posterior urethral valves. toscopic insertion of a balloon/clip/plug to occlude Other possible causes can be urethral atresia and the trachea, which helps in growth of the lungs cloacal abnormalities. The perinatal mortality can by preventing egress of the lung fluid. Although be as high as 90%, primarily due to pulmonary initial reports from CHOP (Children’s Hospital of hypoplasia. 50% of survivors develop renal im- Philadelphia) and UCSF (University of California, pairment. The rationale of in-utero treatment is San Fransisco) did not show significant benefit, to increase amniotic fluid volume, prevent pul- recent results from the Eurofetus trial and a large monary hypoplasia and restore bladder & renal Belgium trial of 210 patients (using in-utero balloon function. The candidates for intervention are removal late in gestation) have been promising. fetuses with severe disease, decreased amniotic Survival for left CDH is reported to improve from fluid, but preserved renal function. Various param- 25% to 49% and right sided CDH 0% to 35%. eters have been used to ensure adequate renal A multicenter randomised phase 2 trial (TOTAL- functioning, like urinary biochemical parameters Tracheal Occlusion to Accelerate Lung Growth) is and echogenicity of kidneys on ultrasound, how- presently underway to assess this approach in ever, none are known to be reliable. Therapeutic cases with moderate pulmonary hypoplasia. The approaches are: removal of the intratracheal balloon can be done a) Vesico-amniotic shunt placement- this is done through an EXIT procedure (Ex-utero intrapartum with ultrasound guidance, under local anaesthesia. therapy), which involves an intrapartum procedure A double pigtailed catheter is introduced connect- to secure fetal airway, while the baby maintains ing the bladder with the amniotic cavity. More than perfusion through utero-placental circulation or 300 shunts have been placed at various centres through ultrasound/fetoscopic guided removal in across the world in the last 25 years. Forty percent late third trimester. (Hedrick, 2013; Jani et al., 2012; neonatal survival has been reported in past stud- Dekoninck et al., 2011) ies, but up to 50% cases develop renal impairment 5. Fetoscopic selective laser coagulation of later in life. Recent studies have raised ques- placental vessels (SLPC): This procedure is used tions about the methodologies of previous reports. primarily for treatment of twin-to-twin transfu- A randomised control trial PLUTO (Percutaneous sion syndrome (TTTS). TTTS occurs in 10-15% of Shunting for Lower Urinary Tract Obstruction) to mononchorionic twin pregnancies due to abnormal assess this approach was initiated, but had to placental communications between the circulation be abandoned due to poor recruitment. (Ruano, of the twins. This leads to one of the twins acting as 2011) a donor twin, whereby it starts pumping blood into b) Fetal cystoscopy- this involves direct visual- circulation of the co-twin, and the other twin be- isation of the bladder for identifying the cause comes the recipient. This causes growth restriction of obstruction. If posterior urethral valves are & oligohydramnios in the donor, and overgrowth causative, these can be disrupted using laser ful- & polyhydramnios in the recipient. The donor is guration, guidewire perforation and hydroablation. at risk of hypovolemia & renal failure, while the Although initial reports have shown good neonatal recipient is at risk of heart failure. Overall mortality survival rates, more trials are required to assess is 80-100% and only 40-50% of survivors are neuro- the effectiveness of this approach. (Ruano, 2011) logically normal. The therapeutic approach to this 4. Congenital diaphragmatic hernia repair: condition is to interrupt the abnormal placental This condition is associated with a neonatal mortal- communicating vessels. A fetoscope is inserted ity of 50-70%, resulting from pulmonary hypoplasia into the uterine cavity, a laser fibre (Nd:Yag or and pulmonary hypertension. In utero therapy is diode) is introduced through the operative channel proposed to be useful by reducing the pressure ef- and the abnormal placental vessels coagulated. fects on thoracic organs. Selection criteria for fetal The Eurofetus trial concluded in 2004 that this surgery are a) liver in thorax, b) gestation less than procedure is the treatment of choice in TTTS (Stage 25 weeks, and c) low lung: head ratio ( 25%). The 2 or beyond in severity) detected between 18-26 surgical approach was initially through< hystero- weeks gestation. The procedure is reported to tomy, followed by correction of the diaphragmatic have neonatal survival rates of 75-85% and nor- defect. This was not shown to show any survival mal neurological outcome in 75-94% of survivors benefit in randomised trials. However, minimally (Khalek et al., 2013a). A five year follow up of invasive approach using a fetoscope, FETO (Fe- these babies reported favourable cardiac function tal Endoscopic Tracheal Occlusion), has shown in majority, 2% severe intellectual disability and

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18% borderline intellectual disability. pre-clinical stage. 6. Others: Fetal lung lesions are amenable to In Utero Gene Therapy: The introduction in utero therapy by resection for solid lesions or of a• deficient gene has the potential to prevent shunt placement for cystic lesions (Khalek et al., disease prior to the onset of irreversible organ 2013b). Cardiac lesions esp. outflow tract obstruc- damage. In view of relative immunodeficient state tion can be approached by valvuloplasty of aortic and small size of fetus, the success of gene uptake & pulmonary valves (Arzt et al., 2011). Fetal tumors and integration into large proportion of cells is like sacrococcygeal teratomas can be excised by also likely to be better. The procedure involves open surgery or their blood supply can be occluded ultrasound guided introduction of the transgene by ablation or embolisation. Table 2 shows the into a fetal cavity or organ of interest. Presently, pathophysiology and therapeutic approaches for this approach has been tried in rodent models surgically correctable fetal conditions. (Deprest et of various metabolic, central nervous system & al., 2010) musculoskeletal diseases. However, though tran- sient gene expression has been attained, sustained Novel therapies in the pipeline (Pearson benefit has not been found. Due to the potential for teratogenic effects & germline transmission, et al., 2013) presently, human trials are not considered ethical.

In Utero Stem cell therapy: Due to the rela- Conclusions tive• immunodeficient state of the fetus, introduc- tion of stem cells may lead to better engraftment. The development of various fetal treatment strate- This formed the basis of various IUHSCT (In Utero gies offers hope for in utero management of fetal Hematopoietic Stem Cell Transplantation) trials in disorders and birth of a healthy baby for families mouse models. However, most achieved only low who are confronted with the scenario of an anoma- level chimerism, and significant host response was lous fetus. However, this field is still in its infancy, seen. Few human trials showed benefit in Severe and more experience from multiple centres is Combined Immunodeficiency syndrome. However, needed before it can be hailed as standard of care for all other disorders, this approach is in the for fetal pathological states.

Table 2 Fetal surgical therapies.

Fetal Pathophysiology Rationale for in Therapeutic Evidence of use condition utero therapy approach Congenital Pulmonary Timely reversal of * Open surgery * Not useful diaphragmatic hypoplasia and pulmonary * Fetoscopic * Large trials show hernia pulmonary hypoplasia and tracheal survival benefit, hypertension prevention of occlusion randomised trial pulmonary commenced hypertension Lower urinary Progressive renal Urinary diversion * * Variable results, tract damage by prevents obstructive Vesico-Amniotic randomised trial obstruction obstruction uropathy and shunt underway Pulmonary restores amniotic * Cystoscopic * Novel technique, hypoplasia by fluid volume posterior requires further oligohydramnios urethral valve study ablation Neural tube Damage to exposed Covering exposed Open surgery- Large trial showed defects neural tube; CSF leak, spinal cord, cessation Closure of spinal survival and long leading to Chiari of leakage preventing defect term benefit malformation and hydrocephaly and hydrocephalus reversing cerebellar herniation

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Twin-to-twin Intertwin transfusion Bichorionization Fetoscopic Large trials have transfusion leads to stops intertwin Selective Laser shown definite oligopolyhydramnios transfusion, reverses Fulgration of survival benefit, sequence, cardiac failure, Placental Vessels treatment of hemodynamic delays delivery choice for 18-26 changes; obstetric weeks, stage I complications TTTS > Sacrococ- High output cardiac Cessation of steal * Open or Few case reports cygeal failure by phenomenon fetoscopic teratoma arteriovenous Reversal of cardiac excision of shunting failure; tumor Fetal anemia by Prevention of * Ablation or tumor growth and/or polyhydramnios embolisation of bleeding within a feeding vessels tumor Thoracic Pulmonary Prevention of * Open surgery- *Few case reports space- hypoplasia pulmonary Excision of solid 60% survival rate occupying (space-occupying hypoplasia and lesions *Few case series lesions mass) cardiac failure *Ultrasound 70-85% survival Hydrops by impaired guided rate venous return placement of (mediastinal thoraco-amniotic compression) shunts for cystic lesions Cardiac Critical lesions Prevention of Ultrasound Technical success malformations causing irreversible hypoplasia or arrest guided of 60-70% in aortic hypoplasia or of progression of valvuloplasty valvuloplasty. damage damage Further trials required Amniotic Progressive Prevention of limb Fetoscope Few case reports bands constrictions causing deformities and guided excision irreversible function loss of bands- neurological or scissors or laser vascular damage or electric current Fetus Discordant Selective feticide to Selective feticide Many case series acardiacus anomalies: where improve chances of of anomalous show survival and discordant one fetus can be a the other fetus; fetus- benefit for co-twin anomalies threat to the other, avoidance of Intracadiac Results better for or to avoid termination of entire saline/potassium dichorionic twins termination of entire pregnancy chloride pregnancy injection under ultrasound guidance Coagulation/ab- lation/ligation of umbilical cord vessels

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References 8. Jani JC, et al. Fetal surgery for severe congen- ital diaphragmatic hernia? Ultrasound Obstet 1. Adzick NS, et al. A randomized trial of prenatal Gynecol 2012; 39: 7-9. versus postnatal repair of myelomeningocele. 9. Khalek N, et al. Fetoscopic laser therapy for N Engl J Med 2011; 364: 993-1004. twin-to-twin transfusion syndrome.Semin Pe- 2. Arzt W, et al. Fetal surgery for cardiac lesions. diatr Surg 2013; 22; 18-23. Prenat Diagn 2011; 31: 695-8. 10. Khalek N, et al. Management of prenatally 3. Bebbington MW, et al. Open fetal surgery for diagnosed lung lesions. Semin Pediatr Surg myelomeningocele. Prenat Diagn 2011; 31: 2013; 22: 24-29. 689-94. 11. Moise KJ Jr. Management of rhesus alloimmu- 4. Dekoninck P, et al. Results of fetal endoscopic nization in pregnancy. Obstet Gynecol 2008; tracheal occlusion for congenital diaphrag- 112:164-76. matic hernia and the set up of the randomized 12. Pearson EG, et al. Stem cell and genetic thera- controlled TOTAL trial. Early Hum Dev 2011; pies for the fetus. Semin Pediatr Surg 2013; 22: 87: 619-24. 56-61. 5. Deprest JA, et al. Fetal surgery is a clinical 13. Ruano R. Fetal surgery for severe lower uri- reality. Semin Fetal Neonatal Med 2010; 15: nary tract obstruction. Prenat Diagn 2011; 31: 58-67. 667-74. 6. Hedrick HL. Management of prenatally diag- 14. van Mieghem T, et al. Minimally invasive fetal nosed congenital diaphragmatic hernia. Semin therapy. Best Pract Res Clin Obstet Gynaecol Pediatr Surg 2013; 22: 37-43. 2012; 26: 711-25. 7. Hui L, et al. Prenatal pharmacotherapy for fetal 15. Ville Y. Fetal therapy: practical ethical consid- anomalies: a 2011 update. Prenat Diagn 2011; erations. Prenat Diagn 2011; 31: 621-627. 31: 735-43.

PhotoQuiz - 4 Contributed by: Dr Prajnya Ranganath Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad Email: [email protected]

This 6 years-old intellectually normal male child was brought for evaluation of short stature and joint deformities. Skeletal radiographs (taken at 3 years of age) showed typical findings. Identify the condition.

Answer on page 231

130 Chapter 20

Newborn with Down Syndrome: Care and Counseling

Shubha R Phadke, Rekha Gupta Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow

Correspondence to: Dr Shubha R Phadke Email: [email protected]

The birth of a child in a family is a very exciting, diagnosis was kept hidden from them. Delaying enjoyable and happy moment for the parents. the diagnosis also leads to loss of an opportunity to Everybody expects a normal, healthy child. Parents get karyotype done and offer genetic counseling, dream about the child and make plans for the especially as some babies with Down syndrome future throughout the pregnancy. With such ex- may die during the neonatal period. pectations, when a child with a malformation or a is born, it shatters the lives of the par- ents. Of course, the family wants to treat and help 1. When to disclose the diagnosis of the baby to the best of their ability. Birth defects Down syndrome in a neonate? like cleft lip, meningocele, and encephalocele are externally obvious and the parents get a fair idea of the problem. In case of internal defects like cardiac The best time is as early as possible. Usually, it anomalies or duodenal atresia the symptoms of should be done within the first 2-3 days of life. If the baby make the parents realize that there is the neonate develops any problems due to some something wrong with the baby. But what about associated malformation then the possibility needs Down syndrome? Most of the neonates with Down to be discussed immediately with the family. syndrome do not have external malformations. The characteristic facial dysmorphism which is ob- 2. Who should talk to the family? vious to a pediatrician may not raise any concern in the parents’ minds. Hence, breaking the news that the neonate has a serious handicapping condition A senior experienced consultant should take the is a very difficult task for the neonatologist and the responsibility of breaking the news as it is a major obstetrician. and serious problem for the family. It is important Some clinicians think that it may not be proper as the news should be given with great sensi- to tell the family immediately that the baby has tivity and the clinician should be confident and a problem. Nobody is comfortable with the chal- comfortable in answering questions of the family lenging task of breaking the bad news to the happy and should also be capable of dealing with the family. The justification of such clinicians is that disturbed psychological situation. A pediatrician let the family enjoy the child birth and over the or a neonatologist and the obstetrician should sit next few months some other pediatrician may together with the parents and tell the diagnosis. disclose the diagnosis of Down syndrome to the family or the parents themselves may notice the developmental delay and other problems and may 3. To whom should the news be told? consult a doctor. But there are many valid reasons to disclose the diagnosis of Down syndrome in the Preferably both the parents should be told together neonatal period. that their newborn baby has Down syndrome. If The most important reason is that it provides there is some doubt in the diagnosis as in cases of an opportunity to investigate the baby for ma- premature babies, then the possibility of Down syn- jor malformations like tracheo-oesophageal fistula, drome may be conveyed to the family and detailed cardiac malformation, cataract, duodenal atresia, counseling can wait till confirmation by karyotyping gastrointestinal malformation, etc. Secondly, most or other quicker methods like fluorescence in situ of the parents feel hurt if they realize that the hybridization or QF-PCR.

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4. How to break the bad news? ents due to the birth of a child with Down syndrome. The severity of mental handicap needs As mentioned earlier, breaking the news that the more explanation. Most of the children with Down almost normal looking neonate is likely to have syndrome are moderately retarded. They learn mental handicap is a great challenge to the pedia- to talk, walk and do self care and some simple trician. Most pediatricians may face this situation repetitive jobs. It is reassuring to parents that some time in their lives. Even to an experienced children with Down syndrome can lead a happy clinical geneticist this task is an ordeal that he or and useful life. But most of them need lifelong she would like to avoid. However, though it is supervision at home and work. There should be a a difficult task, it has to be done by somebody. truthful but positive approach. There is no right way to break the bad news, but The immediate need to investigate for internal some ways are worse than others. Care has to anomalies like cardiac defects, cataract, etc should be taken to understand the state of mind of the be clearly told; absence of any malformation may parents. Most of the parents remember very well relieve the parents’ anxiety to some extent. Positive the moment and the words of the doctor when aspects like no immediate risk to life, no physical they were told that the baby has Down syndrome. handicap are usually helpful. Need for regular The family should be given prior intimation that surveillance for hypothyroidism and hearing prob- the clinician wants to discuss some issues about lems can be mentioned. Rare possibilities like the baby. leukemia, atlantoaxial dislocation, and Alzheimer Sit in privacy with both the parents and prefer- disease need not be told. Information about ably the baby as well. In India, more often than not, early infant stimulation programs, patient support grandparents or other relatives are also involved. groups and relevant literature for parents should They can be a good source of support to the be given. family during such a stressful period. The language should be simple. The conversation should start 6. Risk of recurrence in sibs on a personal note, like enquiring the name of the baby. During the course of discussion the baby These issues discussed in subsequent visits, after should be referred by name as “your baby” and the karyotype report is available. Information not words like “Mongol babies” or “these mentally about prenatal screening and prenatal diagnosis retarded children”. There are a few recommenda- is useful. If the baby has free trisomy 21 (47 tions on how best to deliver the diagnosis of Down chromosomes with 3 copies of chromosome 21), syndrome to the parents (see suggested reading). then the risk of recurrence in the next pregnancy The physician should be caring and empathetic. of the mother is usually 1% and in this situation He or she should assess and acknowledge the the karyotypes of the parents are not needed. If reaction of the parents to stress and help them the baby has Down syndrome due to translocation accordingly. There should be enough time for of chromosome 21 to the other chromosome 21 questions. Follow up visits are needed to reinforce or any other chromosome, then the parental kary- information and help the family to cope up with otypes from peripheral blood are needed to give the situation and slowly accept the reality. the risk of recurrence. If one of the parents is a carrier of a balanced translocation involving chro- 5. What should be told? mosome 21, the risk of recurrence varies from 5% to 100% depending upon the type of translocation The information about Down syndrome should and the parent involved. The other situation when include chromosomal etiology, associated major the karyotypes of the parents may be needed problems like mental handicap and malformations for genetic counseling is when a child with Down in some cases. Genetic etiology and chromosomal syndrome dies before karyotyping could be done. nature may need some explanation in the layman’s language. It needs to be made clear that even Conclusions though Down syndrome is genetic in nature, in most of the cases it is not inherited and usually Facing a neonate with Down syndrome is an expe- there is no similarly affected member in the family. rience by itself as is the first meeting with his/her Information about the general population parents, which every obstetrician, neonatologist prevalence may help to avoid guilt in the par- and pediatrician will have to undergo a few times

132 Chapter 20 in life. This article may help the physicians to does the child with any congenital defect need prepare for the situation and to deal with it with medical management; the parents also need a lot confidence. In this era of prenatal screening for of emotional support along with all the possible Down syndrome, birth of children with Down syn- and latest information about the nature of the ab- drome may decrease. However, seeing a neonate normality, etiology, prognosis, possible treatment with Down syndrome after negative results of and genetic counseling. prenatal screening is much more devastating for the family. Counseling such a family is a more Suggested reading difficult task. Empathy, good communication and adequate time are the important requisites for the 1. Dent KM and Carey JC. Breaking difficult news success of counseling. in a newborn setting: Down syndrome as a A similar situation needs mention here and that paradigm. Am J Med Genet C Semin Med is prenatally detected case of Down syndrome. Genet. 2006 ;142C:173-179. Skotko BG, et al. Breaking the news of a fetus being affected with Prenatal diagnosis of Down syndrome: how Down syndrome after a prenatal diagnostic test is best to deliver the news. Am J Med Genet A; just as difficult as disclosing the diagnosis to the 149A: 2361-2367. family of an affected neonate. Though usually the 2. Skotko BG. Mothers of children with Down pregnancy is terminated in case Down syndrome is syndrome reflect on their postnatal support. prenatally detected, the decision of termination is Pediatrics 2005; 115: 64-77 painful for the family and leads to a great sense of loss. The counseling should be done with senstivity 3. Skotko BG, et al. Postnatal diagnosis of Down and care should be taken to minimize emotional syndrome: synthesis of the evidence on how trauma to the family. best to deliver the news. Pediatrics 2009; 124: It should always be remembered that not only e751-758. PhotoQuiz - 5 Contributed by: Dr Prajnya Ranganath Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad Email: [email protected]

This 1.5 months-old male child was brought for evaluation of abnormal skull shape, facial dysmorphism and limb anomalies. Identify the condition.

Answer on page 231

133 GeNeToon

Contributed by: Dr Prajnya Ranganath Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad Email: [email protected]

GeNeToon

Contributed by: Dr Shubha Phadke Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow Email: [email protected]

134 Section 4 Mendelian Disorders

Chapter 21

Spinal Muscular Atrophy

Dhanya Lakshmi N1, Girisha KM2 1Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad 2Department of Medical Genetics, Kasturba Medical College, Manipal University, Manipal

Correspondence to: Dr Girisha KM Email: [email protected]

Introduction SMA 0, SMA I, SMA II, SMA III and SMA IV. SMA 0 has prenatal onset and presents with neonatal hypo- Spinal muscular atrophy (SMA) is a common in- tonia, respiratory difficulty, facial diplegia and life herited neuromuscular disease in children with a expectancy of less than 6 months. SMA I (Figure 1) frequency of 1 in 10000 livebirths. The frequency of (Werdnig-Hoffman disease), the most common and heterozygous carriers has been estimated to be 1 the severest variety, accounts for about half of the in 40 to 60. (Prior et al., 2007). It is a disease where patients with SMA. The disease usually manifests spinal cord shows severe loss of motor neurons in by 6 months of age and affected infants will never the anterior horn region manifesting with muscle achieve independent sitting. Profound hypoto- weakness of varying severity. Significant advances nia and weakness hinders achievement of normal in understanding the molecular pathogenesis has head control and they commonly present as floppy resulted in better care of the families from the babies. They may demonstrate a bell shaped tho- point of view of clinical genetics. This article dis- rax with paradoxical respiration. Most succumb to cusses the practice of clinical genetics with respect the disease, mainly due to respiratory compromise to spinal muscular atrophy. by one to 2 years of age. SMA II has onset by 7-18 months of age and children usually sit, but never stand and walk. They can have postural tremors of the fingers. The life expectancy extends to late childhood. SMA III (Kugelberg-Welander disease) is a mild form of the disease with onset beyond 2 years of age. These individuals are able to sit, stand and walk independently. However, adults have weakness of variable severity with some being able to perform all activities independently whereas others requiring support for walking. Some patients may have calf muscle hypertrophy. Most of them survive into adulthood with varying degrees of severity. SMA IV has onset in second or third decade of life and affected individuals have normal life expectancy. The other striking features in SMA are normal intellect and presence of fasciculations. Over long term, kyphoscolio- sis and contractures may develop, increasing the Figure 1 A floppy infant with SMA1. morbidity. It is important to realize the spectrum of clinical severity of SMA and consider this as a differential diagnosis in any individual demon- Clinical features strating muscle weakness of lower motor neuron origin. Fasciculations, if clinically observed favors The classical features of spinal muscular atrophy the diagnosis of SMA in a child with hypotonia and are hypotonia and muscle weakness. Depending muscle weakness. Depending on the age of onset on the phenotype, the disease is divided into

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Figure 2 Diagram showing location of SMN genes on and the difference of a single nucleotide on exon 7. of symptoms, several conditions can be considered and long durations coupled with decreased recruit- as differential diagnosis for SMA. The differen- ment. Skeletal muscle shows atrophic fibers with tial diagnoses to be considered in a hypotonic islands of group hypertrophy on biopsy. (Buchthal infant are congenital myopathy, congenital muscu- et al., 1970). Molecular testing, now being easily lar dystrophies, peroxisomal disorders, congenital available at several centers, the diagnosis can be myaesthenic syndromes, central hypotonia as in quickly established by testing DNA obtained from Prader Willi syndrome and infantile Pompe dis- peripheral blood. This obviates the need for ease. In childhood onset of symptoms, Duchenne/ electromyography and the invasive muscle biopsy. Becker muscular dystrophy, Hexosaminidase A de- They still have their role when the mutation is not ficiency, peripheral neuropathies and limb girdle detected in a floppy child. muscular dystrophies may be considered in differ- ential diagnosis. In adult onset type, spinal and bulbar muscular atrophy and amyotropic lateral Genetic basis sclerosis are considered as differential diagnoses. Survival motor neuron gene (SMN) was identified as the causative gene for spinal muscular atrophy. Investigations (Lefebvre et al.,1995). SMN gene is located on the long arm of chromosome 5 (5q13) (Figure 2). Creatinine kinase levels may be normal or mildly It is present in multiple copies in humans, one elevated. Earlier, electromyography and muscle telomeric copy (SMN1) and several centromeric biopsy were used to confirm the diagnosis. Elec- copies (SMN2). These genes contain nine exons tromyography shows features suggestive of dener- and eight introns. These two genes differ in 5 vation with spontaneous activity of positive sharp nucleotides, with just one difference in the coding waves, fibrillation and occasional fasciculations. region. SMN1 gene encodes a protein with 294 Motor unit action potential shows high amplitudes aminoacids which is expressed in all tissues. All

136 Chapter 21 patients with SMA have at least one copy of SMN2. and a review of clinical features should be done. SMN2 has a C to T transition at position six of exon Myopathies (congenital and metabolic), muscular 7 (as compared to SMN1). This leads to alternate dystrophies and neuropathies (central or periph- splicing of SMN2 and exclusion of exon 7 from the eral) need to be considered as alternate diagnosis. mRNA. The resultant protein is non-functional and In the presence of typical features of SMA and neu- is rapidly degraded. Only about 10% of the product rogenic electromyography, if only a heterozygous of SMN2 is properly spliced and leads to a normal deletion of exon 7 is identified, a point mutation SMN protein. This low level of expression leads to in the other SMN1 allele may be considered. Iden- survival in embryonic life, but is not sufficient for tification of intragenic mutation in SMN1 requires the survival of motor neurons in the spinal cord. In sequencing of the long range PCR product or milder forms of the disease, the degree of normal subclone of SMN1. splicing may be up to 50% and may contribute to Next generation sequencing technology (tar- the milder phenotype of these individuals. But geted gene panel testing/ exome analysis) may be variation in the copy number of SMN2 alone does used to look for variants in genes causing a similar not completely explain the phenotypic diversity. phenotype in case no mutations are identified in SMN1. Molecular diagnosis

Diagnosis in the proband: In more than 98% 1 2 3 4 5 6 of individuals• with SMA, homozygous mutation in SMN1 can be identified (Hahnen et al.,1995). The most common type of mutation is homozygous deletion of SMN1 (the deletions vary in size but almost always includes exon 7) accounting for 163bp 95%-98% of the cases and rearrangement or point 42bp mutation in the rest (2-5%). The sensitivity of the test for homozygous deletion is 95% and specificity 205bp is almost 100% (Lefebvre et al.,1998). This test was previously done by a polymerase Lane 1: Negative control (Without DNA) chain reaction to amplify the exon 7 followed by Lane 2: Positive control differentiation of SMN1 and SMN2 by the suscep- Lane 3: Patient tibility to digestion by a restriction endonuclease Lane 4: NC (Dra I) due to the single nucleotide difference (PCR- Lane 5: Undigested product RFLP) (Figure 3). Homozygous deletion of exon 7 of SMN1 confirms the diagnosis of SMA. However Lane 6: 100bp ladder this method does not detect heterozygous deletion Patient is positive for SMA in SMN1 and cannot be used for identification of healthy carriers. Now the gold standard of diagnosis for SMA is Multiplex Ligation Probe Amplification(MLPA), Figure 3 Molecular testing for detection of ho- which is based on a kit containing several probes mozygous deletion of exon 7 using for SMA critical region, including probes for SMN1 PCR-RFLP. The exon 7 is amplified using and SMN2 and other genes like NAIP, GTF2H2, primers that create a restriction site N-cadherin-like, CDH6 and RAD17 genes in the after amplification of exon 8, but not SMA critical region or other autosomes. This test exon 7 due to the nucleotide differ- provides information on SMN1 and SMN2 copy ence. Hence the affected individual (P) numbers, aiding in diagnosis of healthy carriers as will have amplification of only exon 8, well as aides in genotype phenotype correlation which will be cleaved into 149bp and (Figure 4). 39bp fragments. Normal individuals (N) will have a PCR product of 188bp Interpretation of the test: Homozygous from exon 7 which is not cleaved and deletion• of exon 7 confirms the diagnosis of two cleaved fragment from exon 8. UD SMA. In the absence of homozygous/ heterozygous indicates undigested product of PCR. deletion, other diagnoses need to be entertained

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Figure 4 MLPA results of affected patient and a healthy carrier of SMN 1 deletion.

Carrier detection: Carrier frequency of SMA can almost lead to normal life with no significant is estimated• to be 1 in 40 to 60. The PCR-RFLP disability in carrying out the routine activities. technique used for diagnosis of patients with SMA As there is SMN2 gene in all patients with cannot be used for carrier detection. Homology deletion of SMN1, interest is on enhancing the between SMN1 and SMN2 complicates this tech- expression of SMN2 in tissues in patients with nique as the PCR for SMN1 frequently amplifies SMA. Several approaches are being studied to- SMN2 as well. Hence the current strategy is to use wards this goal. These involve inclusion of exon MLPA to quantify the SMN1 and SMN2 copies and 7 in transcripts of SMN2, enhancing the promoter this can detect the carrier status for heterozygous activity of SMN2, modulation of protein transla- deletion. tion and prevention of degradation of SMN2 gene product. Various drugs are under different stages Prenatal diagnosis of clinical trial (Lunn et al.,2008) Molecules like albuterol, phenylbutyrate and valproic acid have Prenatal diagnosis in carrier couple is done by shown some improvement either at the cellular chorionic villi sampling at 10-12 weeks of gesta- level or of muscle strength, but their clinical use tion. The technique used for the diagnosis in the is still far from reality. Phase III clinical trial using proband is applied here as well. It is essential Antisense oligonucleotide therapy aiming to alter to exclude the contamination of fetal sample by SMN2 splicing to increase the transcripts with exon maternal cells during sampling. 7 is on going and the results have not yet been published. Gene therapy with viral vector aided Treatment and prognosis delivery of SMN1 is also being investigated.

Currently the treatment is only supportive. Phys- Genetic counseling scenarios iotherapy and respiratory therapy improves the outcome, especially in milder types of SMA. Older Child with SMA type 1 or 2: When a child is individuals with type 3 and 4 disease have slower suspected• to have SMA type 1 or 2, the prognosis progression and require psychological support in for good neuromuscular function and survival is addition. At one end of the spectrum of the poor. All cases need to have molecular testing. This disease, the children with type 1 have relent- requires about 2ml EDTA anti-coagulated blood. less progression leading to death usually by their The sample can be sent to the laboratory at room second birthday; those with minimal weakness temperature. If the child has a critical illness, it is

138 Chapter 21 essential to preserve the sample. The sample can homozygous deletion of exon 7, can have normal be collected even postmortem and kept in refrig- results for SMN1 carrier testing. This could be erator till it is shipped to the laboratory. When the because in 4% of carriers, two copies of SMN1 are diagnosis is confirmed by molecular testing (usu- seen on a single chromosome, which is misdiag- ally homozygous deletion of SMN1), it is implied nosed, as non-carriers (a false negative test). In 2% that both the parents are carriers of the deletion. of individuals with SMA, de novo deletion of exon 7 As the condition is inherited in autosomal recessive of one SMN1 allele can occur. mode, the risk of recurrence in subsequent siblings Population screening for carrier status: The is 25%. The parents are offered prenatal testing of American College of Medical Genetics has rec- chorionic villi sampling at 10-12 weeks of gestation • ommended carrier testing of all couples in all in all subsequent pregnancies. populations in view of the high frequency of the Older children and adolescents with SMA 3 or carrier state (Prior et al., 2008). Such testing 4: •The disease is less severe here. The phenotype should be voluntary and be done only with in- varies widely in SMA3 and SMA4. Hence, predicting formed consent with adequate support for genetic the course of the disease is difficult. They are counseling. advised to have good physiotherapy and occupa- tional therapy to make the best use of existing muscle power. The confirmation of diagnosis by References molecular testing can be done by MLPA. 1. Buchthal F, Olsen PZ. Electromyography and Adults with SMA: Adults with SMA may want muscle biopsy in infantile spinal muscular atro- to• know if the condition would recur in their phy. Brain 1970; 93:15-30. offspring. Here the risk of recurrence ofthe 2. Lefebvre S, et al. Identification and characteriza- condition in the child of an adult with SMA is 1 in 25 tion of a spinal muscular atrophy-determining (taking the carrier frequency to be 1 in 50). Again gene. Cell 1995; 80: 155-165. carrier testing of the spouse will be of great help 3. Hahnen E, et al. Molecular analysis of candi- in determining the need for providing prenatal date genes on chromosome 5q13 in autosomal diagnosis. recessive spinal muscular atrophy: evidence History of previous infant death due to SMA, of homozygous deletions of the SMN gene in no molecular• tests done: Carrier testing by MLPA unaffected individuals. Hum Mol Genet 1995; 4: should be offered to both the partners. This 1927-1933. helps in offering the recurrence risk to the couple 4. Lefebvre S, et al. The role of the SMN gene and to consider prenatal testing in subsequent in proximal spinal muscular atrophy.Hum Mol pregnancies. It is essential to emphasize to the Genet 1998; 7: 1531-1536. couple that many congenital myopathies and mus- 5. Lunn MR, Wang CH. Spinal muscular atrophy. cular dystrophies may mimic SMA1 and present Lancet 2008; 371: 2120-2133. as hypotonic infant. However, if fasciculations are 6. Prior TW. Carrier screening for spinal muscular documented in the earlier floppy infant or in the atrophy. Genet Med 2008; 10: 840-842. electromyogram, the diagnosis of SMA1 is more 7. Prior TW. Spinal muscular atrophy diagnostics. J likely. About 6% of parents with a child with Child Neurol. 2007; 22: 952-956.

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139 Chapter 22

Duchenne Muscular Dystrophy

Udhaya H Kotecha Zydus Hospital, Ahmedabad; Apollo Hospital, Ahmedabad; Medgenome Laboratories, Bangalore

Correspondence to: Dr Udhaya H Kotecha Email: [email protected]

Abstract ognizing this will prevent delays in the diagnosis. b) Discussing the available diagnostic methodolo- With an incidence of 1 in 3500 affected males, gies and importantly their differences, limita- Duchenne Muscular Dystrophy (DMD) is the most tions and utility. common muscular dystrophy and is inherited in an c) Providing a brief outline on the management X-linked recessive pattern. Though this disorder –which will enable uniform care to all thus has been known for two centuries, delays in its helping improve quality of life. diagnosis and non-uniform practice of care exist d) Outlining the genetic basis, recurrence risks and even to date. Decades of research has led to bet- prenatal diagnosis. ter understanding of the pathophysiology of DMD e) Providing an overview of the current novel which in turn has resulted in newer therapeutic approaches for treatment. advances. While these treatment strategies are yet to reach the clinic, they surely have placed an added responsibility on the treating physician to Clinical Phenotypes ensure early diagnosis and appropriate manage- A boy less than 5 years of age presenting with ment so that maximum benefit can be ensured proximal muscle weakness and bilateral calf hy- when these therapies are available. This review pertrophy and ‘who is one amongst many affected aims at bridging diagnostic gaps by describing males’ on the maternal side embodies the text- various signs and symptoms of DMD followed by book description of DMD. This typical scenario is the currently utilized diagnostic approaches. Also encountered only in 70% of the cases (Leet et discussed is the staged use of glucocorticoids, the al., 2014). A high index of clinical suspicion is need of multidisciplinary management and the necessary to diagnose the other atypical though current arenas which are being explored for the not uncommon presentations which include: cure of this devastating disease. a) Sporadic cases of DMD that account for one Introduction third of the patients affected with DMD and are usually recognized after onset of clinical Duchenne muscular dystrophy is recognized as symptoms (Mukherjee et al., 2003). the most common muscular dystrophy with an b) Asymptomatic elevation of CPK that could be incidence of 1 in 3500-5000 males (CDC, 2007). i) noted when investigations are performed for Though still incurable, light seems to be emerging an unrelated reason ii) when an asymptomatic at the end of this long dark tunnel with many child is investigated due to a positive family new therapies in the making (Malik et al., 2012). history or iii) as a part of newborn screening While these treatment strategies are yet to reach (Moat et al., 2013; Mendell & Lloyd-Puryear, the clinic, they have placed an added responsibility 2013). on the treating physician to ensure early diagnosis c) Unexplained elevation of serum transaminases and appropriate management so that maximum – consideration of muscular dystrophy is im- benefit can be ensured when these therapies are portant as this could prevent unnecessary eval- available. Delayed diagnosis and inconsistent care uation for liver dysfunction (McMillan et al., in DMD has been reported and this review at- 2011). tempts to bridge these gaps by (Ciafaloni et al., d) Impaired cognition or seizures in patients with 2009): symptoms suggestive of DMD- a particular be- a) Describing various clinical presentations – rec- havior spectrum abnormality ranging from low

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IQ to an autism spectrum disorder and attention Investigations and diagnosis deficit can occur in 20% of Duchenne patients. Epilepsy is observed in 10% of cases (Perumal et 1) Creatine kinase levels (CK): The first test to be al., 2013). performed on clinical suspicion of DMD is a e) Manifesting carrier females. While females serum CK level. An elevated serum CK (up to are typically unaffected, mild proximal muscle 100-200 times normal) is commonly observed in weakness has been reported in 8-10% of cases patients with DMD. Elevation is maximum in the (Soltanzadeh et al., 2010). initial stages of DMD followed by a decline later due to fibrous replacement of muscle. Also an Inclusive of the above scenarios, the term dys- increased CK is only present in approximately trophinopathy also includes the allelic milder 30% of carrier females thus making it an un- disorder Becker Muscular Dystrophy as well as suitable test for carrier detection (Zatz & Otto, dystrophin-related cardiomyopathy (Muntoni et 1980). al., 2003). 2) Molecular diagnosis: Molecular testing for confir- mation is often provided as a two or three tier When to suspect the disorder? testing. Proximal muscle weakness brings the disorder to a) Detection of deletions and duplications: parental notice. This is commonly evident as Deletions in the dystrophin gene are re- Difficulty in getting up from sitting or squat- sponsible for 65-70% of the cases while duplications are seen in 5-10%. Evaluation • ting position (patient gets up by supporting himself on his legs and thighs, a maneuver for these is possible via Multiplex-probe- named as Gower’s sign) dependent-amplification (MLPA) (Verma et Difficulty in climbing stairs al., 2012; Sansovic et al., 2013). Before the MLPA technique was developed and became • Development of a waddling gait widely available, most laboratories used to • An inability to jump or hop or do multiplex PCR assay. Even now, many • Tripping while running labs use multiplex PCR for testing for exonic • Other observations include a delay in walking, deletions. However, multiplex PCR can be • tip-toe walking, presence of muscle stiffness or cramps or prominent calf muscles. used for studying deletions in only the ‘hot spot’ regions (within exons 1-20 and 45-50) Disease course and cannot detect duplications. In addition, unlike MLPA, multiplex PCR cannot detect After its onset at 3-5 years of age, the disease carrier status. follows a relentlessly progressive course culminat- b) DMD Gene sequencing: As elucidated ing in wheelchair dependency by the age of 13 above, MLPA results would be normal in years. While the skeletal muscles bear the major 30% of affected individuals, thus necessitat- brunt of the disorder, cardiac, respiratory and ing further gene sequencing for detecting smooth muscles are also affected albeit at a later point mutations and small genomic rear- stage (Yiu & Kornberg, 2008). Death occurs in rangements (Laing et al., 2011). Whole the early teens due to respiratory failure which is gene sequencing has become less expensive accentuated by development of scoliosis (Roberto and faster with the availability of the Next et al., 1976). The inclusion of glucocorticoids along generation sequencing technology. with multidisciplinary care has gone a long way in changing this natural history and prolonging 3) Muscle biopsy: With current advances in ge- the life expectancy. Along with increments in life netic testing the need for muscle biopsy is span, available evidence concludes that usage of decreasing (Na et al., 2013). It has been an steroids leads to better quality of life due to delay important practice parameter till recently and is in wheelchair dependency, scoliosis and hyper- still availed to in atypical situations and hence is trophic cardiomyopathy and better preservation of briefly discussed here. It is important that the pulmonary function (Moxley et al., 2010). biopsy be performed at centers where facilities Becker muscular dystrophy is a milder allelic are available for immunohistochemistry (IHC) or version of DMD with a slower rate of progression, immunoblotting (IB). If not, then a part of the preserved ambulation till the second decade and muscle biopsy sample should be preserved in survival into the fourth decade (Emery, 2005). liquid nitrogen and shipped on dry ice. While

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routine histopathology will demonstrate dystro- which in some cases is very short lasting. With phy, it is only through IHC/IB that the type of loss of ambulation the dose of steroids needs to dystrophy can be ascertained. Thus IHC/ IB are be scaled down. If however, a patient presents central to the diagnosis and need to be per- for the first time in the non ambulatory stage, itis formed on every patient undergoing biopsy (Wei still worthwhile considering glucocorticoids so as to et al., 2014). It is essential to remember that reduce the need of scoliosis surgery and preserv- biopsy results are not diagnostic and should be ing pulmonary function. An updated immunization followed by molecular diagnosis. schedule with special emphasis on varicella vaccine is essential before starting steroids. Principles for The need for molecular diagnosis is often investigating adverse events are similar to other questioned. The utility is multifold including i) diseases where long term steroids are utilized definitive management - allows confident discus- (Bushby et al., 2010). sion for use of corticosteroids in the treatment ii) carrier detection iii) prenatal testing iv) genotype- Multidisciplinary care: Though disease pro- phenotype correlation v) application of newer gression• is curtailed with steroids, it cannot be treatment strategies like exon skipping and non- avoided and a careful modus operandi to discern sense mutation read-through. involvement of other organ systems should be in place. The cardiac and respiratory muscles Management are the two major systems whose involvement is There are two intertwined branches of dys- fatal and screening via ECG, echocardiography and trophinopathy management: Pharmacological pulmonary function tests should begin after 6-8 management and Multidisciplinary care. years of age. Similarly wheel chair dependency ac- celerates development of scoliosis which increases Pharmacological management: Corticos- pulmonary burden and careful management for teroids are currently the only class of drugs • the same is important. A close association with with proven clinical benefit. Both prednisolone/ experts in above fields will allow for comprehensive prednisone and deflazacort have been used with patient care. In addition to physical limitations, the equal success, the former being used in nations disorder has a major psychosocial impact on the where deflazacort is not available. While the ad- patient as well as the entire family – constant en- verse effect profiles are essentially similar, weight couragement, discussing options to keep the child gain is more common with prednisolone /pred- as independent as possible (includes rearrange- nisone while cataracts occur more frequently with ments at home, wheelchair options, splints and deflazacort. Both medications have been tested orthosis, home tutoring, use of electronic devices) at different doses and the recommended initiating and interaction with other parents faced with the dose for ambulatory patients is 0.75mg/kg for similar challenge play a major role in coping with prednisone and 0.9mg/kg for deflazacort (Bushby the disorder (Grimm et al., 2012). et al., 2010). Nearly 29 different therapeutic regimens are in use and include daily, alternate Genetic Counseling day, 10 day on -10 day off and high dose weekend regimens (Griggs et al., 2013). While decline of The entire dystrophinopathy spectrum is an X muscle function is said to occur earlier in the non linked recessive condition. This translates into daily regimens, the adverse effect profile is lower males being preferentially affected, with females in the same (Ricotti et al., 2013). The pros and being carriers in a majority. In 1/3 cases there is cons of each regimen should be weighed on an no family history and the mother tests negative for individual basis. the mutation detected in her affected son-implying The next pertinent issue is when to start that either it is due to a de novo mutation or pharmacological management – to make decision germline mosaicism. The risk of recurrence varies making easier, motor performance has been di- according to the maternal carrier status. In obligate vided into three stages: making progress, plateau cases the risk of recurrence is 50% if the fetus is and decline. The commonest practice is to wait for male. In sporadic cases even in the absence of a the ‘plateau phase’ usually around 4-6 years of age detectable genetic mutation in the mother, there when the child has stopped making any progress exists a 8-20% recurrence risk due to germline mo- but not yet started to fall downhill. Parental saicism (Heldermann-van den Enden et al., 2009; observations, routine follow up visits and timed Murugan et al., 2013). Daughters have a 50% risk performance tests help recognition of this stage of being a carrier and the most sensitive method of

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detection is investigation for the mutation present management, detect carrier females and provide in her family (Esposito et al., 2013). prenatal diagnosis. Ensuring uniform care with Prenatal diagnosis is possible in families where optimum usage of steroids and multidisciplinary mutations are known through chorionic villous care will contribute to improving the quality of life sampling from 10 weeks onwards. In cases where in affected patients. genetic testing in the proband is pending, one can resort to linkage analysis provided the pedigree is References informative and at least two affected members are available, Linkage involves detection of the high risk 1. Bogdanovich S, et al. Functional improvement chromosome using Short Tandem Repeat markers of dystorphic muscle by myostatin blockade. and carries with itself a 5% risk of recombination Nature 2002; 420(6914): 418-421. which may lead to diagnostic errors (Esposito et al., 2. Bushby K, et al. Diagnosis and management 2013; Maheshwari et al., 2000). of Duchenne muscular dystrophy, part 1: di- agnosis, pharmacological and psychological Newer drugs and emerging therapies management. Lancet Neurol 2010; 9: 77-93. Research targeting various aspects of Duchenne 3. Bushby K, et al. Diagnosis and management muscular dystrophy is being explored and many of Duchenne muscular dystrophy, part 2: im- compounds are currently in phase I or II clinical plementation of multidisciplinary care. Lancet trials (Pichavant et al., 2011). Current therapies in Neurol 2010; 9: 177-189. the pipeline include: 4. Center for Disease Control and Prevention. Prevalence of Duchenne/Becker muscular dys- a) Inducing dystrophin expression – either through trophy among males aged 5-24 years-four exon skipping using anti sense oligonucleotides, states, 2007. MMWR Morb Mortal Wkly Rep read through of stop codons, introducing a 2009; 58: 1119-1122. functional dystrophin molecule employing viral 5. Ciafaloni E, et al. Delayed diagnosis in vectors or by upregulating utrophin expression duchenne muscular dystrophy: data from (Hoffmann et al., 2011). Recently, exon 51 the Muscular Dystrophy Surveillance, Tracking skipping therapy (eteplirsen; EXONDYS 51) has and Research Network (MD STARnet). J Pediatr been approved for clinical use by the US Food 2009; 155: 380-385. and Drug Administration (FDA). 6. Deconinck N, Dan B. Pathophysiology of b) Muscle regeneration and replacement- my- Duchenne muscular dystrophy: current hy- oblast transfer, mesangioblast transfer and potheses. Pediatr Neurol 2007; 36: 1-7. stem cell transfer are all probable modalities 7. Emery H. Duchenne and Other X-linked mus- to achieve this. Besides this, pharmacologi- cular dystrophies. In: Emery and Rimoin’s cal agents which can help muscle regeneration Principles and Practice of Medical Genetics are also being explored and include myostatin Fifth Edition. Elsevier 2005; pp 2911-2928. inhibitors and IGF-1 (Mouly et al., 2005). 8. Esposito G, et al. Prenatal molecular diag- c) Modulation of signaling pathways- Modification nosis of inherited neuromuscular diseases: of nitric oxide signaling pathways to augment Duchenne/ Becker muscular dystrophy, my- its effect has shown to be beneficial in improv- otonic dystrophy type 1 and spinal muscular ing cardiac and skeletal performance in mouse dystrophy. Clin Chem Lab Med 2013; 51: models (Guerron et al., 2010). 2239-2245. d) Inhibiting fibrosis- Fibrosed muscle signifies ir- 9. Griggs RC, et al. Corticosteroids in Duchenne reversible destruction and prevents transfer of muscular dystrophy: major variations in prac- effective therapy- hence inhibiting the same tice. Muscle Nerve 2013; 48: 27-31. is an attractive and advantageous idea and is 10. Grimm T, et al. Risk assessment and genetic plausible through myostatin and transforming counseling in families with Duchenne muscular growth factor inhibition (Bogdanovich et al., dystrophy. Acta Myol 2012; 31: 179-183. 2002). 11. Guerron AD, et al. Functional and molecular effects of arginine butyrate and prednisone on Conclusion muscle and heart in the mdx mouse model Utilizing the above outlined diagnostic method- of Duchenne Muscular Dystrophy. PLoS One ologies it is possible to detect cases of DMD 2010; 5(6):e11220. earlier, thus making it possible to institute early 12. Heldermann-van den Enden AT, et al. Re-

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currence risk due to germline mosaicism: 27. Na SJ, et al. Clinical, immunohistochemical, Duchenne andd Becker muscular dystrophy. Western blot and genetic analysis in dys- Clin Genet 2009; 75: 465-472. trophinopathy. J Clin Neurosci 2013; 20: 13. Hoffmann EP, et al. Restoring dystrophin ex- 1099-1105. pression in Duchenne muscular dystrophy 28. Pandey GS, et al. Re-evaluation of reading progress exon skipping and stop codon read frame –shift hypothesis in Duchenne and through. Am J Pathol 2011; 179: 12-22. Becker Muscular dystrophy. Neurol India 2003; 14. Laing NG, et al. Molecular diagnosis of 51: 367-369. duchenne muscular dystrophy: past, present 29. Perumal AR, et al. Neuropsychological Pro- and future in relation to implementing thera- file of Duchenne Muscular Dystrophy. Appl pies. Clin Biochem Rev 2011; 32: 129-134. Neuropsychol Child 2013 Nov 26 (epub). 15. Leet, et al. Differences in carrier frequency 30. Pichavant C, et al. Current status of pharma- between mothers of Duchenne and Becker ceutical and genetic therapeutic approaches to muscular dystrophy patients. J Hum Genet treat DMD. Mol Ther 2011;19: 830-840.Roberto 2014; 59: 46-50. R, et al. The natural history of cardiac and 16. Magri F, et al. Genotype and phenotype char- pulmonary function decline in patients with acterization in a large dystrophinopathic co- Duchenne muscular dystrophy. Spine 1976; 36 hort with extended follow up. J Neurol 2011; (5): E1009-1017. 258:1610-1623. 31. Ricotti V, et al. Long term benefits and adverse 17. Maheshwari M, et al. Prenatal diagnosis of effects of intermittent versus daily glucocor- Duchenne muscular dystrophy. Natl Med J ticoids in boys with Duchenne muscular dys- India 2000; 13:129-131. trophy. Neurol Neurosurg Psychiatry 2013; 84: 18. Malik V, et al. Emerging Drugs for Duchenne 698-705. muscular dystrophy. Expert Opin Emerg Drugs 32. Sansovic I, et al. Improved detection of dele- 2012;17: 261-277. tions and duplications in the DMD gene using 19. McMillan HJ, et al. Serum transaminase levels the multiplex ligation-dependant probe ampli- in boys with Duchenne and Becker muscular fication (MLPA) method. Biochem Genet 2013; dystrophy. Pediatrics 2011; 127: e132-6. 51: 189-201. 20. Mendell JR, Lloyd-Puryear M. Report of 33. Sansovic I, et al. Improved detection of dele- MDA muscle disease symposium on newborn tions and duplications in the DMD gene using screening for Duchenne muscular dystrophy. the multiplex ligation-dependant probe ampli- Muscle Nerve 2013; 48:21-26. fication (MLPA) method. Biochem Genet 2013; 21. Moat SJ, et al. Newborn bloodspot screening 51: 189-201. for Duchenne muscular dystrophy:21 years ex- 34. Soltanzadeh P, et al. Clinical and genetic char- perience in Wales (UK). Eur J Hum Genet 2013; acterization of manifesting carriers of DMD 21: 1049-1053. mutations. Neuromuscul Disord 2010; 20: 22. Mouly V, et al. Myoblast transfer therapy: is 499-504. there any light at the end of the tunnel? Acta 35. Verma PK, et al. Utility of MLPA in mutation Myol 2005; 24:128-133. analysis and carrier detection in Duchenne 23. Moxley R, et al. Change in natural history muscular dystrophy. Indian J Hum Genet of Duchenne Muscular Dystrophy with long 2012;18: 91-4. term corticosteroid treatment: Implications 36. Wei X, et al. Targeted next-generation sequenc- for management. J Child Neurol 2010; 25(9): ing as a comprehensive test for patients with 1116-1129. and female carriers of DMD/BMD: a multi- 24. Mukherjee M, et al. De novo mutations in spo- population diagnostic study. Eur J Hum Genet radic deletional Duchenne muscular dystrophy 2014; 22:110-118. (DMD) cases. Exp Mol Med 2003; 35:113-117. 37. Yiu EM, Kornberg AJ. Duchenne Muscular Dys- 25. Muntoni F, et al. Dystrophin and mutations: trophy. Neurol India 2008; 56: 236-247. one gene, several proteins, multiple pheno- 38. Zatz M, Otto PA. The use of concomitant serum types. Lancet Neuro 2003; 2: 731-740. pyruvate kinase and creatine phosphokinase 26. Murugan SM, et al. Carrier detection in for carrier detection in Duchenne’s muscu- Duchenne muscular dystrophy using molec- lar dystrophy through discriminant analysis. J ular methods. Indian J Med Res 2013; Neurol Sci 1980; 47: 411-417. 137:1102-1110.

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Fragile-X Revisited - Novel Testing, Screening and Treatment Strategies

Priyanka Srivastava1, Shagun Aggarwal2 1Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 2Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad

Correspondence to: Dr Priyanka Srivastava Email: [email protected]

Fragile X syndrome (FXS) (MIM #30955) is among in pre-pubertal children (Figure 1). Mental retar- the common human single gene disorders, and is dation varies from mild to profound retardation the leading cause of inherited cognitive disability with males being more severely affected. Female and also the second most common cause of ge- carriers of full mutations may have long face and netically associated intellectual disability following variable level of cognitive dysfunction (Figure 2). trisomy 21. The name refers to a cytogenetic Male carriers of premutation may suffer from: FX- marker on X chromosome at Xq27.3, a “fragile site” TAS (Fragile X-associated tremor/ataxia syndrome) in which the chromatin fails to condense prop- which is a condition affecting balance, tremor and erly during mitosis. FXS is caused by a dynamic memory in males above the age of 50 years. Ad- mutation which involves an unstable expansion of ditionally, female premutation carriers may suffer a trinucleotide CGG repeat at the 5′-untranslated from FXPOI (Fragile X-associated primary ovarian region (UTR) of the fragile X mental retardation insufficiency) which can lead to infertility and early 1 (FMR1) gene, located at Xq27.3. The silencing menopause. of the FMR1 gene causes deficiency of the fragile X mental retardation protein (FMRP), leading to the classical FXS phenotype. It is inherited as an X-linked dominant disorder with reduced pen- etrance. Important factors that influence disease expression are gender (completely penetrant in males and 50% penetrance in females), the num- ber of CGG repeats and degree of methylation. The complex pattern of inheritance poses an extraordi- nary challenge for accurate diagnosis and genetic counseling of affected families.

Clinical Phenotype Figure 1 Two brothers with Fragile X syndrome. Note conspicuous absence of any facial The classical Fragile X syndrome is characterized dysmorphism. (These pictures were by intellectual disability, speech and communica- provided by Dr Shubha Phadke, Depart- tion problems. Behavioral challenges include poor ment of Medical Genetics, SGPGIMS, concentration, autistic-like behaviors such as an Lucknow). unusual fixed interest in some things and dislike for change in routine. People with Fragile X Genetics of Fragile X syndrome syndrome have a stronger likelihood of suffering from anxiety and hyperactivity. Physical features The name of the syndrome comes from its location show marked heterogeneity. Characteristic fea- on the X chromosome. The gene for fragile X tures such as long face with prominent mandible, syndrome (the FMR1 gene) was identified in 1991. large ears and macro orchidism are seldom noticed Under particular laboratory conditions (use of folic

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Table 1 Table showing the CGG repeat size and associated phenotype.

Category CGG repeats Methylation of FMR1 Female Male Stable 6 to 45 Unmethylated Not affected Not affected Gray zone 45∼ to 55 Unmethylated Not affected Not affected Pre-mutation ∼55 to ∼200 Unmethylated Usually not affected/ Usually not ∼ ∼ FXPOI/ FXTAS affected/ FXTAS Full mutation 200 Completely 50% affected All affected > methylated ∼ Full mutation Different Methylated in cells Variable presentation All affected but with mosaicism cells with full mutation, higher for repeat size showing unmethylated in cells functioning different with premutation/ number of normal number of repeats repeats Full mutation 200 Variable methylation Variable presentation All affected but with mosaicism > in different cells higher for methylation functioning Full mutation 200 No methylation Variable presentation All affected but unmethylated > mild MR

FXPOI: Fragile X-associated primary ovarian insufficiency FXTAS: Fragile X-associated tremor/ ataxia syndrome acid/ thymidine deficient cell-culture medium), the terminal end of the long arm of the X chromosome with the mutated allele can appear broken or fragile. The majority ( 99%) of mutations in the FMR1 gene affect the> length of a CGG repeat at the promoter region of the gene. On the basis of the size of the CGG repeat and its effect on gene expression, two classes of mutations have been identified in the FMR1 gene: ‘premutations’ and ‘full mutations’. In normal unaffected individuals, the number of repeats ranges from 6 to 55 copies. In clinically unaffected (carriers) individuals, alleles in Figure 2 A pedigree with Fragile X syndrome the so called premutation range may have between showing X linked inheritance. Note 55 and 200 copies of the repeat (Figure 3). In variable intellectual abilities in sisters individuals with full mutation, the repeat number with full mutation. ranges from 200 and above. In affected individuals with a full mutation, the Testing for Fragile X syndrome expanded repeats in the 5’ untranslated region of the FMR1 gene and the 5’ region surrounding the In view of great variability in the presentation, clin- promoter of the FMR1 become de novo methylated ical diagnosis is extremely difficult. Hence testing rendering it transcriptionally inactive. This leads to for fragile X syndrome is indicated in all individu- deficiency of the gene product FMRP. FMRP (FMR als with intellectual disability without an obvious protein) binds to specific mRNAs and has an im- cause. Numerous diagnostic methods have been portant role in the regulation of protein synthesis developed for fragile X syndrome, including cyto- at neuronal synapses. Its absence leads to dis- genetic, Southern blot, polymerase chain reaction turbances in synaptic function and dendritic spine (PCR), methylation specific PCR (ms-PCR), and im- morphology, contributing to the clinical phenotype. munohistochemical analyses.

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Figure 3 a) Mutation, Premutation and normal allele size in Fragile X gene. b) Diagrammatic rep- resentation of Southern blot for Fragile X analysis. Note: NF-Normal female, NM-Normal male, PCF-Premutation carrier female, FMM-Full mutation in male, FCFM-Female carrier of full mutation.

1. Polymerase chain reaction (PCR) specific with expanded repeats (Figure 4). This technique for the CGG trinucleotide repeat region of FMR1 can distinguish between normal, intermediate size, has high sensitivity for FMR1 repeats in the normal premutation & full mutation alleles. However, the and lower premutation range (typically 100 to exact size of the repeats cannot be ascertained, 120 repeats; varies by testing laboratory).≤ How- which poses difficulty in prognostication during ever, traditional FMR1-specific PCR is less sensitive counseling. It is a rapid way which has been to larger premutations and fails to amplify full advocated to be useful for routine diagnostic use, mutations. carrier screening as well as newborn screening. 2. Southern blot analysis detects all FMR1 al- leles including normal, larger-sized premutations, Treatment Strategies full mutations and in addition determines methyla- tion status of the FMR1 promoter region. Abnormal There is currently no cure for Fragile X syndrome, hypermethylation of FMR1 is the cause of tran- but once it is diagnosed there are a number scriptional silencing and is critical to assess for of management strategies that can help many premutation and full mutation alleles. Traditional children and adults: PCR plus Southern blot analysis has been the “gold standard” for FMR1 molecular diagnosis. Supportive care: A combination of educa- 3. Detection by Antibody: More recently, an tional,• medical and behavioral management tech- FMRP antibody test has been developed to mea- niques have been demonstrated to improve the sure expression in lymphocytes. This can detect outcomes for affected individuals and their fami- the full mutation in males. It is not useful in mosaic lies. The child’s educational program should in- males or females as some FMRP is still formed. clude occupational, physical and speech therapies, 4. Methylation status can be assessed by introduced as early as possible. These therapies PCR-based methods independent of measuring can be very effective when designed to meet the the number of CGG repeats. needs of an individual in an early intervention and school program. A multidisciplinary team, made 5. Sequence analysis: Very few individuals up of parents, doctors, pediatricians, educational ( 1%) with fragile X syndrome have been identified specialists, psychologists, speech therapists, ge- with an intragenic FMR1 mutation. ∼ neticists, occupational therapists and nurses are 6. TP-PCR: Triplet Repeat Primed PCR (TP-PCR) required for appropriate ongoing management for is a novel technique for rapid detection of Fragile these patients. X mutations, premutaions as well as methylation status. This technique uses a set of three primers Symptomatic Treatment: Specific Manifes- to amplify the FMR1 promoter with CGG repeats. tations• like ADHD, aggressive behavior, anxiety, Capillary electrophoresis of PCR product shows autistic symptoms, mood disorders and seizures presence of peaks beyond a threshold in cases can be managed using medications like CNS stim-

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Figure 4 TP-PCR chromatogram of a male with full mutation showing characteristic stuttered peaks beyond 200 base pair threshold. ulants, antipsychotics, SSRIs (Selective serotonin allosteric modulators currently in clinical trials in reuptake inhibitors), anticonvulsants, etc. as per FXS (see http://www.clinicaltrials.gov) in- the symptoms. Use of medications for behavior clude STX107 (Seaside Therapeutics, phase I trial should be judicious. Individuals with FXS have in- initiated in the United States), AFQ056 (Novartis, creased risk for seizures, with rates of 13% to 18% phase II trial recently completed in France, Italy, and for boys and 5% for girls. Seizures in FXS are easily Switzerland), and RO4917523 (Hoffman-LaRoche, controlled with a single anticonvulsant. Treatment phase II trial initiated in the United States). Phase involves use of a range of anticonvulsant medi- II trial of AFQ056 confirmed its safety but did cations; however, because FXS is associated with not show any significant benefit on the Aberrant hypotonia, loose connective tissue, and cognitive Behavior Checklist. and behavioral problems, these issues should be 2. AMPA receptor positive modulators (am- taken into account while the choice of medication pakine): CX516 in a 4 week trial in adults with is being considered. FXS did not show any associated improvement in memory, language, or attention/executive function Novel Targeted Treatments (Berry et al, 2006). Similarly, trial of riluzole in 6 adults with FXS, did not show clinical benefit. 1. mGluR5 antagonists: FMRP is an RNA-binding 3. GABAB receptor agonists: Lithium targets protein that modulates dendritic maturation and multiple intracellular signaling pathways, which synaptic plasticity through a mechanism involving have been linked to Gp1 mGluR signaling. A particularly the inhibition of group 1 mGluR me- pilot trial of Lithium treatment on 15 patients diated dendritic protein synthesis. It has been with FXS was found to have positive effects on suggested that mGluR5 antagonists would be an behavioral adaptive skills and cognitive measures. effective treatment for FXS. Early animal trials on GABA-B receptor agonists such as baclofen inhibit mice models of Fragile X showed promising re- glutamate release, and subsequent postsynaptic sults following which human trials using various mGluR5 activation. Baclofen has been shown to compounds with mGluR5 antagonistic actions are reduce audiogenic seizures in Fmr1 KO mice. A underway. Recently, a phase II clinical trial was phase II study was conducted using arbaclofen completed, in which 12 adult patients with FXS (also known as STX209), the R-isomer of baclofen. received a single dose of fenobam to assess drug This study has been completed and its results are safety, pharmacokinetics, and a small number awaited. of cognitive and behavioral effects. In this trial, 4. Minocycline: A tetracycline analog, which fenobam was reported to reduce behavioral abnor- has shown beneficial effects on dendritic spine malities and improve cognitive performance. In a morphology and phenotypic benefits in mice mod- separate study, three young adult patients with FXS els. It is believed to act by inhibiting matrix were treated with acamprosate, a drug with mGluR metalloproteinase-9 (MMP-9). A Phase II clinical antagonist properties that is approved for mainte- trial did not show any statistical benefit in treated nance of abstinence from alcohol. In all three pa- individuals. tients, acamprosate was associated with improved 5. Enhancement of cholinergic function in linguistic communication and global clinical benefit. the brain through administration of the acetyl- Other highly potent and selective mGluR5 negative cholinesterase inhibitor donezepil was associated

148 Chapter 23 with improvement on measures of cognition and preventing birth of another affected child. Also behavior in at least two Phase II trials. premutation carriers can be afflicted with various phenotypes, and counseling and intervention for Genetic Counseling these can be provided. In addition, the health care provider should consider extended family DNA Genetic counseling for fragile X syndrome is com- testing, with the help of a genetic counselor or ge- plex and it is advisable to consult a geneticist to neticist, and follow-up care with a multidisciplinary ensure correct interpretation of all positive fragile treatment team. Some recent studies have also X test results and the provision of appropriate advocated population screening of reproductive advice to the family. Mothers of all individuals age women for carrier status of Fragile X premu- with an FMR1 full mutation are carriers of FMR1 tation/full mutation. This was based on reports premutation. Mothers and their female relatives on premutation carrier rates ranging from 1 in who are premutation carriers are at increased risk 100 to 1 in 250 in various populations. Offering for FXTAS and POI; those with a full mutation carrier screening to women, preconceptionally or may have findings of fragile X syndrome. All early during pregnancy would help in primary pre- carriers are at increased risk of having offspring vention of Fragile X syndrome. The psychosocial with fragile X syndrome, FXTAS, and POI. Males aspects, cost-effectiveness and feasibility of such with premutations are at increased risk for FXTAS. a screening program are still being evaluated, Males with FXTAS will transmit their FMR1 premu- and presently no guidelines are available for car- tation expansion to none of their sons and to all of rier screening of low risk pregnant/non-pregnant their daughters, who will be premutation carriers. women. Carrier testing for at-risk relatives and prenatal testing for pregnancies at increased risk are pos- Conclusions sible if the diagnosis of an FMR1-related disorder has been confirmed in a family member. The Fragile X syndrome is the most common inherited risk to offspring of a premutation carrier female cause of mental impairment among children. Con- varies as per the number of repeats and figures firmation of clinical diagnosis by genetic testing are available for predicting accurate recurrence helps in providing early and appropriate clinical risk. All male fetuses found to have full mutation care, prenatal diagnosis services and extended on prenatal testing are likely to be affected. How- carrier screening to the family. Although no defini- ever, predicting the phenotype of a female fetus tive treatment for Fragile X syndrome is available detected to have full mutation is difficult because at present, various new molecular therapies are of the highly variable clinical presentation. under trial and raise hope for better management of these patients. Screening for Fragile X References A robust screening method is needed to accom- modate future directions of early intervention and 1. Bagni C, et al. Fragile X syndrome: causes, anticipation. Thus, to identify individuals with diagnosis, mechanisms, and therapeutics. J Clin FXS and FMR1-associated disorders and to provide Invest 2012; 122: 4314-4322. early intervention services for children, an FMR1 2. Hill MK, et al. A systematic review of population sensitive and specific screening method for both screening for fragile X syndrome. Genet Med males and females is needed. Results of fragile X 2010;12: 396-410. DNA testing allow accurate genetic counseling to 3. Saul RA, et al. FMR1-Related Disorders. 1998 be provided. Carrier testing for at-risk individuals Jun 16 [Updated 2012 Apr 26]. In: Adam MP, and prenatal testing empowers families to make Ardinger HH, Pagon RA, et al., editors. GeneRe- informed reproductive decisions. Newborn screen- views® [Internet]. Seattle (WA): University of ing for Fragile X has recently been introduced in Washington, Seattle; 1993-2017. some populations and initial results show that it 4. Sorensen PL, et al. Newborn screening and has been well accepted. The rationale for newborn cascade testing for FMR1 mutations. Am J Med screening is that it helps in providing early interven- Genet A 2013 161A: 59-69. tion for infants by the pediatrician or family care 5. Zhou Y, et al. Robust fragile X (CGG)n genotype provider. In addition, this helps in identification classification using a methylation specific triple of carrier females and provides opportunity for PCR assay. J Med Genet 2004; 41:e45.

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MECP2 Gene-Related Disorders Meenakshi Lallar, Shubha R Phadke Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow

Correspondence to: Dr Shubha R Phadke Email: [email protected]

Abstract 1. Rett syndrome (OMIM 312750)

Classic Rett syndrome is a neurodevelopmental – Classic # disease in females caused by mutations in the Classical Rett syndrome is an X-linked neurodevel- MECP2 gene. With recent advancements in molec- opment disorder with a prevalence of 1 per 10,000 ular techniques, point mutations and small and girls. The most important feature to diagnose Rett large deletions/duplications have been identified syndrome is regression after a period of normal in the MECP2 gene associated with varying disease development and postnatal microcephaly (Neul et phenotypes in both males and females. This al., 2010). review on MECP2 gene-related disorders discusses The revised diagnostic criteria given in 2010 are the various disease phenotypes associated with as listed in Table 1 (Neul et al., 2008). MECP2 gene mutations. MECP2 gene (OMIM No. 300005) is located on Table 1 Diagnostic criteria for Rett syndrome. chromosome X (Xq28) and has been known since long to be the causative gene for Rett syndrome Inclusion criteria Exclusion criteria in females. But with the advent of new advanced 1. Secondary / 1. Brain injury molecular techniques, new point mutations and postnatal secondary to trauma, copy number changes are being increasingly recog- microcephaly asphyxia and infections nized as the cause of different intellectual disability 2. A period of 2. Grossly abnormal phenotypes of varying severity in both males and regression followed psychomotor females. Due to the phenomenon of X inactivation by recovery or development in the in females, MECP2-related genetic diseases have a stabilization first six months of life. phenotypic spectrum varying from being clinically silent to severe neurodevelopmental syndromes. 3. Partial or complete Earlier it was thought that MECP2 disorders are loss of acquired lethal in males but now it is being increasingly purposeful hand skills recognised that MECP2 accounts for a clinically and stereotypic hand significant proportion of intellectual disability phe- movements (Figure 1). notypes in males. 4. Partial or complete The MECP2-gene related disorders include: loss of acquired spoken language 1. Rett syndrome – classical and atypical 5. Gait abnormality In a patient with Rett-like features but no re- 2. Severe neonatal onset encephalopathy with gression, the patient should be followed up till the microcephaly in males age of 5 years, and in case there is no evidence of regression by 5 years, the diagnosis of Rett syn- 3. MECP2 duplication syndrome drome should be questioned. The other important features include- seizures, failure to thrive, scolio- 4. Intellectual disability associated with manic sis and osteopenia. Autistic features, apnea and depressive psychosis, pyramidal signs, bruxism are also observed but are more indicative parkinsonian features, and macro-orchidism of atypical Rett syndrome. Differential diagnoses (PPM-X syndrome) include atypical Rett syndrome and other MECP2

150 Chapter 24 related disorders, Angelman syndrome, cerebral the next pregnancy are rare, prenatal testing can palsy and autism. be provided in the next pregnancy considering the possibilities of germline mosaicism and carrier mother with skewed X inactivation.

Rett syndrome variants / Atypical Rett syn- drome:• Atypical Rett syndrome/ variants include Rett syndrome patients with neuroregression and postnatal microcephaly which do not fulfil all the criteria given above for the diagnosis of classical Rett syndrome and have more atypical features. Presence of congenital microcephaly, severe pro- gression, preservation of speech, milder presen- tation and late onset are some of the variations which differentiate these cases from classic Rett syndrome. Genetically also, only around 40-50% have MECP2 gene mutation and other important genes include CDKL5 and FOXG1. Revised diag- nostic criteria for atypical Rett syndrome are given below in Table 2 (Neul et al., 2010). There are three main atypical Rett syndrome variants: 1. Preserved speech- Zapella variant- As the name suggests, this is a milder form of Rett syndrome with onset of regression at 1-3 years and simple Figure 1 A girl with Rett syndrome showing the speech recovers by five years of age. Other typical hand movement. features include- better retention of hand function, milder intellectual disability and autistic features. Patients suspected to have Rett syndrome fol- The important features of classic Rett syndrome lowing detailed clinical evaluation need to be like seizures, failure to thrive, scoliosis and mi- confirmed by molecular testing. The first step crocephaly are less frequently observed. Gene is sequencing of the MECP2 gene (exons 1 to involved is MECP2 in majority of cases (30-50%) 4), which identifies pathogenic mutations in more (Bienvenu et al., 2000) than 80% of classical Rett syndrome patients (Bi- 2. Early seizure variant- Hanefeld Variant- The envenu et al., 2000). In the remaining patients, main diagnostic features of this variant are early deletion and duplication analysis identifies partial onset of seizures, at less than five months, and and whole MECP2 gene deletions in around 8-10% before neuroregression. The important features patients with classical Rett syndrome (Hardwick et of classic Rett syndrome are also less frequently al., 2007). Treatment is symptomatic and multidis- observed in this variant. Mutations are mainly ciplinary. Surveillance for development of scoliosis found in the CDKL5 gene and very rarely in the and ECG finding of prolonged QTc is required. MECP2 gene. Genetic counseling and prenatal diagnosis: 3. Congenital variant-Rolando Variant- There is se- More than 99 percent of cases of classical Rett • vere psychomotor developmental delay, seizures, syndrome cases are simplex cases, resulting from regression and severe postnatal microcephaly as a de novo pathogenic variant and in these cases early as four months. Typical features found are the risk of recurrence in the next pregnancy is the autonomic system abnormalities, stereotypical negligible. However, germline mosaicism has been tongue movements and jerky limb movements. reported. In very rare cases the mother can Mutations are mainly found in the FOXG1 gene and be a carrier of the MECP2 gene mutation but very rarely in the MECP2 gene. might not have any clinical features due to an extremely favourable X chromosome inactivation. An approach to the genetic testing of classic In such very rare cases, the risk of recurrence is Rett and atypical Rett syndrome is shown in Figure 50%. Hence, though chances of recurrence in 2.

151 Chapter 24

Table 2 Diagnostic criteria for atypical Rett syndrome.

Essential Criteria Any two out of for diagnosis following four Any five out of eleven supportive criteria main criteria Secondary/ Partial or complete Breathing Bruxism when Impaired sleep Postnatal loss of acquired disturbances awake pattern microcephaly purposeful hand when awake skills A period of Stereotypic hand Abnormal Peripheral Scoliosis/Kypho- regression followed movements muscle tone vasomotor sis by recovery or disturbances stabilization Partial or complete Growth Small cold hands Intense eye loss of acquired retardation and feet contact spoken language Gait abnormality Inappropriate Diminished laughing/ response to pain screaming spells

Figure 2 Investigative approach to a case with possible MECP2-related phenotype.

2. Severe neonatal onset chromosome, no MECP2 produced), it is usually lethal in males. In the rare surviving males, the encephalopathy with microcephaly most common clinical presentation is the so-called (OMIM 300673) severe neonatal-onset encephalopathy with micro- cephaly, abnormal tone, involuntary movements, # severe seizures, breathing abnormalities and death Rett syndrome occurs due to heterozygosity for before two years of age. (Kankirawatana et al., pathogenic MECP2 variants in females. As males 2006) are hemizygous for MECP2 mutations (single X

152 Chapter 24

3. MECP2 duplication syndrome (OMIM thick vermillion). It may rarely have associated eye and limb abnormalities. Males are all affected and 300260) females are either mildly affected (learning disabil- ity and dysmorphism) or unaffected (El-Hattab et MECP2 microduplication syndrome is a severe # al., 2015). There is 0.5Mb duplication on the Xq28 neurodevelopmental syndrome seen exclusively in region, from intron 22 homologous region 1 to males. The females are asymptomatic carriers. intron 22 homologous region 2 (due to inversion Infantile hypotonia is a predominant feature, which and non-allelic recombination between low copy is usually the first presenting sign as severe feeding repeats in this region). The phenotypic effects abnormalities within few weeks of birth. Grad- are due to increased dosage effects of the CLIC2 ually hypotonia fades way to spasticity especially and RAB39 genes in the 0.5Mb duplicated segment. of lower limbs. There is delay in motor and Testing involves identification of the duplicated language milestones. Around 70% males have region by MLPA, interphase FISH or cytogenetic no speech, and one third never learn to walk. microarray. Mild facial dysmorphism like brachycephaly, mid face retrusion, large ears, and depressed nasal Genetic counseling: Till date all affected bridge is observed. There is an increased predis- cases• have been inherited from carrier mothers position to infections, commonest being recurrent and de novo cases, though theoretically possible, respiratory tract infections. Refractory seizures have not been described. So, if the mother is a develop in 50% of affected males. Other features carrier of the 0.5Mb Xq28 duplication (mild affected include gastrointestinal dysfunctions, developmen- or unaffected), 50% of the sons and 50% ofthe tal regression and autistic features. Management daughters will inherit the same, and such males will remains symptomatic and most affected males die be affected and females might be mildly affected by the age of 25 years. or unaffected. In females, the X chromosome In MECP2 microduplication syndrome there is harbouring the 0.5Mb Xq28 duplication is not pref- duplication of the region encompassing the MECP2 erentially inactivated, as there is random skewing gene usually 0.3 to 4Mb in size and it can be of X chromosomes. identified easily with Multiplex Ligation Probe The other important differential diagnosis is Amplification (MLPA) and cytogenetic microarray. alpha Thalassemia X linked intellectual disability Larger duplications of more than 8 Mb, found in syndrome, which can be diagnosed by molecular five percent cases, can be identified by karyotype. testing of the ATRX gene. MRI and EEG show nonspecific changes. It shows complete penetrance in males. Females are always 4. MECP2-associated intellectual asymptomatic carriers unless there is X- disability, autism and parkinsonian translocation. Many a times, the female carriers might develop psychiatric illnesses. features, and macroorchidism (OMIM Genetic counseling: Mothers of all affected 300055 / PPM-X syndrome) # males• are always carriers of MECP2 microduplica- tion, though very rare de novo cases have been Recently, many case reports and studies have reported. All carrier females are unaffected due shown that MECP2 mutations, especially mis- to extremely skewed X inactivation. All mothers sense mutations, are associated with non-lethal being carriers, there is 50% risk of males being intellectual disability, autistic features, psychiatric affected and 50% risk of the female offspring being manifestations, pyramidal signs, parkinsonism like asymptomatic carriers in each pregnancy. features etc. in males. The phenotype can be One other disorder that needs to be mentioned modified by karyotypic abnormalities like 47,XXY here, mainly because of close proximity of the and somatic mosaicism. MECP2 gene might be an region to MECP2 on X chromosome, is the Xq28 important cause of intellectual disability in males duplication syndrome (OMIM # 300055). It in- after fragile X syndrome (2.8% vs 1.3%) (Gomot volves a 0.5 Mb region containing 11 genes but et al., 2003). Also, milder intellectual disability, not MECP2. This is a recently recognised X-linked autistic features etc., without any Rett-like features intellectual disability syndrome characterised by might manifest with MECP2 mutations in females, developmental delay and intellectual disability, be- probably due to skewed X inactivation. So, in havioural defects, obesity, subtle dysmorphism all males and females with intellectual disability, (tall forehead, puffy eyelids, wide nasal bridge, the importance of MECP2 gene testing is being

153 Chapter 24 increasingly recognized (Villard, 2007). tion/deletion: new cases and literature review. BMC Med Genet 2015; 16:12. Conclusion 3. Gomot M, et al. MECP2 gene mutations in non-syndromic X-linked mental retardation: phenotype-genotype correlation. Am J Med MECP2 gene-related disorders vary in clinical phe- Genet A 2003;123A:129-139. notype from classic Rett syndrome and atypical 4. Hardwick SA, et al. Delineation of large deletions Rett syndrome to non-syndromic intellectual dis- of the MECP2 gene in Rett syndrome patients, ability. MECP2 gene should be tested for point including a familial case with a male proband. mutations and deletion/duplications in Rett pheno- Eur J Hum Genet. 2007;15: 1218-1229. types, males with neonatal onset encephalopathy, 5. Kankirawatana P, et al. Early progressive en- and males and females with intellectual disability cephalopathy in boys and MECP2 mutations. or autism. Neurology 2006; 67:164-166. 6. Neul JL, et al. Specific mutations in methyl-CpG- References binding protein 2 confer different severity in Rett syndrome. Neurology 2008; 70: 1313-1321. 1. Bienvenu T, et al. MECP2 mutations account for 7. Neul JL, et al. Rett syndrome: revised diagnostic most cases of typical forms of Rett syndrome. criteria and nomenclature. Ann Neurol 2010; 68: Hum Mol Genet 2000; 9:1377-1384. 944-950. 2. El-Hattab AW, et al. Clinical characteriza- 8. Villard L. MECP2 mutations in males. J Med Gen tion of int22h1/int22h2-mediated Xq28 duplica- 2007; 44: 417-423.

PhotoQuiz - 6 Contributed by: Dr Prajnya Ranganath Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad Email: [email protected]

This fetus was brought for autopsy evaluation following termination of pregnancy due to multiple anomalies detected in the antenatal sonogram. The autopsy findings are shown. Identify the condition.

Answer on page 232

154 Chapter 25

Marfan Syndrome: Recent Advances in Diagnosis and Management

Prajnya Ranganath1, Gaurava Kumar Rai2 1Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad 2Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow

Correspondence to: Dr Prajnya Ranganath Email: [email protected]

Introduction of the ribs leading to chest deformity (pectus excavatum or carinatum) and vertebral column Marfan syndrome (OMIM # 154700) is a heritable deformity (scoliosis/ kyphosis/ lordosis) are the disorder of fibrous connective tissues, which af- common skeletal features of the disorder. Cran- fects multiple systems and has significant clinical iofacial features include a long and narrow face, variability. The estimated world-wide prevalence downward slanting palpebral fissures, malar hy- of Marfan syndrome is one in 5,000 to 10,000 poplasia, micro/ retrognathia, high arched palate individuals (Dietz, 2017). The condition is named and crowding of teeth (Figure 1) (Dietz, 2017). after Antoine Marfan, a French paediatrician who Cardiovascular manifestations are the major first described the typical clinical features ina cause of serious morbidity and early mortality in five year old girl in 1896 (Gott, 1998). It hasan Marfan syndrome and include dilatation of the autosomal dominant pattern of inheritance and aorta, aortic dissection and rupture, mitral valve is caused by heterozygous mutation in the gene prolapse/ regurgitation, tricuspid valve prolapse, encoding fibrillin-1FBN1 ( ) (Dietz, 2017). and enlargement of the proximal pulmonary artery (Dietz, 2017). Clinical Features Other systemic features of the disorder include dural ectasia (stretching of the dural sac in the lum- Cardinal manifestations of Marfan syndrome in- bosacral region), skin striae, hernias, lung bullae volve the ocular, skeletal, and cardiovascular sys- and spontaneous pneumothorax. tems but other systemic abnormalities such as pulmonary, cutaneous, and neurological abnor- Genetics of Marfan syndrome malities also occur as a part of the syndrome (Dietz, 2017). Marfan syndrome is caused by heterozygous mu- Myopia is the most common ocular feature tation in the FBN1gene (OMIM 134797) which seen in Marfan syndrome. Ectopia lentis i.e. encodes the fibrillin-1 protein. FBN1 is located on displacement of the lens from the centre of the the long arm of (15q21.1) and is pupil, though a hallmark feature of the disease, is one of the largest genes in the human genome with seen in only around 60% of affected individuals. 65 exons spanning more than 600kb. Fibrillin-1 Retinal detachment, glaucoma, and early cataract is a matrix glycoprotein made up of 2,871 amino formation are the other eye anomalies associated acids and has five structurally distinct regions; with this condition (Dietz, 2017). the largest region comprising about 75% of the Skeletal system involvement is character- protein is made up of cysteine-rich EGF (epider- ized by bone overgrowth and joint laxity. mal growth factor)-like repeats and the other 4 Dolichostenomelia (disproportionately long ex- regions include a unique amino-terminal stretch tremities compared to the trunk, manifesting of basic residues, an adjacent second cysteine-rich as increased arm span-to-height ratio and de- region, a proline-rich domain, and the carboxy creased upper-to-lower segment ratio), arachn- terminus. Fibrillin-1 is an important component of odactyly (thumb sign and wrist sign), overgrowth microfibrils which are present in elastic and non-

155 Chapter 25

Figure 1 A. 10 year old boy with Marfan syndrome, B. Close up of the patient’s face, C. Arachnodactyly, D. Positive thumb sign, E. Positive wrist sign. elastic tissues and play an important role in the variable expressivity). Although there are no defi- formation and homeostasis of the elastic matrix, nite genotype– phenotype correlations, mutations in matrix-cell attachments and in the regulation of in the central portion of the gene in exons 24–32 some growth factors especially TGF- (transform- have been reported to be associated with the most ing growth factor beta). FBN1 gene훽 mutations severe and rapidly progressive form of Marfan result in production of abnormal mutant forms syndrome termed “neonatal Marfan syndrome”. of fibrillin 1, disruption of normal microfibrillar On the whole, mutations causing in-frame loss or assembly and weakening of the connective tissue. gain of central coding sequences through dele- Over 1,000 mutations have been reported in the tions, insertions, or splicing errors are found to FBN1 gene; these are distributed throughout the be associated with more severe disease and mu- sequence of the gene and there is no reported tations that create a premature termination codon ethnic preponderance of any specific mutations. and result in rapid degradation of mutant tran- Different types of mutations in the FBN1 gene scripts are reported to have milder phenotypes, which result in Marfan syndrome include nonsense probably because mutant protein forms with a mutations, in-frame or out-of-frame deletions/ dominant negative effect have more severe con- insertions, splice site mutations that alter the sequences on microfibrillar assembly. Mutations splice consensus sequence or alter splicing, mis- affecting C-terminal propeptide processing have sense mutations that create or destroy a cysteine been found to be associated with predominant residue and missense mutations that affect con- skeletal manifestations (Dietz, 2017). served residues in the EGF-like domain consensus Other disease phenotypes associated with FBN1 sequence (http://omim.org/entry/134797). gene mutations are the Marfan syndrome is an autosomal dominant syndrome, the MASS phenotype (myopia, mitral disease characterized by high penetrance but valve prolapse, aortic enlargement, and nonspe- marked clinical variability (including intrafamilial cific skin and skeletal features), isolated aortic

156 Chapter 25 aneurysm, isolated skeletal features of Marfan syn- dated, are not applicable in children or necessitate drome, familial ectopia lentis, Shprintzen-Goldberg expensive and specialised investigations and also syndrome (consisting of skeletal findings of Mar- that they do not take into account the variable clin- fan syndrome with ocular hypertelorism, cran- ical expression and extended differential diagnosis iosynostosis, other craniofacial abnormalities and of Marfan syndrome. A revised Ghent nosology cognitive impairment), autosomal dominant Weill- was therefore formulated in 2010, which gives Marchesani syndrome (characterized by ectopia more weight to the cardiovascular manifestations lentis, microspherophakia, short stature, brachy- and in which aortic root aneurysm and ectopia dactyly and the absence of vascular manifesta- lentis are the cardinal clinical features (Loeys et al., tions), stiff skin syndrome (an autosomal dominant 2010). As per the revised Ghent criteria, diagnosis form of congenital scleroderma) and two skeletal of Marfan syndrome can be established if (Loeys et dysplasias (Geleophysic dysplasia-2 and acromicric al., 2010; http://www.marfan.org/dx): dysplasia) (Dietz, 2017). A. In the absence of a family history of Marfan Some patients with many but not all clinical syndrome there is: features of Marfan syndrome are termed to have Marfan–like syndrome or Marfan syndrome type Aortic root enlargement (Z score ) and II. Most of these patients do not have ectopia • one of the following: ≥ 2.0 lentis or prominent dolichostenomelia. Loeys- Dietz syndrome (LDS) is another disorder which – Ectopia lentis shares significant phenotypic overlap with Marfan – A pathogenic FBN1 mutation syndrome; it is an autosomal dominant condi- – A systemic score tion which has the craniofacial features of Marfan Ectopia lentis and a FBN1≥ 7 mutation previ- syndrome along with pectus deformity, scoliosis, ously associated with aortic enlargement arachnodactyly, joint laxity, dural ectasia, and • aortic root aneurysm with dissection, but does B. In the presence of a family history of Marfan not have prominent dolichostenomelia or ectopia syndrome in a first-degree relative there is: lentis. LDS can have additional features such as hypertelorism, broad or bifid uvula, cleft palate, Ectopia lentis learning disability, Chiari I malformation, blue • Systemic score sclerae, exotropia, craniosynostosis, cervical spine instability, talipes equinovarus, soft and translu- • Aortic root enlargement≥ 7 (Z score in cent skin, easy bruisability and generalized arterial • years or in years) ≥ 2 ≥ 20 tortuosity. Both Marfan-like syndrome and LDS are ≥ 3 < 20 caused by mutations in TGFBR1 or TGFBR2 genes, The systemic score is calculated using Table 1 which encode the type I or type II receptors for (http://www.marfan.org/dx). transforming growth factor (TGF- ). The type II Genetic testing receptor (TGFBR2) functions훽 as a transmembrane훽 serine/threonine kinase and is required for the anti-proliferative activity of TGF-beta, whereas the Genetic testing of Marfan syndrome using con- type I receptor (TGFBR1) mediates the induction of ventional Sanger sequencing technique is difficult several genes involved in cell-matrix interactions because of the large size of the gene. The absence of mutational hot-spots in the gene, lack of any eth- (Dietz, 2017; http://omim.org/entry/190181 ; nic preponderance of mutations and requirement http://omim.org/entry/190182). of analysing all 65 exons make genetic diagnosis expensive and time consuming. However, with the Diagnosis advent of new high throughput next-generation sequencing strategies it has become feasible to The Ghent criteria which are used for making a sequence DNA of the whole coding region of the definitive diagnosis of Marfan syndrome were orig- FBN1 gene to detect potentially pathogenic vari- inally developed by an international group in 1996 ants. Capture of the complete FBN1 sequence, and comprise a set of major and minor manifesta- including the promoter region, introns and ′ ′ tions in different body systems. It was perceived untranslated region5 followed by high-throughput3 that some of the diagnostic criteria in the original sequencing of the whole of the FBN1 gene has Ghent’s nosology have not been sufficiently vali- enabled detection of intronic and regulatory region

157 Chapter 25

Table 1 Features used for calculation of the systemic score in the revised Ghent nosology (Dietz, 2017; Loeys etal., 2010; http://www.marfan.org/dx).

Feature Value Wrist and thumb sign 3 Wrist or thumb sign 1 Pectus carinatum deformity 2 Pectus excavatum or chest asymmetry 1 Hindfoot deformity 2 Pes planus 1 Pneumothorax 2 Dural ectasia 2 Protrusio acetabulae 2 Reduced upper segment/lower segment and increased arm span/height ratios 1 Scoliosis or thoracolumbar kyphosis 1 Reduced elbow extension 1 Three of 5 facial features (dolichocephaly, downward slanting palpebral 1 fissures, enophthalmos, retrognathia and malar hypoplasia) Skin striae 1 Myopia 1 Mitral valve prolapse 1 mutations. The development of RNA sequenc- nificant aortic regurgitation is present. Annual ing could facilitate sequencing of FBN1 transcripts ophthalmologic evaluation is required to monitor using RNA from appropriate cell types (for exam- for lens dislocation, glaucoma and cataracts (Dietz, ple fibroblasts derived from skin biopsy or aortic 2017). smooth muscle cells). RNA sequencing would also allow discovery of abnormal transcription start The treatment is largely symptomatic and no sites, splice variants and miRNA differences that definite curative therapy for the disease is available may affect FBN1 mRNA levels or function. at present. Monitoring, prevention and treatment of cardiovascular complications form the most im- portant part of management of Marfan syndrome. Management and Surveillance Beta blocker therapy is initiated at the time of diagnosis to reduce hemodynamic stress on the Marfan syndrome requires multidisciplinary man- aortic wall. Surgical repair of the aorta is indicated agement involving a clinical geneticist, cardiologist, once aortic diameter approaches 5.0 cm in adults cardiothoracic surgeon, orthopaedician and oph- or older children, the rate of increase of the aortic thalmologist. Following an initial detailed eval- diameter approaches 1.0 cm per year, or there is uation by each concerned specialist at the time progressive aortic regurgitation. Composite aortic of diagnosis, subsequent regular surveillance is valve graft or valve-sparing aortic root replacement essential to monitor for the disease-related com- are required in case of aorta dissection or rupture. plications. Yearly echocardiographic examinations Afterload reducing agents are added for congestive are indicated when the aortic dimension is rel- heart failure. Mitral valve repair/ replacement is atively small and the rate of aortic dilation is done for mitral regurgitation. Clinical trials are now relatively slow but more frequent examinations being conducted to study the utility of TGF- an- are required when the aortic root diameter ex- tagonists such as Losartan in controlling abnormal훽 ceeds 4.5 cms in adults, the rate of aortic dilation aortic root growth in Marfan syndrome patients. exceeds approximately 0.5 cm per year, and sig- Ocular problems can often be controlled with

158 Chapter 25 eyeglasses alone. Lens dislocation may require tion in the rate of aortic root enlargement (Brooke laser treatment or surgical replacement. Surgical et al., 2008). Thereafter, more studies have been intervention may be required for severe skeletal conducted in small cohorts of patients either com- anomalies e.g. repair of severe pectus excava- paring the efficacy of losartan versus beta blocker tum or vertebral column stabilization for severe therapy (BBT) or studying the combined effect of kyphoscoliosis (Dietz, 2017; Jondeau, 2014). losartan and BBT on the cardiovascular complica- tions of Marfan syndrome and most of them have Genetic counseling found that losartan provides more effective protec- tion to slow the progression of aortic root dilation Marfan syndrome has an autosomal dominant (Pees et al., 2013; Chiu et al., 2013). Groenink and pattern of inheritance. In approximately 75% of colleagues have recently reported the results of a cases, the condition is inherited from an affected large, randomised, controlled multicentre clinical parent and in the remaining around 25% it occurs trial, wherein a total of 233 operated and un- due to a de novo mutation in the proband (Dietz, operated adults with Marfan syndrome underwent 2017). If a parent is affected, the risk of recurrence randomization to either losartan ( ) or no in each sib of a proband is 50%. If neither parent additional treatment ( ) and푛 = the 116 primary is clinically affected, the proband usually has ade endpoint considered was푛 = 117 aortic dilatation rate novo mutation and therefore the recurrence risk in as determined by magnetic resonance imaging. the sibs is not significant; however, in rare cases, Losartan treatment was found to reduce the dilata- due to germline mosaicism in either parent, the re- tion rate of the aortic root and in patients who had currence risk in the sibs may be above the general already undergone aortic root replacement it was population risk. The children of an individual with seen to reduce the dilatation rate of the aortic arch Marfan syndrome are at 50% risk of inheriting the (Groenik et al., 2013). mutant allele and the disorder. Prenatal testing With more and more studies showing promising for pregnancies at increased risk is possible if the results, TGF-beta antagonists such as losartan are disease-causing mutation in the family is known. likely to soon become the mainstay of treatment for the life-threatening cardiovascular complica- tions of Marfan syndrome. Newer therapeutic strategies Key Messages Studies into the molecular pathogenesis of Mar- fan syndrome have led to elucidation of the fact Marfan syndrome is a hereditary connec- that some of the phenotypic manifestations, espe- • tive tissue disorder affecting multiple body cially the aortic root dilatation, result from excess systems. activation of TGF-beta, a cytokine involved in cel- lular proliferation, migration and programmed cell Establishment of a definitive diagnosis of death. Systemic administration of TGF-beta an- • Marfan syndrome is based on the revised tagonists was found to reduce or prevent many Ghent nosology. disease manifestations including aortic aneurysm, emphysema and myxomatous valve disease in Pathogenesis of Marfan syndrome has not fibrillin-1 deficient mice. Angiotensin II recep- • been fully elucidated but fibrillin-1 gene mu- tor blockers such as Losartan decrease TGF-beta tations are believed to exert a dominant signalling and therefore their potential therapeu- negative effect and lead to excessive TGF- tic benefits in Marfan syndrome are now being signaling. 훽 extensively studied. High throughput molecular techniques such Losartan was demonstrated to halt abnormal • as whole genome, transcriptome as well as aortic root growth and prevent aortic aneurysm in exome sequencing and gene expression stud- a mouse model with a fibrillin-1 mutation by Dietz ies using microarrays are being explored to and colleagues in 2006 (Habashi et al., 2006). This understand the molecular mechanisms be- was found to be the result of both a reduction hind the high level of phenotypic variability in in hemodynamic stress and antagonism of TGF- Marfan syndrome. signalling in the vessel wall. Subsequently, a small훽 cohort study of losartan in 18 pediatric patients Newer therapeutic strategies especially those with Marfan syndrome showed a significant reduc- • targeting the TGF- signalling pathways are 훽 159 Chapter 25

being developed for management of the life- one hundred years later. Md Med J 1998; 47: threatening cardiovascular complications of 247-252. the disease. 5. Groenink M, et al. Losartan reduces aortic dilatation rate in adults with Marfan syndrome: References a randomized controlled trial. Eur Heart J 2013; 34: 3491-3500. 1. Brooke BS, et al. Angiotensin II blockade and 6. Habashi JP, et al. Losartan, an AT1 antagonist, aortic-root dilation in Marfan’s syndrome. N prevents aortic aneurysm in a mouse model of Engl J Med 2008; 358: 2787-2795. Marfan syndrome. Science 2006; 312: 117-121. 2. Chiu HH, et al. Losartan added to β-blockade 7. Jondeau PG. Marfan syndrome. At http: therapy for aortic root dilation in Marfan syn- //www.orpha.net. Accessed on February 12, drome: a randomized, open-label pilot study. 2014. Mayo Clin Proc 2013; 88: 271-276. 8. Loeys BL, et al. The revised Ghent nosology for 3. Dietz H. Marfan Syndrome. 2001 Apr 18 [Up- the Marfan syndrome. J Med Genet 2010; 47: dated 2017 Oct 12]. In: Adam MP, Ardinger HH, 476-485. Pagon RA, et al., editors. GeneReviews® [In- 9. Pees C, et al. Usefulness of losartan on the size ternet]. Seattle (WA): University of Washington, of the ascending aorta in an unselected cohort Seattle; 1993-2017.Available from: https:// of children, adolescents, and young adults with www.ncbi.nlm.nih.gov/books/NBK1335/ Marfan syndrome. Am J Cardiol 2013; 112: 4. Gott VL. Antoine Marfan and his syndrome: 1477-1483.

PhotoQuiz - 7 Contributed by: Dr Prajnya Ranganath Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad Email: [email protected]

This 38 years-old male patient presented with complaints of chronic renal disease, hypertension, history of one episode of stroke and skin lesions (shown in the pictures). His 45 years-old elder brother was similarly affected and his 17 years-old nephew had similar skin lesions andvortex keratopathy. Identify this condition.

Answer on page 232

160 Chapter 26

Disorders of the RAS-MAPK Pathway: Prototype of Pathway Disorders with Overlapping Phenotypes

Prajnya Ranganath1, Dhanya Lakshmi N1, Shubha R Phadke2 1Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad 2Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow

Correspondence to: Dr Prajnya Ranganath Email: [email protected]

Introduction cell differentiation and apoptosis. RAS genes are homologues of rat sarcoma virus genes. Four A molecular pathway may be defined as a series of main RAS genes are known: HRAS (Harvey rat molecular interactions that generates a particular sarcoma oncogene homologue), KRAS (Kirsten rat end product or leads to a certain cellular func- sarcoma oncogene homologue), ERAS (Embryonic tion. A signal transduction pathway is a molecular stem cell expressed RAS) and NRAS (Neuroblas- pathway that ‘transduces’ an extracellular signal toma RAS oncogene). RAS genes encode the Ras into changes in gene transcription within the cell; proteins which are guanosine nucleotide bound the external signal in the form of an extracellular proteins that cycle between an active GTP-bound ligand binds to a surface receptor, resulting in and an inactive GDP-bound conformation. The the activation of intracellular signalling molecules Ras proteins are upstream members of the RAS – to modify the transcription factor activity in the MAP kinase pathway. The RAS – MAPK pathway nucleus and alter transcription of specific genes. gets activated when an extracellular ligand such Many of the developmental processes in eukaryotic as a growth factor binds to a tyrosine kinase organisms including human beings are now known receptor such as the epidermal growth factor re- to be controlled by evolutionarily conserved signal ceptor. The receptor undergoes dimerisation and transduction pathways. Disturbances in these auto-phosphorylation and then binds to the SH2 pathways can affect these normal developmental domain of the adaptor protein GRB2 (growth factor processes and because a disturbance at any level receptor bound protein 2), through the SHP2 pro- of the pathway can lead to disruption of the com- tein which is encoded by PTPN11 (protein tyrosine mon final end-product or function, defects in any phosphatase non receptor type 11). This results of the involved molecules can produce similar or in the recruitment of the GRB2-bound SOS (ho- overlapping phenotypes. mologue of the Drosophila Son of Sevenless gene) Many pathway disorders with overlapping phe- protein to the plasma membrane. In this location, notypes are known but perhaps the most ex- SOS comes into close proximity to the membrane tensively described are the disorders related to bound Ras proteins, catalyses the conversion of the RAS – MAPK pathway, also known as the the Ras- attached GDP to GTP and thereby acti- neuro-cardio-facial cutaneous syndromes (Lopez- vates the Ras proteins. Activation of Ras results Rangel., 2007). Ciliopathies and some of the in the sequential phosphorylation and activation disorders related to the sonic hedgehog pathway of RAF, MEK (MAPK extracellularly regulated kinase (SHH-GLI-PTCH), FGF pathway and WNT pathway and MAPK (mitogen activated protein kinase). The are also known to have some similarity in their activated MAP kinase further acts on many nuclear manifestations. and cytosolic substrates including transcription factors and other signalling molecules and thereby RAS – MAPK pathway influences expression of many important genes involved in cell growth and proliferation. Signalling The RAS – MAPK pathway is an important signal through this cascade is terminated when the Ras transduction pathway that controls the expression attached GTP is hydrolysed to GDP through GTPase of genes involved in cell proliferation, cell survival, activating proteins (GAPs) such as neurofibromin

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Figure 1 RAS – MAP kinase Pathway.

(NF1) & p120 GAP (Denayer et al., 2008) (Figure 1). matosis type 1 and neurofibromatosis type 1 like syndrome (). As a group, these Germline mutations involving any of the genes disorders constitute one of the largest known mal- encoding any of the molecules involved in this path- formation syndromes with an incidence of 1 in way can affect normal developmental processes 1000 individuals (Rauen, 2013). and many of the resulting syndromes are known to have overlapping phenotypes. In fact, the genetic basis of some of these disorders was traced to this Disorders related to the RAS – MAPK pathway following perception of the similarity in pathway their phenotypes to diseases already known to be related to this pathway. The diseases now known Noonan Syndrome: Noonan syndrome (NS) to be related to the RAS – MAP kinase pathway, is a• syndrome characterised by short stature, mild also known as the neuro-cardio-facial cutaneous mental retardation, typical mildly coarse facies (NCFC) syndromes or , include Noonan (down-slanting palpebral fissures, hypertelorism, syndrome, cardio-facio-cutaneous (CFC) syndrome, thick eyelids with ptosis, epicanthal folds, blue or Costello syndrome, LEOPARD syndrome (Noonan bluish green irises, low set posteriorly rotated ears syndrome with multiple lentigenes), neurofibro- with fleshy helices), a broad webbed neck anda

162 Chapter 26 deformed chest (superior pectus carinatum and phatase activity and activation of the RAS-MAPK inferior pectus excavatum) with low set, widely pathway. After this initial identification of the in- spaced nipples (Figure 2). Fifty to eighty percent volvement of the RAS – MAP kinase pathway in the of cases have congenital heart disease of which causation of Noonan syndrome, other researchers pulmonary valvular stenosis is the commonest started investigating the possibility of involvement (20-50%), followed by hypertrophic cardiomyopa- of other molecules related to this pathway in cases thy (HOCM) (20-30%), atrial and ventricular septal of Noonan syndrome without PTPN11 mutations. defects, branch pulmonary artery stenosis and Germline KRAS mutations were first reported by . Ocular abnormalities may Schubbert et al in 2006 and have been found to be present in as many as 95% of cases and cause disease by dysregulation of the RAS-MAPK include strabismus, refractive errors and nystag- signalling via two distinct pathogenic mechanisms: mus. Associated abnormalities include cutaneous decreased GTP-ase activity and interaction of KRAS abnormalities such as follicular hyperkeratosis and with the guanosine nucleotide ring resulting in in- increased cafe au lait spots, genitourinary abnor- creased GTP/GDP dissociation, both of which cause malities such as cryptorchidism, delayed puberty accumulation of the active (GTP bound) form of and mild renal anomalies, coagulopathy, lym- RAS. (Schubbert S et al.,2006) Other genes involved phedema and cystic hygroma or hydrops in fetal in Noonan syndrome are the SOS1 gene and RAF1 life. Affected individuals also have a predisposi- gene, the mutations in which were first reported by tion to malignancies especially myeloproliferative Roberts et al. (2007) and Pandit et al. & Razzaque disorders and juvenile myelo-monocytic leukemia et al. (2007) respectively. (Roberts et al., 2007; (JMML). Pandit et al., 2007).The mutations reported in both genes are mainly missense gain – of – function mutations which cause enhanced activation of the RAS- MAPK pathway. A significant degree of genotype – phenotype correlation has been found for Noonan syndrome. Patients with PTPN11 mutations are more likely to have pulmonary stenosis (HOCM less prevalent), a greater degree of short stature, coagulopathy, pectus deformity & cryptorchidism. Specifically, mutations at codons 61, 71, 72 & 76 are associated with an increased risk of JMML. KRAS mutations produce a more atypical phenotype with more severe mental retardation. SOS1 mutations are associated with more ectodermal abnormalities Figure 2 Two cases with Noonan syndrome with (i.e. skin changes), more cardiac septal defects, the typical facies, neck webbing and less of short stature and no increased risk of pectus deformity. malignancy Noonan syndrome with RAF1 muta- tions have a significantly high association with Noonan syndrome has an autosomal dominant HOCM. BRAF and MAP2K1 mutations cause classical pattern of inheritance with a significant degree of Noonan phenotype, but with florid ectodermal variable expressivity. In 30 – 75% of cases it is manifestations. found to be inherited from a parent and in the rest the mutations occur de novo. PTPN11 as the Cardio-Facio-Cutaneous Syndrome: Cardio- causative gene for Noonan syndrome accounts for facio-cutaneous• (CFC) syndrome is characterised up to 50% of cases. SOS1(13%), RAF1(5%), RIT1(5%), by typical cranio-facial features which are coarser KRAS( 5%), NRAS( 1%), BRAF ( 1%) and MAP2K1 are than in Noonan syndrome, cardiac defects and the other< genes< implicated< in causing the same ectodermal changes. The typical facies includes phenotype. Most of the mutations in these genes a high forehead with bitemporal narrowing, hy- are mis-sense, gain of function mutations. PTPN11 poplasia of the supra-orbital ridges, hypertelorism, mutations were first identified in 2001 by Tartaglia telecanthus, down-slanting palpebral fissures, epi- et al; these mutations disrupt an auto-inhibitory in- canthal folds, ptosis, a short nose with a depressed teraction between the SH2 domain and the catalytic nasal bridge and anteverted nares, low set, pos- phosphatase domain resulting in enhanced phos- teriorly rotated ears and a deep philtrum with

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Figure 3 Child with cardio-facio-cutaneous syndrome with the typical facial changes, dry hyperkeratotic skin and sparse hair. Hyperkeratotic papules on back may be appreciable. She was operated for atrial septal defect and pulmonary stenosis. cupid’s bow lip. Cardiac anomalies in the CFC genes in CFC syndrome cases are also all missense syndrome include pulmonic stenosis, atrial and gain of function mutations which cause increased ventricular septal defects, hypertrophic cardiomy- activation of the MAP kinase in the RAS signalling opathy, valvular defects and rhythm disturbances. pathway. Dermatologic abnormalities typically present in- clude xerosis, ichthyosis, hyperkeratosis of the Costello syndrome: Costello syndrome (CS) arms, legs and face, eczema, hemangiomas, cafe- is characterised• by short stature, a typical cranio- au-lait macules, erythema, lentigines and ulery- facial appearance which includes coarse facies, thema ophryogenes (inflammatory keratotic facial relative macrocephaly, curly, sparse, fine hair, papules) (Figure 3). Hair may be curly, woolly, epicanthic folds, a wide nasal bridge, a short full brittle and sparse. The nails may be broad and dys- nose and thick lips and cutaneous anomalies such trophic. Ocular complications include strabismus, as loose soft skin with increased pigmentation, nystagmus and refractive errors. Patients also facial and perianal papillomata. Cardiac anoma- usually have short stature and skeletal deformities lies are associated with CS, the most common such as a short neck, pectus deformity, kyphosis being hypertrophic cardiomyopathy; other cardiac and scoliosis. Mild to severe cognitive delay and defects include valvular pulmonic stenosis and mental retardation may be present with or without rhythm disturbances. Musculoskeletal anomalies seizure disorder, hydrocephalus, cortical atrophy in CS include diffuse hypotonia, joint laxity, ulnar and agenesis of the corpus callosum. Usually there deviation of wrists and fingers, spatulate finger is no association with malignancies. pads, tight Achilles tendon, positional foot defor- mity, kyphoscoliosis and pectus deformity. Mild to CFC syndrome occurs due to an autosomal severe developmental delay/ mental retardation is dominant de novo mutation; none of the cases usually present with or without seizures and hydro- reported so far were inherited from a parent. cephalus. Patients with CS have a predisposition The genes involved are the BRAF gene (75-80%), to tumours especially solid tumours like rhab- MAP2K1 & MAP2K2 genes (10-15%) and KRAS gene domyosarcoma, neuroblastoma and transitional ( 5%). Mutations in the BRAF, MAP2K1 & MAP2K2 cell carcinoma of the bladder. genes< were first reported by Rodriguez – Viciana et al. in 2006 and KRAS mutations by Niihori et Costello syndrome occurs due to an autosomal al. in 2006; both sets of workers discovered these dominant mutation and almost all cases reported genetic defects by studying the RAS pathway, which so far have been due to de novo mutations. Be- they suspected could be involved in view of the cause of the phenotypic overlap between Noonan great degree of phenotypic similarity of the CFC syndrome and CS, Aoki et al first investigated the syndrome with Noonan and Costello syndromes, genes downstream from PTPN11 in the MAP – ERK both of which were known to be associated with signalling pathway in CS cases. They sequenced germline mutations in the RAS pathway (Rodriguez- the entire coding region of all the 4 RAS genes Viciana et al., 2006). As in the case of Noonan and found mutations in the HRAS gene (Aoki et syndrome, mutations in BRAF, MAP2K and KRAS al., 2005). Subsequently, HRAS mutations have

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Figure 4 Disorders related to germ-line mutations in the RAS-MAP kinase pathway. been found in 85–90% of all cases of Costello in 5%. PTPN11 mutations in cases of LEOPARD syndrome. G12S is the most prevalent substitution syndrome were first reported by Digilio et al.& and G12A is the second most common. (Denayer Legius et al. in 2002; they studied the NS gene et al.,2008) These mis-sense gain of function muta- PTPN11 in cases of LEOPARD syndrome because of tions derange the RAS GTP-ase activity and cause the phenotypic similarity between the two (Digilio constitutive activation of the RAS-MAPK pathway. et al., 2002). However, the PTPN11 mutations associated with LEOPARD syndrome are loss of LEOPARD Syndrome: LEOPARD syndrome function mis-sense mutations which diminish the consists of Lentigines, ECG abnormalities, Ocular • catalytic activity of SHP2, unlike in NS. How loss of hypertelorism, Pulmonic stenosis, Abnormal gen- function mutations result in a similar phenotype italia, Retardation of growth and sensorineural as gain of function mutations (NS) remains to be Deafness. Lentigines are present in thousands elucidated (Denayer et al., 2008). RAF1 mutations typically on the face, neck & upper trunk with first reported by Pandit et al are gain of function sparing of the mucosa; cafe noir patches (large, missense mutations (Pandit et al., 2007) . darkly pigmented skin patches) may also be seen. Additional features are a variable degree of mental Neurofibromatosis type 1: Neurofibromato- retardation, skeletal deformity & hypertrophic car- sis• type 1 (NF1) is a neuro-cutaneous syndrome diomyopathy. Facies are similar to that of Noonan with some typical features that include pigmen- syndrome but milder. LEOPARD syndrome is in- tation abnormalities of the skin such cafe au lait herited in an autosomal dominant manner and has spots and axillary & groin freckling, Lisch nodules variable expressivity. Mutations may occur de novo, in the iris and a predisposition for the development may be inherited from an affected parent or there of nerve related tumours – especially cutaneous or may be germline mosaicism. Mutations have been subcutaneous benign neurofibromas, optic nerve found in the PTPN11 gene (90%), RAF1 gene ( 5%), gliomas, spinal cord tumours, neurosarcoma and BRAF and MAP2K1. The cause remains unidentified< malignant peripheral nerve sheath tumours. Ad-

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Table 1 Clinical features of disorders related to the RAS-MAPK pathway.

FEATURE NS CFCS CS LS NF1 NF1LS Facies Mildly coarse More coarse More coarse Mildly Mildly Mildly facies, than in NS; than in CFCS; coarse: coarse & coarse & hyper-telorism ‘Noonan-like’ wide forehead, ‘Noonan ‘Noonan ‘Noonan ptosis, facies with depressed like’ like’ in like’ in epicanthal bitemporal nasal bridge, some some folds, low set constriction full cheeks, cases cases posteriorly low set rotated ears, posteriorly clear blue rotated ears irises with thick lobes Mental Normal IQ to Moderate to Mild to Normal IQ Learning Learning retardation mild MR severe MR moderate MR to mild MR difficulty, difficulty, mild MR in mild MR some in some Cardiac PS, HOCM, PS, ASD, VSD, HOCM, PS, PS, HOCM, PS in PS defects ASD, VSD HOCM, ECG rhythm ECG some reported changes disturbances changes in 1 case Cutaneous Increased cafe Hyper- Deep palmar Multiple Cafe-au- Cafe-au- changes – au – lait keratotic, dry and plantar lentigines lait lait macules skin; creases, loose (in thou- macules, macules, ulerythaema & soft skin, sands) & axillary axillary ophryogenes facial & cafe noir freckling, freckling sparse, curly, perianal patches juvenile friable hair papillomata xantho- granuloma Short stature Present Present Present Present Present Present Macro- Usually absent Present Present Usually Present in Present cephaly absent many in many Pre- Present Absent Present ? Present ? disposition to mailgnancy

NS - Noonan syndrome; CFCS - Cardiofaciocutaneous syndrome; CS - Costello syndrome; LS - LEOPARD syndrome; NF1 - Neurofibromatosis type 1 syndrome; NF1LS - Neurofibromatosis type 1 like syndrome (Legius syndrome). ditional features include an increased risk of de novo. The gene involved is NF1 which encodes solid tumours such as rhabdomyosarcoma, neu- neurofibromin, a GTPase activating protein, which roblastoma and gastrointestinal stromal tumours, closely interacts with the RAS – MAPK pathway scoliosis and other bony anomalies like long bone by hydrolysing the Ras- attached GTP to GDP and bowing, pseudoarthrosis and sphenoid bone dys- terminating the signalling cascade (Figure 1). While plasia, learning disability, mild to moderate mental majority of patients have intragenic mutations retardation, vascular lesions like vascular ectasias within NF1, around 5% of cases have microdele- and CNS aneurysms, short stature and macro- tions involving the region of the NF1 gene. (Denayer cephaly. et al., 2008) NF1 is an autosomal dominant condition. Mu- Neurofibromatosis type 1 like syndrome tations may be inherited from a parent, may (Legius• syndrome): Neurofibromatosis type 1 like occur through germline mosaicism or may arise syndrome (NF1LS) consists of multiple cafe-au-lait

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Table 2 Ciliopathies: Note the common features namely polydactyly, renal cysts, retinal involvement and central nervous system malformations in varying combinations.

DISEASE GENE INHERITANCE PHENOTYPE Alstrom Syndrome ALMS1 AR Early onset obesity, congenital retinal dystrophy, deafness, dilated cardiomyopathy, hyperinsulinaemia, hypertriglyceridemia Bardet Biedl More than AR + oligogenic Obesity, cone-rod dystrophy, polydactyly, Syndrome 20 loci hypogonadism, renal malformations and cognitive deficits More than AR Cerebellar vermis hypoplasia, brainstem 30 loci malformation, episodic hyperpnoea/ apnoea, ataxia, global developmental delay, polydactyly, retinitis pigmentosa, renal cysts Meckel – Gruber More than AR CNS malformations especially occipital Syndrome 10 loci encephalocele, renal cysts, hepatic cysts, polydactyly Orofaciodigital OFD1 X-linked Malformations of face, oral cavity and digits, syndrome type 1 polycystic kidney disease Nephronophthisis Around 20 AR Medullary cystic kidney disease loci Senior Loken Around 9 AR Nephronophthisis and retinal degeneration syndrome loci AD Polycystic kidney PKD1 & AD Adult onset polycystic kidney disease disease PKD2 AR Polycystic kidney PKHD1 AR Infantile onset polycystic kidney disease, disease polycystic liver disease Leber congenital Around 18 AR Retinal dystrophy amaurosis loci Short-rib thoracic Around 17 AR Narrow thorax, polydactyly, hepatorenal cystic dysplasia with or loci changes without polydactyly spots, axillary freckling, macrocephaly, a Noonan- mental retardation or learning disabilities, car- like facial appearance and learning difficulties. diac defects (particularly pulmonary valve stenosis Although the phenotype closely resembles NF1, and hypertrophic cardiomyopathy), facial dysmor- some typical NF1 features such as Lisch nod- phism, short stature, relative macrocephaly, skin ules in the iris, neurofibromas and CNS tumours abnormalities and an increased risk for malignancy are absent. These cases do not have NF1 or (Table 1). This similarity is easily explained by the NS mutations. Heterozygous loss-of-function their being related to molecular defects involving mutations in the SPRED1 gene (Sprouty Related hyper-activation of a common pathway (Figure 4). Protein with EVH1 domain) have been identified However, the reasons for the significant pheno- in these cases. Spred-1 is a negative regulator of typic variation, inspite of the overlap, between RAF and truncating mutations result in increased these conditions are not fully understood. The RAF1-kinase activity and increased phosphorylated possible mechanisms for the differences in pheno- levels of MEK and MAPK. type could be the differential expression patterns Thus, all the neuro-cardio-facial-cutaneous of mutated genes, different mechanisms of dis- (NCFC) syndromes share considerable phenotypic turbing RAS signalling through specific interactions similarity which includes a variable degree of between effector and regulatory proteins for differ-

167 Chapter 26 ent mutants, involvement of feedback mechanisms signal transduction pathways and thereby in the that affect only upstream molecules (like RAS) but development of many tissue types including the not downstream molecules and the possible ef- kidney, brain, liver, eyes and bone. Many of the fects of the effector molecules on pathways other ciliopathies have hepato-renal cystic changes and than the MAPK pathway (Denayer et al., 2008). retinal degeneration (Table 2) (Adams et al., 2008). While germline mutations have been reported These prototypal pathway disorders demon- in the RAS genes in many of the NCFC syndromes, strate that ‘lumping’ of overlapping phenotypes gain of function somatic mutations in RAS genes, can provide an important clue to the molecular especially KRAS, are found in 20 – 30% of all etiology of diseases. This approach of analysing tumours. However, the somatic KRAS mutations overlapping clinical phenotypes for a common reported in malignancies are different from the etiological pathway can be used to study other germline KRAS mutations found in Noonan and syndromes that share similar clinical features. CFC syndromes. The explanation for this could be that the mutations associated with malignancies References are so strongly activating that they are incompat- ible with normal embryogenesis and cause early 1. Adams M, et al. Recent advances in the molec- embryonic lethality. ular pathology, cell biology and genetics of ciliopathies. J Med Genet 2008; 45: 257-267. 2. Aoki Y, et al. Germline mutations in HRAS proto- Molecular Diagnosis oncogene cause Costello syndrome. Nat Genet Molecular diagnosis can be done by Sanger se- 2005; 37: 1038-1040. quencing of the implicated gene, based on clinical 3. Denayer E, et al. Clinical and molecular aspects diagnosis or by targeted panel testing using next of RAS related disorders. J Med Genet 2008; 45: generation sequencing technology. Molecular di- 695-703. agnosis is essential for counseling and providing 4. Digilio MC, et al. Grouping of multiple- prenatal diagnosis. lentigines/LEOPARD and Noonan syndromes Knowledge of the pathway defects involved in on the PTPN11 gene. Am J Hum Genet 2002; 71: the causation of these disorders provides potential 389-394. molecular targets for therapy. Tyrosine kinase 5. Lopez-Rangel E. Overlapping clinical pheno- inhibitors are already being used successfully in types: the road to identifying dysmorphology cancer therapy e.g. the RAF-inhibitor sorafenib signalling pathways and their associated risks. is beneficial in renal cell and hepatocellular car- Clin Genet 2007; 71: 43-44. cinoma. In vitro studies have shown a response 6. Pandit B, et al. Gain-of-function RAF1 mutations to MEK inhibition for different mutated proteins cause Noonan and LEOPARD syndromes with found in the CFC syndrome. (Senawong et hypertrophic cardiomyopathy. Nat Genet 2007; al.,2008). Mouse studies have shown that learning 39: 1007-1012. problems caused by NF1 knockout can be reversed 7. Rauen KA. The RASopathies. Annu Rev Genomics by farnesyl-transferase inhibitors and statins that Hum Genet 2013; 14: 355-369. inhibit RAS hyperactivation by reducing the level 8. Roberts AE, et al. Germline gain-of-function mu- of isoprenylated RAS. Clinical studies assessing tations in SOS1 cause Noonan syndrome. Nat the effect of statins on cognition in NF1 patients Genet 2007; 39: 70-74. are underway. If these studies show any ef- 9. Rodriguez-Viciana P, et al. Germline mutations fect, this type of pharmacological treatment might in genes within the MAPK pathway cause cardio- also be studied in the larger group of RAS-MAPK facio-cutaneous syndrome. Science 2006; 311: syndromes. 1287-1290. 10. Schubbert S, et al. Germline KRAS mutations Other Groups of Pathway Disorders cause Noonan syndrome. Nat Genet 2006; 38: 331-336. Ciliopathies, which are disorders of primary cilia, 11. Senawong T, et al. Germline mutations of MEK constitute another important example of pathway in cardio-facio-cutaneous syndrome are sensi- disorders with a significant degree of overlap in tive to MEK and RAF inhibition: implications for their phenotypes. Cilia are vital cellular com- therapeutic options. Hum Mol Genet 2008; 17: ponents that play a significant role in diverse 419-430.

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Inherited Disorders of DNA Repair

Lekshmi Nair, Surya Prabha B, Prajnya Ranganath Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad

Correspondence to: Dr Prajnya Ranganath Email: [email protected]

Abstract Base excision repair (BER): This is carried out• by lesion-specific DNA glycosylases which re- move the wrong base by cleaving the N-glycosidic DNA repair disorders are inherited disorders re- bond linking the base to its corresponding de- sulting from defective DNA repair mechanisms of oxyribose, leading to the production of an abasic the cell, most of which have an autosomal recessive site. pattern of inheritance. Majority of these disorders are associated with immunodeficiency states and Mismatch repair (MMR): This mechanism an increased predisposition to malignancies. This is mostly• involved in post-replication correction article reviews the underlying molecular pathology of wrongly incorporated bases which have es- of these conditions and the clinical approach to the caped the proof-reading activity of replication relatively common, well characterized disorders in polymerases and in correction of insertion/dele- this group. tion loops which result from polymerase slippage during replication of repetitive DNA sequences. MMR includes three main components - a recog- Introduction nition step where incorrectly paired bases are recognized; an excision step where the error- DNA repair disorders are inherited disorders that containing strand is degraded resulting in a gap; result from defects in the DNA repair mechanisms and a repair synthesis step, where the gap is filled of the cell. These disorders usually arise from by the resynthesis of DNA. mutations in the ‘caretaker genes’ that protect Nucleotide excision repair (NER): NER in- the genome from damage due to radiation, free volves the steps of DNA damage recognition, local radicals and exogenous chemicals or are involved • opening of the DNA helix around the lesion, ex- in the correction of replication errors. There is cision of a short single-strand segment of DNA a very high predisposition to cancer and immune spanning the lesion, and sequential repair syn- deficiency in most of these conditions. Majority of thesis and strand ligation. Global genome NER these disorders are transmitted in an autosomal (GG-NER) is responsible for repair of DNA lesions recessive manner. throughout the genome, while Transcription cou- pled NER (TC-NER) is involved in correcting defects Normal DNA Repair Mechanisms on the coding strand of actively transcribed genes. Double-strand break repair (DSBR): DSBR DNA damage can occur due to a number of envi- occurs• through both homologous recombination ronmental agents including ionizing and ultraviolet (HR) and non-homologous end joining (NHEJ). In radiation, free radicals, and exogenous chemicals HR-mediated repair, the normal, undamaged sister such as alkylating agents and polycyclic aromatic chromatid is used as a template, whereas in NHEJ hydrocarbons. Errors in DNA replication can direct ligation of the broken ends occurs. NHEJ also lead to base mismatch and insertion/deletion mediated repair is much more prone to errors of bases. DNA repair mechanisms protect the when compared to HR-directed double stranded integrity of the genome by repairing the DNA dam- break repair. age and correcting the replication errors (Clancy, Figure 1 provides an overview of the DNA repair 2008; Dexheimer, 2013). The major DNA repair mechanisms. The genes that code for the various mechanisms found in mammalian cells are: proteins involved in each of these DNA repair

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Table 1 Proteins involved in the DNA Repair mechanisms.

DNA Repair Mechanism Proteins/ protein complexes involved Base excision repair DNA glycosylases, APEX1, XRCC1, PNKP, TDP1, APTX, DNA polymerases β, δ and ε, FEN1, PCNA, Replication factor subunits, Poly (ADP-Ribose) polymerases. Mismatch repair MutSα (MSH2-MSH6 heterodimer), MutSβ (MSH2-MSH3 heterodimer), MutLα (MLH1-PMS2 heterodimer), MutLβ (MLH1-PMS2 heterodimer), MutLγ (MLH1-MLH3 heterodimer), Exonuclease 1, PCNA, Replication factor subunits. Nucleotide excision repair XPC-Rad23B-CEN2 complex, DDB1-XPE complex, CSA, CSB, TFIIH, XPB, XPD, XPA, RPA, XPG, ERCC1- XPF, DNA polymerase δ or ε. Double strand break repair: - Homologous recombination Mre11-Rad50-Nbs1 complex, CtIP, RPA, Rad51, Rad52, BRCA1, BRCA2, Exonuclease1, BLM-TopIIIα complex, GEN1-Yen1 complex, Slx1- Slx4 complex, Mus81/Eme1. - Non-homologous end-joining Ku70-Ku80 complex, DNA-PKc complex, XRCC4-DNA ligase IV complex, XLF. mechanisms are listed in Table 1 (Dexheimer, Clinical features 2013). Most of the inherited disorders of DNA repair are associated with a strong predisposition to malig- nancies (Tomaszewska et al., 2006). Additionally, many of them have associated immunodeficiency, which could be attributable to the fact that DNA repair proteins are involved in joining the double- stranded breaks between the variable (V), diversity (D), and joining (J) segments of the lymphocyte immunoglobulin and antigen receptor genes. Fea- tures of ‘early aging’ (progeroid characteristics) are also seen in many of the disorders in this group. The clinical features of some of the common DNA repair defects are listed hereunder. Cockayne Syndrome: Cockayne syndrome (CS)• is a spectrum of disorders. Patients with CS type I (classic or moderate type) have growth Figure 1 DNA repair mechanisms. retardation, global developmental delay and mi- crocephaly since infancy, and thereafter develop cachectic dwarfism, cutaneous photosensitivity, Defects in DNA Repair Mechanism pigmentary retinopathy, cataracts, sensorineural hearing impairment, and progressive central and Germline mutations in the genes that code for the peripheral nervous dysfunction including demyeli- various proteins involved in the DNA repair path- nating peripheral neuropathy and leukodystrophy ways (listed in Table 1) result in different disorders, (Figure 2a & 2b). CS type II (severe or early-onset CS) which are referred to as inherited disorders of DNA manifests as growth failure from the intrauterine repair. The relatively common DNA repair disor- period with severe postnatal neurologic impair- ders along with their molecular basis are listed in ment and profound global developmental delay. Table 2. There is accelerated development of kyphoscolio-

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Table 2 Common Inherited Disorders of DNA Repair.

Disorder Genes Molecular mechanism associated Disorders of Nucleotide Excision Repair Cockayne ERCC8 The proteins encoded by ERCC6 and ERCC8 help in syndrome ERCC6 transcription-coupled nucleotide excision repair (TC-NER) which removes ultraviolet radiation (UV)-induced pyrimidine dimers and other transcription-blocking lesions from the transcribed strands of the active genes. Mutations in these genes impair the TC-NER mechanism (Citterio et al., 2000). Xeroderma XPA The nucleotide excision repair genes XPB and XPD partially pigmentosum XPB/ ERCC3 unwind the DNA in the region of the damage for further XPC processing. The XPF product makes a single-strand nick at the 5’ XPD/ ERCC2 side of the lesion and the XPG product makes a similar nick on XPE/ DDB2 the 3’ side, releasing a region of approximately 30 nucleotides XPF/ERCC4 containing the damage. The resulting gap is filled by DNA XPG/ ERCC5 polymerase using the other (undamaged) strand as a template XPV/ POLH in a process involving proliferating cell nuclear antigen (PCNA). Mutations in these genes impair the global genome NER mechanisms (Arlett et al., 2006). Trichothiodys- ERCC2/ XPD XPB and XPD encode subunits of the TFIIH (transcription factor II trophy ERCC3/ XPB H) complex, which is included in nucleotide excision repair, in GTF2H5 RNA polymerase (RNA pol I & pol II) transcription initiation and MPLKIP regulation, and in cell cycle control. TFIIH opens the RNF113A double-stranded DNA around the defect, through the helicase GTF2E2 activity of XPD and the ATPase activity of XPB, the core subcomplex associates with NER-specific factors, including XPA, and mediates incision/excision of the damaged oligonucleotide. Mutations in these genes impair the global genome NER mechanisms. Cerebro-oculo- ERCC6 Similar mechanism as described above for Cockayne syndrome facial-skeletal ERCC2 and . (COFS) ERCC5 syndrome ERCC1

Disorders of DNA Mismatch Repair Hereditary MLH1 Germline pathogenic variant in a mismatch repair gene causes nonpolyposis MSH2 microsatellite instability (MSI) and disrupts the mismatch repair colorectal MSH6 (MMR) pathway carcinoma PMS2 (Lynch EPCAM Syndrome)

Muir Torre Syndrome – subtype of Lynch Syndrome

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Disorders of Double Strand Break Repair Ataxia ATM The ATM protein assists cells in recognizing double stand DNA telangiectasia breaks and activates enzymes which repair the breaks in the strand. Without this protein, cells become unstable and undergo apoptosis. Nijmegen NBN The NBN protein product nibrin directs the NBN/Mre11/Rad50 breakage (MRN) complex to the sites of double-strand breaks and syndrome interacts with ATM kinase to coordinate cell cycle arrest with DNA repair. Mutation in the NBN gene impairs the formation of the MRN complex (Carney et al., 1998). Others Bloom RECQL3 Syndrome The RecQ helicases unwind the double helix of the DNA molecule during replication as well as DNA repair. Without these helicases, the cell is not able to efficiently repair the DNA Rothmund- RECQL4 damage caused by ultraviolet light and other damaging agents. Thomson Mutations in these genes lead to an increase in the frequency of syndrome sister chromatid exchanges (around 10 times higher than Werner RECQL2 average) and increase in the frequency of chromosome syndrome breakages (Monat, 2010; Friedrich et al., 2010).

Fanconi FANCA, FANCB, The proteins produced by these genes form the FA pathway Anemia FANCC, FANCD2, which is involved in the repair of DNA damage caused by FANCE, FANCF, interstrand cross links (Dutrillaux et al., 1982). FANCG, FANCI, BRCA2, BRIP1 sis and other joint abnormalities. CS type III (mild or late-onset CS) has normal growth and cognitive development with late onset symptoms. Man- agement includes , antispasticity medications, use of sun screens and sun glasses for skin and lens/retina sensitivity, dental care, and management of hearing loss and cataract.

Xeroderma pigmentosum: Xeroderma pig- mentosum• (XP) presents with freckles over of the face during infancy, excessive sun sensitiv- a) b) ity (sunburn with blistering, persistent erythema) with marked, sunlight-induced ocular involvement (photophobia, keratitis) and high risk of sunlight- Figure 2 a) Cachectic look, deep-set eyes and induced cutaneous neoplasms like basal cell car- cutaneous scars in a patient with Cock- cinoma, squamous cell carcinoma and melanoma ayne syndrome; b) Evidence of cerebral (Greenhaw et al., 1992). Around one-fourths of the atrophy and white matter hyperinten- affected individuals have acquired microcephaly, sities in MRI Brain FLAIR image of the progressive hearing loss, and cognitive impair- patient. ment. Death is commonly due to skin cancer and neurologic degeneration. XP associated with Ataxia Telangiectasia: Classic ataxia- severe neurologic impairment, stunted growth and telangiectasia• (A-T) also known as Louis Bar syn- delayed secondary sexual development is called drome manifests as progressive cerebellar ataxia, the DeSanctis-Cacchione variant. oculomotor apraxia, choreoathetosis, telangiec-

172 Chapter 27 tasias of the conjunctivae, and immunodeficiency structive pulmonary disease, diabetes mellitus and causing frequent infections (Lin et al., 2014). There increased risk of malignancies are also associated is increased risk of hematological malignancies. with this condition. The management is usually The initial symptom is usually cerebellar ataxia symptomatic and supportive. which starts around 1 to 4 years of age and majority of them become wheelchair bound by adolescence. A-T patients are highly sensitive to ionizing radiation, like medical X rays. The non- classic forms of A-T include adult-onset A-T and A-T with early-onset dystonia. Apart from physical therapy and supportive care, monitoring for severe infections and malignancies and aggressive treat- ment of infections, with IVIG wherever required, is recommended. Fanconi Anemia: People with this condition may• have bone marrow failure, physical abnormal- ities like radial ray defects, short stature, organ defects like malformed or absent kidneys, other defects of the urinary tract, gastrointestinal abnor- malities, cardiac defects and an increased risk of certain malignancies. Skin may show hyperpig- Figure 3 Loss of subcutaneous fat and sun- mentation or café-au-lait spots. Other symptoms sensitive lesions on the face in a patient are small abnormally shaped eyes and malformed with Bloom syndrome. ears with hearing loss. They may have abnor- mal internal and external genitalia and could be Nijmegen breakage syndrome: Nijmegen infertile. Central nervous system abnormalities breakage• syndrome manifests with growth retar- include hydrocephalus and microcephaly. There dation, short stature, progressive reduction in head is excessive risk of developing malignancies like circumference and developmental delay. There is acute myeloid leukemia and tumors of skin, gas- associated progressive cognitive decline. Humoral trointestinal system, genital tract and tumors of immunodeficiency results in recurrent respiratory head and neck. Patients with symptomatic anemia tract infections (Chrzanowska et al., 2012). There may require blood transfusions. Erythropoetin and is increased risk of malignancies like colony stimulating factors could increase the blood and also predisposition to tumors such as medul- count and give temporary relief, but bone marrow loblastoma and glioma. The immunodeficiency transplantation is usually required to definitively and malignancies often result in early deaths. manage the aplastic anemia. Caution is required Affected females may develop premature ovarian in the management of these patients as they have failure. Treatment is supportive and symptomatic. an increased risk of adverse events and toxicity IVIG can be given for severe humoral immunod- related to chemotherapeutic and immunosuppres- eficiency causing frequent infections. Vitamin E sive therapies. Though most types of Fanconi and folic acid supplementation are recommended. anemia have an autosomal recessive inheritance For malignancies, standard anti malignancy agents pattern, RAD51-related FA has an autosomal dom- and hematopoietic stem cell transplantation are inant inheritance pattern and FANCB-related FA is preferred. Periodic follow-up to monitor growth inherited in an X-linked manner. and development and management of frequent Bloom syndrome: Bloom Syndrome mani- infections is required for these patients. fests• as severe intrauterine and postnatal growth Hereditary nonpolyposis colorectal carci- deficiency, loss of subcutaneous fat, short stature noma• (Lynch syndrome): Patients with Lynch and sun-sensitive skin lesions of the face (Figure syndrome develop colorectal cancer and other 3). Gastroesophageal reflux is common and causes HNPCC-typical tumors like stomach, small in- recurrent respiratory tract infections. Learning testine, endometrial carcinomas, cervical, breast disability is often seen. Affected males are usu- cancers, bladder cancers (Lynch & Lynch, 2005). ally infertile, while females may be fertile but Periodic surveillance of people with HNPCC with usually have premature menopause. Chronic ob- colonoscopy and surgical removal of the tumor

173 Chapter 27 is the available management at present. The moval of cataracts and treatment for malignancies Muir-Torre variant of Lynch syndrome presents as per standard protocols. Use of sunscreens with with neoplasms of the skin like sebaceous ade- both UVA and UVB protection is recommended to nomas, sebaceous carcinomas, and keratoacan- prevent skin cancers and calcium and vitamin D thomas along with the other internal malignancies supplements are recommended for osteopenia. usually associated with Lynch syndrome. Unlike the other DNA repair disorders, Lynch syndrome has an autosomal dominant pattern of inheritance and presymptomatic screening of at-risk family members is essential, for appropriate surveillance and presymtomatic therapeutic intervention of the other mutation – carrying family members (Hegde et al., 2014). Trichothiodystrophy: Trichothiodystrophy (TTD)• is characterized by brittle, sulfur-deficient Figure 5 Poikiloderma congenita and radial ray hair that shows a typical trichorrhexis nodosa defect (bilateral hypoplastic thumbs) pattern on light microscopy and an alternating in a patient with Rothmund-Thomson light and dark banding pattern called ‘tiger tail syndrome. banding’ under polarizing microscopy (Figure 4). Patients with TTD can have a wide variety of clin- : Werner syndrome is ical features including cutaneous manifestations, characterized by signs of premature and neurologic impairment, and growth abnormalities. • predisposition to cancer. Symptoms usually start Ichthyosis, intellectual disability, reduced fertility, after the second decade of life with graying of hair, ocular abnormalities, short stature, and recurrent excessive hair loss, voice change, short stature infections are associated features. The disorder and scleroderma-like skin lesions. Later, the pa- has both photosensitive and non-photosensitive tients develop cataract, hypogonadism, diabetes, forms. Unlike the other DNA repair disorders, ulcers of skin, atherosclerosis and patients with TTD have not been reported to have by around 30 years. Myocardial infarction and a predisposition to cancer. malignancies are the common causes of death. The mainstay of management is control of dia- betes mellitus, use of cholesterol-lowering drugs for abnormal lipid profile, treatment of skin ulcers, surgical treatment of cataracts and management of malignancies as per standard protocols. Diagnosis

The diagnosis of these condition is established Figure 4 ‘Tiger tail banding’ seen in polarizing by a combination of clinical examination findings, microscopy in a patient with Trichoth- laboratory investigations, and definitive molecular iodystrophy. genetic testing. Dysmorphology evaluation and detailed systemic examination may offer impor- Rothmund-Thomson syndrome: This condi- tant clues to the diagnosis as mentioned in the tion• is associated with poikiloderma congenita, individual disease sections above. growth retardation, alopecia, photosensitivity, nail Chromosomal breakage studies have conven- dystrophy, teeth anomalies, cataract and hypog- tionally been used for the diagnosis of disorders onadism. Skeletal anomalies such as radial such as Fanconi anemia and Nijmegen breakage ray defects, ulnar defects, absent or hypoplastic syndrome, which are associated with an increased patella, and/ or osteopenia may occur in these number of cross linking agent-induced chromoso- patients (Figure 5). They have an increased risk mal breakages. For this study, T lymphocytes in a of skin cancers and other malignancies. Man- peripheral blood sample are cultured in the pres- agement includes pulsed dye laser to treat the ence of a cross-linking agent such as Mitomycin-C telangiectatic component of the rash, surgical re- or Diepoxybutane at varying concentrations, af-

174 Chapter 27 ter which chromosomal aberrations are quantified testing and prenatal diagnosis may be offered to in metaphase spreads. The chromosomal aber- at-risk families, as applicable, once the underlying rations seen include chromatid gaps, chromatid disease-causing mutations are identified in the breaks, tri and quadri radials, and complex inter- proband. change figures (Figure 6). More than ten breaks per cell is usually considered significant and is strongly Management suggestive of a chromosomal breakage syndrome (Anneke et al., 2012). There is no definite disease-specific curative ther- apy available for these disorders at present. Symp- tomatic and supportive treatment remains the mainstay of management of these disorders. Ag- gressive treatment is essential for the recurrent infections resulting from immunodeficiency and in- travenous immunoglobulin (IVIG) may be required in severe infections. Surveillance for and early detection and early surgical/ medical management for malignancies is required for most of these con- ditions. Conditions like Fanconi anemia and Ataxia telangiectasia are associated with higher toxicity and adverse reactions to chemotherapy and ra- diotherapy and HSCT-related immunomodulatory regimens; therefore, caution and appropriate dose- adjustment is required for the same. Conditions with photosensitivity and sunlight-damage such Courtesy: Dr Shubha R Phadke, Professor & Head, as Xeroderma pigmentosum, Cockayne syndrome Department of Medical Genetics, SGPGIMS, Lucknow. and Rothmund-Thomson syndrome require use of Figure 6 Mitomycin-C induced chromosomal sunscreens and protective clothing, and avoidance of sun exposure. breakage study showing chromosomal aberrations including chromatid gaps, Newer Therapeutic Strategies: Gene ther- chromatid breaks, tri and quadri radi- apy• trials are ongoing for Fanconi anemia and als. a Phase I study of the antioxidant quercetin in children with Fanconi anemia is also currently un- Flow cytometry is now a preferred alternative derway. In one study on patients with Xeroderma test for studying chromosomal breakages, since it pigmentosum, the bacterial DNA repair enzyme T4 does not require setting up of cultures and has a endonuclease V in a topical liposome-containing rapid turn-around time. It also eliminates the need preparation has been found to reduce the fre- for technical expertise and rules out intra-observer quency of new actinic keratoses and basal cell variations seen in scoring. It is based on the princi- carcinomas (Yarosh et al., 2001), but this treat- ple that cells are arrested at late S/early G2 phase ment has not yet been approved by the US Food of the cell cycle when exposed to cross linking and Drug Administration (FDA). Oral vismodegib, agents and therefore the fraction of arrested cells an inhibitor of the hedgehog pathway, has been can be taken as a measure of their sensitivity to approved by the FDA for treatment of metastatic cross linking agents. basal cell carcinoma or locally advanced basal cell Definitive diagnosis is most often possible carcinoma that has recurred following surgery or in through molecular genetic testing of the known individuals not fit for surgery or for radiation ther- associated genes. As most of these disorders are apy. For ataxia telangiectasia, antioxidants (vitamin genetically heterogeneous and the causative genes E or alpha-lipoic acid) are recommended, although are large genes, next generation sequencing-based no formal testing for efficacy has been conducted multigene panel testing is the preferred modal- in affected individuals. Stop codon read-through ity for molecular diagnosis, unless the patient is with aminoglycosides and Antisense morpholino from an ethnic population, with a high frequency oligonucleotides have shown some therapeutic carrier rate for a specific mutation, where a tar- benefit in animal models of A-T. Steroid therapy geted testing may be applied. Presymptomatic with dexamethasone or betamethasone, especially

175 Chapter 27 when delivered by loading into erythrocytes, has syndrome (NBS). Orphanet J Rare Dis 2012; 7: been found to be beneficial in reducing the neuro- 13. logical manifestations of A-T and Phase III clinical 5. Citterio E, et al. ATP-Dependent Chromatin trials are ongoing for this modality (Leuzzi et al., Remodeling by the Cockayne Syndrome B DNA 2015). Repair-Transcription-Coupling Factor. Mol Cell Biol 2000; 20: 7643-53. Genetic Counseling 6. Clancy S. DNA damage & repair: mecha- nisms for maintaining DNA integrity. Nature As most of the conditions in this group have an Education 2008; 1:103. autosomal recessive pattern of inheritance, the 7. Dexheimer TS. DNA repair pathways and mech- parents will usually be obligate carriers and the anisms. In: DNA Repair of Cancer Stem Cells recurrence risk in siblings of affected individuals Mathews LA et al. (eds.). doi: 10.1007/978-94- would be 25%. Lynch syndrome has an autosomal 007-4590-2_2, © Springer Science + Business dominant pattern of inheritance with a 50% risk Media Dordrecht 2013. of recurrence in each offspring and sibling of 8. Dutrillaux B, et al. The cell cycle of lymphocytes an affected individual and therefore appropriate in Fanconi anemia. Human Genet 1982; 62: pre-symptomatic testing and surveillance of at-risk 327-32. family members is essential. Female heterozygous 9. Friedrich K, et al. WRN mutations in Werner carriers of ataxia telangiectasia-related ATM gene syndrome patients: genomic rearrangements, mutation and the Fanconi anemia-related FANCD2, unusual intronic mutations and ethnic-specific FANCN and FANCJ gene mutation have increased alterations. Hum Genet 2010; 128:103-11. susceptibility to breast cancer and should be rec- 10. Greenhaw GA, et al. Xeroderma pigmentosum ommended surveillance for the same. In addition, and Cockayne syndrome: overlapping clini- heterozygous carriers of ATM gene mutation can cal and biochemical phenotypes. Am J Hum have increased radiation sensitivity and increased Genet1992; 50: 677-89. risk of toxicity of radiotherapy and higher-dose di- 11. Hegde M, et al. ACMG technical standards agnostic radiation exposure and should be warned and guidelines for genetic testing for inherited about the same. colorectal cancer (Lynch syndrome, familial adenomatous polyposis, and MYH-associated Conclusion polyposis). Genet Med 2014;16: 101-16. 12. Leuzzi V, et al. Positive effect of DNA repair disorders are a genetically heteroge- erythrocyte-delivered dexamethasone in neous group of diseases and accurate diagnosis of ataxia-telangiectasia. Neurol Neuroimmunol these conditions can help in appropriate surveil- Neuroinflamm 2015; 2: e98. lance and management of not just the affected 13. Lin DD, et al. Cerebral Abnormalities in Adults individuals but also their family members. with Ataxia-Telangiectasia. AJNR Am J Neurora- diol 2014; 35: 119-23. References 14. Lynch HT, Lynch JF. What the physician needs to know about Lynch syndrome: an update. 1. Anneke B, et al. Diagnosis of Fanconi Ane- Oncology (Williston Park) 2005; 19: 455-63. mia: Chromosomal Breakage Analysis. Anemia 15. Monnat RJ Jr. Human RECQ helicases: Roles 2012: 238731. doi: 10.1155/2012/238731. in DNA metabolism, mutagenesis and cancer 2. Arlett CF, et al. Clinical and cellular ionizing ra- biology. Semin Cancer Biol 2010; 20: 329-39. diation sensitivity in a patient with xeroderma 16. Tomaszewska A, et al. Chromosome instabil- pigmentosum. Br J Radiol 2006; 79: 510-7. ity syndromes. Pol Merkur Lekarski 2006; 20: 3. Carney JP, et al. The hMre11/hRad50 protein 577-81. complex and Nijmegen breakage syndrome: 17. Yarosh D, et al. Effect of topically applied T4 linkage of double-strand break repair to the endonuclease V in liposomes on skin cancer cellular DNA damage response. Cell 1998; 93: in xeroderma pigmentosum: a randomised 477-86. study. Xeroderma Pigmentosum Study Group. 4. Chrzanowska KH, et al. Nijmegen breakage Lancet 2001; 357: 926-9.

176 Chapter 28

Distal Arthrogryposis

Siddram J Patil Centre for Molecular and Metabolic Diagnostics & Research, Narayana Hrudayalaya Hospitals, Bangalore, India

Correspondence to: Dr S J Patil Email: [email protected]

Introduction Definition and Diagnostic criteria

Congenital multiple joint contractures involving According to Bamshad et al., DA are defined as two or more body areas are collectively termed disorders predominantly affecting the limbs in the as arthrogryposis. Based on the extent of joint form of congenital joint contractures in two or involvement (proximal and/or distal joints), neu- more body areas without any underlying neuro- rological involvement, and involvement of other logic and/or muscle pathology (Bamshad et al., parts of the body/other organs the etiology and 1996). prognosis of arthrogryposis differs (Hall, 1985). Various etiologies of arthrogryposis include sin- gle gene mutations, chromosomal anomalies, and intrauterine environmental factors (e.g. fetal crowding, failed termination of pregnancy) (Hall, 1985; Hall, 2012). Distal arthrogryposis (DA) are a subset of arthrogryposis characterized by con- genital joint contractures predominantly affecting joints of the hands and feet (distal joints) with or without associated anomalies (Bamshad et al., 1996). Here we discuss DA – its types, clinical presentation, inheritance and genes, prognosis and genetic counseling.

Figure 2 Ulnar deviation of fingers with con- tractures, faint flexion creases and ad- ducted thumb.

A diagnosis of DA should be considered in any child with predominant involvement of joints of the hands and feet, presenting with upper extremity features of clenched fingers at birth (Figure 1), ulnar deviation of fingers, flexion contractures of fingers (Figure 2), and hypoplastic and/or absent flexion creases, and lower extremity features of clubfoot, calcaneovalgus deformities, metatarsus varus and/or vertical talus (prominent heels or rocker bottom feet) (Figure 3). There might be Figure 1 Clenched hand at birth. variable involvement of proximal joints of both the upper limb and lower limb. Based on the asso-

177 Chapter 28 ciated anomalies in systems other than skeletal code for contractile proteins of fast-twitch muscle system (face, eye, spine and other organ systems), fibers (Bamshad et al., 1996). DAs are further classified into subtypes (Bamshad et al., 1996). Classification The clinical features of DAs show marked in- terfamilial and intrafamilial variability. In some Based on the above definition and diagnostic cri- families a carrier might be completely unaffected teria, 10 types of DA have been identified. Table or might have mild clinical manifestations like 1 summarizes the clinical features and genetics of absent palmar flexion creases, toe contractures, different types of DA. mild flexion contractures of fingers noticed at birth etc. As suggested by Bamshad et al. the above mentioned definition/diagnostic criteria (two ma- Clinical features jor diagnostic criteria involving the upper limb and lower limb) of DA should hold true for at Majority of DAs are inherited as autosomal domi- least one member of the affected family and for nant disorders with variable clinical features (Hall, other family members mild clinical features can be 1985; Bamshad et al., 1996). DA type 1 is classified considered for the diagnosis. Further, molecular into type 1A and 1B based on the chromosomal genetic testing might help to confirm the diagnosis locus, otherwise clinically they are indistinguish- in mildly affected individuals and obligate carriers able. In DA type 1, around 98% of the cases (Bamshad et al., 1996). have typical manifestations of the hand and 88% have typical manifestations of the feet. The face is normal and there are no associated anomalies (Beals, 2005). DA type 2 is classified into 2A and 2B, which are differentiated by the facial fea- tures. Facial features of DA2A include deep-set downslanting eyes, hypertelorism, small mouth, puckered lips, H-shaped chin dimple, small nose, long philtrum, and mild micrognathia (Stevenson et al., 2006). Some of the other features in DA2A include scoliosis, hip dislocation, strabismus and cryptorchidism. Facial features in DA2B include triangular face, downslanting eyes, prominent na- solabial folds, small mouth and pointed chin. Variable clinical features include proximal joint involvement, short stature and short webbed neck (Beals 2005; Stevenson et al., 2006). In a recent article by Beck AE et al. mutations in four genes (TNNI2, TPM2, TNNT3 and MYH3) have been found to cause both DA1 and DA2B; therefore, authors Figure 3 Rocker bottom feet. suggest that DA1 and DA2B should be considered as a single disorder representing a spectrum of Incidence manifestations. On sequencing of these four genes, mutations were found in 29% of DA1 cases Incidence of arthrogryposis is around 1 in 3000 and 40% of DA2B cases (Beck et al., 2013). births (Hall, 1985). DA type 3 is characterized by finger contrac- tures and cleft palate. Variable features include Inheritance and Genes hip dislocation, patellar dislocation, talipes equino- varus, hearing impairment, scoliosis and limitation Most of the DAs are inherited as autosomal in elbow joint movements (Bamshad et al., 1996; dominant disorders with both interfamilial and Beck et al., 2013). intrafamilial variability (Hall, 1985; Bamshad et DA type 4 manifests with finger contractures al., 1996). Some of the DAs are also inherited and scoliosis. Variable features include intellectual as autosomal recessive disorders (McMillin et al., disability and limitation of elbow joint movements 2013). Most genes causing DA are the ones that (Bamshad et al., 1996; Beck et al., 2013).

178 Chapter 28

DA type 5 is inherited as both an autosomal an abnormal electroretinogram. Some cases are dominant and autosomal recessive disorder. Along known to have pulmonary hypertension secondary with finger contractures and feet involvement, eye to restrictive lung disease (Bamshad et al., 1996; findings are peculiar to patients with DA5. Variable Beck et al., 2013). Based on the eye and other eye features include ophthalmoplegia, strabismus, findings, DA5 has been classified into four subtypes ptosis, pigmentary maculopathy, keratoconus and (DA5A-D) (McMillin et al., 2013). DA5D is inherited

Table 1 Clinical characteristics, inheritance and the known causative genes of different types of DA.

Type of DA Clinical features Intelligence Synonyms Inheritance Genes DA type 1 Typical involvement Normal – AD# TNNI2, TMP2, (1A & 1B) of hands and feet TNNT3, MYH3, MYBPC1 (Beck et al., 2013; Gurnett et al., 2010) DA type 2A Typical involvement Normal Freeman-Sheldon AD MYH3 (Toydemir of hands and feet, syndrome et al., 2006) typical facies* DA type 2B Typical involvement Normal Sheldon-Hall AD TNNI2, TMP2, of hands and feet, syndrome TNNT3, MYH3 typical facies* (Beck et al., 2013; Gurnett et al., 2010) DA type 3 Finger contractures, May have Gordon AD – cleft palate, intellectual syndrome talipoequinovarus disability DA type 4 Finger contractures, May have – AD – scoliosis intellectual disability DA type 5 Finger contractures, Normal – AD, AR ECEL1 (AR) ophthalmoplegia, 휃 (McMillin et al., ptosis 2013) DA type 6 Finger contractures, Normal – AD – sensorineural hearing loss DA type 7 Trismus, Normal Trismus-pseudo- AD MYH8 (Toydemir camptodactyly on camptodactyly et al., 2006) dorsiflexion of wrist syndrome DA type 8 Typical involvement Normal – AD – of hands and feet, multiple pterygia, typical facies* DA type 9 Finger contractures, Normal Congenital AD FBN2 arachnodactyly, contractural external ear arachnodactyly deformity (Beals syndrome)

* For facial description see text. # AD – Autosomal dominant. Autosomal recessive. TNNI2 – Troponin I; TMP2 - Tropomyosin 2; TNNT3 – Troponin휃 T3; MYH3 – Myosin heavy chain 3; MYBPC1 – Myosin-binding heavy protein C; MYH8 - Myosin heavy chain 8; FBN2 – Fibrillin 2.

179 Chapter 28 as an autosomal recessive disorder. inheritance, the risk of recurrence in siblings of an DA type 6 is characterized by distal limb con- affected individual is 25%. Availability of next gen- tractures and sensorineural hearing loss (Bamshad eration sequencing-based multigene panel testing et al., 1996; Beck et al., 2013). has made molecular genetic testing for DA more DA type 7 is characterized by inability to open accessible and affordable. the mouth and flexion of fingers on dorsiflexion of Prenatal diagnosis can be achieved by ul- the wrist. Variable clinical features include talipes trasound (less accurate) and molecular genetic equinovarus, hip involvement, short leg muscles testing, if the underlying gene mutation is known and short stature (Bamshad et al., 1996; Beck et (more accurate). al., 2013). Author suggests readers the recent article on In DA type 8, along with typical manifestations in Arthrogryposis: Diagnostic approach to etiology, clas- the hands and feet, patients present with multiple sification, genetics and general principles by Dr Judith pterygia, scoliosis with vertebral segmentation de- G Hall published in E J Med Genet (2014) for updated fects, facial features, short neck and short stature. clinical approach and list of genes. Facial features include ptosis, downslanting eyes, low set ears, and high arched palate. It is an References autosomal dominant disorder, with most cases oc- curring sporadically due to de novo mutations. The 1. Bamshad M, et al. A revised and extended other conditions which present with similar clini- classification of the distal arthrogryposes. AmJ cal features include autosomal recessive multiple Med Genet 1996; 65: 277-281. pterygium syndrome (Escobar syndrome), X-linked 2. Beals RK. The distal arthrogryposes: a new multiple pterygium syndrome and a lethal form classification of peripheral contractures. Clin (Bamshad et al., 1996; Beck et al., 2013). Orthop Relat Res 2005; 435: 203-210. DA type 9 is characterized by finger contrac- 3. Beck AE, et al. Spectrum of mutations that tures and crumpled ears. Variable clinical features cause distal arthrogryposis types 1 and 2B. Am include scoliosis, limitation of elbow and hip joint J Med Genet A 2013; 161: 550-555. movements, valvular heart disease and talipes 4. Gupta PA, et al. Ten novel FBN2 mutations in equinovarus (Bamshad et al., 1996; Beck et al., congenital contractural arachnodactyly: delin- 2013). DA9 is caused by FBN2 (fibrillin 2) gene eation of the molecular pathogenesis and clin- mutations, with most of the mutations occuring ical phenotype. Hum. Mutat 2002; 19: 39-48. in exon 23 to exon 34. Some patients with the 5. Gurnett CA, et al. Myosin binding protein C1: severe form of DA9 can simulate neonatal Marfan a novel gene for autosomal dominant distal syndrome (Gupta et al., 2002). Crumpled ear is arthrogryposis type 1. Hum Mol Genet 2010; used as a hallmark to identify patients with DA9. 19: 1165-1173. 6. Hall JG. Genetic aspects of arthrogryposis. Clin Management and Genetic counseling Orthop Relat Res 1985; 194: 44-53. 7. Hall JG. Arthrogryposis (multiple congenital Management of patients with DA should involve contractures) associated with failed termina- various specialties (clinical genetics, orthopedics, tion of pregnancy. Am J Med Genet A 2012; neurology, pediatrics and physiotherapy). Joint 158A: 2214-2220. contractures and skeletal complications (scoliosis) 8. McMillin MJ, et al. Mutations in ECEL1 cause are managed by physical therapy and/or surgery. distal arthrogryposis type 5D. Am J Hum Genet Patients requiring surgery might face difficult intu- 2013; 92: 150-156. bation and are at risk of malignant hyperthermia 9. Stevenson DA, et al. Clinical characteristics and (documented in some of the subtypes). Inheritance natural history of Freeman-Sheldon syndrome. is autosomal dominant in most of the cases. Mu- Pediatrics 2006; 117: 754-762. tations can occur de novo (sporadic), in which case 10. Toydemir RM, et al. Mutations in embryonic the risk of recurrence in siblings is negligible. If myosin heavy chain (MYH3) cause Freeman- the mutation is inherited from one of the parents, Sheldon syndrome and Sheldon-Hall syn- the risk of recurrence is 50% for the siblings. drome. Nat Genet 2006; 38: 561-565. Molecular genetic testing and identification of the 11. Toydemir RM, et al. J. Trismus- causative gene mutation help in providing an ac- pseudocamptodactyly syndrome is caused by curate risk of recurrence and appropriate genetic recurrent mutation of MYH8. Am J Med Genet counseling. In cases with autosomal recessive A 2006; 140: 2387-2393.

180 Chapter 29

Osteogenesis Imperfecta: An Update

Vartika Giri, Priyanka Srivastava, Meenakshi Lallar and Shubha R Phadke ∗ Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India

Correspondence to: Dr Shubha R Phadke Email: [email protected]

Abstract sification by Sillence had four types depending on the clinical features and the severity. These groups Osteogenesis imperfecta (OI) is a rare, heritable can be distinguished as type I (mild OI, blue sclerae, systemic disorder of bone and connective tissue autosomal dominant inheritance), type II (lethal that has varying phenotypic heterogeneity -varying perinatal OI, autosomal recessive inheritance, later degree of bone fragility, blue sclerae, dentinogen- subdivided in II-A, -B, and -C based on radiographic esis imperfecta, short stature, scoliosis, and joint features, type III (progressively deforming autoso- hyperextensibility. For decades only autosomal mal recessive inheritance), and type IV (dominantly dominant forms caused by COL1A1 and COL1A2 inherited OI with normal sclerae). Aglan et al., 2012 genes were recognized. But since past decade proposed the scoring system for the assessment of with advancement in molecular technologies new clinical severity including 5 major criteria, namely, genes have been identified leading to recognition average number of fractures per year, motor mile of autosomal recessive OI. Currently there are stones, long bones deformities, length/height stan- around 17 genes for OI. The classification of OI dard deviation score and z-score of the mineral thus is now based on genetic analysis. In the bone density. following review we aim to discuss the different types of OI, classification based on molecular basis, Autosomal Dominant Osteogenesis different genes causative of OI and their patho- physiology and mechanisms. An approach to a Imperfecta suspected case of OI, differentials and the most recent management and therapeutic options for Most families of OI show a dominant pattern of OI are discussed. inheritance. OI type I to OI type IV account for 90% of all OI cases. Chu et al. in 1983 first reported Introduction the presence of an internal deletion of about 0.5 kb in one allele for pro-α1 (I) chain (COL1A1) in a Osteogenesis Imperfecta (OI) or “brittle bone dis- patient with OI. Since then about 1500 mutations ease” is a rare phenotypically and genetically in these genes have been known. Ninety percent heterogeneous group of heritable connective tis- of the cases are caused by heterozygous mutations sue disorders characterized by easy fracturing of in COL1A1 or COL1A2 genes. There is no genotype bones, with or without bony deformities, blue – phenotype correlation and mutations in either sclera, joint laxity and dentin defect. It shows COL1A1 or COL1A2 can cause any of the four types wide range of clinical presentation varying from of OI. In spite of identifying these causative genes a lethal perinatal form to a mild disorder (Figure in 1980s; the etiology of remaining 10% cases con- 1). Very mild case may only become evident in tinued to remain unknown for long. Recurrences adulthood, manifesting as premature osteoporo- in the families with severe types OI like type II or sis. The incidence of OI varies from approximately III were attributed to germ line mosaicism. One 1/15,000–20,000 (Marini et al., 1988) though no more gene for autosomal dominant varieties of data is available from India. OI other than COL1A1 and COL1A2 is IFTM5 for OI The earliest known patient with OI dates from V (Cho et al., 2012). OI type V has distinguish- about 1000 BC and appears to be an Egyptian in- able radiographic features of hypertrophic callus fant, evidence of which can be drawn from studying formation and ossification of introsseus membr- the remains of Egyptian mummy. The classical clas- ane of forarm bones. In one report from India,

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CRTAP gene mutation in homozygous form

Figure 1 Patients with Osteogenesis Imperfecta, showing clinical variability, characteristic clinical and radiological features. It has been observed that bisphosphonates may homozygous mutation in PLOD2 gene. exacerbate callus hyperplasia, and may therefore In addition two genes on X chromosome are have to be used with caution in patients with type identified as etiologies of OI; increasing the number V osteogenesis imperfect (Ranganath et al., 2016). of genes for OI to 18. Loss-of-function mutations in There are other phenotypes also which are caused PLS3 encoding plastin-3 were discovered as a cause by COL1A1 and COL1A2 mutations namely; caf- of one form of X-linked osteoporosis with fractures fey disease (infantile cortical hyperostosis), classic (van Dijk et al., 2013). Recently, Lindert et al., Ehlers-Danlos syndrome, EDS arthrochalasia type, 2016 identified an X-linked form of osteogenesis and osteoporosis. imperfecta in two independent pedigrees. Pheno- typic inheritance pattern, linkage analysis and next Autosomal Recessive Osteogenesis generation sequencing (NGS) were used to localize Imperfecta the causative gene in each family to MBTPS2 at Xp22. Recent developments have generated a new The first 4 subtypes of OI (clinical phenotypes) paradigm for OI and it has been found that a are caused by COL1A1 and COL1A2 genes. Further proportion of cases of OI are caused by the defects numbering upto XVII types (showing clinical over- in the genes which are involved in regulation of the lap with initial four subtypes) is included in OMIM synthesis of type I collagen pro alpha polypeptide based on the causative genes and all of them chains, proteins involved in type I collagen pro- are autosomal recessive inheritance except OI V. cessing in the endoplasmic reticulum and proteins Data from India shows that COL1A1 and COL1A2 involved in osteoblast function. These cases of mutations contribute to only 70% of cases with OI OI are inherited in autosomal recessive fashion. (Stephen et al., 2014). High level of consanguinity is The first gene for autosomal recessive OIwas probably the main contribute to the higher preva- identified in 2006 (Morello et al., 2006). This was lence of autosomal recessive OI. A small number CRTAP gene encoding cartilage-associated protein of cases with recessive OI are reported from India (CRTAP) and was found to be the cause of OI type (Stephen et al., 2015). The products of the genes VII. Presently, thirteen genes of recessive OI have for recessive types of OI are mostly enzymes which been identified (Table 1). CREB3L1 gene causes involves in the post translational modification of autosomal recessive form (OI XVI) and this is due the pre pro type I collagen produced from COL1A1 to the contiguous gene deletion on chromosome and COL1A2 genes. The functions of these genes 11p11 encompassing CREB3L1) (Symoens et al., include lysyl 3 hydroxylation, collagen processing 2013). Bruck syndrome is a syndromic form of and maturation, collagen stability and bone forma- OI associated with joint contractures caused by tion and homeostasis. More genes are still getting

182 Chapter 29 identified. These disorders being very rare most in osteoblast development like CREB3L1 and WNT1 of the publications report only a few cases. There also cause OI. CRTAP, LEPRE1 and PPIB are important is no large scale case series involving study of proteins of the complex involved in hydroxylation multiple genes. of propyl 3 complex which plays a critical role for As can be seen in table 1, the functions of the the proper collagen helix formation in the cell. genes causing OI are collagen synthesis, modifi- Improved understanding of functional pathways cation, folding and crosslinking. In addition to of collagen is paving ways to different treatment mutations in COL1A1 and COL1A2 genes, defects strategies.

Table 1 Classification of classic dominant and new recessive forms of Osteogenesis Imperfecta.

OI Defective Gene MIM Protein Name Protein function Clinical phenotype Type (Reference) No. Autosomal Dominant inheritance I COL1A1 (Mottes 120150 Collagen type I et al., 1990) alpha chain II COL1A1 or 120150 Collagen type I COL1A2 120160 and type II al- Alter the structure or (Mottes et al., pha chain re- quantity of type I Formation of triple 1990) spectively collagen and cause a helix of type I III COL1A1 or 120150 Collagen type I skeletal phenotype collagen COL1A2 120160 and type II al- ranging from (Mottes et al., pha chain re- subclinical to lethal 1990) spectively IV COL1A1 120150 Collagen type I or COL1A2 120160 and type II al- (Mottes et al., pha chain re- 1990) spectively V IFITM5 614757 Bone- Osteoblast formation Characterized by calci- (Cho et al., restricted in early mineraliza- fication of the forearm 2012) Ifitm-like (BRIL) tion stage interosseous membr- ane, radial head dislo- cation and hyperplas- tic callus formation Autosomal recessive inheritance VI SERPINF1 172860 Pigment- Inhibits osteoclast Characterized by re- (Becker et al., epithelium maturation by stimul- duced bone minerali- 2011) derived factor ating osteoprote- sation (PEDF) gerin (OPG) expres- sion VII CRTAP 605497 Cartilage- Components of the (Morello et al., associated collagen prolyl 2006) protein (CR- 3-hydroxylation Severe to lethal bone TAP) complex which plays dysplasia with VIII LEPRE1 610339 Prolyl- a critical role for the rhizomelia (Cabral et al., 3-hydroxylase proper collagen helix 2007) 1 (P3H1) formation in the cell IX PPIB 123841 Cyclophilin B Isomerisation of pep- Severe to lethal bone (van Dijk et al., (CypB) tidylprolyl bonds, cru- dysplasia with rhi- 2009) cial for proper colla- zomelia gen folding

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X SERPINH1 600943 Heat-shock Recognize and help Results in moder- (Christiansen et protein 47 maintain the folded ately severe form al., 2010) (HSP47) state of type I procol- of OI, characterized lagen trimer by osteopenia, bone fragility and skeletal deformities XI FKBP10 607063 FK506 binding Effects on procolla- Results in moder- (Alanay et al., protein, 65kDa gen through collagen ately severe form 2010) (FKBP65) modifying enzymes of OI, characterized by osteopenia, bone fragility and skeletal deformities XII SP7 606633 Osterix, tran- Important role in Characterized by re- (Lapunzina et scription factor bone formation current fractures, mild al., 2010) Sp7 (SP7) bone deformations, generalized osteo- porosis and delayed teeth eruption XIII BMP1 112264 Bone morpho- Functions as the Characterized by nor- (Asharani et al., genetic protein procollagencarboxy- mal teeth, faint blue 2012) 1 (BMP1) (C)-proteinase for sclera, sever growth types I to III procol- deficiency, borderline lagen; Play key role osteoporosis in ECM assembly and tissue patterning XIV TMEM38B 611236 Transmem- Functions as a mono- Characterized by vari- (Volodarsky et brane pro- valent cation channel; able degrees of sever- al., 2013) tein 38B affect Ca2+ home- ity of multiple frac- (TMEM38B) ostasis in the ER tures and osteope- nia, with normal teeth, sclera and hearing XV WNT1 164820 WNT1 Activates expression Characterized by (Keupp et al., of several genes im- early-onset recurrent, 2013) plicated in bone for- bone deformity, sig- mation nificant reduction of bone density, short stature XVI CREB3L1 616215 Old astrocyte An endoplasmic Characterized by os- [contiguous specifically in- reticulum-stress teopenia and sponta- gene deletion duced sub- transducer that alters neous fractures on chromosome stance (OASIS) the transcription of 11p11 target genes involved encompassing in developmental CREB3L1] process, differentia- (Symoens et al., tion or maturation 2013) upon mild ER-stress XVII SPARC 182120 Secreted pro- Expressed by os- Progressive osteo- (Mendoza- tein, acidic, teoblasts; binds to porosis due to defect Londono et al. cysteine-rich collagen type I and in bone formation 2015) (SPARC) other matrix proteins

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Bruck PLOD2 601865 Lysyl hydroxy- It encodes lysyl hy- Characterized by con- Syn- (Puig-Hervas et lase (LH2) droxylase 2 which genital contractures of drome al., 2012) also has a role in the the large joints (syn- hydroxylation of col- dromic lagen telopeptide ly- OI) sine X-linked inheritance X-linked MBTPS2 300294 Site-2 metallo- Sterol control of tran- Characterized by bow- reces- (Lindert et al., protease (S2P) scription and en- ing of upper and lower sive 2016) doplasmic reticulum extremities, prenatal OI (ER) stress response fractures and scoliosis X-linked PLS3 300131 Plastin-3 PLS3 is an actin- Characterized by de- osteo- (van Dijk et al., binding/bundling creased bone mineral porosis 2013) protein density (BMD)

Approach to a Case of OI mise and ambiguous genitalia or normal female external genitalia in most individuals with a 46,XY Clinical diagnosis of a most of the cases is relatively karyotype. easy and straightforward. Radiological evidence • Achondrogenesis - extremely short limbs with of decreased bone density along with history of short fingers and toes, hypoplasia of the thorax, repeated fractures and / or fractures with trivial protuberant abdomen, and hydropic fetal appear- trauma suggests the diagnosis. Presence or ab- ance caused by the abundance of soft tissue sence of blue sclera, dentiginous imperfecta, large relative to the short skeleton. open fontanelles, wormian bones and pre-senile Family history is important in the evaluation of deafness helps in the diagnosis and clinical classi- a case with suspected OI, as milder/mosaic/asymp- fication. Other causes of decreased bone density tomatic forms are difficult to detect clinically. A need to be ruled out by associated findings and three generation pedigree should be drawn and investigations (Table 2). the family should be asked for history of recurrent Differential diagnoses for antenatal cases: easy fractures, short stature, presenile deafness In utero, short and undermineralised bones, frac- in any of the family members. Presence of con- tures can be appreciated in utero and lead to sanguinity suggests the possibility of an autosomal suspicion of OI. The following conditions need to recessive disorder. In some cases the definitive be ruled alongwith– diagnosis of OI may be difficult and confirmation • Hypophasphatasia - characterized by defec- by mutation testing is essential. tive mineralization of bone and/or teeth in the presence of low activity of serum and bone alkaline Molecular Diagnosis phosphatase. Clinical features range from stillbirth without mineralized bone at the severe end to Mutation detection confirms the diagnosis and is pathologic fractures of the lower extremities in essential for genetic counseling regarding risk of later adulthood at the mild end. recurrence and preventing recurrence by way of • Thanatophoric dysplasia- neonatal lethal prenatal diagnosis. The heterogenic etiology and short-limbed dwarfing condition, well-ossified large sizes of genes makes mutation detection spine and skull, platyspondyly, ventriculomegaly, a complex process. The causative gene mostly narrow chest cavity with short ribs, polyhydram- cannot be predicted based on the clinical features nios, and bowed femurs (TD type I), cloverleaf skull as there is no genotype phenotype correlation. (kleeblattschaedel) (often in TD type II; occasionally Presence of hypertrophic callus and ossification of in TD type I) and/or relative macrocephaly. interosseous membrane is characteristic of OI type • Campomelic dysplasia- skeletal dysplasia char- V for which only one causative mutation (c.-14C T acterized by distinctive facies, Pierre Robin se- in IFITM5 gene) is reported in all the cases. The> quence with cleft palate, shortening and bowing of severity of the presentation also does not provide long bones, and club feet. Other findings include any clue the causative gene. Initially when COL1A1 laryngotracheomalacia with respiratory compro- and COL1A2 were the only known genes, collagen

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Table 2 Differential diagnosis postnatal.

Disorder Clinical Features Investigations Comments Pseudoglioma Bone fragility and Ophthalmological evaluation Intelligence is usually -Osteoporosis fractures, Narrow normal syndrome diaphysis, hypoto- (OMIM 259770) nia and eye abnor- malities that lead to vision loss Hypophoshata- Wide sutures, Decreased serum alkaline Perinatal, infantile, child- sia poorly formed phosphatase, hypercalcemia, hood and adult forms teeth, metaphy- hypercalciuria are known seal cupping, poorly formed teeth, bony spurs Disorders of Joint contractures, Osteolysis of carpal and May have renal dysfunc- osteolysis- Ha- pain in joints, gin- tarsals, acro-osteolysis tion jdu Cheney Syn- gival hyperplasia drome, etc. Thalassemia & Hepatosplenome- Anemia, high level of fetal Non-transfusion depen- other chronic gal, hemolytic or presence of dent thalassemia may hemolytic ane- facies abnormal hemoglobin present with fractures mias on trivial trauma and de- creased bone density Battered child Disturbed family Ruling out other causes is nec- High level of suspicion is situation, marks essary. Normal BMD. necessary of injury analysis was done to decide the possible causative Management gene. Later, DHPLC (Denaturing High Performance Liquid Chromatography) was used as a screening The basic serum chemistries- serum calcium, phos- technique to identify the location of mutation and phate, vitamin D and alkaline phosphatase are then the specific exons were sequenced. Such type normal in patients with OI. However these values of screening was essential as both the genes are might be slightly elevated after a fracture. But as large genes with 51 and 52 exons in COL1A1 and the patients started on bisphonates, grow older, COL1A2 respectively. Identification of newer genes have sedentary lifestyle due to bony deformities for OI has increased the complexities of molecular and fractures, the calcium and vitamin D levels fall. diagnosis. Next Generation Sequencing (NGS) is This can lead to delayed healing of fractures and of great help in this disorder associated with a increased bone fragility. So, it is recommended large number of genes, some of which have a to supplement calcium and vitamin D in patients large number of exons. A panel of all OI genes or with OI according to the age group. There is still sequencing of all genes of clinical relevance (Exome no “optimal treatment” for OI, both for limiting or sequencing) is the test of choice. The studies on preventing fractures and pain relief and improved the detection rates and relative contribution of var- mobility. Also, critical assessment of treatment ious genes to OI are still not available. Published outcomes is limited by the small numbers of par- data about mutations in genes other than COL1A1 ticipants in clinical trials and the short duration of and COL1A2 is limited worldwide and hence, more many trials, which is frequently limited to 1 or 2 information about phenotypes of recessive OI is years of observation. The following table provides needed. the list of various treatment modalities (Table 3).

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Table 3 Various treatment strategies. but there was no significant decrease in number of fractures (Orwoll et al., 2014). 3. Denosumab (anti-RANK-ligand antibody)- Existing treatment strategies: The RANK, RANKL complex regulates bone- 1. Bisphosphonates remodeling cycles by regulating osteoblast/osteo- 2. Teriparatide clast coupling and osteoclast differentiation. RANK 3. Denosumab is present on the osteoclast precursor, and RANKL New therapeutic approaches: produced by the osteoblast is part of the TNF 1. Antisclerostin antibody superfamily and, along with the soluble decoy re- 2. Cathepsin K antibody ceptor osteoprotegerin, are essential regulators of 3. Transforming growth factor-β osteoclast development and function. Denosumab 4. Prenatal and postnatal transplantation of is a human monoclonal antibody to RANKL; studies mesenchymal stem cells involving age-related osteoporosis have showed Multidisciplinary management the efficacy of denosumab in reducing signaling via 1. Orthopaedic treatment RANK, leading clinically to prevention of bone loss 2. Rehabilitation (Hoyer-Kuhn et al., 2014). Due to poor response to Bisphsphonates in OI Type VI, Denosumab was tried with success in Existing treatment strategies: OI Type VI leading to BMD increase, normaliza- 1. Bisphosphonates- Bisphosphonates, oral • tion of vertebral shape, and decrease in fracture and intravenous decrease osteoclastic bone re- rate. Denosumab treatment also improved BMD soprtion. Various studies have shown variable and longitudinal bone growth in two children efficacy of bisphosphonates on decreasing the with COL1A1/A2 mutations previously treated with fracture frequency and symptomatic relief of pain. Bisphosphonates. The variability of response may be due to differ- ential effects of bisphosphonates on various types New therapeutic approaches: of OI; the issue which may be solved with studies •1. Antisclerostin antibody - Sclerostin is a on mutation proved cases of OI. For example, negative regulator of bone formation released we found that bisphosphonates increased callus from osteocytes that modulates osteoblast activity formation in a case with type V OI (Dwan et al., acting through Wnt/β-catenin pathway. Preclin- 2014). ical studies have demonstrated that treatment The findings of the 2014 Cochrane review (Dwan with antisclerostin monoclonal antibody acts as et al., 2014) for oral and intravenous Bisphosph- osteoanabolic therapy improves bone mass and nates in OI can be summed up as follows- bone strength, and enhances repair of fractures in a. The oral or intravenous bisphosphonates in- animal models. crease bone mineral density in children and adults 2. Cathepsin K antibody - Cathepsin K is highly with OI, the effect not being different with the expressed in osteoclasts and is an essential en- different bisphosphonates. zyme involved in the degradation of type I collagen b. It is unclear whether oral or intravenous in the organic bone matrix. In an animal model, bisphosphonate treatment consistently decreases the cathepsin K monoclonal antibody (Odanacatib) fractures, though there is no increased fracture effectively suppressed bone resorption. A phase III rate. randomized, placebo-controlled trial assessed the c. The studies included in the Cochrane review effect of Odanacatib on fracture risk over 5 yearsof do not show bisphosphonates conclusively im- treatment in women with osteoporosis, has shown prove clinical status (reduce pain; improve growth increase in BMD and a significant reduction in the and functional mobility) in people with OI. risk of fractures. Applicability to the collagen defect 2. Teriparatide- Teriparatide is human recom- in OI remains to be determined. binant parathyroid hormone, which increases bone 3. Transforming growth factor-β - TGF-β is mass by increasing osteoblast bone formation. It produced by osteoblasts and acts to coordinate is highly effective in the treatment of age-related bone remodelling by coupling osteoblasts and osteoporosis. osteoclasts in the process of bone remodelling. The few randomized trials of teriparatide in OI TGF-β is secreted predominantly in an inactive patients show increased BMD, especially in Type latent form and is deposited into the bone ma- 1 OI than in individuals with OI types IV and III, trix. It has been reported that excessive TGF-β

187 Chapter 29 signaling is a mechanism of OI in both recessive other varieties, shortening and bending of femora and dominant OI mouse models. Also, treatment may be seen in some cases and may be in the later of mice with the anti-TGF-β neutralizing antibody part of pregnancy. However, normal length and 1D11 corrected the bone phenotype and improved shape of long bones in a fetus cannot rule out OI. lung abnormalities in both recessive and dominant forms of OI. Conclusion 4. Prenatal and postnatal transplantation of mesenchymal stem cells - Severe to lethal Last decade has improved understanding of genet- forms of OI may be diagnosed in utero by ultra- ics of OI due to identification of many more genes sonography starting at the 16th week. Following for OI. NGS based diagnostics has provided simple prenatal and postnatal cell transplantation in OI strategy in clinical settings and is also identifying improvement was seen in linear growth and frac- new genetic etiologies in research settings. Auto- tures were reduced in number, in fetus, neonatal somal recessive OI is probably more common in and later life. In humans, improvement of linear India due to high prevalence of consanguinity and growth and reduction of fracture rate followed makes molecular diagnosis of each case essential prenatal and postnatal cell transplantation in OI. as the risk of recurrence is 25% and phenotype Additionally, prenatal transplantation of allogeneic is usually severe in recessive varieties of OI. New MSCs in three OI pregnancies indicated that it has treatments may provide specific drug for specific appeared to be safe. A clinical trial in human type of OI and improve the outcome. pregnancy is currently in progress. (Westgren et al., 2015). References Multidisciplinary management: Orthopedic management• might be necessary in cases of severe bone deformity impairing function, with recurrent 1. Alanay Y, Avaygan H, Camacho N, et al. Mu- fractures and nonunion of fractures. To date, there tations in the gene encoding the RER protein are no physiotherapeutic treatment protocols avail- FKBP65 cause autosomal-recessive osteogen- able for children and adults with OI. A recent study esis imperfecta. Am J Hum Genet. 2010 Apr investigated a rehabilitation approach combining 9;86(4):551-9. resistance training, body-weight-supported tread- 2. Asharani PV, Keupp K, Semler O, et al. At- mill training, and neurodevelopmental treatment tenuated BMP1 function compromises osteo- associated with side-alternating whole-body vibra- genesis, leading to bone fragility in humans tion in 53 individuals with OI (ages 2.5–24.8 years) and zebrafish. Am J Hum Genet. 2012 Apr for 6 months within a period of 12 months of 6;90(4):661-74. treatment. There was improvement of mobility 3. Becker J, Semler O, Gilissen C, et al. Exome between, and also an increase in lean mass and sequencing identifies truncating mutations in BMD was observed (Hoyer-Kuhn et al., 2014). Fur- human SERPINF1 in autosomal-recessive os- ther studies are needed to address the role of teogenesis imperfecta. Am J Hum Genet. 2011 rehabilitation in OI patients. Mar 11;88(3):362-71. 4. Cabral WA, Chang W, Barnes AM, et al. Prolyl 3-hydroxylase 1 deficiency causes a recessive Genetic Counseling and Prenatal metabolic bone disorder resembling lethal/se- Diagnosis vere osteogenesis imperfecta. Nat Genet. 2007 Mar;39(3):359-65. Bisphosphonates has given some relief to some 5. Cho TJ, Lee KE, Lee SK, et al. A single recurrent patients of OI; though for many cases the life con- mutation in the 5 -UTR of IFITM5 causes osteo- tinues to be painful and handicapping. Molecular genesis imperfecta′ type V. Am J Hum Genet. diagnosis can differentiate between OI inherited 2012 Aug 10;91(2):343-8. in dominant or recessive fashion and accurate 6. Christiansen HE, Schwarze U, Pyott SM, et al. risk of recurrence can be provided to the fami- Homozygosity for a missense mutation in SER- lies. Mutation based prenatal diagnosis can be PINH1, which encodes the collagen chaperone provided at early gestation and to all families. protein HSP47, results in severe recessive os- In situations without molecular diagnosis, ultra- teogenesis imperfecta. Am J Hum Genet. 2010 sonographic based diagnosis before 20 weeks of Mar 12;86(3):389-98. gestation for case with lethal variety of OI. For 7. Chu ML, Williams CJ, Pepe G, et al.. Internal

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deletion in a collagen gene in a perinatal lethal 17. Orwoll ES, Shapiro J, Velth S, et al. Eval- form of osteogenesis imperfecta. Nature. 1983 uation of Teriparatdide treatment in adults Jul 7-13;304(5921):78-80. with Osteogenesis Imperfecta. J Clin Invest. 8. Dwan K, Phillipi CA, Steiner RD, et al. 2014;124(2):491–498. doi:10.1172/JCI71101. Bisphosphonate therapy for osteogenesis 18. Puig-Hervás MT, Temtamy S, Aglan M, et imperfecta. Cochrane Database Syst Rev. al. Mutations in PLOD2 cause autosomal- 2014;7:CD005088. recessive connective tissue disorders within 9. Hoyer-Kuhn H, C. Netzer, Koerber F, Schoe- the Bruck syndrome–osteogenesis imper- nau E, et al. Two year’s experience with fecta phenotypic spectrum. Hum Mutat. 2012 Denosumab for children with Osteogenesis Oct;33(10):1444-9. Imperfecta type VI. Orphanet Journal of rare 19. Ranganath P, Stephen J, Iyengar R, et al. diseases. 2014;9;145. Worsening of Callus Hyperplasia after Bisphos- 10. Keupp K, Beleggia F, Kayserili H, et al. Mutations phonate Treatment in Type V Osteogenesis Im- in WNT1 cause different forms of bone fragility. perfecta. Indian Pediatr. 2016 Mar;53(3):250-2. Am J Hum Genet. 2013 Apr 4;92(4):565-74. 20. Stephen J, Girisha KM, Dalal A, et al. Mutations 11. Lapunzina P, Aglan M, Temtamy S, et al. Iden- in patients with osteogenesis imperfecta from tification of a frameshift mutation in Osterix consanguineous Indian families. Eur J Med in a patient with recessive osteogenesis imper- Genet. 2015 Jan;58(1):21-7. fecta. Am J Hum Genet. 2010 Jul 9;87(1):110-4. 21. Stephen J, Shukla A, Dalal A, et al. Mutation 12. Lindert U, Cabral WA, Ausavarat S, et al. spectrum of COL1A1 and COL1A2 genes in MBTPS2 mutations cause defective regulated Indian patients with osteogenesis imperfecta. intramembrane proteolysis in X-linked osteo- Am J Med Genet A. 2014 Jun;164A(6):1482-9. genesis imperfecta. Nat Commun. 2016 Jul 22. Symoens S, Malfait F, D’hondt S, et al. De- 6;7:11920. ficiency for the ER-stress transducer OASIS 13. Marini JC. Osteogenesis imperfecta: com- causes severe recessive osteogenesis imper- prehensive management. Adv Pediatr. fecta in humans. Orphanet J Rare Dis. 2013 Sep 1988;35:391-426. Review. 30;8:154. 14. Mendoza-Londono R, Fahiminiya S, et al. Re- 23. van Dijk FS, Zillikens MC, Micha D, et cessive osteogenesis imperfecta caused by al. PLS3 mutations in X-linked osteoporo- missense mutations in SPARC. Am J Hum sis with fractures. N Engl J Med. 2013 Oct Genet. 2015 Jun 4;96(6):979-85. 17;369(16):1529-36. 15. Morello R, Bertin TK, Chen Y, et al. CRTAP is 24. Volodarsky M, Markus B, Cohen I, et al. A required for prolyl 3-hydroxylation and muta- deletion mutation in TMEM38B associated with tions cause recessive osteogenesis imperfecta. autosomal recessive osteogenesis imperfecta. Cell. 2006 Oct 20;127(2):291-304. Hum Mutat. 2013 Apr;34(4):582-6. 16. Mottes M, Cugola L, Cappello N, et al. Seg- 25. Westgren M, Götherström C. Stem cell trans- regation analysis of dominant osteogene- plantation before birth – a realistic option for sis imperfecta in Italy. J Med Genet. 1990 treatment of osteogenesis imperfecta? Prenat Jun;27(6):367-70. Diagn. 2015;35(9):827-832.

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Six Lethal Skeletal Dysplasias Which a Pediatrician Should Never Miss Sheela Nampoothiri Department of Pediatric Genetics, Amrita Institute of Medical Sciences & Research Centre, Cochin, Kerala, India

Correspondence to: Dr Sheela Nampoothiri Email: [email protected]

Lethal in Greek denotes ‘death bearing’. Lethal dysplasias present with short limbs or narrow thorax and lead to pulmonary hypoplasia. They are included in the classification of genetic bone disorders (Warman et al., 2011). Availability of a lot of photographs of radiographs in atlas of skeletal dysplasias help in radiological diagnosis (Schumacher et al., 2013; Spranger et al., 2002). The incidence is around 1:5000 live births and these fetuses die in utero or shortly after birth or can present as non-immune hydrops. Antenatal detection of almost all lethal dysplasias is possible by 20 weeks of gestation but osteogenesis imper- fecta (OI) type II can be identified even by 14 weeks of gestation. During an anomaly scan if all long bones are less than 4SD below the mean, then careful evaluation of other parameters should be done to rule out lethal dysplasias (Krakow et al., 2009; Krakow, 2015). Ultrasound markers of lethal dysplasias include,

1. Thoracic circumference (TC) less than 5th percentile of mean for gestational age Figure 1 Thanatophoric dysplasia type I - In- 2. Thoracic circumference (TC)/ Abdominal cir- fantogram showing marked bowing of cumference (AC) 0.89 (narrow thorax) femur and humeri, narrow thorax with 3. Femur length/AC 0.16 < short ribs, wafer thin vertebral bodies 4. Femur length/foot 0.8 < / platyspondyly (thin arrow), telephone < receiver shaped femur (thick arrow) and The other parameters to be seen are: trident acetabulum.

1. Increased nuchal translucency (NT) in first trimester For confirmation of specific types of skeletal 2. Poor mineralization of bones dysplasias an infantogram and lateral radiograph 3. Fracture or bent bones of spine are essential following termination of 4. Poor mineralization of skull and compressible pregnancy. Detailed clinical evaluation of fetus skull should be done for associated anomalies (cleft 5. Vertebral anomalies lip, cleft palate, polydactyly, facial dysmorphism, 6. Associated anomalies-cleft lip/cleft palate, dimples in the skin and, deformity of skull e.g. polydactyly, etc. clover leaf skull in thanatophoric dysplasia type II).

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A clinical photograph and fetal autopsy and storage Type I and III (these two types cannot be dis- of DNA for molecular confirmation are extremely tinguished clinically) (Figure 2): They present with helpful for delineating the type which is extremely long thorax, very short ribs, trident acetabulum, important for counseling as the risk of recurrence platyspondyly and postaxial polydactyly. These are is up to 25% in autosomal recessive disorders (e.g. due to mutations in DYNC2H1 gene which is the Short rib polydactyly (SRP) syndrome) (Taylor et al., same gene responsible for asphyxiating thoracic 2015). Six cases of lethal skeletal dysplasia are dystrophy. illustrated here. Type II: Fusion of metacarpals and short oval Thanatophoric dysplasia tibia are two differentiating features from I and III. Type IV: Tibia is normal. Polydactyly is unlikely. Thanatophoric dysplasia type I presents with se- Prenatally SRP, Ellis van Creveld and Jeune vere platyspondyly (wafer thin vertebrae), small Asphyxiating Thoracic dystrophy are indistinguish- thorax, telephone receiver shaped femur and tri- able. dent acetabulum (Figure 1). In Type II, clover leaf shaped skull is the differentiating point from type I and the long bones are not as curved as in type I. Hypophosphatasia This is an autosomal dominant disorder and both types are due to mutation in the FGFR3 gene. Many cases of thanatophoric dysplasias are labeled as This condition can be suspected prenatally by achondroplasia during antenatal scans but a very 14 weeks of gestation. All bony structures are important clue is that radiological features for sonolucent. The classical features include absent achondroplasia will manifest only after 24 weeks skull ossification, large metaphyseal defects in long of gestation. bones extending to diaphysis, lack of ossification of most of the vertebrae, absent ulna or fibula and SRP syndrome (Short rib polydactyly very slender few ribs (Figure 3). Spurs arising from forearm and legs which are covered by skin are syndrome) good clues and are well seen with 3 dimensional ultrasound scan. Serum alkaline phosphatase level There are 4 different types: is undetectable in fetal blood sample. The defect is in ALPL gene. I Saldino Noonan II Majewski III Verma Naumoff IV Beemer Langer (polydactyly is unlikely)

All are autosomal recessive.

Figure 3 Hypophosphatasia - Severe hypomin- eralisation, osteochondral spurs from forearm (thin arrow) and legs, shorten- Figure 2 Short-rib polydactyly syndrome type 1 ing of all long bones, very slender ribs and 3: note the narrow thorax with with fractures, absent ulna (thick arrow) short ribs and postaxial polydactyly and fibula and unossified vertebrae and (arrow). skull

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Figure 4 Osteogenesis imperfecta type II - severe beading of the ribs (thin arrow), thick short crumpled shafts of long bones in older fetuses due to continuous fracture & reunion (thick arrow).

Osteogenesis Imperfecta Type II

Typical features include absent ossification of skull, beaded ribs (due to repeated fractures and re- union) and hypomineralistaion of long bones with severe angulation (Figure 4). In type II, they have severe angulation of tibia which is not a feature in type III (non lethal). In older fetuses the ribs are broad due to repeated fracture and reunion. The perinatally lethal types are caused by COL1A1 / COL1A2 which are transmitted as an autosomal dominant manner due to a de novo mutation in all cases. There are many newly described genes which are transmitted in autosomal recessive man- ner leading to perinatally lethal OI (CRTAP, LEPRE1, SEERPINH1, P3H1)

Campomelic dysplasia

Angulation of femur and tibia are the most promi- nent features. Angulation of femur is at junction of upper1/3 and lower 2/3 and for tibia it is between the upper 2/3 and lower 1/3. Angulation of tibia can be seen as a spike under the skin. Skin dimples Figure 5 Campomelic dysplasia - Angulation of give a clue regarding the underlying bowing of femur is at the junction of upper 1/3 long bones in the lower limb. They have only 11 and lower 2/3 and for tibia at the junc- pairs of ribs. Another feature is absent ossification tion of the upper 2/3 and lower 1/3. of pedicle of thoracic vertebrae. Wing of the The right wing of the scapula is missing scapula is missing and they have very short bowed (thin arrow) and left scapula is absent fibula (Figure 5). Abnormal male genitalia with sex (thick arrow), fetus has only 11 pairs of reversal is seen in fetuses with 46,XY karyotype. ribs. This disorder is caused by heterozygous dominant mutation in the SOX 9 gene.

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Achondrogenesis Type IB: They do not present with beaded ribs. Other features include well ossified vertebral Type IA: Beaded ribs due to healing fractures, pedicles with unossified vertebral bodies, better absent ossification of vertebral bodies, severe ossification of skull, short long bones and crescen- micromelia, absent ossification of skull, and cres- tic iliac wings. There is significant widening of the centic iliac wings characterize this condition. This is interpedicular distance in both cervical and lumbar an autosomal recessive disorder due to mutations region giving the “Cobra head appearance” (Figure in TRIP1. 6). This is an autosomal recessive disorder caused by mutations in DTDST gene. Type II: has well ossified skull, absent ossifica- tion of vertebral body, ossified vertebral pedicles, handle bar clavicles and cupping and spiking of fe- mur. This autosomal dominant disorder is caused by mutations in COL2A1. Accurate identification of lethal dysplasias help in genetic counseling and would help the clini- cian to choose the right molecular test for future prenatal diagnosis

Acknowledgments

I thank Prof. Shubha Phadke and Dr. Girisha KM for providing the radiographs shown in Figure 6 and Figure 2 respectively.

References

1. Krakow D, Lachman RS, Rimoin DL. Guidelines for the prenatal diagnosis of fetal skeletal dys- plasias. Genet Med 2009; 11: 127-133. 2. Krakow D. Skeletal dysplasias. Clin Perinatol 2015; 42: 301-319. 3. Schumacher R, Seaver L H, Spranger J. Fetal : A Diagnostic Atlas. 2013. Springer- Verlag, Berlin Heidelberg. 4. Spranger JW, Brill PW, Poznanski AK.: Bone dys- plasias: An Atlas of Genetic Disorders of Skeletal Development. 2nd Edn, Oxford University Press. Oxford, 2002: pp 95-99. Figure 6 Achondrogenesis IB - The thorax is 5. Taylor SP, et al. Mutations in DYNC2LI1 dis- small with short ribs with expanded rupt cilia function and cause short rib poly- and cupped ends. The crescentic iliac dactyly syndrome. Nat Commun 2015; 7092. wings (arrow) and cobra head appear- doi: 10.1038/ncomms8092 ance of vertebral column are good 6. Warman ML, et al. Nosology and classification clues for diagnosis. of genetic skeletal disorders: 2010 revision. Am J Med Genet A 2011; 155A: 943-968.

193 GeNeToon

Contributed by: Dr Prajnya Ranganath Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad Email: [email protected]

GeNeImage Contributed by: Dr. Dhanya Lakshmi N Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad Email: [email protected]

194 Section 5 Recent Advances in Medical Genetics

Chapter 31

Management of Lysosomal Storage Disorders: The Current Scenario Prajnya Ranganath, Dhanya Lakshmi N Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad, India

Correspondence to: Dr Prajnya Ranganath Email: [email protected]

Introduction the natural course of the disease and significant reduction in residual disease. Lysosomal storage disorders (LSDs) are a group of inborn errors of metabolism (IEM) characterized Enzyme Replacement Therapy by the intra-lysosomal accumulation of complex macro-molecules. Lysosomes are membrane – Enzyme replacement therapy (ERT) involves exoge- enclosed compartments within the cytosol of most nous administration of sufficient amounts of the cell types, which contain acidic hydrolytic enzymes deficient enzyme to effectively clear the accumu- involved in the degradation of certain lipids, pro- lated substrate in the patient. Early results were teins, sugars and other substrates. LSDs usually disappointing for ERT as effective targeted delivery occur due to deficiencies of these lysosomal en- of the synthetic enzyme to the lysosomes could zymes but can also result from defects in key not be achieved. But with the discovery of man- lysosomal membrane proteins, proteins involved nose 6-phosphate receptors on the cell surface of in lysosomal enzyme trafficking or lysosomal en- macrophages, an effective target for the entry of zyme activator proteins. The most common causes the exogenous enzyme was found and ERT became of morbidity and mortality in LSDs are due to a feasible therapeutic option. The recombinant neurological, visceral, cardiovascular and skeletal enzymes are modified by post-translational linkage involvement. At the present time, around 70 differ- of oligosaccharides containing mannose residues ent LSDs are known, with a cumulative prevalence and in some cases, these glycosylated enzymes of around 1 in 5000 live births (Meikle et al., 1999; are additionally phosphorylated at these mannose Fuller et al., 2006; Penati et al., 2017). residues. These enzymes can then bind either to In the past decade, there have been several mannose receptors (MR) present on the plasma advances in the development of therapeutic strate- membrane of mononuclear cells, such as mono- gies for lysosomal storage disorders because of cytes and macrophages, or to the M6P receptor better understanding of the pathophysiology of (M6PR) present on the plasma membrane of many these group of disorders. The various modali- other cell types. Following internalization, the ties tried in the treatment of metabolic disorders enzyme is transported in a vesicle to the pre- in general include substrate restriction (reduc- lysosomal/endosomal compartment (Rosenfeld et ing the load on the affected pathway), control al., 1982). The first successful ERT developed for of endogenous production of substrate, acceler- Gaucher disease paved the way for ERT for other ation of removal of substrate, stimulation of the LSDs. The Food and Drug Administration (FDA) residual enzyme, enzyme replacement and bone has approved the use of ERT for Gaucher disease, marrow transplantation. Many of these modalities Pompe disease, Fabry disease and mucopolysac- e.g. hematopeotic stem cell transplant, enzyme charidoses (MPS) types I, II, IVA and VI and a replacement therapy, pharmacological chaperone number of clinical trials and animal model studies therapy and gene therapy aim to restore the are currently underway for the development of cellular and tissue levels of the missing enzyme. ERT for other LSDs also. The most important draw- The ultimate goal of treatment of LSDs by any of backs of ERTs are the cost, the need for weekly or these methods is reduction in storage of substrate biweekly infusion regimes, their limitation in not within the cells or organs, favorable alteration in being able to reach the central nervous system

195 Chapter 31 and few other tissues due to physiological factors substrate precursor synthesized to a level that can such as the blood brain barrier/ absence of the be effectively cleared by the mutant enzyme with mannose 6-phosphate receptors and the immuno- residual hydrolytic activity, to prevent substrate ac- logical responses to therapy. Research is ongoing cumulation. It could be used as the main treatment to develop newer strategies of enzyme replace- modality in mild to moderate forms of the disease ment to overcome these barriers and limitations, with less severe enzyme deficiency or it could be such as use of recombinant enzymes designed to given as an adjunct to enzyme replacement ther- target receptors involved in trans-cellular transport apy in patients with very low residual functional of substances across the endothelium in the blood enzyme levels. This modality of treatment has an – brain barrier, use of chimeric enzymes targeted advantage of increased bioavailability especially in to alternative receptors of endocytosis such as the CNS. A potential concern regarding the use transferrin receptor or insulin receptor and central of SRT is its non-specificity i.e. the substrate nervous system (CNS)-directed viral and non-viral whose production is blocked or limited might be gene therapy (Muro, 2010; LeBowitz et al., 2004; important in other metabolic processes also and Osborn et al., 2008) limiting its synthesis could adversely affect other metabolic pathways. Bone Marrow Transplantation Substrate reduction therapy is already ap- proved for clinical use in Gaucher disease Type 1 The goal of bone marrow (BMT) or hematopoi- and in Neimann-Pick Type C (Cox et al., 2000; etic stem cell transplantation (HSCT), which was Parenti et al., 2013). In Gaucher disease, the first therapeutic approach introduced, is to re- N-butyldeoxynojirimycin (Miglustat) administered place enzyme-deficient macrophages with marrow- orally has been shown to improve the biochemical, derived donor macrophages that can act as an on- visceral, hematological and bone markers of the going source of normal enzyme. Since the donor disease with very few adverse effects (Hollack et derived cells which produce the enzyme are able al., 2009). Even though Miglustat crosses the blood to migrate to the brain, HSCT has the potential to brain barrier it has not been found to be effective in improve neurological symptoms. Though HSCT can neuronopathic Gaucher disease. In Neimann Pick modify disease progression and improve survival in type C, Miglustat was shown to improve neurologi- some LSD patients, it is not curative. The chances cal symptoms like horizontal saccadic movements, of a successful engraftment are significantly in- ambulation and swallowing (Parenti et al., 2013). creased when an HLA matched sibling donor is A more potent and specific substrate inhibitor, used as opposed to an unrelated mis-matched Eliglustat has got approval in 2015 for commercial donor. The extent of benefit of HSCT also depends use and is now suggested as the first line therapy on the degree of clinical involvement and the age for Gaucher disease type I (Stirnemann et al., 2017). of the patient at the time of transplantation. HSCT This drug can be used only after CYP2D6 genotyp- prior to onset of neurological involvement may ing of the patient to assess the metabolizer status. prevent or delay the neurological manifestations of Adverse effects with this drug are uncommon and the LSD, but does not reverse the existing cognitive mild. impairment. The major limitations of HSCT are Genistein and rhodamine B have been tried for the cost and the considerable degree of morbidity substrate reduction in MPS, but the results have and mortality due to complications such as graft not been consistent. SRT is being tried in preclinical rejection, inter-current infections and graft–ver- studies for other LSD’s like Fabry disease, Sandhoff sus–host disease (Malatack et al., 2003). HSCT is disease and Pompe disease. indicated in the treatment of MPS I and has been shown to ameliorate visceral symptoms in MPS VI, late onset Krabbe disease and metachromatic Small molecule pharmacological leukodystophy. However, the clinical outcome is chaperone therapy determined by the phenotype and the severity of the disease at the time of HSCT. Amongst the newer approaches being developed for treatment of LSDs is chaperone therapy Substrate Reduction Therapy wherein small molecules, designed to enhance the residual enzyme activity by protecting the Substrate reduction therapy (SRT) aims to restore mutant enzyme from misfolding and degradation metabolic homeostasis by limiting the amount of in the cell, are used. This form of therapy is

196 Chapter 31 more beneficial for patients with mutations that do not directly affect the enzyme activity or cat- alytic site but primarily cause enzyme misfolding defects (Desnick & Schuchman, 2002). Chaper- ones can be administered orally, do not need mannose phosphate for delivery into cells and are non-immunogenic. They can reach therapeutic concentrations in the CNS. But the chaperones identified so far also are potential inhibitors ofthe target enzyme. Chaperone therapy is useful only in patients harboring particular mutations located in certain specific protein domains. Migalastat (1-deoxygalactonorijimycin) for Fabry disease was the first drug to be introduced in this group and it has been approved for use in patients above 16 years in the European Union, for cases with amenable mutations (Mehta and Hughes, 2017). Research is ongoing to develop a similar approach for Gaucher disease and Pompe disease.

Gene therapy Figure 1 The different levels of intervention for Gene therapy has the potential to become the the management of lysosomal storage most effective therapeutic strategy for lysosomal disorders and the various therapeutic storage disorders. Gene therapy is intended to strategies which act at each of these increase or restore the levels of the defective en- levels. zyme in the patient’s cells by delivering the normal copy of the defective gene to the recipient. LSD’s are ideal candidates for gene therapy because they Gaucher disease (GD) are monogenic, cross correction of enzyme defi- ciency is possible and a relatively small increase The recombinant glucosylceramidase enzyme in enzyme activity may be sufficient for clinical preparations currently available commercially benefit. The underlying principle of gene therapy for ERT in Gaucher disease are imiglucerase is somatic gene transfer using vectors capable of (Cerezyme) produced in Chinese hamster ovary integrating the exogenous gene sequences into the cells, velalglucerase alfa (VPRIV) manufactured in a host genome for prolonged enzyme expression. human cell line and taliglucerase, produced using Both viral and non-viral vectors are being tried carrot cells. Each formulation is modified to expose for gene therapy. Different viral vectors like the the alpha-mannosyl residues for enhanced uptake herpes virus, adeno virus, adeno associated virus, by macrophages. Regular intravenous infusions lenti virus and retro viruses have been studied in of the recombinant enzyme in a dose of 20 – 60 animal models. Gene transfer can be achieved by U/kg once in 2 weeks, have been demonstrated to an ex vivo or in vivo approach. Another pioneering be safe and effective in reversing the features re- strategy based on increasing the levels of cytosolic sulting from hematologic and visceral involvement, molecules that control intra-cellular trafficking of in reducing bone pain and the frequency of bone vesicles, is being developed; this approach would crises and in significantly improving the quality of be more effective in the treatment of storage disor- life (Figure 2). However, the currently available ERT ders, such as Niemann-Pick disease type C, which does not reverse or ameliorate the neurological are caused by defective lysosomal transporters manifestations of type 2 GD and its benefit in (Muro, 2010). improving the neurological symptoms of type 3 GD The different levels at which the interventional is also doubtful (Pastores and Hughes, 2015). strategies outlined above act are diagrammatically Miglustat has been found to significantly reduce represented in Figure 1. The specific therapeutic the liver and spleen volume in mild to moderate options available for some of the lysosomal storage forms of GD, but has only a modest benefit in disorders are mentioned below. hematological and bone involvement. Bone mar-

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(Courtesy: Dr Shubha Phadke. Professor & Head, Department of Medical Genetics, SGPGIMS, Lucknow)

Figure 2 Three children with Type I Gaucher disease receiving Enzyme Replacement Therapy. The graphs show the improving trends in weight, height and haematological parameters and reduction in liver and spleen size over a one-year follow up period after initiation of ERT. row transplantation is an option for patients with Niemann – Pick disease (NPD) chronic neurologic GD and progressive disease despite ERT. Successful engraftment can correct Variable results have been reported with BMT in the metabolic defect, improve blood counts, re- Niemann- Pick disease. While it has been found duce the increased liver and spleen volume and to reduce the increased liver and spleen volumes, stabilize neurologic and bone symptoms (Pastores stabilization of the neurologic symptoms following and Hughes, 2015). BMT has not been reported. A Phase I enzyme Eliglustat has gained marketing approval in replacement therapy (ERT) trial in adults with NPD 2015 and in a follow-up study of 400 odd patients, B is underway at present (Wasserstein and Schuch- it has been shown to have comparable efficacy man, 2015). Published clinical trials evaluating the to Miglustat. It has significant effect on amelio- efficacy of Miglustat in Neiman Pick Type Cshow ration of bone symptoms and is now suggested that Miglustat is expected to slow the progress as the first line drug for Gaucher disease type1 of neurological symptoms in patients with a later (Stirnemann et al., 2017). onset of disease (Santos-Lozano et al., 2015). Clinical trials for a chaperone drug isofagamine for type 1 GD are also currently underway (Steet et Mucopolysaccharidoses (MPS) al., 2007). Some positive effects have been demon- strated in treatment with ambroxol (Stirnemann et Bone marrow transplantation or hematopoietic al., 2017). stem cell transplantation (HSCT) has been found to

198 Chapter 31 benefit patients with Hurler (MPS IH), Maroteaux- acquisition of motor skills (Leslie and Bailey, 2017). Lamy (MPS VI), and Sly (MPS VII) syndromes. In some countries, newborn screening for Pompe However, unequivocal benefit with HSCT for the disease has been started so that treatment can neurological and skeletal manifestations of Hunter be initiated before the child manifests symptoms. (MPS II), Sanfilippo (MPS III), and Morquio (MPS Children put on ERT from the neonatal period IV) syndromes has not been reported. In MPS I, onwards have shown very good results with near- HSCT has been shown to increase the survival, re- normal development. Infusion reactions and even duce facial coarseness and hepato-splenomegaly, anaphylaxis can occur with this ERT, especially improve hearing and stabilize and improve cardiac in patients who are negative for cross-reactive function. However, it has not been found to immunologic material (CRIM). Immunomodulation reduce/ reverse the skeletal manifestations and protocols are now being used either prior to initi- corneal clouding. The effect of BMT on neuropsy- ation of therapy or early in the treatment course chological symptoms is variable and is related to in CRIM-negative patients. These protocols mostly the age and intellectual capacity of the child at the include rituximab and additional drugs such as time of the engraftment; in children undergoing mycophenylate mofetil, methotrexate or sirolimus BMT before onset of significant developmental (Elder et al., 2013). ERT helps stabilize respira- delay, it may slow the course of cognitive decline. tory functions and motor ability in patients with Children showing significant cognitive impairment late-onset Pompe disease. prior to undergoing BMT do not appear to benefit developmentally (Clarke, 2016). Fabry disease ERT is clinically available for MPS types I (Aldu- razyme), II (Idursulfase/ Elaprase), IVA (Elosulfase The two ERTs available for Fabry disease are alpha) and VI (Naglazyme). These are given as Fabrazyme (algalsidase beta) and Replagal (algalsi- slow intravenous infusions once a week. ERT has dase alpha). Both are given as slow intravenous been shown to produce significant improvement infusions once in two weeks (1 mg/kg/dose for al- in pulmonary function and the six-minute walk test galsidase beta and 0.2 mg/kg/dose for algalasidase performance, reduce urinary glycosaminoglycan alpha). ERT has been found to reduce the plasma excretion, reduce the liver and spleen volume, and GL-3 (globotriaosylceramide) concentration by up improve growth and joint mobility and decrease to 50%, improve the cardiac function, stabilize the sleep apnea. However, it does not have any effect renal function, reduce pain in the extremities and on the neurological features, as it cannot cross improve the quality of life in patients with Fabry the blood – brain barrier. Various approaches disease (Mehta and Hughes, 2017). A PEGylated to deliver ERT to the CNS are currently being form of recombinant alpha glucosidase enzyme researched; these include CSF instillation of en- with a longer circulating half-life is now in the zyme via direct intra-thecal injection, continuous Phase II trial stage. pumps or microcapsule implants and production Phase III trials for the pharmacologic chaper- of chimeric recombinant proteins designed to one (1-deoxygalactonojirimycin – Migalastat) are cross the blood-brain barrier (Clarke, 2016; Scarpa, underway for Fabry disease and it has received 2015). marketing approval in the European Union in 2016 (Mehta and Hughes, 2017). Pompe disease With availability of ERT there is increasing awareness about the disease amongst clinicians The ERT available for Pompe disease is Myozyme and many more cases of Fabry disease, which is (alglucosidase alfa) and Lumizyme. Myozyme (al- a disease difficult to diagnose clinically, are being glucosidase alfa) was approved in 2006 by the FDA picked up. It is important to investigate patients for infantile-onset Pompe disease and Lumizyme with renal, cardiac and cerebro-vascular disease was approved in 2010 for patients older than eight for Fabry disease at a young age and also to screen years with late-onset Pompe disease. ERT is given asymptomatic relatives of patients with Fabry dis- as a slow intravenous infusion 20-40 mg/kg/dose ease so that treatment can be offered to affected once in two weeks. When initiated before the age individuals in the early stages of disease. Though of six months and before the need for ventilatory Fabry disease has an X-linked recessive pattern of assistance, it has been found to improve survival, inheritance, it has been observed that many carrier improve ventilator-independent survival, reduce females may have varying degrees of involvement the cardiac mass and significantly improve the of organs and may also be candidates for ERT.

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Metachromatic leukodystrophy (MLD) References

Bone marrow therapy is presently the only avail- 1. Clarke LA. Mucopolysaccharidosis Type I. 2002 able therapy that has some benefit in treatment Oct 31 [Updated 2016 Feb 11]. In: Adam MP, of the central nervous system manifestations of Ardinger HH, Pagon RA, et al., editors. GeneRe- MLD, especially in the slowly progressing late-onset views. Seattle (WA): University of Washington, forms of MLD i.e. juvenile and adult MLD. The best Seattle; 1993-2017. Available from: https:// outcomes are observed in individuals undergoing www.ncbi.nlm.nih.gov/books/NBK1162/ BMT before the onset of symptoms. BMT has been 2. Cox T, et al. Novel oral treatment of Gaucher’s found to slow central nervous system disease pro- disease with N-butyldeoxynojirimycin (OGT gression but does not alleviate peripheral nervous 918) to decrease substrate biosynthesis. Lancet system manifestations (Krivit et al., 1999). Effective 2000; 355: 1481-1485. ERT that can cross the blood-brain barrier has 3. Desnick RJ, Schuchman EH. Enzyme replace- not been developed yet, but recombinant human ment and enhancement therapies: lessons Arylsulfatase A enzyme is available, and as animal from lysosomal disorders. Nat Rev Genet 2002; studies have suggested that it may be a useful 3: 954-966. supplement to other type of therapies, clinical 4. Elder ME, et al. B-cell depletion and im- testing of recombinant human enzyme has been munomodulation before initiation of enzyme started (Fluharty, 2014). replacement therapy blocks the immune re- sponse to acid alpha-glucosidase in infantile- Globoid cell leukodystrophy (Krabbe onset Pompe disease. J Pediatr 2013;163: 847-854 e1. disease) 5. Fuller M, et al. Epidemiology of lysosomal stor- Hematopoietic stem cell transplantation (HSCT) in age diseases: an overview. In Fabry Disease: pre-symptomatic infants and older individuals with Perspectives from 5 Years of FOS. A. Mehta, mild symptoms has been shown to improve and M. Beck, and G. Sunder-Plassmann, editors. preserve cognitive function but has not been found Oxford PharmaGenesis, 2005, Oxford. 9-20. to be helpful in preventing progressive involvement 6. Hollak CE, et al. Miglustat (Zavesca) in type of the peripheral nervous system. The identifica- 1 Gaucher disease: 5-year results of a tion of newborns with Krabbe disease by newborn post-authorisation safety surveillance pro- screening would help in early HSCT before onset gramme. Pharmacoepidemiol Drug Saf 2009; of neurologic damage (Krivit et al., 1999). ERT for 18: 770-777. Krabbe disease is being tried in animal models. 7. Krivit W, et al. Bone marrow transplan- tation as effective treatment of central Combination therapy for LSDs nervous system disease in globoid cell leukodystrophy, metachromatic leukodystro- Most of the therapeutic strategies that have de- phy, adrenoleukodystrophy, mannosidosis, veloped been developed for treatment of LSD’s, fucosidosis, aspartylglucosaminuria, Hurler, when used in isolation fail to provide the ultimate Maroteaux-Lamy, and Sly syndromes, and aim of cure (Macauley, 2016). Hence various Gaucher disease type III. Curr Opin Neurol preclinical studies in animal models are being tried 1999; 12: 167-76. in treatment of LSD’s. By optimizing routes of 8. LeBowitz JH, et al. Glycosylation-independent delivery, therapeutic timing, and targeting sec- targeting enhances enzyme delivery to lyso- ondary disease mechanisms, combination therapy somes and decreases storage in mucopolysac- represents the future for LSD treatment. charidosis type VII mice. Proc Natl Acad Sci USA Newer advances in nanotechnology are also 2004, 101: 3083-3088. now being made use of in development of strate- 9. Leslie N, Bailey L. Pompe Disease. 2007 Aug 31 gies for organ targeting, controlled circulation, [Updated 2017 May 11]. In: Adam MP, Ardinger effective intra-cellular trafficking and release of HH, Pagon RA, et al., editors. GeneReviews. lysosomal enzymes (Muro, 2010). With all these Seattle (WA): University of Washington, Seat- newly emerging approaches, it is hoped that com- tle; 1993-2017. Available from: https://www. prehensive treatment modalities would become ncbi.nlm.nih.gov/books/NBK1261/ available for all the lysosomal storage disorders in 10. Lukina E, et al. Improvement in hematolog- the not-so-distant future. ical, visceral, and skeletal manifestations of

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Gaucher disease type 1 with oral eliglustat 19. Pastores GM, Hughes DA. Gaucher Disease. tartrate (Genz-112638) treatment: 2-year re- In: Pagon RA, Adam MP, Ardinger HH, et sults of a phase 2 study. Blood 2010; 116: al., editors. 2000 Jul 27 [Updated 2015 Feb 4095-4098. 26]. GeneReviews. Seattle (WA): University 11. Macauley SL. Combination Therapies for Lyso- of Washington, Seattle; 1993-2017. Avail- somal Storage Diseases: A Complex Answer able from: https://www.ncbi.nlm.nih. to a Simple Problem. Pediatr Endocrinol Rev gov/books/NBK1269/ 2016; 13 (Suppl 1): 639-648. 20. Rosenfeld MG, et al. Biosynthesis of lyso- 12. Malatack JJ, et al. The status of hematopoietic somal hydrolases: their synthesis in bound stem cell transplantation in lysosomal storage polysomes and the role of co- and post- disease. Pediatr Neurol 2003, 29: 391-403. translational processing in determining their 13. Martino S, et al. Expression and purification of subcellular distribution. J Cell Biol 1982, 93: a human, soluble Arylsulfatase A for Metachro- 135-143. matic Leukodystrophy enzyme replacement 21. Santos-Lozano A, et al. Niemann-Pick disease therapy. J Biotechnol 2005; 117: 243-51. treatment: a systematic review of clinical trials. 14. Mehta A, Hughes DA. Fabry Disease. In: Pagon Ann Transl Med. 2015; 3: 360. RA, Adam MP, Ardinger HH, et al., editors. 2002 22. Scarpa M. Mucopolysaccharidosis Type II. 2007 Aug 5 [Updated 2017 Jan 5]. GeneReviews. Nov 6 [Updated 2015 Mar 26]. In: Adam MP, Seattle (WA): University of Washington, Seat- Ardinger HH, Pagon RA, et al., editors. GeneRe- tle; 1993-2017. Available from: https://www. views. Seattle (WA): University of Washington, ncbi.nlm.nih.gov/books/NBK1292/ Seattle; 1993-2017. Available from: https:// 15. Muro S. New biotechnological and www.ncbi.nlm.nih.gov/books/NBK1274/ strategies for treatment of lyso- 23. Steet R, et al. Selective action of the iminosugar somal storage disorders. Wiley Interdiscip Rev isofagomine, a pharmacological chaperone Nanomed Nanobiotechnol 2010; 2: 189-204. for mutant forms of acid-beta-glucosidase. 16. Osborn MJ, et al. Targeting of the CNS in Biochem Pharmacol 2007; 73: 1376-1383. MPS-IH using a nonviral transferrin-alpha-L- 24. Stirnemann J, et al. A Review of Gaucher iduronidase fusion gene product. Mol Ther Disease Pathophysiology, Clinical Presentation 2008, 16: 1459-1466. and Treatments. Int J Mol Sci 2017; 18: 441. 17. Penati R, et al. Gene therapy for lysosomal stor- 25. Wasserstein MP, Schuchman EH. Acid Sphin- age disorders: recent advances for metachro- gomyelinase Deficiency. 2006 Dec 7 [Updated matic leukodystrophy and mucopolysaccarido- 2015 Jun 18]. In: Adam MP, Ardinger HH, sis I. J Inherit Metab Dis 2017; 40: 543-554. Pagon RA, et al., editors. GeneReviews. Seat- 18. Parenti G, et al. New strategies for the treat- tle (WA): University of Washington, Seattle; ment of lysosomal storage diseases (review). 1993-2017. Available from: https://www. Int J Mol Med 2013; 31:11-20. ncbi.nlm.nih.gov/books/NBK1370/

Klippel Feil Anomaly & Occipital Haemangioma

Prenatal USG showed short neck with fusion defects of cervical GeNeImage vertebrae and a mass outside the skull in the occipital region ? Contributed by: lipoma. Prenatal MRI suggested Dr. Shubha R Phadke intracranial communication of Department of the mass. Postnatal CT scan Medical Genetics, confirmed cervical vertebral Sanjay Gandhi defects & demonstrated a small Postgraduate Institute hole in the occipital bone. of Medical Sciences, Lucknow Histology of the mass showed Email: hemangiomatous anomaly. [email protected]

201 Chapter 32

Genome Editing: The ‘CRISPR’ Way

Annapurna Gupta1, Meenal Agarwal2 1Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 2Clinical Genetics Division and Medical Genetics Laboratory, GenePath Dx, Causeway Healthcare Private Ltd., Pune 2(Formerly: Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow)

Correspondence to: Dr Meenal Agarwal Email: [email protected]

What is Genome Editing? techniques mainly differ in their mechanism of recognizing target genomic sequences, capability Genomic editing in a simple language, is creation of simultaneous multiplexing to various genomic of a desired alteration in a genome, typically targets, potential of off-target effects and the ease leading either to, 1) correction of a mutation or of their in-vitro genetic engineering. The com- 2) causing an alteration in gene expression at parison in characteristics of these three genome specifically targeted genomic sites. The various editing technologies is presented in table 1. Of components for eukaryotic genome editing, typi- these three main genome editing technologies, cally include a nucleotide or amino acid sequence, CRISPR-Cas9 system has evolved to be the most which identifies the targeted genomic site anda powerful technology among all mainly because of nuclease, which produces double stranded DNA its ease of use, flexibility, unprecedented speci- breaks (DSBs). These DSBs are then repaired by ficity, versatility for clinical and research scenarios using cellular intrinsic DNA repair machinery. If and adaptability of recent research questions and these broken DNA ends are joined end to end technical advancements. (nonhomologous end joining repair, NHEJ), it leads to either insertion or deletion of few nucleotides, CRISPR- cas9 System which often disrupts the reading frame and leads to frame shift mutation. Being error prone, usually CRISPR-Cas9 system stands for Clustered, Regularly it causes the knocking down of a functioning gene. Interspaced, Short Palindromic Repeats and an en- Another method for repair of DSBs is homology donuclease Cas9 (CRISPR- associated) system. This directed repair (HDR), in which, breaks are repaired method of genome editing has been adopted from by complementary pairing to a template strand. adaptive immune system of bacteria and archea This mechanism of DSBs repair are accurate and against invasion of foreign nucleic acids derived theoretically desired gene modifications are pos- from phages and plasmids. Almost all bacterial sible by providing an artificially synthesized DNA genomes have CRISPR-Cas9 loci. As the name construct as “cut and paste” mechanism (Sander et suggests, these loci consist of clustered, direct al., 2014; Manjunath et al., 2013). palindromic repeats, which are spaced by inter- spersed nucleotide sequences (Figure 1). The Various Methods for Genome Editing repeats are typically 21-47 nucleotides long and are identical at a single locus. The interspersed Zinc Finger Nuclease (ZFNs), Transcription Activator nucleotides sequences are called as spacers and Like Effectors nucleases (TALENs) and Clustered, are derived from foreign viruses. Spacer sequence Regularly Interspaced, Short Palindromic Repeats- is transcribed into crisper RNA (crRNA). CRISPR Cas (CRISPR-Cas9) are the three most commonly locus also contain DNA sequences, which code used genome editing technologies for complex for a complementary transactivating crisper RNA eukaryotic genomes (Sander et al., 2014; Wei et (tracrRNA) and various Crisper Associated genes al., 2013). All these methods are based on combi- (cas), which code for nucleases. These crRNA hy- nation of mechanisms to identify genome targets bridize with complementary tracrRNA and together with great specificity and creating DSBs. These as double strand, they recognise the complemen-

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Table 1 Comparative features of three important genome editing systems.

Character Zinc finger nuclease Transcription Clustered regularly (ZFN) activator like interspaced short effector-nucelases palindromic repeat (TALEN) (CRISPR-Cas9) Type of nuclease Fok1 nuclease Fok1 nuclease Cas9 DNA identifying Based on DNA- protein Based on Based on Watson crick mechanism interaction. DNA-protein base pairing between DNA binding domain is interaction. RNA and target DNA. composed repeats of 30 DNA binding domain 20 nucleotide long crRNA amino acids. is composed of which typically consists of 3-6 repeats per ZFN, each repeats of 34 specific nucleotide domain recognises 3 aminoacids which sequence flanked by base pairs. recorgnize one repetitive sequences. nucleotide. Target sequence should be preceded by NGG sequence called as protospacer adjacent motif (PAM). Mechanism of Two ZFN binds to Same is ZFN crRNA hybridizes with producing DNA opposite sides of tracrRNA and activates double strands targeted DNA sequence cas9 nuclease which repair with space of 5-7 produces DSB nucleotides where Fok1 nuclease dimerises and produces double strand break in the space Efficiency 0-12% 0-75% 0-80% Off target effects + + +++ Multiplexing Difficult Difficult Easy Ease of construction Difficult Difficult Relatively very easy tary foreign nucleotide sequences. There are three 1) High throughput functional genomic types of CRISPR-Cas immune systems. Out of them screening: By producing gene disruption or knock- only type II system (adapted from Streptococcus ing down of expression of a gene, this technology pyogenes), which uses cas9 as a nuclease, is used as can produce knock out animal models to study a method of genome editing. That’s why, it is called functions of genes. These technologies seem to as CRISPR-Cas9 system. Cas9 nuclease has HNH be immense value in the era of whole genome nuclease domain and the RuvC-like domain which sequencing, where establishing the functional sig- generate DSBs (Sander et al., 2014; Manjunath et nificance of a genetic variant seems to be of utmost al., 2013; Wei et al., 2013). importance. 2) Gene therapy: CRISPR-Cas9 mediated Potential Uses Of Genome Editing genome editing has successfully shown the per- Methods manent correction of deleterious mutations in cell cultures, animal models and human derived tis- Genome editing technologies have vast range of sue cell lines and induced pluripotent stem cells utilities from research in understanding gene func- (iPSCs). This iPSCs are important resource of tion to gene therapy and drug discovery leading research, which are created by reprogramming to 3500 publications, 1000 US patents and one of already differentiated human cells by specific ex-vivo> clinical trial till date.> transcription factors, by a technique described by

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Takahashi and Yamanaka (Takahashi et al., 2006). genetic modification of highly repetitive loci like These iPSCs can be studied for the efficiency and/ telomeres and chromatin remodelling in live cells, or any genomic off target effects of genome editing known as dynamic imaging. system. These genetically modified iPSCs might 4) Drug discovery: Genome editing tech- be used for autologous transplantation mainly for nologies are being used in various studies for therapy of genetic conditions which are candi- identification of molecular drug targets. Here dates for hematopoietic stem cell transplantation. they are used in large scale high throughput gene Alternatively human immune T-cells can be repro- disruption in in-vitro cell culture and/ or patient grammed in-vitro for immunotherapy of various derived cell cultures and iPSCs. cancers. 5) Others: The genome-editing technologies are widely being used in agricultural and livestock

TracrRNA Cas9 industries.

Cas9 CRISPR-Cas9 System Construction and Modifications

Cas9 In laboratory, the original CRISPR-Cas9 system is slightly modified to be used as genome editing tool. crRNA and transcrRNA can be constructed together as single guide RNA (gRNA). Multiple gRNAs can also be multiplexed in a single construct to target multiple genomic sequences in single experiment. Also these gRNAs can be combined with another DNA template, which can be used for HDR, to induce gene correction. NHEJ HDR Challenges in Genome Editing Figure 1 Schematic representation of CRISPR- Cas9 system. Blue bars represent Technologies the array of identical repeats. Red, yellow and green bars represent the The various challenges in application of genome various spacers/interspersed elements. editing technologies include the safe and efficient Black oval is cas9 and blue bar is delivery of genome editing components to the tar- transactivating crRNA (tracrRNA). Ma- geted cells, the overall efficiency of genome editing, ture crisper RNA (spacer with repeats) off-target effects and long term regulation of gene hybridizes with tracrRNA and along with function. Various ways of delivery which have been cas9 makes a ribonucleoprotein com- used include plasmid transfection of CRISPR-Cas9 plex. This complex binds to targeted system with same vector encoding gRNA and Cas9 20 nucleotide genomic sequence and protein, viral transduction (i.e. lentivirus) direct produces double strand breaks (DSBs). delivery of Cas9 mRNA and gRNA, direct deliv- These DSBs are repaired by either er- ery of Cas9 protein (Cas9/gRNA ribonucleoprotein ror prone nonhomologous end joining complex) electroporation, lipid nanoparticles and (NHEJ) or precise Homology directed microinjection etc. Every delivery system has its repair (HDR). own advantages and disadvantages. Off-target effect mainly are comprise of introduction ofDSBs 3) Studying the epigenetic modification and at genomic sites other than the targeted. The chromatin remodelling: Nucleases, which are identification of these off-target effects canbe used in genome editing methods can be made predicted and/ or diagnosed by the combina- inactive by introducing mutation in one of their tion of bioinformatic approaches (predicting the functional domains, which in turn, might just tag a homologous genomic sites) and whole genome/ desired genomic site. These deactivated nuclease, transcriptome next generation sequencing. The can then control the expression of endogenous various approaches which are being used to reduce genes, as well are important source to learn epi- these off-target effects include the use of modified/

204 Chapter 32 more specific Cas9 nuclease enzyme, limiting the (Manjunath et al., 2013). duration of Cas9 activity and the use of inducible First report of genome editing method was Cas9 enzyme. These nucleases are modified at based on using ZFN and then followed by TALEN their functional domains, so that they produce nick and CRISPR-Cas9 system. Genome editing is her- only in single DNA strand and is called as nick- itable and hence does not require the repeated ase. Two gRNAs bind to adjacent genomic regions therapy like short interfering RNA (siRNA) and only on sense and antisense strands respectively and one time therapy is sufficient. Trials are going paired nickases produce independent breaks. In on using gene editing of patient derived CD4+ case there is an off target effect, there will be break /CD34 +/ hematopoietic stem cells and induced in one strand only which will be repaired by base pluoripotent stem cells followed by autologous excision repair method using homologous strand transplantation back in patients. Early results as a template. This is very rare that two adjacent are positive in terms of persistence of genetically genomic regions are having off target sites. This modified cells in the circulation and no serious modification decreases the off target by 50-100 side effects. However the true efficacy canonly folds. be determined after interruption of HAART and Examples of various Applications of CRISPR- after the final results of these trials. Also genome Cas9 System in research publications: Following editing system has been used to eliminate already research publications are few success stories of integrated HIV proviral DNA. Ebina et al in 2013 CRISPR-Cas9 system: in targeted the long terminal repeats (LTR) of in- Role of CRISPR/Cas9 in repairing of CFTR tegrated HIV viral genome using CRISPR-Cas9 and defect: In a recent study Schwank et al used, showed the blocked expression of genes (Ebina et CRISPR-Cas genome editing approach, to correct al., 2013). d508 mutation in the cystic fibrosis transmem- Role of CRISPR to cure liver disorder: In a brane conductance regulator gene (CFTR). They recent study published in Nature Biotechnology isolated and expanded adult intestinal stem cells Yin et al had used CRISPR-Cas9 genome editing from two patients affected with cystic fibrosis approach to cure hereditary tyrosinemia in mouse (Schwank et al., 2013). Genomic editing was done models (Yin et al., 2014). The genetic correction using CRISPR-Cas9 system and homology medi- was observed in 1/250 liver cells. Strong posi- ated repair of d508 mutation. The authors also tive selection and further expansion of genetically demonstrated the functionality of gene in in-vitro corrected cells also contributed to the success epithelial organoid system. This article provides story. the proof of concept that CRISPR-Cas9 system can be used as effective gene therapy method in various single gene disorders where no curative Conclusion treatment is available. Role of CRISPR in HIV therapy: After the CRISPR-Cas9 system has revolutionised the area of introduction of highly active antiretroviral therapy genome editing mainly because of ease of labora- (HAART) HIV is now considered a chronic disease tory construction, targeting multiple genomic sites which requires prolonged treatment. However simultaneously and application in broad areas. HAART is associated with relapse of disease as Future challenges involve invention of efficient de- soon as treatment is stopped, high cost of treat- livery systems and reducing off target effects and ment, commitment of the family and treating increase in efficient homology mediated repair. clinicians and potential of emergence of HIV re- sistance. CCR5 is coreceptor on CD4+ cells which References is essential for HIV entry inside these cells. A 32 base pair deletion in single exon of CCR5 gene 1. Ebina H, et al. Harnessing the CRISPR/Cas9 produces the frame shift mutation and disrupts system to disrupt latent HIV-1 provirus. Sci the gene function. The famous berlin patient, Rep 2013; 3: 2510. who was having HIV with lymphoma was cured 2. Manjunath N, et al. Newer gene editing tech- by bone marrow transplantation from a person nologies toward HIV gene therapy. Viruses 2013; who was harbouring this homozygous deletion in 5: 2748-2766. CCR5 gene. Hence disruption of CCR5 gene by 3. Sander JD, Joung JK. CRISPR-Cas systems for gene silencing methods appeared to be attractive editing, regulating and targeting genomes. Nat model as a method of gene therapy of HIV patient Biotechnol 2014; 32: 347-355.

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4. Schwank G, et al. Functional repair of CFTR by 126: 663-676. CRISPR/Cas9 in intestinal stem cell organoids of 6. Wei C, et al. TALEN or Cas9 - rapid, efficient cystic fibrosis patients. Cell Stem Cell 2013; 13: and specific choices for genome modifications. J 653-658. Genet Genomics 2013; 40: 281-289. 5. Takahashi K, Yamanaka S. Induction of pluripo- 7. Yin H, et al. Genome editing with Cas9 in tent stem cells from mouse embryonic and adult adult mice corrects a disease mutation and fibroblast cultures by defined factors. Cell 2006; phenotype. Nat Biotechnol 2014; 32: 551-553. PhotoQuiz - 8 Contributed by: Dr Prajnya Ranganath Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad Email: [email protected]

This test was performed to confirm the diagnosis in a 11 months-old female child who presented with growth failure, developmental delay, cleft palate and Tetralogy of Fallot. Name the test and the condition. (Acknowledgements: Dr Rajitha P, Dr Ashwin Dalal. Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics, Hyderabad)

Answer on page 232

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Next Generation Sequencing in New Born Screening - Current Insights

Deepika Delsa Dean, Sarita Agarwal Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS), Lucknow, India

Correspondence to: Dr Sarita Agarwal Email: [email protected]

Abstract aptitude, DNA sequencing underwent major im- provements making it possible to sequence a large Newborn screening (NBS) program refers to a number of samples in parallel which was not quite nation-wide or state-wide program that identifies possible by Sanger sequencing. The emergence of and treats newborns with rare congenital condi- Next generation sequencing (NGS) in 2005 met the tions before the onset of symptoms, preventing key shortcomings of Sanger sequencing in being premature death and serious disability in thou- more cost effective, rapid, and requiring lesser sands of newborns. Following the great success of amount of DNA. Clinical implementation of NGS for Next Generation Sequencing (NGS) technology in disease characterization in individual patients was the clinical diagnosis of genetic disorders, a lot of found to be highly fruitful (Worthey et al., 2011; expectations have been raised among researchers, Lupski et al., 2010; Liew et al., 2013). clinicians and the public for its implementation NGS, in view of its present achievements in in the newborn screening program (NBS). But in the field of diagnosis, has heightened the expec- view of the ethical, legal and social issues revolving tations of the scientific community and clinicians around the use of genome sequencing approaches in incorporating it in routine clinical practice and in health-care and programs it is more recently in mass screening programs like necessary to address these issues beforehand to NBS (new born screening).This review will focus on avoid its long term failure. This review will focus on the realized and expected benefits of using NGS in the realized and expected benefits of using NGS in the state NBS program and will also highlight the state NBS program and will also highlight the major major limitations that have to be considered for hurdles and practical difficulties that have tobe materialization of such a program. considered for materialization of such a program. Extension of NBS in the Genomic Era

Introduction NBS is an essential, preventive public health program established internationally in order to Till date Sanger sequencing has been the gold identify disorders in newborns that was started standard for DNA sequencing. Using this tech- almost 55 years ago. It began as a method for nology a major foray called the Human Genome pre-symptomatic diagnosis and preventive treat- project started in 1990 and lasted for 13 long years ment for one disorder Phenylketonuria (PKU) in wherein $3 billion was expended to determine the newborns and later, in the 1990s, with the intro- whole human genome sequence. But in spite of duction of a much cheaper and reliable analytical the known usefulness of DNA sequence analysis technique Tandem mass spectrometry, more than at that time it was beyond imagination for the 30 different metabolic disorders were added to clinicians to think about sequencing every patient’s the screening panel in neonates which led to a genome to find possible variants underlying the significant expansion of NBS. The initial guidelines concerned disease due to practical limitations of followed for including any disorder in the neonatal this technology i.e. being expensive and time screening program were based on the Wilson and consuming. Thereafter, with continuous advance- Jungner criteria (Laine et al., 2013) that emphasized ment in the research methodology and scientific on conditions that are considered as an important

207 Chapter 33 health problem with well understood natural his- recent reduction in the cost and time required for tory and requiring immediate medical intervention sequencing the whole genome and the promise in order to prevent serious and permanent illness, it holds both for research and health care have and for which there is an available treatment. drawn a significant momentum around the idea of Currently with expanded NBS most babies are using NGS in a state-run mass screening program screened at birth for between 30 and 50 genetic for NBS. But before this thought could be objec- disorders, primarily by using tandem mass spec- tified we must recognize all the challenges that trometry (MS/MS) and many of these disorders do might interfere in attaining this vision. not fit into the classical paradigm of NBS. Inthis way, newborns are being screened even for condi- Challenges to be Faced Before the tions that do not present as emergencies and may not be immediately life-threatening, but could ben- Establishment of NGS-NBS Program efit from treatment with prophylactic antibiotics or if their screening might have additional benefit to NBS, as discussed before, is a state-run mass parents for reproductive purposes (Grosse et al., screening program that aims to identify serious, 2006). Thus, right from the beginning, expansion treatable disorders in asymptomatic newborns of NBS has been an attempt to provide maximum that require immediate medical intervention. To benefit to the child and the family, through use carry out newborn screening NGS can be used in of the growing knowledge about genetic disorders a better way. Being a high throughput technology and technology. Now when we are in the Genomic it can scan the entire sequence of the newborn’s era where progress is taking place in an ever genome to produce a huge amount of information quickening pace, many of the seemingly unreal- about target and off target disorders, information istic visions are beginning to materialize. The of which may or may not be desired, and may not require clinical intervention, but it stands as a

Table 1 Challenges to be addressed before implementation of Next generation sequencing in newborn screening.

ISSUES KEY QUESTIONS DATA STORAGE Who will be responsible for maintenance and governance of large amount of data generated? REAL COST Keeping in mind the costs for data analysis, family notification, follow ups and confirmatory testing, what should be the cost per testand the total cost for screening? ETHICAL CONSIDERATION How to report uncertain results to parents and how to do follow up once the newborn is discharged from the health center? Whether to report back results for late onset disorders to parents as it hampers the child’s autonomy to know or not to know? Who will be responsible for the privacy of stored data and results until maturation of the child? How to settle the issue of sharing results regarding serious, pre- ventable genetic disorders with at-risk close relatives, if parents decline to give consent? Will the test information result in prejudice against individuals with genetic disorders? INSURANCE DISPUTES Will test information make it difficult for an individual to obtain health insurance as predisposition to a genetic disease might be considered as a pre-existing condition? MEDICAL MALPRACTICE Whether clinicians are properly trained to interpret data generated by genome sequencing? Will hospital administrators be sued over testing errors?

208 Chapter 33 modern choice over existing technology. In view of the newborn period identified that majority of the the significant demands in achieving the NBS ob- respondents felt that presently WGS should not be jectives, NGS is expected to bring both these ends used in NBS and if it were to be used, it should not (NBS with modern technology) together. However, be mandatory. They considered that accurate in- this definitely calls for making changes in the cur- terpretation of the result, more extensive consent rent policy, ethics and legal considerations (Table process, pre and post-test counselling, comparable 1). cost and turnaround time must be achieved before using NGS in NBS (Ulm et al., 2015). Howard et NGS-NBS: Whole Genome, Exome or Gene al. in 2015 have suggested to perform targeted Panel:• At the most basic level, before establish- analysis of genes that are clearly involved in a spe- ment of NGS-NBS, the first thing to be sorted out cific disease with effective and accepted preventive is how is NGS to be used in a screening program? or therapeutic intervention (Howard et al., 2015). NGS being a most versatile tool can be imple- However the choice of panel of genes to be tested mented in different ways to provide information will depend upon the epidemiological prevalence about the entire genome or only the gene coding which is not uniform across the world. region or certain target genes in a selected panel known to be involved in disease pathogenesis. Data storage and retrieval: cost and privacy: Sequencing the genome of all the newborns will Generally exome sequencing has an edge over • generate a huge amount of data and proper data sequencing of the entire genome, owing to its analysis and storage will be required. The cost low cost and easy interpretation in terms of dis- of the TMS-based NBS procedure (2011) in the ease. But the exome covers only 1% of the human European Union ranges from € 0.46 per newborn genome: thus any DNA variation in the non-protein to € 43.24 which is much lower in comparison coding region will obviously be missed. Further- to the cost that will be needed for sequencing more, exome capturing by hybridization can in- neonate genome and analyzing the data (Frank et troduce substantial amount of coverage variability al., 2013). The real budget for the entire process that will have impact on comparative analyses. of screening by applying genomic sequencing is Also, copy number and structural variations (CNVs far more than the proposed $1000 as it does and SVs), as well as some insertions, deletions and not include the cost of data analysis, family no- block substitutions are difficult to detect in exome tification and follow-ups and confirmatory testing capture data (Belkadi et al., 2015; Meynert et al., (Mardis et al., 2010). On an average 353,000 2014). These studies highlight the technical upper babies are born per day around the world and the hand of whole genome sequencing over exome economic feasibility of sequencing, analyzing and sequencing in providing an intrinsically richer data maintaining the vast amount of data generated is of polymorphisms outside the coding region and questionable. In the case of late onset disorders or disclosing genomic rearrangement. With the steep a fatal disease the information regarding the result reduction in the cost of DNA sequencing in recent of sequencing must be given to the child after he years (Wetterstrand, 2013; Young, 2014), the main becomes mature and decides to know his disease economic benefit of using exome sequencing is status. Fully guaranteeing the governance and nullified as more sequence information now could privacy of this information until being disclosed be obtained by using WGS, thereby cost effectively is not possible (Chadwick et al., 2013). However, supporting the use of WGS in NBS. To explore the as the speed at which technology is progressing, possibility of establishing WGS-NBS a study was in the near future it is likely that more advanced performed recently, which compared the screen- cost effective sequencing technology will emerge, ing results of 1,696 infants by the state-run NBS thus there might actually be no need to store such program and whole genome sequencing for 27 information and sequencing could be done when disorders. Though WGS yielded fewer false pos- desired at a later age. But this option will invalidate itive results as compared to TMS-based NBS, the the much boasted utility of NGS at an early age frequency of results with uncertain significance and using the information generated for aiding was quite high. The conclusion of the study was in the future. that WGS might be used in complementation with the present TMS-based NBS assays (Dale et al., Variants of unknown clinical utility: Incorpo- 2015). Conversely, a recent online survey to ana- ration• of NGS in NBS will increase the number of lyze the professional opinion of genetic counselors uncertain variants simultaneously increasing the about the use of whole genome sequencing in burden on the parents and the care providers

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(Cooper and Shendure, 2011). Additionally, it will Society of Human Genetics, the Human Genome also lead to an upsurge in the pressure on the Organization Committee on Ethics, Law and Soci- laboratory and clinicians to determine the clinical ety, the PHG Foundation and the P3G International validity of the variant at the earliest. A combination Pediatric Platform have recommended that un- of variants might be detected in some newborns solicited findings which lead to a preventable or which may never lead to occurrence of disease. treatable health problem should be communicated This will cause consequences of over-treatment (Howard et al., 2015). Such ethical issues need to in an otherwise healthy child and will result in be considered and it is advisable to counsel the unnecessary psychological and financial burden on parents about such consequences before the test the family. On the other hand, as sequencing is not is performed. In case of untreatable diseases, it totally free from error and also there are chances is recommended that the information must not be that some variations might get missed depending given to the parents but to the child at the proper on the sequencing platform used, an infant may age after consent (Shannon, 2014). get deprived of early diagnosis and ameliorative Need for clinicians trained in genetics: A or preventive therapy (Knoppers et al., 2013; Clark large number of variants are identified in sequenc- et al., 2011). Interpretation of results might also • ing the newborn genome and the clinical relevance vary among the laboratories and there might exist of most of them is not so straight forward. Rel- discrepancy in assigning a variation as pathogenic atively few doctors receive significant training in or inconsequential hence causing under-diagnosis genetics and related molecular sciences, and thus or over-diagnosis of disease. Keeping all this in lack the background needed to effectively interpret mind it is advisable to determine whether or not to the results of a genetic test. What to report back return uncertain results to the parent.and whether to the parents is often a difficult judgment call for to store the data until validation of these variants these clinicians and if this issue is not addressed and to then notify it to the parents Also, as the before extending NBS to NGS, it may increase the status of variants of unknown significance keeps on number of cases of medical malpractice, where changing and is mostly reclassified over time, it is a physician can be held responsible for not be- important to plan follow-up procedures and family ing able to detect or disclose the genetic risks notification wisely without raising anxiety among preceding the eventual manifestation of the ge- the parents. The follow up procedure in a country netic disorder. Though not in context of newborn like India will become even more problematic as screening, but a recent case in Connecticut in which most of the families come from remote areas and a woman sued her physician for failing to warn her are often impossible to trace again. that her family history of breast cancer also implied a possible genetic risk for ovarian cancer (Downs Unsolicited findings: Although the main em- v. Trias, 2012) provides some insight into the phasis of the NBS program is centered around • bigger picture of NBS upgradation and its pitfalls. what is most beneficial for the child and its One factor that can help reduce liability risks is to expansion to NGS is expected to maximize the improve the knowledge and training of physicians benefits to the child’s health, exome or whole on genetics-based healthcare. But unluckily, most genome sequencing can often reveal probabilistic medical schools have only recently started training information about the relatives in the extended students in genetics, and many physicians feel family also. The primary concern of the clinician that they are not well trained to address genetic is to decide on how to return these results to the issues (Richard et al., 2011).On the other hand, parents. Though the information of an adult onset the fear of missing important genetic information disease may not be required for the child, it might and being held for medical malpractice might force still have clinical implications for the parents or the physician and the policy makers to return relatives. It is a conflicting situation for the clinician more positive results to the parents for which the to decide whether to disclose it to the concerned follow up results may be normal but still it can at-risk individual as it may hamper the child’s right have negative psychological impact on the parents to an open future. Many attempts have been (Hewlett et al., 2006; Johanna et al., 2012). made to categorize these unsolicited findings and decide which of these to be disclosed, but not Informed consent: NBS is usually conducted in the screening context (Bredenoord et al., 2011; without• an explicit consent because it is seen to be Berg et al., 2011). More recently the Public and in the best interest of the child’s health. However, Professional Policy Committee of the European for genetic screening informed consent is the ut-

210 Chapter 33 most requirement and the case is no different in newborn period, the NICHD (National Institute of genetic NBS also. The biggest concern in obtaining Child Health and Human Development) and the parental consent is that who should convey the NHGRI (National Human Genome Research Insti- complicated genetic counselling to the parents tute), both parts of the NIH (National Institutes of and get the informed consent? Are the nurse Health) had launched 4 pilot programs in the year and physician well trained for this or a genetic 2013 and allotted a fund of $25 million to four counselor must be appointed for this purpose? grantees over five years. These grantees include: Will it be possible to give such facilities in smaller Brigham and Women’s Hospital, Boston where the hospitals and medical centers where most of the genome sequence-based screening for childhood babies are born? Would an information brochure risk and newborn illness will be studied in both sick be sufficient for resolving parental queries regard- and healthy infants by employing whole genome ing genetic screening and whether most of the sequencing; Children’s Mercy Hospital, Kansas city parents, who have minimal genetic testing experi- where the researchers are examining the benefits ence, can actually understand the complex genetic and risks of using rapid genomic sequencing tech- information (Harvey, 2014)? A survey was recently nology in the NICU population and trying to return carried out on parents’ opinion of whole-genome the results in 50 hours; University of California, sequencing for newborns, if it were offered by San Francisco are conducting exome sequencing newborn screening programs or pediatrician ser- utilizing newborn blood spots for disorders that vices. In both scenarios, 70% of parents expressed are or are not currently screened for in NBS interest in whole-genome sequencing, citing test with an agenda to improve and expand NBS; and accuracy and the ability to protect a child from University of North Carolina at Chapel Hill that is developing a disease as important factors in their performing exome sequencing of healthy infants decision-making process. But rest of the parents and infants with known disorders. These projects expressed no interest in newborn WGS and were are presently ongoing and in the years ahead we concerned about the “privacy of results”, “potential may look forward to finding more realistic answers for results to be used to discriminate against their to the current ambiguity regarding the application child,” and that results could be used for research of genomic sequencing in NBS. (Goldenberg et al., 2014). Thus the major threat for genetic NBS is that some parents might completely opt out of NBS due to fear that the detection Conclusion of certain genetic variations in their newborn can jeopardize obtaining health or life insurance, or Implementation of NGS in NBS would require even school acceptance and future employment stretching the benefits related to NBS i.e. from (Landau et al., 2014). This might have serious what is good for the infant, to what might be consequences for an infant who has a disorder potentially good for the infant, to what might be that needs immediate medical intervention. good for the family (e.g., reproductive benefit or health benefits for family members), or to what might be beneficial for the society at large (re- Testing the Ground Reality of NGS search), thus in a way diluting the primary goal of the screening program. Presently, in view of the For implementation of a robust technology like haze surrounding the use of WGS or WES in NBS, NGS in a mass newborn screening program, the it seems not likely to fit within the available public main focus should not be just technologically bi- health-care system due to the practical, financial ased; it should also be tested for its long and and ethical challenges that are making this vision short term impact on the family and the child. difficult to achieve. Though the outcomes ofthe The crucial question here is whether large-scale pilot projects on large-scale assessments of the genomic sequencing can provide useful medical risks and benefits of genome sequencing for new- information beyond what current newborn screen- borns will aid in designing the guidelines for NBS ing is already providing and at what economical expansion in the near future, as of now the topic and emotional cost? To address these issues of newborn genome sequencing as a public health and to analyze the technical, clinical, practical initiative remains contentious. It is recommended and ethical aspects of genomics research in the that such a program could be conducted but as a

211 Chapter 33 commercial supplement with consent. in newborn screening? A statement on the continued importance of targeted approaches References in newborn screening programmes. Eur J Hum Genet 2015; 23:1593-1600. 1. Belkadi A, et al. Whole genome sequencing is 14. Johanna L, et al The impact of false-positive more powerful than whole-exome sequencing newborn screening results on families: a for detecting exome variants. Proc Natl Acad qualitative study Genet Med 2012; 14:76-80. Sci U S A. 2015; 112:5473-5478. 15. Knoppers BM, et al. The Human Genome Or- 2. Berg JS, et al. Deploying whole genome se- ganisation: towards next-generation ethics. quencing in clinical practice and public health: Genome Med 2013; 5:38. meeting the challenge one bin at a time. Genet 16. Laine FR, et al. Technical report: ethical and Med 2011; 13:499-504. policy issues in genetic testing and screen- 3. Bredenoord AL, et al. Feedback of individual ing of children. American College of Medical genetic results to research participants: in fa- Genetics and Genomics. Genet Med. 2013; vor of a qualified disclosure policy. Hum Mutat 15: 234–245. Landau YE, et al. Genomics in 2011; 32:861-867. newborn screening. J Pediatr 2014; 164:14-19. 4. Chadwick R, et al. Imagined futures: capturing 17. Liew WK, et al. Clinical application of whole- the benefits of genome sequencing for society. exome sequencing: a novel autosomal re- 2013, doi: 10.13140/RG.2.1.5153.5521. cessive spastic ataxia of Charlevoix-Saguenay 5. Clark MJ, et al. Performance comparison of sequence variation in a child with ataxia. JAMA exome DNA sequencing technologies. Nat. Neurol 2013; 70:788-791. Biotechnol 2011; 29:908-914. 18. Lupski JR, et al. Whole-genome sequencing in a 6. Cooper GM. and Shendure J. Needles in stacks patient with Charcot-Marie-Tooth neuropathy. of needles: finding disease-causal variants ina N Engl J Med 2010; 362:1181-1191. wealth of genomic data. Nat Rev Genet 2011; 19. Mardis ER. The $1,000 genome, the $100,000 12:628-640. analysis? Genome Med 2010; 2:84. 7. Dale LB, et al. Utility of whole-genome se- 20. Meynert AM, Varian detection sensitivity and quencing for detection of newborn screening biases in whole genome and exome sequenc- disorders in a population cohort of 1,696 ing. BMC Bioinformatics 2014; 15:247. neonates. Genet Med 2015; 18:221-230. 21. Richard RS, et al. Addressing Gaps in Physician 8. Frank M, et al. Genome sequencing: a sys- Education using Personal Genomic Testing. tematic review of health economic evidence. Genet Med 2011; 13:750. Health Econ Rev 2013; 3:29. 22. Shannon R. Newborn screening in the ge- 9. Goldenberg AJ, et al. Parents’ interest in whole- nomics era. J Law Biosci.2014; 1:1-9. genome sequencing of newborns. Genet Med 23. Ulm E, et al. Genetics professionals’ opinions 2014; 16:78-84. of whole-genome sequencing in the newborn 10. Grosse SD, et al. From public health emergency period. J Genet Couns. 2015; 24:452-463. to public health service: the implications of 24. Wetterstrand KA, DNA Sequencing Costs: Data evolving criteria for newborn screening panels. from the NHGRI Genome Sequencing Program. Pediatrics 2006; 117:923-929. 2013.Available at https://www.genome.gov/ 11. Harvey LL. Newborn screening: the genomic 27541954/dna-sequencing-costs-data/. challenge. Mol Genet Genomic Med 2014; 25. Worthey EA, et al. Making a definitive diag- 2:81-84. nosis: successful clinical application of whole 12. Hewlett J, et al. A Review of the Psychosocial exome sequencing in a child with intractable Effects of False-Positive Results on Parents and inflammatory bowel disease. Genet Med 2011; Current Communication Practices in Newborn 13:255-262. Screening. J Inherit Metab Dis 2006; 29:677. 26. Young S. Does Illumina Have the First $1,000 13. Howard HC, et al. Whole genome sequencing Genome? MIT Technol Rev 2014.

212 Section 6 Heart-to-Heart Talk

HeartToHeart Talk The Burden of Diagnosis Shubha R Phadke Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh

Correspondence to: Dr Shubha R Phadke Email: [email protected]

I asked the resident to find out if Baby of Malini reported mutation and the data showed very good (name changed) had undergone the bone marrow coverage of this gene region indicating that this transplantation. When the resident doctor called was a reliable report and this was very likely to be the father of the baby, we got the sad news that the disease-causing mutation in this family. The bone marrow transplant was not done and the mutation was confirmed by Sanger sequencing in baby had died the previous day. This distressing the infant as well as his mother, who was a carrier news brought back the memories of the diagnostic of the same mutation. The family now had to journey which the family had travelled with us, the be counselled about this new development in the medical geneticists, or rather we had travelled with diagnosis and about the fact that the actual diag- the family. The story started more than a year ago. nosis was not NKH. It was difficult and confusing The couple, Malini and her husband, had been for the family to be now assigned a new diagnosis, referred for prenatal counseling and testing in view but with appropriate and detailed counseling they of history of death of their two previous offspring were able to understand. due to an undiagnosed illness: one child had died All these events are described in a few lines during the neonatal period and the other had died here but involved multiple travels for the family at 6 months. Malini was now 3 months pregnant. from their home to our hospital – a distance of There were no medical records available of the 800 km, high costs of the tests, multiple discussions affected children and the couple mentioned that on investigations, their possible results, uncertain- the babies had not been thoroughly investigated. ties and the diagnostic plans ahead. The puzzle There was no consanguinity. The possibility of of the previous two infant deaths was solved, genetic metabolic disorders was considered and but the outcome for this child was poor. The the family was told that in the absence of a solution came in the form of a problem. In the definite diagnosis, prenatal diagnosis cannot be visit when the mutation reports became available provided just on the basis of the suspicion of a and were discussed, the baby was noted to have genetic disorder. They were told to get newborn tachypnoea. Investigations revealed that he had screening for inborn errors of metabolism through severe pneumonia - this was the first significant tandem mass spectrometry (TMS) done for this episode of infection in this immunodeficient infant. baby on the 4th day after birth. The couple came After having been explained the need for intensive back to us after the delivery and TMS was done care treatment for the present episode of severe for the baby (male neonate, yet unnamed, and pneumonia, the parents decided to get the child hence, referred to as Baby of Malini). The report hospitalized in their hometown. A few weeks later, showed a high level of glycine. The CSF glycine we got a call from the parents that the baby was level was done, which supported the diagnosis discharged and was better. The option of bone of non-ketotic hyperglycinemia (NKH). The baby marrow transplant was explained to them and was around 3 months old by this time and was our last communication with the family was when asymptomatic. We explained the implications they were travelling with the baby to New Delhi and prognosis of NKH and explained the need for pursuing this option- we gave them informa- for further molecular genetic testing to confirm tion about the pediatric haematologist and bone this diagnosis. NGS (next generation sequenc- marrow transplant specialist in Delhi. We were ing)-based panel for genetic disorders of infantile hoping that the dramatic diagnosis achieved after onset was ordered as it covers all three genes for the long, costly and cumbersome investigational non-ketotic hyperglycinemia. To our surprise, the journey would lead to an equally dramatic cure result showed a hemizygous mutation in the IL2RG and happiness for the family, but sadly it did not gene, suggesting the diagnosis of severe combined happen. immunodeficiency. This is a known, previously Though each genetic disorder is given a single

213 HeartToHeart Talk

OMIM number, in reality, each case and each family sons. Continued communications about the tests affected even with the same genetic disorder is ordered, possible results and their implications different. The diagnostic challenges faced bythe and counselling helps greatly in getting the de- clinician and the counselling issues are huge. But sired cooperation of the patient and families and far more enormous are the agony, the anguish, lessens the anxieties associated with the diagnostic the anxiety created by the large number of in- odyssey. The treating physicians and geneticists vestigations, and the financial and psychological need to take the patient and family along and burden on the families, while travelling through respect their autonomy as well. The educational this journey full of uncertain stops and uncertain and economic status of the patients and their destinations. Many times, one may not reach the families does not necessarily determine their abil- destination of cure or may not be able to fulfil the ity to understand the test results, interpret them prospect of having an unaffected child. Next gen- correctly and take appropriate decisions. Many eration sequencing has made diagnosis possible such families teach the doctor coping strategies, in situations where, until a few years ago, there humane values and strength of character. Stability seemed to be no hope of identifying the cause. But in adversity is indeed the most important virtue to the flip side of these genomic techniques are the sail the seas of life and for doctors and families uncertainties about the pathogenicity of the novel dealing with genetic disorders, to sail through the variations detected, which make understanding of unchartered expanse of the genome! the implications even more difficult for layper-

PhotoQuiz - 9 Contributed by: Dr Prajnya Ranganath Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad Email: [email protected]

This 10 years-old girl was brought for evaluation of short stature and history of two fractures following minimal trauma. Skeletal radiographs showed typical findings. Identify the condition.

Answer on page 232

214 HeartToHeart Talk Looking Beyond The Vision of a Blind Man Shagun Aggarwal,… Shubha R Phadke Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh

Correspondence to: Dr Shubha R Phadke Email: [email protected]

Big things come in small packages. And the earnest request made us search for laboratories most wonderful surprises spring up where we least across the globe which was doing the PAX6 gene expect. As doctors we usually have patronizing at- testing and finally the test was done in a laboratory titudes towards our patients. We are always trying in Thailand. The family made no pretensions of to help them, trying to communicate so much of being poor or helpless, and without much ado medical jargon, all in good faith and all the time arranged for the amount needed. They followed thinking we know what’s best for them. But we all up sincerely and luckily a mutation was identified have moments when we are humbled by some of in all the affected members of the family. Next, our patients. I had the fortune of being the genetic they came when the elder son’s wife was 8 weeks resident in charge of one such very special family. pregnant. We told them that prenatal diagnosis This family consisted of a Class IV government would be possible. Then we worked to devise a employee, his two sons and his wife. His courteous PCR based test to detect the point mutation which and smiling demeanor was very endearing. He was found in that family. After hard work over 2-3 had been blind since the age of 18 years, and in weeks, we were able to devise a simple RFLP-PCR the absence of medical services, had no clue about based test for the particular mutation. But sur- the cause of his visual handicap. He had been prisingly, there was no communication from the happily going on with his life, taking his handicap family. We waited a little more, finally curiosity got in his stride, till his elder son was born. The child the better of us and we called them up. The father had congenital nystagmus which made the parents sounded very apologetic and said that he would suspect that something was wrong with his eyes. come to meet us. So, next morning he turned After many inconclusive consultations with local up with his wife in tow. His hands were folded doctors, the child was taken to an ophthalmologist as he said “We are sorry for the inconvenience where a diagnosis of aniridia was made. The caused to all of you. We talked to our son and younger brother and the father were both also daughter-in-law and the daughter-in-law says she’s diagnosed to be suffering from the same condition. not interested in getting the test done. We’ve tried The father had become blind due to superimposed talking to both of them but we can’t force our complications like glaucoma, corneal opacification decision on them.” We were surprised at the stand and cataract. This child, who was in his teens at taken by this man. In a country where the falling that time, luckily had no other associated problems gender ratio and endless atrocities on women and his vision was correctable with glasses. His across all socio-economic strata bear testimony younger brother however was not so fortunate and to the disadvantage that being a woman is, the had associated problems in the form of corneal respect given to the newly wedded daughter-in-law opacification and subsequently developed blind- in this poor family was indeed amazing. This family ness. This family had come to our department with a disease associated with such a debilitating in 2002 with the diagnoses of aniridia for genetic handicap as blindness had such a positive attitude consultation. They were told about the genetic that they were ready to take the 50% risk of similar nature of this condition and were informed about illness in the unborn child. The daughter-in-law the 50% risk of transmission to the progeny of the had the courage to say no, the parent-in-laws had two boys. They were also informed that a genetic the wisdom to let their children make their own test may be possible from outside India and this decisions. The family was such an inspiring exam- can help in providing prenatal diagnosis. After this ple of the happy Indian family we all like to boast first consultation, they returned after a gapof7 about. They let love & respect rule their lives. And years when the elder son was married. It is to their that truly was the secret of their happiness, their credit that they had remembered all the scientific togetherness against all odds. Their resilience & information correctly and understood the need for willingness to take on all challenges that life threw timely genetic consultation. They requested us to at them was indeed very moving. It has continued arrange for genetic testing of the elder son. Their to inspire me in my difficult moments till date.

215 HeartToHeart Talk Amniotic Cavity Full of LOVE ! Shubha R Phadke Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh

Correspondence to: Dr Shubha R Phadke Email: [email protected]

Two of my old patients turned up in the OPD or optimism? I do not know… today. I was seeing them in person for the first Case 2: She is an eleven month old chubby time! I had seen these patients ‘in utero’ while baby on the shoulders of the mother. She had doing ultrasonographic examination. One was undergone a surgery. Some more is planned for diagnosed as holoprosencephaly at 28 weeks of the future. Her mother is a nursing staff in our gestation and case two was diagnosed as tricuspid hospital. One afternoon after finishing her morn- atresia at 18 weeks of gestation. ing duty she had come for a routine malformation Case 1: A twenty one day baby was qui- scan at 18 weeks of gestation. The USG showed etly sleeping in her grandmother’s lap under the tricuspid atresia. I myself was disturbed. She was watchful eyes of the mother. After the baby was alone. She was more than just a patient for me diagnosed to have holoprosencephaly at 28 weeks as she is working in our hospital. Rather than of gestation the father came home from Hong prolonging her anxiety for a day, I gave her the Kong. The mother wanted him with her to face news. I called up the cardiologist and got her fetal the reality of the birth of a baby with such a major Echocardiography done to confirm the diagnosis. birth defect. The baby was born at 34 weeks The seriousness of malformation and the need of of gestation. As advised, the couple brought the major cardiac after birth were discussed. baby for karyotyping within the first few weeks of As the pregnancy was less than 20 weeks she had life. The holoprosencephaly was clearly seen in the an option of medical termination of pregnancy as CT scan done after birth. They re-discussed the well. She did not express her feelings at all. issues about prognosis of holoprosencephaly. The Next day she told me that she had discussed situation was not very pleasant but they had not the problem with her family in Kerala and nobody let their joy of child birth get dampened by it. The was in favor of termination. We did not get much baby was named, new clothes were bought. The follow up later. However, today she was here with baby’s small head was covered with a colorful cap. the 11 month old baby. The baby was delivered There was no undercurrent of blaming each other in a tertiary care hospital in south India, received which we sense many a times when a child with the care of specialists, underwent surgery and was a birth defect is born. I informed them that the doing well. Today I could see a proud and happy karyotype was normal. The father told that soon mother. My subtle attempts to understand the the baby and the baby’s mother would be joining feelings of the mother were not successful. I did him in Hong Kong. The parents and grandparents not get any idea about how she had felt during the were not overtly disturbed or anxious. I am not rest of the pregnancy. Had she been apprehensive saying that they were not sad but had accepted about child birth, the expected medical problems the situation and were ready to live with it. Not and surgeries? What was her source of strength to much was said. I reminded them about the risk face all the ordeal and uncertainty which she could of recurrence and early prenatal diagnosis. The have easily avoided by termination? The only thing father told that he may think about gene analysis which I understood was that she was sure about at a later date. The grandmother had some queries her decision to continue the pregnancy and was about feeding. Some of her other questions she ready to face any eventuality. And today she was left unsaid. I understood that she wanted some happy… positive news but she almost knew that the an- These cases are not rare but a little uncommon swers were not going to be very much hopeful. in India where in people’s minds the threshold for At last the grandmother asked me to measure the termination of pregnancy is a little low. Medical head circumference and whether it had grown over termination of pregnancy is not possible legally the last 3 weeks. beyond 20 weeks of gestation. This made the Was it complete acceptance that had brought situation difficult in case I. In case 2, the decision peace in this family or was it a thin thread of hope ‘not to terminate’ was probably based on religious

216 HeartToHeart Talk beliefs but the decision was firm and there was illness, the mother and the family puts in all efforts no dilemma. This gave the mother strength to to help the baby and provide the best possible face the problems. Similarly, in the first case, the treatment. The love tries to form a shield/a family knew that there was no other option but to comfortable cocoon for the baby. Mother’s love is continue the pregnancy. This must have helped unique. It accepts the child as it is. Can it not also in acceptance of the situation. If any birth defect accept the child in the womb as it is? is diagnosed after birth or a neonate develops an PhotoQuiz - 10 Contributed by: Dr Prajnya Ranganath Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad Email: [email protected]

This 5 months-old female infant was brought for evaluation of feeding difficulty and joint deformities. On examination, she was found to have typical facial dysmorphism and multiple joint contractures. Identify the condition.

Answer on page 232

GeNeToon

Contributed by: Dr Shubha Phadke

Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow Email: [email protected]

217 HeartToHeart Talk Short – on sensitivity ! Divya Aggarwal, Shubha R Phadke Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh

Correspondence to: Dr Shubha R Phadke Email: [email protected]

Jayant and Rashmi (names changed) were mar- The younger one, Anuj, was 34 years old, had com- ried for a year now. It was a dream come true pleted a masters degree, was soon to be married for Rashmi’s three brothers- Anil, Anuj and Vimal and was the principal of a Hindi medium school. (names changed). It was a perfect marriage and The youngest, Vimal, was 28 yrs old, unmarried, the couple looked beautiful together. Rashmi was and had started his own business after completing young, slim, tall and pretty as a ‘picture’. Jayant his graduation. All three seemed hardworking, well was a handsome young man and well settled settled adults, with no physical or mental health financially. The brothers were so content in having problems. They were very pleasant to talk to with found the “perfect” match for their only sister. They very amiable personalities and were well adjusted gave a lavish reception to the boy’s family at the to their lives and heights. Outside of the clinical wedding, gave expensive gifts, and did everything setting in which we found ourselves, no normal they could do, just to make sure that their princess person would have given them a second look or would not have to listen to anything hostile in the thought of them as being “defective” in any way. family which was much more well to do than their Jayant was aghast at the idea of having his baby own. look like his brother in laws. He curtly stated Everything went well and one year after the again and again without any respect towards his marriage, Rashmi had conceived. Everything was in-laws’ sensibilities that he would not want his going fine till 10 weeks of pregnancy when her child to carry forward the uncle’s genetic defects. gynaecologist took the family history. She coun- He was so obsessed with the idea of bringing his seled the couple regarding the probability of a “perfect” child into this world, that he appeared hereditary disorder running in Rashmi’s family, blind to all other people and their feelings. Rashmi the possibility of the fetus being affected by this maintained a stoic silence through this. Human genetic disorder and referred the couple to our beings are such wonderful creatures with such outpatient department. myriad ways of reacting to the same emotions The couple did not waste anytime in visiting our and thoughts. What could be poison for one OPD. Jayant looked very upset and anxious. Rashmi man, could be different for another. All this talk maintained her calm. As we started taking the instead of bringing about any negative reaction history, we came to know that the genetic disorder from Rashmi’s brothers, made them feel as “guilty for which the gynaecologist had referred was short as hell”. They were shattered and stood there with stature in Rashmi’s three brothers. Jayant did most their heads down, although they were in no way of the talking. He said that because Rashmi was so responsible for the couples’ predicament. Looking tall and beautiful, he and his family did not think at them being tortured and humiliated by Jayant’s that there could even be the slightest possibility endless verbal rant made us feel sorry for the poor of the disorder getting transmitted to his children. folks. We counseled them that any further opinion from Now regarding their reason for having come to our side could be given only after examining at us. We evaluated them in detail. Anil’s height was least one of the brothers. The three brothers were 138 cms, just about half a feet shorter than the staying in different parts of the country, sowe lower limit of normal of an average Indian adult asked them to call the eldest one Anil, who was male. Anuj’s height was 144 cms and Vimal’s height staying closest to Lucknow. was 153 cms. Apart from this, there were no After 3 days, to our utter surprise, the couple skeletal deformities and no systemic abnormalities walked into the OPD with all the three brothers- leading to any physical, sexual or mental disability. Anil, Anuj and Vimal who had come from different Since the short stature was proportionate with cities. All of them were short in height but propor- no deformities and no associated features, along tionate. The eldest, Anil, was 38 years old, married, with a history of Anuj and Vimal having received had 2 children and was a teacher by profession. few Growth hormone injections at 18 years of

218 HeartToHeart Talk age, we took the opinion of our endocrinology or the guardian should give consent for detailed consultant for possibility of Growth hormone de- evaluation. Sometimes this is not possible due ficiency. A clinical diagnosis of Isolated Growth to lack of co-operation of the affected individual hormone deficiency was made. It is a genetically or his/ her guardians. In this family the affected heterogeneous condition known to have autoso- brothers were very co-operative and came all the mal dominant, autosomal recessive and X-linked way on the request of their sister and her husband. forms of inheritance. Known genes involved in But what we observed was that the young brother- the genetic etiology of isolated growth hormone in-law was so engrossed with the possibility of deficiency include those that encode growth hor- having a ‘suboptimal’ or an ‘abnormal’ child like his mone (GH1), growth-hormone-releasing hormone brother- in -laws that he did not realize that many receptor (GHRHR) and transcription factor SOX3 of his words were hurting and humiliating to the (leading to XR inheritance). However, mutations intelligent, successful and well-adjusted brothers- are identified in a relatively small percentage ofpa- in- law. Respect towards other human beings is the tients, which suggests that other, yet unidentified, basic quality of civilized and good ‘human’ beings. genetic factors are involved. Jayant crossed his limits a number of times while Genetic counseling was provided for the family. talking about ‘short people like his brothers- in- The couple were told that the chances of their law’ and was not apologetic about it. He did not baby having short stature due to growth hormone realize that each of them had some points better deficiency would range from 0-25%. Prenatal diag- than him. His approach to the situation lacked nosis could be provided only if the exact genetic the sensitivity the situation demanded. Auton- basis was identified in affected members ofthe omy should not be the most valuable principle of family. The chances of detection of a mutation in bioethics, even if it is the most dominant feature of the family and the cost was also explained to them. human behavior (e.g. selfishness). If it is, we arrive The case illustrated and emphasized the un- at a society with lack of concern for the poor and willingness of the society to accept disability even sick. Instead, we need to balance selfishness with in its most subtle form and the taboo associated sufficient altruism, to make a true loving society. with it. With the advent of prenatal diagnosis, We can refocus our concerns to consider the best we now have the ‘right to know’ and are implored interests of the individual and the family and not to make ‘informed choices’ about our health and just the autonomy of the individual. Genetic free- of those we love. Because of the emphasis on dom is not unconditional freedom, because part of responsibility towards one’s own health, within the concept of autonomy must be recognition of high modernity, as well as notions of perfection, other people’s autonomy, or values. parents are rapidly being faced with an obligation I would like to end with a quote that sums up the to use these technologies for not only the future high aspirations and zest for perfection of parents of their child but also to secure their own future. regarding their unborn baby in the modern society Perfection which earlier happened laboriously over with the availability of modern technologies. generations through the process of Darwin’s “nat- ural selection” has now been put in our hands. ”Dear Designer Baby, Your mother and I created We now have the tools to choose what constitutes you but then decided to give you a little help by “perfect” and what would only be “good” or worse inserting some desired genes. We thought you “bad”. Whom to give life to and whom to wither should look as nice as possible, so you’re quite away ? Such power in human hands needs to be handsome now. We thought it might help if you tempered with virtue and grace. were a little smarter than others, and so you are. The other issue which needs to be addressed And you should be slim, not fat. We love you, so we is the sensitivity towards the proband. Diagnosis made you a better person. Hope you like yourself. of the proband is of paramount importance for Love, Dad.” –Austin E. Sakong, Woodbridge NJ.) genetic counseling. The proband may not be This case reminded all of us to talk with the sib or offspring of the consultand and his/ great sensitivity to and about ‘different’ people her consent is important before getting access to whom we geneticists see every day as a part of his/ her medical records and samples. He/ she our clinical work !

219 HeartToHeart Talk Unacceptable Truth or Acceptable Lie !! Ashwin Dalal Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics, Hyderabad

Correspondence to: Dr Ashwin Dalal Email: [email protected]

Life is full of situations in which it is difficult daughters and explain them about the disease and to differentiate between what is correct or wrong. regarding prenatal diagnosis after their marriage. Circumstances and emotions can compel sane Two years later Ms B presented to us as people to act in most insane fashion. primigravida with 8 weeks pregnancy for prenatal This anecdote is in relation to a patient of diagnosis. She told us that her father had informed Huntington disease. Huntington disease is an adult her that the report was normal but it was better to onset, neurodegenerative, triplet repeat disorder do prenatal diagnosis just to rule out possibility of inherited in autosomal dominant fashion. The dis- disease in fetus. She showed us her report (done ease is caused due to expansion of CTG nucleotide in our laboratory) which showed repeats in normal repeats in HD gene and risk of transmission to range. We were perplexed why her father had told offspring is 50%. The patient presented as45 her to undergo prenatal diagnosis inspite of normal year old female with involuntary movements and repeats. However when we reviewed our records cognitive decline. She was diagnosed as Hunting- we found that Ms B had inherited the expanded ton disease based on molecular test which showed allele from her mother. Further inspection of the 48 repeats as against normal of less than 31 report revealed the shocking truth that the report repeats. We counselled the couple regarding the available with the patient was a colour photocopy progressive nature of disease, absence of treat- of original report with number of repeats changed ment and 50% probability of both her daughters to normal. Thanks to digital technology, it was a being affected. Further we informed them that perfect copy! It is then that it dawned on us that the although both the daughters are symptom free, father in his overprotectiveness had manipulated presymptomatic diagnosis can be done to detect the report to avoid mental anguish in his daughter whether they are likely to develop the disease in before her symptoms started. However even after future. In absence of definite treatment to alleviate his misadventure of manipulating the report, the the symptoms of disease, the main reason for father made sure that she would undergo prenatal presymptomatic diagnosis in adult onset disorders diagnosis as advised by us during counseling. We is to use this information for prenatal diagnosis to explained the whole situation to Ms B and her hus- prevent transmission of the disease in subsequent band. They both understood the facts regarding generation. Presymptomatic diagnosis is always the disease in Ms B, likelihood of transmission to done in adults (not in children below 18 years) and fetus and absence of any known preventive treat- after a detailed pre-test counseling regarding the ment. They both expressed sympathy towards the psychosocial implications of the test. father and appreciated his efforts to get prenatal The father brought both is daughters aged 26 diagnosis inspite of his efforts to hide the truth years (Ms A) and 28 years (Ms B) for presymp- from his daughter. Prenatal diagnosis revealed tomatic testing. The molecular test done after that the fetus had inherited the expanded allele detailed pre-test counseling revealed that Ms A from Ms B and the couple opted for termination of had normal repeats whereas Ms B had inherited pregnancy. the increased repeats from her mother and hence This anecdote raises certain important ethical was likely to develop the disease at a later age. questions. Was the father right in hiding the truth The father came to enquire for the report and was from his daughter? Was the father’s overprotec- very disappointed (especially since he had seen the tiveness or affection for his daughter impinging on suffering of his wife) since one of his daughters was the daughter’s right to know her mutation status? affected and was likely to suffer the same grue- Who was right and who was wrong? These ques- some fate of an untreatable degenerative disease, tions do not have a definite “Yes” or “No” answer just like his wife. The father took the reports from but such experiences enrich our experience as ge- us since the daughters were staying at a different neticists to look at the emotional and psychosocial location. He said he would give the reports to his aspects of genetic diseases.

220 Section 7 Genetic Exercises

Exercises

Genetic Counseling Exercises

Dhanya Lakshmi N1, Shubha R Phadke2 1Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Hyderabad 2Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow

Exercise 1 Sangita (25 year) is 8 weeks pregnant and has 2. How do you confirm whether she is a carrier come for genetic counseling with her mother, Prat- of DMD or not? ibha, as she had a brother (Somesh) who died 5 3. What are the chances that Samruddhi and years ago at the age of 22 years. He was diagnosed Supriya are carriers of DMD? Will you offer to have Duchenne muscular dystrophy. Sangita them carrier testing? has two sisters, Samruddhi (30 years) and Supriya (32 years). Samruddhi has two sons, 8 years 4. What other tests will you offer to the family? and 6 years, who are apparently normal. Supriya 5. What is the term used to denote Sangita? has a 12-year-old son and an 8-year-old daughter, 6. Siddhant’s Hb A2 is 5.5%. What will you do who are apparently normal. Sangita’s husband, next? Siddhant is 30 year old and has a brother, Subodh (32 years)who has a daughter (Twinkle – 2 year Answer: old) with thalassemia major (Homozygous for IVS 1-5G>C mutation in HBB gene). Subodh’s wife, 1. 50% Shobhana is the daughter of Siddhant’s maternal 2. Multiplex ligation probe amplification to look uncle. Sangita’ parents, Pratibha and Prahlad for carrier state of deletions or duplications are unrelated. Pratibha has a living brother [50 in dystrophin gene. year] who has a 30-year-old daughter. There is history that Pratibha had 2 brothers who had a 3. The chance is 50%. They can be offered muscle problem, which was progressive, and they carrier testing after pretest counseling. became bed ridden and died around 20 years of 4. Carrier state of thalassemia by HPLC to look age. On asking leading question, Pratibha told that for hemoglobin variants probably they had prominent calf muscles. 5. Consultand 1. What are the chances that Sangita may be 6. Carrier testing for hemoglobin variants for carrier of DMD? Sangeeta.

Exercise 2 Shika is a 25-year-old female who has just given If you were the Pediatrician, birth to a baby boy. Her family celebrated the birth of a baby boy and named him Siddharth. Shikha 1. What would you tell Shikha? has completed her Masters degree in biology and 2. What do you think is the apt time to talk to is a high school teacher. Except that Siddharth Shikha? was sleeping for long hours, Shikha found him well. On routine examination on 2nd day of life, 3. Which specific investigation would you like to the Pediatrician found that Siddharth has upslant do? of eyes, epicanthal folds and 5th digit clinodactyly bilaterally. He also has a systolic murmur in left After 10 days, the couple came back with lower sternal border. Siddharth to collect the report of the test.

221 Exercises

The test shows the following: 5. Shikha asks you whether this has happened because of some problem in her. She wants to know whether she and her husband should get tested. What will you reply? 6. Will you advice prenatal testing in subsequent pregnancy?

Answer:

1. Counsel about Down syndrome 2. As soon as diagnosis is made 3. Kayotyping from peripheral blood of the baby 4. That the child has three copies of chromo- some 21. 5. Since it is non disjunction, there is no need to get the chromosomal testing for parents. 4. What would you tell Shikha and family re- garding the report ? 6. Yes

Exercise 3 Sonu, 8 years old, has frequent falls and diffi- 1. Is there a chance that Riya will have an culty in getting up from squatting position since 5 affected child? years of age. His maternal uncle, Ravi, was bed ridden by 15 years and expired at 20 years with 2. What test would you recommend before shortness of breath. counseling the couple? Sonu’s maternal aunt, Poonam, has a daughter, Riya, who is 20 years old and is engaged to Naveen. Answer: Riya and Naveen have come for genetic counseling to enquire the possibility of them having a child 1. Yes like Sonu. They have got a report of Sonu’s 2. MLPA to look for deletions and duplications in which it is written that no deletion has been in dystrophin gene for Sonu. If deletions/ identified in the checked 10 exons by multiplex duplications are identified, carrier analysis PCR. can be done in Riya by the same technique.

Exercise 4 1. What is the risk of the maternal uncle being a carrier of Beta Thalassemia? 2. How will you counsel him? 3. Which tests should be offered to them to diagnose Beta thalassemia carrier status? c 4. What medical risk he can have if he is found to be a carrier? 5. Do you recommend pre- pregnancy testing of the prospective wife for carrier status of beta P thalassemia? The proband has beta thalassemia major. 6. What is the chance that his wife, who is not a

222 Exercises

blood relative is also a carrier? Sources of lab errors: 7. What will you do if both the partners are found to be carriers of thalassemia? Falsely low HbA2 level can occur in iron Answer: 훽 • deficiency anemia. In this situation cor- rect iron deficiency and then repeat the 1. In the above situation there is a 50% chance test. of the maternal uncle being a carrier. Falsely high HbA2 levels in megaloblastic 2. Thalassemia major is an autosomal reces- • anemia and HbE disease/carriers. sive disorder due to mutations in the beta 훽 Carriers can have normal A2 levels globin gene on . • with microcytosis or without microcy- The carrier frequency in the Indian population tosis (Silent carrier) varies from 3-11%, being common in Sindhis and Punjabi Khatris. 4. A carrier of a thalassemia mutation is not a Approximately 1 in 1000 couple is at a risk of patient and will have no medical illness. having a child with Thalassemia major. 3. The investigations to identify his carrier sta- 5. If on testing, uncle comes out to be a carrier tus: then it is essential to ask for consanguinity when he gets married. Also, it is essential to Red cell indices-microcytic, hypochromic screen his wife before pregnancy is planned. • Hb electrophoresis for HbA, A2, F bands 6. The chance that his wife, who is not a blood • and any other abnormal Hb e.g. HbE, HBS relative is also a carrier depends on the population frequency of carrier status. Quantitative HbA2 estimation, If HbA2 • >3.5, suggestive of carrier status 7. If she is also a carrier then the risk of them Serum Iron & TIBC levels – as severe Iron having an affected child is 25%. Mutations in • deficiency anemia can mask a high A2 both husband and wife should be identified level. before prenatal diagnosis can be offered.

Exercise 5 Family with two affected individuals with Answer: Hemophilia A 1. Hemophilia A is an X-linked recessive disor- der where males manifest the disease and I 1 2 3 4 5 females are carriers. If the female is a carrier there is 50% risk of her male fetus being affected. II 1 2 3 4 5 6 In the above pedigree I-3 is an obligate carrier C as she has two affected sons. If proband’s maternal grandmother is a car- 1. What is the chance that II-6 is a carrier of rier, the risk of proband’s maternal aunt I-4 Hemophilia A? being a carrier would be 50%. If I-3 has a de 2. How will you prove the carrier status of the novo mutation, the risk of her sister I-4 being consultand (II-6)? a carrier would be 0%. 3. What will do you do if proband’s mutation is If she is a carrier, then her daughter II-6 also not identified? has a 50% risk of being a carrier. 4. Which technique was previously used to 2. Mutation analysis of F8 gene can be done detect carrier status and provide prenatal in the affected individual, followed by carrier diagnosis? detection in at-risk-females.

223 Exercises

3. Inversion mutation is responsible for the dis- further sequence analysis of the F8 gene ease in 45% severely affected patients. It can would be required and if that is negative, be detected by various techniques like long testing for exonic deletions or duplications range PCR, inverse PCR or Southern blot. If through MLPA has to be done. the common intron 22 inversion mutation or 4. Linkage analysis intron 1 inversion mutation is not detected,

Exercise 6 Severe bilateral hydronephrosis with oligohy- minimum of 2 weeks. The use of kary- dramnios in a 28 weeks old fetus otyping in this scenario with advanced gestational age is to avoid active inter- 1. What will be the plan of management? vention if karyotype is abnormal as the 2. How will you provide counseling? prognosis is poor. Associated extra renal malformations 3. What is the current recommendation on ter- present in 35% cases e.g. neural tube mination of pregnancy beyond the fetal age • defects, skeletal dysplasia, gastrointesti- of viability (28 weeks)? nal anomalies. Answer: Simpson-Golabi-Behmel syndrome, • Beckwith-Wiedemann syndrome and 1. Points to note: Meckel-Gruber syndrome.

Severe B/L Hydronephrosis: AP diameter 2. Counseling : of the renal pelvis >20 mm with severe • Most of the conditions can be surgically calyceal dilation treated after birth. Oligohydramnios • Detailed examination and investigation • 28 wks fetus • of the neonate after delivery by a Pedia- • Causes of B/L hydronephrosis: trician and Pediatric Surgeon is essential. Severe oligohydramnios is a poor prog- Posterior urethral valves • nostic sign. It results in pulmonary • Bilateral pelviureteric junction (PUJ) ob- hypoplasia and neonatal death in many • struction cases or neonatal chronic failure or Rarely urethral agenesis, ureterovesicu- chronic renal failure in infancy and child- hood. Prognosis for life and renal • lar obstruction function is thus poor with severe oligo- Rule out: hydramnios.

Exposure to teratogenic agents like 3. In India, as per the Medical Termination of • Busulphan, Vitamin A analogs, alcohol, Pregnancy (MTP) Act, termination of preg- maternal diabetes. nancy is not legally permitted beyond 20 Chromosomal disorder (associated in weeks of gestation. Delivery at 28 weeks • 10% of cases) XXY, Trisomy 21,13,18. can result in a live birth with additional Reporting time for karyotyping will be a complications of prematurity.

Exercise 7 A pregnant woman with a previous child with 2. What are the causes of microcephaly? microcephaly 3. What investigations will you recommend in the proband to establish the diagnosis? 1. How will you confirm the diagnosis of micro- 4. How will you provide counseling and risk of cephaly in the proband? recurrence to the family?

224 Exercises

5. Is there an option for providing prenatal diag- 4. The recurrence of microcephaly varies from nosis and what are the limitations of prenatal 3-25% in the siblings of the child with mi- diagnosis? crocephaly depending on the etiology. In this family it may be autosomal recessive as there is consanguinity and the recurrence risk could be as high as 25%. If the child with mi- I crocephaly does not have CNS malformations and his cytogenetic testing is normal with syn- dromic, metabolic and environmental causes of microcephaly ruled out, then microcephaly 1 2 3 4 5 6 II can be genetic and inherited in an autosomal recessive form in this family. 5. With this possibility of recurrence, II-3 and his wife are counseled regarding possibility of prenatal diagnosis by ultrasound examina- III 1 2 P tion of the fetus by serial measurements of head circumference and abdominal circum- ference from 18 weeks gestation. However, Answer: prenatal diagnosis of microcephaly by USG 1. Microcephaly is defined as an occipito-frontal has many limitations and is not sensitive circumference (OFC)<-3 SD below the mean enough. Diagnosis may not be possible till nd rd for the age and gender. late 2 or 3 trimester or may not be de- tectable prenatally at all. It depends on the 2. Microcephaly could be due to genetic and severity of microcephaly. non-genetic causes. 3. In this situation it is essential to try and Diagnostic Criteria for antenatal diagnosis identify the cause of microcephaly in the of microcephaly proband (III-I). For this the child needs to be examined in detail and appropriate investi- Head circumference < -3 SD for mean for a gations like MRI brain, development and IQ • particular gestation assessment, eye evaluation, hearing evalua- But reliably diagnosed on a single observation tion and neurological evaluation need to be if < -5 SD done. If a syndrome is suspected, further • evaluation is planned based on the syndrome Head circumference to abdominal circumfer- suspected. The first line of investigation rec- • ence ratio < -3 SD ommended for non syndromic microcephaly Serial measurements to establish the trend is chromosomal microarray. • or lack of growth of the head are essential Exercise 8 A family with a child with bilateral retinoblas- toma treated and cured - risk of recurrence in the next pregnancy

1. Is this a familial case or sporadic? 2. What is the difference in risk of recurrence in familial and sporadic cases? 3. What is the empiric risk of recurrence in case of sporadic case? P P 4. Can you provide prenatal diagnosis for the family?

225 Exercises

Answer: Sporadic: One mutation has occurred as a de novo event or a new germline mutation dur- 1. Sporadic ing gametogenesis and the second mutation 2. Familial: occur in the somatic retinal cells. 44% of spo- radic bilateral retinoblastoma are hereditary If there is family history of a similarly affected and 56% are non hereditary. family member, then it is a familial/hereditary form of retinoblastoma. In this case, one 3. The empiric recurrence risk to sibs of a mutation is already present in the germline proband with sporadic bilateral retinoblas- and most or all of the somatic cells. The toma is 2%. second event occurs spontaneously. The 4. Yes. Analysis for mutation in RB gene in prevalence of familial Retinoblastoma is 10%. the patient and if needed in parents can give The recurrence risk for sibs and offspring is more definitive information and option of 45%. prenatal diagnosis.

Exercise 9 5-year-old Bhavish has intellectual delay and disorder? autistic features which the parents recognized 4. What is the risk of having another child with from 2 years of age. Now the parents are planning autistic spectrum disorder? for pregnancy and want to know the risk of having another child with similar illness. Answer: 1. What is the recommended first line of inves- 1. Chromosomal microarray tigation in Bhavish? 2. What additional investigation would you like 2. Fragile X mutation analysis to do in Bhavesh? 3. MECP2 gene mutation testing 3. If the proband was a girl, then what addi- 4. 3-10% for non-syndromic autistic spectrum tional testing is indicated in autistic spectrum disorder.

Exercise 10 20 year old Mukesh has progressive proximal 2. The variant has to be confirmed by Sanger muscle weakness and bilateral calf muscle hy- sequencing in the proband and his parents pertrophy. He did not have any deletions or duplications in dystrophin gene in MLPA. His sister 3. The chance that his sister is an asymptomatic is getting married and wants to know the risk of carrier is 50%. If she is not opting for con- having a child with similar illness as her brother. sanguineous marriage, the chance that her Their parents are first cousins. partner would be a carrier for such a rare 1. What is the test of choice in Mukesh? genetic variant is very less. If her partner is not a carrier for the same variant, there is no 2. A novel homozygous variant was identified in risk to her offspring even if she is a carrier of CAPN3 gene in Mukesh. What is the next that rare variant. step? 3. What is the risk of his sister having a child 4. The sister can get the carrier testing done. with similar illness? If she is not a carrier, no further work up is 4. How will you counsel the sister? needed. If she is a carrier, her prospective partner can be tested for the same variant. If Answer: he is also a carrier for the same variant, then 1. A panel testing of all genes implicated in the chance of having an affected offspring is proximal myopathy/ muscular dystrophy by 25% and prenatal diagnosis can be provided using next generation sequencing technique by targeted mutation analysis of fetal DNA.

226 CrossWord Puzzle 1

Contributed by: Dr. Dhanya Lakshmi N Department of Medical Genetics, Nizam's Institute of Medical Sciences, Hyderabad Email: [email protected]

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20 Across Down 1. 'G” in G Banding stands for 4. Criteria for diagnosis of Marfan syndrome 2. A Syndrome with cardiac and upper limb 5. Phenomenon in which a single gene is malformations responsible for a number of distinct and 3. Mucopolysaccharidosis which can present as seemingly unrelated phenotypic effects non immune fetal hydrops 8. Substrate reduction therapy was first tried in 6. 55-200 CGG repeats in FMR1 gene this LSD 7. PCR based molecular method which uses 9. The first line of investigation for Rett fluorescent labeled primers to amplify syndrome is sequencing of this gene specific DNA markers which are polymorphic 10. The first disorder targeted for newborn 12. Curated collection of structural variation in screening human genome 11. Deletions and duplications in dystrophin gene 13. Single gene disorder with multicystic resulting in Duchenne muscular dystrophy can dysplastic kidneys, encephalocele and be identified by this method polydactyly 15. Father of Medical Genetics 14. Traditional classification of osteogenesis 16. Distortion of a normally developed structure imperfecta caused by mechanical forces usually in the 17. Triplet repeat disorders can be diagnosed latter half of gestation and most by this method 18. Recent efficient gene editing system 19. LSD inherited in X linked manner 20. First line of investigation for intellectual disability Answers on page 230 CrossWord Puzzle 2 1 Contributed by: Dr. Dhanya Lakshmi N 2 Department of Medical Genetics, Nizam's Institute of Medical Sciences, Hyderabad Email: [email protected]

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3. In RNA not DNA 6. Lyon X inactivation 9. Jumping genes 10. Affects less than 200000 Americans 19 or less than 1 in 2000 Europeans 11. expression of blood group antigens 12. Cats eye, but no ears Down 20 14. An extended family 1. plot A graph of all P values for an 16. Evolution island association between a trait and all 19. A virus whose genome has been of SNP's in GWAS segment engineered to express a 2. expressed in heterozygous state DNA of interest 4. structure at centromere to which 20. postion occupied by a gene on a spindle fibres are attached chromosome 5. Fundamental unit of chromatin organisation 7. increasing severity 8. explores epigenetic patterns 13. disomy two chromosomes from same parent 15. Not a snake, not a ladder 17. The beginning 18. Mendel The Monk Answers on page 230 CrossWord Puzzle 3

Contributed by: Dr. Dhanya Lakshmi N Department of Medical Genetics, Nizam's Institute of Medical Sciences, Hyderabad Email: [email protected]

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3. Coined the term 'genetics' 1. The tribe with highest twinning rate 6. Phenomenon in which a single gene is 2. Homozygote is more seriously affected responsible for a number of distinct and 4. First inborn error of metabolism seemingly unrelated phenotypic effects 5. Change in DNA;but not in ACTG 12. Complete protein complement of a cell 7. Replication of DNA 13. He set up the famous Fly Room 8. No male to male transmission 14. Both homologs of a chromosome pair 9. A genetic disease with lower risk of in a diploid offspring being derived cancer and diabetes from a single parent 10. Trait that exists in a population in at 16. First mammal to be cloned least two phenotypes, neither of which 17. I have cells from two zygotes occurs at a frequency of less than 1% 18. Most frequent pathological mutations 11. Curated collection of structural in the human genome variation in human genome 19. Type of allele masked by another allele 15. One gene masking the phenotype 20. Study of organisation of entire genome of another

Answers on page 230 Crosswords

Answers to CrossWord Puzzle 1 Across: Down: 4. Ghent 15. Victor McKusick 1. giemsa 12. DGV 5. Pleiotropy 16. Deformation 2. Holt oram 13. Meckel gruber 8. Gaucher 18. CRISPR cas9 3. Sly 14. Sillence 9. MECP2 19. Fabry 6. premutation 17. TPPCR 10. PKU 20. microarray 7. QFPCR 11. MLPA Answers to CrossWord Puzzle 2 Across: Down: 3. Uracil 12. Waardenburg 1. Manhattan 8. Encode 6. Mary 14. Kindred 2. Dominant 13. Uniparental 9. Transposons 16. Galapagos 4. Kinetochore 15. DNA 10. Orphan 19. Vector 5. Solenoid 17. AUG 11. Codominance 20. Locus 7. Anticipation 18. Gregor Answers to CrossWord Puzzle 3 Across: Down: 3. Bateson 16. Dolly 1. Yoruba 8. X linked 6. Pleiotropy 17. Chimera 2. Semidominant 9. 12. Proteome 18. Substitution 4. Alkaptonuria 10. Polymorphism 13. Morgan 19. Recessive 5. Epigenetic 11. DGV 14. Uniparental disomy 20. Genomics 7. Semiconservative 15. Epistasis

GeNeToon

Contributed by:Dr Shubha Phadke Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow Email: [email protected]

230 PhotoQuiz

Answer to PhotoQuiz 1 Mucolipidosis type II (I cell disease) (OMIM # 252500) Mucolipidosis type II, also known as I cell disease, is an autosomal recessive lysosomal storage disorder characterized by coarse facies, growth retardation, psychomotor retardation, skeletal abnormalities and cardiomegaly. It is caused by a defect in the phosphorylation and intralysosmal localization of lysosomal enzymes, which leads to very significant elevation of the levels of these enzymes in the plasma. Itresults from homozygous or compound heterozygous mutations in the GNPTAB gene on chromosome 12q23.2. Answer to PhotoQuiz 2 Noonan syndrome (OMIM # 163950) Noonan syndrome is an autosomal dominant disorder characterized by short stature, typical facial dysmorphism (including hypertelorism, downslanting palpebral fissures and low-set, posteriorly rotated ears), and congenital cardiac malformations. The most common cardiac defects seen in Noonan syndrome are pulmonic stenosis and hypertrophic cardiomyopathy but many other types of cardiac lesions can also occur. Other features include chest and spine deformities, webbed neck, mild intellectual disability, cryptorchidism, and bleeding diathesis. Noonan syndrome is a Rasopathy and results from heterozygous mutation in the PTPN11 gene in around 50% of cases. The other genes found to be associated with Noonan syndrome are SOS1, RAF1, RIT1, KRAS, NRAS, BRAF, and MAP2K1. Answer to PhotoQuiz 3 type 2 (OMIM # 607624) Griscelli syndrome (GS) is a rare autosomal recessive disorder characterized by hypopigmentation of the skin and hair, presence of large clumps of pigment in hair shafts and an accumulation of melanosomes in melanocytes. GS type1 (OMIM # 214450), wherein hypomelanosis is associated with primary neurologic impairment, is caused by mutations in the MYO5A gene on 15q21.2. GS type 2 (OMIM # 607624), wherein immunologic impairment and hemophagocytosis are associated with the hypomelanosis, is caused by mutations in the RAB27A gene on 15q21.3. GS type 3 (OMIM # 609227), characterized by hypomelanosis with no immunologic or neurologic manifestations, can be caused by mutations in the melanophilin MLPH (on 2q37.3) or MYO5A genes. Answer to PhotoQuiz 4 Mucopolysaccharidosis type IV (OMIM # 253000 & 253010) Mucopolysaccharidosis (MPS) type IV, also known as , is an autosomal recessive lysosomal storage disorder characterized by short stature, dysostosis multiplex, significant skeletal deformities, mild coarsening of facies and variable degree of corneal clouding and hepatosplenomegaly. Affected individuals have normal intelligence. MPS IVA is caused by homozygous orcompound heterozygous mutations in the GALNS gene on chromosome 16q24, which result in deficient activity of the lysosomal enzyme galactosamine-6-sulfate sulfatase. MPS IVB is caused by homozygous or compound heterozygous mutations in the GLB1 gene on chromosome 3p22, which result in deficient activity of the beta-galactosidase enzyme. Answer to PhotoQuiz 5 Apert syndrome (OMIM # 101200) Apert syndrome also known as acrocephalosyndactyly type 1, is an autosomal dominant disorder char- acterized by craniosynostosis, typical facial dysmorphism (including a towering skull (turribrachycephaly), and severe syndactyly of the hands and feet. The syndactyly in many cases can lead to the ‘mitten-hand’ deformity. Apert syndrome results from heterozygous mutation in the FGFR2 gene and majority of cases have either one of the pathogenic variants p.Ser252Trp or p.Pro253Arg.

231 PhotoQuiz

Answer to PhotoQuiz 6 Meckel syndrome (OMIM # 249000 & 12 others) Meckel syndrome, also known as Meckel-Gruber syndrome, is an autosomal recessive condition characterized by occipital encephalocele, polydactyly, and polycystic kidneys. There may be other associated anomalies such as cleft lip/palate, cardiac and genital anomalies, central nervous system malformations, and liver fibrosis. Meckel syndrome belongs to the group of primary ciliopathies and is genetically heterogeneous. At present 13 different genes are known to be associated with Meckel syndrome.

Answer to PhotoQuiz 7 Fabry disease (OMIM # 301500) Fabry disease is an X-linked lysosomal storage disorder that results from deficient activity of the enzyme alpha-galactosidase A with resultant intra-lysosomal accumulation of globotriaosylceramide (GL-3) within the cells. The clinical features include periodic episodes of severe pain in the extremities (acroparesthesia), vascular cutaneous lesions (angiokeratomas), corneal and lenticular opacities, and proteinuria. Chronic renal disease, cardiac and/or cerebrovascular disease are the major causes of morbidity and mortality. Heterozygous females have milder symptoms and a later age of onset than males. It is caused by a monoallelic mutation in the GLA gene present on the chromosome Xq22.1.

Answer to PhotoQuiz 8 Technique - Multiplex ligation-dependent probe amplification (MLPA) The Multiplex ligation-dependent probe amplification (MLPA) technique is a DNA-based dosage study. It is a modified form of multiplex polymerase chain reaction (PCR) wherein probes complementary to specific regions of the genome are hybridized to the region, get ligated, are amplified and arethereafter analysed through capillary electrophoresis. MLPA can be used to detect genomic copy number variations (microdeletions and microduplications) as well as intragenic exonic deletions or duplications. Condition - 22q deletion syndrome 22q deletion syndrome also known as DiGeorge syndrome or velocardiofacial syndrome, is a microdeletion syndrome characterized by congenital cardiac defects (especially conotruncal anomalies), palatal abnormalities, immune deficiency, hypocalcemia, growth impairment and mild intellectual impairment/ learning difficulties.

Answer to PhotoQuiz 9 Pycnodysostosis (OMIM # 265800) Pycnodysostosis is an autosomal recessive disorder characterized by short stature, , acroosteolysis of the distal phalanges and increased bone fragility with predisposition to fractures. Typical craniofacial features of this condition include large skull with wormian bones, delayed closure of the anterior fontanelle, a small mandible and delayed eruption of primary and secondary dentition. It is caused by homozygous or compound heterozygous mutations in the CTSK (cathepsin K) gene.

Answer to PhotoQuiz 10 Freeman-Sheldon syndrome (OMIM # 265800) Freeman-Sheldon syndrome, also known as the Whistling face syndrome, is a severe form of distal arthrogryposis (DA2A) characterized by multiple congenital joint contractures, distinctive facies (microstomia with a whistling appearance of the mouth, puckered lips and an H-shaped dimple on the chin), and club foot. Majority of the cases are autosomal dominant and are caused by heterozygous mutation in the MYH3 gene.

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GAUCHER DISEASE POMPE DISEASE MPS I DISEASE FABRY DISEASE NIEMANN PICK - B DISEASE

Enlarged liver and spleen “Floppy” appearance in Coarse facial features Severe burning pain in Enlarged liver & spleen infants or young children hands & feet Delayed or stunted growth grow Early onset joint Bleeding manifestations in children stiffness/claw-hand Intolerance to heat & cold Unexplained deformities/contractures Skeletal abnormalities & Easy bruising and bleeding Cardiomyopathy Inability (or decreased Growth delays Corneal clouding (leading ability) to sweat Anemia and Progressive respiratory to light sensitivity or Thrombocytopenia muscle weakness or impaired vision) Red, purple spots on skin insufficiency (angiokeratomas) Unexplained Bone pains Recurrent respiratory Progressive Limb-girdle infections (including Evidence of early renal Unexplained Avascular involvement (nephropathy) necrosis of femur muscle weakness (in late- sinuses & ears) onset cases) History of recurrent hernia History of stroke in young repair in young age age

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