Mapping Genes Causing Syndromic and Non- Syndromic Human Hereditary Skin Disorders

by

Khadim Shah

Department of Biochemistry Faculty of Biological Sciences Quaid-i-Azam University Islamabad, Pakistan 2018

Mapping Genes Causing Syndromic and Non- Syndromic Human Hereditary Skin Disorders

A thesis submitted in the partial fulfillment of the requirements for the degree of Doctor of Philosophy

in

Biochemistry

by

Khadim Shah

Department of Biochemistry Faculty of Biological Sciences Quaid-i-Azam University Islamabad, Pakistan 2018

Declaration

I hereby declare that the work presented in this thesis is my own effort,

except where otherwise acknowledged, and that the thesis is my own

composition. No part of this thesis has been previously published or

presented for any other degree or certificate

Khadim Shah

DEDICATION

Dedicated to my Late Grandfather

Whose love and support was matchless from my childhood to mid of my Doctorate study

(May Allah Bless his Soul with Peace)

List of Contents

CONTENTS Page No ACKNOWLEDGMENTS I LIST OF FIGURES III LIST OF TABLES VI LIST OF ABBREVIATIONS VIII ABSTRACT XIII Chapter 1 1 INTRODUCTION 1

Human Skin 2 Development of the Skin 3 Skin Appendages 5 Hair Follicle 6 Teeth 7 Nail 7 Sweat Glands 8

Genetic Skin Disorders 8 9 Classification of Ectodermal Dysplasias 9 Hypohidrotic Ectodermal Dysplasia 10 Pure Hair and Nail type Ectodermal Dysplasia 11 Ectodermal Dysplasia of Hair, Nail and Teeth Type 11 Ectodermal Dysplasia of Nail and Teeth Type 12 Isolated Congenital Micronychia/Anonychia 12 Isolated Congenital Nail Clubbing 12 Isolated Nail Dystrophy 13 14 Xeroderma Pigmentosum 14 Trichothiodystrophy 15 Ichthyoses 15

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders List of Contents

Nonsyndromic Ichthyoses 16 Common Ichthyoses 16 Vulgaris 16 X-linked Recessive Ichthyosis 17 Autosomal Recessive Congenital Ichthyoses 17 17 Congenital Ichthyosiform Erythroderma 18 Harlequin Ichthyosis 18 Keratinopathic Ichthyoses 19 Erythrokeratoderma 19 Syndromic Ichthyoses 20 Keratitis Ichthyosis Deafness Syndrome 20 20 Ichthyosis Follicularis with Atrichia and Photophobia 21 Dorfman-Chanarin Syndrome 21 Sjögren-Larsson syndrome 21

Epidermolysis Bullosa 22 Simplex 22 Dystrophic Epidermolysis Bullosa 23 Junctional Epidermolysis Bullosa 23 24

Hypotrichosis 24 Autosomal Recessive Isolated Hypotrichosis 24 Atrichia with Papular Lesions 25 Localized Autosomal Recessive Hypotrichosis 1 25 Localized Autosomal Recessive Hypotrichosis 2 26 Localized Autosomal Recessive Hypotrichosis 3 26 Hypotrichosis with Recurrent Skin Vesicles 26 Autosomal Recessive Wooly Hair 27 Autosomal Dominant Isolated Hypotrichosis 27 Hypotrichosis Simplex 27

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders List of Contents

Autosomal Dominant Wooly Hair 28 Autosomal Dominant Monilethrix 28 Marrie Unna Hereditary Hypotrichosis 28 Syndromic Forms of Hypotrichosis 29 Autozygosity Mapping and Mutation Analysis 30 Chapter 2 32

MATERIALS AND METHODS 32 Ethical Approval and Study Subjects 32 Blood Sampling and Genomic DNA Extraction 32 Phenol-chloroform Method 32 DNA Extraction using Kits 33 Polymerase Chain Reaction (PCR) 33 RNA Extraction and cDNA Synthesis 34 Real Time Polymerase Chain Reaction 34 Mapping Candidate Genes 35 Human Genome Scan 35 Mutation Analysis 37 Sanger Sequencing 37 Whole Exome Sequencing 37 Restriction Enzyme Essay 38 Computational Analysis 38

RESULTS AND DISCUSSION 51 Chapter 3 51

ECTODERMAL DYSPLASIA 51 Family A 52 Family History and Clinical Features 52 Genetic Mapping and Mutation Analysis 52 Restriction Fragment Length Polymorphism (RFLP) Analysis 53 Computational Analysis 53

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders List of Contents

Family B 54 Family History and Clinical Features 54 Genetic Mapping and Mutation Analysis 54

Family C 55 Family History and Clinical Features 55 Genetic Mapping and Mutation Analysis 55 Family D 56 Family History and Clinical Features 56 Genotyping and Sequence Analysis 56 Family E 57 Family History and Clinical Features 57 Whole Genome Scan 57 Whole Exome Sequencing (WES) 57

Discussion 58 Chapter 4 75

TRICHOTHIODYSTROPHY 75 Family F 76 Family History and Clinical Features 76 Genome Wide Scan and Mutation Analysis 76 Family G 77 Family History and Clinical Features 77 Genotyping and Mutation Analysis 77 Discussion 77

Chapter 5 88

HEREDITARY ICHTHYOSIS 88 Family H 89 Family History and Clinical Findings 89 Genetic Mapping and DNA Sequencing 89 Family I 90 Family History and Clinical Features 90

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders List of Contents

Genotyping and Sequencing 91 Family J 91 Family History and Clinical Features 91 Genotyping and Sequencing 92 Discussion 92 Chapter 6 106 EPIDERMOLYSIS BULLOSA 106 Family K 107 Family History and Clinical Features 107 Genotyping and Sequencing 107 Family L 108 Family History and Clinical Features 108 Genotyping and Sequence Analysis 108

Discussion 108

Chapter 7 116

HEREDITARY HAIR LOSS DISORDERS 116 Family M 117 Family History and Clinical Features 117 Genotyping and Sequence Analysis 117 Expression Analysis 117 Family N 118 Family History and Clinical Features 118 Genome Scan 118 Exome Sequencing 119 Family O 119 Family History and Clinical Features 119 Genotyping and Sequencing 119

Family P 120 Family History and Clinical Features 120 Genotyping and Sequencing 121

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders List of Contents

Family Q 121 Discussion 122 Chapter 8 140

CONCLUSION 140 Chapter 9 143

REFERENCES 143

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders Acknowledgments

ACKNOWLEDGMENTS

ALL praises to Almighty Allah, the omnipotent, the most compassionate, who bestowed me with the potential and ability to complete the present work. Without Allah’s divine help, I would not have been able to achieve anything in my life. All respects to Holy Prophet Hazrat Muhammad (P.B.U.H) the most perfect among all human beings ever born on the surface of the earth, who is forever a source of guidance and knowledge for humanity as a whole.

First of all, I am obliged to express my sincere gratitude to my honorable supervisor Prof. Dr. Wasim Ahmad, whose scholastic guidance, kind interest and valuable suggestions proved crucial in the smooth completion of my PhD studies and related research. His continuous support patience, motivation, and immense knowledge helped me in all the time of research and writing of this thesis. On this occasion, I present my heartfelt thanks to the chairman department of Biochemistry QAU, Prof. Dr. Muhammad Ansar, who provided us a perfect, amicable and constructive research environment.

I have no words to explain my gratitude to Prof. Dr. Richard A Spritz for great support, valuable guideline line and very frank interactions during my IRSIP research in his lab in the University of Colorado Denver, USA, who provided me access to the laboratory and research facilities. My sincere thanks also go to Dr. Tracey M Ferrara, Dr. Diana Dills, Dr. Shaikh Tamim, Dr. Joanne Cole, Genevieve Andersen, Shelly Fortner and Paulene Segura Holland, Benedict Villamil for their support, care and help in six month of research period in USA.

I am also thankful to Dr. Sulman Basit, Assistant Professor, Taibah University Madinah, Saudi Arabia, for his valuable support and help in human genome scan and exome sequencing.

I would like to express thanks to the higher education commission (HEC) of Pakistan for providing me financial backing IRSIP fellowship during my PhD studies.

I have to appreciate the friendly and cooperative attitude of my lab seniors and fellows Dr. Saad Ullah Khan, Dr. Syed Irfan Raza, Dr. Abid Jan, Dr. Abdul Aziz, Dr. Irfanullah, Dr. Raja Husain Ali, Muhammad Umair, Farooq Ahmad, Shoaib Nawaz, Shabir Hussain, Khurrum Liaqat, Asmat Ullah, Shazia Khan,

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders I Acknowledgments

Saba Mehmood and Abida Akbar during the entire period of my PhD studies. I am also thankful to all my lab juniors, specifically: Mehboob Ali, Sarmad Mehmood, Wajid Amin, Nouman, Soahil Ahmad, Abdullah, Naseebullah, Zohaib Gillani, Hammal Khan, Mujahid Khan, Naila Shinwari, Sidra Habib, Surrya Hamayun Kifayat Ullah, Amjid, Laila Akbar and Pashmina Wiqar for the respect they gave to me and for their moral support. I would like to acknowledge the clerical staff of the Department of Biochemistry especially to Mr. Tariq, Mr. Fayaz, Mr. Saeed, Mr. Shehzad and other staff members for their support and help. I also thank the research volunteers who participated in this study. I wish and pray to Allah to give me strength and resources to relieve the pains of affected families and do something for the betterment of humanity. I am indeed ineffable to mention my appreciation to all of my family members especially my loving father for his unstinting support and encouragement. Besides, the love of my sweet mother and grandmothers proved a beacon of light at every step of my life. I am unable to find words which can express my feelings of thanks for my uncle and brothers for their valuable support and encouragement.

Khadim Shah

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders II List of Figures

LIST OF FIGURES

Figure No Title Page No

Cross section of human skin, showing layers and structures Figure 1.1 5 of the skin

Pedigree drawing and clinical presentation of affected Figure 3.1 64 individuals in family A

Microsatellite markers generated haplotypes, Sanger sequencing chromatograms of SLURP1 and presentation of Figure 3.2 65 agarose gel showing restriction enzyme analysis in family A

Computational analysis of SLURP1 (p.Met1?) variant in Figure 3.3 66 family A

Figure 3.4 Pedigree diagram and clinical presentation of family B 67

Genotypes from exome data and Sanger sequencing Figure 3.5 68 chromatograms of SLCO2A1 gene in family B

Figure 3.6 Pedigree and clinical manifestations of family C 69

Haplotypes and sequence chromatogram of RSPO4 gene in Figure 3.7 70 family C

Figure 3.8 Pedigree and clinical presentation of family D 71

Haplotypes and sequence chromatogram of FZD6 gene in Figure 3.9 72 family D

Presentation of pedigree and clinical features of affected Figure 3.10 73 individuals in family E

Sanger sequencing chromatogram of ERCC5 gene in Figure 3.11 74 family E

Pedigree and clinical pictures of affected individuals in Figure 4.1 82 family F

Microscopic investigation of hair and 2D Figure 4.2 83 echocardiography in affected member IV-2 in family F

Haplotypes generated using SNP markers and MPLKIP Figure 4.3 84 variant in family F

Continued....

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders III List of Figures

Pedigree and clinical pictures of affected individuals in Figure 4.4 85 family G

Haplotypes generated by genotyping microsatellite Figure 4.5 86 markers in family G on chromosome 7p14.1

Sequence chromatogram of MPLKIP gene in family F and Figure 4.6 87 G

Figure 5.1 Pedigree and clinical pictures of family H 98

Haplotypes generated using microsatellite markers on Figure 5.2 99 chromosome 12q12-q14.1 in family H

Genotypes from exome data and Sanger sequence Figure 5.3 100 chromatograms of the KRT83 gene in family H

Presentation of pedigree and clinical features of affected Figure 5.4 101 individuals in family I

Genotypes extracted from exome data and Sanger Figure 5.5 102 sequencing chromatograms of ALDH3A2 gene in family I

Pedigree outline of family J with Chanarin-Dorfman Figure 5.6 103 syndrome

Figure 5.7 Clinical pictures of family J 104

Figure 5.8 Sanger sequence analysis of ABHD5 in family J 105

Figure 6.1 Presentation of pedigree and clinical pictures of family K 111

Haplotypes generated by genotyping microsatellite Figure 6.2 112 markers in family K on chromosome 17q21

Figure 6.3 Sanger sequence analysis of KRT14 gene in family K 113

Pedigree presentation and clinical pictures of affected Figure 6.4 114 individuals in family L

Haplotypes and Sanger sequencing chromatogram of Figure 6.5 115 PLEC gene in family L

Pedigree drawing and clinical presentation of affected Figure 7.1 129 individuals in family M

Figure 7.2 Analysis of Sanger sequencing of BTAF1 in family M 130

Continued....

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders IV List of Figures

Comparative expression analysis of KRT81, EDA2R, Figure 7.3 FGF5, ALDH3A2 and AR genes in normal and affected 131 individuals in family M

Pedigree and clinical features in affected individuals in Figure 7.4 132 family N

Figure 7.5 Sequence chromatogram of C3orf52 gene in family N 133

Figure 7.6 Pedigree diagram and clinical presentations of family O 134

Figure 7.7 Analysis of Sanger sequencing of MTUS1in family 135

Pedigree and clinical features of affected individuals in Figure 7.8 136 family P

Presentation of autozygous region on chromosome 22 and Figure 7.9 137 Sanger sequence of SGSM1 gene in family P

Pedigree drawing and presentation of clinical features Figure 7.10 138 observed in affected individuals in family Q

Figure 7.11 Sequence analysis of DCAF17 gene in family Q 139

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders V List of Tables

LIST OF TABLES

Table No Title Page No

List of microsatellite markers used for genotyping of the Table 2.1 40 genes/loci involved in hereditary skin disorders

Table 2.2 List of primers used for PCR amplification of PLEC1 gene 42

Table 2.3 List of primers used for PCR amplification of SLURP1 gene 43

Table 2.4 List of primers used for PCR amplification of KRT1 gene 44

Table 2.5 List of primers used for PCR amplification of KRT2 gene 44

List of primers used for PCR amplification of MPLKIP Table 2.6 45 gene

Table 2.7 List of primers used for PCR amplification of KRT14 gene 45

Table 2.8 List of primers used for PCR amplification of HPGD gene 45

Table 2.9 List of primers used for PCR amplification of FZD6 gene 46

Table 2.10 List of primers used for PCR amplification of RSPO4 gene 46

List of primers used for PCR amplification of HR and Table 2.11 47 U2HR genes

Table 2.12 Primers used for PCR amplification of SMARCB1 gene 48

Primers used for PCR amplification of exon # 11 of Table 2.13 48 ERCC5 gene in family E

Primers used for amplification of exon # 1 of ALDH3A2 Table 2.14 48 gene in family I

Primers used for PCR amplification of exon # 5 of KRT83 Table 2.15 49 gene in family J

Primers used for PCR amplification of exon # 6 of ABHD5 Table 2.16 49 gene in family H

Primers used for amplification of exon # 1 of DCAF17 gene Table 2.17 49 in family Q

Primers used for amplification of exon # 1 of SLCO2A1 Table 2.18 49 gene in family B

Continued....

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders VI List of Tables

Primers used for amplification of targeted exons in BTAF1, Table 2.19 CCD74, CDC40, COL9A2, IBTK, LOXL4, PHF14, RGS10, 49 RPA2, SAA1 and SCE31B genes in family O

Table 2.20 List of primers used for qPCR in family N 50

List of SLURP1 sequence variants reported for mal de Table 3.1 63 Meleda type of PPK

Clinical features observed in affected members in family F Table 4.1 80 and G

Two-point and multipoint LOD scores obtained with SNP Table 4.2 markers flanking MPLKIP gene on chromosome 7p14 in 81 family F

Clinical features observed in affected individuals of family Table 5.1 97 H

List of variants extracted from exome data of family H, Table 5.2 these were homozygous in affected individuals (III-3, III-7, 97 III-8) and heterozygous in their parents (II-1, II-3) List of variants identified in exome data of family I, these Table 5.3 were homozygous in affected and heterozygous in their 97 parents. List of potential variants identified in exome data of family Table 7.1 128 M List of rare variants extracted from exome data of Table 7.2 128 autozygous region in family O List of variant extracted from exome data of autozygous Table 7.3 128 region on chromosome 22q11.23, in family P

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders VII List of Abbreviations

LIST OF ABBREVIATIONS Abbreviation Description % Percentage °C Degree Centigrade 3D Three Dimensional A Adenine ABCA12 ATP Binding Cassette Subfamily A Member 12 ABCA5 ATP Binding Cassette Subfamily A Member 5 ABHD5 Abhydrolase Domain Containing 5 ADHED Autosomal Dominant HED AEC Ankyloblepharon Ectodermal Defects cleft-lip/palate AFs Anchoring Fibrils AI Amelogenesis Imperfecta ALDH1A3 Aldehyde Dehydrogenase 1 Family Member A3 ALDH3A2 Aldehyde Dehydrogenase 3 Family Member A2 ALOX12B Arachidonate 12-Lipoxygenase, 12R type ALOXE3 Arachidonate Lipoxygenase 3 AnnoDB Annotation Database APCDD1 Adenomatosis Polyposis Down-regulated-1 APMR Alopecia with Mental Retardation AR Androgen Receptor ARCI Autosomal Recessive Congenital Ichthyoses Arg Arginine ARHED Autosomal Recessive HED ARWH Autosomal Recessive Woolly Hair Asn Asparagine AT2 Angiotension II ATGL Adipose Triglyceride Lipase ATP6V1B2 ATPase H+ Transporting V1 Subunit B2 BG1 Beijing Genomic Institute BLAST Basic Local Alignment Search Tool BMP Bone Morphogenetic Protein bp Base Pairs BTAF1 B-TFIID TATA-Box Binding Protein Associated Factor 1 BWA Burrows-Wheeler Aligner C Cytosine C3orf52 Chromosome 3 Open Reading Frame 52 CADD Combined Annotation Dependent Depletion CCDS Consensus Coding Sequence CDH3 Cadherin 3 cDNA Complementary Deoxyribonucleic Acid CDS Chanarin-Dorfman Syndrome CDSN Corneodesmosin CERS3 Ceramide Synthase 3 CIE Congenital Ichthyosiform Erythroderma cM centiMorgan CNS Central nervous system Continued....

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders VIII List of Abbreviations

COL9A2 Type IX Alpha 2 Chain CTSC Cathepsin C CYP4F22 Cytochrome P450 Family 4 Subfamily F Member 22 Cys Cysteine dbSNP Database of SNPs DCAF17 DDB1 and CUL4 associated factor 17 DD Dentin Dysplasia DDB2 damage specific DNA binding protein 2 DEB Dystrophic Epidermolysis Bullosa del Deletion DEPC Diethyl Pyrocarbonate DI Dentinogenesis Imperfecta DNA Deoxyribonucleic Acid dNTP Deoxynucleoside triphosphate DSC3 Desmocollin-3 DSG4 Desmoglein-4 DSP DTCS Dye Terminator Cycle Sequencing EB Epidermolysis Bullosa EBS Epidermolysis Bullosa Simplex EBS-MD EBS With Muscular Dystrophy EDA1 Ectodysplasin A1 EDA2R Ectodysplasin A2 Receptor EDAR Ectodysplasin-A Receptor EDARADD EDAR-Associated Death Domain EDs Ectodermal Dysplasias EDTA Ethylenediaminetetraacetic Acid EGFR Epidermal Growth Factor Receptor EPS8L3 Epidermal Growth Factor Receptor Pathway Substrate 8-Like 3 ERCC Excision Repair Cross-Complementing EVC EvC ciliary Complex Subunit 1 ExAC Exome Aggregation Consortium FALDH Fatty Aldehyde Dehydrogenase FDFT1 Farnesyl-Diphosphate Farnesyltransferase 1 FGF Fibroblast Growth Factor FLG fs frameshift FZD6 Frizzled Class Receptor 6 g Gram G Guanine GATK Genome Analysis Toolkit GJA1 Gap Junction Protein, Alpha 1 GJB2 Gap Junction Protein Beta 2 HBD Homozygous by Descent HED Hypohidrotic Ectodermal Dysplasia HF Hair Follicle hg19 Human Genome 19 HGMD Human Gene Mutation Database HI Harlequin Ichthyosis Continued...

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders IX List of Abbreviations

HJMD Hypotrichosis with Juvenile Macular Dystrophy HOXC13 Homeobox C13 HPGD Hydroxyprostaglandin Dehydrogenase HR Hairless HYPT Hypotrichosis IBTK Inhibitor of Bruton Tyrosine Kinase ICNC Isolated Congenital Nail Clubbing IFs Intermediate Filaments Ile Isoleucine IND Isolated Nail Dystrophy ins Insertion IRS Inner Root Sheath IV JUP Junction kb Kilo Base kDa Kilo Dalton KPI Keratinopathic Ichthyoses KPK Khyber Pakhtunkhwa KRT1 Kertain-1 KRT14 Kertain-14 KRT2 Kertain-2 KRT25 25 KRT74 Keratin 74 KRT81 Keratin 81 KRT83 Keratin 83 LAH Localized Autosomal Recessive Hypotrichosis Leu Leucine LIPH Lipase-H LIPN Lipase Family Member N LOD Logarithm of Odds LOR Loricrin LOXL4 Like 4 LPAR6 Lysophosphatidic Acid Receptor 6 M Molar MAF Minor Allele Frequencies MDM mal de Maleda MEDOC Mendelian Disorders of Cornification Met Methionine MgCl2 Magnesium Chloride min Minute mL Millilitre mM Millimolar MPLKIP M-phase Specific PLK1 Interacting Protein MSX1 Msh Homeobox 1 MTUS1 Associated Scaffold Protein 1 MUHH Marie Unna Hereditary Hypotrichosis MYOF Myoferlin nAChR Neuronal Nicotinic Acetylcholine Receptor NaCl Sodium Chloride Continued...

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders X List of Abbreviations

NAD Nicotinamide Adenine Dinucleotide NCBI National Centre for Biotechnology Information NER Nucleotide Excision Repair Ng Nanogram NIPAL4 NIPA Like Domain Containing 4 OMIM Online Mendelian Inheritance in Man ORS Outer Root Sheath p Short Arm Of Chromosome p63 Tumor Protein p63 PCR Polymerase Chain Reaction PG prostaglandin PGE2 prostaglandin E2 Phe Phenylalanine PHNED Pure Hair and Nail Ectodermal Dysplasia PK Proteinase K PKP1 1 PLEC PLK1 Polo Like Kinase 1 pmol picomole PNPLA1 Patatin Like Phospholipase Domain Containing 1 POLH DNA Polymerase Eta PPK Hereditary Palmoplantar Keratoderma Pro Proline PSEK Progressive Symmetric Erythrokeratoderma PVRL1 Poliovirus Receptor-like 1 PVRL3 Poliovirus Receptor-Related 3 q Long Arm of Chromosome qPCR Quantitative Polymerase Chain Reaction RB Recovery Buffer RDEB Recessive Dystrophic Epidermolysis Bullosa REEP4 Receptor Accessory Protein 4 RNA Ribonucleic Acid RPA2 Replication Protein A2 RPL21 Ribosomal Protein L21 rpm Revolutions Per Minute RSPO4 R-spondin 4 SDR9C7 Short Chain Dehydrogenase/Reductase Family 9c, Member 7 SDS Sodium Dodecyl Sulphate Sec Seconds Ser Serine SERPINB7 Serpin Family B Member 7 SGSM1 Small G Protein Signaling Modulator 1 SHH Sonic Hedgehog SLCO2A1 Solute Carrier Organic Anion Transporter Family Member 2A1 SLS Sjögren-Larsson syndrome SLURP1 Secreted Ly6/Plaur Domain Containing 1 SNP Single Nucleotide Polymorphism SNRPE Small Nuclear Ribonucleoprotein Polypeptide E STS Steroid Sulfatas Continued...

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders XI List of Abbreviations

SULT2B1 Sulfotransferase Family 2B Member 1 T Thymine Taq Thermus aquaticus TBE Tris Borate EDTA TBP TATA-Binding Protein TE Tris-EDTA TEMED N, N, N, N-Tetramethylethylenediamine TGF Transforming Growth Factor TGM1 Transglutaminase 1 Tm Melting Temperature TNF Tumour Necrosis Factor TP63 Tumour Protein p63 Tris Tris(hydroxymethyl)aminomethane Trp Tryptophan TTD Trichothiodystrophy TTDN1 Nonphotosensitive Trichothiodystrophy TTMP TPA Induced Trans-Membrane Protein UTR Untranslated Region UV Ultra Violet v/v Volume/volume Val Valine VCF Variant Call FORMAT WES Whole Exome Sequencing WNT10A Wingless-type MMTV Integration Site Family, Member 10A WWS Woodhouse-Sakati Syndrome XLHED X-linked HED XLRI X-linked Recessive Ichthyosis XP Xeroderma pigmentosum α Alpha β Beta μL Micro-Litre μM Micro-Molar

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders XII Abstract

ABSTRACT

Genetic defects in complex processes of embryonic development and postnatal maintenance of the skin and its appendages result in clinically and genetically heterogeneous group of skin disorders. Due to nonspecific presentation, variable clinical manifestations and highly overlapping phenotypes, the diagnosis of skin disorders is a challenging job for the clinicians and geneticists. However, recent advances in molecular biology technologies notably whole exome sequencing (WES) and microarray have incredibly accelerated identification of genes involved in inherited diseases.

The research work, presented in this dissertation, described clinical and molecular characterization of seventeen families of Pakistani origin (A-Q) segregating various forms of syndromic and non-syndromic skin disorders. This included one with palmoplantar (A), three with nail disorders (B-D), one with xeroderma pigmentosum (E), two with non-photosensitive trichothiodystrophy and mitral regurgitation (F-G), three with different types of ichthyosis (H-J), two with epidermolysis bullosa (K-L), and five with different types of hair abnormalities (M-Q).

In all seventeen families, combination of at least two or three techniques, including microsatellite/SNP genotyping and Sanger/Exome sequencing, led to the establishment of linkage on human chromosomes and detection of potential disease causing variants in different genes. This included fourteen novel variants in 13 different genes (SLURP1, SLCO2A1, ERRC5, MPLKIP, KRT83, ALDH3A2, ABHD5, PLEC, BTAF1, C3orf52, MTUS1, SGSM1, DCAF1) and previously reported three variants in 3 genes (RSPO4, FZD6, KRT14). Disease causing variants, identified in six genes (KRT83, SLCO2A1, BTAF1, MTUS1, C3orf52, SGSM1), are the first report of involvement of such genes in causing skin disorders. Pathogenicity of disease causing variants were tested and verified by various bioinformatics tools (SIFT, PolyPhen, MutationTaster, MutationAssessor, GERP++, phyloP). Non-polymorphic nature of the variants was validated by screening large number of ethnically matched control individuals and by searching various databases. In a couple of cases, qPCR was used to monitor effect of the variants on expression of other genes. In addition, where necessary, protein modelling studies were

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders XIII

Abstract performed to identify location of the mutations in the protein and its possible effect on structure and functions of the protein.

The work presented in the dissertation resulted in the following publications.

1. Shah K, Ansar M, Khan FS, Ahmad W, Ferrara TM, Spritz RA (2017). Recessive progressive symmetric erythrokeratoderma results from a homozygous loss-of-function mutation of KRT83 and is allelic with dominant monilethrix. Journal of Medical Genetics 54: 186-189. 2. Shah K, Ferrara TM, Jan A, Umair M, Khan S, Ahmad W, Spritz RA. Homozygous SLCO2A1 translation initiation codon mutation in a Pakistani family with recessive isolated congenital nail clubbing (ICNC) (2017). British Journal of . doi: 10.1111/bjd.15094. 3. Shah K, Ali RH, Ansar M, Lee K, Chishti MS, Abbe I, Li B, Smith JD, Nickerson DA, Shendure J, Coucke PJ, Leal SM, Ahmad W (2016). Mitral regurgitation as a phenotypic manifestation of nonphotosensitive trichothiodystrophy due to a splice variant in MPLKIP. BMC Medical Genetics 17: 13. 4. Shah K, Nasir A, Shahzad S, Khan S, Ahmad W (2016). A novel homozygous mutation disrupting the initiation codon in the SLURP1 gene underlies mal de Meleda in a consanguineous family. Clinical and Experimental Dermatology. 41: 675-679. 5. Raza SI, Navid AK, Noor Z, Shah K, Dar NR, Ahmad W, Rashid S (2017). GLY67ARG substitution in RSPO4 disrupts the WNT signaling pathway due to an abnormal binding pattern with LGRs leading to anonychia. RSC Advances 7:17357- 17366. 6. Shah K, Mehmood S, Jan A, Abbe I, Ali RH, Khan A, Chishti MS, Lee K, Ahmad F, Ansar M, University of Washington Center for Mendelian Genomics, Nickerson DA, Bamshad MJ, Coucke PJ, Santos‐Cortez RL, Spritz RA, Leal SM, Ahmad W (2017). Sequence Variants in Nine Different Genes Underlying Rare Skin Disorders in Ten Consanguineous Families. International Journal of Dermatology (Under Review). 7. Ahmad F, Shah K, Muhammad D, Basit S, Wakil SM, Umair M, Ramzand K, Ahmad W (2017). Novel autosomal recessive LAMA3 and PLEC1 mutations underlie non-hertz junctional epidermolysis bullosa and epidermolysis bullosa simplex with Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders XIV

Abstract muscular dystrophy in two consanguineous families. Clinical and Experimental Dermatology (Under Review). 8. Shah K, Jan A, Ahmad W, Umair M, Irfanullah, Basit S, Ahmad W. A novel start loss variant in DCAF17 underlies Woodhouse-Sakati Syndrome phenotypes in a large consanguineous family (In Preparation). 9. Shah K, Umair M, Ahmad F, Ali G, Nawaz G, Jhon P, Ferrara TM, Spritz RA, Ahmad W. A heterozygous missense sequence variant in BTAF1 gene miss-regulate transcription and results in progressive patchy hair loss from the scalp (In Preparation). 10. Shah K, Ali G, Jhon P, Irfanullah, Ahmad F, Basit S, Ahmad W. C3orf52 is a probable candidate gene for autosomal recessive hypotrichosis in large Pakistani family. (In Preparation). 11. Shah K, Hussain S, Raza SI, Basit S, Ahmad W. Missense sequence variant in SGSM1 gene underlies unexplored phenotypes of hypertrichosis, macrocephaly, facial deformities, cardiac and urinary defects in Pakistani kindred. (In Preparation). 12. Shah k, Irfanullah, Ahmad F, Umair M, Basit S, Ahmad W. Complete hair loss in large consanguineous Pakistani family results from mutation in MTUS1 gene in a large consanguineous family. (In Preparation).

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders XV

Chapter 1 Introduction

INTRODUCTION

Genetic disorders including hereditary skin diseases are common in Pakistan due to the high rate of consanguinity (Hamamy, 2012). However, lack of basic knowledge about genetic disorders, proper genetic counseling and carrier testing is not accredited in Pakistan. Genetic diagnosis of inherited skin disorders is important to figure out the disease pathogenesis, expected complications, and to allow specific genetic counseling. Due to complex phenotypes associated with several skin disorders, a multidisciplinary approach is required with step by step clinical, physical and molecular examinations to reach an exact diagnosis of skin disorders.

As a matter of fact, diagnosis of human hereditary skin disorders is a challenging job for the clinicians and geneticists due to nonspecific presentation, variable clinical manifestations, highly overlapping phenotypes and lack of recognition as a discrete clinical entity. Since genetic variations are involved in human disease, particularly, rare Mendelian disorders, identification of genetic variants in individuals is the central goal of genetic studies (Stranneheim and Wedell, 2016). Recent advances in molecular genetic techniques notably whole exome sequencing (WES) and microarray have incredibly accelerated the identification of genes involved in inherited diseases. These state-of-the-art technologies have facilitated investigators to understand the complex mechanisms involved in regulating the skin and its associated appendages. Molecular characterization and diagnosis have exposed multiple causative genes for many syndromic and non-syndromic skin disorders, providing practical benefits for patients with rare genetic skin disorders in terms of accurate diagnosis, better genetic counseling and availability of DNA-based prenatal diagnosis. Still there aroused new challenges in clinical interpretation of the large number of emerging genomic variants, genotype-phenotype correlations, and ultimately its application to precision medicine. Nevertheless, these emerging technologies will soon give converge, with far-reaching implications for the elucidation of genetic disease and health care.

Pakistani population is a goldmine for genetic skin research due to its high rate of consanguinity and extended pedigrees. However, less attention has been paid to this field due to lack of knowledge among affected families about the disorder, the intermittence of skin disorders, lack of research expertise and unviable facilities in

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 1 Chapter 1 Introduction this field. Recently, some advancement has been made in identification of novel variants and genes for monogenic skin disorders.

Human Skin

Skin covers the entire surface of the human body and the principal site of interaction with the environment. Skin is the largest and highly specialized organ that is about 2.5 mm thick and weighs approximately 5-6 kilograms. Skin serves as epidermal barrier of defense that aids the body in immune surveillance, UV protection, thermoregulation, sweating, lubrication and pigmentation, sensory perception, control of insensible fluid loss and protection of various stem cell niches in the skin. In addition to bearing important appendages, due to its flexibility and responsiveness, skin is a master in the art of self-defense – proving that a tissue need not to be hard in order to be tough (Ross and Christiano, 2006).

Skin is basically composed of two layers; the epidermis and dermis, parted by a . Beneath the dermis lays a fatty subcutaneous layer known as hypodermis. These layers vary in their function, thickness and strength (Chuong et al., 2002).

Epidermis is the outer layer, which is nourished and maintained by dermis. The epidermis is a stratified, squamous epithelium that consists primarily of keratinocytes in progressive stages of differentiation from deeper to more superficial layers. Differentiation of keratinocytes gives rise to several distinguishable layers as they move outwards and progressively differentiate into five major strata. These are stratum basale, stratum spinosum, stratum granulosum, stratum lucidum and stratum corneum from bottom to top, respectively (Lai-Cheong and McGrath, 2009). The stratum lucidum is restricted to the skin of palms and soles. The stratum basale is a continuous layer that is generally one cell thick, however, may be 2-3 cells thick in glabrous skin and hyper-proliferative epidermis (McGrath et al., 2004). In addition to keratinocytes, epidermis also hosts several other cell populations, including , which donate pigment to the keratinocytes, Langerhans and Merkel cells, with immunological and sensory functions, respectively. Keratinocytes are linked to each other by several types of cellular junctions which are responsible for mechanical, biochemical and signaling interactions between the cells. These are desmosomes, adherens junctions, tight junctions and gap junctions.

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 2 Chapter 1 Introduction

Dermis is biochemically complex layer with shock absorbing capacity, lying immediately below the stratified epidermis. Dermis can be differentiated into an upper ‘papillary’ and a lower ‘reticular’ portion. They differ in cell number, composition of connective tissues, and stream of nerves and blood vessels. Unlike epidermis, the origin of dermis is mesoderm and the main cell population is fibroblasts. Dermis consists of fibrous and amorphous extracellular matrix, secreted by dermis cells. The extracellular matrix is a mixture of bioactive macromolecules including , , , fibulins, fibrillins, integrins, proteoglycans and the enzymes involved in their processing (Tobin, 2006).

Hypodermis (subcutaneous) is a deeper continuation of the dermis comprising loose connective tissue and adipose cells creating a deep layer of variable thickness. However, no adipose tissue is found in hypodermis of the eyelids, clitoris or penis (Clark, 1985).

Development of the Skin

The skin originates by the juxtaposition of two embryonic layers: prospective epidermis and mesoderm; the embryonic endoderm also plays a role in skin development (De Robertis et al., 2000). In addition to dermis development, mesoderm is also essential for inducing differentiation of the epidermal structures.

The development of epidermis depends on complex interplay of Notch and Wnt signaling with β-, Lef1 and Notch peptide. Signals from the Sonic hedgehog pathway and bone morphogenetic proteins (BMPs) has important role in early embryogenesis, particularly in defining whether cells have an ectodermal or neural fate. Specifically, BMP signaling promotes ectodermal development, while sonic hedgehog promotes the development of neural tube and central nervous system (CNS). Thus complex cross talk of BMP signaling, sonic hedgehog, fibroblast growth factors (FGFs) and regulatory control mechanisms from the Wnt pathway, underlies the preliminary development of epidermis (Altman and Brivanlou, 2001; Fuchs and Raghava, 2002).

The epidermis develops from embryonic ectoderm and remains a single layer of undifferentiated cells till three weeks of embryonic age. Between four and six weeks the stratum basale and the periderm appeared. A middle layer starts to appear by the gestational age of eight to eleven weeks. The fetus develops one or more intermediate

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 3 Chapter 1 Introduction epidermal layers between twelve to sixteen gestational weeks. The periderm is a temporary embryonic layer that serves as the first barrier to the embryo's physical environment. It exists throughout the entire stratification process and sheds off at 17th gestational week, when it is replaced by corneocytes. The cells of intermediate layers initially proliferate, but then lose their proliferative ability and differentiate into spinous keratinocytes. By fifteen weeks, the filaggrin granules are visible in keratinocytes. Development of the spinous and granulosum epidermal layers marks the formation of the stratified epidermis and enables the establishment of the outer cornified layer of the skin (McGrath et al., 2004).

The embryonic dermis of five to six weeks old fetus is just collection of mesenchymal cells with abundant glycogen, with no distinction of dermal from subdermal tissue. In the gestational age of six to eight weeks, the cellular mesenchyme passes through several changes: the cytoplasm extends in size, rough endoplasmic reticulum (RER) and Golgi apparatus become prominent, the extracellular collagen fiber increases in diameter and blood vessels become more organized. The dermis at this stage is called cellular dermis (Smith et al., 1986). The time between ninth to thirteenth weeks is transition stage for dermis, where cellular dermis transforms to fibrous dermis. Transition stage is marks by an increase in the accumulation of fibrous collagen, increase in the diameter (25-40 nm) of individual fibers. As a result the density of dermal cells decreases as they are moved apart by the accumulating fibrous matrix. Concurrently, the cells tend to lose their rounded or stellate shape and random orientation. The dermal-epidermal junction becomes complex by the expression of genes coding for hemi-desmosomes, anchoring fibrils and fine structural elements that involved in attachment of the epidermis to the dermis. From thirteenth week onward, the developing epidermal appendages extend deeply into the dermis and connective tissue sheaths appeared around the vessels and nerves at all levels of the dermis. The deep boundary of the reticular dermis is further defined by the accumulation of adipose cells in the subcutaneous connective tissue. Till twentieth week of gestational age, the histologic features of the adult dermis become evident (Smith et al., 1986).

The p63 protein is indispensable regulator of skin development; alterations in the p63 pathway result in a number of genodermatoses in which the skin and its appendages underlie abnormal development (Zhou and Aberdam, 2013).

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 4 Chapter 1 Introduction

Figure 1.1: Cross section of human skin, showing layers and structures of the skin (http://cnx.org/contents/[email protected])

Skin Appendages

Morphogenesis of ectodermal appendages depends on inductive epithelial– mesenchymal interactions mediated by a conserved set of signaling molecules. Despite the adult appendages variances in shape, function, number, and regenerative ability, ectodermal appendages share multiple features during development (Biggs and Mikkola, 2007b). Organogenesis of the skin appendages proceeds via initiation, morphogenesis, and differentiation stages, followed by patterning. Initiation of organogenesis is marked by the appearance of a local epithelial thickening, a placode, which subsequently invaginates to produce a bud. These early developmental stages require many of the same genes and signaling circuits. Consequently, mutations in these genes often cause similar phenotypes in several skin appendages. After the bud stage, these organs adopt diverse patterns of epithelial growth, reflected in the usage of more divergent genes in each (Mikkola, 2007b).

The organogenesis of skin structure including hair, teeth, nails and sweat glands is regulated by crosstalk of different tissues; the epidermis defines the class of epidermal

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 5 Chapter 1 Introduction appendages expressed, while the dermis specifies their size and surface pattern distribution. The epithelial–mesenchymal interactions is mediated by comparatively limited number of signaling pathways including the Wnt, hedgehog, TGF, FGF and TNF families and executed by their downstream transcriptional regulators (Mikkola, 2007).

Hair Follicle

Humans have up to five million hairs follicles. There are three types of hair: lanugo hair, which is shed soon after birth; vellus hair, which is fine hair distributed mostly over the body; terminal hair, which is longer and coarser. Besides protecting the body against coldness and wetness, hair is the sign of strength, power and beauty in human beings. Externally, hair is a thin flexible tube of fully keratinized dead epithelial cells, whereas inside the skin, it is a part of individual living hair follicles (Lai-Cheong and McGrath, 2013).

Typical hair shaft comprises three parts: the cuticle, which is the outer covering of the hair; the cortex, the middle layer, which provides strength, color and texture to the hair shaft; the medulla, the central layer, present only in thick hair. The follicle is the essential growth structure of hair, surrounded by outer root sheath (ORS), a reservoir of multipotent stem cells. ORS surrounds the inner root sheath (IRS), which comprises three layers: Henle’s layer, Huxley’s layer, and cuticle layer. The base of the hair follicle consists of the dermal papilla, which is supplied with blood vessels and sensory nerves. At the base of hair bulb resides the hair papilla, the site where the growing hair originates.

The earliest development of the hair rudiments occurs at about ninth week of gestation in the upper lip, chin and regions of the eyebrow (McGrath et al., 2004). The initial step is the formation of the hair placode in response to inductive (Wnt) and inhibitory (BMP) signals. Further dermal messages instruct the placodes to make the hair follicles (HFs). After the initial follicle morphogenesis, the HF continues in growth phase (anagen) generating the first pelage approximately 2 weeks after birth. The HF undergoes cyclic transformations, with the anagen phase terminating with the remodeling catagen phase followed by a resting telogen phase (Duverger and Morasso, 2008). Most hereditary hair loss disorders are implicated with mutations in the genes involved in HF development, morphogenesis and hair follicle cycling.

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Teeth

Teeth are specialized structural components of the craniofacial skeleton and composed of three distinct components: enamel, dentin, and cementum. Humans have three basic tooth shapes: incisors, canines and multicuspids (molars, premolars). Their shape is defined in early developmental stages by variable expression of different genes in different region of the mesenchyme of the jaw (Hu and Simmer, 2007). Anatomically a tooth has three parts: crown, root and the pulp cavity. The crown and the root consist of two layers of hard substance surrounding the dental pulp. Human has two sets of teeth: primary and secondary teeth; primary teeth are also called deciduous teeth which start to erupt around the age of six months, while, the permanent teeth appear in the age 6-7 years.

In humans, the formation of primary dentition begins at around sixth week of gestation age, progressively erupting between six months and 2.5 years of age, with development usually complete by around 3 years. In addition to a series of epithelial- mesenchymal interactions, several signaling pathways like FGF, BMP, WNT and SHH ligands, their receptors, inhibitors and transcription factors play a key role in tooth development (Thesleff, 2003; Mikkola, 2007a,b). Several genetic, epigenetic and environmental factors contribute to dental abnormalities. The isolated genetic defect in in dentin is called dentinogenesis imperfecta (DI) or dentin dysplasia (DD), while the inherited enamel defects are termed as amelogenesis imperfecta (AI). Till date, several genes have been implicated with inherited dental conditions, which are involved in the morphogenesis and development or contribute to structure of teeth (Cabay, 2014).

Nail

Nail is a keratinous plate like structure grows on the dorsal surface of each digit, to protect the soft tissues of the distal digits from environmental assault and injury. The nail apparatus also enhance the sensory perception of finger-tips to fine manipulate small objects with refined dexterity. It is also a tool for scratching and grooming, and can be used as a natural weapon. Anatomically, nail unit demonstrates four distinct but collaborated parts enlisted as the nail matrix, nail bed, nail plate and nail folds. The nail plate consists of a number of hard and soft , embedded in an amorphous matrix. Nail plate is synthesized by the matrix epithelial cell and grows

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 7 Chapter 1 Introduction over the nail bed continuously throughout life. The matrix also has melanocytes which donating pigment to keratinocytes (Baran et al., 2012).

Nail development initiates at ninth week of gestation and is completed during the fifth month of gestation with the development of the toenails lagging approximately one month the fingernails. Nail development involves several signaling proteins for proper epithelial-mesenchymal interaction, including bone morphogenetic protein-4 (BMP4), fibroblast growth factor-4 (FGF4), Wnts and Sonic hedgehog (Shh). Mutations in the genes related to these pathways might lead to nail dysplasia (Baran et al., 2012).

Sweat Glands

Sweat glands are highly active miniorgans of skin that fulfill a variety of functions. They are coiled tubular glands that play a crucial role in the maintenance of homeostasis and regulate body temperature. Sweat glands are mainly divided into two types namely eccrine gland and apocrine glands. Eccrine glands are found throughout the body and are important for regulating the body temperature, as they produce a watery mixture of salts, antibodies and metabolic wastes, known as sweat. Apocrine glands (scent glands) are found in the dermal or hypodermal regions but the secretary ducts of these glands are mostly open into hair follicle while their secretions are more viscous than that of eccrine glands (Serri et al., 1963).

Eccrine glands start to develop on the palms and soles at about third month of gestation, while rest of the body develops eccrine as well as apocrine glands in fifth month of gestation. In addition to other signaling pathways, EDA pathway plays a major role in sweat glands formation. Ectodysplasin-A (EDA), EDA-receptor (EDAR) and EDAR-associated death domain (EDARADD) are the components of EDA-pathway. Mutations in these genes result in hypohidrotic/anhidrotic ectodermal dysplasia, characterized by hypohidrosis/anhidrosis, hypodontia and hypotrichosis (Keller et al., 2011).

Genetic Skin Disorders

The term genodermatoses is used for inherited monogenic, polygenic and chromosomal skin conditions. Genodermatoses are a heterogeneous group of rare disorders, account for one third of all monogenic diseases (Scriver et al., 2001). Genodermatoses display a wide spectrum of phenotypic outcomes; including abnormality of keratinization and pigmentation, alopecia, ichthyosis, epidermolysis

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 8 Chapter 1 Introduction bullosa and ectodermal dysplasia. These disorders are rare, might be limited to the skin and its appendages, and may be a part of a multi-system pathology causing significant complication. Online Mendelian Inheritance in Man (OMIM) database has described 560 unique disorders affecting the skin and its associated appendages with 501 distinctive protein encoding genes and their phenotypic representations (Feramisco et al., 2009; Sadreyev et al., 2009). Approximately 16% of cutaneous disorders are linked with two or more mutated genes and 18% of genes are related with two or more ectodermal dysplasias (Sadreyev et al., 2009). Association of many genes and disease phenotypes manifests unknown functional relations between disease phenotypes, proteins and signaling pathways.

Ectodermal Dysplasia

Ectodermal dysplasias (EDs) are genetically heterogeneous and clinically diverse group of rare inherited disorders characterized by defects in two or more ectodermal structures, at least one of these involving defects in hair, teeth, nails or sweat glands (Itin, 2014). A number of signaling molecules coordinate in the formation and function of ectodermal structures. Defects in these molecules are mainly involved in the pathogenesis of ectodermal dysplasias. EDs are termed pure if the defects are limited to classical ectodermal appendages (hair, teeth, nail, sweat glands), while the associated ectodermal dysplasia display defects in other tissues in addition to classical ectodermal appendages. Other associated structures are thymus, anterior pituitary, adrenal medulla, cornea, external ear, melanocytes, conjunctiva and lacrimal duct as well as central nervous system (Irvine, 2009). Till date, approximately 200 different types of EDs are reported in the literature, however; only about 80 of them are associated with their candidate genes (Itin, 2014).

Classification of Ectodermal Dysplasias

Recent advances in molecular genetics have increasingly elucidated the molecular defects in inherited conditions; almost monthly new diseases belonging to the ectodermal dysplasias are reported. This needs a comprehensive classification and categorization of these disorders to help dermatologists in precise diagnosis and proper management of specific skin conditions (Itin, 2014).

Previously, Pinheiro and Freire-Maia, (1994) classified EDs into two groups on the basis of clinical description. Group A consists of ectodermal dysplasias of at least two

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 9 Chapter 1 Introduction classic ectodermal structures without associated abnormality. Group B consists of ectodermal dysplasias of at least one of the four ectodermal appendages (hair, nail, teeth, and sweat glands) plus one other associated anomaly. In this classification, they used numbers for the basic affected ectodermal appendage like 1, 2, 3 and 4 for hair, teeth, nails, sweat glands, and 5 for other associated ectodermal anomalies of the lips, ears, or dermatoglypics, respectively. Later, Freire-Maia et al. (2001) classified EDs into 11 subgroups on the basis of the clinical physiognomies. They also distinguished ‘pure EDs’, which are exclusively characterized by ectodermal signs, and ED syndromes, in which ectodermal derivatives defects are associated with other malformations in different organs or tissues.

Based on functional perspectives of the identified genes, Lamartine (2003) classified EDs into four main subclasses including cell-cell communication and signaling [EDA (MIM 300451), EDAR (MIM 604095), EDARADD (MIM 606603)], cell adhesion [CDH3 (MIM 114021), PVRL1 (MIM 600644), PKP1 (MIM 601975)], transcription regulation [P63 (MIM 603273), EVC (MIM 604831)] and development [MSX1 (MIM 142983), SHH (MIM 600725)]. Priolo (2009) arranged EDs into two groups, based on molecular-genetic data and corresponding clinical findings. Group 1 includes the disorders which result from defective epithelial-mesenchyme interactions; the subsequent clinical phenotype is hypoplasia or aplasia of structures derived from the ectoderm. Group 2 represents the disorders in which there is abnormal function of a structural protein in the cell membrane. Clinically, these disorders are characterized by abnormalities in skin such as palmoplantar keratoderma.

Hypohidrotic Ectodermal Dysplasia

Hypohidrotic ectodermal dysplasia (HED) is the most common form of ED, characterized by hypotrichosis, hypohidrosis/anhidrosis, hypodontia/anodontia, and defects in nails. Other associated features include saddle nose, low-set ears, immunodeficiency, and atopic dermatitis (Lu and Schaffer, 2008; Trzeciak and Koczorowski, 2016). HED shows all possible pattern of Mendelian inheritance; however, X-linked HED (XLHED; MIM 305100) is the most common type. The XLHED is caused by sequence variants in EDA (MIM 300451) gene, located on chromosome Xq12–q13.1. The gene EDA codes for ectodysplasin-A, a type 2 transmembrane protein belonging to the tumor necrosis factor (TNF) family (Kere et al., 1996). The autosomal recessive and dominant forms of HED are comparatively

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 10 Chapter 1 Introduction rare, caused by mutations in EDA-receptor (EDAR; MIM 604095), located on chromosome 2q11–q13 and EDAR-associated death domain (EDARADD; MIM 606603), located on chromosome 1q42.2–q43 (Monreal et al., 1999; Headon et al., 2001). EDAR is a type 1 transmembrane protein and belongs to the TNF receptor superfamily, while EDARADD is an adopter of the death domain of EDAR. These proteins activate nuclear factor-kappa B (NF-kB) path way for initiation, morphogenesis, and differentiation of ectodermal structures.

According to Human Gene Mutation Database (HGMD) Professional 2017.1, a total of 312 pathogenic variants in the EDA have been associated with XLHED, whereas 60 variants in the EDAR and 10 in the EDARADD have been identified as a cause of ARHED and ADHED from different ethnicities around the world.

Pure Hair and Nail type Ectodermal Dysplasia

Pure hair and nail ectodermal dysplasia (PHNED; MIM 602032, 614931, 614929) is a rare genetic condition, characterized by defect in hair (hypotrichosis, brittle hair) and dystrophy of the nails (onychodystrophy/micronychia) with no other associated abnormality. The patients with PHNED have been reported with variable severity of hypotrichosis and nail dystrophy. Three genes have been associated with PHNED in patients with different ethnic backgrounds. These are keratin 85 (KRT85; MIM 602767), homeobox C13 (HOXC13; MIM 142976) and keratin 74 (KRT74; MIM 608248); all of the three are located on chromosome 12q12–q14.1 (Naeem et al., 2006; Lin et al., 2012; Raykova et al., 2014). To date, two pathogenic sequence variants in KRT85 have been implicated with PHNED phenotypes (Naeem et al., 2006; Shimomura et al., 2010a). Recently, Raykova et al. (2014) identified a missense variant in KRT74 gene in patients with PHNED phenotypes. According to HGMD Professional (2017.1), six sequence variants in HOXC13 have reported in the pathogenesis of PHNED.

Ectodermal Dysplasia of Hair, Nail and Teeth Type

Ectodermal dysplasia of hair, nail and teeth type (MIM 602401) is a rare congenital disorder characterized by defect in development of hair, fine/sparse scalp and body hair, dystrophic nails, and irregular to missing teeth (Tariq et al., 2008). Other associated features include erythematous lesions of the facial region and hyperhidrosis with thickened soles and palms (Megarbane et al., 2004). Tariq et al. (2008) mapped

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 11 Chapter 1 Introduction the disease locus for ectodermal dysplasia of hair, nail and teeth type to chromosome 18q22.1–q22.3 in large Pakistani kindred. The candidate gene is still to be discovered.

Ectodermal Dysplasia of Nail and Teeth Type

Ectodermal dysplasia of nail and teeth type is also termed as Witkop syndrome (MIM 189500), characterized by missing teeth and poorly formed nails, especially of toenails. This condition is inherited in apparent autosomal pattern (Witkop, 1965; Altug-Atac and Iseri, 2008). Jumlongras et al. (2001) reported a nonsense variant in the MSX1 (MIM 142983) in a three generation pedigree segregating Witkop syndrome’s phenotypes. The features of Witkop syndrome were also observed in Msx1-/- mouse model ratifying that MSX1 is the causative gene for Witkop syndrome (Jumlongras et al., 2001). According to HGMD professional 2017.1, a total of 53 pathogenic sequence variants are reported in MSX1 gene.

Isolated Congenital Anonychia/Micronychia

Isolated congenital anonychia/micronychia (MIM 206800) is a rare inherited nail disorder characterized by hypoplastic/aplastic fingers and toes nails. The disorder is inherited in autosomal recessive pattern. Anonychia/micronychia is caused by mutations in the R-spondin 4 (RSPO4; MIM 610573), located on chromosome 20p13 (Bergmann et al., 2006; Blaydon et al., 2006). The gene RSPO4 consists of five exons, codes for 234 amino acids protein of R-spondin family. Like other members, the RSPO4 contains an N-terminal signal peptide, two furin-like domains, a thrombospondin type-1 domain, and a C-terminal region with low-complexity (Kim et al., 2006). The furin-like domains are required for activation and stabilization of β- catenin (Kazanskaya et al., 2004), the signaling protein required for proper differentiation of nails (Takeo et al., 2013). According to HGMD Professional 2017.1 seventeen sequence variants in RSPO4 have been reported in pathogenesis of anonychia/micronychia.

Isolated Congenital Nail Clubbing

Isolated congenital nail clubbing (ICNC; MIM 119900) also called Isolated digital clubbing is a rare disorder of nails, characterized by bilateral, symmetric enlargement of the nail plate and terminal segments of fingers and/or toes resulting from excessive proliferation of connective tissue between the nail matrix and distal phalanx. Loss of normal angle between the nail and posterior nail fold is associated with a shiny,

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 12 Chapter 1 Introduction hypoplastic, thick-ended, long, broad nail (Shah et al., 2016b-present study). The thumbs are almost always involved, though some fingers or toes may be spared. Autosomal recessive as well autosomal dominant modes of inheritance have been described for ICNC. Mutations in two genes encoding components of the prostaglandin degradation pathway have been associated with nail clubbing; hydroxyprostaglandin dehydrogenase (HPGD; MIM 601688) (Tariq et al., 2009) and solute carrier organic anion transporter family member 2A1 (SLCO2A1; MIM 601460) (Shah et al., 2016b-present study). HPGD encodes prostaglandin degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH), while the protein product of SLCO2A1 is the principal prostaglandin transporter (Kanai et al., 1995; Tai et al., 2002). Till date, a homozygous sequence variant in HPGD (Tariq et al., 2009) and two sequence variants in SLCO2A1 have been described for ICNC (Seifert et al., 2012; Shah et al., 2016b-present study).

Isolated Nail Dystrophy

Isolated nail dystrophy (IND; MIM 614157) is genetically heterogeneous and clinically diverse group of rare disorders characterized by onychauxis, onycholysis of fingers and toes nails, while some individuals exhibit claw-shaped fingernails. Autosomal recessive as well autosomal dominant modes of inheritance have been reported for isolated nail dystrophy. Mutations in two genes have been implicated with IND including collagen, type vii, alpha-1 (COL7A1; MIM 120120) (Sato- Matsumura et al., 2002) and frizzled 6 (FZD6; MIM 603409) (Fröjmark et al., 2011). In addition, two loci have also been mapped on chromosome 17p13 (Krebsová et al., 2000) and chromosome 17q25.1-25.3 (Rafiq et al., 2004) for the IND phenotypes. The gene COL7A1 is located on chromosome 3p21.31 and codes for collagen VII polypeptide, the main constituent of anchoring fibrils which are located in the skin below the basal lamina at the dermal-epidermal basement membrane zone (Ryynänen et al., 1992). The gene FZD6 located on chromosome 8q22.3 encodes for a member of frizzled family receptor proteins which is involved in negative regulation of the canonical Wnt/β-catenin signaling pathway, and positively regulates non-canonical Wnt pathway (Golan et al., 2004). According to HGMD Professional 2017.1, thirteen sequence variants have been identified in FZD6 causing isolated nail dystrophy. So far, only two sequence variants in COL7A1 have been described with IND (Sato- Matsumura et al., 2002).

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Palmoplantar Keratoderma

Hereditary palmoplantar keratodermas (PPKs) are genetically heterogeneous and phenotypically diverse group of keratinization disorders, characterized by hyperkeratosis of palms and soles. PPKs are described in all possible pattern of Mendelian inheritance as well as mitochondrial transmission (Has and Technau-Hafsi, 2016). PPKs belong to group-2 of ectodermal dysplasia (Priolo, 2009). Based on the pattern of epidermal involvement, PPKs may be classified as diffuse, focal, and punctate. The term diffuse PPK is used for the uniform involvement of the palmoplantar surface. Focal PPK includes localized hyperkeratosis, mostly over pressure points, may be nummular or striate. Punctate PPK describes multiple scattered, discrete round lesions of small keratotic papules on palmoplantar surface. PPKs may be isolated or syndromic, based on the extent of tissues involved. Isolated PPKs are limited to the thickness of palmoplantar skin, while syndromic cases include defect in other ectodermal structures or involvement of extra-cutaneous tissues in addition to palmoplantar skin thickness (Schiller et al., 2014). In Syndromic PPKs the palmoplantar thickness might be associated with ectodermal dysplasias, ichthyosis, epidermolysis bullosa, , pityriasis rubra pilaris, and (Has and Technau-Hafsi, 2016).

On molecular bases, PPKs may arise due to defects in keratin intermediate filaments, desmosomes, gap junctions, water channels, epidermal growth factor receptor (EGFR) signaling (Has and Technau-Hafsi, 2016). So far, twenty-five genes have been reported in pathogenesis of PPKs (Schiller et al., 2014).

Xeroderma Pigmentosum

Xeroderma pigmentosum (XP) is a rare hereditary condition characterized by over- sensitivity to ultra violet radiations. XP exhibits variable prevalence across the globe in different races with apparent autosomal recessive mode of inheritance. Individuals with xeroderma pigmentosum may develop blistering, persistent erythema and -like pigmentation and on sun exposed regions, ocular and neurological manifestations. Some cases of XP are also reported with acquired microcephaly, muscular impairment and progressive sensorineural hearing loss (Schäfer et al., 2013). The variation in clinical features are dependent on several

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 14 Chapter 1 Introduction factors including climates, exposure to sunlight, skin color, smoking, stage of diagnosis, the mutant gene and nature of the mutation (Lehmann et al., 2011).

XP is a genetically heterogeneous disease, which can be caused by pathogenic sequence variants in several genes of xeroderma pigmentosum complementation group including XPA (MIM 611153), XPB/ERCC3 (MIM 133510), XPC (MIM 613208), XPD/ERCC2 (MIM 126340), XPE/DDB2 (MIM 600811), XPF/ERCC4 (MIM 133520), XPG/ERCC5 (MIM 133530), and XPV/POLH (MIM 603968). The proteins XPA through XPG are involved in nucleotide excision repair (NER) pathway, while XPV is involved in translesion synthesis (Lehmann et al., 2011).

Trichothiodystrophy

Trichothiodystrophy (TTD) is an autosomal recessive disorder characterized by dry and brittle hair. Other associated features include intellectual disability, dwarfism, microcephaly, abnormal facial features, premature aging, ichthyosis, nail dystrophies, infertility and proneness to respiratory infections (Faghri et al., 2008). TTD is divided into two types i.e. photosensitive (TTD1) and nonphotosensitive (TTDN1) based on the presence or absence of clinical and cellular photosensitivity, respectively. Mostly, the photosensitive TTD is caused by sequence variants in nucleotide excision repair pathway genes. These are excision repair cross-complementing 2 (ERCC2; MIM 126340) (Takayama et al., 1997), excision repair cross-complementing 3 (ERCC3; MIM 133510) (Weeda et al., 1997) and general transcription factor IIH subunit 5 (GTF2H5, MIM 608780) (Giglia-Mari et al., 2004). While sequence variants in M- phase specific PLK1 interacting protein (MPLKIP; MIM 609188) have been reported in TTDN1 phenotypes (Nakabayashi et al., 2005; Shah et al., 2016a-present study). The exact function of MPLKIP is still to be discovered, however, its nuclear localization signifies its role as a transcriptional regulator of genes relevant for metabolic pathways that are central to the outcome of TTDN1 phenotypes (Nakabayashi et al., 2005). According to HGMD professional 2017.1, a total of 93 sequence variants in ERCC2, 16 in ERCC3, 5 in GTF2H5 and 17 sequence variants in MPLKIP have been described, so far.

Ichthyoses

Inherited ichthyoses are diverse group of genetic conditions characterized by defective cornification of skin. Ichthyoses may be transmitted in autosomal dominant,

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 15 Chapter 1 Introduction autosomal recessive and X-linked fashion. The diverse clinical attributes are characterized by scaling of skin, often with other associated cutaneous and non- cutaneous manifestations. The associated manifestations include erythroderma, palmoplantar keratoderma, hypohidrosis, and recurrent infections (Marukian and Choate, 2016).

Sequence variants in more than fifty genes have been implicated with ichthyoses, and these disrupt cellular pathways including lipid biosynthesis, DNA repair, adhesion and desquamation in addition to many other pathways. Even with numerous pathways involved in pathogenesis, each features interrupted barrier function (Schmuth et al., 2013). On the bases of pathophysiology, clinical manifestations and mode of inheritance a consensus classification for ichthyoses has been established by Oji et al. (2010). According to this classification system ichthyoses can be divides into two main types: 1) nonsyndromic forms, with cutaneous features only, and 2) syndromic forms, with association of extra-cutaneous organs.

Nonsyndromic Ichthyoses

On the bases of mode of inheritance, rate of incidence and molecular involvement, nonsyndromic ichthyoses are arranged into four groups: 1) common ichthyoses, 2) autosomal recessive congenital ichthyoses (ARCI), 3) keratinopathic ichthyoses (KPI) and 4) other forms (e.g. erythrokeratodermia) (Takeichi and Akiyama, 2016).

Common Ichthyoses

The term common ichthyoses is used for ichthyosis vulgaris (IV) and X-linked recessive ichthyosis (XLRI).

Ichthyosis Vulgaris

Ichthyosis vulgaris (MIM 146700) is the common most type of ichthyosis with an approximate incidence of 1/250 births (Marukian and Choate, 2016). The clinical features of ichthyosis vulgaris include generalized xerosis characterized by dry and rough skin, most evidently on theextensor regions of the extremities, abdomen and chest. In most cases, and palmoplantar hyperlinearity are associated with IV. The condition is inherited in semi-dominant fashion. Pathogenic sequence variants in the gene, filaggrin (FLG; MIM 135940), are associated with IV. FLG has an indispensable role in the differentiation of epidermis and establishment of the skin

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 16 Chapter 1 Introduction barrier (Smith et al., 2006). According to HGMD professional 2017.1, ninety-three pathogenic sequence variants have been described in the gene, FLG, so far.

X-linked Recessive Ichthyosis

X-linked recessive ichthyosis (MIM 308100) is the second most prevailing type of hereditary ichthyosis that affects 1/2000 to 1/6000 males worldwide (Lykkesfeldt et al., 1984). In adults, the XLRI is characterized by large polygonal, plate like scales those are tightly fixed to the skin. XLRI is results from sequence variant in steroid sulfatase (STS) gene, located on chromosome Xp22.31. STS is a membrane-bound microsomal enzyme, involved in metabolism of estrogens, androgens and cholesterol (Alperin and Shapiro, 1997). According to HGMD professional 2017.1, 68 sequence variants (mostly large deletions) in STS gene have been described in the pathogenesis of XLRI.

Autosomal Recessive Congenital Ichthyoses

Autosomal recessive congenital ichthyoses (ARCI) encompass a diverse group of inherited disorders of cornification with approximate incidence of 1 in 200,000 births (Richard et al., 1993). ARCI has three subtypes: 1) lamellar ichthyosis (LI), 2) congenital ichthyosiform erythroderma(CIE) and 3) harlequin ichthyosis(HI). To date, pathogenic sequence variants in twelve genes have been associated with ARCI including transg1utaminase 1 (TGM1; MIM 190195), 12R-1ipoxygenase (ALOX12B; MIM 603741), 1ipoxygenase 3 (ALOXE3;MIM 607206), ATP binding cassette subfamily A member 12 (ABCA12; MIM 607800), cytochrome P450, fami1y 4, subfami1y F, po1ypeptide 22 (CYP4F22; MIM 611495), NIPA-1ike domain containing 4 (NIPAL4; MIM 609383), short chain dehydrogenase/reductase family 9C member 7 (SDR9C7; MIM 609769), lipase N (LIPN; MIM 613924), ceramide synthase 3 (CERS3; MIM 615276), patatin-1ike phospho1ipase domain-containing protein 1 (PNPLA1; MIM 612121) and suppression of tumorigenicity 14 (ST14; MIM 676797) (Shigehara et al., 2016; Bastaki et al., 2017). Significant allelic and locus heterogeneity exist in ARCI.

Lamellar Ichthyosis

Lamellar ichthyosis (MIM 242300) is characterized by variable hyperkeratosis and palmoplantar keratoderma, thick and dark gray or brown scales on face, trunk, flexor areas and scalp, ectropion of eyelids, hypohidrosis, alopecia and erythema. Mostly

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 17 Chapter 1 Introduction infants are covered with a collodion membrane at birth (Akiyama et al., 2003). Other associated features include photophobia, abnormal dentition and blepharitis. The LI patient’s skin histology shows prominent hyperkeratosis with only a small number of parakeratotic cells and normal or mildly thick granular layer. The stratum corneum (SC) is mostly thicker in LI as compare to CIE (Akiyama et al, 2003).

Sequence variants in nine genes have been implicated with LI which include ABCA12, ALOXE3, ALOX12B, CERS3, CYP4F22, NIPAL4, PNPLA1, LIPN and TGM1. Of these, mutations in TGM1 are described frequently (Takeichi and Akiyama, 2016; Wakil et al., 2016). The gene TGM1 encodes for 817-amino-acids protein called transglutaminase-1, a member of transglutaminase family. TGM1 is a Ca2+-dependent, membranebound enzyme that plays role the development of the cornified cell cover (Farasat et al., 2009). Almost 180 sequence variants have been identified in the TGM1 gene in pathogenesis of ARCI (HGMD professional 2017.1. Accessed, 20th June, 2017).

Congenital Ichthyosiform Erythroderma

Congenital ichthyosiform erythroderma (CIE) is also called epidermolytic hyperkeratosis (MIM 113800), a congenital ichthyosis, inherited in autosomal recessive mode that is characterized by widespread scaling and erythroderma without blister formation. Children born with CIE usually have a collodion membrane (Williams and Elias, 1985). Development of erythroderma and scaling often associated with ectropion, eclabium, keratoderma, and nail dystrophy (Akiyama et al, 2003). Histology of patient’s skin sample shows hyperkeratosis, a usual or mildly thickened stratum granulosum, minor acanthosis and irregular parakeratosis (Akiyama, 1998). Sequence variants in ABCA12, ALOXE3, CERS3, ALOX12B , CYP4F22, NIPAL4, LIPN, , TGM1 and PNPLA1 have been implicated in pathogenesis of CIE.

Harlequin Ichthyosis

Harlequin ichthyosis (MIM 242500) is phenotypically the most harsh form of hereditary ichthyosis, sometime life threatening. Clinically HI is characterized by thick, large scales with severe eclabium, ectropion and flattened ears. Scarce development of skin occurs in utero; hair canal hyperkeratosis take place in the second trimester and typical ultra-structural defects such as atypical lamellar granules

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 18 Chapter 1 Introduction are contemporary in the epidermis of affected fetus (Dale and Kam, 1993). HI consequences from null mutations in ABCA12 gene that encodes for a member of lipid transporter proteins. ABCA12 performs lipid transport in epidermal keratinocyte lipid transporter and defects in ABCA12 leads to loss of lipid barrier in skin, resulting in to HI (Akiyama et al., 2005). HGMD 2017.1 professional (Accessed; 20th June, 2017), reported a total of 112 sequence variants in ABCA12 linked with different forms of ARCI.

Keratinopathic Ichthyoses

Keratinopathic ichthyoses (KI) is the broad term used for different types of ichthyoses results from pathogenic sequence variants in keratin genes. Keratinopathic ichthyoses include epidermolytic ichthyosis (EI), annular epidermolytic ichthyosis (AEI), superficial epidermolytic ichthyosis (SEI), autosomal recessive epidermolytic ichthyosis (AREI), ichthyosis Curth-Macklin (ICM), congenita1 reticu1ar ichthyosiform erythroderma (CRIE), epidermo1ytic nevi (EN) and congenita1 reticu1ar ichthyosiform erythroderma (CRIE). Mostly KI conditions are transmitted with autosomal dominant mode, while some autosomal recessive cases are also reported. Sequence variants in KRT1 (MIM 139350), KRT2 (MIM 600194) and KRT10 (MIM 148080) have been implicated with pathogenesis of keratinopathic ichthyoses (Takeichi and Akiyama, 2016). According to HGMD 2017.1 professional, a total of 60 sequence variants in KRT1, 18 in KRT2 and 60 in KRT10 have been associated with different forms of KI.

Erythrokeratoderma Erythrokeratodermas represent a minor type of ichthyosis featured by the appearance of well demarcated, erythematous, hyperkeratotic skin plaques. The erythrokeratodermas are classified clinically into two groups; progressive symmetric erythrokeratoderma (PSEK; MIM 602036) and erythrokeratodermia variabilis (EKV; MIM 133200), with considerable phenotypic overlap between PSEK and EKV. Till date, sequence variants in five genes have been reported in pathogenesis of erythrokeratoderma including gap junction protein alpha 1 (GJA1; MIM 121014) that encodes for 43 (Takeichi et al., 2016); gap junction protein beta 3 (GJB3; MIM 603324) that encodes connexin 31 (Richard et al., 1998); gap junction protein beta 4 (GJB4; MIM 605425) encoding connexin 30.3 (van Steensel et al., 2009); loricrin (LOR; MIM 152445) (Ishida-Yamamoto et al., 1997) and keratin 83 (KRT83;

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 19 Chapter 1 Introduction

MIM 602765) (Shah et al., 2017-present study). Gap junctions are formed by connexin proteins, featured by four trans-membrane domains, two extracellular loops, one intracellular and cytoplasmically located carboxyl and amino termini (Spray, 1998). Loricrin is the main component of the cornified epidermal cell envelope, synthesized in the upper granular layer of epidermis (Ishida-Yamamoto and Iizuka, 1998). KRT83 was initially described as a type II keratin weakly expressed in hair cortex. However, in rat and sheep KRT83 is expressed in whole skin. Recently a homozygous frameshift sequence variant in KRT83 is reported in pathogenesis of progressive symmetric erythrokeratoderma in large Pakistani kindred (Shah et al., 2017-this study). According to HGMD professional 2017.1; a total of 21 sequence variants in GJB4, 37 in GJB3, 103 in GJA1 and 10 sequence variants in LOR have been described. Syndromic Ichthyoses

Accompanied by cutaneous involvement, at least one or more other organ systems are also affected in syndromic ichthyoses. Consistent with the genes involved, several abnormalities in the CNS, skeletal system, endocrine system, cardiovascular system can be noticed in syndromic ichthyoses. The syndromic forms of ichthyosis include keratitis ichthyosis deafness (KID) syndrome(MIM 148210), Netherton syndrome(MIM 256500), ichthyosis follicu1aris with photophobia and atrichia (MIM 308205), Dorfman-Chanarin syndrome (MIM 275630) and Sjögren-Larsson syndrome (MIM 270200).

Keratitis Ichthyosis Deafness Syndrome

Keratitis ichthyosis deafness syndrome (MIM 148210) is an autosomal dominant syndromic form of rare ichthyosis, characterized by palmoplantar hyperkeratosis with leather grain-like keratoderma, ichthyosiform scaling, alopecia, nail dystrophy, sensorineural hearing loss and progressive ocular manifestation. The disease results from sequence variants in GJB2 gene (MIM 121011) (van Steensel et al., 2002). According to HGMD 2017.1 professional, a total of 387 pathogenic sequence variants have been reported in GJB2 gene.

Netherton Syndrome

Netherton syndrome (MIM 256500) is a rare autosomal recessive form of syndromic ichthyosis clinically featured by ichthyosiform dermatosis, hair shaft abnormalities

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 20 Chapter 1 Introduction and atopic diathesis (Chavanas et al., 2000). Netherton syndrome results from sequence variants in SPINK5 (MIM 605010) gene, mapped on chromosome 5q31- q32. SPINK5 coding protein, 15-domain serine protease inhibitor, expressed in stratum spinosum, tonsils, thymus, parathyroid glands, hair follicles and the trachea. According to HGMD 2017.1 professional, a total of 84 pathogenic sequence variants have been reported in SPINK5 gene, so far.

Ichthyosis Follicularis with Atrichia and Photophobia

Ichthyosis follicularis with atrichia and photophobia (IFAP; MIM 308205) is a rare X- linked recessive syndromic ichthyosis, featured by defects in brain, alopecia, ectodermal dysplasia, skeletal anomalies, eye and ear defects, cleft palate, renal abnormalities and mild scales over skin. Pathogenic sequence variants in MBTPS2 (MIM 300294) are associated with IFAP (Oeffner et al., 2009). MBTPS2 encodes site-2 protease (S2P), a membrane-embedded zinc metalloprotease, plays role in the activation of the signal protein, associated with sterol control of transcription and the endoplasmic reticulum stress response (Sakai et al. 1996). According to HGMD 2017.1 professional, a total of 24 pathogenic sequence variants have been reported in MBTPS2 gene.

Dorfman-Chanarin syndrome

Dorfman-Chanarin syndrome (MIM 275630) is a rare autosomal recessive neutral lipids storage disease, marked by inborn defects in liver, muscle, and central nervous system and ichthyosiform erythroderma due to accumulation of neutral lipids in these organs. Pathogenesis of Dorfman-Chanarin syndrome has been associated with sequence variants in abhydrolase domain containing 5 (ABHD5; MIM 604780) gene. ABHD5 functions as an acyltransferase for the synthesis of phosphatidic acid, an important intermediate in membrane and storage lipid metabolism. According to HGMD 2017.1 professional, a total of 36 pathogenic sequence variants have been reported in ABHD5 gene for pathogenesis of Dorfman-Chanarin syndrome.

Sjögren-Larsson syndrome

Sjögren-Larsson syndrome (SLS; MIM 270200) is a rare form of autosomal recessive syndromic ichthyosis, with estimated worldwide prevalence of 0.4/100000 live births (Gordon, 2007). SLS is characterized by diffuse generalized ichthyosis, spasticity and neurological complications. Pathogenic sequence variants in aldehyde dehydrogenase

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 21 Chapter 1 Introduction

3 family member A2 (ALDH3A2; MIM 609523) result in SLS phenotypes (Laurenzi et al., 1996). ALDH3A2 is located on 17p11.2 and encodes for (FALDH) fatty aldehyde dehydrogenase. FALDH is a NAD dependent enzyme of microsome that carries out the conversion of long-chain aliphatic aldehydes into fatty acids (Kelson et al., 1997). FALDH deficiency results in aberrant oxidation of long-chain fatty aldehydes to fatty acids with subsequent accretion of fatty aldehyde precursors such as fatty alcohols. Thus the cutaneous and neurological symptoms appear. According to HGMD professional 2017.1, 101 pathogenic sequence variants in ALDH3A2 have been identified for SLS phenotypes.

Epidermolysis Bullosa

Cutaneous basement membrane zone (BMZ) has several anchoring molecules that are indispensible for stabilization of the dermis-epidermis association. These include hemidesmosomes, anchoring fibrils and anchoring filaments which make an inter- connecting net spreading among intracellular milieu of basalkeratinocytes through the dermal epidermal basment membrane to the underlying dermis. Irregularities in the structure of the network may consequence in fragile skin at the cutaneous BMZ level. The phenotype of this condition is epidermolysis bullosa (EB), a diverse class of genodermatoses with highly variable clinical severity, characterized by skin blisters formation as a result of minor injury. In addition to skin blisters, different extracutaneous manifestations are also reported which include scarring alopecia, corneal erosions, enamel hypoplasia, development of esophageal structures, erosions in the tracheal epithelium, congenital pyloricatresia and muscular dystrophy. On the bases of phenotypes, EB may be classified into four types: 1) EB simplex (EBS), 2) dystrophic EB (DEB), 3) junctional EB (JEB) and 4) Kindler syndrome (Fine et al., 2014).

Epidermolysis Bullosa Simplex

EB simplex (EBS) is the common most type of epidermolysis bullosa, making almost 70% of all cases, and comparatively milder than the other types. In EBS the blisters occur in epidermis. Mostly, EBS is inherited with autosomal dominant mode, with sequence variants in (KRT5; MIM 148040) and (KRT14; MIM 148066) genes (Fine et al., 2014). KRT5 and KRT14 dimerise to produce intermediate filaments, which contribute to the structure, flexibility and strength of

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 22 Chapter 1 Introduction the of keratinocyte. When defective, they are prone to physical stress, resulting in the rupture of basal keratinocytes and following blistering of the epithelium. Sequence variants in plectin (PLEC; MIM 601282) gene have been implicated with autosomal recessive EBS associated with extracutaneous anomalies including muscular dystrophy (EBS-MD; MIM 226670) and pyloric atresia (EBS-PA; MIM 612138). PLEC encodes for a cytoskeletal linker protein associated with intermediate filaments, various subplasma membrane–cytoskeleton and membrane- cytoskeleton junctional complexes in epithelia, muscles and fibroblasts. Consistent with its numerous binding partners, sequence variants in plectin results in pleiotropic phenotypes (Wiche, 1998). According to HGMD professional 2017.1, a total of, 141 sequence variants in KRT5, 103 sequence variants in KRT14 and 87 sequence variants in PLEC have been described in pathogenesis of EBS.

Dystrophic Epidermolysis Bullosa

Dystrophic epidermolysis bullosa (DEB) account for 25% of the cases of EBs that is characterized by formation of blisters underneath the lamina densa of the cutaneous BMZ. Other associated findings in DEB include blistering and scaring of gastrointestinal tract, corneal erosion, dystrophic nails and scaring alopecia (Fine et al., 1999). Autosomal dominant as well recessive DEB is caused by sequence variants in collagen, type vii, α-1 (COL7A1; MIM 120120) gene, mapped on 3p21.1 (Christiano et al., 1993, 1994). COL7A1 is a major constituent of anchoring fibrils, which are located below the basal lamina at the dermal-epidermal BMZ of the skin. According to HGMD professional 2017.1, a total of 721 sequence variants are reported in COL7A1 gene for DEB. The disease phenotypes severity defends on the position and nature of the mutation. In addition to pathogenic sequence variants, polymorphic variations also contribute to phenotypic variability (Titeux et al., 2008).

Junctional Epidermolysis Bullosa

Junctional epidermolysis bullosa (JEB) is the most severe and rarest types of EB, representing 5% of the cases and characterized by blister development in the basement membrane. Sequence variants in the genes encoding components of hemidesmosome-anchoring filaments are reported for JEB. Based on the severity of phenotypes, JEB can be grouped into two types: JEB generalised severe and JEB generalised intermediate. Till date, sequence variants in seven genes have been

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 23 Chapter 1 Introduction reported in pathogenesis of JEB, which belong to [LAMA3 (MIM 600805), LAMB3 (MIM 150310), LAMC2 (MIM 150292)], integrin [ITGA3 (MIM MIM 605025), ITGB4 (MIM 147557), ITGA6 (MIM 147556)] and collagen [COL17A1 (MIM 113811)] families of protein (Yenamandra et al., 2017).

Kindler Syndrome

Kindler syndrome (MIM 173650) was included in epidermolysis bullosa in the third consensus meeting for the classification of EB (Fine et al., 2008). This is a rare type of featured by acral blistering and light-sensitivity in childhood which is followed by cutaneous atrophy and progressive . Other associated features include nails dystrophy, webbing of the digits, gingivitis, labial leukokeratosis, and esophageal, vaginal and urethral mucosal lesions leading to stenosis (Jobard et al., 2003). Kindler syndrome is caused by mutations in fermitin family member 1 (FERMT1; MIM 607900) gene (Jobard et al., 2003). The FERMT1 codes for KIND1 protein, involved in regulation of keratinocytes adhesion as well manage proliferation and differentiation of cutaneous stem cells.

Up to now, 68 mutations in the FERMT1 have been reported in in the pathogenesis of Kindler syndrome (HGMD professional 2017.1 Accessed; 20th June, 2017).

Hypotrichosis

Hypotrichosis represents a group of clinically heterogeneous conditions characterized by scarce to complete missing of hair from the scalp and other body parts. Hypotrichosis might be an isolated entity or may be associated with abnormalities in other tissues and organs. The associated abnormalities include intellectual disability, hearing impairment, retinal degeneration and defects in nails and skin. Hypotrichosis are inherited in either autosomal recessive or autosomal dominant fashion.

Autosomal Recessive Isolated Hypotrichosis

Over the past few years, several genes and loci for isolated autosomal recessive form of hypotrichosis have been mapped to different human chromosomes. The genes described for non-syndromic autosomal recessive hypotrichosis include HR (hairless; MIM 225060) (Ahmad et al., 1998), DSG4 (desmoglein-4; MIM 607892) (Kljuic et al., 2003), LIPH (lipase-H; MIM 607365) (Kazantseva et al., 2006), lysophosphatidic acid receptor 6 (LPAR6; MIM 609239) (Pasternack et al., 2008), desmocollin-3

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 24 Chapter 1 Introduction

(DSC3; MIM 600271) (Ayub et al., 2009) and DSP (desmoplakin; MIM 125647) (Jan et al., 2015). Recently, mutation in KRT25 (Keratin-25; MIM 616646) has been described for autosomal recessive wooly hairs (Ansar et al., 2015). In addition, two loci for autosomal recessive hypotrichosis have been mapped to chromosome 7p21.3- p22.3 (Basit et al., 2010) and chromosome 10q11.23-22.3 (Naz et al., 2010).

Atrichia with Papular Lesions

Atrichia with popular lesions (APL; MIM 209500) is featured by localized or generalized irreversible loss of hair mostly associated with diffused numerous keratinus folicular papules particularly on the scalp, face and extremities. APL is inherited in pseudo-dominant or autosomal recessive pattern (Damste and Prakken 1954; Zlotogorski et al., 2002a,b). Pathogenic sequence variants in hairless (HR; MIM 225060) gene have been associated with APL phenotypes in different ethnic groups around the globe (Ahmad et al., 1998; Zlotogorski et al., 1998). HR gene is located to the short arm of chromosome 8 (8p21) and encodes for a putative transcription corepressor of several nuclearreceptors, with highest expression in brain, epidermis and hair follicle.

HGMD Professional 2017.1 (Acessed, 20th June 2017) has documented a total of 52 mutations in the HR as a cause of APL from various populations around the globe.

Localized Autosomal Recessive Hypotrichosis 1

In affected individuals of localized autosomal recessive hypotrichosis 1 (LAH1; MIM 607903) scalp hair may be present at birth but after sparse hair re-grow after ceremonial shaving (Rafique et al., 2003). The affected individuals exhibit sparse to completely absent eyebrows and eyelashes: however, hair of pubic and axillary regions are normal (Rafiq et al., 2003; John et al., 2006b). Beard hair in affected male individuals are normal: however hairs over extremities are absent (Rafiq et al., 2003; Schaffer et al., 2006). DSG4 (MIM 607892) is the causative gene for LAH1 (Kljuic et al., 2003), located on 18q12.1 (Whittock and Bower, 2003). DSG4 encodes for desmoglin-4, a member of desmosomal cadherin superfamily which are involved in cell-cell adhesion and has critical role in proper differentiation and development of hair follicle. In human, DSG4 has prominent expression in the inner layers of the hair follicle specially in inner root sheath, lower hair cuticle and the cortex region of the hair shaft and in human (Bazzi and Getz, 2006). According to HGMD Professional

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 25 Chapter 1 Introduction

2017.1, so far, fifteen mutations have reported in the DSG4 gene for pathogenesis of LAH1.

Localized Autosomal Recessive Hypotrichosis 2

Localized autosomal recessive hypotrichosis 2 (LAH2; MIM 604379) is characterized by sparse wooly hair to fine thin hair on the scalp (Kazantseva et al., 2006; Ali et al., 2007). Affected individuals exhibit sparse to absent eyelashes and eyebrows, and sparse hair in auxiliary regions and other body parts. However, beard hairs often appear normal in affected males. Genome wide autozygosity mapping revealed the disease locus on chromosome 3q26.33-q27.3, in consanguineous Pakistani kindred (Alsam et al. 2004). Further molecular analysis identified LIPH as a causal gene in the locus, LAH2 (Kazantseva et al., 2006). The protein product of LIPH is phospholipase A1 that has been designated for conversion of phosphatidic acid to acyllysophosphatidic acid, a ligand of the lysophosphatidic acid receptor. So far, 26 pathogenic sequence variants have been identified in the gene, LIPH, in pathogenesis of LAH2 (Stenson et al., 2017).

Localized Autosomal Recessive Hypotrichosis 3

Just like LAH2, affected individuals with localized autosomal recessive hypotrichosis 3 (LAH3; MIM 278150) show fragile, slow growing tightly curled wooly hairs. Hairs re-grow sparsely after ceremonial shaving that shed gradually and replaced with curly, thin light colored hair. Eyebrows and eyelashes, and pubic and axillary hairs are sparse normal (Wali et al., 2007a; Azeem et al., 2008; Shimomura et al., 2008). Whole genome autozygosity mapping had previously revealed a chromosomal segment 13q14.11–q21.32 for LAH3 (Wali et al., 2007a), followed by the identification of pathogenic sequence variants in LPAR6 gene, located in LAH3 locus (Pasternack et al., 2008; Shimomura et al., 2008). Both, LIPH and LPAR6 are expressed in the IRS of hair follicles, and are associated with the same pathway of differentiation and hair growth regulation (Pasternack et al., 2009). So far, a total of 26 mutations in the LPAR6 have been reported (Stenson et al., 2017).

Hypotrichosis with Recurrent Skin Vesicles

Ayub et al. (2009) described a unique form of hypotrichosis in a consanguineous Afghani kindred, the scalp hair in affected individuals were normal at birth but shed within few weeks, however, the hair regrew after ritual shaving. Vesicles were

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 26 Chapter 1 Introduction observed in affected individuals over scalp and other body parts which were filled with watery fluid. The disease was mapped to chromosome 18q, upon sequencing the candidate genes within the autozygous region, a missense sequence variant was found in DSC3 gene. The protein product of DSC3 is a member of desmosome family, which helps to maintain the tissue integrity by keeping adjacent cells connected through desmosome-junctions (Thomason et al., 2010). To date, only one missense homozygous sequence variant has been reported in DSC3 gene in pathogenesis of hypotrichosis (Ayub et al., 2009).

Autosomal Recessive Wooly Hair

Ansar et al. (2015) described autosomal recessive wooly hair in two Pakistani families; the affected individuals exhibited tightly curled scalp hairs. No other ectodermal or systemic abnormality was observed. Upon linkage mapping and exome analysis, they identified a pathogenic missense variant in KRT25 (MIM 616646) segregating with autosomal recessive wooly hair in both the families.

Autosomal Dominant Isolated Hypotrichosis

The autosomal dominant forms of isolated hair abnormalities have been implicated with defects in several proteins that play role in the development and maintenance of hair.

Hypotrichosis Simplex

Hypotrichosis simplex is an autosomal dominant condition, featured by thin scalp hair without any other associated anomaly (Shimomura et al., 2010c), however, eyelashes, eyebrows and other body hairs remain normal (Levy-Nissenbaum et al. 2003). Heterozygous sequence variants in APCDD1 (adenomatosis polyposis coli downregulated 1; MIM 607479) (Shimomura et al., 2010c), CDSN (corneodesmosin; MIM 602593) (Levy-Nissenbaum et al. 2003), SNRPE (small nuclear ribonucleoprotein polypeptide E; MIM 128260) (Pasternack et al., 2013) and RPL21 (ribosomal protein L21; MIM 603636) (Zhou et al,. 2011) have been described in pathogeneses of autosomal dominant hypotrichosis simplex. According to HGMD Professional 2017.1, only one missense variant in APCDD1, one missense variant in RPL21, two missense variants in SNRPE and fifteen sequence variants in CDSN have been reported, till date.

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 27 Chapter 1 Introduction

Autosomal Dominant Wooly Hair

Wooly hair represents a disorder of hair’s shaft, characterized by thin and tightly curled hair, which stop growing after a few inches. The microscopic analysis of wooly hair shows trichorrhexis nodosa and tapered end (Petukhova et al. 2009). Sequence variants in KRT71 (keratin-71; MIM 608245) (Fujimoto et al., 2012) and KRT74 (keratin-74; MIM 608248) (Shimomura et al., 2010b) have been associated with autosomal dominant wooly hair. According to HGMD 2017.1 professional, only one sequence variants in KRT71 and four sequence variants in KRT74 have been described in pathogenesis of autosomal dominant wooly hair.

Autosomal Dominant Monilethrix

Autosomal dominant monilethrix (MIM 158000) is characterized by beaded hair. The hair of affected individuals are normal hair at birth but shortly after birth the hair become fragile and brittle, results in varying degrees of dystrophic alopecia (Zlotogorski et al., 2006). Sequence variants in type II hair keratins: KRT81 (keratin- 81; MIM 602153) (Winter et al., 1997), KRT83 (keratin-83; MIM 602765) and KRT86 (keratin-86; MIM 601928) (van Steensel et al., 2005) have been identified in individuals with autosomal dominant monilethrix. The protein products of these genes are expressed in hair follicles (Rogers et al., 1997). According to HGMD 2017.1 professional, only five sequence variants in KRT81, four sequence variants in KRT83 and eleven sequence variants in KRT86 have been reported, till date.

Marrie Unna Hereditary Hypotrichosis

Marie Unna hereditary hypotrichosis (MUHH) is a hair loss condition transmitted with autosomal dominant pattern and characterized by sparse to absent hair at birth, development of coarse hair in early childhood with gradual hair loss results in varying degrees of alopecia in adults (Cai et al., 2009; Duzenli et al., 2009). MUHH has been implicated with sequence variants in U2HR (upstream open reading frame of the HR; MIM 146550) located on 8p21 and epidermal growth factor receptor pathway substrate 8-like 3 (EPS8L3; MIM 614989), located on chromosome 1p13.3. The protein product of U2HR negatively regulates translation of the HR gene (Wen et al., 2009). EPS8L3 belongs to EPS8 proteins family, which are involved in epidermal growth factor receptor (EGFR) pathway. EGFR signaling is indispensable for hair growth initiation (Murillas et al., 1995). According to HGMD 2017.1 professional,

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 28 Chapter 1 Introduction only one sequence variant in EPS8L3 and 18 in U2HR have been implicated with pathogenesis of MUHH.

Syndromic Forms of Hypotrichosis

In addition to non-syndromic hair loss disorders, several syndromes have been described in which hair abnormality occur in association with heterogeneous clinical features such as neurological, cardiac and ocular anomalies and defects in other ectodermal structures (Headon and Overbek, 1999; Monral et al., 1999; Sprecher et al., 2001; John et al., 2006a; Wali et al., 2006; Wali et al., 2007b). Hair loss disorders have also been described in association with some rare genetic conditions such as hypogonadism and neuroendocrine (Alazami et al., 2008; Nousbeck et al., 2008).

The neurological manifestation with hair loss is characterized by alopecia and mental retardation (APMR) syndrome. Four loci for such phenotypes have been described in the literature: APMR1 on chromosome 3q26.33–q27.3 APMR2 on chromosome 3q26.2–q26.31 , APMR3 on 18q11.2–q12.2 (John et al., 2006a; Wali et al., 2006, 2007b) and APMR4 on chromosome 1p31.1–p22.3 (Tzchach et al., 2008). Recently, Sailani et al. (2017) identified a missense variant in alpha-2-hs-glycoprotein (AHSG; MIM 138680) in large Iranian kindred, segregating with APMR phenotypes.

Hair loss disorder accompanied by ocular manifestation is represented by hypotrichosis with juvenile macular dystrophy (HJMD; MIM 601553), characterized by macular dystrophy of the retina with sparse hair (Sprecher et al., 2001); and ED, ectrodactyly and macular degeneration syndrome (EEM syndrome, MIM 225280) (Kjær et al., 2005). Both HJMD and EEM syndrome are implicated with sequence variants in cadherin 3 (CDH3; MIM 11402) gene. CDH3 belongs to cadherin proteins family, which are essential membrane glycoproteins involved in calcium-dependent cell-cell adhesion (Sprecher et al., 2001). So far, 29 mutations in CDH3 have been identified for pathogenesis of HJMD, EEM syndrome and isolated hypotrichosis (Stenson et al., 2017).

Woodhouse Sakati syndrome is a multisystem disorder characterized by hypotrichosis, diabetes mellitus, hearing impairment, hypogonadism, extrapydamidal manifestations and learning disabilities (Alazami et al., 2008; Ali et al., 2016). Sequence variants in ddb1 and cul4 associated factor 17 (DCAF17; MIM 612515)

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 29 Chapter 1 Introduction have been identified as the cause of the disorder in multiple families of Woodhouse Sakati syndrome phenotypes. DCAF17 encodes for a nuclear protein, expressed in brain, liver and skin that correlates with the organ involvement in WSS patients. According to HGMD 2017.1 professional, a total of 12 sequence variants in DCAF17 have been implicated in pathogenesis of WWS.

Autozygosity Mapping and Mutation Analysis

Genetic linkage analysis or autozygosity mapping localize genes with respect to genetic markers based on the fact that alleles at two nearby loci on the chromosome tend to co-segregate during meiosis. Linkage analysis relies on the principles that affected siblings from consanguineous marriages have a higher likelihood of sharing homozygous genomic regions due to identity by decent (IBD). Almost, 1/16 part of the siblings DNA from first cousin marriage is expected to be homozygous. Thus, random homozygous stretch of chromosome would be present between the siblings. However, in case of recessive disorders a common homozygous region (autozygous) in a disease locus would be expected among all the affected individuals. The genetic markers that are commonly used in linkage analysis are single nucleotide polymorphism (SNPs) and microsatellite repeats number variations. Microsatellite markers are highly polymorphic and have higher mutation rate than SNPs, so highly informative. SNPs occur more frequently in the genome, so provide coverage for smaller homozygous region. Recent advances in molecular technologies have made SNPs-genotyping possible with short time and affordable price.

Identification of the pathogenic sequence variants in Mendelian disorders is of great interest to clinicians and biologist for molecular diagnosis and understanding the disease mechanism and function of the gene. Sanger sequencing is the technique of choice for sequencing candidate genes, while, next generation sequencing is the state- of-the-art technology for searching the pathogenic variant in the entire coding regions (whole exome sequencing) or whole genome (whole genome sequencing). Whole exome sequencing (WES) is more cost-effective and frequently used for analysis of Mendelian disorders. However, whole genome sequencing (WGS) can detect variants in DNA sequences outside of exome, frequently used in analysis of complex disorders.

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 30 Chapter 1 Introduction

The present study was focused on clinical and molecular characterization of different Pakistani families segregating syndromic and non-syndromic forms of skin disorders. For the current study eighteen Pakistani families were recruited from different rural areas. These included five families with ectodermal dysplasia, three with ichthyosis, two with epidermolysis bullosa, two with trichothiodystrophy and six with hair loss disorders.

Objectives of the study included assessment of the inheritance pattern of the disease phenotypes followed by identification of disease causing genes and sequence variant in affected individuals. Linkage analyses were carried out using microsatellite and SNP markers while the pathogenic sequence variants were hunt down using chain termination sequencing in combination with whole exome sequencing.

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 31 Chapter 2 Materials and Methods

MATERIALS AND METHODS

Ethical Approval and Study Subjects

Prior to start the study, approval was obtained from the Institutional Review Board (IRB), Quaid-i-Azam University (QAU), Islamabad, Pakistan. In total, seventeen families segregating various types of skin disorders such as ectodermal dysplasia, ichthyosis, epidermolysis bullosa and hair loss disorders were incorporated in the present study. Different resources were used to identify and ascertain these families. All the families were visited at their residence and clinical information was recorded using a standardized questionnaire. The families were educated in their local languages and written informed consents were obtained from the affected members and/or their guardians for clinical and molecular analysis of the disorders they were suffering with and presentation of the data including photographs for publications. Multiple members in each family were interviewed for obtaining information relevant to the consanguineous nature, clinical history and construction of the pedigree. Pedigrees were drawn according to the protocol described by Bennett et al. (1995). For phenotypes documentation photographs of the affected individuals were obtained using a high-resolution digital camera. Clinical investigations were carried out in local government hospitals under the supervision of medical officers and dermatologists.

Blood Sampling and Genomic DNA Extraction

From all those participated in the study, peripheral blood samples of 4-6 mL were collected in 10 ml EDTA containing vacutainer set (BD Vacutainer® K3 EDTA, Franklin Lakes NJ, USA), by venipuncture with the help of 5 mL (BD 0.60 mm X 25 mm) and 10 mL (BD 0.8 mm X 38 mm 21) syringes, attached usually with a butterfly (BD Vacutainer, Franklin Lakes NJ, USA).

Extraction of the genomic DNA from the whole blood was carried out either using organic (phenol-chloroform) method described by Sambrook and Russell (2001) or using commercially available kits.

Phenol-chloroform Method

In phenol-chloroform method of DNA extraction, 0.75 mL blood and equal volume of solution A (10mM Tris pH 7.5, 1% v/v Triton X100, 5 mM MgCl2, 0.32 M Sucrose)

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 32 Chapter 2 Materials and Methods was mixed in a 1.5 mL tube (GEB, Torrance, CA, USA) and kept at 20-30°C for 15- 20 min. The centrifugation was carried out at 13000 rpm for 60 seconds, supernatant was discarded and pellet was resuspended in 500 μL of solution A. Again, centrifugation was carried out and the pellet was resuspended in 500μL of solution B (2 mM EDTA pH 8.0, 10 mM Tris pH 7.5, 400mM NaCl), 25 μL of 10% SDS and 10-15 μL of PK (proteinase K) and kept overnight at 37°C. On the following day 500 μL mixture of equal volumes of solution C and D [chloroform: isoamylalcohol (24:1 ratio)] was added to the microcentrifuge tube, after mixing uniformly centrifugation was carried out at 13000 rpm for 10 min. Two clear phases were observed; the upper one was picked and placed into a new microcentrifuge tube. Again, 400 μL solution D was added to the microcentrifuge tube, and after several times inverting, the tube was centrifuged at 13000 rpm for 10 min. Again, the upper layer was transferred to a new microcentrifuge tube and 55 μL of sodium acetate (3M, pH 6) and 0.5 mL chilled isopropanol were added to precipitate the DNA. After inverting several times the tube was centrifuged at 13,000 rpm for 10 min. After discarding the supernatant the DNA pellet was washed with chilled 70% ethanol. Centrifugation of the tube was again carried out for 10 minutes at 13,000 rpm. Ethanol was removed and the DNA pellet was dried in vacuum concentrator at 30/45°C. Tris-EDTA (TE) buffer was added to the dried DNA pellet and allowed to incubate at 37C for 12-24 hours. On the next day the DNA was run the on 1% agarose gel and also quantified using Fluorometer (Invitrogen Inc.TM, CA, USA).

DNA Extraction using Kits

In addition to phenol-chloroform method, extraction of genomic DNA from blood was also carried out using commercially available kits i.e. GenEluteTM blood genomic DNA kit (Sigma-Aldrich MO, USA), PureGenomeTM Tissue DNA Extraction Kit (Merck, KGaA, Darmstadt, Germany) and GeneJET Genomic DNA Purification Kit (Thermo Fisher scientific, Vilius, Lithuania), following the manufacturers protocol.

Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) was carried out in 200 μL tubes, containing 15- 50μL reaction volume, comprising 35-55 ng human genomic DNA, 2.5-5μL 10X reaction buffer (750 mM Tris-HCl pH 8.8, 25 mM (NH4)2SO4), 1.5-2 mM MgCl2

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 33 Chapter 2 Materials and Methods

(Sigma-Aldrich, CA, USA), 1 U Platinum™ Taq Polymerase (Thermo Fisher Scientific, UK), 200 μM of each deoxynucleoside triphosphate (dNTP) and 4-10 pmol of forward and reverse primers. The reaction volume was raise to 25-50 μL with PCR water. PCR conditions used for DNA amplification included first denaturation at 95°C for 7-10 min, followed by 35 cycles of amplification each consisting of three steps: 30-60 seconds at 96°C for DNA denaturation into single strands, 30-60 sec at 52-62°C for primer annealing and 30-60 seconds at 72°C for extension. Finally, 5 minutes extension incubation was performed at 72°C to synthesize any unextended strand.

RNA Extraction and cDNA Synthesis

For RNA extraction, 3mL of the whole blood sample was collected in Tempus™ Blood RNA Tube. RNA was extracted from the blood using Tempus™ RNA Isolation kit (Applied Biosystem, Foster City, CA, USA). For RNA extraction the manufacturer’s protocol was followed. To make RNA free of DNA traces, the RNA sample was treated with DNase-1. RNA was quantified through Qubit® 2.0 Fluorometer (InvitrogenTM, CA, USA) and stored at -70C. cDNA synthesis, using purified RNA as template, was performed with RevertAid First strand cDNA Synthesis Kit (Thermo Fisher scientific, CA, USA). Equimolar working dilutions were prepared for all the RNA samples. Reverse transcription-PCR reaction was performed in PCR-tubes, comprising 25 μL reaction volume [4 μL RNA, 5μL 5X reaction buffer,1 μL oligo-dT/random-hexamer primer, 1µL dNTPs 10 mM], 1μL RiboLock (20U/µL) and 0.8 μL RevertAid reverse transcriptase (200U/µL), and the final volume was raised to 25μL with DEPC water. Incubation of the reaction mixture was carried out for one hour at 48C. In case of random hexamer primed synthesis, first incubation was performed at 30°C for 5 min followed by 60 min at 48°C. The cDNA was stored at -20°C for further analysis.

Real Time Polymerase Chain Reaction

In family N, to analyze the significance of alteration (p.Leu1829Val) in transcription factor BTAF1, quantitative PCR (qPCR) was carried out for exploring the differential expression of five genes (AR, EDA2R, ALDH1A3, KRT81, FGF5). Real time PCR reaction was carried out using 2X Maxima SYBR Green/ROX qPCR Master Mix

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 34 Chapter 2 Materials and Methods

(Fermentas, Vilnius, Lithuania) in ABI 7500 theromocycler (Applied Biosystems, CA USA). The qPCR was carried out in 200 µL tubes containing 25 µL reaction volume [1 µL cDNA, 1 µL forward and reverse primers, 12.5 µL SYBR Green MasterMix and 9.5µL DEPC-treated water]. The thermal cycler conditions used for qPCR included initial denaturation for 10 minutes at 96°C, followed by 39 cycles of amplification each consisting of three steps: denaturation at 96°C for 45 seconds, primer annealing at 55-58°C for 45 seconds and amplification at 68°C for 45 seconds. The data was collected at step 3 (amplification) and analyzed for differential expression between normal and affected individuals using the ABI-7500 software (Applied Biosystems, CA USA).

Mapping Candidate Genes

The families, presented in the dissertation, were initially subjected to autozygosity mapping using microsatellite markers flanking the genes involved in causing different forms of skin disorders. Amplification of the microsatellite markers was performed out following standard PCR protocol. For resolving the PCR amplified products, 8% nondenaturing polyacrylamide gel was used. The polyacrylamide gel was prepared using 13.5mL 30% acrylamide solution (29:1 ratio of acrylamide and Methylenebisacrylamide) (MERCK, Darmstadt, Germany), 5 mL 10X TBE (Tris- Borate EDAT), 25 μL TEMED (N, N, N’, N’-Tetra methylenediamine) 400-450 μL of 10% ammonium persulphate and volume was raised to 50mL with distilled water. The gel mixture was transferred to assembled glass plates, held apart by spacers of 1.5mm depth. For polymerization, the gel was kept for 25-35 minutes at room temperature. Before loading the gel, PCR-amplified products were mixed with 4-6 loading dye and electrophoresis was carried for 2-3 hours at 120 volts. For visualization on UV Transilluminator (Biometra, Gottingen, Germany) the gel was stained with ethidiumbromide (8 μg/mL) solution. For taking gel photographs Kodak DC 290 gel documentation system (Kodak, Japan) was used and genotypes were assigned by visual inspection.

Human Genome Scan

After exclusion from candidate genes the families were subjected to whole genome genotyping with SNP based DNA Microarray, using either GeneChip Mapping 250 K

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 35 Chapter 2 Materials and Methods

NspI array (Affymetrx, Santa Clara, CA, USA) or InfiniumHD assay (Illummina Inc., San Diego, CA, USA).

Autozygosity mapping with AffymetrixGeneChip Mapping 250K Nsp1 array was carried out using 200-300 ng of the genomic DNA. The genomic DNA was digested with Nsp1 restriction enzyme followed by ligation to the adapters, using T4 DNA ligase. Amplification of the Adaptor-ligated DNA fragments was performed using universal primers that are complementary to the adaptor sequences. Purification of the PCR amplified products were performed on a CleanUp plate and eluted by Recovery buffer (RB). The PCR-amplified products Fragmentation was carried out with fragmentation Reagent (0.05 U/μL DNase 1) at 37°C for 40 min. The DNA fragments were labeled at 37°C followed by hybridization to the array. After washing, the array was stained on an automated Fluidic device and scanning was performed with GeneChip® Scanner 3000 7G. The allele of each SNP was analyzed by the BRLMM algorithm merged in Affymetrix Genotyping Console. A call-rate of >98% was obtained across the entire sample.

Genome scan through HumanCoreExome BeadChip (Illumina, USA) was carried out using a total of 0.25 μg DNA from all the available individuals in respective families. After denaturation, the genomic DNA was neutralized and amplified at 37°C for 22 hours. The amplified DNA was fragmented using flourous multiple system reagent. The fragmented DNA was precipitated and suspended in RA1 (resin iron) for one hour at 48°C. DNA sample was hybridized on to the BeadChip for 20 hours at 48°C. After washing the Beadchip, single base extension and staining was carried out. Finally, the BeadChip was coated, desiccated and scanned on Scanner (Illumina, Huntsville, USA). The SNP genotypes were generated through the Illumina Genome Studio software from bead intensity data.

HomozygosityMapper (Seelow and Schuelke, 2012) and AutoSNP (http://www.insilicase.com/Guide/AutoSNPa.aspx) were used to find out autozygous regions. Linkage analysis was carried out assuming full penetrance, mutation carrier frequency of 0.001 and autosomal recessive mode of inheritance. EasyLINKAGE Plus (Hoffmann and Lindner, 2005) program was used for Two-point and multi-point LOD scores calculation.

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 36 Chapter 2 Materials and Methods

Mutation Analysis

Once linkage was established in a family, a candidate gene located within the linkage autozygous region was sequenced through chain termination reaction method. Candidate genes selected within the autozygous region were based on their relevance to the phenotypes observed in the particular skin disorder, function of the gene, sub- cellular localization and tissue specific expression of the protein product.

Sanger Sequencing

For amplification of the coding regions and splice sites of the targeted genes, primers were designed from intronic regions of the genes (Table 2.3-2.20). To amplify the genomic regions of interest, standard PCR reaction, as described earlier, was carried in 200 μL tube containing 25 μL reaction mixture. 1.5-2% agarose gel was used for the analysis of the PCR amplified products, followed by purification of the amplified products using commercially available kits such as Fermentas GeneJET™ PCR Purification Kit (MBI Fermentas, Life Sciences, UK) and GeneJET PCR Purification Kit (Thermo Fisher Scientific, Vilius, Lithuania), according to the protocol provided by manufacturers. For Sanger sequencing the purified PCR products were subjected to cycle sequencing using BigDye TM Terminator for ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA) or DTCS (Dye terminator cycle sequencing) for CEQ8800 DNA sequencer (Beckman Coulter, Inc, USA). For searching the variants, sequenced data was aligned to reference genomic sequence via BioEdit (version 7.2). The reference sequences of candidate genes were obtained from Ensemble Genome browser. The co-segregation of the possible pathogenic variants was tested in all the available individuals of the respective families.

Whole Exome Sequencing

Whole exome sequencing was carried out either at University of Washington Center for Mendelian Genomics, USA or Beijing Genomics Institute (BGI), Hong Kong. At University of Washington Center for Mendelian Genomics, capturing of the sequence was carried out in solution with the Roche NimbleGen SeqCap EZ Human Exome Library v2.0. While at BGI, Agilent Sure Select target enrichment chemistry was used for exome capture. Briefly, Illumina HiSeq sequencer was used for exome sequencing. With an average read depth of 75X. After quality control, BWA (Burrows-Wheeler Aligner) was used for Fastq files alignment to the hg19 human

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 37 Chapter 2 Materials and Methods reference sequence (Li and Durbin, 2009). Re-alignment of indel, base qualities re- calibration, and variant calling and detection were performed using the GATK (Genome Analysis Toolkit) to produce variant calling files (VCF) (McKenna et al., 2010). Annotation of the VCF files were carried out with AnnoDB and SeattleSeq137, which integrates ENSEMBL, RefGene, ESP6500, dbSNP, 1000 Genomes Project data and ExAc databases. In silico analysis of rare variants was carried out by using online tools such as SIFT, PolyPhen2, Mutationtaster2 and MutationAssessor and conservation was assessed using GERP++12 and phyloP.

Restriction Enzyme Essay

In family A, the sequence variant c.2T>C in the SLURP1 gene abolished a restriction site for BsrD1 enzyme. Taking its advantage, BsrD1 (Thermo Fisher Scientific, Vilius, Lithuania) was used to confirm the non-polymorphic nature of the variant in 250 ethnically matched control individuals. PCR amplification of exon-1 of the SLURP1 gene in DNA of affected, normal and carrier individuals was carried out following standard method. After purification of the amplified products, these were incubated at 55°C for 9 hours with BsrD1 restriction enzyme. The incubated products were separated on 2% agarose gel and the results were analyzed under UV transilluminator (UVP Inc., San Gabriel, USA).

Computational Analysis

To analyze the effect of sequence variants on protein structure and function, 3D structure of normal as well mutant proteins were generated through comparative modeling approach using MOE 2011-12 (http://www.chemcomp.com). Primary sequences for targeted proteins were retrieved through Ensemble genome browser and subjected to BLAST search against protein data bank to find a suitable template. Templates were chosen on the basis of high sequence identity and query coverage values directed from NCBI. The 3D structures of the proteins were energy minimized using VEGA ZZ release 2.0.8 and refined by adding charges and missing atoms using KoBaMIN server (Rodrigues et al., 2012) and UCSF Chimera (Pettersen et al., 2004).

To monitor the comparative interactions of normal and mutated proteins with their target molecules ZDOCK (Chen et al, 2003) server was used. Molecular docking analysis was performed with active and passive receptor binding residues as input parameters. These parameters indicated the potential binding regions of binding Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 38 Chapter 2 Materials and Methods molecules which were analyzed through literature. ZDOCK score is weighted as sum of the following three terms: optimized desolvation energy, grid-based shape complementarity and electrostatics energy. For ZDOCK, the more negative score indicate the more reliable interactions.

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 39 Chapter 2 Materials and Methods

Table 2.1: List of microsatellite markers used for genotyping of the genes/loci involved in hereditary skin disorders Cytogenetic S. No Gene(s) Marker cM Position D1S2676 63.04 D1S2613 65.97 GJB3 (MIM 603324), 1 1p34.3 D1S2656 66.34 GJB4 (MIM 605425) D1S472 68.26 D1S186 70.05 D1S2345 157.06 D1S2346 158.66 2 LOR (MIM 152445) 1q21.3 D1S305 159.20 D1S2777 160.41 D1S2624 161.76 D2S1371 218.77 D2S2210 219.39 3 WNT10A (MIM 606268) 2q35 D2S173 219.91 D2S104 219.91 D2S433 221.68 D6S1668 18.14 D6S1640 20.26 4 DSP (MIM 125647) 6p24.3 D6S1547 20.85 D6S1674 22.28 D6S309 22.28 D6S1706 124.29 D6S287 124.75 5 GJA1 (MIM 121014) 6q22.31 D6S1608 125.27 D6S1712 125.65 D6S979 127.29 D7S2846 58.35 D7S555 59.75 6 MPLKIP (MIM 609188) 7p14.1 D7S2541 60.90 D7S521 61.51 D7S3043 62.11 D8S1714 113.20 D8S545 115.75 7 FZD6 (MIM 603409) 8q22.3 D8S276 115.91 D8S385 117.06 D8S1096 117.19 D8S1741 164.27 SLURP1 (MIM D8S1729 165.80 8 606119),PLEC1 (MIM 8q24.3 D8S1727 168.86 601282) D8S1744 168.86 D8S1751 170.10

Continued....

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 40 Chapter 2 Materials and Methods

Cytogenetic S. No Gene(s) Marker cM Position D8S373 171.32 D8S2334 171.32

D8S1925 171.32 D8S1926 172.62 D11S4082 96.95 D11S1780 97.60 9 CTSC (MIM 602365) 11q14.2 D11S1367 98.13 D11S4175 98.75 D11S1358 98.75 D12S331 56.87 D12S1065 57.90 D12S1301 59.20 D12S85 61.69 D12S2196 63.82 D12S1633 65.52 D12S1712 67.04 Type II Keratin Genes 10 12q13.13 D12S270 67.84 Cluster D12S398 68.15 D12S1622 70.52 D12S1990 72.00 D12S1691 73.71 D12S104 74.31 D12S1056 75.09 D12S83 75.25 D13S1316 0.00 D13S175 0.55 11 GJB2(MIM 121011) 13q12.11 D13S633 3.10 D13S115 6.16 D13S292 8.75 D17S1788 64.18 D17S1818 65.53 Genes D17S1563 67.94 12 Cluster 17q21.2 D17S902 69.32 JUP (MIM 173325) D17S930 69.99 D17S920 70.65 D18S814 89.47 D18S42 89.47 13 SERPINB7 (MIM 603357) 18q21.33 D18S392 89.99 D18S969 91.20 D18S465 91.77 D20S103 2.10 D20S105 2.82 D20S117 2.82 14 RSPO4 (MIM 610573) 20p13 D20S199 4.92 D20S906 6.02 D20S170 7.30 Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 41 Chapter 2 Materials and Methods

Table 2.2: List of primers used for PCR amplification of PLEC1 gene

PLEC1_EX1F GCTGTCACACCCTGTAGCG 60.5 1 729 PLEC1_EX1R CTGACCACTCCAACCACTCC 60.6 PLEC1_EX2F TGTGTCTGCACGGTGGT 57.9 2 259 PLEC1_EX2R CAGAGATGAAAGGTGAGCAC 55.9 PLEC1_EX3F CCAGCACCCACTCTGTAGATC 59.7 3 289 PLEC1_EX3R GGTTGAGCTGGATTCCAAG 58.2 PLEC1_EX4/5F AGGGTGAACCTGGGATGAG 59.9 4, 5 580 PLEC1_EX4/5R ACAGATCCAGTCCAGGGTTG 60.0 PLEC1_EX6F CCTGGGTGCTGAGCTACC 59.4 6 463 PLEC1_EX6R GAGATCAAGTGGTGGTGCTTC 59.7 7, 8, PLEC1_EX7/8/9/10F CATGGTTGCTGGGATGTGT 60.4 895 9, 10 PLEC1_EX7/8/9/10R CACCAGACCTGGGACAGC 60.3 11, PLEC1_EX11/12F GCTGTCCCAGGTCTGGTG 60.3 617 12 PLEC1_EX11/12R CTGGCTTTAGGGCTGCA 58.6 13, PLEC1_EX13/14F AGGAAGCGGCTGAGGGT 60.9 727 14 PLEC1_EX13/14R AACAGATGAGACGGTGAGGTC 59.2 15, PLEC1_EX15/16F GGAGTGACGAGGTGGGT 56.7 515 16 PLEC1_EX15/16R GCTCCCAGCAAACTCGA 58.5 17, PLEC1_EX17/18F CCCTATGGTCAGAGACCAGTG 59.6 488 18 PLEC1_EX17/18R GGCTCCTGATTGGAGCTG 59.5 PLEC1_EX19F TGCCACTCCTTCCTCAGT 56.7 19 486 PLEC1_EX19R CTTCGGCTGACCTCTACAC 55.9 20, PLEC1_EX20/21F GCCTGTCCCAGCTATACCTC 58.8 615 21 PLEC1_EX20/21R CAGGGCTACAGTCAGCGTC 59.6 22, PLEC1_EX22/23F CTCCGTGTGCTTCCTGGT 59.8 626 23 PLEC1_EX22/23R GGGAACACATGTGGGTCAC 59.6 24, PLEC1_EX24/25F CAGCCACCACTACCAGCAG 60.5 663 25 PLEC1_EX24/25R CCCTCCTGCAAGGTCACAG 61.8 PLEC1_EX26F GTGGCTTGGGTTCCATTC 58.9 26 556 PLEC1_EX26R AGGGGTCTGCACTCTCG 57.2 27, PLEC1_EX27/28F GTCGCCCAGTTGCTTGA 59.5 487 28 PLEC1_EX27/28R CCCCTAAACCCTCACATGG 60.2 29, PLEC1_EX29/30F GGCTTTCCCTGAGCCTG 59.4 495 30 PLEC1_EX29/30R CCTCAGGCAGCTCCTGTG 60.7 PLEC1_EX31F1 TGTCTGAGTGAACTGTGCC 55.5 31 851 PLEC1_EX31R1 ACGTGTTCCTCCTGCAG 55.4 PLEC1_31F2 GAGCTGCAGAGCAAACG 56.7 31 636 PLEC1_31R2 CCTCTGCTTGGACTTCTCG 58.7 PLEC1_31F3 CGGCAGGAGCTGGAAG 59.1 31 797 PLEC1_31R3 TTCAGCCGCTCGACTTC 58.7 PLEC1_31F4 AGCGCGTGCAGAAGAGC 61.6 31 739 PLEC1_31R4 CGCACCGAGAAGAGCTC 58.2 Continued......

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 42 Chapter 2 Materials and Methods

PLEC1_31F5 AGAAGAACCTGCTGGACGA 58.5 31 490 PLEC1_31R5 AAGCCCTGCGTCTCCTC 59.0 PLEC1_31F6 CACGCGACTCAAGGCTG 60.3 31 527 PLEC1_31R6 AGCACCCATCACCCACC 60.3 PLEC1_32F1 AGCTCAAGTCTGAGGAGGTAC 55.3 31 806 PLEC1_32R1 CAACAGCCCTGCGATACTG 60.4 PLEC1_32F2 AGGAGCTGCAGCGGTTG 61.3 32 727 PLEC1_32R2 GTGGCCTTCTCAAAGACGTC 59.9 PLEC1_32F3 CCACTCACGGATAAGGCTG 59.3 32 859 PLEC1_32R3 TGCTGGTCTCCTCATCCA 58.8 PLEC1_32F4 TGTGACAGGGTACAGGGAC 56.8 32 685 PLEC1_32R4 CTCAAACTGGGCTCTGCT 56.8 PLEC1_32F5 ATCGTGGAGGAGGTGGA 57.3 32 627 PLEC1_32R5 GTTCATCTCCTCACTGAAGTAGC 57.2 PLEC1_32F6 AGCACCATCTCCCTCTTC 55.0 32 660 PLEC1_32R6 GGTCTCGAGAGAGATGATCC 56.2 PLEC1_32F7 CATCGAGATCATTGAGAAG 51.3 32 768 PLEC1_32R7 GACAGTGGCAGGAGCAG 56.2 PLEC1_32F8 CTACACGCAGCTGCTCAG 56.8 32 679 PLEC1_32R8 GTCGGTCAGGATCTCGTTC 58.1 PLEC1_32F9 CATCGACCCTGAGGAGAG 56.6 32 711 PLEC1_32R9 AGCGTCTCCGTGTCCAG 58.4 PLEC1_32F10 CTCTCCATCACCGAGTTC 53.7 32 633 PLEC1_32R10 GCAGGTGAGGTACTTGG 51.9 PLEC1_32F11 GGAGGTGCAGTACCTGACC 58.1 32 583 PLEC1_32R11 CACCACTTGGGAGGAAGAC 58.0

Table 2.3: List of primers used for PCR amplification of SLURP1 gene

SLURP1_EX1F TTACCACGTTCCTGACTCAC 56.1 1 427 SLURP1_EX1R CACAGACTTGAATGCTGCAT 57.4 SLURP1_EX2F ATGCAGCATTCAAGTCTGTG 57.4 2 369 SLURP1_EX2R AAGGAGGGAGGCACTTGG 60.2 SLURP1_EX3F TGAACAGGTCACAGTCAGAGG 58.9 3 415 SLURP1_EX3R AGTGAGCTGTGCCACAGC 58.6

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 43 Chapter 2 Materials and Methods

Table 2.4: List of primers used for PCR amplification of KRT1 gene

KRT1_1F1 CTGACTCTAGAAGACCAAGCCC 58.2 1 395 KRT1_1R1 CAAAGCCACTACCACGTCC 58.2 KRT1_1F2 AGTCGGAGTCTTGTTAACCTTG 57.5 1 574 KRT1_1R2 TAACACCATTCACAAGCATACATC 58.9 KRT1_2F AATGAGGCACAGTTCTCCAT 57.2 2 413 KRT1_2R TGTCAAACTGATGTGCCTCT 56.8 KRT1_3F AGGCAGTGATAGGCAATGC 58.8 3 368 KRT1_3R CTCCACTTACTATGTAAGGTGCTG 57.3 KRT1_4F ACACTCCCTTGATGGTGCTA 58.2 4 445 KRT1_4R CCTGCAAGACATAATAGGTTAGTGA 58.8 KRT1_5F CTTACAGCACTCTACCAAGCAG 57.0 5 451 KRT1_5R AGAACTCAGCATGGCACAG 57.5 KRT1_6F GAGTCCTTGTACCAGAGCAAG 56.7 6 433 KRT1_6R GCATTCTGGAAACTAGCATAGTC 57.3 KRT1_7F ATTATTGGCCTCACTGGAGT 56.2 7 452 KRT1_7R CTTCCTCCTGCTCCGTG 57.9 KRT1_8F CTCCGTGTTTGTACTCTTGCTG 60.0 8 395 KRT1_8R TACCTCCAGAGCCGTAGCC 60.4 KRT1_9F GTGAGTGTGTGTAAGTACAAGTCGA 58.4 9 625 KRT1_9R CTCCGGTAAGGCTGGGA 59.7

Table 2.5: List of primers used for PCR amplification of KRT2 gene

KRT2_1F1 CACCTAGTTGGCAGGTATATAAGG 58.3 1 521 KRT2_1R1 CCTAAACCTCCAACACCACC 59.3 KRT2_1F2 GACCAAGAGCATCTCCATTAGTG 60.1 1 497 KRT2_1R2 TACTGCGGTACACCACTGG 58.1 KRT2_2F GAGGTCCTGAAGCAGCAC 56.9 2 402 KRT2_2R CAACTGGGAGATGAATGGC 59.0 KRT2_3F TGTGAGGAAGACAGTGAGAC 53.4 3 367 KRT2_3R CCAAACTGGCTGCTTAGACAC 59.9 KRT2_4F ATGGACGTAGGCATGAAGTC 57.6 4 418 KRT2_4R GATGGCAGATTGAAGTCAGC 58.4 KRT2_5F TGCTTAGTCTGAGGGTCTGC 58.2 5 359 KRT2_5R CAATAACCTTACTGTACACTTTCCAG 57.6 KRT2_6F CTGGACCAGTAACCAGCTCTC 59.0 6 351 KRT2_6R GGATCGATACATGCTAGAGTGC 58.8 KRT2_7F GCGGCAAGTCTTAGCACAG 59.8 7 500 KRT2_7R ATGTCAGTTCTCAGTTGTCAGGA 58.9 KRT2_8F GACATGCAAGGTTGTGCA 57.6 8 415 KRT2_8R CGGAACTGGACCCTCTACC 59.5 KRT2_9F GGTGTGGTCCTGTTCCATG 58.8 9 598 KRT2_9R ACTTGCTGCCAGTTAGAGGTAC 57.7

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 44 Chapter 2 Materials and Methods

Table 2.6: List of primers used for PCR amplification of MPLKIP gene

MPLKIP-1F CGTACGGGAGCAGTCACTC 59.4 1 327 MPLKIP-1R TTCCGGAAGTCACTCAACTTC 59.3 MPLKIP-2F AATGTGATTCCCGCTAACC 57.5 2 389 MPLKIP-2R CAATCAAAGTCATCATCTTTGG 57.2

Table 2.7: List of primers used for PCR amplification of KRT14 gene

KRT14_1 F TTACCCGAGCACCTTCTC 1 649 KRT14_1R GCATGAATTGTTCCCAGA KRT14_ 2F TGGGCTGCTATGGTCAAG 2 325 KRT14_2R ATGCACCTATCCTGGTACTGG KRT14_3F AAGCCCTCGCTGTGATCA 3 479 KRT14_3R TTTGGACAGGGTCTAGCCT KRT14_4F AGAGAAGGGAGAGTCGAGATAG 4 351 KRT14_4R AGAATGCCATTCACACCAG KRT14_5F AATGGTTCTTCACCAAGGTG 5 368 KRT14_5R CAGTAGCGACCTTTGGTCTC KRT14_6F CTCAGCATGGTAGGAATAGTGC 6 478 KRT14_6R GGAGGGTCTTACCATCTCTGG KRT14_7F ACGCCCAGTGAGTCTTG 7 267 KRT14_7R GGAAACTGGGTGACAGCAC KRT14_8F GCCAGTTTGAGTTTCCTCAC 8 365 KRT14_8R GAGAGAGGCGAGAATTATGC

Table 2.8: List of primers used for PCR amplification of HPGD gene

HPGD-1F CAAAGATCGCGAAGCTTG 58.2 1 471 HPGD-1R ACTTCTGAGGTGTGCTCAC 53.4 HPGD-2F GTGAGCACACCTCAGAAGTG 56.9 2 446 HPGD-2R GCTATTGGGCTGTCAGAAGG 59.8 3 HPGD-3F CCAAGCTGCCAGATTGATG 60.4 555 HPGD-3R GCCAATCCCTGAGTTAAGC 59.3 HPGD-4F GGCAAACCCAAAGAATCCAGG 64.9 4 570 HPGD-4R GGAGTCTCACCACAACCTTTG 56.6 HPGD-5F GGCTACTGAGTTTCACAAAGC 56.8 5 571 HPGD-5R GGCCTATTGCATCTTGCATTTC 62.9 HPGD-6F CATTGTTACATAGCTGGGAGG 57.3 6 439 HPGD-6R CTCCCAGAGAGTTTGCCAAAC 59.1 HPGD-7F CCTGCCAAAATGATGGAAGG 62.6 7 625 HPGD-7R TACAACCTAGCCTTTGGTCC 56.8

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 45 Chapter 2 Materials and Methods

Table 2.9: List of primers used for PCR amplification of FZD6 gene

FZD6-2F ACTCTGCACTTTGACTCCAC 55.3 2 508 FZD6-2R ACACAACTTGAAGAAATCGG 55.3 FZD6-3F CATAAGTCTGATAGAGGGAGA 51.1 3 417 FZD6-3R TGAAAGCTTCTCACTAACATC 52.3 FZD6-4F1 CTCTTCCCATCAATCATGAA 56.0 4 731 FZD6-4R1 CAGCATGAAACCACACTGCT 59.9 FZD6-4F2 GGTGACACTGTTGTCCTAGG 55.5 4 710 FZD6-4R2 CCTTACCTATGGCTCTTGTA 52.9 FZD6-5F GTGTTGCACTTAGAGCATGC 57.1 5 377 FZD6-5R GATTTGAACAGTTCCTTGG 53.2 FZD6-6F AGTGACTAAGGATTTTTGGC 53.6 6 601 FZD6-6R GCTTTCCAAATGTGTTATGC 55.8 FZD6-7F CTGGTTAGGGGTGAAACTCA 57.6 7 381 FZD6-7R CAGTATAACAGTAGGCAATGC 52.8

Table 2.10: List of primers used for PCR amplification of RSPO4 gene

RSPO4_1F AACGCCCTCACTAGACCTG 57.9 1 393 RSPO4_1R CCATCTTAAGTGAGGTTGAGACTC 58.2

RSPO4_2F TACTATCCCAATGAGACCATCTC 57.2 2 409 RSPO4_2R CTCAGAGTCTGGGCTTAGACAT 57.2

RSPO4_3F CTTCAAAGGAGACACTGAGTC 54.1 3 453 RSPO4_3R GTTTTGCAGTTACTATGTGGAG 54.3

RSPO4_4F TTATAAATAACTCTGCTCCTAGCAGGT 59.4 4 543 RSPO4_4R TCCTAATGTATTTGGGACTGAAC 57.2

RSPO4_5F CTCTGTCCTCTCATTGCAGAGA 59.7 5 290 RSPO4_5R AGAAAGGGAAGGTAGACTGACAG 58.1

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 46 Chapter 2 Materials and Methods

HR_2F1 GATGGTTATGCTCCAGGGAC 59.3 2 435 HR_2R1 TATGCTCAGGCATCAGGG 58.2 HR_2F2 GAGGAAGGTCAACTGGCTG 58.3 2 412 HR_2R2 CGAGCAACTTTGCTAGGC 57.3 HR_3F1 CCTTCCTTCTTGCTTGTCAG 57.6 3 566 HR_3R1 TGTTCACTTCCTCGCTGG 58.4 HR_3F2 GGAACCTTGGGTACCAGC 57.9 3 605 HR_3R2 GGTCCACTCATAAAGCCTACAG 57.8 HR_4/5F GCTCTGAGTGTGGATGGG 57.5 4/5 610 HR_4/5R GTCTAGGAGCTGGCAGTGTG 58.6 HR_16F ACGTGAAGCCTTCCATTG 57.1 6 301 HR_6R ATGACCACAGGCTTGCAG 58.2 HR_7/8F CTGACCTTAACCTGTGATTACC 55.4 7/8 506 HR_7/8R AAAGGTCAGCCATTTGCAG 58.8 HR_9F GTGGGTTCTGTTGAATTGTG 56.3 9 222 HR_9R TCCTGCCTCAAACTCTGG 57.3 HR_10F GAAGAGAGGGAAGAGCAGC 56.8 10 399 HR_10R GCGATAATGCTGTCCAGG 57.7 HR_11F GAATACACATGGCCTTCGC 59.0 11 526 HR_11R AGGGCAGTAGAACAGCTCG 58.2 HR_12/13F CCGAGCTGTTCTACTGCC 57.0 12/13 611 HR_12/13R GAGTACCAGGGACCACGG 58.8 HR_14F GTTTGCCCTCTGGGAGTTAC 58.6 14 288 HR_14R CGAGATGACAGGCAGACAG 57.9 HR_15/16F TCCATGTTGAGGCTGGTC 58.0 15/16 538 HR_15/16R GACTGGACGAGCTTCTAGGG 59.0 HR_17F CATTAGAGGAAGCGAACCTC 56.7 17 309 HR_17R AGGGTGGGATCTGCTATGTC 58.9 U2HR_1F GGGCACCCGCTCCCTAGC 67.0 18 230 U2HR_1R TCTCCCGCTCTGTCTGCTCA 64.8

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 47 Chapter 2 Materials and Methods

Table 2.12: Primers used for PCR amplification of SMARCB1 gene

SMARCB1_Ex1F TCCTCTACACCACGACTTGG 58.7 1 612 SMARCB1_Ex1R GAAATCCCAGGTCGATGAG 58.0 SMARCB1_Ex2F CTGTTGCTTGATGCAGTCTG 58.1 2 419 SMARCB1_Ex2R GACAGTGGACCACCAATGG 59.8 SMARCB1_Ex3F ATGCGAGGACCTTGATGTG 59.6 3 383 SMARCB1_Ex3R AGATCGTGCCACTGCACTC 60.0 SMARCB1_Ex4F GAGCCTGACAGAGGTACAGTG 57.5 4 386 SMARCB1_Ex4R GTTCAAAGGCGAGGAGGTAC 58.8 SMARCB1_Ex5F CTCAGCTGTTAGCTGACAAGC 58.1 5 448 SMARCB1_Ex5R ATCCACAGTGAACAGCAACAG 58.8 SMARCB1_Ex6F GGTTGTCCTCTCCTGCATC 58.1 6 474 SMARCB1_Ex6R TTACTATGAGAACTCAGCCTATGC 57.0 SMARCB1_Ex7F CTGGAAGGACAAGGACCAC 58.0 7 537 SMARCB1_Ex7R GGTGCTTCTAACCCTACCTG 56.4 SMARCB1_Ex8F CCCTTGAGGTTCAGGTGAC 58.0 8 388 SMARCB1_Ex8R GGTACTCTGTCTCAGAGACCATG 57.9 SMARCB1_Ex9F GTTCCCACCCCTACACTTG 57.8 9 316 SMARCB1_Ex9R GCTCAACAAATGGAATGTGTG 59.0

Table 2.13: Primers used for PCR amplification of exon # 11 of ERCC5 gene in family E

ERCC5-11F AATGCATTACATGAAGTGGTAGG 58.1 11 415 ERCC5-11R GCAATTTCCATCAATGCATC 58.9

Table 2.14: Primers used for amplification of exon # 1 of ALDH3A2 gene in family I

ALDH3_1F TCCGACTGGCAGTGGGACT 64.3 1 494 ALDH3_1R CTGTATCGCTGGAGATCATACTCC 61.3

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 48 Chapter 2 Materials and Methods

Table 2.15: Primers used for PCR amplification of exon # 5 of KRT83 gene in family J

KRT83_5F GAGAAGCCCAGATAAGTGAG 54.1 5 220 KRT83_5R TTAGGGATCAGAATCCCTAGAC 56.4 Table 2.16: Primers used for PCR amplification of exon # 6 of ABHD5 gene in family H Tm Amplicon Exon Primer Primer sequence (5′→3′) (°C) Size (bp) ABHD5_6F GTGACAGTGCAATGCATACTC 56.8 6 367 ABHD5_6R TGCAGGTATGGCTCAGATGA 60.4

Table 2.17: Primers used for amplification of exon # 1 of DCAF17 gene in family Q

DCAF17_1F CCTCGAAATTCGAAGGCAG 60.9 1 406 DCAF17_1R CGTAAAACTAAAAGCAGCTGGTG 60.3

Table 2.18: Primers used for amplification of exon # 1 of SLCO2A1 gene in family B

SLCO2A1_1F TCTCCATCCCCTCCACTCAC 62.4 1 659 SLCO2A1_1R TGGCTCCGGCAGACAGAAG 65.0

Table 2.19: Primers used for amplification of targeted exons in BTAF1, CCD74, CDC40, COL9A2, IBTK, LOXL4, PHF14, RGS10, RPA2, SAA1 and SCE31B genes in family O

BTAF1_38F CCCATCACTTGAAGTACTCA 53.5 38 318 BTAF1_38R GAAATGTAACTGAACTAGCAGCA 56.5 CCDC74A_6F AGCTGTGGAATACCAACCTC 56.6 6 313 CCDC74A_6R AGAGGCTCTTGGTGGAGAC 56.9 CDC40_14F ACCATAAGCAGTACAGTGTCATC 55.9 14 383 CDC40_14F TTATACAAATAATGGCACAGTGAC 56.0 COL9A2_2F GCCTGCTATACAGAAGATTCTC 55.1 2 488 COL9A2_2R ATGAGGCCTCGCTGTGAGCTG 67.4 IBTK_9F GTGTGTGTGTGTGTGTGTGTAGG 59.9 9 438 IBTK_9R TCATGTTACCAGGTCTGCTACAGC 62.8 LOXL4_4F GGCTTAGACAAGCTGAGGTTC 58.2 4 411 LOXL4_4R TGTCGACAGGCACCTCGCT 65.6 PHF14_13F ACATTACCATCTTGGATGTCTGG 60.1 13 584 PHF14_13R TCAAATGTAGTGATTCGATATGCCT 61. 0

Continued….

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 49 Chapter 2 Materials and Methods

RPA2_3F ACCAGCCTGTCTACACCCT 57.1 3 614 RPA2_3R CCCACAATAGTGACCTAGGT 54.1 SAA1_3F CCTTTCTCAGCATCAGTATTCC 58.0 3 597 SAA1_3R ACTCACTCCTACCATCCACTCAG 59.7

Table 2.20: List of primers used for qPCR in family N

7 ALDH1A3_7F ACCCTCAGATCAACAAGATC 54.0 256 9 ALDH1A3_9R CAGGTCTACTCTGAGTTTGTCAG 55.9 5 EDA2R_5F CTCTGATCCAGTACCAGCTAG 54.3 206 6 EDA2R_6R CACTAGTCGAGCTGCAGTC 54.6 5 FGF5-1F ATGAGCTTGTCCTTCCTCCTC 59.8 343 5 FGF5-1R CTTCGTGGGATCCATTGACT 59.9 3 KRT81_3F AGCAACAGCTGAGAACGAGT 57.8 258 5 KRT81_5R GTGACAATGTCGTCATACTGTG 56.5 1 AR_1F CATCCTGGCACACTCTCTTC 58.4 218 1 AR_1R AGGGTACCACACATCAGGTG 58.3

F Forward Primer, R Reverse Primer, bp base pairs, Tm Melting Temperature, °C Degree Centigrade, qPCR Quantitative Polymerase Chain Reaction

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 50 Chapter 3 Ectodermal Dysplasia

ECTODERMAL DYSPLASIA

Ectoderm is one of the basic embryonic structures which differentiate into the neuroectoderm that prefigured the nervous system, and the ectoderm, which forms the entire embryonic surface and the epidermis, epidermal appendages and tooth enamel. In addition to classical ectodermal structures (hair, nail, teeth, sweat glands), ectoderm also gives rise to nervous system, eyes, ears, and nose, as well as the mammary, eccrine and pituitary glands (Sadler, 2008). During embryonic development, a complex interplay of signals take place between ectoderm and mesoderm for the formation of various structures, therefore it is possible that ectodermal disorders may extend to mesodermal structures such as musculoskeletal and genitourinary systems (Priolo, 2009).

Ectodermal dysplasias (EDs) are clinically diverse and genetically heterogeneous group of rare inherited disorders with estimated prevalence of 7/10,000 births (McKusick et al., 1998). EDs are characterized by defects in ectoderm derived tissue, however, these diseases are sometime associated with imperfections in the mesoderm derived structures (García-Martín et al., 2013). All possible pattern of Mendelian inheritance are described for EDs transmission. The classification of ectodermal dysplasia is complex; several classification systems have come and gone in order to accommodate clinical and genetic findings in the disease. On the bases of clinical phenotypes, Pinheiro and Freire-Maia (1994) classified EDs into two groups: pure EDs, defects in classic ectodermal structures only; associated EDs, defects in extra- ectodermal tissues in association to ectodermal structures. Lamartine (2003) arranged EDs into four groups on the bases of functional perspectives of the involved genes; cell-cell communication and signaling, cell adhesion, transcription regulation and development. Priolo (2009) arranged EDs into two groups, based on molecular and genetic facts and parallel clinical information. Group 1 represents the disorders which result from defective epithelial-mesenchyme interactions, while Group 2 includes the disorders with abnormal function of a structural protein in the cell membrane.

Recent advances in molecular genetics technologies gradually elucidated the molecular defects in ectodermal dysplasia. So far, more than two hundred different clinical and pathological conditions have been defined as ectodermal dysplasias

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 51 Chapter 3 Ectodermal Dysplasia

(EDs). Nevertheless, only about eighty of these have been described at the molecular level with their causal genes (Itin, 2014).

This chapter describes five consanguineous families (A-E) with clinical phenotypes representing different types of ectodermal dysplasias. Linkage analysis and DNA sequencing identified disease causing variants in five different genes.

Family A

Family History and Clinical Features

A four generation consanguineous family A (Figure 3.1a) was recruited from Lower Dir district of Khyber Pakhtunkhwa (KPK) province of Pakistan. For this study, peripheral blood samples were collected from five affected (IV-1, IV-2, IV-3, IV-4, IV-6) and four unaffected individuals (III-1, III-2, III-3, IV-5) were collected for this study. Parents of the affected were phenotypically normal and undergone consanguineous marriage.

Clinical features of affected individuals were consistent with those reported earlier for mal de Meleda (MDM) phenotypes. Ages of the patients were thirty to sixty years at the time of the study; however, the disease phenotypes appeared soon after birth. The disease appeared in the form of redness on palms and soles, developed into demarcated transgrediens palmoplantar keratoderma with small lesions, hyperhidrosis and malodour (Figure 3.1b-e). Nails presented thick and curved shape with parrot beak like appearance, which was not reported in the disease previously. Features such as hyperkeratosis over elbows, knees, perioral regions, and lower legs; joint contractures and stiffness, and brachydactyly reported previously in patients with MDM were not detected in affected individuals of the family. Affected individuals seemed intellectually normal, and they had normal hairs and teeth.

Genetic Mapping and Mutation Analysis

In family A, genotyping was carried out in nine individuals including four unaffected (III-1, III-2, III-3, III-5) and five affected (IV-1, IV-2, IV-3, IV-4, IV-6), using microsatellite markers. Based on clinical phenotypes, observed in the family, six genes were tested for linkage including WNT10A (MIM 606268), DSP (MIM 125647), CTSC (MIM 602365), JUP (MIM 173325), SERPINB7 (MIM 603357) and SLURP1 (MIM 606119) (Table 2.1).

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 52 Chapter 3 Ectodermal Dysplasia

Analysis of microsatellite markers (D8S1741, D8S1729, D8S1727, D8S1744, D8S1751, D8S373, D8S2334, D8S1925, D8S1926) revealed linkage in the family to gene, SLURP1, located on chromosome 8q24.3. Haplotype analysis marked the linkage intervals between microsatellite markers D8S1741 (centromeric) and D8S1926 (telomeric), encompassing 5.46 MB homozygous region on 8q24.3 (Figure 3.2a). Following linkage in the family, the gene, SLURP1, was sequenced in all individuals. Sequence analysis identified a homozygous sequence variant (c.2T>C; Chr8:143823802T>C) in exon 1 of the SLURP1 gene in all five affected family members. Three healthy individuals (III-1, III-2, III-3) were heterozygous carrier for the variant; however, the member IV-5 was homozygous normal (Figure 3.2b-d). This variant alters translation initiation codon of SLURP1 (c.2T>C; p.Met1Thr). There are two possible effects of this variant; either it abolishes translation of the SLURP1 polypeptide in affected individuals or it result in use of next initiation codon ATG at codon position 17 leading to production of a truncated protein missing first 16 amino acids.

Restriction Fragment Length Polymorphism (RFLP) Analysis

The sequence variant (c.2T>C), identified in the family, abolished a restriction site for enzyme BsrD1. The BsrD1 restriction analysis of PCR-amplified exon-1products revealed a single DNA band of 427 bp in affected, three DNA bands (427bp, 301bp, 126bp) in carriers of the family and two bands (301bp, 126bp) in control individuals (Figure 3.2e). The non-polymorphic nature of the sequence variant was verified by restriction enzymes analysis using genomic DNA of 250 ethnically matched control individuals.

Computational Analysis

Using homology modeling, three-dimensional models of normal and mutated SLURP1 protein (p.Meth1Thr) were predicted (Figure 3.3a, b) and assessed by online structure-analysis tools. Ramachandran plot revealed that approximately 91% residues in the model lie in permissible regions of torsion angles. Analysis of the wild type and mutant SLURP1 with ProSA plot showed Z scores of 0.74 and 3.35, respectively, indicating no major deviation from the scores calculated for proteins of similar size (Figure 3.3c, d). Using molecular docking analysis, binding of normal SLURP1 was detected to neuronal nicotinic acetylcholine receptor (nAChR) through several

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 53 Chapter 3 Ectodermal Dysplasia residues, including Trp5, Val7, Leu10, Leu11, Val12, Trp15, Met51, Phe63, Ile85, Leu90, Ile91, Phe92 and Phe95. The resultant truncated protein is likely to be deficient in seven residues (Trp5, Val7, Leu10, Leu11, Val12, Trp15) involved in binding to a7-nAChR, and will thus lose its target (Figure 3.3f, g).

Family B

Family History and Clinical Features

Pedigree of family B represents segregation of isolated congenital nail clubbing (ICNC) in an apparent pseudodominant inheritance pattern (Figure 3.4a). The family had five individuals affected with ICNC in two successive generations. For the present study blood samples were collected from six individuals including three affected (III-2, IV-1, IV-3) and three normal (II-3, III-1 and III-4).

Affected members of the family exhibit isolated congenital nail clubbing (ICNC) with onset during early childhood (Figure 3.4b-e). Severity of clubbing varied not only among affected members but also in different digits in the same individual. One of the affected members IV-3 showed dermatophytic onychomycosis in three clubbed finger nails. Abnormalities involving heart, bones, joints, perspiration, skin etc. were not found in the affected members.

Genetic Mapping and Mutation Analysis

Initially, based on nail phenotypes similarity, showed by affected individuals of the family, with those reported previously in patients with ICNC, the gene HPGD (MIM 601688) was directly sequenced in one of the affected individual. However Sanger sequencing results didn‟t detect any potential pathogenic variant. Thereafter, whole- exome sequencing of three affected (III-2, IV-1, IV-3) and two unaffected obligate heterozygotes (II-3, III-1) of the family members was performed. Autozygosity analysis of the exome sequence data using AgileVCFMapper (Watson et al., 2015) defined a 41.19 Mb region of extended SNP autozygosity on chromosome 3q12.2-q23 (100.62 Mb to 141.81 Mb) (Figure 3.5a). Within this region all SNPs were homozygous in all the affected individuals of the family, while, two obligate carriers were heterozygous. Across the entire exome, only three exonic variants were found homozygous in the three affected individuals, and heterozygous in their parents. The minor allele frequency (MAF) < 0.01 of the variant among reported South Asian Exome Aggregation Consortium (ExAC) alleles. Among these, only the variant

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 54 Chapter 3 Ectodermal Dysplasia chr3:133748646T>C was located within the region of extended autozygosity on chromosome 3q12.2-q23. This variant altered the translation initiation codon of SLCO2A1 (c.1A>G; p.Met1Val) and was thus predicted to abolish translation of the SLCO2A1 polypeptide. Online prediction bioinformatics tools including SIFT, PolyPhen2, GERP++ and phyloP predicted the variant to be probably damaging. The variant was neither reported in ExAc nor 1000 genome database. Co-segregation of this variant with the disease phenotypes was verified by Sanger sequence analysis of the DNA from all six available individuals of the family.

Family C

Family History and Clinical Features

Family C, comprising of four generation consanguineous pedigree (Figure 3.6a), was recruited from district Mardan in KPK province, Pakistan. Blood samples from three affected (IV-1, IV-2, IV-3) and four unaffected individuals (III-2, III-3, IV-4, IV-5) were collected for DNA analysis. Analysis of the Pedigree confirmed autosomal recessive mode of inheritance of the disease. Genetically heterozygous parents were phenotypically normal.

Affected individuals of family C showed complete loss of finger- and toe-nails, which is consistent with anonychia. No defect was observed in structure and appearance of the nail bed, however, the matrix of nail was swollen (Figure 3.6b, c). Affected individuals showed normal teeth and sweat glands. Skin related abnormalities such as ichthyosis, palmar keratosis, hyperhidrosis and any type of pigmentation were not apparent in members of the family. Clinical laboratory investigations such as granulocyte function and WBCs count were normal. Limb and girdle tissues were also normal. Nails in Heterozygotes were completely normal and indistinguishable from healthy individuals.

Genetic Mapping and Mutation Analysis

Based on phenotypic consistency with previously reported cases of anonychia, in family C linkage was tested to RSPO4 gene on chromosome 20p13 using microsatellite markers (D20S103, D20S105, D20S117, D20S199, D20S906, D20S170). Four of the microsatellite markers (D20S105, D20S117, D20S199, D20S906) were found homozygous in affected and heterozygous in unaffected individuals, while, two markers (D20S103, D20S170) were heterozygous in all the

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 55 Chapter 3 Ectodermal Dysplasia individuals. Thus, linkage was established on chromosome 20p13, harboring RSPO4 gene (Figure 3.7a). Subsequently, Sanger sequencing of RSPO4 in affected individuals identified a reported 26bp deletion (-9- +17del26) containing initiation codon (ATG) in exon 1. The variant was homozygous in all the three affected individuals, obligate carrier parents were heterozygous and unaffected individuals were homozygous for wild type allele (Figure 3.7b-d).

Family D

Family History and Clinical Features

Family D (Figure 3.8a), with four generation pedigree, was collected from district Rawalpindi, Punjab, Pakistan. Pedigree analysis revealed three affected individuals in the fourth generation. Parents of affected individuals were phenotypically normal. Blood samples of seven individuals including two affected (IV-4, IV-6) and five normal members (III-3, III-4, III-5, IV-5, IV-7) were collected for this study.

Detailed clinical investigation was carried out at Combined Military Hospital (CMH) Rawalpindi, Pakistan. The clinical features of onychauxis, hyponychia, and onycholysis in finger and toes were found (Figure 3.8b, c), which were consistent with non-syndromic congenital nail disorder 10. At the time of investigation, ages of affected individuals were 15 to 35 years. They had normal perspiration, teeth and hair. Absence of palmoplantar hyperkeratosis in affected members ruled out diagnosis of pachyonychia congenita.

Genotyping and Sequence Analysis

Homozygosity mapping was carried out on the DNA samples of two affected (IV-4, IV-6) and five normal members (III-3, III-4, III-5, IV-5, IV-7) with microsatellite markers (Table 2.1) linked to FZD6 gene at chromosome 8q22.3. Analysis of microsatellite markers revealed an autozygous region of 5.22 Mb (Figure 3.9a) at this chromosomal region. Establishing linkage in the family was followed by Sanger sequencing FZD6 gene in all members. Analysis of the sequence data revealed a previously reported missense variant c.1265G>A (p.Gly422Asp), which was homozygous in the two affected (IV-4, IV-6), heterozygous in the parents (III-3, III-4) and two normal individual (III-5, IV-7), while, one normal individual (IV-5) was homozygous for wild type allele (Figure 3.9b-d).

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 56 Chapter 3 Ectodermal Dysplasia

Family E

Family History and Clinical Features

Family E, segregating another called xeroderma pigmentosum, was recruited from district Lower Dir, KPK, Pakistan. The family had a consanguineous pedigree of four generations with two affected individuals in the last generation (Figure 3.10a). Pedigree analysis presented autosomal recessive pattern of the disease transmission. Phenotypically normal parents were first cousins.

At the time of study, ages of affected individuals varied from seven to twelve years. They had restricted to facial region (Figure 3.10b, c), which was consistent with a condition called xeroderma pigmentosum. Variation in the disease severity was observed among the affected members. Affected female individual (IV- 1) was comparatively more severely affected than her brother. Their parents also reported seasonal variation of development of the pigmentation in affected individuals. Hyperpigmentation on rest of the body was not observed. Affected individuals presented normal hair, nails and teeth. There was no history of cancer and visual defects in the family.

Whole Genome Scan

In order to perform genotyping quickly and efficiently, DNA from all available individuals of family E was subjected to genome wide scan using Infinium® HumanCoreExome Array (Illumina, USA), which interrogates >500,000 SNP markers. Due to the small size of this family, whole genome scan identified three large homozygous regions, including: a 35.12 Mb region (chr1:32.13-67.25 Mb) on chromosome 1p35.2-p31.3; a 16.82 Mb region (chr8:0.16-16.99 Mb) on 8p23.3-p22; and a 14.71 Mb region (chr13:100.38-115.09 Mb) on 13q32.3-q34. A maximum LOD score of 1.93 was obtained at each of these three homozygous regions.

Whole Exome Sequencing (WES)

Following linkage analysis in family E, DNA of affected individual (IV-1) was used in whole exome sequencing at the University of Washington, Centre for Mendelian Genomics. Analysis of the exome data identified a missense mutation, chr13:103519115C>T (c.2453C>T; p.Ala818Val) in the ERCC5 gene within the autozygous region on chromosome 13q32.3-q34. Sanger sequencing verified the co-

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 57 Chapter 3 Ectodermal Dysplasia segregation of the variant (ERCC5 c.2453C>T) with disease phenotype in the family (Figure 3.11). Analysis with all bioinformatics tools confirmed pathogenicity of the variant. This variant was not found in 250 in-house ethnically matched chromosomes. Moreover, this variant was reported in the ExAC database, with a very low MAF (minor allele frequency) of 0.000061 in south Asians.

Discussion

This chapter of the thesis describes clinical and molecular investigation of five families (A-E) with different ectodermal dysplasia conditions. Expert dermatologists carried out clinical investigation in each family. Phenotypes of family A were consistent with MDM type of hyperkeratosis. Affected individuals in three families (B, C, D) exhibit defects in nail structure. While affected individuals in family E displayed pigmentation defect.

In family A, affected individuals were diagnosed with transgrediens palmoplantar hyperkeratosis compatible with MDM. Establishment of the linkage was followed by cycle sequencing. Sequencing results showed a homozygous missense variant (p.Met1Thr) in SLURP1 gene. The missense variant (p.Met1Thr) is the second variant targeting initiation codon in the SLURP1 gene. So far, seventeen pathogenic sequence variants including fourteen missense, one deletion and four splice sites have been identified in the SLURP1 gene from nineteen different countries (Table 3.1).

SLURP1 is a member of Ly6 superfamily of protein which is highly conserved from to humans to snakes (Eckl et al., 2003). In human, several Ly6 protein family members are reported. In humans SLURP1 is the first known secreted Ly6-related protein. Its expression in humans has been reported in skin, stomach, exocervix, gums, and esophagus with highest level in keratinocytes especially in palms and soles (Mastrangeli et al., 2003; Favre et al., 2007; Adeyo et al., 2014). In MDM patients, SLURP1 is nearly untraceable in the skin and sweat (Favre et al., 2007). Being a secreted protein, SLURP1 expression has been reported in aerodigestive epithelia, blood, lymphocytes, stomach, uterus, cornea, sweat, saliva and urine (Grando, 2008), and rat spinal cord neurons (Moriwaki et al., 2009). In presence of Ca++, SLURP1 mRNA expression is upregulated by epidermal growth factor while down regulated by retinoic acid and interferon‐γ (Mastrangeli et al., 2003).

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SLURP1 is arranged into three exons and encoding 103 amino acids protein, secreted LY6/PLAUR-related protein1. It has a signal polypeptide chain and three domains in its 3D structure and five disulfide bridges that are indispensible for the proper folding and function of the protein (Ploug et al., 1994, Shen and Chou, 2007). One of the possible effects of the variant (p.Met1Thr) is use of next initiation codon ATG at position 17 producing a truncated molecule missing first sixteen amino acids. The truncated protein can cause inaccurate peptide targeting in family A (Beuret et al., 1999). PolyPhen2 and SIFT also predicted that replacement of methionine by threonine residue damage function of the protein.

In family B, analysis of the WES data revealed a novel sequence variant (p.Met1Val) in SLCO2A1 gene. Based on extreme rarity, this variant was considered the probable cause of ICNC phenotypes in the family. According to HGMD professional 2017.1, so far, 47 sequence variants are described in the gene, SLCO2A1. However, only one heterozygous nonsense variant has been described in pathogenesis of isolated nail clubbing (Seifert et al., 2012).

SLCO2A1 codes for solute carrier organic anion transporter family member 2A1, the principal prostaglandin transporter. SLCO2A1 is widely expressed in various tissues, functioning as an organic anion transporter that mediates selective uptake of prostaglandin (PG) across the plasma membrane. Both isolated and syndromic forms of nail clubbing are thought to result from elevated levels of prostaglandin E2 (PGE2) due to its reduced degradation, which is a two-step process requiring carrier-mediated uptake across the plasmamembrane and cytoplasmic oxidation in the lung (Endo et al., 2002; Nomura et al., 2004). SLCO2A1 is the principal carrier responsible for PGE2 uptake in the lung (Kanai et al., 1995), while the cytoplasmic oxidation of PGE2 is performed by 15-PGDH (Ferreira and Vane, 1967; Tai et al., 2002). Thus, there is considerable overlap of phenotypes associated with SLCO2A1 and HPGD mutations both in humans and in knockout mice (Chang et al., 2010).

SLCO2A1 is a multi-pass cell membrane protein with 12 transmembrane domains and only one known transcript (CCDS3084.1). The mutation is expected to be a protein null, abolishing the translational initiation codon, and the SLCO2A1 gene lacks other potential inframe translational starts; indeed the second methionine is not until codon 169. It thus seems difficult to reconcile homozygous total loss of SLCO2A1 function resulting in recessive ICNC versus heterozygous loss of function resulting in

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 59 Chapter 3 Ectodermal Dysplasia dominant ICNC. Furthermore, why does homozygous total loss of SLCO2A1 function result only in ICNC in family A, whereas homozygous truncating mutations result in more severe primary hypertrophic osteoarthropathy or pachydermoperiostosis phenotypes in other families? It is possible that truncated SLCO2A1 polypeptides do not entirely lack function and exert toxic or neomorphic effects. Alternatively, it is possible that there are common second-site modifiers that modulate the phenotypic effects of loss of SLCO2A1 function.

In family C, the only clinical feature observed was anonychia. Based on phenotypic consistency with previous reports, linkage was tested to the gene, RSPO4, located on chromosome 20p13. After establishing linkage in the family, RSPO4 gene was subjected to Sanger sequencing in affected members. Sequence analysis identified 26bp deletion (-9-+17del26) including initiation codon (ATG) in exon 1 of the RSPO4 gene. Under the present scenario, a possibility of use of next ATG codon for translation initiation cannot be ruled out. This would however lead to production of a truncated protein lacking the first sixteen residues encoding putative signal peptide. Finally, the 26bp deletion in family resulted in a frame-shift and premature termination codon. In case, if translation of the mutant transcript occurred, it is expected to produce a 220 residues truncated protein that would lack any features of RSPO4 protein.

RSPO4 belongs to R-Spondin family of secretary proteins; contain a transmembrane domain, a basic C-terminal tail domain and two Furin-like cysteine rich domains at the N-terminal (Kazanskaya et al., 2004). R-Spondins have been shown to activate WNT/β-catenin signaling by binding to leucine rich-repeat containing G protein- coupled receptor (LRG) superfamily. WNT/β-catenin pathway is evolutionarily conserved and plays a central role in early development by the regulating the morphogenesis, proliferation and motility of cells (Cadigan and Nusse, 1997). Initiation of WNT signaling is also critical for β-catenin stabilization and the following T-cell/β-catenin factor dependent expression of WNT target genes. In the absence of WNT, β-catenin is subsequently degraded through ubiquitin-dependent pathway, while, stabilization of β-catenin is required for development of nail (Takeo et al., 2008).

Affected members in family D showed onychauxis, hyponychia, and onycholysis of finger- and toenails. This condition is also termed as “claw shaped nails”. Since

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 60 Chapter 3 Ectodermal Dysplasia hyperkeratosis of palms and soles was not observed in affected individuals, therefore this ruled out diagnosis of another similar condition called pachyonychia congenita. Candidate gene homozygosity mapping revealed linkage in the family to the gene, FZD6, located on chromosome 8q22.3. Subsequently, Sanger sequencing of the exons and splice junction sites of FZD6 gene was carried out. Analysis of the sequence data revealed a missense variant (p.Gly422Asp), which was earlier reported in a Pakistani kindred with similar phenotypes (Raza et al., 2013). Sanger sequencing of all available individuals confirmed cosegregation of the mutation with the disease phenotype family D.

FZD6 belongs to frizzled family of proteins, whose molecules act as receptors for Wnt pathway ligands. Frizzleds are cross membrane proteins consist of seven transmembrane domains and an N-terminal extracellular cysteine-rich domain. Interaction of frizzled proteins with Wnt ligands result in activation of disheveled proteins, Wnt target genes, nuclear accumulation of b-catenin and inhibition of GSK- 3 kinase (Clevers, 2006). Wnt–FZD signaling is essential for a number of developmental processes including tissue morphogenesis, differentiation and regeneration (Yamamoto et al., 2008). Aberration in the structure and function of FZD6 leads to defect in the ectodermal tissues such as nails dystrophy (Fröjmark et al., 2011) and cleft lip and palate phenotypes (Cvjetkovic et al., 2015).

Family E with pigmentation disorder showed feature of xeroderma pigmentosum. Homozygosity mapping and linkage analysis in the family identified three autozygous regions on chromosome 1p35.2-p31.3, 8p23.3-p22 and 13q32.3-q34, with equal LOD score of 1.93. Exome sequencing followed by confirmation with chain termination sequencing revealed a homozygous missense variant (p.Ala818Val) in the ERCC5 gene, located within the autozygous region on chromosome 13q32.3-q34.

ERCC5 encodes for 133.1 kDa protein, xeroderma pigmentosum G (XPG) which contains an N region, a spacer, an I-region and two C-terminal nuclear localization signals (Constaninou et al., 1999). XPG is one of seven proteins described in nucleotide excision repair pathway, the flexible DNA repair mechanism that eliminates defect in the DNA which is produced by UV radiations and other chemical mediators. The catalytic role of XPG in nucleotide excision repair pathway is well understood: Using its structure-specific endonuclease activity, XPG make cuts in the damaged DNA strand 3/ to the defect near the junction between the unpaired damaged

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 61 Chapter 3 Ectodermal Dysplasia strand and downstream undamaged duplex DNA (Lalle et al., 2002). Independent from its catalytic activity, the XPG is also required by XPF-ERCC1 heteroduplex for 5/ incision (Staresincic et al., 2009). XPG also perform an essential role in transcription by stabilizing the structure of transcription factor II-H, which has role in transcription as well DNA repair (Ito et al., 2007; Schafer et al., 2013). The pathogenic missense variant (p.Ala818Val), revealed in the family, was predicted to disturb the DNA repair (Schafer et al., 2013), and produce XP phenotypes in affected individuals. So far, twenty-five sequence variants in the gene, ERCC5, have been reported in pathogenesis of XP and XP/Cockayne syndrome complex phenotypes (Yang et al., 2017). The truncated mutations mostly result in disruption of NER as well aberration of gene transcription and result in XP/Cockayne syndrome complex phenotypes (Ito et al., 2007), nevertheless, individuals with missense variants develop only xeroderma pigmentosum phenotypes (Emmert et al., 2002).

In this chapter of the dissertation, five consanguineous families with defects in embryonic ectoderm derived structures were evaluated at clinical and molecular level. Genetic analysis identified five pathogenic sequence variants including three novels and two previously known. Use of bioinformatics tools such as SIFT, PolyPhen2, Mutationtaster2, MutationAssessor, GERP++ and phyloP predicted that the variants are probably damaging. In addition, in family A, molecular docking analysis also predicted disruption of binding of the mutant variant to its target. The study presented in this chapter will extend the preexisting human genomic database and may help to develop a community-generated knowledge base that will significantly improve diagnosis, management and understanding of ectodermal dysplasia and related conditions.

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Table 3.1: List of SLURP1 sequence variants reported for mal de Meleda type of PPK

S. Mutation Type Protein Changed References No 1 c.1A>C p.Met1Leu Eckl et al. 2003 Missense 2 c.2T>C p.Met1Thr (Shah et al., 2016) this study 3 c.43T>C Missense p.Trp15Arg Eckl et al. 2003 c.58 + 1 4 Splice-site Altered splice-site Wajid et al. 2009 G>A 5 c.58 + 1G>C Sakabe et al., 2014 Altered splice-site 6 c.58 + 5G>C Splice-site Nellen et al. 2015

7 c.82delT Deletion p.Cys28fs32Term Fischer et al. 2001 Muslumanoglu et al. 8 c.129C>A Missense p.Cys43Term 2006 9 c.212G>A Missense p.Arg71His Favre et al. 2007 10 c.212G>C Missense p.Arg71Pro Nellen et al. 2009 c.178 + 1 11 Splice-site Altered splice-site Fischer et al. 2001 G>A 12 c.229T>C Missense p.Cys77Arg Charfeddine et al. 2003 13 c.244C>T Missense p.Pro82Ser Gruber et al. 2011 14 c.256G>A Missense p.Gly86Arg Eckl et al. 2003 15 c.256G>C Missense p.Gly86Arg Eckl et al. 2003 16 c.280T>A Missense p.Cys94Ser Zhao et al., 2014 17 c.286C>T Missense p.Arg96Term Fischer et al. 2001 18 c.293T>C Missense p.Leu98Pro Yerebakan et al. 2003 19 c.296G>A Missense p.Cys99Tyr Marrakchi et al. 2003

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Figure 3.1: Pedigree drawing and clinical presentation of affected individuals in family A. (a) Pedigree of family A with five affected individuals (IV-1, IV-2, IV-3, IV-4, IV-6) in the fourth generation. Individuals who were available for the study are shown with asterisks. (b-e) Clinical presentation of mal de Maleda phenotypes in affected members. (b, c) Affected individual IV-2 had demarcated transgrediens hyperkeratosis on the hands with erythematous lesion and dystrophic nails (b) and transgrediens hyperkeratosis on the hand with restricted boundaries (c); affected individual IV-1 had hyperkeratosis of the feet with dystrophic nails and fissures over the toes (d); and affected member IV-4 had mild hyperkeratosis over dorsa of the feet (e).

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 64 Chapter 3 Ectodermal Dysplasia

Figure 3.2: Microsatellite markers generated haplotypes, Sanger sequencing chromatograms of SLURP1 and presentation of agarose gel showing restriction enzyme analysis in family A. (a) Haplotypes construction for genotyped microsatellite markers on chromosome 8q24.3 in individuals of family A; Genetic distance (cM; centi-morgans) is according to the Rutgers combined-linkage physical map (build 36.2.38). (b-d) Nucleotide sequence of a missense variant (c.2T>C, p.Met1?) in the SLURP1 gene in (b) unaffected control, (c) heterozygous carrier and (d) affected individual; arrows indicate positions of the variant. (e) BsrD1 restriction enzyme analysis and PCR amplification of the resulting products of 427 bp showed three DNA bands (126, 301, 427 bp) in carriers of this mutation, a single DNA band of 427 bp in individuals with MDM and, two bands (126 and 301 bp) in normal controls. MWM, molecular weight marker.

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 65 Chapter 3 Ectodermal Dysplasia

Figure 3.3: Computational analysis of SLURP1 (p.Met1Thr) variant in family A. (a, b) Modeled structure of (a) normal and (b) mutant SLURP1 protein; orange, purple and grey represent helix, beta-sheet and coil, respectively. (c, d) Protein model quality scores of (c) normal and (d) mutant SLURP1; the Z scores of both are represented in the plots by the large black dots on the left of the plots. (e, f) Surface view of (e) the interacting region „e‟ and (f) the interacting region „f‟ of the complex. (g) The α-7 nicotinic acetylcholine receptor and normal SLURP1 interface; the nAChR–SLURP1 complex is represented as a ribbon diagram, with nAChR and SLURP1 shown in cyan and green, respectively. Interacting residues are depicted in atomic pictures, and H bonds are indicated by black lines with the distances shown in angstroms (Å).

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Figure 3.4: Pedigree diagram and clinical presentation of family B. (a) Pedigree of family B segregating isolated nail clubbing. Squares symbolize males and circles females; filled symbols symbolize affected and unfilled unaffected individuals; double lines represent cousin marriage and cross symbols represent deceased individuals. Samples of the individuals, labeled by asterisks, were available for the study; arrows indicate the individuals analyzed by exome sequencing. (b-e) Clinical features of affected family members; (b) nail clubbing of hands in affected individual IV-1; (c, d) nail clubbing of hands and feet of IV-3, with nail dystrophy (dermatophytic onychomycosis) in three digits of the left hand; (e) nail clubbing of hands of III-2.

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Figure 3.5: Genotypes from exome data and Sanger sequencing chromatograms of SLCO2A1 gene in family B. (a) Chromosome 3 genotyping extracted from exome sequence data of five family members using AgileVCFMapper. Upper three lanes showing family members III-2, IV-1 and IV-3; lower two lanes are family members II-3 and III-1. The 41.19 Mb autozygous region of 3q12.2-q23 is shown. (b-c) Sanger sequencing analysis of SLCO2A1 in family B; (b) The homozygous wild-type allele in unaffected individual (III-4); (c) heterozygous SLCO2A1 (c.1A>G, p.Met1Val) mutant and wild-type alleles in obligate heterozygotes (II-3, III-1); (d) homozygous SLCO2A1 c.1A>G (p.Met1Val) mutant allele in affected individuals (III-2, IV-1, IV-3).

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Figure 3.6: Pedigree and clinical manifestations of family C. (a) Pedigree of family C segregating anonychia in autosomal recessive manner. Filled symbols represent affected and unfilled unaffected individuals. Double lines indicate cousin marriage while crossed line deceased individuals. The individuals recruited for the study are shown with asterisks. (b-c) Clinical pictures of affected individual (IV-2) representing anonychia in fingers of both hands.

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Figure 3.7: Haplotypes and sequence chromatogram of RSPO4 gene in family C. (a) For genotyped members, RSPO4 closely linked microsatellite markers haplotypes on chromosome 20p13 are given below each symbol. The linkage interval is marked by D20S103 and D20S842. Genetic distance in cM is shown according to the Rutgers linkage-physical map (Build 36.2). (b-d) Sanger sequence analysis of RSPO4 gene in family C. (b) The homozygous wild-type allele in unaffected individual (IV-5); (c) heterozygous RSPO4 c.-9+17del26 mutant and wild-type alleles in heterozygotes (III- 2, III-3, IV-5); (d) homozygous RSPO4 c.-9+17del26 mutant allele in affected individuals (IV-1, IV-2, IV-3).

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Figure 3.8: Pedigree and clinical presentation of family D. (a) Pedigree representing autosomal recessive inheritance pattern of the disorder in family D. Squares symbolize males and circles females; filled symbols indicate affected individuals and unfilled unaffected individuals; paired lines represent cousin marriage; cross symbols indicate deceased individuals. Samples of the individuals labeled by asterisks were available for the study. (b, c) Finger- and toe-nails in affected individual (IV-6) with claw shaped appearnce.

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Figure 3.9: Haplotypes and sequence chromatogram of FZD6 gene in family D. (a) Allele pattern generated with microsatellite markers, closely linked to FZD6 on chromosome 8q22.3, is given below each individual. (b-d) Sanger sequencing results of FZD6 gene shows (b) homozygous wild type allele in unaffected individual (IV-5), (c) mutant FZD6 c.1265G>A and wild type alleles in four normal individuals (III-3, III-4, III-5, IV-7) and (d) homozygous mutant allele (p.Gly422Asp) in affected individuals (IV-4, IV-6).

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Figure 3.10: Presentation of pedigree and clinical features of affected individuals in family E. (a) Four generation consanguineous pedigree of family E, segregating XP in autosomal recessive pattern. Filled symbols indicate affected, unfilled indicate unaffected, double lines indicate consanguineous union, crossed symbols indicate deceased individuals. (b) Affected girl in family with hyperpigmentation spread over facial regions and (c) affected brother (IV-2) with comparatively less severe hyperpigmentation than his sister (IV-1).

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Figure 3.11: Sanger sequencing chromatogram of ERCC5 gene in family E. (a) Homozygous wild type allele in individual IV-3, (b) mutant and wild type alleles in carrier individuals (III-4, III-5); (c) homozygous mutant allele (p.Ala818Val) in affected individuals (IV-1, IV-2).

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 74 Chapter 4 Trichothiodystrophy

TRICHOTHIODYSTROPHY

Trichothiodystrophy (TTD) is an autosomal recessive disorder characterized by dry and easily broken brittle hair, result due to deficiency of sulfur. Associated features of TTD vary widely; mild cases may include only hair and in more severe cases additional features of intellectual disability, dwarfism, microcephaly, abnormal facial features, premature aging, ichthyosis, nail dystrophies, infertility and proneness to respiratory infections have been reported (Faghri et al., 2008). TTD is divided into two forms i.e. photosensitive (TTD1-3) and nonphotosensitive (TTDN1; MIM 234050) based on the occurrence of photosensitivity. Most of the photosensitive TTD patients have mutations in nucleotide excision repair pathway (NER) genes. Mutations in ERCC2 (MIM 126340) cause TTD1 (MIM 601675) and are also responsible for causing cerebrooculofacioskeletal syndrome (MIM 610756) and xeroderma pigmentosum group D (XPD; MIM 278730) (Broughton et al, 2001). Variants in ERCC3 (MIM 133510) cause not only TTD2 (MIM 616390) but also xeroderma pigmentosum complementation group B (XPB; MIM 610651) (Weeda et al., 1997). Homozygous variants in GTF2H5 (MIM 608780) underlie TTD3 (MIM 616395) (Giglia-Mari et al, 2004). These genes translate for different subunits of the general transcription factor IIH (TFIIH), which is also involved in global genome repair and transcription-coupled repair. For nonphotosensitive TTDN only causal variants in the MPLKIP gene (MIM 609188) have been reported (Nakabayashi et al., 2005). MPLKIP consists of two coding exons on chromosome 7p14.1 that encodes a nucleus restricted Plk1-interacting protein, and as ubiquitous expression in brain, heart, lung, placenta, epidermis and hair follicles, among others (Nakabayashi et al., 2002, 2005).

In this chapter of the thesis, two unrelated consanguineous families (F and G) with nonphotosensitive type of trichothiodystrophy are presented. Detail clinical evaluation of multiple affected individuals was carried out by a group of expert clinicians. To find out the molecular bases of the disease, homozygosity mapping and exome sequencing were performed in both families.

Mapping of Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 75 Chapter 4 Trichothiodystrophy

Family F

Family History and Clinical Features

Family F (Figure 4.1), with autosomal recessive pedigree, was recruited from Pashto speaking tribal area. For the current study blood samples were obtained from eight memebers including four normal (III-1, III-2, IV-3, IV-5) and four affected (IV-1, IV-2, IV-4, IV-6). Due to the unavailability of patients’ father, blood sample of their paternal uncle (III-1) was collected. All affected individuals were born full-term. At the time of samples collection, ages of affected individuals were 23-38 years. They presented features of TTDN such as hair abnormalities, premature ageing, repeated sinus infection, slurred speech and mild intellectual disability. The stature of affected members was within the normal range. Polarized light microscopic analysis of hair shafts revealed irregular structure and fractures through the hair shafts and tiger tail pattern, the prominent feature of TTD (Figure 4.2c), which indicate low content of sulfur. In none of the affected individuals showed UV sensitivity and ichthyoses. Common facial features among affected individuals included curved noses with bent nasal bridges and apparently flat malar regions with either mandibular retrognathia or prognathia (Figure 4.1b-i). In some of the affected individuals additional features were found including nails dystrophy, teeth and eyes abnormalities (Table 4.1). All affected individuals showed high arch palate, while only individual IV-2 showed corneal opacity upon clinical checkup and he also had a history of epilepsy (Table 4.1, Figure 4.1b). Cardiac investigation revealed mitral regurgitation in all the affected individuals.

Genome Wide Scan and Mutation Analysis

Human genome scan and linkage analysis identified a 16.17Mb extended autozygous region flanked by rs6959715 and rs12718947 SNP markers on chromosome 7p14 (Figure 4.3), with 3.26 maximum multipoint LOD score. Analysis of the exome data of affected individual IV-1 revealed 63 homozygous variants within the autozygous region. Of these variants (Table 4.3), only a splice site variant (c.339+1G>A) in the MPLKIP gene was not found in ExAc and 1000-genome databases. The CADD scaled score of this variant was 18.51, signifying that the variant is probably damaging. Co-segregation of the variant with disease phenotypes was confirmed by Sanger sequencing DNA of all the available members in the family. The variant

Mapping of Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 76 Chapter 4 Trichothiodystrophy c.339+1G>A was not found in 218 in-house exomes and in 284 Sanger sequence data from unrelated non-TTDN Pakistani individuals.

Family G

Family History and Clinical Features

Family G with four generation pedigree (Figure 4.4a), segregating TTDN in autosomal recessive manner, was collected from district Lower Dir, KPK, Pakistan. Clinical investigation of affected individuals revealed hair defects such as slow growing fragile hair on scalp with patchy loss, sparse eyelashes and eyebrows, recurrent chest infections and mild intellectual disability (Figure 4.4b-f). Affected individuals IV-3 and IV-4 were below 1 and 2 standard deviations, respectively, from the mean age-related height of children in Pakistani (Aziz et al., 2012). No UV sensitivity or ichthyosis was observed. Affected individual IV-5 also presented thick and dystrophic nails in some of her fingers. Another affected individual IV-4 showed high arch palate and hypodontia with irregular teeth (Table 4.1).

Genotyping and Mutation Analysis

Based on phenotypic similarity with family F, linkage in family G was tested to MPLKIP gene. Microsatellite markers were typed in DNA samples of four normal (III-1, III-2, IV-1, IV-2) and two affected individuals (IV-3, IV-4). Haplotype analysis of microsatellite markers (D7S2209, D7S2846, D7S2497, D7S555, D7S2541, D7S521, D7S691, D7S2428, D7S2436) identified linkage in the family to the gene, MPLKIP, located on chromosome 7. Subsequently, MPLKIP gene was sequenced from DNA of all available members of the family. Analysis of the Sanger sequence data revealed a splicesite variant c.339+1G>A, identified earlier in family F. Two affected members (IV-3, IV-4) were homozygous for the mutant allele, normal sibling (IV-2) and obligate carriers (III-1, III-2) were heterozygous, while, one unaffected brother (IV-1) was homozygous for the wild type allele (Figure 4.6).

Discussion

This chapter described clinical and genetic investigation of two unrelated Pakistani kindreds segregating TTDN in autosomal recessive manner. Clinical features including intellectual disability, hair anomalies, and proneness to infections noted in affected members of these two kindreds were consistent with those observed

Mapping of Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 77 Chapter 4 Trichothiodystrophy previously (Nakabyashi et al., 2005; Botta et al., 2007; Heler et al., 2015). Features variability such as facial characteristics, nail dysplasia, tooth irregularities, epilepsy and ocular manifestations were noted in affected members of the two families. Particularly all affected members in the two kindreds had mitral regurgitation which was most possibly due to cardiomyopathy, a condition not reported previously in TTDN patients.

Sequence analysis of affected individuals in both families identified a splice site variant (c.339+1G>A) in MPLKIP gene, located on chromosome 7p14.1. So far at least seventeen kindred and probands with non-photosensitive trichothiodystrophy carrying sequence variants in the gene, MPLKIP, have been described. Of these, only one mutation was missense, found in an Amish kindred, while all others were truncating deletions found in individuals of Moroccan, European and Middle Eastern descent (Nakabayashi et al., 2005; Botta et al., 2007; Heller et al., 2015).

To find out the result of the splice site variant (c.339+1G>A), identified in two families here, a minigene assay was performed (Shah et al., 2016-this study). The assay indicated abnormal splicing due to loss of the canonical splice site, which resulted in retention of the intervening intron of 969bp. The subsequent transcript featured with a frame shift probably to result in formation of a truncated MPLKIP protein of 131 residues, as compared to its counterpart wild type with 179 amino acids.

The exact function of MPLKIP is yet to be discovered, however, its localization in nucleus suggests its role as a transcription regulator of genes related to the metabolic pathways that are central to the outcome of TTDN1 (Nakabayashi et al., 2005). The protein binds to PLK1, serine/threonine kinase polo like kinase 1, which has significant roles in cell division such as mitotic entry, centrosome development and partition, kinetochore attachment, spindle pole integrity and cytokinesis (Zhang et al., 2007). The MPLKIP colocalization with PLK1 all through mitosis and inhibition or overexpression of MPLKIP consequences in nuclear disintegration and distorted mitotic spindles, signify the regulatory role of MPLKIP for PLK1 in cell cycle (Zhang et al., 2007). MPLKIP Phosphorylation by CDK1 at several serine or threonine amino acids is essential for interactions of MPLKIP with PLK1 (Winkles and Alberts, 2005). Moreover both MPLKIP and PLK1 has co-expressed in a number of tissues that are involved in TTDN including skin, brain, heart and lungs (Nakabyashi et al., 2002,

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2005). The miss splicing of MPLKIP due to the splice site variant as validated by the splice assay (not shown) confirmed formation of a frame shift transcript due to retention of intron. The transcript with premature termination signal at codon 132 is predicted to result in the loss of a putative phosphorylation site at Ser133. The loss of the phosphorylation site is likely to cause the TTDN pathogenesis in these families. Taken together this evidence recommends that non-functional MPLKIP due to protein truncation and deficient phosphorylation might lead to loss of interactions between MPLKIP and PLK1, which plays a role in development of various organs including brain, skin and heart.

In conclusion we have reported a novel splice donor site variant (c.339+1G>A) in the MPLKIP gene, segregating in autosomal recessive manner in families showing TTDN phenotypes. This study expands both phenotypic and allelic spectra of MPLKIP- related non-photosensitive TTD, to include a splice variant that causes cardiomyopathy as part of the TTDN phenotype.

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Table 4.1: Clinical features observed in affected members in family F and G

Mapping of Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 80 Chapter 4 Trichothiodystrophy

Table 4.2: Two-point and multipoint LOD scores obtained with SNP markers flanking MPLKIP gene on chromosome 7p14 in family F

Two Point LOD score Physical Multipoint LOD Marker name 0 0.01 0.05 0.1 0.2 0.3 0.4 Location Score rs1229014 39039535 -Infinity -Infinity 0.3166 0.858 0.9548 0.8234 0.5361 0.1945 rs6959715 39123415 -Infinity -Infinity 0.3166 0.858 0.9548 0.8234 0.5361 0.1945 rs4723836 39318380 3.2543 2.3551 2.3122 2.1366 1.9082 1.4188 0.882 0.3242 rs7807596 40826291 3.2574 2.3551 2.3122 2.1366 1.9082 1.4188 0.882 0.3242 rs7455525 41683897 3.2582 1.5587 1.524 1.3867 1.2176 0.8846 0.5537 0.2327 rs6943150 42503307 3.2587 2.3551 2.3122 2.1366 1.9082 1.4188 0.882 0.3242 rs10951731 43452028 3.2586 1.4516 1.4219 1.3024 1.152 0.8464 0.5328 0.2228 rs4724335 44978600 3.2592 1.4516 1.4219 1.3024 1.152 0.8464 0.5328 0.2228 rs2040863 45298128 3.2593 2.3551 2.3122 2.1366 1.9082 1.4188 0.882 0.3242 rs2965083 46044637 3.2594 2.3551 2.3122 2.1366 1.9082 1.4188 0.882 0.3242 rs4580949 46892451 3.2592 1.4516 1.4219 1.3024 1.152 0.8464 0.5328 0.2228 rs7797887 47686618 3.2593 2.3551 2.3122 2.1366 1.9082 1.4188 0.882 0.3242 rs12056015 48379052 3.2592 2.3551 2.3122 2.1366 1.9082 1.4188 0.882 0.3242 rs12666784 49403393 3.259 2.3551 2.3122 2.1366 1.9082 1.4188 0.882 0.3242 rs9642409 50995828 3.2585 1.4516 1.4219 1.3024 1.152 0.8464 0.5328 0.2228 rs10215689 51908082 3.2585 1.7957 1.7518 1.5777 1.3636 0.9486 0.5534 0.1998 rs6945518 52595003 3.2583 2.3546 2.3121 2.1383 1.9119 1.4259 0.8928 0.3424 rs6967345 53156438 3.2577 1.7957 1.7518 1.5777 1.3636 0.9486 0.5534 0.1998 rs7810981 53467855 3.2214 1.4516 1.4219 1.3024 1.152 0.8464 0.5328 0.2228 rs12718947 55292571 -Infinity -Infinity 0.3166 0.858 0.9548 0.8234 0.5361 0.1945 rs11974040 56066329 -Infinity -Infinity 0.3166 0.858 0.9548 0.8234 0.5361 0.1945

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Figure 4.1: Pedigree and clinical pictures of affected individuals in family F. (a) Four generation pedigree of the family with four affected individuals. Filled symbols signify affected and unfilled unaffected members. Double line represents consanguineous union, and crossed symbols represent deceased individuals. (b-i) Clinical pictures of affected members in family F. (b) Individual IV-2 has corneal bulging and cataract and on the left eye, sparse eyebrows, absent eyelashes, (c) patchy hair loss on scalp and (d) dystrophic toenail with grooved ridges. (e) Affected individual IV-4 has sparse eyebrows, eyelashes, facial and (f) leg hair, and (g) dystrophic nails with grooved ridges in toes. (h-i) Affected individual (IV-6) with absent eyelashes, and sparse eyebrows, facial and scalp hair.

Mapping of Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 82 Chapter 4 Trichothiodystrophy

Mapping of Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 83 Chapter 4 Trichothiodystrophy

Figure 4.3: Haplotypes generated using SNP markers and MPLKIP variant in family F. Positions of the SNP markers, indicated, are according to Rutgers combined- linkage physical map (build 36.2.38). Arrow indicates the individual analyzed by exome sequencing.

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Figure 4.4: Pedigree and clinical pictures of affected individuals in family G. (a) Pedigree diagram of the family segregating TTDN in autosomal recessive manner. Individuals available for the study are depicted with asterisks. (b-c) Affected individual IV-3 has (b) sparse eyebrows and eyelashes, crooked beaked nose, flat malar eminences, and retrognathia, (c) patchy hair loss on scalp, (d) and nail dystrophy. (e-f) Affected individual IV-4 has (e) sparse hair over scalp and (f) sparse eyebrows and eyelashes, and hypodontia. (g) 2D echocardiogram from IV-4 shows mitral regurgitation.

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Figure 4.5: Haplotypes generated by genotyping microsatellite markers in family G on chromosome 7p14.1. The markers D7S2846 and D7S2428 are flanking the linkage interval.

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Figure 4.6: Sequence chromatogram of MPLKIP gene in family F and G. (a) Homozygous wild type allele in unaffected individuals in family F (IV-3, IV-5) and family G (IV-1); (b) heterozygous wild and mutant alleles in carrier individual of family F (III-1, III-2) and family G (III-1, III-2, IV-2); (c) homozygous mutant allele in affected individuals of family F (IV-1, IV-2, IV-4, IV-6) and G (III-3, IV-4).

Mapping of Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 87 Chapter 5 Hereditary Ichthyosis

HEREDITARY ICHTHYOSIS

Skin provides physical, chemical and biological protection from the external environment. The skin also provide as an inside-outside barrier to avoid dehydration by regulating trans-epidermal water loss. The principal physical barrier section is stratum corneum, which is contains hydrophilic corneocytes acting as ‘blocks’ and lipophilic intercellular lipid bilayers acting as ‘mortar’. Corneocytes possess a cornified envelope, composed of several structural proteins such as loricrin, involucrin, filaggrin, cystatin and small proline-rich protein, cross-linked by transglutaminase enzymes (Proksch and Jensen 2012; Gittler et al., 2013). Moreover, the nucleated layers of epidermis also contribute to skin protection through tight junctions and desmosomes. Intercellular lipids also have important roles in regulating skin permeability barrier property, keeping skin hydration and providing chemical and biological protection. Disruption in the barrier function of skin results in various skin conditions such as ichthyoses, dermatitis and psoriasis (Gittler et al., 2013).

Ichthyoses are genetically heterogeneous and clinically diverse group of Mendelian disorders of cornification (MEDOC) characterized by localized or generalized scaling and erythema and may result in significant morbidity and mortality. So far, sequence variants in more than fifty genes have been identified in pathogenesis of ichthyoses; despite diverse function, these conditions share a common pathobiologic feature of increased transepidermal water loss (Boyden et al., 2017). MEDOC may be grouped into syndromic and non-syndromic forms, based on existence or absence of extra cutaneous phenotypes. On the bases of clinical findings, Oji et al. (2010) classified ichthyoses into five types: (1) common ichthyoses (X-linked recessive ichthyosis and ichthyosis vulgaris), (2) autosomal recessive congenital ichthyoses (harlequin ichthyosis, lamellar ichthyosis and congenital ichthyosiform erythroderma), (3) keratinopathic ichthyosis, and (4) other minor types (erythrokeratoderma, loricrin keratoderma and peelin skin disease).

Due to the high rate of consanguinity the prevalence of autosomal recessive types of ichthyoses is higher in developing countries like Pakistan. In this chapter of the thesis, three families (H-J) with cornification disorders were clinically and genetically/molecularly evaluated. Genetic analysis in these families identified a novel candidate gene and two novel sequence variants in two other genes.

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Family H

Family History and Clinical Findings

Family H, with cornification disorder, was recruited from Punjab province, Pakistan. The four generation pedigree contains four affected individuals, segregating the disorder in autosomal recessive manner (Figure 5.1a). There were no documented inbreeding loops, but the family hailed from a rural region in Punjab province where marriages within community are very common. In all cases disease onset was within the first two years of life. Affected family members exhibited symmetric lichenified hyperkeratotic plaques over the arms, legs, hands, feet, face, under the axillae, elbows and knees; accentuated creases over the knees and elbows; multiple peridigital constrictions on the dorsa of the fingers; palmoplantar hyperkeratosis with thick nails on the fingers and toes; and joint stiffening of the hands, feet, knees, and elbows (Figure 5.1b-g and Table 5.1), consistent with a condition called progressive symmetric erythrokeratoderma (PSEK). Moreover, the affected individuals also showed hyperkeratotic scales over legs and arms (Figure 5.1g). All family members had normal perspiration, hair and teeth, hearing, and intelligence.

Genetic Mapping and DNA Sequencing

To search for the gene underlying PSEK in the family, initially a candidate gene approach following autozygosity mapping using microsatellite markers was performed. Microsatellite markers located in the closest regions of genes including LOR, GJA1, GJB2, GJB3, GJB4, type I and type II keratin gene clusters were typed in both affected and unaffected family members (Table 2.1). Analysis of genotyping data and haplotype showed autozygosity of markers in the region of the type II keratin gene cluster, spanning a 13.5 cM segment flanked by D12S1653 and D12S104, on chromosome12q12-q14.1 (Figure 5.2). Two genes, KRT1 and KRT2, located within the autozygous region, were Sanger sequenced, but didn’t find any pathogenic variant in affected members of the family.

For more comprehensive analysis, whole-exome sequencing was carried out using DNA samples of three affected (III-3, III-7, III-8) and two unaffected obligate heterozygotes (II-1, II-3) of the family. Homozygosity mapping of the exome sequence data using AgileVCFMapper detected a single extended genomic segment within which all SNPs were homozygous in the three affected family members and

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 89 Chapter 5 Hereditary Ichthyosis heterozygous in the two obligate heterozygotes. The 15.56 cM segment on chromosome 12q12-q14.1 corresponded to the same 13.5 cM region identified here earlier in this family by autozygosity mapping using microsatellite markers (Figure 5.2).

Analysis of the exome data revealed only three exonic variants those were homozygous in the three affected individuals, heterozygous in the two obligate heterozygotes, and had a minor allele frequency (MAF) < 0.01 in South Asian ExAC alleles (Table 5.2). Among these three variants, two were within the region of extended homozygosity on chromosome 12q12-q14.1. One was a single-base insertion c.364_365insA (chr12: 57324205G>GT; MAF = 0.0002423), in SDR9C7 gene, encoding short chain dehydrogenase/reductase family 9C, member 7. The other variant identified was also a frameshift (c.811delA; p.Ser271fs), a single-base deletion (chr12:52710746CT>C) in KRT83 gene. All the available bioinformatics tools predicted that these variants are probably damaging.

Sanger sequencing of DNA from all 10 available individuals of the family confirmed the co-segregation of both variants (chr12:57324205G>GT; chr12:52710746CT>C) with the disease phenotypes. These variants were not found in our in-house exome sequence data from 50 additional unrelated Pakistani individuals with non-PSEK phenotypes. Nevertheless, the SDR9C7 and KRT83 variants are reported in the ExAc database, with a very low MAF of 0.00024 and 0.00018 in Southeast Asians, respectively.

Family I

Family History and Clinical Features

Family I, was collected during a field visit to Pashto speaking tribal area of Pakistan. The four generation consanguineous pedigree had three affected members (IV-1, IV- 2, IV-3) (Figure 5.4a). The heterozygous parents were phenotypically normal. Affected individuals were born full-term. They had diffuse generalized scales on the trunk, neck, and extremities with varying intensity. Facial regions were clear of scales (Figure 5.4b-d). In all of three affected individuals hyperkeratosis was more prominent as compare to scale on their trunks. Early global neurodevelopmental delay was observed in them, especially in language, speech, and motor milestones. Their receptive language was apparently better than expressive language. Like most

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 90 Chapter 5 Hereditary Ichthyosis reported cases, spasticity of lower limb was observed in all three affected individuals with variable severity i.e. elder brother (IV-1) did not walk until he was two and half years old and still face difficulty in walking. Another affected individual (IV-2) was unable to sit until 12 months of age and to stand independently. The youngest affected member was unable to sit and stand at the time of examination. Forelimbs of the patients were fully functional. Their hair, nail and teeth were also normal.

Genotyping and Sequencing

In family I, exome sequencing was performed in five members including three affected (IV-1, IV-2, IV-3) and two unaffected (III-2, III-3). Autozygosity mapping of the exome sequence data using AgileVCFMapper revealed a 16.17 Mb homozygous region on chromosome 17p13.2-p11.2 (chr17:4.43-21.14 Mb) (Figure 5.5a). Analysis of the exome data revealed three exonic variants that were: (a) homozygous in the three affected individuals and heterozygous in the two obligate heterozygotes, and (b) had MAF <0.01 in the ExAC database. Of the five variants, only one variant chr17:19552294C>A (c.10G>T; p.Glu4*) in the ALDH3A2 gene was located within the autozygous region on chromosome 17p13.2-p11.2. This variant was predicted to result in a truncated ALDH3A2 protein. Co-segregation of the variant with disease phenotype in the family was confirmed by Sanger sequencing DNA of all the available members (Figure 5.5).

Family J

Family History and Clinical Features

Family J, with six affected individuals, was sampled from District Swat, KPK, Pakistan. This was a five generations consanguineous pedigree (Figure 5.6) segregating Chanarin-Dorfman syndrome in autosomal recessive manner. For this study, blood samples of thirteen individuals including six affected (V-1, V-2, V-3, V- 10, V-11, V-12) and seven normal (IV-3, IV-4, IV-7, IV-8, V-4, V-9) were collected. Clinical features including congenital ichthyosiform erythroderma, dry and brownish scaly skin over most part of the body, and black spots as a result of deposition of neutral lipids were found in affected members (Figure 5.7a-f). Three affected individuals (V-2, V-3, V-11) had more severe phenotypes. Other ectodermal structures (hair, nail, teeth) were not affected.

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Genotyping and Sequencing

In family J, whole genome scan was carried out at Washington Center for Mendelian Genomics using SNP microarray. Analysis of the genotyping data identified a single large homozygous region of 9.71 Mb (chr3:40.50-50.21 Mb) on chromosome 3p22.1- p21.31, flanked by rs112300086 and rs946039787 SNP markers, with maximum multipoint LOD score of 5.01. Subsequently, WES was carried out on DNA sample of an affected individual (V-1). Exome data analysis revealed a novel homozygous deletion chr3:43759225delA (c.836delA; p.Gln279Argfs*14), which resulted in a frameshift in the ABHD5 gene. Chain termination sequencing of the variant in all available members verified its co-segregation with the Chanarin-Dorfman syndrome within the family (Figure 5.8).

Discussion

Investigation of three families (H-J), displaying features of cornification disorders, is presented in this chapter. Analysis of genotyping and exome sequencing data identified a novel candidate gene and two novel sequence variants, involved in generating ichthyosis phenotypes, in the families. Literature reviewed revealed that occurrence of erythrokeratoderma in association with ARCI, observed in family I, is probably the first case of its nature ever reported. The patients in the family presented symmetric lichenified hyperkeratotic plaques on arms and legs; palmoplantar hyperkeratosis with thick nails on the fingers and toes; generalized hyperkeratosis and accentuated creases over the knees and elbows; multiple peridigital constrictions on the dorsa of the fingers; and joint stiffening of the hands, feet, knees, and elbows. Homozygosity mapping and exome sequencing revealed two candidate sequence variants: chr12:52710746CT>C (MAF= 0.00018) in KRT83 and chr12:57324205G>GT (MAF = 0.0002423) in SDR9C7 gene. Unexpectedly, both variants were segregating with the disease phenotypes in the family. Based on the reported phenotypes resulting from keratins mutations, the KRT83 sequence variant (chr12:52710746CT>C) was suggested for causing recessive progressive symmetric erythrokeratoderma. However, recently reported findings of involvement of SDR9C7 in producing ARCI phenotypes (Shigehara et al., 2016; Karim et al., 2017), it seems highly likely that the associated scaly skin phenotypes, observed in family I here, might be due to a frameshift variant (chr12:57324205G>GT) detected in the SDR9C7 gene. Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 92 Chapter 5 Hereditary Ichthyosis

Keratins are a large family of structural proteins, mainly found in epithelial tissues (Coulombe et al., 2002). Humans have 54 keratin genes, grouped into type I (n = 28; acidic, with low molecular weight) and type II (n = 26; neutral to basic, with high molecular weight), and arranged in two clusters on chromosomes 17q21.2 and 12q13.13, respectively. Keratins form heteropolymeric filaments containing type I and type II keratin heterodimers, expressed in a cell type-specific manner, and mutations in keratin genes lead to variety of genodermatoses. KRT83 was initially described as a type II keratin weakly expressed in hair cortex (Langbein et al., 2001). However, in rat and sheep KRT83 is expressed in whole skin (Nanashima et al., 2008; Yu et al., 2011). Similarly, immunohistochemistry of human skin demonstrates strong expression of KRT83 in skin keratinocytes (Petryszak et al., 2014).

The KRT83 c.811delA (p.Ser271fs) frameshift, identified here, would result in premature translational termination at KRT83 codon 274, in the segment encoding the L2 linker region of the central rod domain. Frameshift mutations in other keratin genes have been shown to result in non-production of the corresponding polypeptide and mRNA (Corden et al., 1998; Gripp et al., 2013), apparently due to NMD (nonsense mediated decay) of mRNA; thus, the heterozygous KRT83 frameshift apparently lacks dominant-negative effects. Heterozygous missense variants of KRT83, c.1219G>A (p.Glu407Lys) and c.1252G>A (p.Glu418Lys), have been reported in two families with the autosomal dominant hair disorder monilethrix (MIM 158000) (van Steensel et al., 2005, 2015). In the family segregating p.Glu407Lys substitution, one of the three affected members exhibited additional feature of mild follicular hyperkeratosis. However, affected members of family H here did not show any hair abnormality, not even within the hyperkeratotic plaques. Together, these findings indicate that at least some cases of autosomal dominant monilethrix and autosomal recessive PSEK are allelic resulting from heterozygous missense substitutions and homozygous loss-of-functions of KRT83, respectively. Complete lack of KRT83 may destabilize the epidermal cytoskeleton, resulting in erythrokeratoderma, whereas abnormal hair characteristic of monilethrix are specifically associated with missense substitutions that alter KRT83 function.

The gene SDR9C7 codes for short chain dehydrogenase/reductase family 9C member 7, which is associated with vitamin A metabolism. Vitamin A down-regulates corneodesmosomal proteins and consequently elevates desquamation of stratum

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 93 Chapter 5 Hereditary Ichthyosis corneum. Accordingly, an absence of vitamin A metabolizing enzyme might decelerate the normal cycle of keratinocytes proliferation, differentiation and desquamation, thus leading to appearance of hyperkeratosis. (Shigehara et al., 2016). The c.364_365insA variant results in a frame-shift and a downstream premature termination codon (p.Thr122AsnfsTer4), predicting an absence of functional mRNA due to NMD and absence of SDR9C7 protein. The hypothesis of PSEK phenotypes resulting from sequence variant in the KRT83 gene and apparent associated phenotypes due to SDR9C7 variant, is strengthened by reports of sequence variants in the SDR9C7 gene in Lebanese population (Shigehara et al., 2017) and documentation of the variant (c.364_365insA), identified here, in another family of Pakistani origin (Karim et al., 2017) producing only hyperkeratosis without PSEK phenotypes.

In family I due to lack of clear association of phenotypes, observed in affected members, with reported cases of ichthyosis, direct WES was performed on DNA samples of three affected members (IV-1, IV-2, IV-3) and their parents (III-2, III-3). Analysis of the exome data revealed a novel nonsense mutation chr17:19552294C>A (c.10G>T; p.Glu4*) in the ALDH3A1 gene. The available bioinformatics tools such as SIFT, PolyPhen2, GERP++ and phyloP predicted the variant to be probably damaging. Chain termination sequencing confirmed co-segregation of the mutation with disease phenotypes in family I.

The ALDH3A2 gene is mapped on chromosome 17p11.2 and arranged into eleven exons, encoding for fatty aldehyde dehydrogenase (FALDH), a protein of 485 amino acids residues (Chang and Yoshida, 1997; Rogers et al., 1997). FALDH is a microsomal NAD dependent enzyme that executes oxidation of longchain aliphatic aldehydes into fatty acids (Kelson et al., 1997). FALDH deficiency results in aberrant oxidation of long-chain fatty aldehydes to fattyacids, which results in accretion of fatty aldehyde precursors, such as fatty alcohols, or the formation of aldehyde modified macromolecules is suggested to influence the normal development of stratum corneum and myelin, thus the cutaneous and neurological symptoms appear. So far, 101 pathogenic variants including missense, nonsense, splicing, deletions, insertions, indels and complex rearrangements in the ALDH3A2 gene have been implicated with Sjögren-Larsson syndrome (SLS) (HGMD Professional 2017.1). Patients with loss of function mutations in the ALDH3A2 are more severe as compared those with missense mutations (Willemsen et al., 2001). The nucleotide

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 94 Chapter 5 Hereditary Ichthyosis change c.10G>T abolishes the translational immediately after its start (p.Glu4*), and expected to abolish the ALDH3A2 protein in family members homozygous for this mutation. The absence of this variant in ExAc and 1000 genome databases, and unavailability of the variant in 50 in-house exome data base together support causality of this mutation for SLS in the family. Moreover, its wide expression is consistent with a number of phenotypes associated with ALDH3A2 mutation.

In family J homozygosity mapping identified 9.71 Mb autozygous region on chromosome 3p22.1-p21.31 with a significant maximum multipoint LOD score of 5.01. WES revealed a novel homozygous mutation (c.836delA) in the ABHD5 gene. Sanger sequencing of all available members confirmed co-segregation of the mutation with the disease.

ABHD5 gene mapped on chromosome 3p21.33, encodes a/b-hydrolase domain containing protein 5, which belongs to the esterase, lipase and thioesterase sub-family, characterized by alpha/beta-fold domain and catalytic triad contained a nucleophile, a histidine and an acid (Ollis et al., 1992; Zhang et al., 1998; Heikinheimo et al., 1999; Nardini and Dijkstra, 1999). ABHD5 has been reported for co-activating adipose triglyceride lipase (ATGL), which hydrolyzes triacylglycerol to yield fatty acid and diacylglycerol (Lass et al., 2006). It is highly conserved among the different species. Pathogenic sequence variants abrogate lipolysis and induced a systemic accretion of lipids particles, which is characteristic of Chanarin-Dorfman syndrome (CDS). The broad spectrum of expression of this protein explains clinical phenotypes of CDS. The novel homozygous variant (c.836delA), identified in family J, produces a truncated protein with missing histidine residue at 327 position in catalytic triads necessary for ABHD5 lipase/esterase/thioesterase activity, consequently leads to extremely compromise or completely loss of ABHD5 catalytic activity.

In summary, KRT83 is identified as a novel gene in pathogenesis of PSEK in family H, extending the genetic heterogeneity of erythrokeratoderma. Moreover, the additional phenotypes of ichthyosis are probably owing to the sequence variant (c.364_365insA) in SDR9C7 gene. Also, this study extends the KRT83 role to skin in addition to its function in hair. In families I and J novel mutations in the ALDH3A2 and the ABDH5 were identified for Sjögren-Larsson syndrome and Chanarin- Dorfman syndrome, respectively. This study extends the preexisting human genomic

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 95 Chapter 5 Hereditary Ichthyosis database and may help to develop a genotype-phenotypes correlation and genetic counseling of the respective families.

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 96 Chapter 5 Hereditary Ichthyosis

Table 5.2: List of variants extracted from exome data of family H, these were homozygous in affected individuals (III-3, III-7, III-8) and heterozygous in their parents (II-1, II-3) Chr Position Gene Effect Fre cDNA Protein 9 6592178 GLDC Missense 0.0001 c.1447G>A p.Asp483Asn

12 52710746 KRT83 Frameshift 0.00018 c.811delA p.Ser271fs 57324205- p.Thr122_Lys 12 SDR9C7 Frameshift 0.00003 c.364_365insA 57324206 123fs

Table 5.3: List of variants identified in exome data of family I, these were homozygous in affected and heterozygous in their parents

Chr Position Gene Effect Frequency cDNA Protein 1 235973192 LYST Missense 0.000024 c.926G>A p.Arg309Gln 2 195452535 MUC20 Missense NR c.36655T>G p.Leu12219Val 17 19552294 ALDH3A2 Nonsense NR c.10G>T p.Glu4* Chr= chromosome, Fre= frequency

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Figure 5.1: Pedigree and clinical pictures of family H. (a) Pedigree of the family segregating PSEK in autosomal recessive pattern. Squares represent males and circles females; filled symbols represent affected members and unfilled unaffected. Samples of the individuals labeled by asterisks are those which were available for the study; arrows indicate the individuals analyzed by exome sequencing. (b-g) Clinical features of PSEK in family H; (b) lichenified patches on arms (c) striate palmar hyperkeratosis, and (d) dorsal hyperkeratotic plaques, peridigital constrictions, and thick fingernails in lll-3. (e) Palmar hyperkeratosis, thick fingernails, and (f) hyperkeratosis on leg in lll-8. (g) Dorsal hyperkeratosis and peridigital constrictions in lll-7.

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 98 Chapter 5 Hereditary Ichthyosis

Figure 5.2: Haplotypes generated using microsatellite markers on chromosome 12q12-q14.1 in family H. Genetic distance (centi-morgans; cM) is according to the Rutgers combined-linkage physical map (build 36.2.38).

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Figure 5.3: Genotypes from exome data and Sanger sequence chromatograms of the KRT83 gene in family H. (a) Genotyping (Chromosome 12) extracted from exome sequence data in five family members using AgileVCFMapper. Upper three lanes showing family members lll-3, lll-7 and lll-8; lower two lanes are family members ll-1 and ll-3. The 12.97 Mb autozygous region on chromosome 12q12-q14.1 is shown. (b-d) Sanger sequence of the KRT83 in family H. (b) homozygous wild-type allele in unaffected members lll-1, lll-5; (c) heterozygous deletion/wild-type alleles in carriers ll-1, ll-3, lll-2, lll-4, lll-6; and (d) homozygous deletion (c.811delA) in cases lll-3, lll-7, lll-8.

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Figure 5.4: Presentation of pedigree and clinical features of affected individuals in family I. (a) Four generation consanguineous pedigree of the family segregating SLS in autosomal recessive pattern. Filled symbols indicate affected, unfilled unaffected, double lines consanguineous union, and crossed symbols deceased individuals. Individuals labeled by asterisks were available for the study, and arrows indicate the individuals analyzed by exome sequencing. (b-d) Clinical features of affected family members: (b) Hyperkeratrosis on anterior side of abdomen and fore limbs, (c) hyperkeratosis and scales on the back and neck of affected individual IV-1; and (d) Hyperkeratosis on anterior side of the body and large polygonal scales on lower limbs of affected individual IV-3 in Family I.

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Figure 5.5: Genotypes extracted from exome data and Sanger sequencing chromatograms of ALDH3A2 gene in family I. (a) Chromosome 17 genotyping extracted from exome sequence data of five family members using AgileVCFMapper. Upper three lanes showing family members IV-1, IV-2 and IV-3; lower two lanes are family members III-2 and III-3. The 16.17 Mb autozygous region of 17p13.2-p11.2 is shown. (b-d) Sanger sequence analysis of the ALDH3A2 gene in family I; (b) A homozygous wild-type allele in unaffected individual (lV-4); (c) heterozygous ALDH3A2 c.10G>T (p.Glu4*) mutant and wild-type alleles in obligate heterozygotes (lll-2, lll-3); and (d) homozygous mutant allele ALDH3A2 c.10G>T (p.Glu4*) in affected individuals (lV-1, lV-2, lV-3).

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Figure 5.6: Pedigree outline of family J with Chanarin-Dorfman syndrome. Squares represent males; circles females; filled symbols affected individuals; and unfilled symbols unaffected individuals. Individuals labeled by asterisks were available for the study; and arrow indicates the individual analyzed by exome sequencing.

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Figure 5.7: Clinical pictures of family J: (a) Black spots on face, neck and ear (b) and hyper-linearity on palms of affected individual V-2; (c) Hands of affected individual V-1 with brownish scales; (d) Brownish scales and black spots on the facial area of affected individual V-12; (e) light brown scales and hyperkeratosis on facial region of and (f) large thin scales on lower leg of affected individual V-10 in family J.

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Figure 5.8: Sanger sequence analysis of ABHD5 in family J; (b) The homozygous wild-type allele in unaffected individual (V-4); (c) heterozygous ABHD5 c.836delA (p.Gln279Argfs*14) mutant and wild-type alleles in obligate heterozygotes (lV-3, lV- 4, lV-7, lV-8); (d) homozygous ABHD5 c.836delA (p.Gln279Argfs*14) mutant allele in affected individuals (V-1, V-2, V-3, V-11, V-12, V-13).

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Epidermolysis Bullosa

Inherited epidermolysis bullosa (EB) is clinically and genetically heterogeneous group of genodermatoses characterized by mucocutaneous fragility following minor mechanical trauma. With an incidence of 8 per 1,000,000 live births, EB ranks among the orphan diseases (Fine et al., 2010). EB results from defects in genes encoding proteins that are involved in maintenance of skin integrity. This include (a) hemidesmosomes which extend from the intracellular milieu of basal keratinocytes to the extracellular matrix of the lamina lucida; (b) anchoring filaments, thread-like structures, which traverse the lamina lucida and extend to the underlying lamina densa; and (c) anchoring fibrils, attachment complexes which extend from the lower portion of the lamina densa to the underlying mesenchyme. EB is inherited with autosomal dominant and autosomal recessive pattern. Mostly the autosomal dominant forms are mild and permit a normal life-span, while, majority of autosomal recessive forms are associated with worse prognosis. Based on extracutaneous tissue involvement EB may be syndromic or isolated. The extra cutaneous tissues mostly affected in the disease are the mucous membranes of mouth, gastrointestinal and genital tract, and skeletal muscles; causing complications such as ulceration, oesophageal strictures and muscular dystrophy (Marinkovich, 2014). Epidermolysis bullosa is classified according to the level of skin cleavage. Epidermolysis bullosa simplex results from defects within basal keratinocytes of the epidermis, in junctional epidermolysis bullosa the cleavage plane is within the basement membrane, while, dystrophic epidermolysis bullosa causes blister formation underneath the lamina- densa of the basement membrane (Fine et al., 2014).

Over the past two decades significant improvement has been made in understanding the clinical and molecular bases of epidermolysis bullosa and translational research has been extended to clinical trials and therapy. This advancement in understanding of the molecular basis of the epidermolysis bullosa has several direct benefits for patients to improve genetic counseling, develop prenatal testing and possible gene and cell therapies (Salam et al., 2014).

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In this chapter of the thesis, two families (K and L) with epidermolysis bullosa are presented. In family K a recurrent mutation in KRT14 was identified, while, genetic analysis of family L revealed a novel sequence variant in the PLEC gene.

Family K

Family History and Clinical Features

Family K, with epidermolysis bullosa simplex was sampled from district Lower Dir, KPK, Pakistan. The four generation pedigree contains eighteen members including three affected in the last generation (Figure 6.1a). Blood samples were collected from three affected (IV-2, IV-3, IV-4) and six unaffected individuals (III-1, III-2, III-3, III-4, IV-5, IV-6). The parents of patients were phenotypically normal. All three affected individuals (IV2, IV-3, IV-4) presented typical features of epidermolysis bullosa simplex. In affected individuals large bullae, pustules, erosion and blisters were more prominent on the dorsum of hands and feet, and mild over limbs, trunk and palmoplantar skin (Figure 6.1b-e). The blisters appeared after mild mechanical stress, and healed with mild scarring. The nails appeared to have onycholysis and would easily detach from the nail bed. Hyperkeratosis over palms and soles were not observed. None of their normal siblings or parents presented any skin or nail manifestations, including skin fragility.

Genotyping and Sequencing

To find out the genetic defect causing autosomal recessive EBS in family K, genotyping was carried out with microsatellite markers flanking KRT5 and KRT14 genes (Table 2.1), previously reported for causing similar phenotypes. Analysis of haplotype established linkage in the family to KRT14 gene, located on chromosome 17q21.2. Subsequently, KRT14 was Sanger sequenced in all available members. Analysis of the Sanger sequencing data identified one base pair deletion c.92delT that probably results in a premature stop codon in exon 1 of the KRT14. Affected individuals (IV-2, IV-3, IV-4) were homozygous for the mutant allele (c.92delT), individuals (III-1, III-2, III-3, III-4, IV-6) were heterozygous for wild type and mutant alleles, while, the individual IV-5 was homozygous for wild type allele.

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Family L

Family History and Clinical Features

Family L, segregating epidermolysis bullosa simplex with muscular dystrophy (EBS- MD; MIM 226670), was sampled from district Upper Dir, Khyber Pakhtunkhwa. It was a five generation consanguineous pedigree comprises sixteen members including two affected (Figure 6.4a). Blood samples were collected from two affected (V-3, V- 4) and three unaffected (IV-1, IV-2, V-1) individuals. Phenotypically normal parents were first cousins. At the time of study ages of affected individuals were 12 to 15 years; however, the disease onset starts in early childhood. Clinical evaluation of affected individuals revealed generalized skin blisters, excoriated and verrucous papules and progressive skin lesions most pronounced on the dorsal sides of hands with post-inflammatory hyperpigmentation. Nails of both hands and toes were found dystrophic. Affected members faced progressive weakness in skeletal muscles. In addition, the younger affected boy also showed hypertrichosis over arms, legs, back and facial region. Enamel and mucous membranes were unaffected.

Genotyping and Sequence Analysis

Based on phenotypic consistency with previous reports, linkage in the family was carried out against PLEC gene, using microsatellite markers (D8S1741, D8S1729, D8S1727, D8S1744, D8S1751, D8S373, D8S2334, D8S1925, D8S1926) linked to PLEC gene. Analysis of microsatellite markers revealed an autozygous region of 6.54 cM (Figure 6.5a) at this chromosomal region. Linkage in the family was followed by Sanger sequencing PLEC gene in all members. Sequence analysis revealed a novel missense mutation c.10909C>T (p.3637Arg>Cys) in the gene, PLEC, which was homozygous in the two affected (V-3, V-4), heterozygous in the parents (III-3, III-4), while, one normal individual (V-1) was homozygous for wild type allele (Figure 6.5b- d).

Discussion

Two Pakistani families (K and L) with epidermolysis bullosa were collected during a field visit to Khyber Pakhtunkhwa province of Pakistan. Upon clinical assessment, EBS was diagnosed in both the families. EBS is the most common type of epidermolysis bullosa, accounting for 70% of cases. Phenotypically EBS may be

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 108 Chapter 6 Epidermolysis Bullosa arranged into three subtypes: EBS-localized, characterized by blistering confined to soles and palms; EBS-generalized intermediate, characterized by widespread blistering, but with milder blisters; EBS-generalized severe, resembling the eponymous EBS-Dowling Meara, characterized by widespread erosions, scarring and clustered blistering, and may involve the mucous membranes, nails and hair. Pathogenic sequence variants in the KRT5 and KRT14 genes mostly cause EBS phenotypes. KRT5 and KRT14 dimerize to form intermediate filaments, which are indispensable for the maintenance of integrity and flexibility of the epidermis against mechanical forces. When compromised, they are susceptible to mechanical stress, leading to the fracture of basal keratinocytes and subsequent blistering of the epithelium. So far, no clear genotype-phenotype association has been identified, however, majority of the severe conditions result from mutations in conserved rod- domains of KRT5 and KRT14, which are crucial for correct assembly of keratin filament. While, the milder phenotypes result from mutations outside of rod domains in these genes. Despite this correlation, other genetic and epigenetic factors also contribute to the extent of phenotypic severity.

In family K, homozygosity mapping and Sanger sequencing revealed a homozygous frameshift variant (c.92delT) in KRT14, which was previously reported in a consanguineous Pakistani kindred with epidermolysis bullosa simplex (EBS) (Batta et al., 2000). However, the phenotypes described by Batta et al, (2000) for the same variant (c.92delT) were mild as compared to those observed in the present family. The difference in severity of the phenotype may be owing to second-site modifiers that affect the function of KRT14 and consequent phenotypes. The c.92delT variant predicted to results in a truncated protein of 116 amino acids residues including 30 identical to the normal KRT14 sequence and 86 residues resulted with changed nucleotide sequence. The truncated protein would likely to result in failure to form heterodimers with keratin 5 and intermediate filaments, which are essential for maintenance of integrity and flexibility of the epidermis against mechanical forces. So far, 103 sequence variants have been described in pathogenesis of autosomal dominant and autosomal recessive EBS (Stenson et al., 2017).

In family L, clinical features such as skin blister, excoriated and verrucous papules, skin lesions and dystrophic nails were consistent with phenotypes previously described as a result of mutations in the PLEC gene (Takizawa et al, 1999; Rouan et

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 109 Chapter 6 Epidermolysis Bullosa al., 2000; Bolling et al., 2010; Chiaverini et al., 2010; Alvarez et al., 2016). However, hypertrichosis, observed in the present family, was never reported previously in cases of EBS-MD.

Autosomal recessive EBS with muscular dystrophy (EBS-MD) has been associated with sequence variants in the plectin gene (PLEC). This gene encodes for a 500 kDa cytolinker protein plectin with eight different isoforms. Plectin contains a central rod domain flanked by two globular domains, which contain several protein interaction sites. The central rod domain is expected to mediate self-association via coil-coil interactions. Plectin has expression in many tissues such as striated muscle, skin and gastrointestinal epithelia. In skin, high level of plectin is reported along the basal pole of basal keratinocytes, where it link hemidesmosomes, keratin IF and the underlying basement membrane layer.

In family L, homozygosity mapping and Sanger sequencing revealed a novel sequence variant c.10909C>T (p.3637Arg>Cys) in exon 32 of PLEC gene. This variant is located in C-terminal globular domain of the protein. The C-terminal globular domain is important for binding to various cytolinker proteins such as , , , and glial fibrillary acidic protein (Castanón et al., 2013). The variant p.3637Arg>Cys might perturb the three dimensional structure of the protein and possibly consequence in weak/no binding of the plectin to the cytoskeletal filaments. So far, total of 87 sequence variants have been described in the PLEC gene for pathogenesis of EBS, EBS-MD and EBS with pyloric atresia (Stenson et al., 2017). A condition of hypertrichosis has not been described previously in patients with PLEC mutations. In the present family, the segregation of hypertrichosis phenotypes with plectin variant c.10909C>T suggests its role in hair development.

In summary, two Pakistani families (K and L) with EBS and EBS-MD were investigated. Genetic mapping and mutation analysis in family K identified a recurrent mutation in the KRT14 gene. While molecular analysis of family L revealed a novel missense variant in the PLEC gene. This study will outspread the genome variation database and extend the phenotypic spectra of EBS. This can also leads to a better understanding of the pathogenesis of EBS, clarify prognosis and assist in prenatal testing.

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Figure 6.1: Presentation of pedigree and clinical pictures of family K. (a) Four generation consanguineous pedigree of family K with three affected individuals. Filled symbols represent affected and unfilled unaffected members. Double line represents consanguineous union, and crossed symbols represent deceased individuals. Individuals available for the study are depicted with asterisks. (b-e) Clinical representation of affected members in family K. (b, c) The blisters, scars and fresh wounds over foot and hands of affected individual IV-2. (d, e) Scars and blisters on the feet of affected individual IV-3 of family K.

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Figure 6.2: Haplotypes generated by genotyping microsatellite markers in family K on chromosome 17q21. The markers D17S2191 and D17S1150 are flanking the linkage interval. Markers positions are according to Rutgers combined-linkage physical map (build 36.2.38).

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Figure 6.3: Sanger sequence analysis of KRT14 gene in family K. (a) Homozygous wild type allele in unaffected individuals in the family (lV-5); (b) heterozygous wild and mutant (c.92delT) alleles in carrier individual of family (lll-1, lll-2, lll-3, lll-4, lV-6); (c) homozygous mutant (c.92delT) allele in affected individuals (lV-2, lV-3, lV-4) of the family.

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Figure 6.4: Pedigree presentation and clinical pictures of affected individuals in family L. (a) Five generation pedigree of family L with two affected individuals (V-3, V-4) in the fifth generation. Individuals available for the study are shown with asterisks. Squares symbolize males and circles symbolize females; filled symbols indicate affected and unfilled unaffected; double lines represent consanguineous union; and crossed symbols represent deceased members. (b-e) Clinical pictures of affected individuals in family L. In affected individual (V-4) (b) scares over facial region, (c) blister and erosion, post inflammatory hyperpigmentation and nail dystrophy on hand, and (d) toe nail dystrophy. (e) Mild blister and scars over hands of affected individual V-3.

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Figure 6.5: Haplotypes and Sanger sequencing chromatogram of PLEC gene in family L. (a) Haplotypes for genotyped microsatellite markers on chromosome 8q24.3 in individuals of the family. The linkage interval is marked by D8S1741 and D8S1926. Genetic distance in centi-Morgans (cM) is according to the Rutgers combined linkage-physical map (Build 36.2.38). (b-d) Sanger sequence analysis of PLEC in family L; (b) The homozygous normal allele in unaffected member (V-1); (c) heterozygous mutant (c.10909C>T) and wild type alleles in heterozygotes (lV-1, lV-2); (d) homozygous mutant allele (c.10909C>T) in affected individuals (V-3, V- 4).

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HEREDITARY HAIR LOSS DISORDERS

Hereditary hair loss disorders comprise a rare group of genetic conditions characterized by hypoplastic or aplastic hair; sparse scalp hair, eyebrows, eyelashes, and body hair. The term “hypotrichosis” (hypo-less, trich-hair) is used for less than normal hair, while, the term “atrichia” is used when an individual is devoid of body and scalp hair. Hereditary hair loss might be an isolated condition or may be associated with abnormalities in other tissues and organs. The associated abnormalities include intellectual disability, hearing impairment, retinal degeneration and defects in nails and skin. Hypotrichosis can be inherited in either autosomal recessive or autosomal dominant pattern. To date, seventeen various types of isolated hair loss disorders have been identified. Eight of these forms segregate in autosomal dominant and nine in autosomal recessive manner. For autosomal recessive hair loss disorders, seven genes (HR, DSG4, DSC3, LIPH, P2RY5, DSP, KRT25) have been identified. While, for autosomal dominant hair loss conditions also seven genes (APCDD1, CDSN, SNRPE, KRT71, KRT72, RPL21, U2HR) have been mapped on different human chromosomes. These genes encode proteins which are involved in development and maintenance of hair. In addition to above cited isolated hair loss disorders, syndromic forms in which hair abnormality is associated with defects in other ectodermal structures, and/or neurological, cardiac and ocular abnormalities are also reported (Headon and Overbeek, 1999; Monreal et al., 1999; Sprecher et al., 2001; John et al., 2006a; Wali et al., 2006, 2007b). Hair abnormalities have also been reported in few rare genetic conditions such as hypogonadism and neuroendocrine manifestations (Alazami et al., 2008; Nousbeck et al., 2008), Coffin–Siris syndromes (MIM 135900), Zimmermann– Laband syndrome (MIM 135500), X-linked syndromic ID type Nascimento (MIM 300860) Wiedemann-Steiner syndrome (MIM 605130) and Cornelia de Lange syndrome (MIM 122470).

In this chapter of the thesis, five families (M-Q) with hair abnormalities are presented. Affected individuals in four families (M, N, O, Q) showed isolated form of hypotrichosis, while affected individuals in family P presented conditions of hypertrichosis, intellectual disability, hearing impairment, macrocephaly, cardiac and urinary complications.

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Family M

Family History and Clinical Features

The family, segregating autosomal dominant patchy hair loss, was recruited from district Skardu in Gilgit-Baltistan region of Pakistan. Three generation pedigree (Figure 7.1a) had fourteen members including six affected (II-2, III-3, III-4, III-5, III-6, III-8). Blood samples were collected from eleven members including aforementioned affected and five normal (II-1, II-4, III-1, III-2, III-7) in the family. Affected individuals aged 30 to 50 years; however, as the family elders described, the phenotypes appeared early in first three years of life. The abnormal condition appeared in the form of patchy hair loss on scalp (Figure 7.1b-d). Mild form of intellectual disability was noted in two affected individuals (III-3, III-6). Eyelashes, Eyebrows and hair on other parts of the body were normal in the affected members. In addition, males carrying hair abnormality had normal moustache and beard. Similarly, hearing, perspiration and teeth were not affected.

Genotyping and Sequence Analysis

Initially, genome wide genotyping was carried out using microsatellite markers was performed but this failed to establish linkage in the family. Therefore, in order to search for the disease causing variants, exome sequencing was carried out on DNA samples of eight members including six affected (II-2, III-3, III-4, III-5, III-6, III-8) and two unaffected (II-1, II-4). Exome data was then used in genotyping using AgileVCFMapper. After excluding the presence of any autozygous region, the exome data was searched for heterozygous variants. Analysis of the data revealed four such sequence variants (SEC31B c.400C>G, BTAF1 c.5485C>G, MYOF c.565C>T, RPA2 c.166C>A) (Table 7.1). These were found in heterozygous state in affected (II-2, III- 3, III-4, III-5, III-6, III-8) and absent in two unaffected members (II-2, II-4). Further analysis revealed absence of these four variants in public databases (ExAc and 1000 genome). Analysis of Sanger sequencing revealed co-segregation of only the missense variant (BTAF1 c.5485C>G, p.Leu1829Val) with the disease phenotype in the family (Figure 7.2).

Expression Analysis

In order to assess effect of the BTAF1 variant (p.Leu1829Val) on transcription of the genes, related to the development and maintenance of hair, Real time PCR was

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 117 Chapter 7 Hereditary Hair Loss Disorders carried out using DNA of a normal (II-1) and three affected individuals (III-3, III-4, III-5). Analysis of the qPCR data revealed that the variant in the BTAF1 gene associated with down regulation of five other genes (AR, EDA2R, ALDH1A3, KRT81, FGF5) (Figure 7.3). Miss-regulation of these genes was previously reported in BTAF1 mutant mouse as well (Wansleeben et al., 2011).

Family N

Family History and Clinical Features

Family N was a five generation pedigree (Figure 7.4a), belonged to Muzaffarabad, Azad Jammu & Kashmir. The pedigree had thirty eight members including six affected. For this study, blood samples were collected from ten individuals including five affected (IV-2, IV-4, IV-6, IV-8, V-5) and five unaffected (III-5, IV-1, IV-5, IV- 7, V-4). Clinical evaluation revealed presence of hypotrichosis of variable degree in affected individuals of the family. Affected individual (IV-6) had sparse scalp hair, sparse eyelashes and eyebrows since birth. He had less dense beard, and missing hair on rest of the body parts (Figure 7.4b, c). Affected individual (V-5) had normal scalp hair, eyebrows and eyelashes; however, hairs were not found on rest of his body. Affected female individuals (IV-2, IV-4, IV-8) had sparse scalp hair, sparse eyebrows and eyelashes, and absence of hair on rest of their body parts (Figure 7.4d). Affected individuals of the family had normal nails, teeth and perspiration.

Genome Scan

Initially, attempt was made to find linkage in the family with those genes which were proven involved in causing various types of hair abnormalities. This included five candidate genes (LIPH, P2RY5, HR, DSC3, DSG4) mapped on different human chromosomes. Linkage was carried out by using microsatellite markers located in vicinity of the five genes. Analysis of the haplotypes however excluded the family from linkage to the tested genes. Affymetrix GeneChip Genome-Wide Human SNP 250K array was used for whole genome scan. Analysis of the array data revealed 4.07 Mb (110.35-114.28 Mb) autozygous region flanked by markers rs1462309 and rs2693051 on chromosome 3q13.12-q13.31. All SNPs in the autozygous region were homozygous in affected and heterozygous in normal individuals of the family. Following establishing linkage in the family, two probable candidate genes CD200 (MIM 155970) and PVRL3 (MIM 607147), located within the autozygous region on

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3q13.12-q13.31, were subjected to sequencing using dideoxy chain termination method. However, analysis of the sequence data didn’t reveal any pathogenic variant which could be considered for producing hair abnormality in the family.

Exome Sequencing

After Sanger sequencing failed to detect any potential variant, whole exome sequencing was carried out using DNA sample of an affected individual (IV-6) of the family. Analysis of the exome data revealed 36 sequence variants within the extended autozygous stretch on chromosome 3q13.13-q13.31. Of these, only one coding variant had MAF > 0.01, namely frameshift c.438_442delTTTTA (p.T148Sfs*2) in the gene C3orf52 (MIM 611956) which was neither found in ExAc nor in 1000 genome database. The variant was predicted to be damaging by available bioinformatics tools such as SIFT, PolyPhen, Mutationtaster, MutationAssessor, GERP++ and phyloP. The variant c.438_442delTTTTA was not found in the exome sequence data of 50 unrelated Pakistani individuals with non-hypotrichosis phenotypes. The co- segregation of the variant with disease phenotypes was confirmed by Sanger sequencing the DNA samples of all available members of the family (Figure 7.5).

Family O

Family History and Clinical Features

The consanguineous family O with atrichia was sampled from district Lower Dir, KPK, Pakistan. This was a six generation pedigree (Figure 7.6a) comprising of twenty nine members including five affected (I-2, IV-3, IV-6, IV-9, VI-1). For this study, blood samples were collected from nine members including three affected (IV-3, IV- 9, VI-1) and six unaffected (III-3, IV-1, V-3, V-5, V-6, V-8). At birth affected individuals had normal scalp hair, eyebrows and eyelashes. However, hair loss started in early childhood. At the time of study complete hair loss from scalp, axillae, eyelashes, eyebrows, pubic region and other body parts was reported in affected individuals (IV-3, IV-6, IV-9). One of the affected members VI-1, aged one and half year, presented sparse eyebrows and eyelashes; but lacked hair on scalp (Figure 7.6b, c).

Genotyping and Sequencing On the bases of phenotypes similarities with those reported previously, linkage in the family was tested to HR and DGS4 genes. Genotyping using microsatellite markers

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 119 Chapter 7 Hereditary Hair Loss Disorders was performed to search for the linkage. Analysis of haplotypes revealed linkage in the family to the gene, HR, located on chromosome 8p21.3. Subsequently, HR and U2HR were Sanger sequenced in the family, however, Sequence data analysis didn’t identify any potential pathogenic variant. As a next step to search for the linkage in the family, DNA of all available members was subjected to SNP-based human genome scan. Genotyping revealed single homozygous region of 17.07 Mb (11.83- 28.90 Mb) on chromosome 8p23.1-p12, flanked by rs10086500 and rs10088428 SNP markers. This was followed by exome sequencing using DNA sample of affected individual (VI-1). Analysis of exome data revealed 25 sequence variants within the autozygous region on chromosome 8 (11.83-28.90 Mb). Among those, five variants (c.226_231delCACTCC in FDFT1, c.2495C>T and c.1771A>G in MTUS1, c.18_26delGCGGGGGAT in ATP6V1B2 and c.661C>T in REEP4) were present in the exons and had MAF > 0.01 in ExAc database (Table 7.2). Sanger sequencing, based on dideoxy chain termination, validated co-segregation of the variant c.2495C>T (p.Pro832Leu) in the MTUS1 gene with disease phenotypes in family O (Figure 7.7).

Family P

Family History and Clinical Features

Three generation family pedigree P (Figure 7.8a), resident of Baluchistan province, was found segregating syndromic form of hair abnormality in autosomal recessive pattern. The pedigree comprised of twelve members including four affected (III-1, III-2, III-3, III-4). For DNA extraction and analysis, blood samples were collected from two affected (III-1, III-2) and three unaffected members (II-1, II-2, II-3). Comprehensive clinical examination of the patients was carried out at Combined Military Hospital (CMH) located at Rawalpindi city of the country. Affected individual (III-2), aged seven years, exhibited sparse scalp hair and sparse eyebrows, however, her eyelashes were normal. She also had macrocephaly with prominent forehead and facial deformities (Figure 7.8b, c). Another affected individual (III-1), aged five years, portrayed over dense eyebrows, long eyelashes, and hypertrichosis on facial region, legs and back (Figure 7.8d, e). In addition, both affected individuals (III-1, III-2) had features of profound deafness, speech problems, growth retardation and were below the mean age-based height of Pakistani children (Aziz et al., 2012).

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History of the family, as revealed by the parents, two other children showing similar features passed away at the age of three to eight years. Obligate heterozygous carriers were clinically indistinguishable from healthy individuals.

Genotyping and Sequencing

In order to map the gene, underlying phenotypes observed in the family, SNP-based human genome scan was performed using DNA extracted from blood of five available family members (II-1, II-2, II-3, III-1, III-2). Analysis of the microarray data revealed single autozygous region of 3.18 Mb (22.3-25.41 Mb) on chromosome 22q11.23, flanked by SNP markers rs6003643 and rs7290901. Two genes including SMARCB1 and CD200, located in the candidate region, were sequenced using dideoxy chain termination method. After failing to detect any potential variant in the two genes, as a next step in search of nucleotide variation, whole exome sequencing was carried on DNA sample of an affected individual (III-2). This led to the identification of thirteen variants within the autozygous region on chromosome 22 (Table 7.3). Of these, only chr22:25270390G>A (c.1300G>A, p.Asp434Asn), within the SGSM1 gene, was considered as a rare variant with MAF > 0.000379 in South Asian ExAc alleles. This variant was predicted to be damaging by bioinformatics tools such as SIFT, PolyPhen, Mutationtaster, MutationAssessor, GERP++ and phyloP. Also, the variant was not found in 50 in-house exomes. Sanger sequencing of the gene SGSM1 confirmed co-segregation of the variant chr22:25270390CG>A (c.1300G>A, p.Asp434Asn) with disease phenotype in the family. This was based on the fact that both affected individuals (III-1, III-2) were homozygous for the mutation, parents (II-2, II-3) heterozygous and unaffected (II-1) homozygous for the wild type allele (Figure 7.9b-d).

Family Q

Family Q was first described by another student in the laboratory in his PhD thesis (Chapter 3, Jan A, 2016). It was a four generation pedigree (Figure 7.10a) and had twenty one members including four affected (IV-1, IV-2, IV-3, IV-9). For DNA extraction blood samples were obtained from nine members including aforementioned affected and five unaffected (III-3, III-4, IV-4, IV-7, IV-8). Affected individuals were 9–20 years old at the time of sampling. They presented phenotypic variability of hair loss with sparse and thin hair over scalp. More severe sparseness was observed at

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 121 Chapter 7 Hereditary Hair Loss Disorders frontal, temporal and occipital regions of the scalp (Figure 7.10b-e). In adult affected individuals no beard, moustache, pubic and axillary hair were observed. Affected members were mentally normal and studying in schools and colleges. Features observed in the affected members overlapped with phenotypes presenting Woodhouse Sakati syndrome. Using whole genome homozygosity mapping, an autozygous region of 10.85 Mb was identified on chromosome 2q31.1–q32.2, flanked by rs4148797 and rs7558006 SNP markers. However, the casual gene was not reported by Jan (2016).

The study, carried out by this author, involved exome sequencing on DNA samples of two affected individuals (IV-1, IV-2) in a search to find the disease causing gene. Analysis of exome data of the autozygous region, located on chromosome 2, identified a start loss variant c.1A>G (chr2:172291088A>G) in the DCAF17 gene, earlier reported for Woodhouse-Sakati syndrome phenotypes. This variant was found in homozygous state in exome data of both affected individuals. Previously, this variant (c.1A>G, p.Met1?) was reported only once in heterozygous state in South Asian ExAc database. The co-segregation of the variant with the disease phenotypes in the family was confirmed by Sanger sequencing of exon 1 of DCAF17 gene (Figure 7.11). This variant was not found in 50 chromosomes screened from the same ethnicity group individuals. Bioinformatics tools such as SIFT, PolyPhen, Mutationtaster, MutationAssessor, GERP++ and phyloP predicted the variant as disease causing.

Discussion

This chapter describes clinical and genetic investigation of five families (M-Q), segregating various types of hair abnormalities, are presented in this chapter. In the first family, M, presented here, affected individuals exhibited progressive patchy hair loss from scalp without effecting structure of hair on rest of the body parts. Onset of the disease was noted early in first three years of life. In addition to patchy hair loss, mild intellectual disability was reported in some of the affected individuals of the family. Direct exome sequencing, performed in multiple individuals in the family, identified a heterozygous sequence variant (c.5485C>G, p.Leu1829Val) in the BTAF1 gene. The BTAF1 is an evolutionary conserved protein that belongs to SNF2-like family of ATPase proteins and in association with TBP (TATA-binding protein) forms the B-TFIID complex which is associated in RNA polymerase II transcription. TBP identifies the TATA box within RNA polymerase II (pol II) promoter and assist Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 122 Chapter 7 Hereditary Hair Loss Disorders the recruitment of pre-initiation complex. BTAF1 regulates the assembly and activity of TBP on promoter DNA (Pereira et al., 2004).

BTAF1 expression has been reported in a number of human tissues including hair follicles (Bastian et al., 2008). Wansleeben et al. (2011) carried out detailed experiments to define transcription regulatory role of BTAF1. These authors have demonstrated that the biallelic mutations in the BTAF1 significantly miss-regulate the transcription of several genes including those associated with structure and development of hair, heart and nervous system. The mutant mouse showed severe phenotypes of cardiac edema, disturbed blood circulation, delay in head and brain development (Wansleeben et al., 2011).

The variant (c.5485C>G, p.Leu1829Val) identified in the present study was in heterozygous state and located near the C-terminal end of the protein and therefore expected to generate mild clinical features. Previously, Wansleeben et al. (2011) have reported miss-regulation of five genes (aldh1a3, fgf5, eda2r, ar, ), which are central to the outcome of hair phenotypes, as a result of mutant btaf1 in mouse. Therefore, in the present study, effect of the mutant BTAF1 on expression of the same five genes (ALDH1A3, FGF5, EDA2R, AR, KRT81) was tested. mRNA comparative expression for these five genes was studied in three affected and one normal member of the family. Quantitative PCR analysis revealed down-regulation of these genes (Figure 7.3). The gene, ALDH1A3, encodes for aldehyde dehydrogenase 6, and express in various epithelial and mesenchymal tissues including hair follicles throughout hair cycle. ALDH1A3 is involved in retinoic acid biosynthesis in different layers of hair follicle, sebaceous gland, and inter-follicular epidermis in a hair cycle- dependent manner. The pattern of retinoic acid biosynthesis suggests its role in regulation of hair follicle cycling, differentiation and growth (Everts et al., 2007). The FGF5 encoding protein belongs to the FGF (fibroblast growth factor) family, which plays role in different biological procedures, such as tissue repair, embryonic development, cell growth, morphogenesis, tumor growth and invasion. FGF5 has a prominent role in hair growth and it’s down regulation is reported in alopecia areata (Subramanya et al., 2010). The gene, EDA2R, encodes a transmembrane protein of the TNF-receptor superfamily. This protein binds to the EDA-A2 isoform of ectodysplasin, which has significant role in maintenance of hair and teeth. AR/EDA2R locus has been implicated with female pattern hair loss (FPHL) (Redler

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 123 Chapter 7 Hereditary Hair Loss Disorders et al., 2012). The KRT81, encodes for protein, keratin-81, which is highly expressed in cortex and medulla of hair’s shaft. Mutations in KRT81 have been reported to be implicated with monilethrix (Winter et al., 1997, 1998; Ferrando et al., 2012).

In family N, affected individuals presented a slightly novel pattern of hair loss. Hairs were sparse on scalp and in beard (males), and missing from the remaining parts of the body. Eyebrows and eyelashes were normal. To find out the disease causing gene whole genome scan was carried out using SNP markers. This led to the mapping an autozygous region of 4.07 Mb (110.35-114.28 Mb) on chromosome 3q13.12-q13.31. After failing to detect potential variants in the selected two candidate genes (CD200, PVRL3), located within the autozygous region, exome sequencing was performed in an affected individual (IV-6). Analysis of the exome data identified a homozygous frameshift mutation (c.438_442delTTTTA) in the C3orf52 gene, located within the homozygous region. The C3orf52 spans 33 kb region on chromosome 3q13.2 and composed of six exons. It encodes a predicated membrane protein TTMP (TPA induced trans-membrane protein) (Chan et al., 2005), however, the precise function of the protein is yet to be explored. The C3orf52 protein expression has been reported in a number of tissues such as nerve, endocrine, urogenital and muscles; however, in cutaneous tissues its mRNA exists with high concentration (Duarte and Blackburn, 2017).

The frameshift variant c.438_442delTTTTA (p.T148Sfs*2) is located in exon 4 of the C3orf52 gene and predicted to probably consequence in a truncated polypeptide of 148 residues instead of 217 amino acids normal protein. The variant is predicted to cause instability of the truncated protein or its degradation by nonsense mediated decay leading to complete loss of function of C3orf52. The occurrence of a single autozygous region in a large pedigree, the co-segregation of only one variant with the phenotypes in the family and the rarity of the variant in public databases, together support the causality of the variant for disease phenotypes.

In family O, affected individuals were devoid of hair from the scalp and other body parts. Eyebrows and eyelashes were missing as well. Beard and moustaches were missing in affected male members. Genotyping followed by exome sequencing result in the identification of MTUS1 as the casual factor for disease phenotypes manifested in the family O. Sanger sequencing confirmed co-segregation of the sequence variant

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MTUS1c.2495C>T (p.Pro832Leu) with the disease phenotype in the family. The gene MTUS1 is located on chromosome 8p22-p21.3 (Seibold et al., 2003). Alternative splice site mechanism has been reported in this gene which gives rise to multiple transcript variants encoding different isoforms. The longest transcript (NM_001001924) consists of fifteen exons. It encodes microtubule associated scaffold protein-1, containing a C-terminal domain for interaction with AT2 (angiotension II) receptor and a large coiled-coil region for dimerization. Different transcripts are expected to codes for proteins of different functions. One such transcript encodes a mitochondria related molecule that performs tumor suppressor activity and participates in AT2 signaling pathways. Other transcripts may encode transmembrane or nuclear proteins; however, it is undefined whether they also involved in AT2 signaling pathways. The MTUS1 expression has been reported in several tissues including skin (Duarte and Blackburn, 2017). Moreover, MTUS1 is a candidate cancer suppressor gene and alteration in its expression has been associated with development and progression of different cancers. MTUS1 also plays role in regulation of several cellular processes including proliferation, differentiation, inflammation, DNA repair, senescence and vascular remodeling (Bozgeyik et al., 2017).

Clinical features of hypertrichosis, prominent forehead and extended nasal bridge, neurological anomalies, deafness, cardiac and urinary complications observed in affected members in family P, overlapped with several hypertrichosis syndromes including Cornelia de Lange syndrome (Ramos et al., 2015; Ayerza et al., 2017), Coffin-Siris syndrome (Tsurusaki et al., 2012; Kosho and Okamoto 2014; Zweier et al., 2017) and Barber-Say syndrome (Barber et al., 1982; Zweier et al., 2017). Autozygosity mapping and exome sequencing revealed a missense variant (p.Asp434Asn) in the SGSM1 gene. Sanger sequencing of SGSM1 in all members verified co-segregation of the variant with disease phenotypes in the family.

The gene SGSM1 is located on chromosome 22q11.2 and generates three transcripts; the longest one is composed of 26 exons. The SGSM1 codes for small G protein signaling modulator 1, which is one of the three small G protein signaling modulators (SGSM1/2/3). These proteins have guanine binding capacity with GTPase activity, which has a number of molecular functions such as differentiation, proliferation and other molecular mechanisms within cells (Yang et al., 2007). SGSM interacts with

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RAB and RAP proteins. RAP proteins are involved in various cellular activities, such as cadherin-mediated cell junction formation, integrin-mediated cell adhesion, neuronal cells differentiation and synaptic plasticity and Wnt/β-catenin signaling pathway (Yang et al., 2007). RAB proteins act as molecular switches by interchanging between active and inactive forms. The RAB proteins are localized on several cellular membranes, involved in membrane traffic, synaptic transmission, endosomal regulation, secretion in cytotoxic T lymphocytes and distribution of melanosomes (Yang et al., 2007). SGSM1 mainly expressed in brain and cardiac tissues, however, its considerable expression has been reported in other tissues such as testis, adipose tissues, endocrine, muscles and skin (Fagerberg et al., 2014).

Mutation in ATP-binding cassette, subfamily A, member 5 (ABCA5; MIM 612503) was identified in a family showing features of hypertrichosis as described previously by DeStefano et al. (2014). ABCA5 belongs to ATP-binding cassette (ABC) transporters, which execute translocation of different substrates through membranes. Molecular defects in these molecules result in a number of clinical conditions such as retinal degeneration, neurological manifestations, cystic fibrosis, cholesterol and bile transport defects (Dean et al., 2001). The sequence variant identified in family P possibly alters the structure and function of SGSM1 protein, consequently leads to defects in membrane traffic of specific molecules. Thus, the complex phenotypes of hypertrichosis, macrocephaly, neurological and cardiac manifestation, deafness, and urinary complications appeared.

In family Q, affected members presented sparse and thin hair over scalp. In adult affected individuals no beard, moustaches, axillary and pubic hair were observed. Homozygosity mapping followed by exome sequencing identified a start loss variant (p.Met1?) in the DCAF1 gene. The gene DCAF17 is mapped on chromosome 2q31.1 and encodes for a nucleolar protein with two known transcripts, alpha (NP_001158293.1) and beta (NP_079276.2). In different species the amino acid sequence of the protein is well conserveds (Alazami et al., 2008). In adult human both the isoforms show low ubiquitous expression in various adult tissues including gastrointestinal, endocrine, heart, bone marrow, urogenital systems and skin (Fagerberg et al., 2014). Previously, twelve sequence variants in the DCAF17 gene were reported for Woodhouse-Sakati syndrome (WSS; MIM 241080) including missense, nonsense, frameshifts and splice site variants (Stenson et al., 2017).

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WSS is a rare autosomal recessive condition featured by hypotrichosis, intellectual disability, diabetes mellitus, sensorineural hearing loss, extrapyramidal manifestation and hypogonadism. In WWS phenotypic variability has been reported in different families and even among different individuals within the same kindred (Alazami et al., 2008, 2010; Habib et al., 2011; Abdulla et al., 2015; Ali et al., 2016). Most patients exhibited with mental retardation and delayed puberty; however, hypotrichosis exist in all affected individuals and ranges from sparse hair to complete absence of hair from scalp, eyebrows, eyelashes and other body parts. The occurrence of only hair phenotypes in patients of the present family is probably due to difference in the position and nature of the mutation. The DCAF17 translation initiation codon mutation described here would truly abolish DCAF17 protein, whereas more distal nonsense mutations and frameshifts may produce truncated DCAF17 polypeptides that do not entirely lack function and exert toxic or neomorphic effects. Alternatively, there may be common second-site modifiers that strongly modulate the effects of loss of DCAF17 function and consequent phenotypes.

In summary, five Pakistani families with various hair abnormalities were evaluated at clinical and molecular level. Genetic analysis identified four novel potential candidate genes (BTAF1 in family M, C3orf52 in family N, MTUS1 in family O, SGSM1 in family P) and a novel sequence variant (p.Met1>?) in the DCAF17 gene in family Q. This study extends the genetic heterogeneity of hair conditions that will help clinicians and biologists to better understand the molecular bases underling hereditary hair conditions.

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Table 7.1: List of potential variants identified in exome data of family M Chromosome Position Gene Effect Frequency cDNA Zygosity 10 28233769 RPA2 Missense NR c.166C>A Het 10 93788625 BTAF1 Missense NR c.5485C>G Het 10 95169365 MYOF Missense NR c.565C>T Het 10 102268866 SEC31B Splice site NR c.400C>G Het

Table 7.2: List of rare variants extracted from exome data of autozygous region in family O

Chromosome Position Gene Effect Frequency cDNA Zygosity Inframe 8 11666218 FDFT1 NR c.226_231delCACTCC HOM deletion 8 17573365 MTUS1 Missense 0.000041 c.2495C>T HOM 8 17611546 MTUS1 Missense 0.00236 c.1771A>G HOM Inframe 8 20054931 ATP6V1B2 0.000023 c.18_26delGCGGGGGAT HOM deletion 8 21996199 REEP4 Missense 0.0054 c.661C>T HOM

Table 7.3: List of variant extracted from exome data of autozygous region on chromosome 22q11.23, in family P

Chromosome Position Gene Effect Frequency cDNA Zygosity 22 23964282 DRICH1 Inframe_deletion 0.124002 c.377_379delATG HOM 22 24035970 RGL4 Missense 0.774561 c.721C>T HOM 22 24038847 RGL4 Missense 0.76857 c.1133T>C HOM 22 24,121,378 MMP11 Missense 0.708267 c.113C>T HOM 22 24224655 SLC2A11 Missense 0.34365 c.716G>A HOM 22 24325095 GSTT2 Missense 0.63119 c.385A>G HOM 22 24340938 GSTTP1 Splice variant 0.779153 n.383-5T>C HOM 22 24468386 CABIN1 Missense 0.0613019 c.2558G>A HOM 22 24717850 SPECC1L Missense 1 c.902A>G HOM 22 24761467 SPECC1L Missense 0.9998 c.2851G>A HOM 22 25145453 PIWIL3 Missense 0.926518 c.1252G>A HOM 22 25145471 PIWIL3 Missense 0.672923 c.1234T>C HOM 22 25270390 SGSM1 Missense 0.000262 c.1300G>A HOM Hom= homozygous, NR= Not Reported, Hom= homozygous

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Figure 7.1: Pedigree drawing and clinical presentation of affected individuals in family M. (a) Pedigree showing six affected individuals (II-2, lll-3, lll-4, lll-5, lll-6, lll-8). Filled symbols indicate affected, unfilled unaffected and crossed symbols deceased individuals. Individuals who were available for the study are shown with asterisks. Arrows indicate those analyzed by exome sequencing. (b-d) Clinical presentation of hypotrichosis in affected members of the family. (b, c) Affected individual III-3 showing patchy hair loss on scalp; (d) affected individual III-6 showing thin sparse scalp hair.

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Figure 7.2: Analysis of Sanger sequencing of BTAF1 in family M. (a) Homozygous wild type allele in normal individuals (II-1, II-4, III-7); (b) BTAF1 allele (c.5485C>G) in heterozygous state in affected individuals (ll-2, lll-3, lll-4, lll-5, lll-6, lll-8) of the family.

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Figure 7.3: Comparative expression analysis of KRT81, EDA2R, FGF5, ALDH3A2 and AR genes in normal and affected individuals in family M. Expression of genes in percentage is shown on Y axis and names of the genes on X-axis. purple bars represent unaffected individual (II-1). Green, blue and maroon represent affected individual III-5, III-4 and III-3, respectively. Table below explains the graphical representation.

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Figure 7.4: Pedigree and clinical features in affected individuals in family N. (a) Five generation pedigree of the family with six affected individuals. Filled symbols represent affected and unfilled unaffected members. Double line represents consanguineous union, and crossed symbols represent deceased individuals. Individuals who were available for the study are shown with asterisks, while, arrow indicate those analyzed by exome sequencing. (b-d) Presentation of clinical features observed in affected individuals in the family. (b) Affected IV-6 showing sparse beard and missing hair on chest and and legs (c). (d) Affected female individual (IV- 8) with sparse, short and thin hair on scalp.

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Figure 7.5: Sequence chromatogram of C3orf52 gene in family N. (a) Homozygous wild type allele in unaffected individual (IV-1); (b) heterozygous allele pattern (C3orf52 c.438_442delTTTTA) in carriers (III-5, IV-7, V-4); (c) homozygous mutant allele in affected individuals (lV-2, lV-4, lV-6, lV-8) of the family.

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Figure 7.6: Pedigree diagram and clinical presentations of family O. (a) Six generation pedigree of the family with four affected individuals. Squares symbolize males and circles symbolize females; filled symbols indicate affected and unfilled unaffected indicate members. Samples of the individuals labeled by asterisks are those which were available for the study; Arrow shows the individual analyzed by exome sequencing. Affected individual VI-1 in the family with (b) sparse eyebrows and eyelashes and (c) total hair loss on scalp.

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Figure 7.7: Analysis of Sanger sequencing of MTUS1in family O. (a) Homozygous wild-type allele in unaffected member IV-1; (b) heterozygous mutant and wild-type alleles in carriers V-3, V-5, V-6 and V-8; and (c) homozygous missense variant (c.811delA) in affected members IV-3, IV-9 and VI-1.

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Figure 7.8: Pedigree and clinical features of affected individuals in family P. (a) Four generation pedigree of the family with four affected individuals. Filled symbols indicate affected, unfilled unaffected, double lines consanguineous union, and crossed symbols deceased individuals. Individuals labeled with asterisks were available for the study, and arrow indicate the individual analyzed by exome sequencing. (b-e) clinical features observed in affected individuals in the family: (b, c) Affected individual (III-2) with macrocephaly, prominent forehead and broad nasal bridge. (d, e) Dense eyebrows, long eyelashes, and hypertrichosis on facial region and back of an affected individual (III-1).

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Figure 7.9: Presentation of autozygous region on chromosome 22 and Sanger sequence of SGSM1 gene in family P. (a) Autozygous region of 3.18 Mb (22.3-25.41 Mb) on chromosome 22q11.23 obtained with SNP microarray. (b-d) Sanger sequence analysis of SGSM1 gene: (b) wild type allele in unaffected member II-1; (c) heterozygous mutant/wild type alleles in obligate heterozygous parents (II-2, II-3); and (d) homozygous mutant (SGSM1 c.1300G>A) allele in affected individuals (III-1, III-2).

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Figure 7.10: Pedigree drawing and presentation of clinical features observed in affected individuals in family Q. (a) Four generation pedigree of the family segregating WSS phenotypes in autosomal recessive manner. Individuals available for the study are shown with asterisks, and those analyzed with exome sequencing are shown with arrows. (b-e) Affected individuals lV-1, lV-2, lV-3 and lV-9 showing sparse and curly hair on scalp. Sparseness is more prominent on temporal regions.

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Figure 7.11: Sequence analysis of DCAF17 gene in family Q. (a) upper panel shows homozygous wild type allele in individual IV-8, (b) middle panel show mutant/wild type alleles in carrier individuals lll-3, lll-4, lV-4, lV-7, and (c) the lower panel shows homozygous mutant allele (DCAF17 c.1A>G) in affected individuals lV-1, lV-2, lV-3 and lV-9 in family Q.

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 139 Chapter 8 Conclusion

CONCLUSION

The term consanguineous marriage is used for union between biologically related persons. In medical genetics the consanguineous marriage is defined as a union between a couple related as second cousins or closer, equivalent to a coefficient of inbreeding in their progeny of F0.0156. Due to several geographic, religious, cultural and socioeconomic factors consanguinity is common in communities of North Africa, Middle East and West Asia, while, its prevalence is low in Western countries. Approximately, 690 million people in the world are in consanguineous union (Bittles and Black, 2010). Pakistan is among the countries with highest prevalence of consanguineous marriages, where more than 60% of the total marriages are consanguineous, and of these, about 80% are among first cousins (Bittles, 2001). In Pakistan consanguineous marriages are preferred due to higher understanding between the partners as well other members of their families, as they share several social and economic values. The high rate of consanguinity results in a gradual increase of genome wide homozygosity. Consequently, producing children with genetic defects, by favoring the expression of recessive deleterious alleles inherited from common ancestors, than that of the general population (Kumaramanickavel et al., 2002). Each year, approximately 6% of total births (7.9 million babies around the globe) have severe congenital defects of genetic or partially genetic origin, with highest (8.2%) prevalence in Sudan and lowest (3.97%) in France. While, in Pakistan 7.9% children are born with genetic defects (Christianson et al., 2005).

Monogenic inherited diseases are rare, but ever more affecting millions of individuals worldwide. These conditions may be chronically devastating and life-threating and despite the inimical effects on patients and their families, these diseases have an incredible cost for health-care structures and societies. Unfortunately, effective therapies for these conditions are themselves reasonably rare. Genetic skin disorders account for one third of all monogenic diseases (Scriver et al., 2001). Diagnosis of human hereditary skin disorders is a quite challenging job for the clinicians and geneticists due to overlapping phenotypes and allelic and locus heterogeneity. Recent advances in molecular genetic techniques notably whole exome sequencing (WES) and microarray have incredibly accelerated the identification of genes involved in inherited diseases. These state-of-the-art technologies have helped investigators to

Mapping Genes Causing Syndromic and Non-Syndromic Human Hereditary Skin Disorders 140 Chapter 8 Conclusion comprehend the complex mechanisms involved in regulating the skin and its associated appendages.

In this dissertation seventeen families are described, segregating syndromic and non- syndromic forms of skin disorders. These included five families with ectodermal dysplasia, three with ichthyosis, two with epidermolysis bullosa, two with trichothiodystrophy and five with hair abnormalities.

Genetic mapping and sequencing analysis detected five novel candidate genes which include KRT83 for erythrokeratoderma phenotypes in family H; SGSM1 for syndromic hypertrichosis in family P; BTAF1, C3orf52 and MTUS1 for isolated hypotrichosis in family M, N, O, respectively. Identification of the novel genes in these disorders will be of great interest to clinicians and biologists for molecular diagnosis and understanding the disease mechanism and function of the genes.

For the first time, SLCO2A1 was associated with autosomal recessive ICNC in family B. Previously a single heterozygous sequence variant had reported in SLCO2A1 for ICNC. The first start-loss sequence variant in DCAF17 was identified in family Q for Woodhouse Sakati syndrome. In family F and G, segregating nonphotosensitive trichothiodystrophy (TTDN), a novel splice site variant was identified and mitral regurgitation was reported as a part of TTDN phenotypes. For the first time, mitral regurgitation phenotypes were associated with mutation in MPLKIP gene in our patients of family F and G. Moreover, in four families novel pathogenic sequence variants were found. These included SLURP1 c.2T>C for Mal de Meleda type palmoplantar hyperkeratosis in family A; ALDH3A2 c.10G>T for Sjögren-Larsson syndrome in family I, ABHD5 c.836delA for Chanarin-Dorfman syndrome in family J; and PLEC c.10909C>T in family L. Genetic analysis of three other families (C, D, K) revealed recurrent sequence variants in known genes: RSPO4 c.-9- +17del26 for anonychia in family C, FZD6 c.1265G>A for claw shaped nails in family D and KRT14 c.92delT for epidermolysis simplex in family K. In three of the studied families (A, B, C) translation initiation codon mutations (p.Met1?) were identified in three different genes (SLCO2A1, DCAF17, RSPO4, the major consequence of such mutation, is probably either the absence of the protein or the synthesis of a peptide starting at the next ATG, losing some of its amino acid residues and resulting in defective function.

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Understanding the genetic basis underling human disease is one of the central aims for exploring the human genome. Recent advances in genotyping and DNA sequencing technologies has changed the landscape of rare-genetic-disease research, with casual genes being identified at an accelerating rate. This advancement resulted in identification of ~50% of estimated 7000 Mendelian disorders, and the remaining disease-causing genes will be explored soon. Identification the molecular bases of genetic disorders will significantly improve understanding of the disease mechanism, genotype-phenotype correlation and provide insights into the gene function. In turn, this will be helpful in promoting health in term of improved diagnosis with prognostic implications, effective genetic counseling, prenatal DNA testing and combating the disease. Most of the genetic diseases are not curable, however, early diagnosis can help improve the quality of life or even prolong the lifetime of victims. Current clinical trials on genetic therapies for skin disorders offer the promise of eventual treatments that may give sufferers a life free of ailments. Diagnostic tests can help couples decide whether to risk passing on specific disease-related genes to their children. Tests assist in vitro fertility doctors to specifically select embryos that do not carry the risky gene.

Increase in the rate of disease genes identification means that, in effect, there is an almost equal increase in the number of molecularly defined, readily diagnosable, disease condition. This in turn means the hope of obvious and facile therapeutic approach. An effort made to analyze some of the rare genetic skin diseases, presented here, will contribute in improving care and quality of life of the patients.

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