Descriptive Study on Phenotypes of Genetic Eye Disorders Presented to the Ophthalmo-Genetic Clinic at the Faculty of Medicine Colombo

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Descriptive study on Phenotypes of Genetic Eye Disorders Presented to the Ophthalmo-genetic clinic at the Faculty of Medicine Colombo

KUSHARA NUWANTHI DILSHANIE WEERAPPERUMA

(M.B.B.S. Colombo)

REG NO 25563

DISSERTATION SUBMITTED TO

THE UNIVERSITY OF COLOMBO, SRI LANKA

IN PARTIAL FULFILMENT OF THE REQUIREMENTS OF THE

MASTER OF SCIENCE IN CLINICAL GENETICS

AUGUST 2014

CERTIFICATION

I certify that the contents of this dissertation are my own work and that I have acknowledged the sources where relevant.

…………………………………………

Signature of the candidate

This is to certify that the contents of this dissertation were supervised by the following supervisors:

…………………………….

NAME OF SUPERVISOR

……………………………. …………………………..

Dr Dulika Sumathipala Prof. V.H.W. Dissanayake

ACKNOWLEDGEMENTS

I would like to thank my supervisors Prof. Vajira H.W. Dissanayake, Professor in Anatomy and Medical Geneticist, Human Genetics Unit, Faculty of Medicine, University of Colombo and Dr. Madhuwanthi Dissanayake, Head of the department in the department of Anatomy,

Faculty of Medicine, University of Colombo for their valuable input, guidance and supervision during the study. This research was supported by the NOMA grant funded by

NORAD in collaboration with the University of Colombo, Sri Lanka & the University of

Oslo, Norway.

I would specially thank Dr. Dharma Irugalbandara, Consultant pediatric Ophthalmologist, Dr.

Hiranya Abeysekera, Senior registrar in pediatric ophthalmology and all the medical officers in Ophthalmology Unit, Lady Ridgeway Children’s Hospital, Colombo, for support provided regarding the recruitment of patients. I would also like to thank Consultant Ophthalmologists

Dr.Muditha Kulatunga, Dr. Binara Amarasinghe, Dr. Deepanee Wewalwala, Dr. Mangala gamage, and Dr Manel Pasquel from National eye hospital, Colombo, and Ophthalmology

Units in General Hospitals, for support provided regarding recruitment of patients. I wish to thank all our patients and their families for their participation in this study.

I also wish to acknowledge Miss P.K.D.S. Nisansala and Mrs. S.S.S.M. Bandaranayake of the

Human Genetics Unit, Faculty of Medicine, Univercity of Colombo for their support and assistance.

I thank Lord Buddha for inculcating the ethical background in me and strengthening mind to face challenges. I thank my parents for their confidence, strength and enhancement, throughout my educational pathway. Their expectations motivated me to complete this task.

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TABLE OF CONTENTS

ACKNOWLEDGEMENT...... i DECLARATION…………………………………………………………………. ii LIST OF TABLES …………………………………………………………………… vii LIST OF FIGURES………………………………………………………………….. ix ABSTRACT……………………………………………………………………….. xii PUBLICATIONS AND ABSTRACTS………………………………………… xiv

1.0 Introduction………………………………………………………………….. 1 1.1 Background ……………………………………………………………. 1 1.2 Retinoblastoma …………………………………..…………..………… 2 1.2.1 Diagnosis of retinoblastoma………………………………….. 2 1.2.2 Classification of Retinoblastoma……………………………... 4 1.2.3 Genetics of Retinoblastoma…………………………………... 7 1.2.4 Two hit hypothesis……………………………………………. 9 1.2.5 Place of Chromosome analysis in Retinoblastoma………….. 10 1.2.6 Molecular genetic testing of Retinoblastoma………………... 11 1.2.7 Genetic counseling of Retinoblastoma……………………….. 15 1.2.8 Management of Retinoblastoma……………………………... 20 1.2.9 Surveillance of Retinoblastoma……………………………... 20 1.3 Retinitis pigmentosa…………………………………………….. …….. 22. 1.3.1 Diagnosis of Retinitis pigmentosa …………………………... 22 1.3.2 Genetics and inheritance of Retinitis pigmentosa …………… 22 1.3.3 Clinical Manifestations of Retinitis pigmentosa……………. 30 1.3.4 Molecular genetic testing of Retinitis pigmentosa…………… 36 1.3.5 Prevalence of Retinoblastoma……………………………….. 37 1.4 Stargartd macular dystrophy…………………………………………….. 37 1.4.1 Clinical Manifestations of Stargartd macular dystrophy …….. 38 1.4.2 Genetics and inheritance of Stargartd macular dystrophy ……. 38 1.4.3 Pathophysiology of Stargartd macular dystrophy …………….. 41 1.5 Epithelial Basement Membrane type corneal Dystrophy ………………. 41 1.5.1 Clinical Manifestations of EBMD…………………………… 41 1.5.2 Genetics and inheritance of EBMD…………………………. 42

1.6 Aniridia ………………………………………………………………. 43 1.6.1 Diagnosis of Aniridia ...………………..…………………... 43 1.6.2 Genetics and Inheritance of Aniridia……………...... 44 1.7 Coloboma………………………………………………………………. 46 1.8 Waardenburg syndrome………………………………………………... 48 1.8.1 Diagnostic criteria of Waardenburg syndrome ……………... 48 1.8.2 Clinical Manifestations of Waardenburg syndrome ………… 50 1.8.3 Phenotypes of Waardenburg syndrome ……….……………. 50 1.8.4 Genetics and inheritance of Waardenburg syndrome ………. 54 1.9 Blepherophimosis Ptosis Epicnthus Inversus Syndrome (BPES)……. 55 1.9.1 Clinical features of BPES…………………………………… 55 1.9.2 Diagnostic criteria of BPES ………………………………… 56 1.9.3 Genetics of BPES……………………………………………. 57 1.9.4 Patterns of inheritance of BPES……………………………… 59 1.10 Moebius syndrome…………………………………………………….. 59 1.11 Justification……………………………………………………………. 60 1.12 Objectives……………………………………………………………… 60 2.0 Methodology ………………………………………………………………… 69 2.1 Study design…………………………………………………………… 69 2.2 Subjects……………………………………………..…………..……… 70 2.3 Inclusion criteria………………………………………………………. 71 2.4 Exclusion criteria………………………………………………………. 71 2.5 Obtaining written informed consent…………………………………… 72 2.6 Clinical evaluation…………………………………………………….. 73 2.7 Statistical analysis of data……………………………………………… 74 2.8 Ethical issues relevant to the study…………………………………….. 74

3.0 Results…………………………………………….………………………….. 76 3.1 Retinoblastoma …………………………………..……….…..………... 86 3.2 Retinitis pigmentosa……………………………………………………. 96 3.3 Stargartd macular dystrophy……………………………………………. 104 3.4 Epithelial Basement Membrane type corneal Dystrophy ……………… 105 3.5 Aniridia ………………………………………………………………… 107 3.6 Coloboma………………………………………………………………. 108

3.6.1 Patient 1 with Coloboma (CB1)……………………………… 108 3.6.2 Patient 2 with Coloboma (CB2)….………………………… 108 3.7 Waardenburg syndrome………………………………………………… 109 3.7.1 Patient 1 with Waardenburg syndrome (WB1)………………. 109 3.7.2 Patient 2 with Waardenburg syndrome (WB2)………………. 112 3.8 Blepherophimosis ptosis epicnthus inversus syndrome………………... 113 3.9 Moebius syndrome……………………………………………………. 115 4.0 Discussion…………………………………………………………………… 118 4.1 Retinoblastoma …………………………………..…………….….….. 120 4.2 Waardenburg syndrome (WS1)……………………………………….. 125 4.3 Waardenburg syndrome (WS2)……………………………………….. 130 4.4 Moebius syndrome…………………………………………………….. 133 4.5 Blepherophimosis ptosis epicnthus inversus syndrome………………. 135 4.6 Retinitis pigmentosa…………………………………………………... 137 4.7 Epithelial Basement Membrane type corneal Dystrophy …………….. 139 4.8 Aniridia ……………………………………………………………….. 140 4.9 Coloboma……………………………………………………………… 140 4.10 Stargartd macular dystrophy…………………………………………... 141 5.0 Conclusions………………………………………………………………….. 143 6.0 Limitations…………………………………………………………………… 144 7.0 Recommendations…………………………………………………………… 146

8.0 REFERENCES…………………………………………………………… 147 APPENDIX 1: List of abbreviations……………………………………… 163 APPENDIX 2: Documents used for subject recruitment………………… 164

List of Tables

Table 1.1: Summary of molecular genetic testing used in Retinoblastoma 12

Table 1.2: The probability of germline mutation being present in a proband with RB 18 based on family history and tumor presentation

Table 1.3: Causes of nonsyndromic Retinitis pigmentosa by mode of inheritance 23

Table 1.4: Genes associated with autosomal dominant retinitis pigmentosa 24

Table 1.5: Genes Associated with Autosomal Recessive Retinitis Pigmentosa 27

Table 1.6: Genes Associated with X-Linked RP (xlRP) 30

Table 1.7: Reported mutations in ABCA4 gene that can cause STGD 1 40

Table 1.8: Types of mutations in PAX6 gene that result in aniridia 45

Table 1.9: Major and minor criteria for the diagnosis of Waardenburg syndrome 49

Table 1.10: Phenotypes of Waardenburg syndrome with associated genes/loci and 52 MIM numbers

Table 1.11: FOXL2 Pathogenic Allelic Variants causing BPES 58

Table 1.12: Magnitude of blindness in children according to the region 62

Table 1.13: Magnitude of blindness in children according to the causative factors 63

Table 1.14: Etiological categories in 226 Sri Lankan children attending blind schools 65 with severe visual impairment and blindness

Table 3.1: The consistency of the cohort of patients in the ophthalmo-genetic project 77 and the percentage of patients in each category

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Table 3.2: Summery of ophthalmo-genetic conditions observed within the paediatric 79 group of patients

Table 3.3: Patient numbers and disorder categories 86

Table 3.4: Presenting clinical features of retinoblastoma cohort of patients 87

Table 3.5: Mean age of presentation of bilateral and unilatelal retinoblastomas 89

Table 3.6: Family history positivity in bilateral and unilatelal retinoblastomas 90

Table 3.7: Globe preservation rates of bilateral and unilateral Retinoblastoma 95

Table 4.1: Percentage of patients presented with leucocoria in different countries 121

Table 4.2: Comparisen of clinical presentations of retinoblastomas in different 123 countries

Table 4.3: A comparison of globe preservation and enucleation rates in Bilataral and 123 Unilatral Retinoblastomas in different countries

Table 4.4: Clinical features of Waardenburg syndrome type I (WS1) compared to 126 clinical features of the project patient

Table 4.5: Comparison of clinical features of patient 1, with the reported clinical 128 features of Waardenburg syndrome type

Table 4.6: Comparison of reported clinical features of WS2 with clinical features of 131 project patient who presented with clinical features of WS2

Table 4.7: Comparison of the reported clinical features of WS 1 and WS 2 with the 132 patients who presented to the Ophthalmo-genetic clinic

Table 4.8: Comparison of the clinical features of Moebius syndrome observed in the 134 Dutch cohort with the Sri Lankan patient

Table 4.9: Comparison of clinical features of this patient with the reported cases of 136 BPES 1

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List of Figures

Figure 1.1: Fundus images of two patients with retinoblastomas; Right is a 3 Highly vascular large tumor and the left is a multifocal tumour

Figure 1.2: Cytogenetic location of RB 1 gene at 4 position 13q14.2 in the long arm of chromosome number 13

Figure 1.3: Retinal photographs illustrating multifocal 5 and unifocal retinoblastomas

Figure 1.4: Retinal photographs illustrating macular tumor (photograph on the lft) 6 and peripheral (photograph on the right) retinoblastomas

Figure 1.5: Retinal photographs of early detected retinoblastoma on the left and 7 late presentation of retinoblastoma on the right

Figure 1.6: Diagrammatic representation of the molecular-genetic mechanisms that 10 result in non-hereditary and hereditary Retinoblastoma (Rb)

Figure 1.7: Optical Coherence Tomography (OCT) report from a retinitis 31 pigmentosa patient

Figure 1.8: Fundus photograph of an eye genetics project patient 32

Figure 1.9: Optical Coherence Tomography (OCT) report of an ophthalmo-genetic 36 clinic patient with retinitis pigmentosa demonstrating macular thinning

Figure 3.1: Number and percentage of patients with different ophthalmo-genetic 78 disorders

Figure 3.3: Summery of ophthalmo-genetic conditions observed within the 80 paediatric group of patients

Figure 3.4: Summery of ophthalmo-genetic conditions observed within the adult 81 group of patients

Figure 3.5: Distribution of bilateral and unilateral eye involvement in the paediatric 82 and adult groups of patients and the collective cohort

Figure 3.6: Number and percentages of patients depending on the family history 83 positivity

Figure 3.7: Number of patients with positive family history in each disorder 83

Figure 3.8: Classification of ophthalmo-genetic project patients according to the 84 type of genetic eye disorder

Figure 3.9: Illustration of the non malignant conditions referred to ophthalmo- 85 genetic clinic

Figure 3.10: Common Clinical presentations of retinoblastomas 88

Figure 3.11: Two thirds of the retinoblastomas are unilateral and one third is 88 bilateral

Figure 3.12: Diagrammatic representation of family history positivity of the 90 retinoblastoma group of patients

Figure 3.13: Family history positivity in bilateral and unilatelal Retinoblastomas 91

Figure 3.14: Family tree of the family with mother and child (RT1) affected with 92 Retinoblastoma

Figure 3.15: The family tree of a child (RT2) with positive family history of 93 retinoblastoma and other malignancies which are caused by mutated RB1

Figure 3.16: Recurrence of retinoblastoma in a defaulted patient. This patient had 94 to undergo unilateral enucleation

Figure 3.17: Globe preservation rates of bilateral and unilateral Retinoblastoma 95

Figure 3.18 Clinical of presentation of Retinitis Pigmentosa in the series of 96 ophthalmo-genetic project patients Figure 3.19: The pedigree of the family with brother and sister affected with 97 Retinitis Pigmentosa

Figure 3.20: Family tree of the family with four siblings. Out of four two are 98 affected with Retinitis Pigmentosa

Figure 3.21: Retinal photo grafts of the patient with atypical retinitis pigmentosa 99

Figure 3.22: Family pedigree of the patient with atypical retinitis pigmentosa 100

Figure 3.23: Family tree of the Retinitis Pigmentosa patient who had episodes of 101 red eye

Figure 3.24: Fundus appearance of a patient with Retinitis Pignemtosa 102

Figure 3.25: Family pedigree of the 38 year old Retinitis Pigmentosa patient with 103 one year old child

Figure 3.26: Family tree of the family with two siblings affected with Stargardt 104 disease (ST1 and ST2 patients)

Figure 3.27: Family tree of the family with epithelial basement membrane type 106 corneal dystrophy

Figure 3.28: Family tree of the family with father and son affected with Aniridia 108

Figure 3.29: The child showing clinical features of Waardenburg Syndrome 109

Figure 3.30: Family tree of patient 1 (WB1) 110

Figure 3.31: Ophthalmic features of the WS1 patient; Hypopigmented right iris and 111 retina is in comparison with the normal iris and the retina in the left eye

Figure 3.32: Clinical features of the patient with Waardenburg syndrome type 2 112 (WB2)

Figure 3.33: Eye features of the lady with BPES. Bilateral blepharophimosis, 113 partial ptosis and epicanthus inversus are exhibited.

Figure 3.34 Family tree of the family with Blepherophimosis ptosis 114 epicnthus inversus syndrome

Figure 3.35: Congenital amelia, convergent strabismus and expressionless face 116

Figure 3.36: Facial dysmorphism in the child with clinical features of 117 Moebius syndrome

Figure 4.1: Graphical representation of Percentage of patients presented with 122 leucocoria in different countries

Figure 4.2: A comparison of globe preservation and enucleation rates in Unilatral 124 Retinoblastomas in different countries

Figure 4.3: A comparison of globe preservation and enucleation rates in Unilatral 125 Retinoblastomas in different countries

Abstract

Introduction:

Genetic eye disorders cause significant visual impairment to the affected individuals and burden families. Limited data is available regarding genetic and heritable visual impairment in Sri Lankan children and adults. Hence we present an initial study done to explore the phenotypes of genetic eye disorders seen in Sri Lanka.

Methods:

Clinical genetic evaluation, genetic counseling and genetic risk assessment of 59 patients seen at the ophthalmo-genetic clinic of the Faculty of Medicine Colombo

Results: 54 patients (92%) had isolated eye disorders, while the remaining 5 patients had syndromic phenotypes. Isolated eye disorders include, retinoblastomas 40 (68%), retinitis pigmentosa (RP) 8 (14%), Stargartd disease 2 (3%), isolated Aniridia 2 (3%), isolated

Coloboma 1 (2%) and Epithelial Basement Membrane type corneal Dystrophy (EBMD) 1

(2%). Those with significant eye involvement as part of rare Ophthalmo-genetic syndrome include; Waadernberg syndrome with heterochromia iridis 2 (3%), Mobius syndrome with defective abduction of eyes 1(2%), Blepharophimosis, Ptosis, Epicanthus Inversus Syndrome

(BPES) 1 (2%) and Edward syndromic baby with a Coloboma 1 (2%). Fourteen patients had a positive family history (24%), while 45 were simplex cases (86%).

Discussion:

Phenotypes detected in retinoblastomas, BPES, Aniridia, EBMD and Stargardt disease are similar to phenotypes in the reported literature. Some patients in the Retinitis pigmentosa,

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Waardenburg syndrome and Moebius syndrome categories exhibited atypical clinical features remaining within the diagnostic criteria. Although autosomal dominant (BPES, Aniridia,

EBMD) and autosomal recessive (Stargartd disease, RP) inheritance patterns were detected in this cohort, the majority (86%) were simplex cases. Reduced penetrance, variable expressivity, de novo mutations, (germline) mosaicism and two hit hypothesis are the possible explanations for negative family history. Genetic counseling issues, genetic risk assessment and prevention are discussed depending on each case or category.

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Publications and abstracts

 The abstracts accepted for the SAARC International Ophthalmology Conference which

will be held from 28th to 30th of September.

1. Clinical Presentations, Heredity and Globe Preservation Rates of Retinoblastoma in

Sri Lanka; K.N.D. Weerapperuma, H. Abeysekera, D. Irugalbandara, M.M. Dissanayke,

V.H.W. Dissanayake; Oral presentation

2. Phenotypic characterization of retinitis pigmentosa patients presenting at an

ophthalmo-genetic clinic in Sri Lanka ; K.N.D. Weerapperuma, M.M. Dissanayke, W.

Wijayasiriwardana, B. Amarasinghe , V.H.W. Dissanayake: Poster presentation

3. Waardenburg syndrome: A rare genetic eye disorder with iris heterochromia

associated with marked facial dysmorphism and pectus excavatum: Case report :

K.N.D. Weerapperuma, D. Wewalwala, M.M. Dissanayake, V.H.W. Dissanayake; Poster

presentation

4. Genetic Evaluation and Counseling of a Sri Lankan Family with Autosomal

Dominant Blepharophimosis, Ptosis, and Epicanthus Inversus Syndrome Type I :

K.N.D. Weerapperuma, M.M. Dissanayke, A. Sithambarapillai, V.H.W. Dissanayake;

Poster presentation

5. Moebius syndrome: A rare neuro-developmental disorder restricting ocular

movements, causing marked facial dysmorphism and amelia: Case report; K.N.D.

Weerapperuma, H. Abeysekera, D. Irugalbandara, M.M. Dissanayke, V.H.W.

Dissanayake; Poster presentation

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1. Introduction

1.1 Background

Ophthalmo-genetic disorders are the group of eye disorders caused by genetic etiology. The responsible genes differ according to the disorder phenotype and the population concerned.

The severity of this group of disorders ranges from the simple hypopigmenation of the structures of the eye to blindness. These disorders may involve the interior structure of the eye causing defective vision or may involve ocular muscles interfering with eye movements.

Genetic eye disorders occur as isolated (exclusive) eye disorders (Eg. Aniridia) only affecting the eyes or as part of complex syndrome (Waardenberg syndrome), resulting from a genetic defect.

Clinical presentation of genetic eye disorders differs depending on the disorder phenotype.

The ophthalmo-genetic disorder may be unilateral or bilateral, Symetrical or asymmetrical, familial or sporadic. The disease outcome, prognosis and the genetic risk varies depending on the disorder phenotype.

High percentage of ophthalmo-genetic disorders is congenital (Eg. Coloboma), but minority is late onset (Eg. Retinitis pigmentosa). Therefore majority of the patients with ophthalmo- genetic disorders present within the paediatric age group, but presentations during the adulthood is not uncommon.

Significant proportion of patients with genetic eye disorders exhibits a familial occurrence depending on the disorder and the population, while the rest of the patients are simplex cases.

The pattern of inheritance of a familial disorder phenotypes are established by analyzing the

1 pedigree. Both the familial and sporadic categories of ophthalmo-genetic disorders carry a recurrence risk specific to the patient concerned. Estimation of genetic risk is complex in ophthalmo-genetic conditions, but highly important with regards to the preventive aspect.

Described under subtopics are the ophthalmo-genetic conditions seen in Sri Lanka.

1.2 Introduction to Retinoblastoma (RB)

The rare childhood malignancy retinoblastoma (RB) serves as one of the most important models in modern cancer genetics, since the study of its familial and sporadic occurrence has lead to the identification of the first tumor suppressor gene. Retinoblastoma is the most common ophthalmic malignancy in children under the age of five years. The incidence of retinoblastoma is 1:15,000 to 1:20,000 live births [1, 2].

Clinical presentation of retinoblastoma is usually due to leucocoria , strabismus and red eye.

Uvitis, proptosis and glaucoma are rare presentations.

1.2.1 Diagnosis of retinoblastoma

The accepted method of clinical diagnosis of retinoblastoma is usually established by examination of the fundus of the eye using indirect ophthalmoscopy. Fundus imaging,

Computed Tormography or Magnetic Resonance Imaging and ultrasonography are used to support the diagnosis and stage the tumor. See figure 1.1 for fundus images of two patients with retinoblastomas.

Figure 1.1: Fundus images of two patients with retinoblastomas; Right is a highly vascular large tumor and the left is a multifocal tumour. (Photographs of eye genetics research patients referred from the eye unit of Lady Ridgeway hospital)

Histological diagnosis is usually achieved by Fine Needle Aspiration Biopsy (FNAC) of eye ball aspirates or microscopic examination of enucleated eye.

Mutations in both alleles of the RB1 gene are a prerequisite for this tumor to develop. Figure

1.2 illustrates the cytogenetic location of RB 1 gene in the long arm of chromosome number

13[3].

Figure 1.2: Cytogenetic location of RB 1 gene at position 13q14.2 in the long arm of chromosome number 13.

1.2.2 Classification of Retinoblastoma

Rb can be classified as unilateral or bilateral, hereditery or sporadic, unifocal or multifocal, macular or peripheral tumour.

 Unilateral / Bilateral

 Retinoblastoma may be unilateral or bilateral depending on single or both eyes are

affected.

 Unifocal / Multifocal

 Retinoblastoma is said to be multifocal when more than one tumor is present in one eye;

where as it is said to be unifocal if only a single retinoblastoma tumor is present in one

eye. Please see figure 1.3 for retinal photograph of multifocal and unifocal

retinoblastomas.

Figure 1.3: Retinal photographs illustrating multifocal (left) and unifocal (right) retinoblastomas. (Photographs of eye genetics research patients referred from the eye unit of

Lady Ridgeway hospital)

 Hereditary RB / sporadic (nonhereditary RB)

 In sporadic (nonhereditary RB) Both RB1 gene mutations occur in somatic cells and are

not passed over to their offspring.

 Individuals heterozygous for a RB1 allele mutation have a germline mutation and thus

have a heritable predisposition to RB. They also have an increased risk of developing

non-ocular tumors such as osteosarcomas and clear cell carcima of the lung.

 Macular or peripheral tumour

 Depending on the location of the tumour it can be classified as macular tumour or

peripheral retinoblastoma. Shown in figure 1.4 are retinal photographs of Macular and

peripheral retinoblastomas

Figure 1.4: Retinal photographs illustrating macular tumor (photograph on the left) and peripheral (photograph on the right) retinoblastomas. (Photographs of eye genetics research patients referred from the eye unit of Lady Ridgeway hospital)

An important goal of medical care is prevention, either by preventing the manifestation of retinoblastoma and of secondary malignancies related to the same predisposition or of their reaching an advanced stage of exacerbation. Figure 1.5 exhibits an early detected retinoblastoma and a late presentation of retinoblastoma. This can only be achieved by the close collaboration of several medical specialists. The clinical geneticist can contribute by offering genetic counseling, including risk estimates for offspring of affected individuals.

Figure 1.5: Retinal photographs of early detected retinoblastoma on the left and late presentation of retinoblastoma on the right. (Photographs of eye genetics research patients referred from the eye unit of Lady Ridgeway hospital)

1.2.3 Genetics of Retinoblastoma

Molecular Genetic Pathogenesis

RB1 is the only gene in which mutations are known to initiate retinoblastoma. RB1 gene is located in the long arm of Chromosome 13in region 42.2 from 48,877,887bp to

49,056,122bp.[3] RB1 gene belongs to endogenous ligands family of genes that encodes for endogenous ligand proteins.

Normal allelic variants

Twenty-seven exons are transcribed and spliced into a 4.7-kb mRNA. There is no indication of functional alternative splicing. No frequent polymorphic sites within the 2.7-kb open reading frame are known, but there are intronic variants and two highly polymorphic microsatellites (Rb1.20, Rbi2) and one minisatellite (RBD). [4]

Normal gene product

RB1 encodes a ubiquitously expressed nuclear protein that is involved in cell cycle in Gap1 to Synthesis phase transition regulation. The RB protein is phosphorylated by members of the cyclin-dependent kinase system prior to the entry into S-phase. Following phosphorylation, the binding activity of the pocket domain is diminished, resulting in the release of cellular proteins. [5].

Pathologic allelic variants

More than 2500 distinct point mutations have been identified in DNA of leucocytes individuals with retinoblastoma or in tumors, 1400 have been described. The majority of RB1 mutations results in a premature stop codon, mainly through single base substitutions, frameshift mutations, or splice mutations. Mutations have been found scattered throughout exon 1 to exon 25 of RB1 and its promoter region. In a single family, a possible disease- causing variant in exon 27 was identified [6]. Recurrent mutations are observed at 14 methylated CpG dinucleotides. Other types of pathogenic allelic variants are gross rearrangements and deletions [7, 8, 9 ].

Abnormal gene product

The majority of mutant alleles (if expressed) code for proteins that have lost cell cycle- regulating functions. Retention of partial activities has been observed in proteins resulting from mutant alleles that are associated with low-penetrance retinoblastoma [10, 11, 12].

1.2.4 Two-hit hypothesis

The exploring genetic basis of retinoblastoma has provided valuable insights into the genetic basis of cancer. A two-hit model, was proposed by Knudson, based on the finding that children with bilateral retinoblastoma developed multifocal, bilateral tumors at an earlier age than children with unilateral, unifocal tumors [13]. According to the two-hit hypothesis, two events are necessary for the retinal cells to transform into malignancy. The first DNA mutational event can be constitutional (germline / inherited) and would then be present in all cells in the body. The second event results in the loss of the remaining normal allele and occurs within a particular retinal cell or cells with dysregulation of the cell cycle [14].

In the sporadic, nonheritable form of retinoblastoma, both mutational events occur within a single retinal cell after fertilization (somatic events), resulting in unilateral retinoblastoma.

Unilateral multifocal retinoblastoma can develop if the first mutation occurs during development such that more than one retinal cell contains this hit. Conversely, not all children with unilateral disease represent somatic events. Overall, 85% of children with unilateral disease represent somatic events, but 15% represent the hereditary form with constitutional mutations in the RB1 gene [15]

The RB1 gene was localized and then cloned based on rare children with retinoblastoma who carry a cytogenetically visible constitutional deletion at chromosome 13q14.2 [16]. Even in cases of bilateral retinoblastoma, the majority of children do not have a family history of the disease, and disease is a result of de novo mutations in the RB1 gene that primarily occur during spermatogenesis [17]. This suggests that mutations in the retinoblasto ma gene locus

(RB1) occur more commonly during spermatogenesis or that the paternal chromosome in the early embryo is at a higher risk for mutation. Studies of retinoblastoma tumors in both the hereditary and nonhereditary forms reveal that the second hit or mutation frequently results in

9 loss of heterozygosity of polymorphic markers that flank the RB1 gene in the tumor. Loss of heterozygosity can arise from allelic loss, mitotic recombination, or loss of whole chromosome 13.

In non-hereditary Rb, both mutations (first and second mutation) occur in somatic cells

(somatic mutations). (Figure 1.6)

In hereditary Rb, only the second mutation is a somatic event. Independent second mutations give rise to independent tumor foci (multifocal Rb tumors). The first mutation is inherited via the germline (Either a new germline mutation or a mutant allele inherited from a parent who has the mutation). (Figure 1.6) In constitutional cells, the affected individual is heterozygous for the mutant allele. [18, 19]

Figure 1.6: Diagrammatic representation of the molecular-genetic mechanisms that result in non-hereditary and hereditary Retinoblastoma (Rb). The development of

Retinoblastoma is initiated if both alleles of RB1 are mutated (rb rb).

Non Hereditary Retinoblastoma

First and second mutations are acquired RB, RB rb,rb

1.2.5 Place of Chromosome analysis in Retinitis pigmentosa

Cytogenetic analysis of blood leucocytes is used to detect deletions or rearrangements involving 13q14.1-q14.2, which are present in nearly 5% of individuals with unilateral Rb

10 and 7.5% of individuals with bilateral Rb. [20] Cytogenetic resolution at the 600-650 band level is essential and 30 metaphases should be analyzed in order to detect mosaic aberrations that are present in about 1% of individuals with Rb. [21]These chromosomal rearrangements are usually associated with developmental delay. Since cytogenetic rearrangements are infrequent in retinoblastomas and the mutation is in the gene level in most cases molecular genetic testing methods are preferred for RB1 mutation detection. But cytogenetic tests are of value if RB is associated with developmental delay or other systemic or dysmorphic features where a large rearrangement is suspected. These tests are also of value in instances where somatic or germline mosaicism is suspected, which might be detected only in proportion of leucocytes.

1.2.6 Molecular genetic testing in Retinoblastoma

Since RB1 is the only gene in which mutations are known to initiate retinoblastoma, analysis of RB1 gene detects considerable percentage of germline mutations. Please see table 1.1 for a summary of molecular genetic testing methods used in retinoblastoma.

Linkage analysis.

Linkage analysis using highly informative microsatellite markers tightly linked to RB1 can be used in two instances: [4]

 To track the mutant allele in families with more than two affected individuals

 To determine if an individual at risk in a family with only one affected individual has

inherited either RB1 allele present in the affected individual.

Table 1.1: Summary of molecular genetic testing used in Retinoblastoma (Gene rewiews;

Retinoblastoma , Rushlow et al 2009, Zhang et al 2008, Zeschnigk et al 2000 [4, 27,28]

Gene Test Method Mutations Detected Mutation Symbol Detection Frequency RB1 Sequence analysis / mutation Single-base substitutions, 70%-75% scanning small intragenic deletions, insertions Targeted mutation analysis Panel of recurrent point 25% mutations Sequence analysis of RNA from Deep intronic splice <5% blood mutations, gross rearrangements Gross deletion Deletion / Exonic, multiexonic, and 16% / duplication duplication whole-gene deletions along analysis analysis with large insertions, rearrangements FISH Submicroscopic deletions and >8% translocations Heterozygosity De novo submicroscopic 8% testing germline deletions Methylation analysis Hypermethylation of the 10%-12% promoter region

1.2.6. Genetic testing for retinoblastoma

1.2.6.1 Interpretation of molecular genetic test results

1) If molecular genetic test result is available that is used in combination with clinical

presentation and family history to determine the origin of probands’ genotype (a germline

mutation or two somatic mutations) in the RB gene.

2) If a pathogenic RB1 mutation is present in the DNA of leucocytes of a RB patient, he or

she nearly always harbors a germline mutation.

3) Therefore he or she will have 50% risk of passing that pathogenic mutation to his or her

offspring.

4) One of his or her parents will probably have the mutation in her blood leucocytes (With

the exception of rare possibility of germline mutation and germline mossaicism in

parents).

5) Therefore the patient’s siblings will have a 50% chance of harboring the pathogenic

mutation.

6) Thus if a germline mutation is found in a RB patient it is important to screen his or her

siblings for pathogenic germline mutation.

7) If germline pathogenic mutation is found in a non affected individual follow up detailed

eye screening examinations should be done every two months till the age of two years

and every tree months till the age of five years accompanied by full genetic counseling of

the family.

8) If germline pathogenic mutation that is found in a RB patient is not shared by his /her

sibling then the risk of acquiring a RB mutation for the non affected sibling is similar to

that of the general population.

9) If any individual at risk does not have either RB1 allele in common with the affected

relative, the individual's risk of developing retinoblastoma decreases to the risk level in

the general population [22, 23].

10) If pathogenic RB1 mutation identified in tumor tissue is not found in the DNA of

leucocytes, the affected individual has a low probability of having an RB1 germline

mutation. The reason for this finding is blood mossaicism is approximately 20% which

can be detected by molecular genetic analysis. Failure to detect an RB1 pathogenic

mutations in the DNA of leucocytes reduces but cannot eliminate the probability of the

individual having an RB1 mutation in his/her germline.[4]

1.2.6.2 Confirmation of the clinical diagnosis in a proband

A. In individuals with familial or bilateral retinoblastoma, to identify the constitutional RB1

mutation that caused inactivation of the first RB1allele.

1) Molecular genetic testing including sequence analysis and deletion/duplication analysis

(MLPA, FISH, CMA, heterozygosity testing) can be performed on leucocytic DNA to

identify the constitutional RB1 mutation. 90% to 95% of individuals are expected to have

a detectable RB1 mutation in leucocytes.

2) In individuals with bilateral retinoblastoma without positive family history, an oncogenic

RB1 mutation may not be detectable in peripheral blood leucocytes. In those instances,

tumor DNA may be used for investigations.

3) In cases where tumor DNA demonstrates both RB1 alleles in the tumor having an RB1

mutation or hypermethylation of the RB1 promoter region, peripheral blood leucocytic

DNA can be tested for the RB1 mutations identified in the tumor.

4) If neither of the two RB1 mutations identified in the tumor is detected in DNA from

peripheral blood, mutational mossaicism can be assumed. If one of the RB1 mutations

identified in the tumor is a large deletion, testing for mutational mossaicism in peripheral

blood leucocytes can be done using FISH analysis.

B. In individuals with unilateral retinoblastoma without family history of retinoblastoma

(simplex cases) to identify the two RB1 mutations that caused inactivation of both RB1

alleles in the tumor.

1) Molecular genetic testing including sequence analysis, deletion/duplication analysis

(MLPA, FISH, CMA, heterozygosity testing) and methylation analysis is first performed

on tumor tissue. If tumor DNA demonstrates that each of the two tumor alleles have an

RB1 mutation or hypermethylation of the RB1 promoter region, leucocytic DNA can be

tested for the presence of the RB1 mutations identified in the tumor.

2) In about 14% of individuals with unilateral retinoblastoma and no family history of

retinoblastoma , one of the RB1 mutations identified in the tumor is also detected in

leucocytes, either as a heterozygous mutation, or in a mosaic state (indicating a mutation

that occurred after conception) which may or may not be present in the germline of the

proband.

1.2.7 Genetic Counseling of Retinoblastomas

1.2.7.1 Genetic risk to parents of a proband

A. Minority of individuals diagnosed with retinoblastoma have an affected parent

B. Majority of individuals with retinoblastoma have the disorder as the result of a de novo

gene mutation.

C. In some cases one of the probands parent has an RB1 mutation but he/she is not affected

 If one parent or his/ her close relative has had retinoblastoma then the parent has an RB1

cancer-predisposing germline mutation. Therefore there is 50% probability that the

offspring will inherit the mutated allele of the RB1gene.

 If the proband has a cytogenetically detectable chromosome 13 deletion or rearrangement

both the parents should be screened for carrier stages of a balanced chromosome

translocation or rearrangement. The result will reveal each parent’s genetic states. If a

parent has a balanced chromosome translocation or rearrangement then there is 50%

probability of offspring caring the genetic rearrangement. The offspring may be carriers

of the same parental balanced chromosome rearrangement or being affected by the

chromosome 13 deletion or rearrangement. The resulting genotype will depend on the

independent assortment of the chromosomes during carrier parents’ meiosis.

 If there is no family history of RB then Ophthalmic examination of apparently unaffected

parents should be done in order to detect/exclude retinoblastoma, retinoma, and

retinoblastoma-associated eye lesions. If such a lesion is detected, the parent is likely to

have an RB1 cancer- predisposing germline mutation.

 If pathogenic RB1mutation is present in the proband Molecular genetic testing of both

parents is recommended. If a heterozygous mutation is identified in either parent, the

parent is at risk of developing non-ocular second primary tumors and there is 50%

probability of transmitting the pathogenic RB1 mutation to the offspring. In 10% of

simplex cases that have a heterozygous RB1 mutation detected in blood, one of the

probands unaffected parents also has the mutation. [8].

 For approximately 1% of simplex cases that have a heterozygous RB1 mutation detected

in blood, one of the probands unaffected parents has mutational mossaicism, possibly at

levels too low to be detectable by sequencing of DNA extracted from leukocytes, but

detectable by more sensitive methods such as allele-specific PCR [8]. Sibs of the proband

are at risk of inheriting the mutation.

 A parent may have germline mosaicism even in the absence of a detectable mutation in

leukocyte DNA. Therefore, even if the RB1 mutation cannot be detected in leukocyte

DNA from either parent, sibs of the proband should still be tested for the presence of the

proband’s mutation.

 In the cases of presence of mosaic RB1 cancer-predisposing mutation in the proband [8]

molecular genetic testing of the parents is not indicated because the pathogenic RB1

mutation is a result of de novo as a post-zygotic event in the proband.

1.2.7.2 Siblings of a proband

The risk to sibs of a proband depends on the genetic status of the parents.

 If a parent is determined to have a germline RB1 cancer-predisposing mutation either by

positive family history, by an eye examination that reveals a retinoblastoma-associated

eye lesion (retinoma), or by molecular genetic testing that reveals the presence of a

cancer-predisposing RB1 mutation, the risk to each sib of the proband is 50% (or lower if

the parent with the RB1 mutation has mossaicism) of inheriting the cancer-predisposing

RB1 mutation. Given the approximately 99% penetrance of most RB1 cancer-

predisposing mutations, the actual risk for retinoblastoma in these individuals is about

50% (or lower if the parent with the RB1 mutation has mossaicism).

 If neither parent shows the cancer-predisposing RB1 germline mutation that was

identified in the proband, germline mosaicism in one parent is still possible. Thus, each

sibling should be tested for the RB1 mutation identified in the proband.

 If the proband clearly shows mosaicism for an RB1 pathogenic mutation outside tumour

cells such as leukocyte DNA, the mutation arose as a post-zygotic event therefore neither

parent has an RB1 germline mutation. The risk to the siblings is not increased; therefore

testing of siblings for the RB1 mutation identified in the proband is not warranted.

 If molecular genetic testing is not available or is uninformative, empiric risks based on

tumor presentation (on clinical backgrounds) and family history can be used (Table 1.2).

The low, but not negligible, risk to siblings of a proband with a negative family history

reflects the presence of either a germline RB1 mutation with reduced penetrance in one

parent or somatic mosaicism (that includes the germline) for an RB1 mutation in one

parent.

Table 1.2: The probability of germline mutation being present in a proband with RB

based on family history and tumor presentation (Gene Reviews) [2]

Family History Rb Presentation Probability that an RB1

Unilateral Bilateral Germline Mutation is

Multifocal Unifocal Present

Positive - More than one + 100%

affected family member + 100%

+ 100%

Negative - Only one + Close to 100% 3

affected individual in the + 14%-95%

family + ~14%

 If a parent has a cytogenetically detectable balanced chromosome 13 translocation or

rearrangement, the sibs are at increased risk of inheriting an unbalanced chromosome

rearrangement.

1.2.7.3 Offspring of a proband

 If the proband has bilateral RB and a negative family history of RB, the presence of a

germline RB1 pathogenic mutation is assumed and the risk to each offspring of inheriting

the mutation is 50%. Predictive DNA testing in offspring is possible if the cancer-

predisposing RB1 mutation has been identified in the proband.

 If the proband has had unilateral multifocal RB and no family history of RB, recurrence

risk to offspring is lower [8, 24].

 The risk to offspring of a proband with unilateral unifocal disease and a negative family

history is 6%, reflecting the possibility that the proband has mosaicism for a germline

mutation or a germline RB1 mutation associated with milder phenotypic expression.

1.2.7.4 Evaluation of relatives at risk

All the siblings of affected children with RB are screened and followed up with ophthalmological examinations if molecular genetic testing is not available.

Use of molecular genetic testing for early identification of at-risk family members enhances early diagnosis and reduces the need for unnecessary screening procedures in family members who do not harbor the pathogenic mutation [25,26,]. The American Society of

Clinical Oncologists declared RB as a Group 1 disorder in the category of hereditary

19 syndrome for which genetic testing is considered part of the standard management for at risk family members [18].

1.2.8 Management of Retinoblastoma

Management includes treatment of manifestations, cytogenetic and molecular genetic testing, surveillance, evaluation of relatives at risk and genetic counseling. Early diagnosis and treatment of Rb and non-ocular tumors can reduce morbidity and mortality. Treatment options depend on tumor stage, number of tumor foci (unifocal, unilateral multifocal, or bilateral), localization and size of the tumor(s) within the eye, presence of vitreous seeding, and age of the child. Treatment options include enucleation, cryotherapy, laser theraphy, systemic or local ocular chemotherapy and radiotherapy. These methods are usually used in combination, depending on the clinical scenario.

1.2.9 Surveillance

Detection of subsequent Rb after initial diagnosis

Following successful treatment, children require frequent follow-up examination for early detection of newly arising intraocular tumors.

 It is recommended that children detected to have an RB1 germline mutation get an eye

examination done under anesthesia every three to four weeks until age six months, then

less frequently until age three years. Clinic examinations with cooperative children are

performed every three to six months until age seven years, then annually and eventually

biannually.

 Individuals who have unilateral retinoblastoma are at risk of developing tumors in their

normal eye [27].

 If the two RB1 mutant alleles are identified in the tumor and if the individual is shown to

have one of those two mutations in leukocyte DNA (~14% of individuals), the children

are followed as described above. Mosaicism involving more than 15% of blood cells is

molecularly detectable by conventional methods.

 If the RB1 mutant alleles identified in the tumor are not detected in leukocyte DNA, there

is still risk that the individual has low-level mosaicism (involving <15% of blood cells)

for the mutant allele and will develop a tumor in the other eye [8]. This risk is small

enough that examination under anesthesia may be replaced with regular clinical

examination of the eyes, including clinical ultrasound.

1.3 Introduction to Retinitis pigmentosa

Retinitis pigmentosa (RP) is a rare hereditary retinal dystrophy caused by the loss of photoreceptors, characterized by retinal pigment deposits visible on fundus examination.

Abnormalities of the photoreceptors of the pigment epithelium of the retina lead to progressive visual loss.

1.3.1 Diagnosis of Retinitis pigmentosa

Clinical diagnosis is based on the presence of night blindness and peripheral visual field defects, lesions in the fundus, hypovolted electroretinogram traces, and progressive worsening of these signs.

1.3.2 Genetics and inheritance of Retinitis pigmentosa

Mutations in more than 50 different genes or loci are known to cause Retinitis pigmentosa

(RP) [28]. The mode of inheritance of RP is determined by family history. RP can be inherited in an autosomal dominant, autosomal recessive or X-linked manner. Rare digenic and mitochondrial forms also occur. Table 1.3 summarizes the relative proportion of probands with RP by mode of inheritance.

Table 1.3: Causes of nonsyndromic Retinitis pigmentosa by mode of inheritance (Gene

Reviews; Retinitis Pigmentosa [28]

Mode of Inheritance Proportion of all RP Probands

Autosomal dominant RP 15%-25%

Autosomal recessive RP 5%-20%

X-linked RP (xlRP) 5%-15%

Unknown: Simplex RP 40%-50%

Digenic RP Very rare

Genetic counseling depends on an accurate diagnosis, determination of the mode of inheritance in each family, and results of molecular genetic testing.

 Autosomal Dominant RP (adRP)

Autosomal Dominant RP (adRP) shows vertical inheritance in family pedigrees. But family history is not positive always due to de novo mutations.

Table 1.4 summarizes the genes associated with Autosomal Dominant Retinitis Pigmentosa

(adRP).

Table 1.4: Genes associated with autosomal dominant retinitis pigmentosa. (OMIM) [29,

30, 31, 32, 33, 34,]

Estimated Proportion of

Gene adRP Attributed to Protein OMIM

Mutations in This Gene

RHO 20%-30% Rhodopsin 180380, 613731

PRPF31 5%-10% U4/U6 small nuclear 600138, 606419

ribonucleoprotein Prp31

PRPH2 5%-10% 2 Peripherin-2 179605, 608133

RP1 3%-4% 2 Oxygen-regulated protein 1 180100, 603937

IMPDH1 2%-3% 2 Inosine-5'-monophosphate 146690, 180105,

dehydrogenase

PRPF8 2%-3% 2 Pre-mRNA-processing- 600059, 607300

splicing factor 8

KLHL7 1%-2% Kelch-like protein 7 611119, 612943

NR2E3 1%-2% Photoreceptor-specific 604485, 611131

nuclear receptor

CRX 1% Cone-rod homeobox protein 120970, 602225,

PRPF3 1% U4/U6 small nuclear 601414, 607301

ribonucleoprotein Prp3

TOPORS 1% E3 ubiquitin-protein ligase 609507, 609923

Topors

CA4 Rare Carbonic anhydrase 600852, 114760

NRL Rare Neural retina-specific 162080, 613750

leucine zipper protein

ROM1 Rare Retinal outer segment 180721

membrane protein

RP9 Rare Retinitis pigmentosa protein 180104, 607331

RDH12 Unknown Retinol dehydrogenase 608830, 612712

SNRNP200 Unknown U5 small nuclear 601664, 610359

ribonucleoprotein 200 kDa

helicase

AIPL1 Rare Aryl-hydrocarbon- 604392

interacting protein-like 1

BEST1 Rare Bestrophin- 1 607854, 613194

PRPF6 Rare Pre-mRNA-processing 613979, 613983

factor 6

RPE65 Rare Retinoid isomerohydrolase 180069, 613794

GUCA1B 4%-5% in Japan; rare in Guanylyl cyclase-activating 602275, 613827

UK protein 2

FSCN2 3% of Japanese with Fascin-2 607643, 607921

adRP; otherwise rare

SEMA4A 3%-4% in Pakistan Semaphorin-4A 607292, 610282

Here listed are the detected genetic mutations in the responsible genes of autosomal dominant retinitis pigmentosa.

25 o RHO Gene - More than 100 RHO mutations have been identified; one mutation,

(NM_000539.3:c.68C>A (NP_000530.1:p.Pro23His) associated with distinct sectorial

disease, accounts for 12%-14% of adRP [ 35]. o RP1 gene - Out of the reported RP1 mutations, two mutations account for half of adRP

caused by an RP1 mutation: Those are c.2029C>T (p.Arg677Ter) and

c.2285_2289delTAAAT (p.Leu762TyrfsTer17); reference sequences are NM_006269.1

NP_006260.1.[1] o PRPH2 (RDS) - Mutations are are responsible for in clinical phenotypes of RP to

macular degeneration and complex maculopathies. [1] o PRPF31- Mutation of PRPF31, account for 8% of adRP, 2.5% of adRP is caused by

genomic rearrangements of this gene that are detected using deletion/duplication analysis.

Approximately 6% is detected by sequence analysis.[1]

 Autosomal Recessive Retinitis Pigmentosa

Table 1.5 summarizes the Genes Associated with Autosomal Recessive Retinitis Pigmentosa.

Table 1.5: Genes Associated with Autosomal Recessive Retinitis Pigmentosa (arRP)( Based on OMIM data and Gene reviews ; Retinitis Pigmentosa)

Proportion of arRP

Gene Attributed to Protein OMIM

Mutations in This

Gene

USH2A 10%-15% Usherin 608400, 613809

ABCA4 2%-5% 2 Retinal-specific ATP-binding cassette 601691, 601718

transporter

PDE6A 2%-5% Rod cGMP-specific 3',5'-cyclic 180071, 613801

phosphodiesterase subunit alpha

PDE6B 2%-5% Rod cGMP-specific 3',5'-cyclic 180072, 613801

phosphodiesterase subunit beta

RPE65 2%-5% Retinoid isomerohydrolas 180069, 613794

CNGA1 1%-2% cGMP-gated cation channel alpha-1 123825, 613756

BEST1 ≤1% Bestrophin-1 607854, 613194

C2ORF71 ≤1% Uncharacterized protein C2orf71 613425, 613428

C8ORF37 ≤1% Uncharacterized protein C8orf37 614477, 614500

CLRN1 ≤1% Clarin-1 606397, 614180

CNGB1 ≤1% Cyclic nucleotide-gated cation channel 600724, 613767

beta-1

DHDDS ≤1% Dehydrodolichyl diphosphate 608172, 613861

synthetase

FAM161A ≤1% Protein FAM161A 606068, 613596

IDH3B ≤1% Isocitrate dehydrogenase [NAD] 604526, 612572

subunit beta, mitochondrial

IMPG2 ≤1% Interphotoreceptor matrix 607056, 613581

proteoglycan 2

LRAT ≤1% Lecithin retinol acyltransferase 604863, 613341

MAK ≤1% Serine/threonine-protein kinase MAK 154235, 614181

MERTK ≤1% Tyrosine-protein kinase Mer 604705, 613862

NRL ≤1% Neural retina-specific leucine zipper 162080, 613750

protein

PDE6G ≤1% Retinal rod rhodopsin-sensitive cGMP 180073, 613582

3',5'-cyclic phosphodiesterase subunit

gamm

PRCD ≤1% Progressive rod-cone degneration 610598, 610599

protein

PROM1 ≤1% Prominin-1 604365, 612095

RBP3 ≤1% Retinol-binding protein 3 180290

RGR ≤1% RPE-retinal G protein-coupled 600342, 613769

receptor

RHO ≤1% Rhodopsin 180380, 613731

RLBP1 ≤1% Retinaldehyde-binding protein 1 180090, 607475

RP1 ≤1% Oxygen-regulated protein 1 180100, 603937

SPATA7 ≤1% Spermatogenesis-associated protein 7 604232, 609868

TTC8 ≤1% Tetratricopeptide repeat domain 8 608132, 613464

TULP1 ≤1% Tubby-related protein 1 600132, 602280

ZNF513 ≤1% Zinc finger protein 513 613598, 613617

ARL6 ≤1% ADP-ribosylation factor-like protein 6 608845, 613575

NR2E3 Rare; found in nuclear receptor subfamily 2 group E3 604485, 611131

Sephardic Jews in

Portugal

EYS 10%-30% in Spain; Protein eyes shut homolog 602772, 612424

common in China

CRB1 6%-7% in Spain Crumbs homolog 1 600105, 604210

CERKL 3%-4% in Spain Ceramide kinase-like protein 608380, 608381

SAG 2%-3% in Japan S-arrestin 181031, 613758

[28,36, 37, 38, 39, 40]

 X-Linked Retinitis Pigmentosa (xlRP)

Retinal disease in females with xlRP is less severe than that seen in males; in contrast, in adRP males and females are, on average, equally affected. Females heterozygous for a mutation in an X-linked RP-related gene may be unaffected or express mild to severe retinal degeneration. [40, 41]. Table 1.6 illustrates the genes associated with

Table 1.6: Genes Associated with X-Linked RP (xlRP) (OMIM, Gene reviews; Retinitis

Pigmentosa) [Souied et al 1997, Grover et al 2000]

Estimated Proportion of

Gene xlRP Attributed to Mutations Protein OMIM

in This Gene

RPGR 70%-90% 2 X-linked retinitis 300029, 312610

pigmentosa GTPase

regulator

RP2 10%-20% 2, 3 Protein XRP2 300757, 312600

 Digenic RP

Digenic RP is caused by the simultaneous presence of a mutation in PRPH2 and a mutation in ROM1 [42]. Although the same PRPH2 mutation (NM_000322.4:c.554T>C;

NP_000313.2:p.Leu185Pro) was found in all cases reported, three different ROM1 mutations were identified in these families. In a cohort of 215 families with apparent adRP, one family

(0.5%) had digenic RP [29].

1.3.3 Clinical Manifestations of Retinitis pigmentosa

 Night blindness.

Loss of rod function predominates early in the clinical course of RP. The initial symptom of

RP in most of the patients is defective dark adaptation (nyctalopia). Patients explain this as tendency to fall or clumsiness from child hood, or early adulthood.

 Visual acuity.

Central visual acuity is usually preserved until the end stages of RP. Loss of central visual acuity over time correlates with the of macular lesions in the course of the disease[43].

Reduction of central visual acuity can occur at all ages from cystoid macular edema, which occurs in 10%-50% of individuals with RP, depending on the population, the genetic type, and the diagnostic tool. (Figure 1.7)

Figure 1.7: Optical Coherence Tomography (OCT) report from a retinitis pigmentosa patient. Maculer thickness is increased due to macular oedema.

 Fundus appearance of RP.

The fundus appearance in RP usually depends on the stage of the retinal degeneration.

The earliest recognizable changes in the fundus are arteriolar narrowing, fine dust-like intraretinal pigmentation, and loss of pigment from the pigment epithelium.

As photoreceptor deterioration progresses, increasing loss of pigment from the pigment epithelium takes place with simultaneous intraretinal clumping of melanin, appearing as clumps in a bone spicule configuration. (Figure 1.8)

Figure 1.8: Fundus photograph of an eye genetics project patient. Bone spicule-shaped pigment retinal atrophy is seen. Macula is preserved although with a peripheral ring of depigmentation. Retinal vessels are attenuated.

Moderate to severe retinal vessel attenuation and waxy pallor of the optic nerve become apparent in individuals with advanced RP. The cause of the retinal vessel attenuation is unknown, but it is a secondary change and not the primary cause of disease.

In the earliest stages when electroretinography reveals defective rod responses in individuals who may not yet have appreciated symptoms, the fundus usually appears normal. The term retinitis pigmentosa sine pigmento has been used to refer to a normal appearance of the retina despite documented abnormalities of photoreceptor function.

 Posterior subcapsular cataracts

Posterior subcapsular cataracts characterized by yellowish crystalline changes in the visual axis of the posterior lens cortex are common in all forms of RP. Severity of the cataract correlates with the age of the affected individual. Approximately half of individuals with RP eventually require (and benefit from) cataract surgery.

 Dust-like particles in the vitreous

Dust-like particles in the vitreous are present in the great majority of individuals with RP.

These are fine, colorless particles comprising free melanin pigment granules, pigment epithelium, uveal melanocytes, and macrophage-like cells, which are evenly distributed throughout the vitreous. Observation of these particles can be helpful in the diagnosis of early

RP before fundus changes are apparent.

 White dots deep in the retina

White dots deep in the retina at the level of the pigment epithelium are a nonspecific manifestation of pigment epithelial degeneration and account for the retinal appearance termed retinitis punctata albescens, which is a manifestation of RP.

 Hyaline bodies (drusen) of the optic nerve head

Hyaline bodies (drusen) of the optic nerve head occur frequently in RP, may be associated with arcuate visual field loss, and are not diagnostic of a specific subtype.

 Exudative vasculopathy ( Coats-like disease)

Exudative vasculopathy, ( Coats-like disease), is the rare occurrence in individuals with severe or advanced RP of telangiectatic vessels, lipid deposition in the retina, and serous retinal detachment [43].

 Sector RP

Sector RP is a term that has been used to describe changes in one quadrant or one half of each fundus. Most commonly, the inferior and nasal quadrants are symmetrically involved. [28]

The visual field defects are less severe than those of typical RP and correspond to the ophthalmoscopically abnormal retina. Individuals with sector RP may lack symptoms of defective dark adaptation, although widespread abnormalities of rod and cone function are usually detected by electroretinography. The incidence of true sector RP is infrequent. Many forms of RP can present initially with a sectorial distribution over decades, develops into a widespread, diffuse disease. Sectoral changes have been observed in autosomal dominant RP, and in females heterozygous for X-linked RP.

Establishing the Diagnosis

A consensus conference suggested that the diagnosis of retinitis pigmentosa (RP) is established when the following are present [45]:

 Rod dysfunction as measured by

34 o Dark adaptation (elevated rod final threshold)

OR o Electroretinogram (ERG) (nondetectable or severely reduced rod responses, with

prolonged implicit time, often with lesser reduction and prolongation of cone-mediated

responses)

 Progressive loss in photoreceptor function

 Loss of peripheral vision that often is greater superiorly but can involve other regions as

well

 Bilateral involvement that has a high degree of symmetry, with respect to both the

severity and the pattern of visual field loss and retinal changes

The retina is assessed through the following:

 Visualization of retina by direct or indirect ophthalmoscopy and slit lamp examination

 Functional assessment of visual ability by visual acuity, visual fields, and color vision

 Electrophysiologic testing using electroretinography

 Structural assessment of the retina by spectral ocular coherence tomography (OCT) or

adaptive optics (Figure 1.9)

Figure 1.9: Optical Coherence Tomography (OCT) report of an Ophthalmo-genetic clinic patient with retinitis pigmentosa demonstrating macular thinning.

1.3.4 Molecular Genetic Testing

The possibility of finding a RP causing mutation in a RP patient is approximately 50% with genetic testing [46,47].

To identify the molecular basis of RP single gene testing or a RP multi-gene panel may be used.

Single gene testing

Molecular genetic testing for mutations in many RP associated genes is available. Testing of single genes is most efficient if the mode of inheritance is known. The testing is prioritized based on the proportion of mutations in each gene as a cause of RP. [28]

Multi-gene panels

Since RP exhibits locus heretogenisity, when the phenotypic pattern and the family pedigree does not narrow down to a specific gene using a multi-gene panel is effective. Multi-gene panel includes a number of genes associated with RP. [28]

1.3.5 Prevalence of Retinoblastoma

The world wide prevalence of RP is estimated to be 1:3000 to 1:7000 persons, or 14 to 33 per

100,000 [48]. The prevalence in the United States and Europe is approximately 1:3,500 to

1:4,000.[48 ] reported that in Denmark the lifetime risk of developing RP is 1:2500. Similar frequencies are expected in other populations but have not been documented. Prevalence data is not available for Sri Lanka or south Asia.

1.4 Introduction to Stargardt's disease (STGD)

Stargardt's disease (STGD) is a bilateral, symmetrical and progressive macular dystrophy that usually starts between the ages of 6 and 20 years and rapidly leads to loss of central vision

[49]. Although visual acuity is severely reduced, peripheral visual fields remain normal throughout life. STGD is the most common form of hereditary macular dystrophy and accounts for 7% of all retinal dystrophies [50, 51, 52].

1.4.1 Clinical manifestations of STGD

Symptoms include wavy vision, blind spots, blurriness, impaired color vision, and difficulty adapting to dim lighting. Children first notice difficulty in reading, complaining of gray, black, or hazy spots in the center of their vision. Patients complaint of a longer length of time needed to adjust between light and dark environments. [53] On fundoscopy, the fovea may be normal or show nonspecific mottling. The classical oval, snail-slime, or beaten-bronze foveal appearance may be surrounded by yellow-white flecks, which is suggestive of Stargardt’s disease. In some cases, a geographic atrophy with bull’s eye configuration may be seen. [54]

1.4.2 Genetics and Inheritance of STGD

STGD is a heterogeneous disorder that is usually inherited as an autosomal recessive disorder but rarely can present as an autosomal dominant trait with a later onset of clinical symptoms

[49, 53].

Stargardt disease 1 (STGD1) can be caused by homozygous or compound heterozygous mutation in the ABCA4 gene (MIM NO 601691) on chromosome 1p22. STGD1 is inherited as autosomal recessive trait [55].

Stargardt disease 3 (STGD3; MIM NO 600110) is caused by mutation in the ELOVL4 gene

(MIM NO 605512) on chromosome 6q14. STGD3 is inherited as autosomal dominant trait.

[55]

Stargardt disease-4 is caused by mutation in the PROM1 gene (MIM NO 604365) on chromosome 4. STGD 4 is inherited as autosomal dominant trait. [55]

Gerber et al and Allikmets et al determined that the ABCA4 gene contains minimally 50 exons and spans out for 150 kb. Exon sizes range from 33 base pairs to 266 base pairs.

ABCA4 gene is uniquely retina-specific.[ 56,57] Allikmets et al revised the estimate of the size of the ABCR gene to 6,819 base pairs encoding a 2,273 amino acid protein.

The ABCA4 gene produces an ATP-binding cassette superfamily transmembrane protein expressed exclusively in retinal photoreceptors that is involved in clearance from photoreceptor cells of all-trans-retinal aldehyde, a byproduct of the retinoid cycle of vision

[58, 59 ]. The ATP-binding cassette superfamily includes genes whose products are transmembrane proteins involved in energy-dependent transport of a wide spectrum of substrates across membranes.

Mutations in ABCA4 gene alter the normal function of above function of the trans-membrane protein. Table 1.6 illustrates the reported mutations in ABCA4 gene that can cause STGD 1.

Table 1.7: Reported mutations in ABCA4 gene that can cause STGD 1 [OMIM entry]

Gene Mutation ABCA4 Gene GLY863ALA VAL931MET ALA1028VAL LEU2027PHE VAL2050LEU IVS40, G-A, +5 TRP855TER GLU1036LYS 2-BP INS, 3211GT ALA1038VAL IVS13, G-A, -1 TYR340ASP IVS5AS, A-G, -2 ARG212CYS ARG18TRP ARG572GLN LEU541PRO AND ALA1038VAL LEU1940PRO PRO1780ALA ARG943GLN TRP821ARG GLU1122LYS

1.4.3 Pathophysiology of STGD

The genetic defect manifests in the visual phototransduction cycle. The mutations cause the production of a dysfunctional protein adenosine triphosphate-binding cassette transporter, producing defective transport of N-retinylidene-phosphatidylethanolamine from the disk space to the cytoplasm of rods and cones.[53] This leads to a buildup of a toxic metabolite lipofuscin, which causes the photoreceptor and retinal pigment epithelial cells to degenerate by way of membrane permeability, lysosomal dysfunction, and causing detachment of proapoptotic proteins, leading to a cell-death pathway [60]. Central vision loss occurs, while peripheral vision usually is retained [61].

1.5 Introduction to Epithelial Basement Membrane type corneal Dystrophy (EBMD)

Epithelial basement membrane type corneal dystrophy (EBMD)/ map-dot-fingerprint dystrophy (MDFD) is an autosomal dominantly inherited corneal dystrophy with variable expressivity. It is diagnosed clinically by characteristic appearance on ophthalmological examination.

1.5.1 Clinical features of EBMD

Map-dot-fingerprint dystrophy (MDFD) / Epithelial Basement Membrane type corneal

Dystrophy (EBMD) is a bilateral anterior corneal dystrophy characterized by grayish epithelial fingerprint lines, geographic map like lines, and microcysts (seen as dots) on slit- lamp examination [62]. Pathologic studies show abnormal, redundant basement membrane and intraepithelial lacunae filled with cellular debris [63, 64]. Clinical Findings are variable.

10% of patients have recurrent corneal erosions, usually beginning after age of 30, but the

41 disorder is usually asymptomatic in the remaining [65]. 50% of patients presenting with idiopathic recurrent erosions have evidence of MDFD. Laibson and Krachmer

(1975) reported 10 families demonstrating autosomal dominant inheritance of MDFD, with affected children and adults [66].

1.5.2 Genetics and Inheritance of EBMD

Boutboul et al. reported the analysis of 2 families with an autosomal dominant pattern of epithelial basement membrane corneal dystrophies and the analysis of single affected individuals [67]. They identified 2 point mutations in the TGFBI gene at different sites

(601692.0012, 601692.0013), which the sites of mutations are causing several other forms of corneal dystrophy.

TGFBI gene (Transforming Growth Factor, Beta-Induced)

TGFBI gene is located at the long arm of the chromosome 5 at the region 5q31.1 and spans from 135,364,583base pairs to 135,399,506 base pairs. [65]

TGFBI protein is a 68-kD extracellular matrix protein with 4 domains similar to insect fasciclin-1 and a C-terminal arg-gly-asp (RGD) sequence. TGFBI is secreted into extracellular space to bind fibronectin, collagen, and integrins. [68].

Here described are the two detected mutations in TGFBI gene which causes EBMD.

1. TGFBI, LEU509ARG

In affected members of a French pedigree with autosomal dominant epithelial basement membrane corneal dystrophy, Boutboul et al. in 2006 [67] identified heterozygosity for a

1526T-G transversion in the TGFBI gene that caused a leu509-to-arg (L509R) amino acid substitution.

The disorder had occurred in 4 generations of the family in autosomal dominant pedigree pattern.

2. TGFBI, ARG666SER

In a family from Northern Ireland, Boutboul et al in 2006 [67] found that epithelial basement membrane corneal dystrophy was associated with heterozygosity for an arg666-to-ser

(R666S) mutation in the TGFBI gene. This mutation, which arose from a 1998G-C transversion, was transmitted by the unaffected father.

1.6 Introduction to Aniridia

Aniridia is a rare genetic eye disorder in which there is variable degree of hypoplasia or the absence of iris tissue. It may be associated with other ocular changes, some present from birth and some arising progressively over time.

Aniridia is seen in approximately 1.8/100 000 live births [69].The incidence ranges from 1:40

000 to 1:100.000. No significant racial or gender predilection has been described. [70, 71, 72]

1.6.1 Diagnosis

The major diagnostic feature is congenital absence or hypoplasia of the iris; Foveal hypoplasia with reduced visual acuity is almost always present and is usually associated with nystagmus.

1.6.2 Genetics and inheritance of Aniridia

In majority of the cases mutations or deletions of the PAX6 gene is detected. PAX6 gene is located at 11p13. Approximatly two thirds of the cases are familial with autosomal dominant inheritance and high degree of penetrance. [73] Seventy percent of individuals diagnosed with isolated aniridia have an affected parent. [72] Paients with no family history have a de novo gene mutation or deletion.

Loss of function of one copy of the PAX6 gene can be identified in about 90% of Aniridia cases, with intragenic mutations accounting for two-thirds and chromosomal rearrangements for one third of cases [ 74, 75,]. About two-thirds of all cases are familial and show dominant inheritance with high penetrance. [72, 74,] In the remaining sporadic cases, the newly expressed mutation will be dominantly inherited in subsequent generations [70,76].

Molecular and genetic basis of Aniridia

The PAX6 gene is located in chromosome 11 at the region 11p13, spanning 22 kb of genomic

DNA, contains 14 exones. [77] It encodes a transcriptional regulator with two DNA binding domains (a paired domain and a homeodomain) and a transcriptional trans-activation domain.[9,10,11] PAX6 is expressed in the developing eye, multiple brain regions, olfactory bulb, neural tube, gut and pancreas. It is active early in ocular morphogenesis, fulfilling multiple roles in development of the retina, lens, cornea and iris [78, 79]. Its targets include

PAX6 itself and genes encoding other developmental regulators, such as SOX2, cell adhesion molecules and structural proteins including lens crystallins and corneal keratins.[ 77,78,79]

Pathologic allelic variants of PAX6 mutations

Three hundred PAX6 mutations have been identified; 286 are associated with congenital eye malformations; 257 of them cause Aniridia and the reminder 29 cause related ocular phenotypes such as Peters anomaly, foveal hypoplasia, and optic nerve anomalies.[70,80].

Table 1.8 summarizes the types of PAX3 mutations that cause Aniridia.

Table 1.8: Types of mutations in PAX6 gene that result in Aniridia (Nelson LB, Bamiou

DE, Free SL, Sisodiya SM, et al)

Type of mutation Percentage %

Nonsense mutations 39%

Splice mutations 13%

Frameshift deletions and insertions 25%

Inframe insertions and deletions 6%

Missense mutations 12%

Run-on mutations 5%

Most of the above mentioned mutations lead to loss of protein function thereby resulting in aniridia. In the cases of aniria, 94% of all intragenic point mutations lead to the introduction of a premature termination codon , or to C-terminal extensions, or aminoacid substitutions

(missense mutations). [70,80]

1.7 Introduction to Coloboma

Coloboma is defined as a congenital defect in any ocular tissue(s), typically presenting as absent tissue or a gap, at a site consistent with aberrant closure of the optic fissure [81]. It represents an important cause of congenital blindness and visual impairment, estimated to account for 3–11% of blindness in children worldwide [81]. The prevalence varies by population ranging from 4 to 19 per 100 000 live births across Europe [82-86] with higher rates reported in populations with high degrees of consanguinity [86, 87].

Coloboma is an congenital ocular defect resulting from abnormal development of the eye during embryogenesis.[81]The embryonic optic fissure is a transient ventral opening that arises during invagination of the optic vesicle in the formation of the bilayered optic cup. It permits the migration of periocular mesenchymal cells (mostly of neural crest origin) into the developing eye to form the hyaloid artery)[81]. During subsequent growth of the optic cup, the edges of the fissure align and fuse completing formation of the eye globe so that the ventral and dorsal retina become morphologically indistinguishable. In human development, the fissure narrows and begins to close during the 5th week, starting centrally and progressing anteriorly and posteriorly, and is normally completely fused by the end of the 7th week [88].

Failure of fusion can lead to coloboma of one or multiple regions of the inferior portion of the eye affecting any part of the globe traversed by the fissure, from the iris to the optic nerve including the ciliary body, retina and choroid [89].

1.7.1 Genetic basis of coloboma

Clinical, epidemiological and experimental evidence from animal models indicate a strong genetic basis. Recurrence risks for siblings of patients affected with optic fissure closure defects have been suggested to be between 8 and 13% [83]. A recent UK study estimated that approximately 40% of coloboma cases exhibit a genetic cause based on positive family history or suspected familial syndrome [90]. Genes associated with coloboma phenotypes have been identified in at least 20 syndromes, where the coloboma arises as an occasional feature of a complex multisystem birth defect [89, 91-95]; these include dominant mutations in SHH (OMIM 600725) [96], RAX (OMIM 601881) [97], GDF3 (OMIM 606522) and

GDF6 (OMIM 601147) [91, 98] and recessive mutations in STRA6 (OMIM 610745)[99] and

SMOC1 (OMIM 608488) [100]. Additionally, rare cases of non-syndromic coloboma have also been identified in patients with recessive mutations of VSX2 (formerly CHX10; OMIM

142993)[101] and ABCB6 (OMIM 605452) [102] more frequently associated with microphthalmia, dominant mutations of PAX6 (OMIM 607108) generally associated with a range of ocular defects including aniridia, and MAF (OMIM 610210) (cataract and anterior segment dysgenesis) [103-106]. The most commonly identified genetic causes of isolated coloboma, without microphthalmia, are CHD7 (OMIM 608892) mutations associated with

CHARGE syndrome (OMIM 214800) and PAX2 mutations, which cause renal-coloboma syndrome (OMIM 167409) [90,107,108]. However, in the majority of cases, the genetic contribution to ocular coloboma phenotypes remains to be determined [89,91,92,94,98]. The findings to date indicate significant genetic heterogeneity and suggest perturbation at multiple stages of eye development can result in failure of optic fissure closure. A comprehensive understanding of pathogenic genetic changes is required to resolve the molecular etiology of coloboma.

1.8 Introduction to Waardenburg syndrome

Waardenburg syndrome (WS) is a rare genetic disorder characterized by heterochromia iridis,

White forelock, distropia canthorum, sensorineural hearing impairment, congenital leukoderma, depressed or high nasal root and hypoplastic ala of nose. Mutations in

28 genes / loci are associated with various phenotypes of WS. Waardenburg syndrome is named after Waardenburg (1886–1979), a Dutch ophthalmologist, who first described the condition in 1951.[109] Since then, four subtypes (type 1 to 4) with variable expressivity and incomplete penetrance , gene expression of different clinical features have been described.[110,111,112] Overall, the syndrome affects around 1 in 42,000 people. [113]

1.8.1 Diagnostic criteria of Waardenburg syndrome

Diagnostic criteria have been proposed by Waardenburg Consortium [114]. The diagnosis should include two major criteria or one major and two minor criteria for the diagnosis of

WS. Please see table 1.9 for major and minor criteria.

Table 1.9: Major and minor criteria for the diagnosis of Waardenburg syndrome.

(Farrer LA, Grundfast KM, Amos J, Arnos KS, Asher JH, Beighton P, Diehl SR, Fex J, Foy

C, Friedman TB. et al)[114]

Major Criteria Minor Criteria

 Pigmentation abnormality of the iris:

o Complete heterochromia iridum

(irides of different color)  Skin hypopigmentation (congenital o Partial/segmental heterochromia (two leukoderma) different colors in same iris, typically

brown and blue)

o Hypoplastic blue irides, or brilliant

blue irides

 White forelock, hair  Synophrys/medial eyebrow flare hypopigmentation

 Congenital sensorineural hearing loss  Broad/high nasal root

 Dystopia canthorum  Hypoplastic alae nasi

 Affected first-degree relative  Premature gray hair (age <30 years)

1.8.2 Clinical features of Waardenburg syndrome

 Heterochromia iridum

Pigmentation abnormality of the iris is a stricking fature in WS. This could be complete heterochromia iridum when the two irides are of different colour. Partial/segmental heterochromia is present when two different colors appear in the same iris. (usually brown and blue). Irides could be hypoplastic in some cases.

 Hair pigmentation anomaly

The classic white forelock is the most common hair pigmentation anomaly seen in WS

(Figure 1). The white forelock may be present at birth, or appear later, typically in the teen years. The white forelock may become normally pigmented over time. The white forelock is typically in the midline but the patch of white hair may also be elsewhere. Red a nd black forelocks have also been described. The majority of individuals with WS1 have either a white forelock or early graying of scalp hair before age 30 years [114].The hypopigmentation can also involve the eyebrows and eyelashes.

 Leukoderma

Congenital leukoderma (white skin patches) is frequently seen in WS on the face, trunk, or limbs. These areas of hypopigmentation frequently have hyperpigmented borders and may be associated with an adjacent white forelock.

 Congenital sensorineural Hearing loss

The hearing loss in WS is congenital, typically non-progressive, either unilateral or bilateral, and of the sensorineural type. The most common type in WS is profound bilateral hearing loss (>100 dB). The laterality of the hearing loss is variable among and within families.

Various temporal bone abnormalities have been identified in persons with WS and hearing loss [115]. The temporal bone abnormalities include enlargement of the vestibular aqueduct and upper vestibule, narrowing of the internal auditory canal porus, and hypoplasia of the modiolus.

Other clinical features identified in multiple families occasionally are as follows

 Spina bifida [116], a can be explained by neurocristopathy etiology with PAX3 being

expressed in the neural crest, (Kujat et al described the prenatal diagnosis of spina bifida

in a WS family [117]. However Studies by Lu et al indicated that PAX3 SNPs were not

strong risk factors for spina bifida [118].

 Vestibular symptoms including vertigo, dizziness, and balance difficulties, even without

hearing loss [119]

 Intestinal and spinal defects, elevation of the scapula, and cleft lip and palate are the other

associated features of WS [ 110,120]

1.8.3 Phenotypes of Waardenburg syndrome

Waardenburg syndrome has been classified into 4 main phenotypes. WS type 1 is distinguished by the presence of dystopia canthorum. WS type 2 is distinguished from type 1 by the absence of dystopia canthorum. WS type 3 has dystopia canthorum and upper limb abnormalities. WS type 4, also known as Waardenburg-Shah syndrome, has the additional feature of Hirschsprung disease [121,122]. Waardenburg syndrome is genetically

51 heterogeneous. Table 1.10 illusrates the recognized phenotypes and gene loci of the associated gene with each phenotype. [123]

Table 1.10: Phenotypes of Waardenburg syndrome with associated genes/loci and MIM numbers. (Based on OMIM)

Phenotype Gene/Locus

Location Phenotype MIM Gene/Locus MIM

number number

2q 36.1 Waardenburg syndrome, type 1 193500 PAX3 606597

1p21-p13.3 Waardenburg syndrome, type 2B 600193 WS2B 600193

PAX3, WS1,

2q36.1 Waardenburg syndrome, type 3 148820 HUP2, CDHS, 606597

WS3

PAX3, WS1,

2q36.1 Waardenburg syndrome, type 1 193500 HUP2, CDHS, 606597

WS3

Waardenburg syndrome/ocular MITF, WS2A, 3p14-p13 103470 156845 albinism, digenic CMM8

MITF, WS2A, 3p14-p13 Waardenburg syndrome, type 2A 193510 156845 CMM8

8p23 Waardenburg syndrome, type 2C 606662 WS2C 606662

SNAI2, SLUG, 8q11.21 Waardenburg syndrome, type 2D 608890 602150 WS2D

TYR, SHEP3, Waardenburg 11q14.3 103470 CMM8, 606933 syndrome/albinism, digenic OCA1A, ATN

EDNRB,

13q22.3 Waardenburg syndrome, type 4A 277580 HSCR2, 131244

ABCDS, WS4A

EDN3, WS4B, 20q13.32 Waardenburg syndrome, type 4B 613265 131242 HSCR4

SOX10, WS4, 22q13.1 Waardenburg syndrome, type 4C 613266 602229 WS4C, PCWH

Waardenburg syndrome, type SOX10, WS4, 22q13.1 2E, with or without neurologic 611584 602229 WS4C, PCWH involvement

WS type 1 can be distinguished from WS type 2, by the presence of dystopia canthorum in

WS type 1 and absence of dystopia canthorum in WS type 2 [123]. In section 5.4 we present two patients with WS presented to ophthalmo-genetic clinic. Patient 1 has clinical features of

WS type 1 and patient 2 has clinical features of WS type 2A.

1.8.4. Genetics and inheritance of Waardenburg syndrome

WS1 are caused by mutations in the PAX3 gene located on chromosome band 2q36.1 from

223,064,605 base pairs to 223,163,714 base pairs and inherited usually in an autosomal dominant pattern [124,125,126]. Family history may not be positive due to reduced penetrance and de novo mutations. The majority of probands have an affected parent. A minority of probands does not have an affected parent and are presumed to have a de novo mutation. The mutation rate has been estimated at 0.4 per 100,000 [109].

PAX3 gene is a member of the paired box (PAX) family of transcription factors. Members of the PAX family typically contain a paired box domain and a paired-type homeodomain.

These genes play critical roles during fetal development [126].

In contrast to WS1, WS2 is genetically heterogeneous phenotype. Mutations in the microphthalmia-associated transcription factor (MITF) gene, located on chromosome band

3p14.1–p12.3 cause 15% of WS2 [127]. This gene encodes a transcription factor that contains both basic helix-loop-helix and leucine zipper structural features. It regulates the differentiation and development of melanocytes retinal pigment epithelium and is also responsible for pigment cell-specific transcription of the melanogenesis enzyme genes [126].

Other cases of WS2 have been linked to another locus on band 1p; still others remain unlinked to other loci. Table 1.10 summarizes the genes and loci associated with other types of WS [123]. SOX10 point mutations [128] and deletions [129] have been described in about

15% of individuals with WS2. SOX10 is located in chromosome 22 at the region 22q13.1.

[130] indicated that SOX10 mutations had a similar frequency as MITF mutations in individuals with WS2 of Chinese ancestry. [131] and [132] performed functional analysis of

SOX10 mutations. A frameshift mutation showed a dominant negative effect on wild type

SOX10, resulting in a milder WS2 phenotype [131].

1.9 Introduction to Blepharophimosis, Ptosis, Epicanthus Inversus, Syndrome (BPES)

Blepharophimosis , Ptosis, Epicanthus Inversus, Syndrome (BPES) is an eyelid malformation syndrome characterized by four major features: blepharophimosis, ptosis, epicanthus inversus, and telecanthus.

There are two major types of BPES; Type 1 and type 2.BPES type I includes the four major features and premature ovarian failure (POF) where as BPES type II includes only the four major features. [133]

1.9.1 Clinical features

The diagnosis of blepharophimosis syndrome (BPES) is based primarily on the following four clinical findings, which are present at birth [134]:

1) Blepharophimosis

Blepharophimosis refers to narrowing of the horizontal aperture of the eyelids. In normal

adults, the horizontal palpebral fissure measures 25-30 mm; in individuals with BPES, it

generally measures 20-22 mm.

2) Ptosis

Drooping of the upper eyelid causes a narrowing of the vertical palpebral fissure. In

individuals with BPES, ptosis is secondary to dysplasia of the musculus levator palpebrae

superioris.

To compensate for the ptosis, affected individuals use the musculus frontalis, wrinkling

the forehead to draw the eyebrows upward, which results in a characteristic facial

appearance and tilt their head backward into a chin-up position

3) Epicanthus inversus

Epicanthus inversus refers to a skin fold arising from the lower eyelid and running

inwards and upwards.

4) Telecanthus

Lateral displacement of the inner canthi with normal interpupillary distance.[135]

Two types of blepharophimosis syndrome have been described [136]

1.9.2 Diagnosis criteria of two types of BPES syndrome

 BPES type I - Includes the four major features and female infertility caused by premature ovarian failure.

 BPES type II - Includes only the four major features.

1.9.3 Genetics of BPES

FOXL2 gene located at 3q22.3 is the only gene which has been proven to cause BPES.

FOXL2 is a single-exon gene of 2.7 kb. FOXL2 spans out from 138,663,066 base pairs to

138,665,982 base pairs. [137]

FOXL2 gene codes for FOXL2 protein. FOXL2 protein consists of 376 amino acids and belongs to the large family of winged-helix, fork-head transcription factors. Fork-head proteins are present in all eukaryotes and they have important functions in the establishment of the body axis and the development of tissues from all three germ layers in animals. [138].

More than 125 FOXL2 mutations have been detected in individuals with BPES types I and II, demonstrating that both phenotypic features in two systems (eyelid defect and premature ovarian failure) are caused by the pleiotropic effect of a single gene (Please see table 1.13).

More than106 unique iatrogenic FOXL2 mutations have been identified in 206 unrelated families with BPES of different ethnic origins worldwide [139].

Table 1.7: FOXL2 Pathogenic Allelic Variants causing BPES; Gene reviews;BPES [133]

DNA Nucleotide Protein Amino Acid Change Change c.205G>A p.Glu69Lys c.244C>T p.Gln82Ter c.273C>G p.Tyr91Ter c.500_501delTCinsAA p.Phe167Ter (c.500T>A; c.501C>A) c.560G>A p.Gly187Asp c.650C>T p.Ser217Phe c.650C>G p.Ser217Cys c.655C>T p.Gln219Ter See footnote 2 p.Ala221(15_24) 3 c.655C>T p.Gln219Ter c.663_692dup p.Ala225_Ala234dup c.667_702dup p.Ala221_Ala234dup c.684_698dup15 p.Ala230_Ala234dup c.672_701dup p.Ala225_Ala234dup c.804dupC p.Gly269ArgfsTer265 c.841_857dup p.Pro287ArgfsTer75 c.843_859dup p.Pro287ArgfsTer75 c.843_865dup p.His289ArgfsTer75 c.854delC p.Pro285ArgfsTer71 c.855_871dup p.His291ArgfsTer71 c.855_871del17 p.Pro287AlafsTer241 c.305T>C p.Ile102Thr c.1056delG p.Glu352AspfsTer4

1.9.4 Patterns of inheritance in BPES

Blepharophimosis, ptosis, and epicanthus inversus syndrome is generally inherited in an autosomal dominant manner. Autosomal recessive inheritance pattern has been reported in one consanguineous family. [133]

1.10 Introduction to Moebius syndrome

Moebius syndrome (MS) is defined as congenital, nonprogressive facial weakness with limited abduction of one or both eyes (at the Moebius Syndrome Foundation Research

Conference in 2007) [140] Moebius syndrome results from an underdevelopment of VI and

VII cranial nerves. People with Moebius syndrome are born with facial paralysis and inability to move their eyes laterally. [141] [142] Although Von Graefe described a case of congenital facial diplegia in 1880, the syndrome was reviewed and defined further by Paul Julius

Mobius, a German neurologist, in 1888. [143]

The estimated incidence of Moebius syndrome is 2–20 per million live births,with a geographic variation [143]

1.10.1 Features of Moebius syndrome

MS is an extremely rare developmental disorder of the brainstem which causes underdevelopment of VI and VII cranial nerves [141,142]. MS could associate craniofacial, musculoskeletal, cardiovascular and neurological defects which are expressed in variable severity in different individuals [141, 142, 143, 144]. Most patients present in infancy or early childhood. [145]

1.10.2 Genetics and inheritance of Moebius syndrome

MS is a genetically heterogeneous syndrome. To date three main genes which are associated with Moebius syndrome have been suggested. MBS1in chromosome 13 at 13q12.2-q13 region [146,147], MBS2 in chromosome 3 at 3q21-q22 [148] and MBS3 in chromosome 10 at 10q21.3-q22.1regions are of major concern. [149]

Most reported MS cases are sporadic, although Many families affected with autosomal dominantly inherited Moebius syndrome have also been reported [150, 151]. Autosomal recessive and X linked inheritance patterns are rarely reported. [150, 152]

1.11 Justification

1.11.1 The World Health Organization (WHO) estimates that globally about 314 million people are visually impaired; of whom 45 million are blind [153 ]. There are an estimated 1.5 million blind children world-wide [153]. The major causes of blindness include cataract, uncorrected refractive errors, glaucoma, age-related macular degeneration, corneal opacities, diabetic retinopathy, eye diseases in children, trachoma and onchocerciasis[153]. The causes

60 of blindness are cataract 51%, glaucoma 8%, age related macular degeneration 5%, childhood blindness and corneal opacities 4%, uncorrected refractive errors and trachoma 3%, and diabetic retinopathy 1%, and the undetermined causes are 21%[154].

1.11.2 One third of the world’s 45 million blind and half of the world’s 1.5 million children live in South East Asia [155]. Four people out of twelve people who become blind in every minute in the world are in this region [155]. With one quarter of the world’s population and one third of the world’s blind South East Asia has a particularly heavy burden of blindness

[155]. The highest rates of visual impairments are found in the WHO Eastern Mediterranean

Region, with 40.5 per million, and South-East Asia Region (without India) with 48.2 per million. India has 53 cases of visual impairment per million population while China has 55.4 per million[156]. Delayed diagnosis and treatment of eye disorders, due to deficiency of awareness of etiology and phenotypic patterns of eye disorders prevalent in the area, also contribute to this burden in the region.

Lack of counseling on preventable causes of visual impairment and lack of ophthalmological research data on the field worsens the situation putting off the solutions by years.

According to community eye health journal these tables illustrates the magnitude of blindness in children according to the region and causative factors[157] (Table 1.12 and Table 1.13)

Table 1.12: Magnitude of blindness in children according to the region (Community Eye

Health 1998. 11(27): p. 33-34. How Can Blind Children Be Helped? By Foster, A.,) [157]

Magnitude of blindness in children

Region Number of Number of Prevalence Percentage of

children per blind per 1000 blind children

million children

Africa 253 3,300 1.2 24%

India 340 270,000 0.8 20%

Rest of Asia 264 220,000 0.8 16%

China 336 200,000 0.6 12%

Middle East 238 190,000 0.8 14%

Latin America 167 100,000 0.6 8%

Western Economics 168 50,000 0.3 4%

Eastern Europe 77 40,000 0.5 2%

Total 1843 1400,000 0.71 100%

Table 1.13: Magnitude of blindness in children according to the causative factors (Taken from Community Eye Health 1998. 11(27): p. 33-34. How Can Blind Children Be Helped?

By by Foster, A.,) [157]

Causes of blindness in children Site Nunber Percentage of blind of blind Conditions children children Retina 400,000 29% Retinal dystrophies and Retinopathy of

prematurity

Cornea 300,00 21% Vitamin A deficiency, measles, Opthalmia

neonatarum, Traditional eye medicines

Globe 200,000 14% Microphthalmus, Coloboma

Lence 130,000 9% Cataracts and Aphakia

Other 130,000 9% Cortical blindness, Ambloyopia

Optic Nerve 120,000 9% Optic atrophy/ hypoplasia

Glaucoma 70,000 5% Bupthalmus or Glucoma

Uvia 50,000 4% Aniridia and Uvitis

Total 1400,000 100%

1.11.3 As the impact on blindness from acquired causes such as Vitamin A deficiency, infectious diseases and trauma decreases, the inherited ocular diseases have become predominant. Genetic diseases of the eye and syndromes involving the eye continue as a leading cause of blindness in children and adults. This development is prominent in countries with high consanguinity where Sri Lanka is one example due to cultural acceptance of consanguineous marriages. Although individually rare, when considered together as a group

63 of disorders inherited ocular diseases place a heavy burden on the health care system in any country. No studies have been done in Sri Lanka taking inherited eye disorders as a group revealing the inheritance patterns of each disorder and exploring the phenotypes of rare and common eye disorders.

1.11.4 Sri Lanka is a country of approximately 20 million people spread over a geographic area of 65,610 sq. km (25,332 sq. miles). The Island has considerable racial diversity. Out of

20 million citizens in the country 150,000 are blind, 18,000 have refractive errors, 57,000 have cataracts, 15,000 have glaucoma, 6,000 have diabetes related vision defects (diabetic retinopathy), 10,500 have macular degeneration, while a high of 400,000 suffer from low vision[158]. The causes of such sight impairments are avoidable or treatable in most cases

(75 percent) and that includes even congenital blindness due to cataracts and glaucoma which if properly managed can restore vision to affected children[158]. Altho ugh genetic and inherited causes play a major role in ocular disorders such as retinoblastomas and retinitis pigmentosa no nation-wide survey of genetic blindness and visual impairment has been undertaken in the country. In an epidemiological study of eye diseases among children in a health unit area in Sri Lanka 5/1000 children were reported to have congenital eye diseases

[159]. In another study done of children at six schools for the blind in Sri Lanka, 35% of children had hereditary eye diseases (Table 1.14) [160]. The results of these studies further emphasizes the need for hospital based, detailed research should be done on inherited eye disorders.

Table 1.14: Etiological categories in 226 Sri Lankan children attending blind schools with severe visual impairment and blindness (British Journal of Ophthalmology;79:633-

636. Causes of childhood blindness in Sri Lanka: results from children attending six schools for the blind, by Eckstein MB, Foster A, Gilbert CE. 1995) [160]

Etiological category Number Percentages Hereditary disease 79 35.0%

Intrauterine factor 8 3.5%

Perinatal factor 0 0.0%

Childhood factor 12 5.3%

Unknown 127 56.2%

Total 226 100%

1.11.5 The current number of human eye disorders that have a genetic or hereditary component is impressive and continues to grow with time parallel to expanding scientific knowledge and new discoveries. An internet search for the term "eye" on the website "Online

Mendelian Inheritance in Man" yields 1278 entries. The scientific literature reports identification of nearly 500 genes that contribute to hereditary eye disorders. Specific changes or mutations within over 150 of these genes have been cited as having an association with cataracts, glaucoma, retinitis pigmentosa, eye tumors, and corneal and retinal dystrophies. Better understanding of scientific basis would create the groundwork for the future development of treatment strategies for these disorders.

1.11.6 Hereditary ophthalmologic disorders may be isolated or part of a syndrome when associated with other physical findings. Isolated or non-syndromic eye disorders can be

65 inherited in a family in a number of different ways, with the risk for unaffected family members to have a child with an eye disorder being dependent upon the pattern of affected individuals within the family. Examples of non-syndromic hereditary eye disorders include most varieties of retinitis pigmentosa and Stargardt disease. Syndromes that involve eye disease as a component of the condition include Blepharophimosis Ptosis Epicanthus

Inversus syndrome (BPES), and Waardenburg syndrome.

Several isolated eye disorders and genetic syndromes with an ophthalmologic component now have identifiable gene mutations known to cause disease. Specific mutations identified in over 150 genes are associated with isolated hereditary eye disorders and genetic syndromes with an ophthalmologic element.

A greater knowledge of the clinical and molecular features of these disorders is important for accurate diagnosis, appropriate genetic counseling, and application of treatment strategies targeted at the individual's diagnosis and genetic status. This knowledge can only be gained through continued research.

Early diagnosis and effective treatment for complications of inherited eye disorders can prevent blindness.

1.11.7 Inherited eye disorders show variety of inheritance patterns depending on the phenotype.

Eg.1: Retinitis pigmentosa can be inherited in an autosomal dominant, autosomal recessive or

X-linked manner and some digenic and mitochondrial forms have also been described.

[161,162]

Eg.2: Stargardt macular dystrophy also shows autosomal dominant and autosomal recessive inheritance pattern depending on the phenotype which can be revealed by pedigree analysis.[49,53]

Identifying different inheritance patterns with clinical phenotypes will reveal the disease patterns in Sri Lankan population. Studying the common patterns of inheritance could facilitate ophthalmologists to arrive at diagnosis and help them detect carriers and patients with minor degree of illness. This will guide patients to prevent complications, plan their lives to prevent unexpected outcomes and leads to a better clinical and social outcome. For the clinical geneticist this will help to estimate the carrier risk and the disease risk to the patient.

1.11.8 Inherited eye disorders can be caused by various genetic alterations such as deletions, duplications, translocations, inversions or point mutations depending on the eye disorder phenotype. Even without invasive techniques or genetic testing data it is possible to estimate the probable genotype and thereby to predict the genetic risk to family members.

Eg: Phenotype characterization of bilateral and unilateral retinoblastoma is useful estimation of genetic risk [162]. Table 1.2 in the retinoblastoma section illustrates the probability of an RB1 germline mutation being present depending on the patients’ family history and the clinical presentation.

As described above, studying patterns of inheritance and revealing phenotypes of patients of inherited eye disorders will facilitate ophthalmologists to diagnose these disorders at early stages preventing blindness. By referring patients and their families for genetic counseling early health care professionals can further reduce preventable genetic causes of blindness.

Exploring the phenotypic patterns in Sri Lanka will broaden scientific understanding of the disorders which are highly prevalent and less prevalent in the country. Establishment of

67 phenotype genotype correlation could help medical geneticists to design genetic tests methods more suitable for Sri Lanka. If genetic etiology is explored that can lay the framework for treatment strategies; Such as gene therapy or regenerative experiments. Final result of this study will add a drop of knowledge to the vast field of ophthalmo-genetics, which will improve the diagnosis, treatment and prevention strategies of genetic eye disorders.

1.12 Objectives

1) To document inherited eye disorders in Sri Lanka

2) To clinically phenotype patients with different eye disorders based on history, clinical

examination, disease progression, investigation results and response to treatment

3) To compare and contrast the phenotypes of inherited eye disorders seen in Sri Lanka and

inherited eye disorders reported in scientific literature

2. Methodology

2.1 Study design

This is a descriptive cohort study designed prospectively to meet the objectives that describes the clinical genetic aspects of the study.

1) Patients with a clinical diagnosis of any inherited eye disorder were recruited

prospectively

2) This involves patients with exclusive eye disorder or patients who exhibits eye

involvement as part of their syndrome or systemic disorder

3) Patients with inherited eye disorders referred to the Ophthalmo-genetic clinic (of the

Human Genetics Unit in the faculty of Medicine Colombo) from November 2013 to July

2014 were recruited prospectively.

4) Ophthalmologists all over Sri Lanka were informed regarding the study and the genetic

services provided by the Ophthalmo-genetic unit of Faculty of Medicine.

5) Patients were referred mainly from the Lady Ridgeway Hospital Colombo, National Eye

Hospital Colombo and the General Hospitals of Sri Lanka.

6) Detailed complete medical and surgical history was taken with more emphasis on family

history.

7) Family tree was drawn including three generations.

8) Visual acuity was assessed using a Snellen chart and examination of the retina was done

using a direct ophthalmoscope and the slit lamp.

9) Eye evaluation was done by a Consultant Ophthalmologist and arrived at a clinical

diagnosis.

10) Complete systemic examination and clinical genetic evaluation was done on each patient.

11) Investigations performed, treatment given to patients, response to treatment, and the

course of the disorder were documented

12) In consenting subjects photographs of clinical features and photographs of retina were

taken.

13) In relevant families relatives of the participants were also examined.

14) Genetic counseling was performed on each subject and their families.

15) Data were analyzed on clinical background.

2.2 Subjects

Patients with inherited eye disorders referred to the Ophthalmo-genetic unit (of Human

Genetics Unit at the Faculty of Medicine Colombo) from November 2013 to July 2014 were recruited. Patients with inherited eye disorders referred from the Lady Ridgeway Hospital

Colombo, National Eye Hospital Colombo and the General Hospitals of Sri Lanka were recruited.

2.3 Inclusion criteria – Subjects who meet the following criteria were recruited into the study

Subject should fall in to one of the categories of A, B, C, or D AND fulfill 1, 2 or 3 consenting eligibility criteria

A. Patients with clinical diagnosis of any inherited eye disorder or

B. Subjects (patients) with features of any hereditary Eye disorder or

C. Symptomatic relatives of patients with inherited eye disorder or

D. Patients with hereditary systemic disorder or a genetic syndrome with eye

involvement as part of their syndrome or disorder

With

1. Adults (above18) who are mentally and physically fit enough and capable of

providing informed consent or

2. Children less than 18 years, where parent / guardian consent is available or

3. Adults ( above18) who’s mental capacity is diminished due to any reason proxy

consent was taken

2.4 Exclusion criteria

 Non consenting subjects

 Subjects without (features or symptoms of ) hereditary Eye disorders

2.5 Obtaining written informed consent

1. Subjects were given an information sheet which includes details about the study and a

consent form should be read and signed before participating in the study.

2. All subjects were given sufficient amount of time in an appropriate setting to read and

understand the patient information sheet and the consent form

3. If the subjects had any questions regarding the study they were answered before signing

the consent form.

4. All subjects were permitted to withdraw his / her consent to participate in the study at any

time, with no penalty or effect on medical care or loss of benefits and this was clearly

stated in the consent form.

5. If the subject is below the age of 18 proxy consent was taken from the parents or guardian

6. In the case of diminished cognition or psychiatric symptoms where informed written

consent cannot be obtained proxy consent was obtained from guardians.

7. Molecular genetic studies or cytogenetic studies were done only on selected subjects

depending on clinical diagnosis and phenotype only with informed written consent

8. Moreover, all participants were given contact details of the investigators and the contact

details of the Ethics Review Committee (ERC) in case they need to clarify any doubts

about the study.

2.6 Clinical evaluation

1. Complete medical and surgical history including a complete family history was taken

from each participant.

2. The family pedigree included familial traits (background of the concerned condition) up

to three generations where ever possible.

3. Visual acuity was assessed using a Snellen chart and examination of the retina was done

using a direct ophthalmoscope and the slit lamp.

4. Eye evaluation was done by a Consultant Ophthalmologist and arrived at a clinical

diagnosis.

5. Complete systemic examination and clinical genetic evaluation was done on each patient.

6. Investigations performed, treatment given to patients, response to treatment, and the

course of the disorder were documented

7. In consenting subjects photographs of clinical features and photographs of retina were

taken.

8. Complete review of patients’ medical records and available test results were done.

9. The clinical phenotypes, patterns of inheritance and patterns of disease expression were

assessed.

10. Photocopies and soft copies of relevant investigation records were collected from

consenting patients.

11. Relevant investigations done, treatment given to patients and response to treatment and

course of the illness were recorded.

12. When assessing phenotypes variability of disease expression, age of onset, Bilateral or

unilateral disease, severity of symptoms were assessed.

13. In relevant families relatives of the participants were also examined.

14. Genetic counseling was performed on each subject and their families.

15. Data were analyzed on clinical background.

2.7 Statistical analysis of data

Standard descriptive statistics were used to describe the data. Statistical analysis between diseases subgroups were conducted through one way ANOVA (Analysis of variance)

Relevant computer software packages were used for data analysis

2.8 Ethical issues relevant to the study

1) The study was conducted according to the Declaration of Helsinki (2008).

2) The study was built on collaborative links the Human Genetics Unit has established with

local ophthalmologists and medical geneticists.

3) The study has social value because it would contribute to generalizable knowledge in the

field. The study was designed appropriately to ensure scientific validity. The study was

open to all patients with any inherited eye disorder; this therefore has fair participant

selection.

4) Appropriate measures were taken to ensure that consent was obtained in an ethical

manner from all study participants. In the case of subjects bellow 18 years of age proxy

consent was taken from guardians. In the case of mentally incapacitated patients proxy

consent was taken from guardians. All the subjects were informed that any subject have

the wrights to withdraw his/her consent to participate in the study at any time, with no

penalty or effect on medical care or loss of benefits.

5) The patients were interviewed privately in a separate room to ensure privacy and were

able to discuss the study privately with the investigators without the presence of others.

Informed written consent was obtained after providing the necessary information and

giving them time to make a decision in private. The data collection booklet was designed

to ensure confidentiality of information gathered. Soon after collecting the personal

information, the identification page was removed and filled separately. The only

identification number in the rest of the booklet was a coded subject study number which

cannot be linked to an individual without the page containing the personal information

which was kept by the principal investigator under lock and key. The electronic database

containing the data was only had the subject study number thus ensuring confidentiality.

The database and the counter containing the database would be password protected.

These measures ensured that loss of confidentiality was minimized.

6) The benefit to the participants was to receive genetic counselling and was referred for

further management for as appropriate and their follow up in the genetic clinic and

ophthalmology clinic was continuous even after this research is over. Family screening

for potential clinical symptoms helped participants with early diagnosis or exclusion of

possible diagnosis of the familial conditions. By participating in this study the subjects

may contributed to the advancement of generalisable knowledge on the frequency of

distribution of the particular inherited eye disorder in Sri Lanka.

7) Data would be stored for future use after the completion of the study for future studies in

eye genetics. Appropriate consent would be obtained for this purpose and such studies

would be subjected to ethics review prior to commencement.

8) Ethical clearance was taken from the Ethical Clearance Committee of the Faculty of

Medicine Colombo.

9) Administrative clearance was taken from the Director of the National Eye Hospital and

the ethics committee of the National Eye Hospital to recruit patients there.

10) Administrative clearance was taken from the Director of the Lady Ridgeway children’s

Hospital and the ethics committee of the Lady Ridgeway children’s Hospital to recruit

patients there.

3. Results

 59 patients with genetic eye disorders presented to Ophthalmo-genetic clinic at the

Faculty of Medicine Univercity of Colombo, from1.12.2014 to 31.7. 2014.)

 Table 3.1 illustrates the Ophthalmo-genetic conditions referred to clinical geneticist

during the period of one year of research.

 Figure 3.1 summarizes the number and percentage of patients with different Ophthalmo-

genetic disorders during the period of one year of research.

 Referrals are manly from the Lady Ridgeway Hospital, National eye hospitals and

General hospitals of Sri Lanka.

 54 patients (92%) were diagnosed to have isolated eye disorders without systemic

involvement; those are 40 retinoblastoma patients (68%), retinitis pigmentosa 8 patients

(14%), Stargardt disease 2 patients (3%), 2 patients with isolated Aniridia (3%), one

patient with isolated Coloboma (2%) and one patient with Epithelial basement membrane

type corneal dystrophy (2%).

 5 patients were referred to Ophthalmo-genetic clinic due to significant eye involvement as

part of rare genetic syndrome. Those are Waadernberg syndrome two patients with

heterochromia iridis (3%), Mobius syndrome one patient with defective abduction of eyes

(2%), Blepharophimosis ptosis Epicanthus Inversus Syndrome one patient (2%) and

Edward syndromic baby with a Coloboma (2%).

Table 3.1: The consistency of the cohort of patients in the Ophthalmo-genetic project and the percentage of patients in each category

Category of disorders Number of patients Percentage of patients

Retinoblastoma 40 68%

Retinitis pigmentosa 8 14%

Stargartds disease 2 3%

Aniridia 2 3%

Epithelial Basement Membrane 1 2% type Corneal Dystrophy

Coloboma 2 3%

Waadernberg syndrome 2 3%

Blepharophimosis , ptosis, 1 2%

Epicanthus Inversus Syndrome

Mobius syndrome 1 2%

Total 59 100%

Figure 3.1: Number and percentage of patients with different Ophthalmo-genetic disorders

Aniridia, 2, 3% Blepharophimosis Epithelial , ptosis, Coloboma, 2, 3% Basement Epicanthus Membrane type Inversus Corneal Syndrome, 1, 2% Dystrophy, 1, 2% Mobius syndrome, 1, 2%

Stargartds disease, 2, 3%

Waadernberg syndrome, 2, 3% Retinoblastoma, 40, 68% Retinitis pigmentosa, 8, 14%

 49 patients (83%) were children (within the paediatric age range of bellow 18 years) and

10 patients were adults 17%.

 The paediatric patient group consists of 40 patients with Retinoblastoma (68%), 2 patients

with Retinitis pigmentosa (3%), 2 patients with Waardenburg syndrome (3%), 2 patients

with Stargartds disease (3%), one patient with Moebius syndrome (2%), one patient with

coloboma (2%), and one patient with Aniridia (2%). (Figure 3.2)(Table 3.2)

Table 3.2: Summery of Ophthalmo-genetic conditions observed within the paediatric group of patients

Ophthalmo-genetic Disorder Number of patients Percentage of patients

Retinoblastoma 40 82%

Retinitis pigmentosa 2 4%

Waardenburg syndrome 2 4%

Stargartds disease 2 4%

Moebius syndrome 1 2%

Aniridia 1 2%

Coloboma 1 2%

49 100%

 In the paediatric group of patients 45 patients had isolated eye disorders, while 4 patients

had developed significant ophthalmic involvement as part of complex genetic disorder.

 The children who had exclusive (isolated) eye disorders were 40 Retinoblastoma patients,

2 Retinitis pigmentosa patients, 2 Stargartd disease patients and one Aniridia patient.

(Fig:3.3)

 The children with significant ophthalmic involvement as part of complex genetic disorder

were two children with Waardenburg syndrome, one child with Moebius syndrome and

the baby with a Coloboma caused by Edward’s syndrome. (Fig:3.3)

Figure 3.3: Summery of Ophthalmo-genetic conditions observed within the paediatric group of patients

Syndromic and nonsyndromic ophthalmo-genetic conditions in children

Retinitis Stargartds disease pigmentosa, 2, Moebius Retinoblastoma, , 2, 4% syndrome , 1, 2% 40, 82% 4% Aniridia, 1, 2%

Coloboma with Edwards Syndromes, 4, syndrome, 1, 2% 8%

Waardenburg syndrome , 2, 4%

 The adult patient group consists of 6 Retinitis pigmentosa patients, one coloboma patient,

one Aniridia patient, one patient with epithelial basement membrane type corneal

dystrophy and one patient with blepharophimosis epicanthus inversus syndrome (Figure

3.4).

 In the adult group of patients only one patient had developed ophthalmic involvement as

part of a complex genetic whereas all other patient had exclusive (isolated) genetic eye

disorders.

Figure 3.4: Summery of Ophthalmo-genetic conditions observed within the adult group of patients

Figure 3.4: Composition of the adult patient group Blepherophymosi Epithelisl s basement Ptosis membrane type Epicanthus corneal dystrophy inversus 1, 10% Syndrome, 1, 10%

Aniridia, 1, 10% Retinitis pigmentosa , 6, 60%

Coloboma, 1, 10%

 Out of the collective cohort 17 (29%) patients had bilateral eye involvement where as 42

(71%) patients had unilateral eye involvement. (Figure 3.5)

 Out of the paediatric group of patients 17 (35%) patients had bilateral eye involvement

whereas 32 (65%) patients had unilateral eye involvement. (Figure 3.5)

 The whole group of adult patients had bilateral eye involvement 6 (100%); hence none of

the adults exhibited unilateral eye involvement. (Figure 3.5)

Figure 3.5: Distribution of bilateral and unilateral eye involvement in the paediatric and adult groups of patients and the collective cohort.

Percentage of patients with Unilateral and Bilateral eye involvement 100%

90%

80%

70%

60%

50% Unilateral Bilateral 40%

30%

20%

10%

0% Collective cohort Adult Group Paediatric Group

 Out of the collective cohort 14 patients had a positive family history 24%, while 45 were

simplex cases without family history. (Figure 3.6)

Figure 3.6: Number and percentages of patients depending on the family history positivity

Family history

Positive family history, 14, 24% Simplex cases, 45, 76%

 The disorders with positive family history included Retinoblastoma, Retinitis pigmentosa,

Stargardt disease, Aniridia, Epithelial basement membrane type corneal dystrophy and

Blepharophimosis epicanthus inversus syndrome. Figure 3.7 illustrates the number of

patients with positive family history in each category of disorders.

Figure 3.7: Number of patients with positive family history in each disorder

Number of patients with positive family history 4

0 RB RP Stargardt Aniridia BPES EBMD

For the purpose of description the convenient way of categorization of this cohort of patients is to be divided into three groups; the patients with malignancies (Retinoblastoma), the exclusive (isolated) eye disorders and Ophthalmo-genetic syndromes with significant eye involvement.

(Figure 3.8)

Majority of the cohort consists of retinoblastoma patients (40 patients, 68%), 14 patients

(24%) had isolated eye disorders and 5 patients (8%) had Ophthalmo-genetic syndromes.

Figure 3.8: Classification of ophthalmo-genetic project patients according to the type of genetic eye disorder.

Classification based on the type of opthalmo-genetic disorder Syndromes, 5, 8%

Isolated Eye disorders, 14, 24%

Malignant (RB), 40, 68%

Figure 3.9 illustrates the categories and the numbers of patients who participated the ophthalmo-genetic project due to non-malignant ophthalmo-genetic conditions.

Figure 3.9: Illustration of the non malignant conditions referred to ophthalmo-genetic clinic

Number of Patients referred due to non malignant conditions 9

0 RP WS Stargartd Mobius BPES Coloboma Aniridia EBMD

The following sections describe results obtained regarding each disorder.

Patient numbers and disorder categories are stated in table 3.3

The results of the Retinoblastoma group of patients are stated first, followed by the isolated

Ophthalmo-genetic disorders. Ophthalmo-genetic syndrome cases are presented in the latter part of the results section.

Table 3.3: Patient numbers and disorder categories

Patient number Disorder

RT1 to RT40 Retinoblastoma

RP1 to RP8 Retinitis pigmentosa

ST1 and ST2 Stargardt’s Disease

CD1 Corneal dystrophy

AN1, AN2 Aniridia

CB1 and CB2 Coloboma

WB1 and WB2 Waardenburg syndrome

BPES1 Blepherophimosis ptosis epicanthus inversus

syndrome

MB1 Moebius syndrome

3.1 Retinoblastoma

All 40 patients in the retinoblastoma group were aged below five years.

Majority of the retinoblastoma group of patients (15 patients) presented because of the onset of strabismus accompanied by leucocoria which was 37.5% of the RB cohort. Another 15 patients presented due to the onset of isolated leucucoria which was 37.5% of the patients with RB. Four patients presented due to isolated strabismus which is 10% of the RB group.

Only three patients presented with red eye which represents 7.5% of the RB group. Proptosis,

86 fever and crying, pain and crying were rare presentations in each category representing one patient. Table 3.4 summarizes the presenting features of retinoblastoma group. Figure 3.10 is a graphical representation of the common clinical presentations of retinoblastomas.

Table 3.4: Presenting clinical features of retinoblastoma cohort of patients

Number of patients Percentage of patients %

Strabismus with leucocoria 15 37.5

Isolated leucucoria 15 37.5

Isolated strabismus 4 10

Red eye 3 7.5

Proptosis 1 2.5

Fever and crying 1 2.5

Pain and crying 1 2.3

40 100

Figure 3.10: Common Clinical presentations of retinoblastomas

Clinical presentation of retinoblastoma 16

6 Number ofpatients Number 4

0 Srabismus with Isolated white Isolated Red eye Other Leucocoria reflex strabismus

62.5% of the cases (25 patients) had unilateral retinoblastomas, while 37.5% (15 patients) had bilateral RB. Figure 3.11 summarizes the laterality of retinoblastomas.

Figure 3.11: Two thirds of the retinoblastomas are unilateral and one third is bilateral

Retinoblastomas

Unilateral Retinoblastomas 15 (33%) Bilateral Retinoblastomas 25 (67%)

The mean age of onset was 17.3 months in the total retinoblastoma cohort. Bilateral retinoblastomas tend to present earlier with mean age of presentation being 8.7 months, and unilateral retinoblastomas tend to present late with mean age of presentation being 22.5 months. (Table 3.5)

Table 3.5: Mean age of presentation of bilateral and unilatelal retinoblastomas

Number of patients Percentage of Mean age of

patients presentation

Unilateral 25 (62.5%) 22.5 months

Bilataral 15 (37.5%) 8.7 months

Total 40 (100%) 17.3 months

4 patients (10%) had positive family history of malignancies caused by RB1 gene mutations;

36 (90%) did not reveal a positive family history. Figure 3.12 is a diagrammatic representation of family history positivity of the cohort.

Figure 3.12: Diagrammatic representation of family history positivity of the retinoblastoma group of patients

Family history

Patients without a positive 36, 90% family history 4, 10% Patients with a positive 4, 10% family history

Table 3.6 and figure 3.12 illustrate the family history positive individuals in bilateral and unilatelal retinoblastoma categories.

Table 3.6: Family history positivity in bilateral and unilatelal retinoblastomas

Parameter Unilateral Bilataral

Positive family 4 2 (8%) 2 (13%) history

Negative family 36 23 (92%) 13 (87%) history

Total Patients 40 25 (100%) 15 (100%)

Figure 3.13: Family history positivity in bilateral and unilatelal retinoblastomas

Bilataral Positive family history Negative family history

Unilateral

Total

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

Out of the four patients with a positive family history RT1 patient’s mother had unilateral retinoblastoma at the age of two years and the right eye was enucleated. The RT2 patient had a family tree with many relatives affected with RB, osteosarcoma and carcinoma of the lung.

RT3 patient’s paternal grandmother was affected with retinoblastoma. The RT4 patient’s mother has died of a liver cell carcinoma. The case history and family trees of two patients

(RT1 and RT2) with a positive family history is stated below.

RT1 patient presented at three months of age due to bilateral white reflex. Right sided painful red eye developed within a few days which was the presenting feature. Her mother also had right sided retinoblastoma when she was two years of age and her right eye was enucleated.

No other relatives in the family are affected by RB. Figure 3.14 illustrates the family tree of

RT1. In this patient bilateral enucleation was performed due to bilateral retinoblastoma.

Chemotheraphy, radiotheraphy and laser therapy was combined with primary and secondary enucleation. Currently the child is stable.

Figure 3.14: Family tree of the family with mother and child (RT1) affected with

Retinoblastoma.

35y 34y

13y 6y 5y 8 months

III 4y

Figure 3.15 illustrates the family tree of a child with positive family history of retinoblastoma and other malignancies which are caused by mutated RB1 gene (osteosarcomas and clear cell carcinomas) of the lung. This child was delivered at 34 weeks of gestation due to pre- eclamsia by an emergency cessarian section. During screening for retinopathy of prematurity right sided white reflex was detected. On examination under anesthesia right sided inferolateral tumour was detected. The tumour was unilateral unifocal and had no vitreous seeding or secondary deposits. The child was treated with chemotheraphy and lazertheraphy.

Child’s both eye globes were preserved with intact tree dimentional vision. The lower half of the retina is scarred; hence the visual field of the right eye is 66%.

Figure 3.15: The family tree of a child (RT2) with positive family history of retinoblastoma and other malignancies which are caused by mutated RB1 gene

(Osteosarcomas and Clear cell carcinomas) of the lung.

RB Osteosarcoma Clear cell carcinoma lung

II RB

III

This cohort consists of 19 male children (47.5%) and 21 (52.5%) female children. Twenty patients (50% of the tumors) had multifocal tumors while 20 patients (50%) had unifocal tumours.

The first sign of retinoblastoma was detected by parents in 38 patients (95%). The first sign of retinoblastoma was detected incidentally by doctors during examination in 2 patients (5%).

In 4 (10%) cases first contact doctor missed the clinical sign (and therefore diagnosis) at the initial stage. 3 patients (7.5%) defaulted treatment and returned later; Out of these two patient end up with enucleations. Figure 3.16 illustrates a tumour with bad prognosis.

Figure 3.16: Recurrence of retinoblastoma in a defaulted patient. This patient had to undergo unilateral enucleation (Retinal photograph of a patient from the LRH)

Out of bilateral retinoblastomas bilateral enucleation was performed in 2 (13.33%) patients and unilateral enucleation in 11 (73.33%) patients. Bilateral globe preservation was achieved

(and there by three dimensional vision was preserved) in two patients (13.33%). ( Table

3.7)(Figure 3.15)

Of unilateral cases unilateral enucleation was performed in 21(84%) patients. Bilateral globe preservation was achieved (and there by three dimensional visions was preserved) in two patients (13.33%). ( Table 3.7) (Figure 3.17)

Table 3.7: Globe preservation rates of bilateral and unilateral Retinoblastoma

Patients Bilateral Globe Unilateral Globes Bilateral

Preservation Preservation Enucleation

Unilateral cases 25 3 (12%) 22 (88%) -

Bilateral cases 15 2 (33.3%) 11 (73.3%) 2 (33.3%)

Total cohort 40 5(12.5%) 33 (82.5%) 2(5%)

Figure 3.17: Globe preservation rates of bilateral and unilateral Retinoblastoma

100%

90%

80%

70%

60% Globe Preservation 50% Unilataral Enucleation 40% Bilaleral Enucleation

30%

20%

10%

0% Bilateral RB Unilateral RB

3.2 Retinitis pigmentosa

Retinitis pigmentosa group consists of 5 (62.5%) females and 3 (47.5%) males.

Seven (87.5%) patients presented because of the onset of symptoms, while the other patient was detected during screening for visual impairment. Five patients (62.5%) presented due to onset of nyctalopia. Presentations due to appearance of dark spots, or squints were also reported with one patient being in each category (Figure 3.18).

Figure 3.18: Clinical of presentation of Retinitis Pigmentosa in the series of Ophthalmo- genetic project patients.

Number of patients presenting with each presenting symptom 6

0 Nyctalopia Dark spots Strabismus Screening

Subcortical posterior cataract was present in two (25%) of RP patients. All eight patients developed nyctalopia during the course of the illness.

Four patients (50%) out of eight had an affected sibling (positive family histories) .The other four (50%) patients did not reveal a family history. Two patients (25%) ware products of consanguinity. Out of the two consanguineous pedigrees one pedigree was suggestive of autosomal recessive inheritance.

Figure 3.19 illustrates the pedigree of the family with brother and sister affected with RP.

They are products of consanguinity sharing the genes of two common ancestors. Parents of the affected are first cousins.

Figure 3.19: The pedigree of the family with brother and sister affected with Retinitis

Pigmentosa.

III

65y 55y

IV 40y 38y 36y 33y 30y

The onset of nyctalopia started at the age of 22 years, though they had clumsiness from the childhood. Peripheral vision was affected mainly at the onset but over 18 years peripheral and central vision was impaired due to the onset of cataracts. Visual acuity was 6/60 bilaterally,

Ophthalmoscopy and slit lamp examination revealed bone specular pigmentation, attenuated vessels and macular edema.

Mentioned in figure 3.20 is a family of four siblings out of four two are affected with RP.

One presented with night blindness and the other was detected during a screening program.

Figure 3.20: Family tree of the family with four siblings. Out of four two are affected with Retinitis Pigmentosa.

39y 36y

III 17y 14y 11y 4.6y

In this family the parents are non consanguineous and non-related. The possible mode of inheritance is autosomal recessive.

An 18 year old girl was referred to Ophthalmo-genetics unit with the diagnosis of atypical retinitis pigmentosa. At the age of 17 years she developed dark spots in her visual field when looking at objects. Now nyctalopia is also present. Her visual acuity is 6/18 on the right side and 6/9 on the left side without correction. Her best corrected visual acuity is 6/9 on the right side and 6/6 on the left. On slit lamp examination her retina showed bone specular pigmentation which is more prominent on the right side than the left side. Attenuation of

98 arteries are also more prominent on the right than the left side. Vitreous opacity and a small punctuate lenticular opacities are also present. Her retinal photographs are shown in figure

3.21. She has no family history of RP and her parents are non-related. Her family tree is shown in figure 3.22.

Figure 3.21: Retinal photo grafts of the patient with atypical retinitis pigmentosa.

Attenuation of arteries is also more prominent on the right than the left side. Bone specular pigmentation is more prominent on the right side than the left side.

Figure 3.22: Family pedigree of the patient with atypical retinitis pigmentosa. The proband is a simplex case with a negative family history.

46y 40y

III 15 days 19y 18y 12y

A 42 year old lady was referred to genetic unit due to retinitis pigmentosa of asymmetrical eye involvement. She had nyctalopia for five years. Nyctalopia mainly affected peripheral vision but the central vision was intact. She has had two episodes of red eye which responded to treatment with steroids. Her visual acuity was 6/60 on the right eye and 6/24 in the left eye.

Slit lamp examination showed pigmentary deposits in the both eyes, but right eye more than the left. One of her brothers also has retinitis pigmentosa but the other brother does not have any visual impairment. Please see figure 3.23 for the family tree.

100

Figure 3.23: Family tree of the Retinitis Pigmentosa patient who had episodes of red eye.

31y 25y

III 45y 43y 41y

A 38 year old patient was referred to ophthalmo-genetic clinic due to night blindness for thirteen years with reducing visual acuity for last three years which is not corrected by refraction.

His visual acuity was of 6/12 in both eyes; Ophthalmoscopy and slit lamp examinatio n revealed early peripharal bone specular pigmentation, attenuated vessels and Pale waxy optic disc. (Fig: 3.24)

101

Figure 3.24: Fundus appearance of a patient with Retinitis Pignemtosa. Pale waxy disc with early peripheral pigmentation are recognizable.

None of his siblings are affected by RP or any other ophthalmic disorders; His parents and all his parents’ siblings are healthy. Therefore this patient is a simplex (sporadic) case of retinitis pigmentosa (See figure 3.25 for the pedigree).

102

Figure 3.25: Family pedigree of the 38 year old Retinitis Pigmentosa patient with one year old child

I Myocardial Infarction

Renal Disorder

38y 30y

III 1y

Since the pattern of inheritance cannot be determined by the family pedigree in a de novo mutation, estimation of the risk to the child has to be performed after genetic testing. The genetic mutation of the proband has to be detected first and then the child can be tested for the pathogenic mutation in the father. However genetic testing in one year old child is ethically questionable.

103

3.3 Stargadt’s disease

Two Sri Lankan male siblings were referred to Ophthalmo-genetics clinic due to impairment of central vision. These brothers were aged 15 and 11 at presentation and both were schooling. At 7 years of age mother realized that when they read they started holding books very closely. When watching television they wanted to take a closer look at the television.

Over a year they developed difficult to write on a sheet of paper. Gradual blurring of vision and difficulty reading and identifying peoples’ faces in both eyes developed over 3 years.

Parents are non consanguinious and nonrelated. Family tree is given in figure 3.26.

Figure 3.26: Family tree of the family with two siblings affected with Stargardt disease

(ST1 and ST2 patients)

6w 11y 15y

Ocular examination showed unaided visual acuity of 6/36 in the right eye and 6/60 in the left eye. With refraction visual acuity improved up to 6/24 in the right eye and 6/36 in the left eye. The anterior segment examination was not remarkable. Dilated fundoscopy showed

104 macular atrophy and a beaten-bronze appearance with surrounding yellow white flecks in both eyes. The systemic examination was not remarkable. Diagnosis of Stargardts macular dystrophy was made.

3.4 Epithelial Basement Membrane type corneal Dystrophy (EBCD)

A 32 year old male with hereditary corneal dystrophy was referred to the Human Genetic

Unit for genetic evaluation. At the age of 23 he developed abnormal sensation of left eye which progressed in to painful red eye over a few days. After two months same symptoms developed in the right eye as well. The patients parents are non consanguineous and non- related. He gives a positive family history of similar corneal defects. Eight persons in three generations were affected by the same corneal defect. Family tree is illustrated in figure 3.27.

105

Figure 3.27: Family tree of the family with epithelial basement membrane type corneal dystrophy. The pedigree shows autosomal dominant pattern of inheritance.

54y 52y 51y

33y 29y 30y

14y 7y 1y

Initial refraction was 6/9 bilateral and slit lamp examination of the cornea revealed epithelial fingerprint lines, geographic map-like lines, and microcysts on slit-lamp examination.

Diagnosis of map dot fingerprint type corneal dystrophy /epithelial basement membrane corneal dystrophy was made.

106

He developed recurrent corneal ulcers and was treated with antibiotics. As a consequence refraction gradually reduced from 6/9 bilateral to 6/24 bilateral over six months.

Bilateral corneal grafting was performed to reduce the effects of corneal scarring.

3.5 Aniridia

A five year old girl and her 25 year old father with clinical diagnosis of aniridia were referred to Human Genetic Unit for genetic evaluation and counseling. The child had photophobia since ten months of age and her father also had photophobia since childhood. Visual acuity of the father was 6/18 bilaterally and 6/24 bilateraly in the child. Slit lamp examination after dilatation revealed foveal hypoplasia with bilateral aniridia in both the patients. No evidence of keratopathy or cateract was present in the family. Hearing and olfactory sensation was also normal in both the patients. General examination revealed horizontal nystagmus. The rest of the systemic examination in the farther and the child was normal. Neither the child nor the father was a product of consanguinity. Genetic evaluation and pedigree analysis revealed autosomal dominant inheritance pattern showing three affected generations and four affected people in the family. The family tree is mentioned in figure 3.28

107

Figure 3.28: Family tree of the family with father and son affected with Aniridia

39y 21y 25y 21y III

3.6.1 Patient 1 with coloboma (CB1)

A 38 year old male was referred to Ophthalmo-genetic clinic with the diagnosis of bilateral retinocoroidal irish Colobomas associated with horizontal nystagmus. At presentation the visual acuity of the right eye was 6/60 which was the only eye with intact vision. This patient did not have a family history of genetic eye disorders and his parents were non consanguineous and non-related.

3.6.2 Patient 2 with coloboma (CB2)

A 2 months old baby girl was reffered to Human Genetic Unit with the diagnosis of bilateral irish Colobomas associated with dysmorphic features. The baby had microcephaly,

Microphthalmia, micrognathia, low birth weight, ventricular septal defect, patent ductus arteriosus, rocker bottom feet and typical hand posture of first and fourth fingers overlapping the second and third fingers. This patient did not have a family history of genetic eye disorders or genetic syndromes and her parents were non consanguineous and non-related.

108

Cytotogenetic analysis was performed and Edward syndrome due to non dysjunction was detected.

3.7 Waardenburg syndrome (WB)

3.7.1 Patient 1 with Waardenburg syndrome (WB1)

A four year old female child with heterochromia irides was referred to Opthalmo-genetics unit for genetic evaluation and counseling. Parents have noticed colour difference in two eyes, hypopigmented patch of hair since birth and hypopigmented area of skin over the left fore-arm (Figure 3.29).

Figure 3.29: The child showing clinical features of Waardenburg Syndrome.

109

This child was born to non consanguineous non related healthy parents, delivered normally after an uneventful prenatal period. There is no family history of any children with symptoms of syndromes but there is a history of Intra Uterine Death of twin pregnancy. Figure 3.30 illustrates the Family tree of patient WS1.

Figure 3.30: Family tree of patient 1 (WB1)

DM I

44y 34y

Breast CA

III 4y 14y IUD at 24 wks

The Child Health Development Record (CHDR) shows poor weight gain despite normal birth weight of 2.750kg, and normal birth length of 51cm.

On examination complete heterochromia on the right eye with bright blue iris was striking

(Figure 3.29). She showed white forelock over the frontal area, and patch of hypopigmented skin was present over the left fore-arm (figure 3.29). Pectus excavatum chest wall deformity

110 was present. Her facial features were hypertelorism, smooth philtrum, flat nasal bridge, long eye lashes, low set ears, synophrysis with medial eye brow flare, malformed nasal ale and dystonia canthorum.

Slit lamp examination showed a hypopigmented area in the right fundus; (Figure 3.31)

Convergent squint was also present. Her visual acuity was normal being 6/6 bilaterally with normal color vision. Her hearing was reported to be normal after ENT assessment.

Figure 3.31: Ophthalmic features of the WS1 patient; Hypopigmented right iris and retina is in comparison with the normal iris and the retina in the left eye

The child is socially active and friendly though aggressive at times. Can count in all 3 languages and play interactively with other children. The child has normally attained milestones and all areas of development are age appropriate.

111

3.7.2 Patient 2 with Waardenburg syndrome (WB2)

A two year old child with heterochromia of iris, with developmental delay and failure to thrive was referred for genetic evaluation and counseling. Parents have noticed colour difference in two eyes with hypopigmented patch of skin over the back of the neck and trunk since birth. This child was the second child of non consanguineous non related parents and the first child is normal. There is no family history of any children with similar symptoms of syndromes. He was born at term through an emergency cesarean section due to lack of progression of labor, though the antenatal period was uneventful.

On examination complete heterochromia on the right eye with bright blue iris was striking. A patch of hypopigmented skin was present over the back of the neck and thorax. Pectus excavatum chest wall deformity was present. His facial features were hypertelorism, prominant philtrum, flat nasal bridge, micrognathia and low set ears (figure 3.32).

Figure 3.32: Clinical features of the patient with Waardenburg syndrome type 2 (WB2)

112

On ocular evaluation, child was able to look and follow light. He reached for objects held close. Fundus evaluation showed normal fundus in both eyes without any obvious pigmentary changes.

His responses to sounds are satisfactory and the hearing assessment was normal. This child shows developmental delay in areas of gross motor development and speech development.

3.8 Blepherophimosis Ptosis Epicnthus Inversus Syndrome (BPES)

29 year old, Sri Lankan Tamil Lady was referred to Human Genetics Unit by Gynecologist due to premature ovarian failure associated with abnormalities of external eye features since childhood. (Figure. 3.33)

She was born to non consanguineous non related parents. She had small eye lids and narrowing of the eye opening (blepharophimosis) with droopy eyelids (ptosis) since her childhood. She has no history of strabismus, myopia, hyperopia amblyopia or any other visual disturbance.

Figure 3.33: Eye features of the lady with BPES. Bilateral blepharophimosis, partial ptosis and epicanthus inversus are exhibited.

Her growth and development was completely normal and attended menarche at the age of 13 years. She had oligomenorrhea and irregular menstruation since puberty. She got married at

113 the age of 24 and the couple was sub-fertile for five years. She developed primery ovarian insufficiency at the age of 28 years.

There is positive family history of individuals affected by the same disorder. Her father and one of the brothers also had similar eye features which are stated in the family tree illustrated in figure 3.34.

Figure 3.34 Family tree of the family with Blepherophimosis ptosis epicnthus inversus syndrome; three individuals in two generations are affected. The pedigree exhibits autosomal dominant mode of inheritance.

III

34y 33y 29y

Clinical examination confirmed the bilateral blepharophimosis (horizontal palpebral fissure measures 21 mm), bilateral partial ptosis and epicanthus inversus. Temporal displacement of

114 the lower eyelid and lateral displacement of the inferior punctum in the lower eyelid

(telecanthus) were recognized. Interpupillary distance was normal. Her nasal bridge was flat and orbital rim was hypoplastic. (Figure 3.33) She was constantly wrinkling the forehead to draw the eyebrows upwards. Her head posture was slightly tilted backwards to a chin up position to compensate for ptosis. She did not have low-set ears, or short philtrum. Her vision was normal (6/6 bilaterally) and she did not have strabismus or nystagmus. She had normal breast development and normal female body hair distribution. Her intelligence level should range from average to above average being a scientist and a degree holder.

Her karyotype was 46,XX which is compatible with normal female. Her hormonal assays are as follows; FSH – 51.1Miu/ml (High) or 33.20Miu/ml, Estradiol - 145 pg/ml (Normal), LH –

23.1 mIU/ml; signals developing premature ovarian insufficiency.

3.9 Moebius syndrome

A six year old Sri Lankan boy was referred to opthalmo-genetics clinic due to dysmorphic face with marked hypokinesia, difficult abduction of ocular globes and congenital amelia of left forearm and hand (Figure 3.35). He was born to non consanguineous parents and does not have a family history of similar features.

115

Figure 3.35: Congenital amelia, convergent strabismus and expressionless face

The Pre natal period was uncomplicated and the mother had taken Rubella vaccination prior to this pregnancy. The child was born at term at 39 weeks by LSCS and his birth weight was

2.75 kg which is within the normal range. The baby was kept in the baby room for 4 days due to difficulty in breast feeding caused by poor sucking. Baby’s weight started reducing up to

2kg due to poor sucking. Gross motor and speech development was markedly delayed but other areas of development were age appropriate.

General examination revealed convergent strabismus, epicanthal folds, hypertelorism, low-set large ears, depressed nasal bridge, under developed tip of the nose, smooth philtrum, thin upper lip, and thickening of the lower lip, high arched palate and dental crowding. Face lacked expression and left fore-arm and hand were absent. (Figure 3.36)

116

Figure 3.36: Facial dysmorphism in the child with clinical features of Moebius syndrome

Neurologic examination revealed bilateral, symmetrical abducens and facial nerve palsies with generalized hypotonia. On ocular examination, his best corrected visual acuity was 6/18 in both eyes while it was 6/36 in both eyes without correction. There was restricted convergence and extraocular movements were totally restricted in abduction. There was no diplopia at near or distance or any kind of nystagmus in any gaze.

117

4. Discussion

This was the first study done in Sri Lanka describing the ophthalmo-genetic disorders prevalent in the country.

The majority of the patients that presented with ophthalmo-genetic conditions are children

(49 patients 83%), because in a majority of ophthalmo-genetic conditions clinical manifestations start early in life, and some disorders are congenital (Eg: Waardenburg syndrome, Moebius syndrome). But the Retinitis pigmentosa group had a considerable adult representation because of its late onset nature. Epithelial basement membrane type dystrophy also presents late because the clinical manifestations starts after the pediatric age. However late presentations of childhood onset Ophthalmogenetic disorders were also detected (Eg.

Isolated Coloboma - CB1 and Aniridia adult patient - AN1)

Although positive family history was expected in most of the patients clinically diagnosed to have Ophthalmo-genetic disorders family history was positive only in 14 patients (24% of the collective cohort) (Figure 3.5). This may be explained genetically by occurrence of de novo mutations or germline mossaicism in parents or by two hit hypothesis. Negative family history may be explained clinically by reduced penetrance and variable expressivity.

However minor clinical features were not detected in any of the undiagnosed relatives in any of the categories. Molecular genetic testing of pathogenic mutations needs to be performed in the patient and the relatives in order to reveal the origin of the genetic mutations, and to study the descent of pathogenic mutations through generations.

Five patients (8%) had syndromic phenotypes with significant eye involvement. For four syndromic patients (WS1, WS2, and Moebius syndrome, BPES) eye signs (Heterochomia iridum, defective eye movements, blepherophymosis and ptosis) provided the best guide to diagnosing the complex syndromes. Correct diagnosis of the above rare syndromes led to

118 proper management of all the systemic features of the syndrome and genetic risk assessment.

This finding emphasizes the importance of complete physical examination of relevant patients with significant eye signs. However confirmation of clinical diagnosis would be the ideal method of planning the management. Exclusive eye disorders such as S targartds disease, Aniridia, Retinitis pigmentosa, EBMD, Isolated coloboma and retinoblastoma represent 92% of the cohort. Detection of a number of rare disorders in the study population indicate that so called rare disorders may not be rare in Sri Lankan populations and common disorders may be rare in the same population. Therefore a high degree of suspicion for rare disorders is important for clinicians for accurate diagnosis of Ophthalmo-genetic disorders.

However epidemiological studies are the best scientific method of extracting prevalence data of a population, which was not a part of this study.

As stated in the results section the majority (68% of the cohort) of patients were referred due to retinoblastoma. The possible reason is that clinical genetic evaluation and family screening had been emphasized in the RB guidelines [18,19] as an essential part of RB management.

Furthermore family screening and risk assessment is stressed because of its malignant nature with high morbidity rate due to visual loss and considerable mortality rate.

119

4.1 Retionoblastoma

Retionoblastoma is a significant cause of childhood blindness and visual impairment in children less than 5 years of age. The incidence is higher in developing countries, than in developed countries. But scientific data on retinoblastomas in Sri Lanka is limited as this is the initial study investigating the clinical presentations, heredity phenotypes and outcome of retinoblastomas in Sri Lankan children. Lower socioeconomic status and the presence of human papilloma virus sequences in the retinoblastoma tissue have been implicated as the reason for higher incidence of RB in developing countries [164]. But evidence is insufficient to prove that hypothesis.[165]

In the retinoblastoma group of patients family history was positive only in 4 patients (10%).

In a study done in Tunisia the percentage of family history positivity was 9.5% which is almost similar to Sri Lankan value [166]. In a study done in Iran the family history was positive in 5% of the cohort.[167]Possible explanation for the low rate of family history positivity is the two hit hypothesis. This theory explains the autosomal dominantly inherited but recessively expressed nature of RB1 gene. Infective causes such as viruses were proposed as a cause of pathogenesis, because of relatively high prevalence of sporadic Retinoblastoma in developing world, but no evidence has been provided to prove the viral etiology. [164,

165]

Strabismus associated with leucocoria is the most common presentation in Sri Lankan patients. Leucocoria is acceptable as the most common presenting sign in many populations worldwide (Table 4.3) (Figure 4.1) including Sri Lanka, Korea, Malaysia, Pakistan, Tunisia,

Taiwan, Turkey, United States, and Congo; In most instences leucocoria was the most common presenting finding; the reason being white reflex readily recognized by relatives when the child turns the head the child’s eyes are seen in different angles. In some cases

120 leucocoria was missed by first contact doctors. The reason is leucocoria is seen in the non dialated eye in certain angles only (depending on the location of the tumour). Therefore it is important to examine eyes under full dilatation, in each patient who reveals history of leucocoria.

Table 4.1: Percentage of patients presented with leucocoria in different countries

[167,168,169,170,171,172,173,174,]

Country Percentage of the patients presented with

leucocoria

Sri Lanka 75%

Korea 80%

Malaysia 71.8%

Tunisia 80%

Taiwan 71.4%

Turkey 82%

United States 56.2%

Kongo 49%

Pakistan 44%

121

Figure 4.1: Draphical representation of Percentage of patients presented with leucocoria in different countries

Percentage of the patients presented with leucocoria 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

Presentations due to isolated leucocoria, isolated strabismus or red eye were also usual.

Presentations due to proptosis, fever and excessive crying are uncommon but reported. Only one patient in the cohort (2.5%) had proptosis in Sri Lankan patients but in Congolese patients it was 28%. This may be explained by high literacy rate of Sri Lankans seeking health care at the onset of disorder. Table 4.2 compares clinical presentations of retinoblastomas in different countries. Strabismus is the secound commonest presentation of

Retinoblastoma in many populations.

122

Table 4.2: Comparisen of clinical presentations of retinoblastomas in different countries

Sri Lanka Korea Thaivan Iran Turkey leucocoria 70% 45.6% 71.4% 64.8% 61.9%

Strabismus 47.5% 28.3% 14.3% 28.2% 10.7%

Red, 7.5% - 18.4% - - painful, or tearing eye

[167,168,170,172,]

Globe preservation rate in Sri Lanka is in parallel to most of the other developing countries. It has not yet achieved the Globe preservation rates of developed countries. Screening programs of retinoblastomas both genetically and clinically should be implemented to achieve early diagnosis and thereby reducing the morbidity rates. Default rate is considerable (5%) in this cohort which should be handled with improved counseling skills. However it is not as high as in other Asian countries such as Thaiwan where the default rate is 13.5%[ 170].

Table 4.3: A comparison of globe preservation and enucleation rates in Bilataral and

Unilatral Retinoblastomas in different countries

Sri Lanka Iran Singapoor

Unilateral UL 88% UL 75.9% U/L 50%

enucleated BL 73.3% BL 34.3% B/L 18.7%

UL 12% UL 24.1% U/L 50% B/L globe preserved BL 26.7% BL 65.7% B/L 81.3%

123

Figure 4.2: A comparison of globe preservation and enucleation rates in Unilatral

Retinoblastomas in different countries

Singapo or Unilateral Enucleation

Iran

Both Globe preserved Sri Lanka

0% 20% 40% 60% 80% 100%

Sri Lanka has high enucleation rates compared to Singapoor and Iran and this is the same for bilateral as well as unilateral categories. Better sicioechonomic states in Singapoor compared to Sri Lanka and availability of more advanced alternative therapy may be presrving more eye globes in Singapoor. Seeking health care at early stages in tumour in Iran and Singapoor may also contribute for the better globe survival rates.

124

Figure 4.3: A comparison of globe preservation and enucleation rates in bilatral

Retinoblastomas in different countries

Singapoor U/Lenucleated

Iran

B/L globe preserved Sri Lanka

0% 20% 40% 60% 80% 100%

Bilateral retinoblastomas tend to present earlier than the unilateral RB. This was observed in the past retinoblastoma research and provided the basis for the Knudsons two hit hypothesis.

[13,14] The possible explanation is that the majority of bilateral retinoblastomas have a constitutional mutation therefore only one more mutation has to be acquired for the cell to become malignant, whereas unilateral retinoblastomas which are mostly non constitutional has to acquire two constitutional mutations to become malignant[13,14 ]

4.2 Waardenburg syndrome

Patient WS1 meets the diagnostic criteria of WS because she exhibits complete heterochromia, white forelock, patch of hypopigmented skin, medial eyebrow flare and

125 hypoplastic alae nasi. With the presence of dystopia canthorum her clinical diagnosis is WS type 1.

The phenotype of Waardenburg syndrome type I (WS1) is variable even within a family.

[175] Liu et al summarized the penetrance (percentage) of clinical features of WS1 (see Table

3) in 60 individuals with WS1 in his cohort and 210 affected individuals reported elsewhere in the literature. Table 3 compares clinical features that Leu et tals documented in his cohort with the clinical features of the Ophthalmo-genetics project patient.

Table 4.4: Clinical features of Waardenburg syndrome type I (WS1) compared to clinical features of the project patient.

Clinical Finding Percentage of Affected Project patient 1

Individuals

Heterochromic irides 15%-31% Present

Hypoplastic blue irides 15%-18% Present

White forelock 43%-48% Present

Sensorineural hearing loss 47%-58% Absent

Early graying 23%-38% Absent

Leukoderma 22%-36% Present

Medial eyebrow flare 63%-73% Present

High nasal root 52%-100% Absent

[122,175, 176]

126

Dystopia canthorum is the most penetrant feature of WS and is found in 99% of the reported cases [177].

When analyzing WS as a single category variability of reported phenotypic features are as follows; Hearing loss is not a universal feature of WS but penetrance of sensorineural hearing loss has been observed to be 9%-38% of WS. Cutaneous or hair pigmentary defects occur in

17 to 58% of patients. Partial or complete heterochromia iridis occurs in 21–28% of patients with WS, hypoplastic blue iris occur in 14.9–42% of cases [177].

Occasional findings identified in multiple families include cleft lip and palate, spina bifida

[116], and vestibular symptoms [ 119]. However these features were not present in either of the project patients. Table 4.5 compares clinical features of WS1 reported cases in literature with the features in WS1diagnosed project patient.

127

Table 4.5: Comparison of clinical features of patient 1, with the reported clinical features of Waardenburg syndrome type 1 (OMIM entry 193500 and project patient)

Category Subcategory Features Patient 1

Head and Neck Face Smooth philtrum Smooth philtrum Decreased philtrum length Ears Congenital sensorineural Normal hearing deafness Eyes Laterally displaced inner Laterally displaced canthi inner canthi (dystopia canthorum) (95 to dystopia canthorum 99%) Increased intercanthal distance Hypertelorism Blepharophimosis Complete Hypertelorism heterochromia iridis Heterochromia iridis, complete or partial Hypopigmented Hypoplastic iris stoma ocular fundus Hypopigmented ocular fundus Bright blue irides Bright blue irides Synophrys Synophrys Lower lacrimal dystopia Nose Broad, high nasal root Broad nasal root Wide nasal bridge Hypoplastic alae nasi Hypoplastic alae nasi Decreased nasal bone length Mouth Cleft lip/palate Normal Mandibular prognathism Chest Ribs, Sternum, Supernumerary ribs Pectus excavatum Clavicles and deformity Scapulae

128

Genitourinary External Genitalia Absent vagina (rare) Normal (Female) Internal Genitalia Absent uterine adnexa (rare) Normal (Female) Skeletal Skull Aplasia of posterior Normal semicircular canal on CT scan Spine Sprengel anomaly Normal Supernumerary vertebrae Skin, Nails, Hair Skin Congenital partial albinism Congenital partial (leukoderma) on face, trunk, albinism or limbs (leukoderma) on Hypopigmented skin lesions forearm Hair White forelock White forelock White eyelashes and eyebrows Bushy eyebrows Bushy eyebrows Premature graying of Premature graying of hair hair Neurologic Central Nervous Spina bifida (less common) Normal System Myelomeningocele (less common)

In the first patient it is important to predict the risk of developing WS1 in the probands mother’s subsequent pregnancies. Since all the other family members are devoid of signs and symptoms the probands is a simplex case. But exploring the other family members’ genetic status will increase the diagnostic certainty; especially because the penetrance of WS1 is 85%

[178] on clinical grounds. Once the pathogenic mutation in the proband is identified multiplex ligation-dependent probe amplification (MLPA), quantitative PCR, or targeted chromosomal microarray analysis may be applied to identify the genetic status of the family members and to explore the genetic status of the family.

The risk to the siblings of the proband depends on the genetic status of the proband's parents.

If a parent of the proband has a PAX3 mutation, the risk to the siblings is 50%. If neither

129 parent has pathogenic mutations of PAX3 gene the risk to siblings of a proband is low, because there is high probability that the child’s pathology is due to a De novo germline mutation during gametogenesis. It is also possible that (though less probable) child’s pathogenic mutation is due to germline mosaicism. Therefore the genetic risk to the siblings of the proband is not negligible due to the possibility of germline mosaicism [ 179].

Even though the WS1 patient’s pathogenic mutation is not inherited from her parents the disease causing mutation is heritable because she carries a 50% possibility of passing the mutation to her offspring. However the clinical manifestations in the offspring cannot be predicted and range from mild or subclinical features through the classic phenotype of WS.

Patient 2 also fulfills the diagnostic criteria of WS by exhibiting heterochromia iris, broad and flat nasal brigde and leucoderma over the posterior part of the neck and trunk (Fig 3). He does not exhibit dystonea canthorum thus it can be differentiated from WS1. He does not exhibit upper limb abnormalities hence can be differentiated from WS3. He does not have features of Hurshprungs disease therefore it is unlikely to be Waardenburg-Shah syndrome which is getegorized as WS4. Hence the patient should be included into the phenothpe category WS2 [112].

4.3 Waardenburg syndrome type II (WS2)

Arias suggested the existence of 2 types of Waardenburg syndrome based in the presence or absence of dystopia canthorum [112]. Hageman and Delleman presented family data supporting delineation of 2 types: type I, with dystopia canthorum; and type II, without dystopia canthorum [180]. Table 3 compares the clinical features of WS2 project patient with the clinical features of reported WS2 patients.[123]

130

Table 4.6: Comparison of reported clinical features of WS2 with clinical features of project patient who presented with clinical features of WS2 ( OMIM enytry and WS2)

Category Subcategory Features Patient 2

Head and Ears Deafness, congenital sensorineural Normal hearing Neck Dystopia Dystopia canthorum absent canthorum absent Heterochromia iridis Eyes Heterochromia Hypoplastic iris stoma iridis Synophrys

Wide nasal bridge Wide nasal bridge Nose Hypoplastic alae nasi Skin, Congenital partial albinism (leukoderma) on leukoderma) on Nails, Skin face, trunk, or limbs trunk Hair White forelock Hair White eyelashes and eyebrows Absent

Premature graying of hair

WS1 is distinguished from WS2 by the presence in WS1 of lateral displacement of the inner canthi (dystopia canthorum). Sensorineural hearing loss and heterochromia iridum are the two most characteristic features of WS2. Both are more common in WS2 than WS1. White forelock and leukoderma are both more common in WS1 than in WS2. Table 4 compares clinical features of both the project patient with WS with the reported cases of WS1 and

WS2.[125]

131

Table 4.7: Comparison of the reported clinical features of WS 1 and WS 2 with the clinical features of two patients who presented to the Ophthalmo-genetic clinic with features of WS

Affected individuals Project patients Clinical Finding WS1 WS2 WB P 1 WB P 1

Heterochromic irides 15%-31% 42%-54% Present Present

Hypoplastic blue irides 15%-18% 3%-23% Present Present

Sensorineural hearing 47%-58% 77%-80% Absent Absent loss

White forelock 43%-48% 16%-23% Present Absent

Early graying 23%-38% 14%-30% Present Absent

Leukoderma 22%-36% 5%-12% Present Present

High nasal root 52%-100% 0%-14% Present Absent

Medial eyebrow flare 63%-73% 7%-12% Present Absent

As mentioned in the introduction testing for mutations in the microphthalmia-associated transcription factor (MITF) gene, and SOX 10 gene may confirm clinical diagnosis in WS2 patient. Although pattern of inheritance of the reported families of WS2 is autosomal dominant none of the family members of the WS2 patient had clinical features of WS.

132

Therefore this patient is a simplex case. Germline mutation during maternal or paternal gametogenesis or germline mosaicism in a parent may explain the possible mechanism. In case of germline mosaicism the probability of the next child being affected is high. In case of germline mutation during gametogenesis (de novo mutation) the risk of recurrence is low.

Despite the origin (inherited or de novo) of the mutation the offspring of the proband with

WS2 has a 50% chance of inheriting the disease-causing mutation, yet it does not determine the severity of the clinical manifestations.

4.4 Moebius syndrome

MS1 project patient is diagnosed to have Moebius syndrome because he exhibits facial and ocular muscle paralysis due to defective innervations of 6th and 7th cranial nerves. Facial diplegia is noticeable by incomplete eyelid closure during sleep, drooling, and difficulty in sucking.[16] The project patient MB1 also has a history of poor sucking. Inability to make facial expressions and close the mouth is common to most of MS patients including the MS1 project patient. Apart from defective innervations of facial muscles lip abnormalities such as undue prominence and eversion of lower lip also contribute to speech defects.

Verzijl et al. examined 37 Dutch patients with Moebius syndrome in 2003, which is the largest cohort of moebius syndrome reported to date. He defined Moebius syndrome as having, at a minimum, congenital facial weakness with impairment of ocular abduction.

[149]Table 4.6 shows a comparison of features observed in that Dutch cohort of patient with this Sri Lankan patient.

133

Table 4.8: Comparison of the clinical features of Moebius syndrome observed in the

Dutch cohort of patient with the clinical features of Moebius syndrome observed in the

Sri Lankan patient

Clinical feature Dutch Cohort % Sri Lankan patient

Bilateral facial weakness 92 Present Feeding problems at birth 86 Present Tongue hypoplasia 77 Present Nasal dysarthria 76 Present Delayed language development 55 Present Weak bite or absence of jaw rotation 16 Absent during chewing Loss of sensation of the lip, cheek, 10 Absent forehead, and cornea, indicating a partial defect of the sensory root of the trigeminal nerve Relative sparing of the lower half of the 62 lower half of the face face from neurological deficit also affected Duane retraction anomaly 34 Absent Epicanthal Folds 89 Present Flattened Nasal Bridge 81 Present Micrognathia 64 Absent High-arched palate 61 Present Teeth defects 33 Present Hypertelorism 25 Present External ear defects 47 Present Limb deformities 86 Present Motor disabilities 88 Present

Most of the reported cases of MS are simplex cases (sporadic). Out of familial cases the

134 majority showed autosomal dominant pattern of inheritance. X linked and autosomal dominant inheritance was reported rarely.[150,151,152]. Project patient MS1 is also a simplex case without a family history being in the typical group.

4.5 Blepharophimosis ptosis epicanthus inversus syndrome (BPES)

The project patient BPES1 fulfills the diagnostic criteris of BPES type 1 by exhibiting blepharophimosis, ptosis, epicanthus inversus, telecanthus and female subfertility caused by premature ovarian failure.

Phenotype of BPES expressed by this patient is almost similar to BPES 1 cases reported in scientific literature. She exhibits flat nasal bridge and hypoplastic orbital rim that are well documented in literature. (Please see figure 3.2 for eye features). Low-set ears or short philtrum are not exhibited in this patient but are documented findings [133]. This patient’s intelligence is normal. But mental retardation is present in some reported cases of BPES

[134,140]. Table 4.9 summarizes the comparison of clinical features of this patient with the reported cases of BPES 1.

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Table 4.9: Comparison of clinical features of this patient with the reported cases of

BPES 1 [1]

Clinical Feature Reported patients Project patient

Blepharophimosis Present Present

Ptosis Present Present

Epicanthus inversus Present Present

Premature ovarian failure Present Present

Flat nasal bridge Present Present

Hypoplastic orbital rim Present Present

Low-set ears Present Absent

Short philtrum Present Absent

Menarchase Present Present

Secondary sexual characteristics Present Present

Mental retardation or low Present Absent intelligence

This family has autosomal dominantly inherited BPES type 1 which is the most reported pattern of inheritance. When analyzing pedigrees this syndrome is observed to be transmitted to the offspring only by males because syndromic women develop subfertility due to premature ovarian failure and the same is observed in this pedigree where the patient BPES inherited the disorder from her father and she will not be passing the disorder naturally to her offspring due to premature ovarian failure. This lady has developed hypergonadotrophic

136 hypogonadism. But with hormonal replacement and monitoring she might have a rare chance of getting pregnant. Pregnancy has been reported in BPES 1 patient in United States [182].

Since the disorder is autosomal dominantly inherited each pregnancy bears a 50% chance of having an affected child. Alternative reproductive options can be offered to prevent the offspring from getting the pathogenic mutation harbored by the parent. In vitro fertilization in combination with ovum donation is an option. In vitro fertilization in combination with pre implantation diagnostics is another option. Adaption of a child is the other option.

Ovum donation in combination with in vitro fertilization is the most advisable reproductive option currently available in Sri Lanka, because it reduces the probability of concepts with

BPES mutation. Major disadvantages being the high cost and the necessity for advanced scientific technology and optimum level of sterility which is available only at few centers in

Sri Lanka. On the other hand ethical issues may arise due to dual maternity. The ovum donor passes genetic material to the fetus thus considered to be the genetic mother of the fetus while the surrogate mother will be considered as the legal mother of the fetus.

To start with pre implantation diagnostic testing detection of the patient’s genotype is crusial.

These cytogenetic rearrangements occur in 2% of individuals with BPES [139]. But this lady’s karyotype is normal. Molecular genetic testing is the next step of confirming the diagnosis.

Management requires the input of specialists including a clinical geneticist, ophthalmologist, oculoplastic surgeon, endocrinologist, reproductive endocrinologist, and gynecologist.

4.6 Retinitis pigmentosa

The commonest inheritance pattern of autosomal dominant pattern was not seen [28] (as a straight forward inheritance pattern) in any of the families within this series of patients, where as autosomal recessive (50%) is the predominant inheritance pattern in this series (RP1

137 to RP9) of patients. The possible reasons are the autosomal dominance genetic mutations being relatively less frequent in this population because of founder effect, high rate of consanguinity in an isolated population or diagnosed patients with autosomal dominant retinitis pigmentosa not having an offspring due to avoidance of the possibility of an affected offspring. Larger study with extended samples and study periods are recommended to detect the exact frequencies in the Sri Lankan population.

In a study done in Nigeria the commonest mode of presentation was poor vision in 90% of the patients followed by night blindness in 56.7%.[183]In contrast nyctalopia is the commonest presenting symptom in 62.5% of the ophthalmo-genetics series (RP1 to RP8) and all patients developed nyctalopia later (100%). The difference of presenting symptom may be due to early recognition of night blindness and poor vision in dim light by Sri Lankan patients because Sri Lankans have a high literacy rate hence the habit of reading enhancing better understanding explanation and health concern. Headaches were occasional finding common to Sri Lankan and Nigerian cohorts. In a study done at United States, the most common problem noted in 53.3% was headache which is in contrast to Sri Lankan and Nigerian findings where headache is only an occasional finding in RP. [184] Head ache is a non specific symptom which may occur due to various mental, physical and social reasons such as mental stress caused by social complexity which may be less in people who lead simple life styles in Sri Lanka and Nigeria. One RP patient (RP3) had frequent hospital admissions due to episodes of red eye. The red eye responded to steroids each time. This must have caused by inflammation within the eye and the cause may or may not be related to Retinitis pigmentosa. Dual pathology or atypical presentations of RP may explain the condition.

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4.7 Epithelial Basement Membrane type corneal Dystrophy (EBMD)

EBMD1 patient shows the typical clinical findings of epithelial fingerprint lines, geographic map-like lines, and microcysts on slit-lamp examination. Family pedigree also shows autosomal dominant inheritance pattern which is the reported pattern of inheritance in

EBMD. In the family tree eight individuals in three generations are affected. Both males and females are affected and transmit the disorder to the next generation. All generations are affected without skipping generations, exhibiting the usual autosomal dominant pattern of inheritance. In the French pedigree which was described by Boutboul et al. in 2006 [67]

EBMD had occurred in 4 generations of the family in an autosomal dominant pedigree pattern. A man in the third generation had recurrent corneal erosion from the age of 18 years.

At the age of 51 years, visual acuity was decreased and both eyes were successfully treated by phototherapeutic keratectomy. Two of his daughters presented with similar clinical features, with recurrent corneal erosions beginning at the age of 18 years. Corneal maps were observed on slit-lamp examination. The youngest daughter had suffered from spontaneous corneal erosions beginning at the age of 8 years [67]. In the Sri Lankan patient the pattern of inheritance and the course of the disease tend to be similar. ( Figure 3.27) In the Irish family that Boutboul et al [67] studied the mutation was passed over to the daughter by the unaffected father, exhibiting the variable expressivity of the disorder. Variable disease severity was common to the Sri Lankan pedigree as well, because two affected individuals had recurrent episodes leading to corneal scarring while others had only two or three episodes. Age of onset was also highly variable withthin individual in the Sri Lankan family ranging from 16 to 33 years.

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4.8 Aniridia

This family of isolated Aniridia indicates autosomal dominant inheritance pattern which is similar to majority of reported cases of isolated Aniridia worldwide. Since isolated aniridia occurs due to mutations or deletions of the paired box gene 6 (PAX6) which are dominantly inherited in a majority of patients, this family should be harboring a mutation in the PAX6 gene mutations and passing through generations. [72,73] In a minority aniridia occurs as part of the WAGR (Wilms tumor aniridia genital anomalies retardation) contiguous gene syndrome in which the adjacent PAX6 and Wilms tumor (WT1) genes are both deleted (74).

Therefore it is important to exclude WAGR syndrome in any patient with aniridia. In the family which was mentioned above (AN1 and AN2) is devoid of genitourinary abnormalities and mental retardation [74]; therefore WAGR syndrome was clinically excluded.

4.9 Coloboma

Although both syndromic and non syndromic phenotypes of Colobomas were known to have a strong genetic basis neither of the project patients had a positive family history. But in CB2,

Coloboma was associated with Edward syndrome and microphthalmia; hence indicate a genetic etiology. In the patient CB1 Coloboma was idiopathic and isolated because the patient does not exhibit any syndromic features. Although CB1 did not have a positive family history de novo mutations is a possibility; thus genetic testing may confirm the genetic etiology.

Both the project patients (CB1 and CB2) had bilateral involvement of Coloboma. The reported percentages of coloboma patients with bilateral involvement in Scotland and

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Hungary are 42% and 47.5% respectively and 33% in United States. [83, 99, 100] Wider study with more patients is needed to arrive at a conclusion.

4.10 Stargardt’s macular degeneration (STGD)

The clinical picture of two siblings presented with central macular dystrophy is compatible with Stargardt’s macular degeneration which was first described by German ophthalmologist

Karl Stargardt in 1909 [10].

These siblings complained of central visual loss rather than peripheral visual defect which in favor of STGD. They presented during teens which is the typical age of presentation of

STGD1. The pedigree suggests that the condition is autosomal recessively inherited.

Although the rare possibility of autosomal dominant inheritance of other forms of STGD with gonadal mossaicism persists, the clinical picture favours STGD1 (Please see section 3.3).

Tunji S Oluleye reported two Nigerian siblings affected with STGD1; In that report the pedigree had suggested autosomal recessive inheritance pattern and dilated fundoscopy had shown macular atrophy and a beaten-bronze appearance with surrounding yellow white flecks in both eyes which is similar to the fundoscopy findings in Sri Lanken siblings [53].

Other forms of STGD are usually inherited in an autosomal dominant manner. However genetic testing has to be performed for the confirmation of clinical diagnosis. Sequense analysis of entire coding region in combination with deletion duplication analysis has a high possibility of detecting the pathogenic mutation.

Although atypical and unusual phenotypes were detected among the study participants, in general the majority of the ophthalmo-genetic disorder phenotypes observed in Sri Lankan population is similar to the phenotypes observed worldwide. The variations of clinical phenotypes and clinical presentations may be explained by founder effect, de novo mutations, environmental influences and epigenetic mechanisms. This study will build up scientific

141 knowledge on genetic eye disorder phenotypes observed in South Asian population enhancing accurate diagnosis, and guiding for improved treatment options.

This study shows that there is a wide range of genetic eye disorders in Sri Lanka. Although individually rare, collectively genetic-eye disorders place a heavy burden on the health care system. It also causes grate impact on the affected individuals and their families. Most of the patients with Ophthalmo-genetic disorders need to be managed by a multidisciplinary team including an Ophthalmologist, Clinical geneticist, pediatrician, physiotherapist, optician and counselors. This study provides insight into the need for further expansion of genetic service provision in Sri Lanka.

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5. Conclusions

Genetic eye disorders present as part of complex syndrome or isolated eye disorder.

Retinoblastoma, Retinitis pigmentosa, Stargartds disease, Epithelial Basement Membrane type corneal Dystrophy (EBMD) and Aniridia are the isolated genetic eye disorders detected in the cohort. Waadernberg syndrome, Blepharophimosis Ptosis Epicanthus Inversus

Syndrome (BPES) and Mobius syndrome are genetic syndromes with significant ophthalmic involvement. Colomobas present in isolation or as syndromic phenotypes.

Phenotypes detected in retinoblastomas, BPES, Aniridia, EBMD and Stargardt disease are similar to phenotypes in the reported literature. Some patients in the Retinitis pigmentosa,

Waardenburg syndrome and Moebius syndrome categories exhibited atypical clinical features; however they remained within the diagnostic criteria.

The majority (83%) of patients presented with ophthalmo-genetic conditions are within the paediatric age group, which is explained by the congenital and early onset nature of the majority of ophthalmo-genetic conditions. A minority (17%) of patients presented within the adult age category with late onset ophthalmo-genetic disorders.

In the retinoblastoma (68% of the cohort) group of patients the main clinical presentations and phenotypes were similar to the global situation. However Sri Lanka did not achieve the globe preservation rates of developed countries, due to late presentations.

A minority (24%) of patients had a positive family. Although autosomal dominant (BPES,

Aniridia, EBMD) and autosomal recessive (Stargartd disease, RP) inheritance patterns were detected in this cohort, the majority (86%) were simplex cases. Two hit hypothesis, reduced penetrance, germline mosaicism and de novo germline mutations are the possible explanations.

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6. Recommendations

 Clinical genetic evaluation, genetic testing, genetic counseling including risk assessment

should be provided routinely for all the patients and families suffering from ophthalmo-

genetic conditions that are prevalent in Sri Lanka.

 Sri Lanka is an island with isolated populations and high incidence of consanguinity;

hence genotypes and phenotypes of genetic eye disorders may be unique in comparison to

the global situation. Therefore a wider research programs (including population based

screening programs and surveys) should be implemented to explore the phenotypic and

inheritance patterns in Sri Lanka.

 Molecular genetic testing should be provided for the patients and families with

ophthalmo-genetic disorders free of charge by the government because that plays a major

role in genetic risk estimation, counseling and prevention. This is crucial for

retinoblastomas especially for bilateral, multifocal or familial tumors, as part of standard

retinoblastoma management.

 After the genetic tests are implemented for ophthalmo-genetic conditions, it is possible to

explore the genotypes of Sri Lankan populations, eliciting phenotype genotype

correlations leading to better diagnosis and management of patients.

 Maintaining a good data flow and sharing information with the international community

is important to update knowledge on new scientific data, and to apply that to the practice

of medicine in our country.

 Pre-natal diagnosis and pre-implantation diagnostics should be implemented in an

ethically acceptable legal framework in the country. Especially Pre natal diagnosis for

retinoblastomas should be recommended for at risk families to treat congenital

retinoblastomas at the earliest stage of the disorder reducing the morbidity and mortality.

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 A single clinical sign or symptom in the eye might be the presenting feature of a complex

syndrome. Therefore systemic examination should be performed on relevant patients.

 Rare disorders may be common in some populations. Therefore during clinical evaluation

high degree of suspicion of diseases including less common disorders will point towards

accurate diagnosis.

 Awareness programs should be implemented among first contact doctors and general

public because early detection of Ophthalmo-genetic conditions has a better prognosis

and rates of missing diagnosis (especially in retinoblastoma category) and default rate of

treatment is considerable.

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7. Limitations of the study

1. Exploring the phenotyps of Ophthalo-genetic disorders in Sri Lankan patients is

interesting, with a wide variety of phenotypes and frequent atypical and unusual

presentations. The one year time constrain was therefore a limitation.

2. Since no previous research had been done in Sri Lanka on the topic of genetic visual

impairment in the country, lack of local data on the topic was a limitation.

3. Most of the Ophthalmo-genetic disorders are rare disorders; therefore lack of

established guidelines to arrive at a clinical diagnosis was a limitation.

4. Although most of the patients’ relatives were screened for miner manifestations of the

disorder and pre-malignant changes in the eye (in RB), this was not practically

possible in all the relatives.

5. Minority of pedigrees were inconclusive due to early death of parents, late onset

disorders, adoption and false paternity.

6. In a minority of patients distinct clinical features could not be recorded as

photographs were not taken due to unavailability of photo-consent.

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Appendix 1 - List of abbreviations adRP - Autosomal Dominant Retinitis Pigmentosa arRP - Autosomal Recessive Retinitis Pigmentosa

BL - Bilataral

BPES - Blepherophimosis Ptosis Epicnthus Inversus Syndrome

DNA - Deoxyribonucleic acid

EBMD - Epithelial Basement Membrane type corneal Dystrophy

FISH - Fluorescent In Situ Hybridization

MLPA - Multiple Ligation Probe Analysis mRNA – Massenger RNA

OMIM - Online Mendelian Inheritance in Man

Optical Coherence Tomography (OCT)

RB - Retinoblastoma

RNA - Ribonucleic acid

RP - Retinitis pigmentosa

UL – Unilataral

WHO - World health organization

WS - Waardenburg Syndrome xlRP - X-Linked Retinitis Pigmentosa

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Appendix 2: Documents used for subject recruitment

Data Collection Form

Descriptive Study on Phenotypes and Genotypes of Inherited Eye Disorders in Sri Lanka

Subject Study Number -

Name of subject …………………………………………………………………

Date of birth ……/……/……

Address …………………………………………………………………

…………………………………………………………………

Telephone number (Home) ...………………………………………………………………

(Mobile) ………………………………………………………………….

Email …………………………………………………………….

Referring Ophthalmologist ………………………………………………………………….

Date of Referral ……………………………………………………………….

Hospital …………………………………….Ward /clinic:……….

Clinic No/ BHT No ………………………………………………………………

Data Protection and Confidentiality

After completion of this page, ensure that the subject study number is entered on all pages of this booklet. Then detach this page and store separately from the remainder of the booklet.

164

165

Subject Study Number -

Date of entry to study (Date on consent form)

Date of birth

FAMILY TREE

Draw the family tree indicating all illnesses present, document abortions/still births as well.

III

Consanguinity : Yes / No

166

Subject Study Number -

Additional pedigree information

Location in Clinical or other information pedigree

167

Subject Study Number -

(1)Clinical diagnosis -

(2)Clinical history

(3)Examination findings

(4)Investigations

168

Subject Study Number -

(5) Treatment details)

(6) Response to treatment

(7)Course of the illness

(8)Any other remarks

169

(Only for selected subjects)

Subject Study Number -

InvestigationDate - -

Label Volume Storage Comments

K/EDTA vial 1. 1. 1. 2. 2. 2.

COMMENTS

The booklet should be signed when ALL available data have been entered and cross checked with relevant data recorded elsewhere in this booklet.

Signed……………………………………………………………. Date……………………………………………

Investigator/Research Assistant

170

INFORMATION SHEET

DESCRIPTIVE STUDY ON PHENOTYPES AND GENOTYPES OF INHERITED EYE DISORDERS IN SRI LANKA

I am Dr. Kushara Nuwanthi Weerapperuma, attached to Human Genetics Unit, Faculty of Medicine, Colombo. My current designation is post graduate trainee in Clinical Genetics.

I would like to invite you to take part in the research study titled “DESCRIPTIVE STUDY ON PHENOTYPES AND GENOTYPES OF INHERITED EYE DISORDERS IN SRI LANKA”, conducted by myself, under the supervision of

 Prof. Vajira H.W. Dissanayake at the Human Genetics Unit, Faculty of Medicine, University of Colombo and  Dr. Madhuwanthi Dissanayake (Ophthalmologist) Department of anatomy, Faculty of Medicine, University of Colombo. This study will be done at the National Eye Hospital Colombo and Human Genetics Unit, Faculty of Medicine, Colombo.

1. Purpose of the study

The purpose of the study is to explore the clinical features (phenotypes) & inheritance patterns of inherited eye disorders in Sri Lanka and to correlate clinical features with genetic defects.

2. Voluntary participation

Your participation in this study is voluntary. You are free to not participate at all or to withdraw from the study at any time despite consenting to take part earlier. There will be no loss of medical care or any other available treatment for your illness or condition to which you are otherwise entitled. If you decide not to participate you may withdraw from the study at any time by informing us.

171

3. Duration, procedures of the study and participants responsibilities

The study will be conducted over 1 year. We require your permission to ask you questions, examine you, have access to your medical records & discuss your disorder with other relevant doctors (medical geneticists and ophthalmologists). We might require your permission to take photographs or videotape appropriately. B ut those are not essential thus you can deny at any time. We also need your permission to publish the data collected in a scientific journal or scientific presentation. We will not mention your name or any other identifiable information about you when we publish the results. We might need to take 5ml of venous blood from you to do the genetic test. But that is not essential and it will be done only with your permission.

4. Potential benefits

Participation in this study will help you to know the genetic basis of your inherited eye disorder. This will contribute to the increasing of knowledge about genetic eye disorders in Sri Lankan patients. When we know the genetic basis and common inheritance patterns in Sri Lankan patients it will facilitate us to diagnose these patients at early stages, counsel patients with inherited eye disorders and to detect carriers and prevent some of these disorders. It might also help us to design suitable genetic tests to be used in Sri Lanka

5. Risks, hazards and discomforts

We might discover familial eye disorders which can be inherited by your children & siblings; Thus it might increase your anxiety; This will be overcome by proper genetic counseling of the patient & if appropriate the family and at risk individuals. On the other hand we can take steps to prevent expression of such familial disorders if we pick up at risk individuals. Early detection of at risk individuals will prevent complications.

Blood may be drawn to detect the genetic defect causing the disorder. Approximately 5ml of blood will be taken for testing from you. The risk to you by participating in the study is the risk of pain, bruising and infection at the needle prick site. These will be minimized by performing blood drawing under aseptic conditions by trained phlebotomist. Blood will be drawn only from selected subjects only (not all participants). Any patient can deny blood investigations because that is done after written informed consent.

172

6. Reimbursements

There will be no reimbursement for participating in the study, but you will be given a copy of the test results if we do any investigations on you.

7. Confidentiality

Confidentiality of all records is guaranteed and no information by which you can be identified will be released or published. The data collection booklet is designed to ensure confidentiality of information gathered. The electronic database containing the data will have only the subject study number and the database and the computer containing the database would be password protected. These data will never be used in such a way that you could be identified in anyway in any public presentation or publication without your expressed permission.

8. Termination of study participation

You may withdraw your consent to participate in this study at any time, with no penalty or effect on medical care or loss of benefits. Please notify us as soon as you decide to withdraw your consent. However it will not be possible for you to withdraw your consent once the results are sent for publication or once the results are published.

9. Clarification

If you have questions about any of the tests / procedures or information please feel free to ask any of the persons listed below by calling 011 2689 545 or 0714964470

Dr. Kushara Nuwanthi Prof. VajiraDissanayaka

Post graduate trainee Medical Geneticist

Human Genetics Unit Human Genetics Unit

Faculty of Medicine Faculty of Medicine

Colombo Colombo

173

CONSENT FORM

DESCRIPTIVE STUDY ON PHENOTYPES AND GENOTYPES OF INHERITED EYE DISORDERS IN SRI LANKA

(A) To be completed by the participant/guardian

 The participant / guardian should complete the whole of this sheet himself / herself.  Please keep a copy for yourself

1. Have you read the information sheet? YES/NO

2. Have you had an opportunity to discuss this study and ask any questions? YES/NO

3. Have you had satisfactory answers to all your questions? YES/NO

4. Have you received enough information about the study? YES/NO

5. Who explained the study to you? …………………………………………………………

6. Do you understand that you are free to withdraw from the study at any time, without having to give a reason and without affecting your medical care? YES/NO

7. Sections of your medical notes, including those held by the investigators relating to your participation in this study may be examined by other research assistants. All personal details will be treated as STRICTLY CONFIDENTIAL. Do you give your permission for these individuals to have access to your records? YES/NO

8. Do you give permission to take photographs or (Only if necessary) videotape your clinical features? YES / NO

9. Do you agree to have leftover blood samples and DNA be stored for future research into eye genetics? YES/NO

174

10. Do you agree for the samples to be sent abroad? YES/NO

11. Have you had sufficient time to come to your decision? YES/NO

12. Do you agree to take part in this study? YES/NO

Participants’/ Guardian’s signature:…………………………..……………………….Date…………………………… ……

Name (BLOCK CAPITALS):…………………………………………………………………………………… ………………………….

(B) To be completed by the investigator

I have explained the study to the above volunteer and he / she has indicated her willingness to take part.

Signature of investigator:……………………....…………………………………………..Date………… ………………..…….

Name (BLOCK CAPITALS):…………………………………………………………………………………… ………………………….

175

úia;r ^f;dr;=re& m;s%ldj

› ලx m%fõKs.; wCIsfrda. iïnkaO kqo¾Y සහ m%fõKso |

úia;rd;aul m¾fhaIKd;aul wOHhkh

fuu m¾fhaIKh fld

^1& m¾fhaIKfha wruqKq

wOHhkfha m%Odk wruqK jkafka m%fõKs.; wCIsfrda. j, frda. ,CIK meyeÈ,s lsÍu;a" tajd iy cdk úlD;s;d w;r iïnkaOh fidhd ne,Su;a" ta tla tla wCIs frda.h m%fõKs.;j Wreujk wdldrh meyeÈ,s lsÍu;a fõ'

^2& iafõÉPd ^ish leue;af;ka& iyNd.S;ajh

m¾fhaIKh i|yd Tnf.a iyNd.S;ajh wksjd¾h fkdjk w;r leu;s kï iyNd.Sùug fyda iyNd.S fkdùug ;SrKh lsÍu Tn i;= fõ' tfukau m¾fhaIKh i|yd iyNd.Sùug leue;a; m%ldY lsÍfuka miqj fyda m¾fhaIKh lrf.k hk w;ru.§ Tnf.a woyia fjkia jqjfyd;a ´kEu wjia:djl bka bj;a ùug;a mQ¾K whs;sh Tn i;= fõ' tfia m¾fhaIKh w;ru.§ woyia fjkia lsÍu fyda bka bj;a ùu Tnf.a bÈß ffjoH wjOdkh wvqùug lsisfia;a fya;=jla fkdfõ' tys§ Tn bj;a jk nj wmg ±kaùu muKla m%udKj;a fõ'

^3& m¾fhaIK ld, mrdih" m¾fhaIK mámdáh iy iyNd.Sjk Tnf.a j.lSï

m¾fhaIKh jirla mqrd meje;afõ' wOHhkh i|yd Tnf.a frda.h ms

176 i|yd;a" Tnf.a fi!LH ;;a;ajh iy m%fõKs.; frda. ms

iuyr wjia:dj, wmg Tnf.a frda. ,CIK PdhdrEm .;lsÍug fyda ùäfhda .; lsÍug o wjYH úh yels fyhska Tn Bg tlÕ jkafka kï ta i|yd o Tnf.a wjirh ,ndÈh yels fõ' fuh wksjd¾h fkdjk w;r Tng ´kEu wjia:djl m%;slafIam l< yels fõ'

fuf,i Tnf.ka iy m¾fhaIKhg iyNd.Sjk wfkl=;a frda.Ska$mqoa.,hkaf.ka ,nd.kakd o;a; iy tajdfha úoHd;aul miqìu idlÉPd lsÍu i|yd úoHd;aul iÕrdj, m< lsÍu fyda m%o¾Ykh fyda bÈßm;a lsÍu isÿúh yelsh' ta i|yd o wms Tnf.a wjirh n,dfmdfrd;a;= fjuq'

Tnf.a cdk ixhq;sh ±k .ekSug wjYH wjia:dj,§ muKla" wms ñ,s,Sg¾ 5 l reêr mßudjla Tnf.ka ,ndf.k wod, cdk mÍCIKh isÿlsÍu i|yd Tnf.a wjirh n,dfmdfrd;a;= fjuq' fuu mshjr w;HjYH fkdjk w;r Tnf.a wjirh u; muKla isÿflf¾'

tn÷ lsisÿ wjia:djl Tnf.a wkkH;djh fy

~~^4& m¾fhaIKhg iyNd.S ùfuka Tng ,eìh yels m%;s,dN~~

~~m¾fhaIKhg iyNd.Sùfuka Tng Tnf.a m%fõKs;.; wCIsfrda.hg cdk úoHd;aul fya;=j jvd fyd¢ka f;areï.; yelsh' fuu mÍCIKh i|yd Tnf.a iyNd.S;ajh" YS% ,xldfõ m%fõKs.; wCIsfrda. ms~~

~~YS% ,xldfõ m%fõKs.; wCIsfrda.j,g fya;= iy m%fõKs.; jk l%u ms~~

~~177~~

~~^5& m¾fhaIKhg iyNd.S ùu ksid Tng úh yels wjodkï ;;a;aj iy wmyiq;d~~

Tng ygf.k we;s m%fõKs.; wCIsfrda.h Tnf.a ¥ orejkag;a" fidfydhqre fidfydhqßhkag;a yg.ekSfï wjodkula we;s nj fyda wÞ, cdkuh úlD;sh Tjqkag m%fõKslj Wreu ù we;s nj ±k.ekSu Tng udkisl is;a;ejq,la ùug bvla ;sfí'

wmf.a m%fõKs úoHd WmfoaYk fiajdj fj; Tn;a wjYH kï Tnf.a mjqf,a wh;a" fhduq lsÍu u.ska fuu ;;a;ajh u. yrjd f.k jvd hym;a m%;sM, ,nd.; yelsh' úfYaIfhka frda. wjodku iys; mjqf,a mqoa.,hka we;akï frda. j

~~iuyr úg wKql cdk úoHd m¾fhaIK i|yd frda.hg fya;=jQ cdk úlD;sh y÷kd.ekSug ñ,s,Sg¾ myl reêr mßudjla Tnf.ka ,nd .ekSug wjYH ùug bv ;sfí'~~

~~fuys§ reêr idïm, ,nd.kakd ia:dkh wi, iq¿ fõokdjla" r;=ùula fyda wdidok ;;ajhla we;s ùfï wjodkula we;' kuq;a mQ¾K Ôjdkqyß;a WmlrK yd ;;a;aj Ndú;d lsÍfuka yd b;d m~~

~~^6& ^ydks& m%;smQrKh~~

~~wKql cdk úoHd mÍCIK i|yd reêr idïm, ,nd§u fjkqfjka ydksmQrK f.ùula isÿ fkdfõ' kuq;a mÍCIK m%;sM, f,aLkfha msgm;la iyNd.shd ^frda.shd& fj;g ,ndfokq ,efí'~~

~~^7& ryiHNdjh~~

~~Tnf.a ish¨ ffjoH jd¾;dj, ryiHNdjh wm úiska wdrCId lrkq ,efí' Tnf.a wkkH;djh fy~~

mß.Kl.; o;a; YdLdfõ wvx.= jkafka iyNd.slhd ^frda.shdf.a& wkq wxlh muKs' mß.Kl o;a; .nvdj wksjd¾hfhka uqr mo oud (Password Profected) wdrCId iys;j ;efnkq we;' fuu mß.Kl o;a; lsisúfgl;a Tnf.a wkkH;djh fy

~~Ndú;d l< fkdyelsh' fuu o;a; lsisúfgl;a Tnf.a wkkH;djh fy~~

~~^8& m¾fhaIKfhka bj;a ùug Tng wjYH jQjfyd;a~~

Tng ´kEu wjia:djl m¾fhaIKhg iyNd.S ùfuka bj;a úh yelsh' tys§ Tnj lsisÿ oKavkhlg hg;a fkdfõ' ;jo lsisÿ ffjoH wjOdkh wvqùula fyda m%;s,dN wvqùula isÿ fkdfõ' kuq;a Tn m¾fhaIKfhka bj;a jkafka kï fyda Tfí wjirh bj;a lr .kafka kï" ta nj wmg ±kqï §ug ldreKsl jkak' flfia fj;;a m¾fhaIKfha m%;sM, m%isoaO ùug hejQ úg§ fyda m%isoaO l< miq Tfí wjirh wdmiq ,nd .ekSug fkdyels nj i,lkak'

~~^9& ;jÿrg;a meyeÈ,s lsÍu wjYH kï" by; úia;r l< m¾fhaIKh ms~~

~~179~~

~~› ලx m%fõKs.; wCIsfrda. iïnkaO kqo¾Y සහ m%fõKso |~~

~~úia;rd;aul m¾fhaIKd;aul wOHhkh wkque;sh (leue;a;) m%ldY lsÍfï m;%sldj~~

~~(A) m¾fhaIKhg iyNd.S jk mqoa.,hd fyda Ndrlre úiskau msrúh hq;=fõ'~~

~~³ iïmQ¾K m;%sldju m¾fhaIKhg iyNd.S jk mqoa.,hd fyda Ndrlre úiskau msrúh hq;=fõ'~~

~~³ lreKdlr fuu m;%sldfõ msgm;la Tn i;=j ;nd.kak'~~

~~^1& Tn úiska iïmQ¾K f;dr;=re ^úia;r m;%sldj& lshjk ,oo @ Tõ$ke;~~

~~^2& m¾fhaIKd;aul wOHhkh ms~~

~~^3& m¾fhaIKd;aul wOHhkh ms~~

~~^4& m¾fhaIKd;aul wOHhkh ms~~

~~^5& m¾fhaIKd;aul wOHhkh ms~~

~~'''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''~~

~~^6& ´kEu wjia:djl fuu m¾fhaIKd;aul wOHhkfhka Tng bj;aj hd yels nj;a" tfia bj;aùug .kakd ;SrKh ms~~

^7& Tnf.a ffjoH jd¾;d" m¾fhaIKhg iïnkaO wfkl=;a jd¾;d wdÈh" m¾fhaIKhg iïnkaO ffjoHjreka iy ffjoH iydhlhka úiska mÍCIdlr ne,Su i|yd Tnf.a wjirh wjYH fõ' Tnf.a w;sYh fm!oa.,sl f;dr;=rej, ryiHNdjh wm úiska ;rfha wdrCId lrkq ,efí'

~~180~~

~~wmg Tnf.a ffjoH jd¾;dj,g msúiSug wjir ,nd§ug leu;so @ Tõ$ke;'~~

~~^8& Tnf.a frda. ,CIK PdhdrEm .;lsÍug iy ùäfhda .; lsÍug ^wjYH jqjfyd;a muKla& Tn wjir ,nd§ug leu;so @ Tõ$ke;~~

^9& Tn úiska wKql cdk úoHd mÍCIK i|yd ,nd fokq ,nk reêr idïm, ^,nd foa kï muKla& m%fõKs.; wCIsfrda. iïnkaOj wkd.;fha§ isÿ lrk m¾fhaIKd;aul wOHhk i|yd Ndú;d lsÍu i|yd Tnf.a wjirh §ug leu;so @ Tõ$ke;

~~^10& Tnf.a reêr idïm, jeäÿr m¾fhaIK i|yd msgrg heùug isÿ jqjfyd;a ta i|yd wkque;sh ,nd§ug leu;so @ Tõ$ke;~~

~~^11& m¾fhaIKhg iyNd.S ùu ms~~

~~^12& Tn fuu m¾fhaIKd;aul wOHhkhg iyNd.S ùug leu;so @ Tõ$ke;~~

~~m¾fhaIKhg iyNd.S jkakd fyda Ndrlref.a w;aik~~

~~'''''''''''''''''''''''''''''''''''''''''''''''''''''''~~

~~Èkh ''''''''''''''''''''''''''~~

~~iyNd.S jkakdf.a ku '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' ^meyeÈ,s wl=ßka&~~

~~(B) m¾fhaIlhd úiska iïmQ¾K l< hq;=hs' fuu m¾fhaIKd;aul wOHhkh ms~~

~~'''''''''''''''''''''''''''''''''''''''''''''''''''''''~~

~~Èkh '''''''''''''''''''''''''''''''''' m¾fhaIlhdf.a ku '''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' ^meyeÈ,s wl=ßka&~~

~~181~~

~~jfty; mwpf;if~~

~~; மரபியல் கண் FiwghLfspன் பீந ோடைப்; மற்쟁ம் பரம்படரத்ந ோற்றம் த ோைர்போன tpsf;f Ma;T~~

தகோ폁ம்ꯁ ம쏁த்鏁வ பீைத் ில் மனி மரபியல் பிரிவில் ம쏁த்鏁வ மரபியல் 믁鏁 ிடைப்பட்ை மோணவியோக இ쏁க்埁ம் Dr. 埁ஷோரோ ꏁவந் ி வ ீரப்தப쏁மோ ஆகிய

~~என்னோல் இந் ஆய்ퟁ நமற்தகோள்ளப்ப翁கிற鏁.~~

தகோ폁ம்ꯁ கண் டவத் ியசோடையி쯁ம் மனி மரபியல் பிரிퟁ தகோ폁ம்ꯁ டவத்ிய பீைத் ி쯁ம் ைத் ப்ப翁ம் " ; மரபியல் கண் FiwghLfspன் பீந ோடைப் ; மற்쟁ம் பரம்படரத்ந ோற்றம் த ோைர்போன tpsf;f ஆய்வில்" கைந்鏁 தகோள்ள உங்கடள뿁ம் அடைக்கின்நறன்.

~~NkYk; ,e;j Ma;thdJ Nghuhrphpah; t[puh H.W. jp];]ehaf;f (nfhOk;G itj;jpa gPlk;> kdpjkugpay; myF)> kJte;jp jp];]ehaf;f (fz; kUj;Jth;> itj;jpagPlk;> nfhOk;G) MfpNahhpd; jiyikapd; fPo; elhj;jg;glTs;sJ.~~

~~1. Nehf;fk; Ma;tpd; Nehf;fkhdJ kUj;Jt mk;rq;fs;> guk;giu Kiwfs;> kuGhpik fz; NfhshWfis fz;lwptJk; kugZ; FiwghLfis ghpNrhjpj;jYk;.~~

~~2. jd;dhh;t gq;Nfw;G~~

~~. ,~~

~~3. Ma;tpw;fhd gq;Nfw;ghshpd; nghWg;GfSk; nra;KiwfSk; Neu mtfhrKk;~~

~~182~~

~~இந் ஆய்ퟁ ஒ쏁வ쏁ை கோைத் ிற்埁 நமைோக ைத் ப்ப翁ம்.~~

~~, , ,~~

~~போர்டவயிட்翁 எங்க쿁க்埁 உங்களின் சம்ம ம் ந டவப்ப翁கிற鏁. .~~

. இந் கவல்கடள அறிவியல் ஆய்வி ைில் தவளியிை உங்கள鏁 சம்ம ம் எங்க쿁க்埁த் ந டவ. ோங்கள்

உங்கள鏁 தபயடரநயோ அல்ை鏁 நவ쟁 அடையோளம் கோட்ைக்埂羿ய கவல்கடளநயோ தவளியி翁வட விர்ப்நபோம். மரபியல்

~~பரிநசோ டனக쿁க்கோக உங்கள鏁 உங்களிைமி쏁ந்鏁 5 ml இரத் ம் எ翁க்க நவண்羿 ஏற்பைைோம்.~~

4. rhjfkhd gyd;fs; ,e;j Ma;tpy; gq;nfLg;gjd; %yk; cq;fsJ kugZ Kiwapyhd guk;giuahf flj;jg;gLk; fz;NfhshWfis mwpe;Jnfhs;syhk;. ,yq;ifapd; fz;Nehahspfspd; kugZ mbg;gilapyhd fz;NfhshWfis gw;wpa mwpit Nkk;gLj;j ,e;j Ma;T gad;gLj;jg;gLk;. ,jd; %yk; Nehapd; Muk;g fl;lepiyikia vspjhf fz;lwpe;J mjw;Fhpa nghJthd kugZKiwapyhd rpfpr;iria>ghpNrhjidfis mwpKfg;gLj;jKbAk;.

5. Mgj;J fs;> cghijfs;> kw;Wk; mnrsfupaq;fs; ,e;j Ma;tpw;fhf Vwj;jho 5ml msthd FUjp vLf;fg;gl;L ghpNrhjid elhj;jg;gLk;. FUjpj; njhw;Wf;fs;> typ vd;gd gq;Nfw;ghsuhy; vjph;nfhs;sg;gLk; rthy;fshf ,Ue;jhYk;> jFe;j epge;jidapd; fPo;> gapw;Wtpf;fg;gl;l itj;jpa gapw;Wdhpd; cjtpAld; Fiwf;fg;gLk;. NkYk; Fwpg;gpl;l rpy NehahspfsplkpUe;J khj;jpuNk FUjp khjphp ngwg;gLk;.

~~6. rYiffs;~~

~~183~~

~~gq;Nfw;ghsUf;F vt;tpj rYiffSk; toq;fg;glkhl;lhJ. (cjhuzk;: gz cjtpfs;) ghpNrhjid Nkw;nfhs;sg;gbd; mjw;Fhpa Kbtpd; gpujp toq;fg;gLk;.~~

7. ,ufrpaj;jd;ik gq;Nfw;ghsh;fs; gw;wpa tpguq;fs;> ngah; vd;gd ,ufrpakhf Ngzg;gLk;. ,yj;jpudpay; jftywpf;ifapy; gq;Nfw;ghshpd; ngaUf;F gjpyhf Fwpg;gpl;;l vz; toq;fg;gLk;. mDkjpapd;wp vt;tpj Ma;twpf;ifapYk; gqN;fw;ghshpd; ghpNrhjid KbTfs; ntspaplg;glkhl;lhJ.

8. KbT Ma;tpypUe;J ve;NeuKk; gq;nfLg;ghsh; tpyfpf;nfhs;syhk;. ,jw;fhf jz;lg;gzNkh> itj;jpaghpNrhjid ,ilA+WfNsh Vw;gLj;jg;glkhl;lhJ. vdpDk; gq;Nfw;ghshpd; ghpNrhjidKbTfs; tpQ;QhdehNsLfspy; xUKiw ntspaplg;gl;lgpd; tpyFtJ fbdkhFk;.

9. njspTgLj;jy; ,J njhlh;ghd cq;fsJ re;Njfq;fis> tpsf;fq;fis jaf;fkpd;wp> vq;fsplk; ve;NeuKk; Nfl;fyhk;. gpd;tUk; itj;jpa kw;Wk; kugZtpay; itj;jpa mjpfhhpfis njhlh;G nfhz;L cq;fsJ re;Njfq;fSf;F jPh;T fhzyhk;.

~~njhiyNgrp ,y: 011-2689545, 0714964470~~

~~Dr. 埁ஷோரோ ꏁவந் ி நபரோசிரியர்.வஜிர ிசோனோயக~~

~~믁鏁 ிடைப்பட்ை மோணவி ம쏁த்鏁வமரபியைர்,~~

~~மனி மரபியல் பிரிퟁ மனி மரபியல் பிரிퟁ டவத் ியபீைம் டவத் ியபீைம்~~

~~தகோ폁ம்ꯁ தகோ폁ம்ꯁ~~

~~184~~

~~Xg;Gjy; gbtk;~~

~~; மரபியல் கண் FiwghLfspன் பீந ோடைப்; மற்쟁ம் பரம்படரத்ந ோற்றம் த ோைர்போன tpsf;f Ma;T~~

A. Ma;tpy; gq;Nfw;gth; (Nehahsp) / mtuJ ghJfhtyuhy; ,g;gbtk; G+h;j;jp nra;ag;gl Ntz;Lk;.  gq;Nfw;ghsh; / ghJfhtuhy; Kw;whf ,g;gbtk; epug;gg;glNtz;Lk;.  ,j;jhspd; gpujpnahd;iw cq;fsplk; itj;jpUf;fTk;.

~~1. ePq;fs; jfty; mwpf;ifia gbj;jPh;fsh / thrpj;jPh;fsh? (,jw;fhd gpujpia cq;fSld; itj;jpUq;fs;) Mk;/,y;iy~~

~~2. ,e;j Ma;T gw;wp fye;jhNyhrpf;fTk;> tpdhf;fs; Nfl;gjw;Fk; cq;fSf;F re;jh;g;gk; toq;fg;gl;ljh? Mk;/,y;iy~~

~~3. cq;fSila vy;yh tpdhf;fSf;Fk; jpUg;jpfukhf tpilaspj;Js;sPh;fsh?~~

~~Mk;/,y;iy~~

~~4. ,e;j Ma;Tgw;wp NghJkhd jfty;fisg; ngw;Ws;sPh;fsh?~~

~~Mk;/,y;iy~~

~~5. ahh; cq;fSf;F ,ijg; gw;wpa tpsf;fk; mspj;jth;? ………………………..~~

~~6. vt;tpjfhuzKk; ,d;wp> vt;tpj itj;jpa ,ilA+Wk; ,d;wp ve;j re;jh;g;gj;jpYk; ,e;j Ma;tpypUe;J gpd;thq;f KbAk; vd;gij ePq;fs; mwptPh;fsh? Mk;/,y;iy~~

7. cq;fsJ itj;jpaf; Fwpg;gpd; xUgFjp> ,e;j Ma;tpw;fhf cq;fSld; MNyhrpf;fg;gl;l jfty;fs;> kPz;Lk; (rpyrkak;) NtW Ma;T MNyhrfh;fshy; ghPl;rpf;fg;glyhk;. MdhYk; cq;fs; Ratpguq;fs;> vg;NghJk; ,ufrpakhfNt Ngzg;gLk;. ,e;j Ma;T MNyhrfh;fshy; cq;fsJ itj;jpa mwpf;iffs; ghPl;rpf;fg;gLtjw;F rk;kjk; mspg;gPh;fsh? Mk;/,y;iy

~~185~~

~~8. cq;fsJ kUj;Jt mk;rq;fis Gifg;glk; gpbg;gjw;F mDkjp jUtPh;fsh?~~

~~Mk;/,y;iy~~

~~9. fz; kugZtpaYf;fhd vjph;fhy Ma;TfSf;F cq;fs; kpFjpahd FUjpia Nrkpj;J itf;fKbAkh?~~

~~Mk;/,y;iy~~

~~10. cq;fs; FUjpkhjphpia ntspehLfSf;F mDg;Gtjw;F rk;kjpf;fpwPh;fsh?~~

~~Mk;/,y;iy~~

~~11. cq;fsJ Kbit vLg;gjw;F Njitahd Neuk; toq;fg;gl;bUe;jjh?~~

~~Mk;/,y;iy~~

~~12. ,e;j Ma;tpy; gq;Nfw;f rk;kjpf;fpwPh;fsh?~~

~~Mk;/,y;iy gq;Nfw;ghsh; / ghJfhtyh; ifnahg;gk; :- ……………………………… jpfjp :- ………………………………~~

~~………………………………………………………………~~

~~(B) ஆய்வோளரினோல் ꯂர் ிதசய்யப்பை நவண்羿ய鏁~~

இந் ஆய்டவப் பற்றி நமல்埁றிப்பிைப்பட்翁ள்ள பங்நகற்போள쏁க்埁 விளங்கப்பLத் ி뿁ள்நளன். அத்鏁ைன் அவர் இந் ஆய்வில் பங்埁தபற்ற சம்ம ித்鏁ள்ளோர்.

~~டகதயோப்பம்:………………………………~~

~~ிக ி: ………………………………~~

~~………………………………………………………………~~

~~186~~