PHENOTYPE STUDY OF CHILDHOOD ONSET RETINAL DYSTROPHIES ARUNDHATI DEV ...

372 Pages·2016·9.65 MB·English

by  Arundhati Dev Borman

A GENOTYPE – PHENOTYPE STUDY OF
CHILDHOOD ONSET RETINAL
DYSTROPHIES

ARUNDHATI DEV BORMAN
FRCOphth

INSTITUTE OF OPHTHALMOLOGY
UCL

A THESIS SUBMITTED FOR THE DEGREE OF MD(Res)
2015
Declaration

I, Arundhati Dev Borman, confirm that the work presented in this thesis is my own.
Where information has been derived from other sources, I confirm that this has been
indicated in the thesis.

Signed:

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Abstract
Introduction
The childhood onset retinal dystrophies comprise a clinically and molecularly
heterogeneous group of disorders. To date, sixteen genes have been implicated in the
pathogenesis of the spectrum of disorders comprising Leber Congenital Amaurosis
(LCA) and Early Onset Retinal Dystrophy (EORD), accounting for approximately
70% of cases. Although a wide range of phenotypes have been observed within this
spectrum, some genotype – phenotype associations are reported. Further detailed
genotype – phenotype studies will be important for expanding our understanding of
the effects of mutations in these genes on patients and their families. Our knowledge
of the phenotypic effects of mutations in other genes implicated in childhood onset
retinal dystrophies, such as the bestrophinopathies, continues to expand.

Purpose
To undertake detailed phenotypic studies into subjects with molecularly identified
childhood onset retinal dystrophies, and to describe novel phenotypes.

Methods
Affected subjects and their families were recruited from Moorfields Eye Hospital to
an ongoing Study into childhood onset retinal dystrophies. Subjects were examined
clinically and those that were historically recruited to the Study were invited back for
further phenotypic analyses, if their molecular cause was identified. Genetic analysis
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was performed using a variety of methods including DNA microarray analysis,
autozygosity mapping, direct sequencing and whole exome sequencing.

Results
Between August 2008 and August 2011, 201 subjects from 186 families were
recruited into the Childhood Onset Retinal Dystrophy Study, and categorised into 2
cohorts: cohort 1 - the generalised retinal dystrophies, comprising 177 subjects (166
families); and cohort 2 – subjects with a macular phenotype, comprising 24 subjects
(20 families). The molecular cause was identified in 34.5% of subjects in cohort 1 and
25% of subjects in cohort 2. RDH12 accounted for 28% of mutations in cohort 1, 18%
had mutations in CEP290, and 13% had mutations in RPE65. The subjects in cohort 2
with autosomal recessive bestrophinopathy all had bi-allelic mutations in BEST1. The
phenotype associated with the different genes identified was expanded, and focused
on those genes with limited reports of the phenotype, such as SPATA7, LRAT, RGR
and BEST1. The phenotype associated with a gene not previously identified in human
EORD, TUB, was studied, and the features associated with a novel macular phenotype
named Benign Yellow Dot Dystrophy were characterised.

Conclusions
This study has expanded and refined our understanding of the phenotypes associated
with mutations in genes that cause childhood onset retinal dystrophies, and has
identified a novel phenotype. This work will allow accurate prognostic and genetic
counselling to affected families, and provides phenotypic information that will be
important in ascertaining disorders that may be suitable for clinical trials of novel
therapies.
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Table of Contents
DECLARATION.......................................................................................................... 2
ABSTRACT .................................................................................................................. 3
INTRODUCTION ........................................................................................................... 3
PURPOSE ..................................................................................................................... 3
METHODS ................................................................................................................... 3
RESULTS ..................................................................................................................... 4
CONCLUSIONS ............................................................................................................. 4
TABLE OF CONTENTS ............................................................................................ 5
LIST OF FIGURES ................................................................................................... 10
LIST OF TABLES ..................................................................................................... 12
ACKNOWLEDGEMENTS ...................................................................................... 13
1.0 INTRODUCTION ............................................................................................ 14
1.1 HISTORY AND EPIDEMIOLOGY OF LEBER CONGENITAL
AMAUROSIS AND EARLY ONSET RETINAL DYSTROPHY ........................ 15
1.1.1 GENETICS OF INHERITED RETINAL DYSTROPHIES ........................................... 16
1.1.2 HISTORY OF LEBER CONGENITAL AMAUROSIS .............................................. 18
1.1.3 EPIDEMIOLOGY ............................................................................................. 21
1.1.4 SPECTRUM OF CLINICAL FEATURES................................................................ 22
1.2 RETINAL STRUCTURE AND FUNCTION ................................................ 24
1.2.1 ULTRASTRUCTURAL AND MICROSCOPIC STRUCTURE OF THE RETINA ............. 25
1.2.1.1 Photoreceptor cells ................................................................................ 27
1.2.1.2 Neural cells ............................................................................................ 31
1.2.1.3 Supporting cells ..................................................................................... 33
1.2.2 THE RETINAL PIGMENT EPITHELIUM .............................................................. 33
1.2.3 THE PHOTOTRANSDUCTION CASCADE ............................................................ 34
1.2.4 THE VISUAL CYCLE ........................................................................................ 36
1.3 TECHNIQUES FOR PHENOTYPING ......................................................... 39
1.3.1 VISUAL ACUITY ............................................................................................ 39
1.3.1.1 Visual acuity testing in children ............................................................ 42
1.3.2 COLOUR VISION ............................................................................................ 43
1.3.2.1 Ishihara Pseudoisochromatic Plates ..................................................... 43
1.3.2.2 Hardy Rand Rittler Pseudoisochromatic Plates .................................... 44
1.3.2.3 Other tests of colour vision .................................................................... 45
1.3.3 REFRACTIVE ERROR ...................................................................................... 46
1.3.4 PSYCHOPHYSICAL TESTING - PERIMETRY ...................................................... 47
1.3.4.1 Kinetic Perimetry ................................................................................... 48
1.3.4.2 Static Perimetry ..................................................................................... 50
1.3.4.3 Perimetry in children ............................................................................. 50
1.3.5 ADDITIONAL PSYCHOPHYSICAL TESTS ........................................................... 51
1.3.6 ELECTROPHYSIOLOGY ................................................................................... 51
1.3.6.1 The ISCEV standard Electro-Oculogram .............................................. 52
1.3.6.2 The ISCEV standard Electro-Retinogram ............................................. 53
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1.3.6.3 Other electrodiagnostic tests ................................................................. 55
1.3.6.4 Electrodiagnostic testing in children ..................................................... 56
1.3.7 OPTICAL COHERENCE TOMOGRAPHY ............................................................ 57
1.3.8 FUNDUS AUTOFLUORESCENCE IMAGING ....................................................... 59
1.3.9 FUNDUS IMAGING .......................................................................................... 62
1.4 TECHNIQUES FOR MOLECULAR ANALYSIS........................................ 63
1.4.1 LINKAGE ....................................................................................................... 63
1.4.2 GENETIC MARKERS ....................................................................................... 65
1.4.2.1 Restriction Fragment Length Polymorphisms ....................................... 66
1.4.2.2 Microsatellites ........................................................................................ 66
1.4.2.3 Single nucleotide polymorphisms .......................................................... 67
1.4.3 POLYMERASE CHAIN REACTION .................................................................... 68
1.4.4 AGAROSE GEL ELECTROPHORESIS ................................................................. 70
1.4.5 DNA SEQUENCING ........................................................................................ 71
1.4.6 DNA MICROARRAYS .................................................................................... 73
1.4.7 AUTOZYGOSITY MAPPING ............................................................................. 74
1.4.8 APEX MICROARRAY .................................................................................... 76
1.4.9 NEXT GENERATION SEQUENCING ................................................................... 78
1.5 GENETICS OF LEBER CONGENITAL AMAUROSIS AND EARLY
ONSET RETINAL DYSTROPHY ........................................................................... 84
1.5.1 GUCY2D (LCA1) ......................................................................................... 86
1.5.2 RPE65 (LCA2) ............................................................................................. 88
1.5.3 SPATA7 (LCA3) ........................................................................................... 94
1.5.4 AIPL1 (LCA4) .............................................................................................. 96
1.5.5 LEBERCILIN (LCA5) ...................................................................................... 99
1.5.6 RPGRIP1 (LCA6) ...................................................................................... 101
1.5.7 CRX (LCA7) ............................................................................................... 103
1.5.8 CRB1 (LCA8) ............................................................................................. 107
1.5.9 LCA9 LOCUS ............................................................................................... 109
1.5.10 CEP290 (LCA10) ..................................................................................... 110
1.5.11 IMPDH1 (LCA11).................................................................................... 115
1.5.12 RD3 (LCA12) ........................................................................................... 116
1.5.13 RDH12 (LCA13) ...................................................................................... 118
1.5.14 LRAT (LCA14) ......................................................................................... 120
1.5.15 TULP1 (RP14) .......................................................................................... 122
1.5.16 RGR (RP44) .............................................................................................. 124
1.5.17 IQCB1 / NPHP5 ....................................................................................... 126
1.6 AIMS AND OBJECTIVES ............................................................................ 128
1.6.1 AIMS ........................................................................................................... 128
1.6.2 OBJECTIVES ................................................................................................. 128
2.0 MATERIALS AND METHODS ................................................................... 130
2.1 ETHICS / PATIENT SELECTION .............................................................. 131
2.1.1 ETHICAL APPROVAL .................................................................................... 131
2.1.2 PATIENT SELECTION .................................................................................... 131
2.1.3 CONSENT ..................................................................................................... 133
2.2 PHENOTYPING............................................................................................. 134
2.2.3 CLINICAL HISTORY ..................................................................................... 134
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2.2.4 CLINICAL EXAMINATION ............................................................................. 135
2.2.4.1 Visual Acuity ........................................................................................ 135
2.2.4.2 Colour vision ........................................................................................ 136
2.2.4.3 General Ocular Examination ............................................................... 137
2.2.5 FUNDUS AUTOFLUORESCENCE IMAGING ..................................................... 139
2.2.6 OPTICAL COHERENCE TOMOGRAPHY .......................................................... 139
2.2.7 PSYCHOPHYSICAL TESTING: GOLDMANN VISUAL FIELDS ............................. 140
2.2.8 COLOUR FUNDUS PHOTOGRAPHY................................................................. 141
3.3 GENETIC METHODS .................................................................................. 142
3.3.1 EXTRACTION OF DNA ................................................................................. 142
3.3.2 GENOTYPE IDENTIFICATION STRATEGIES ..................................................... 143
3.3.2.1 DNA microarray using the Asper LCA chip ........................................ 143
3.3.2.2 Polymerase chain reaction and candidate gene sequencing ............... 144
3.3.2.3 Autozygosity Mapping .......................................................................... 148
3.3.2.4 Next Generation Sequencing ................................................................ 149
3.3.3 BIOINFORMATICS ........................................................................................ 149
4.0 RESULTS ........................................................................................................ 151
4.1 OVERVIEW OF STUDY ............................................................................... 152
4.1.1 STUDY PERIOD AND PATIENT RECRUITMENT ............................................... 152
4.1.2 DEMOGRAPHICS AND DIAGNOSES ............................................................... 153
4.1.2.1 Cohort 1 ............................................................................................... 153
4.1.2.2 Cohort 2 ............................................................................................... 154
4.2 OVERVIEW OF STUDY: OVERALL MOLECULAR FINDINGS ........ 155
4.2.1 LCA CHIP RESULTS FOR COHORT 1 ............................................................. 155
4.2.2 MOLECULAR DIAGNOSES IDENTIFIED IN THIS STUDY ................................... 159
4.2.2.1 Molecular Diagnoses for Cohort 1 ...................................................... 159
4.2.2.2 Cohort 2 ............................................................................................... 164
4.2.3 DIAGNOSIS BY GENE – COHORT 1 ............................................................... 164
4.2.3.1 AIPL1 ................................................................................................... 164
4.2.3.2 CEP290 ................................................................................................ 164
4.2.3.3 CRB1 .................................................................................................... 165
4.2.3.4 GUCY2D .............................................................................................. 166
4.2.3.5 LCA5 .................................................................................................... 166
4.2.3.6 MERTK ................................................................................................ 167
4.2.3.7 RDH12 ................................................................................................. 167
4.2.3.8 RPE65 .................................................................................................. 168
4.2.3.9 RPGRIP1 .............................................................................................. 169
4.2.3.10 SPATA7, TULP1, LRAT, RGR ........................................................... 169
4.2.4 DIAGNOSIS BY GENE – COHORT 2 ............................................................... 169
4.2.5 SUMMARY OF OVERVIEW OF STUDY ............................................................ 169
4.3 GENOTYPE - PHENOTYPE ASSOCIATIONS ........................................ 171
4.3.1 AIPL1 GENOTYPE - PHENOTYPE ASSOCIATION ............................................ 171
4.3.2 CEP290 GENOTYPE - PHENOTYPE ASSOCIATION......................................... 173
4.3.3 CRB1 GENOTYPE - PHENOTYPE ASSOCIATION ............................................ 177
4.3.4 GUCY2D GENOTYPE - PHENOTYPE ASSOCIATION....................................... 182
4.3.5 LCA5 GENOTYPE - PHENOTYPE ASSOCIATION ............................................. 183
4.3.6 MERTK GENOTYPE - PHENOTYPE ASSOCIATION ......................................... 185
4.3.7 RDH12 GENOTYPE - PHENOTYPE ASSOCIATION .......................................... 187
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4.3.8 RPE65 GENOTYPE - PHENOTYPE ASSOCIATION ........................................... 193
4.3.9 RPGRIP1 GENOTYPE - PHENOTYPE ASSOCIATION ...................................... 200
4.4 SPATA7 PHENOTYPE .................................................................................. 202
4.4.1 CLINICAL HISTORY ..................................................................................... 202
4.4.2 CLINICAL EXAMINATION ............................................................................. 203
4.4.3 FUNDUS AUTOFLUORESCENCE IMAGING ..................................................... 204
4.4.4 OCT IMAGING ............................................................................................. 204
4.4.5 PSYCHOPHYSICAL TESTING: GOLDMANN VISUAL FIELDS ........................... 207
4.4.6 ELECTROPHYSIOLOGICAL TESTING ............................................................. 208
4.4.7 MOLECULAR ANALYSIS ............................................................................... 210
4.4.8 DISCUSSION ................................................................................................. 213
4.5 TUBBY AND TUBBY-LIKE PROTEIN GENOTYPE - PHENOTYPE
ASSOCIATIONS ..................................................................................................... 217
4.5.1 TUB GENOTYPE - PHENOTYPE ASSOCIATION .............................................. 217
4.5.1.1 Clinical History .................................................................................... 218
4.5.1.2 Clinical Examination ........................................................................... 219
4.5.1.3 Fundus Autofluorescence Imaging....................................................... 221
4.5.1.4 OCT Imaging ....................................................................................... 221
4.5.1.5 Psychophysical testing: Goldmann Visual Fields ................................ 223
4.5.1.6 Electrophysiological Testing ............................................................... 224
4.5.1.7 Molecular analysis ............................................................................... 224
4.5.1.8 Collaborative studies ........................................................................... 226
4.5.1.9 Summary of TUB phenotype identified in this study ............................ 226
4.5.2 TULP1 GENOTYPE - PHENOTYPE ASSOCIATION .......................................... 227
4.5.2.1 Clinical History .................................................................................... 227
4.5.2.2 Clinical Examination ........................................................................... 228
4.5.2.3 Fundus Autofluorescence Imaging....................................................... 230
4.5.2.4 OCT Imaging ....................................................................................... 230
4.5.2.5 Psychophysical Testing: Goldmann Visual Fields .............................. 232
4.5.2.6 Electrophysiological Testing ............................................................... 233
4.5.2.7 Molecular analysis ............................................................................... 233
4.5.2.8 Summary of TULP1 phenotype identified in this study ........................ 237
4.5.3 DISCUSSION ................................................................................................. 237
4.6 LRAT PHENOTYPE ...................................................................................... 243
4.6.1 CLINICAL HISTORY ..................................................................................... 243
4.6.2 CLINICAL EXAMINATION ............................................................................. 244
4.6.3 FUNDUS AUTOFLUORESCENCE IMAGING ..................................................... 246
4.6.4 OCT IMAGING ............................................................................................. 246
4.6.5 ELECTROPHYSIOLOGICAL TESTING ............................................................. 252
4.6.6 PSYCHOPHYSICAL TESTING ......................................................................... 253
4.6.6.1 Goldmann Visual Fields ...................................................................... 253
4.6.6.2 Dark Adapted Perimetry ...................................................................... 255
4.6.6.3 Dark adapted spectral sensitivities ...................................................... 256
4.6.6.4 Critical Flicker Fusion ........................................................................ 258
4.6.7 MOLECULAR ANALYSIS ............................................................................... 260
4.6.8 DISCUSSION ................................................................................................. 261
4.7 RGR PHENOTYPE ........................................................................................ 268
4.7.1 CLINICAL HISTORY ..................................................................................... 268
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4.7.2 CLINICAL EXAMINATION ............................................................................. 269
4.7.3 FUNDUS AUTOFLUORESCENCE IMAGING ..................................................... 270
4.7.4 OCT IMAGING ............................................................................................. 270
4.7.5 PSYCHOPHYSICAL TESTING – GOLDMANN VISUAL FIELDS ......................... 271
4.7.6 ELECTROPHYSIOLOGICAL TESTING ............................................................. 272
4.7.7 MOLECULAR ANALYSIS ............................................................................... 273
4.7.8 DISCUSSION ................................................................................................. 274
4.8 CHILDHOOD ONSET AUTOSOMAL RECESSIVE
BESTROPHINOPATHY ........................................................................................ 276
4.8.1 CLINICAL HISTORY ..................................................................................... 277
4.8.2 CLINICAL EXAMINATION ............................................................................. 277
4.8.3 FUNDUS AUTOFLUORESCENCE IMAGING ..................................................... 279
4.8.4 OCT IMAGING ............................................................................................. 279
4.8.5 ELECTROPHYSIOLOGICAL STUDIES .............................................................. 284
4.8.6 MOLECULAR ANALYSIS .............................................................................. 285
4.8.7 DISCUSSION ................................................................................................. 290
4.9 BENIGN YELLOW DOT DYSTROPHY .................................................... 298
4.9.1 CLINICAL HISTORY ..................................................................................... 298
4.9.2 CLINICAL EXAMINATION ............................................................................. 299
4.9.3 FUNDUS AUTOFLUORESCENCE IMAGING ..................................................... 307
4.9.4 OCT IMAGING ............................................................................................. 307
4.9.5 ELECTROPHYSIOLOGICAL STUDIES .............................................................. 310
4.9.6 DISCUSSION ................................................................................................. 310
5.0 DISCUSSION AND CONCLUSIONS .......................................................... 314
5.1 THE CHILDHOOD ONSET RETINAL DYSTROPHY STUDY ................................. 315
5.2 PHENOTYPIC HETEROGENEITY IN CHILDHOOD ONSET RETINAL DYSTROPHIES 318
5.3 THERAPEUTIC OPTIONS FOR CHILDHOOD ONSET RETINAL DYSTROPHIES ........ 324
5.4 ADVANCES SINCE THIS STUDY WAS CONDUCTED ............................................ 327
5.5 CONCLUSIONS AND FURTHER WORK ............................................................... 330
6.0 REFERENCES ............................................................................................... 332
7.0 APPENDICES ................................................................................................. 360
7.1 PATIENT DOCUMENTS .............................................................................. 361
7.1.1 PATIENT INFORMATION LEAFLET ................................................................ 361
7.1.2 CONSENT FORM – ADULT ............................................................................ 365
7.1.3 CONSENT FORM – CHILD ............................................................................. 366
7.1.4 DATA PROTECTION CONSENT FORM ........................................................... 367
7.2 PRIMER SEQUENCES ................................................................................. 368
7.2.1 TULP1 PRIMERS ......................................................................................... 368
7.2.2 RGR PRIMERS ............................................................................................. 369
7.2.3 BEST1 PRIMERS .......................................................................................... 369
7.3 PUBLICATIONS AND ABSTRACTS FROM THIS STUDY ................... 370

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List of Figures
FIGURE 1 – GROSS ANATOMY OF A NORMAL RETINA, RIGHT EYE. ................................. 25
FIGURE 2 – SCHEMA OF THE LAYERS OF THE RETINA .................................................... 27
FIGURE 3 – ROD PHOTORECEPTOR ................................................................................ 29
FIGURE 4 – COMPARISON OF CONE AND ROD PHOTORECEPTOR ANATOMY .................... 30
FIGURE 5 – PROCESSES IN HUMAN ROD AND RPE CELLS ............................................... 37
FIGURE 6 – ETDRS VISUAL ACUITY CHART .................................................................. 41
FIGURE 7 – TESTS OF COLOUR VISION .......................................................................... 44
FIGURE 8 – NORMAL SD-OCT IMAGE ............................................................................ 58
FIGURE 9 – NORMAL FUNDUS AUTOFLUORESCENCE IMAGE, RIGHT EYE. ...................... 61
FIGURE 10 – SPATIAL REPRESENTATION OF 14 DIFFERENT LCA / EORD GENES AND
THEIR PUTATIVE ROLES ......................................................................................... 85
FIGURE 11 - RESULTS OF COHORT 1 SUBJECTS SENT FOR LCA CHIP ANALYSIS .......... 158
FIGURE 12 - GENOTYPES OF ALL SUBJECTS IDENTIFIED WITH VARIANTS IN COHORT 1 160
FIGURE 13 - SUBJECT 2, AIPL1, HYPOMORPHIC EORD .............................................. 172
FIGURE 14 - GRAPHICAL REPRESENTATION OF AGE VERSUS VISUAL ACUITY IN CEP290
RETINOPATHY ..................................................................................................... 176
FIGURE 15 - SUBJECT 10, CEP290, EORD ................................................................. 176
FIGURE 16 - SUBJECT 19, CRB1. DIAGNOSIS EORD ................................................... 179
FIGURE 17 - SUBJECT 15, CRB1, EORD ..................................................................... 180
FIGURE 18 - SUBJECT 16, CRB1 .................................................................................. 181
FIGURE 19 - SUBJECT 20, GUCY2D ............................................................................ 183
FIGURE 20 - SUBJECT 25, LCA5 .................................................................................. 185
FIGURE 21 - SUBJECT 29, MERTK, EORD .................................................................. 186
FIGURE 22 - GRAPHICAL REPRESENTATION OF AGE VERSUS VISUAL ACUITY IN RDH12
RETINOPATHY ..................................................................................................... 190
FIGURE 23 - RDH12 SUBJECTS WITH EORD ............................................................... 191
FIGURE 24 - SUBJECT 35, RDH12, ROD-CONE DYSTROPHY ........................................ 192
FIGURE 25 - SUBJECT 43, RDH12 EORD .................................................................... 193
FIGURE 26 - GRAPHICAL REPRESENTATION OF AGE VERSUS VISUAL ACUITY IN RPE65
RETINOPATHY ..................................................................................................... 197
FIGURE 27 - SUBJECT 48, RPE65, LCA ...................................................................... 197
FIGURE 28 - SUBJECT 54, RPE65, EORD ................................................................... 198
FIGURE 29 - SUBJECT 51, RPE65, LCA ...................................................................... 199
FIGURE 30 - SUBJECT 52, RPE65, HYPOMORPHIC EORD ............................................ 200
FIGURE 31 - SUBJECT 56, RPGRIP1 EORD ................................................................ 201
FIGURE 32 - SUBJECT 1, FAMILY 1 SPATA7 ................................................................ 205
FIGURE 33 - SUBJECT 2, FAMILY 1 SPATA7 ................................................................ 206
FIGURE 34 - SUBJECT 5, FAMILY 3 SPATA7 ................................................................ 206
FIGURE 35 - SUBJECT 10, FAMILY 5, SPATA7 ............................................................. 207
FIGURE 36 – GOLDMANN VISUAL FIELD, SUBJECT 2, SPATA7 ................................... 208
FIGURE 37 - PEDIGREE OF TUB FAMILY ...................................................................... 219
FIGURE 38 - TUB FAMILY: PROBAND (VI.11) ............................................................. 222
FIGURE 39 – TUB FAMILY: BROTHER (VI.10) ............................................................ 222
FIGURE 40 - TUB FAMILY: SISTER (VI.12) ................................................................. 223
FIGURE 41 – GOLDMANN VISUAL FIELD, SUBJECT VI.11, TUB .................................. 224
FIGURE 42 - PEDIGREES OF TULP1 FAMILIES .............................................................. 228
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