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A CLINICAL AND MOLECULAR GENETIC INVESTIGATION OF X-LINKED CONGENITAL STATIONARY NIGHT BLINDNESS LOUISE ELIZABETH ALLEN M.B.,BS.(Lond.) F.R.C.Ophth. Thesis submitted for the degree of Doctor of Medicine at the University of London UMI Number: U591839 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U591839 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 A clinical and molecular genetic investigation of XLCSNB L E Allen Abstract Aims This study examines the clinical and molecular genetic features of X-linked Congenital Stationary Night Blindness (XLCSNB). The aims are to accurately document the phenotype of affected subjects by investigating functional visual deficit, to evaluate possible disease mechanisms and to confirm or refute the veracity of the “complete” and “incomplete” descriptions of phenotype in common clinical usage. In addition, by identifying causative gene mutations in the pedigrees studied, the study investigates the possibility of a genotype-phenotype correlation. Methods Members of fifteen families previously diagnosed as having XLCSNB underwent standardised clinical, psychophysical and electrophysiological phenotypic testing. Comprehensive mutation screening of the genes NYX and CACNA1F was performed for each pedigree. Results Seven different loss-of-function mutations in the NYX gene were identified in 11 families and three mutations in the CACNA1F gene were identified in another three pedigrees. No mutations w ere detected in members of the remaining pedigree and this family was excluded from further study. Electrophysiological and psychophysical evidence of a functioning but impaired rod system was present in subjects from each genotype group, although scotopic responses tended to be more severely affected in subjects with NYX gene mutations. Scotopic oscillator)’ potentials were absent in all subjects with NYX gene mutations whilst subnormal OFF responses were specific to subjects with CACNA1F gene mutations. Conclusions NYX gene mutations were a more frequent cause of XLCSNB than CACNA IF gene mutations in the 15 British families studied. Since evidence of a functioning rod system was identified in the majority of subjects tested, the clinical phenotypes “complete” and “incomplete” do not correlate with genotype. Instead, electrophysiological indicators of inner retinal function, specifically the characteristics of scotopic oscillatory potentials, 30Hz flicker and the OFF response may prove more discriminatory. A clinical and molecular genetic investigation of XLCSNB L E Allen CONTENTS PAGE Abstract 2 Table of Contents 3-7 Table of Illustrations 8-9 Acknowledgements 10-11 Table of Contents Chapter 1 Introduction 12 1.1 Aetiology of nyctalopia 12 1.2 CSNB - historical review 12 1.3 Classification of CSNB 13 1.4 CSNB with abnormal fundus appearance 14 1.4.1 Oguchi’s disease 14 1.4.2 Fundus albipunctatus 14 1.4.3 Flecked retina of Kandori 15 1.5 CSNB with normal fundus appearance 16 1.6 Molecular genetics of the inherited retinal dystrophies 16 1.7 The subject of this thesis 7 1 Chapter 2 Applied retinal anatomy and physiology 20 2.1 Functional anatomy of the retina 20 2.1.1 The cells and layers of the peripheral retina 20 2.2 Topography of the retina 22 2.3 Retinal neurophysiology 22 2.3.1 Rod phototransduction 23 2.3.2 Cone phototransduction 25 2.3.3 Photoreceptor adaptation 25 2.3.4 Transmission from photoreceptors 25 2.3.5 Retinal circuitry of the rod and cone pathways 26 3 A clinical and molecular genetic investigation of XLCSNB L E Allen 2.4 Visual function of the rod and cone systems 26 2.4.1 The effect of retinal position on visual function 26 2.4.2 The effect of light intensity on visual function 27 2.4.3 Spectral sensitivity 27 2.5 Introduction to clinical psychophysical testing 30 2.5.1 Measurement of visual acuity 30 2.5.2 Clinical evaluation of colour vision defects 30 2.5.3 Perimetry 32 2.5.4 Dark adaptometry 34 2.6 Introduction to the ERG 34 2.6.1 Components of the ERG 34 2.6.2 The normal human ERG 35 2.6.3 Factors affecting the ERG 36 Chapter 3 Review of the previous clinical investigation 38 o f CSNB 3.1 Clinical and psychophysical manifestations of CSNB 38 3.1.1 Symptoms 38 3.1.2 Visual acuity 38 3.1.3 Refractive state 38 3.1.4 Extra-ocular movements 39 3.1.5 Colour vision 39 3.1.6 Spectral sensitivity 39 3.1.7 Visual field examination 39 3.1.8 Ocular examination 40 3.1.9 Systemic abnormalities 40 3.1.10 Dark adaptometry 40 3.2 Photochemistry in CSNB 41 3.3 The electrophysiology of CSNB 41 3.3.1 Introduction 41 3.3.2 The Rigg’s type ERG 41 3.3.3 The Schubert-Bornschein type ERG 42 3.3.4 The scotopic threshold response 45 4 A clinical and molecular genetic investigation of XLCSNB L E Allen 3.3.5 Oscillatory potentials 45 3.3.6 30 Hz Flicker response 45 3.3.7 The ON-OFF response 46 3.3.8 The Photopic negative response 46 3.4 Visual evoked potential recording 46 3.5 The ERG in carriers of XLCSNB 47 3.6 Other disorders causing a “negative” ERG waveform 47 3.6.1 Aland Island eye disease 47 3.6.2 Oregon eye disease 48 Chapter 4 Previous genetic investigation of XLCSNB 50 4.1 XLCSNB linkage studies 50 4.2 Identification of the CSNB2 gene - CACNA1F 50 4.2.1 Properties of the gene CACNA1F 51 4.2.2 Structure and function of the L-type calcium channel 51 4.2.3 Mutation analysis of the gene CACNA1F 52 4.2.4 Pathophysiological effect of CACNA1F mutations 52 4.3 Identification of the CSNB1 gene - NYX 53 4.3.1 Properties of the gene NYX 53 4.3.2 Structure and function of nyctalopin 53 4.3.3 Mutation analysis of NYX 54 4.3.4 Pathophysiological effect of NYX mutations 54 4.3.5 The nob mouse model 54 Chapter 5 Summary of Review 5.1 Summary of review and problems remaining 58 5.2 Aims of this project 59 Chapter 6 Investigation of XLCSNB: Methods and protocols60 6.1 Ethical Committee approval 60 6.2 Diagnostic criteria 60 6.3 Identification of pedigrees and patient recruitment 60 6.4 Ophthalmic examination 60 6.5 Psychophysical examination 61 5 A clinical and molecular genetic investigation of XLCSNB L E Allen 6.5.1 Scotopic perimetry 61 6.5.2 Dark adaptometry protocol 61 6.5.3 Spectral sensitivity protocol 62 6.6 Electrophysiological examination 62 6.6.1 ERG protocol 62 6.6.2 STR protocol 64 6.6.3 ON-OFF response protocol 64 6.6.4 VEP protocol 65 6.7 Molecular genetic analysis 65 6.7.1 DNA extraction from blood samples 66 6.7.2 Mutation screening of the genes CACNA1F and NYX 66 Chapter 7 Molecular genetic results 69 7.1 Mutation screening of CA CNA1F 69 7.2 Mutation screening of NYX 70 7.3 Summary of molecular genetic analysis 72 Chapter 8 Clinical examination results 76 8.1 Visual acuity 76 8.2 Refractive error 77 8.3 Nystagmus and strabismus 77 8.4 Slit lamp examination 78 8.4.1 Anterior segment examination 78 8.4.2 Fundoscopy 79 Chapter 9 Psychophysics results 83 9.1 Colour vision 83 9.2 Photopic perimetry 83 9.3 Scotopic perimetry 83 9.4 Dark adaptometry 88 9.4.1 Dark adaptometry in CSNB 1 subjects 88 9.4.2 Dark adaptometry in CSNB2 subjects 88 9.5 Spectral sensitivity 91 6 A clinical and molecular genetic investigation of XLCSNB L E Allen Chapter 10 Electrophysiology 92 10.1 The rod mediated ERG 92 10.1.1 The scotopic threshold response 92 10.1.2 The scotopic ERG 95 10.1.3 The dark adapted standard flash ERG 98 10.1.4 Scotopic oscillatory potentials 98 10.2 The cone mediated ERG 101 10.3 The b-wave to a-wave ratio 105 10.4 The 30Hz flicker response 105 10.5 The ON-OFF response 109 10.6 The photopic negative response 109 10.7 The visual evoked response 113 Chapter 11 Discussion of results 115 11.1 Molecular Genetic Findings 115 11.2 Clinical Examination in XLCSNB 115 11.3 “Complete” vs “Incomplete” clinical forms of XLCSNB 116 11.4 CSNB1 vs CSNB2 117 11.5 Other ERG abnormalities in XLCSNB 119 Chapter 12 Summary 122 References 124 Appendices 136 A.