Leber Congenital Amaurosis and other autosomal recessive retinal dystrophies: A clinical and molecular genetic study Moorfields Eye Hospital Institute of Ophthalmology University College London Mr Phillip Moradi BSc MBChB FRCOphth Supervisors: Professor AT Moore, Professor AR Webster MD thesis to be submitted to the University of London I, Phillip Moradi, confirm that the work presented in this thesis is my own. Information from other sources have been referenced appropriately and indicated clearly. 1 Abstract Leber congenital amaurosis (LCA) and the early onset retinal dystrophies (EORD) are a spectrum of autosomal recessively inherited genetic conditions affecting children who have visual impairment starting under the age of five years. There are currently 19 known genes that account for approximately two thirds of cases. Only two of these 19 genes (IMPDH1 and CRX; not studied in this project) have been found to cause autosomal dominant LCA. This genetic heterogeneity makes the identification of these causative genes expensive and time consuming. Phenotype-genotype correlations are therefore important in directing efforts to determine the molecular cause of disease. The aims of this research project were to recruit and clinically characterise a large panel of LCA and EORD patients and to identify the underlying genetic cause of autosomal recessive disease. Patients were recruited from Moorfields Eye Hospital and Great Ormond Street Hospital. A full clinical examination was carried out. DNA samples were analysed using the Asper Ophthalmics LCA microarray chip and by direct sequencing. Large families, with several affected members, were examined using the Affymetrix gene chip arrays for regions of homozygosity and candidate gene sequencing was performed. DNA samples from 158 patients were obtained and 117 patients were examined clinically. A definitive molecular diagnosis was obtained for 26% of patients. Of the cohort of 158 patients with one or two mutated alleles identified and genotyped: RPE65 accounts for 1% of this cohort, 6% are due to mutations in CRB1, 15% are due to RDH12 mutations and 11% are due to mutations in CEP290. Two families were identified with novel CRALBP mutations. The genotype yield from the period of this research, August 2006- August 2 2008, is lower than that expected with newer technologies in 2014; such as next generation sequencing (NGS) or whole exome sequencing. Useful prognostic information gained will help future patients with these disorders. Patients with a molecular diagnosis may be eligible for clinical trials of gene replacement therapy. 3 Contents Abstract ............................................................................................................................... 2 Contents .............................................................................................................................. 5 1 Introduction .................................................................................................................... 17 1.1 History ......................................................................................................................... 18 1.2 Prevalence and incidence ............................................................................................ 20 1.3 Clinical spectrum ........................................................................................................ 20 1.4 Differential diagnosis .................................................................................................. 21 1.4.1 Clinically similar non-syndromic eye diseases ........................................................ 21 1.4.2 Clinically overlapping syndromic eye diseases ....................................................... 22 1.5 Molecular genetics of LCA and EORD ...................................................................... 24 1.5.1(a) Phototransduction and GUCY2D ......................................................................... 29 1.5.1(b) GUCY2D ............................................................................................................. 31 1.5.2 GUCY2D Genotype-phenotype correlations ............................................................ 33 1.6 The visual cycle and the LCA genes involved (RPE65, LRAT, RDH12) ................... 34 1.6.1 RPE65 ...................................................................................................................... 35 1.6.2a Genotype-phenotype correlations in RPE65 .......................................................... 37 1.6.2b Animal models of RPE65 and RPE65 gene therapy .............................................. 38 1.6.3 LRAT ........................................................................................................................ 39 1.6.4 Genotype-phenotype correlations for LRAT ............................................................ 40 1.6.5 RDH12 ..................................................................................................................... 40 1.6.6 Genotype-phenotype correlations for RDH12 ......................................................... 43 4 1.7 Photoreceptor development and structure (CRX, CRB1) ............................................ 44 1.7.1 CRX .......................................................................................................................... 44 1.7.2 Genotype-phenotype correlations for CRX .............................................................. 46 1.7.3 CRB1 ........................................................................................................................ 46 1.7.4 Genotype-phenotype correlations for CRB1 ............................................................ 48 1.8 Ciliopathies and LCA ................................................................................................. 48 1.8.1 TULP1 ...................................................................................................................... 49 1.8.2 Genotype-phenotype correlations for TULP1 .......................................................... 50 1.8.3 RPGR-Interacting Protein 1 ..................................................................................... 51 1.8.4 Genotype-phenotype correlations for RPGRIP1 ..................................................... 53 1.8.5 Centrosomal protein 290 (CEP290) ......................................................................... 53 1.8.6 Genotype-phenotype correlations ............................................................................ 57 1.8.7 Lebercilin LCA5 ....................................................................................................... 58 1.8.8 Genotype-phenotype correlations for LCA5 ............................................................ 59 1.9 Other LCA disease mechanisms ................................................................................. 61 1.9.1 AIPL1 ....................................................................................................................... 61 1.9.2 Genotype-phenotype correlations for AIPL1 ........................................................... 64 1.9.3 MERTK .................................................................................................................... 65 1.9.4 Genotype- phenotype for MERTK ........................................................................... 66 1.9.5 IMPDH1 ................................................................................................................... 66 1.9.6 Genotype-phenotype correlations for IMPDH1 ....................................................... 67 1.9.7 SPATA7 .................................................................................................................... 68 1.9.8 SPATA7 Genotype-phenotype analysis .................................................................... 69 5 1.10 Other genes associated with LCA ............................................................................. 70 1.10.1 Calcium binding protein 4 (CABP4) ..................................................................... 70 1.10.2 IQ-motif containing protein B1 (IQCB1) .............................................................. 71 1.10.3 NMNAT1 ................................................................................................................ 72 1.10.4 KCNJ13 .................................................................................................................. 73 1.10.5 RD3 ........................................................................................................................ 75 1.10.6 RLBP1 : a gene associated with juvenile RD..........................................................76 1.11 Gene identification strategies to identify disease causing mutations ........................ 77 1.11.1 Genetic markers ..................................................................................................... 77 1.11.2 Restriction Fragment Length Polymorphisms ....................................................... 78 1.11.3 Microsatellite markers ........................................................................................... 78 1.11.4 Single nucleotide polymorphisms (SNPs) ............................................................. 80 1.11.5 Genomic maps ....................................................................................................... 81 1.11.6 Linkage analysis ....................................................................................................