4.2.3.1 AIPL1
Mutations in AIPL1 were identified in 2 subjects in this cohort (Subjects 1 and 2); 1 by the LCA chip (only one variant was identified), and 1 by direct sequencing by Dr Tan, UCL. In the subject in whom the Asper chip identified one variant, Dr Mackay, UCL, identified the second variant by direct sequencing. One subject carried a diagnosis of LCA and one of EORD. The phenotype associated with AIPL1 mutations in this cohort will be discussed in chapter 4.3.
4.2.3.2 CEP290
CEP290 was the most frequent gene identified in subjects with LCA (affecting 13.4%
of subjects with LCA) and in the subjects who were screened across the LCA chip
(identified in 31.0% of subjects sent for LCA chip analysis from UCL). The most frequent mutation identified was the common intronic variant, c.2991+1655A>G, p.Cys998X, occurring in 6 of 40 (15.0%) alleles identified by the LCA chip. This variant is reported to be the most common LCA variant, identified in up to 26% of LCA cases in North Western European populations, but it has a much lower incidence in other populations [247]. All but one of the subjects identified with variants in CEP290 in this cohort (Subjects 3 – 13) were of European origin; the one subject who was not was from South Africa although he is presumed to be of British and Dutch descent. The genotype-phenotype associations for CEP290 will be discussed briefly in chapter 4.3.
4.2.3.3 CRB1
Mutations in CRB1 comprised 9.8% of those identified with mutations in this cohort (Subjects 14 - 19) and the strategies used to identify them included screening using the LCA chip (5 subjects) and exome sequencing (1 subject). This patient (Subject 16) had been screened across the LCA chip prior to exome sequencing, but no variants were identified. The compound heterozygous mutations in CRB1 identified in this subject (c.3655C>T, p.Q1219X and c.1312T>C, p.C438R) were excluded from the 1000 genomes project and exome variant server, and were considered to be novel.
The phenotype associated with CRB1 mutations in this cohort will be discussed briefly in chapter 4.3.
4.2.3.4 GUCY2D
GUCY2D mutations were identified in 5 subjects in cohort 1, all via the LCA chip (Subjects 20 – 24). All 5 subjects carried a diagnosis of LCA. The chip identified two variants in only one subject, but one variant in 4 subjects. Direct sequencing of GUCY2D was performed by Dr Ocaka, UCL, in 2 of these four subjects, which did not identify a second variant. These two subjects had no other affected family members and no family history of consanguinity. GUCY2D has been identified to segregate both in an autosomal dominant and a recessive manner [114, 126-128]; it may be postulated that in this cohort the identification of only one variant in GUCY2D could suggest an autosomal dominant inheritance pattern with a possible de novo mutation in these individuals. However, this is unlikely to be the case as all dominant mutations in GUCY2D have been identified, to date, to cluster in 3 particular codons, 837, 838 and 839 [128]. These were not implicated in any of the GUCY2D subjects identified in this study. The genotype-phenotype associations of GUCY2D will be discussed in chapter 4.3.
4.2.3.5 LCA5
2 subjects were identified with LCA5 mutations. One subject (Subject 25) had undergone autozygosity mapping due to her consanguineous ancestry, which identified a region of homozygosity in which LCA5 resided. Positional gene screening was carried out, which identified the c.439_449dup, p.Glu151X variant in the homozygous state. The molecular analysis in this family was performed by Dr Davidson, UCL. The other subject (Subject 26), identified with compound heterozygous mutations in LCA5, had one variant identified by the Asper chip and the
other variant by direct sequencing by Dr Mackay, UCL. The phenotypes in these individuals will be briefly discussed in chapter 4.3.
4.2.3.6 MERTK
A homozygous mutation in MERTK was identified in one subject by the Asper chip (Subject 29). This subject carried a diagnosis of EORD. The phenotype associated with this MERTK variant will be discussed in chapter 4.3.
4.2.3.7 RDH12
RDH12 was the most frequently identified gene, comprising 27.8% of the genes identified in cohort 1 (Subjects 30 – 46). All 17 subjects identified with RDH12 mutations underwent direct sequencing by Dr Mackay, which identified both alleles in 15 of 17 (88.2%) subjects, and one allele in 2 subjects, both of whom had one allele identified by the LCA chip. The reason for the high proportion of subjects with RDH12 mutations in this series is that the distinctive phenotype associated with RDH12 mutations had recently been described [278], and the majority of these subjects were selected for RDH12 screening based upon their phenotype. A number of novel mutations were identified. These subjects were combined with those with RDH12 mutations that were identified and phenotyped by the previous research registrar, Mr Phillip Moradi, and published [284]. The genotype-phenotype associations for RDH12 identified in this study will be discussed in chapter 4.3.
4.2.3.8 RPE65
Mutations in RPE65 were identified in 13.1% of the subjects in whom a molecular diagnosis was identified (Subjects 48 – 55). The Asper chip identified variants in 2 subjects, but the majority of the RPE65 variants were identified by direct screening at the National Genetics Reference Laboratory (NGRL) in Manchester, part of the Central Manchester University Hospitals NHS Foundation Trust, UK, who at the time of conducting this study, provided a clinical service in which molecular analysis of RPE65 was performed using direct screening. The subjects were selected for RPE65 screening based upon their history and clinical features, and referred directly to the NGRL, who identified variants in 7 of the 8 subjects with mutations in RPE65 ascertained in this study. 6 of these variants were novel. The phenotype associated with mutations in RPE65 will be discussed in chapter 4.3.
There was one RPE65 subject who displayed hypomorphic features (Subject 52), and he was a compound heterozygote, as were 5 of the 7 families identified with RPE65 mutations. However, he was the only subject to harbour a duplication on one allele, c.1067dupA, p.Asn356Lysfs*8, which occurs beyond halfway in the transcript. His other variant, c.1543C>T, p.Arg525Trp occurs late in the transcript, and in silico analysis suggests that it is damaging (SIFT) or probably damaging (PolyPhen2). It is presumed that his mutation leads to some functional protein product. However, comparison of the location of these variants to the other milder cases reported in the literature, which have occurred much earlier in the transcript, do not identify any true correlation between mutation location and phenotypic severity [151, 161].
4.2.3.9 RPGRIP1
2 subjects (Subjects 56 and 57) were identified by the Asper chip to harbour only one variant each in RPGRIP1. This was the same variant in both subjects, c.1447C>T, p.Gln483X. No further molecular screening was carried out in these subjects, and so the second variants were not identified. The phenotypes in these individuals will be briefly discussed in chapter 4.3.
4.2.3.10 SPATA7, TULP1, LRAT, RGR
The variants identified in these genes and the associated phenotypes observed will be discussed in the appropriate chapters (4.4 – 4.7).