Identifying genetic causes of paediatric cataract onset and severity : validation of candidate gene HTR1F and mapping of modifier genes

Paediatric cataract is an opacity of the normally transparent lens in the eye, which occurs at birth (congenital) or develops during childhood. It is a leading cause of irreversible blindness in children. Up to 30% of paediatric cataracts are inherited. Although hundreds of cataract-related genes and loci have been found, genetic aetiology can not be identified in at least 40% of patients with inherited cataracts. Paediatric cataract is a highly heterogeneous disease with variable severity observed in affected individuals with the same causative mutation within a family. Several studies have suggested the involvement of modifier genes in paediatric cataract formation, but such genes have not been identified. This project consists of two studies and the overall aim is to identify causative and modifier genes for paediatric cataract. The first study assessed the biological function of a novel candidate causative gene previously identified in a family with paediatric cataract. The HTR1F gene was knocked out in F0 zebrafish using CRISPR-Cas9 ribonucleoprotein complexes. There was no significant difference in cataract formation between the htrlfknockout group and the control group. Therefore we concluded that knocking out HTRJF in the zebrafish models does not provide evidence for it being a cataract causative gene. Further investigations are warranted to obtain additional evidence. The second study investigated the potential for modifier genes in two extended families (family CRCH13 and family CSA91/110) with autosomal dominant paediatric cataract. The clinical phenotype information for each participant was updated and a high degree of variability for age of diagnosis and age of surgery within each family was noted. Genome-wide multipoint parametric linkage analyses of the affection status of cataract combined with whole-genome sequencing validated the previously reported causative genes. In addition, linkage analysis also indicated the existence of potential modifier genes through the presence of several suggestive linkage peaks. Further study of genes that modify the disease severity was conducted in these two extended paediatric cataract families. Genome-wide multipoint variance components linkage analysis of disease severity classified by age of diagnosis and age of surgery revealed one significant linkage region and two suggestive linkage regions likely containing modifier variants in family CRCH13, but failed to detect any linkage regions in CSA91/110 likely due to limited statistical power. In summary, we evaluated the function of a novel candidate cataract­ causing gene in zebrafish models. In addition, we developed an approach for rapid and highly efficient evaluation of cataract candidate genes in FO zebrafish using CRISPR-Cas9 ribonucleoprotein complexes. Zebrafish cataract assessment criteria were also generated, which to our knowledge, is the first systematic, definitive cataract assessment criteria for the zebrafish model. We confirmed the causative genes in two large paediatric cataract families and identified candidate linkage intervals for genetic modifiers in one of the families. The findings and novel strategies generated from this project can benefit the understanding of the pathogenesis of paediatric cataract and contribute to the identification of novel cataract-causing genes and modifier genes.