Genomic and functional approaches in identification of retinal disease genes and new insights to centrosome and cilia biology in the retina

Abstract

Purpose : Retinal dystrophy (RD) is a significant cause of vision loss. Many of the underlying disease genes and their functions are not known. We are using genomic approaches to identify variants in known disease genes, followed by strategies for novel disease gene identification where required. Our purpose is to provide molecular diagnoses for affected individuals, as well new insights to retinal and cell biology.

Methods : We applied Trusight One Clinical Exome capture (Illumina Inc CA USA), followed by sequencing on the Illumina HiSeq2500. Further genomic investigation was undertaken for known and novel disease gene identification. Variants in known and candidate RD genes were filtered and prioritized in 50 sporadic and familial RD patients. Expression studies were performed in mouse tissues and human cell lines. Functional studies were performed in mutant and control human fibroblasts, and transfected ARPE-19 and HeLa cells.

Results : Pathogenic and likely pathogenic variants were detected in over 70% of our RD cohort. Clear-cut causative variants were identified in the majority of cases, and results also included unexpected findings in CHM, and combinations with variants in more than one RD gene, including EYS and RP1, requiring detailed phenotypic and segregation analysis. We identified a novel retinal disease gene which our analysis implicates in centrosome and cilia biology. Expression analyses showed presence of the encoded protein of this gene in the retinal pigmented epithelium and inner segments of the photoreceptors. Cell-based assays revealed its localization in centrosomes during interphase, and the basal body of the primary cilium in non-dividing cells. Immunocytochemistry on skin fibroblasts revealed a higher percentage of cells with abnormal numbers of centrosomes in affected individuals. In HeLa cells transfected with the mutant construct, there were a higher number of multinucleated cells, further suggesting abnormality of centrosome and cell cycle biology.

Conclusions : Genomic investigation is valuable in RD and this study highlights benefits of genetic and phenotypic multidisciplinary review for accurate diagnosis. Our genomic and functional work has also led to new knowledge with implications for regulation of centrosome biology and impact on the cell cycle and ciliary function in the retina.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.