A Next Generation Sequencing amplicon-based strategy to explore Inherited Retinal Degeneration complexity

Inherited Retinal Degeneration (IRD) are a group of eye disorders, characterized by photoreceptors degeneration which include: Retinitis Pigmentosa, Stargardt disease, Usher Syndrome and Leber congenital amaurosis. The high genetic heterogeneity, the incompleteness of disease specific databases and the elevated number of genes involved in IRD, often hamper the correct molecular diagnosis and patients stratification. To clarify IRD molecular profile, we used a next Generation Sequencing (NGS) strategy and designed a custom AmpliSeq panel (Life Technologies), containing the coding sequences of 72 disease related genes, for a total of 1649 amplicons. An in-house bioinformatic pipeline was optimized to filter synonymous variants and polymorphism and to annotate variants with prediction algorithm (dbNSFP) and disease specific databases (LOVD eye diseases, Retina International, RPGR database). A cohort of 40 samples was selected (29 patients, 11 healthy relatives). They underwent a complete ophthalmologic examination (visual acuity, anterior segment and fundus examination, ERG and/or EOG, OCT), as well as a genetic counselling. Possibly causative mutations were detected in 62% patients (n=18). We found mutations in 8 genes. The most recurrent gene was mutated in 38% (n=7) of patients. The remaining seven genes harboured lower frequencies with just one or two patients mutated. Overall, seven genes were inherited with an autosomal recessive pattern and one gene was X-linked. Of note, less than 21% of variants have been already described in specific databases. These preliminary results highlight the need to further explore the molecular complexity and heterogeneity of RD in order to translate these analyses into clinical practice.