Molecular Diagnose Of Retinal Hereditary Disease Based On Next Generation Sequencing

Retinal inherited diseases are a set of common blindness disease with high clinical and genetic heterogeneity. Retinitis pigmentosa （RP） and Leber Congenital Amaurosis （LCA） are two main genetic forms of retinal disorders. These affected approximately 2,000,000 people around the word.The genetic mechanism of retinal inherited diseases is complex, and the clinical features are diverse and non-specific. It is difficult to identify the type of disease and the pathogenesis of the disease only by the clinical manifestations. In recent years, molecular diagnosis technology based on genetic testing has been gradually applied in clinical practice suggesting that it possess a good auxiliary diagnosis effect. At present, Sanger sequencing and APEX are the main methods for molecular diagnosis. The advantages of these two methods are high accuracy and speed, but they can only detect known mutation sites and the cost is high. Recently, the development of next generation sequencing technology has been favored with its high throughput, wide scale and low cost. Meanwhile, in combination with the developed capture technology, it is more effective to reduce the cost of NGS for a single sample.In this study, capture-NGS technology with a step by step strategy was utilized to systematically identify the putative pathogenic mutations for 553 retinal inherited disease probands, who were from difference parts of the world （including Caucasian and Chinese）. After genomic DNA was extracted and sheared into fragments of 300-500 bp, we completed the library prepare and capture processes. Then the DNA was sequenced on an Illumina Hiseq2000 machine. The obtained data were processed with an in-house bioinformatics pipeline and identified potential pathogenic variants. Sanger sequencing was used to validate these potential mutations. Segregation test was performed if the DNA of proband’s immediate family member was available. In combination with clinic information, the final pathogenic mutations were determined.We identified the potential mutations in 331 probands with the 60.0% solution rate.194 and 54 probands of them were identified with RP and LCA pathogenic genes, respectively. Caucasian and Chinese RP pathogenic mutations in solution rate was similar to that of about 65%, while the solution rate of LCA pathogenic mutation in Chinese （75.0%） was significantly higher than that in Caucasians （66.7%）. In the solved cases, a total of 538 mutations were identified in 82 genes including 401 novel mutations, and 61% of them were missense mutations. In our study, we found that the USH2A, ABCA4, EYS, CRB1 and GUCY2D were high frequency of mutations in Chinese retinal disease probands. Variant c.T2802G（p.C934W） in USH2A is a highly recurrent pathogenic mutation in Chinese RP patients, while the variants in PRPF31 were the most frequent mutations among Hispanic probands from the Miami area. More importantly, the incomplete penetrance and the sex bias were observed in PRPF31 mutation. Especially the variant c.322+4₃22+7del（p.?） in PRPF31 was found in three cases （BLM043, BLM067, BLM101）. We also identified a de novo mutation in SNRNP200 and 4 non-canonical RP genes （WDR19, IMPG1, C21orf2 and CHM） in four RP cases.In summary, genetic testing based capture-NGS technology could achieve the large scale, high through and relatively low cost for sequence determination. Through extensively assaying the pathogenic gene in inherited retinal disease probands from difference regions and ethnic group, we first reported the feature of mutation spectrum in Hispanic RP probands from Miami area. Our results could enrich the mutation spectrum and gene prevalence for retinal disease and provide the theoretical basis for enhancing the knowledge of the pathogenic mechanism of retinal diseases, which promoted the application of molecular diagnostic techniques in clinical practice.