A Report on Molecular Diagnostic Testing for Inherited Retinal Dystrophies by Targeted Genetic Analyses

Abstract

Aim: To test the utility of targeted sequencing as a method of clinical molecular testing in patients diagnosed with inherited retinal degeneration (IRD). Methods: After genetic counseling, peripheral blood was drawn from 188 probands and 36 carriers of IRD. Single gene testing was performed on each patient in a Clinical Laboratory Improvement Amendment (CLIA) certified laboratory. DNA was isolated, and all exons in the gene of interest were analyzed along with 20 base pairs of flanking intronic sequence. Genetic testing was most often performed on ABCA4, CTRP5, ELOV4, BEST1, CRB1, and PRPH2. Pathogenicity of novel sequence changes was predicted by PolyPhen2 and sorting intolerant from tolerant (SIFT). Results: Of the 225 genetic tests performed, 150 were for recessive IRD, and 75 were for dominant IRD. A positive molecular diagnosis was made in 70 (59%) of probands with recessive IRD and 19 (26%) probands with dominant IRD. Analysis confirmed 12 (34%) of individuals as carriers of familial mutations associated with IRD. Thirty-two novel variants were identified; among these, 17 sequence changes in four genes were predicted to be possibly or probably damaging including: ABCA4 (14), BEST1 (2), PRPH2 (1), and TIMP3 (1). Conclusions: Targeted analysis of clinically suspected genes in 225 subjects resulted in a positive molecular diagnosis in 26% of patients with dominant IRD and 59% of patients with recessive IRD. Novel damaging mutations were identified in four genes. Single gene screening is not an ideal method for diagnostic testing given the phenotypic and genetic heterogeneity among IRD cases. High-throughput sequencing of all genes associated with retinal degeneration may be more efficient for molecular diagnosis.

Prioritizing variants of uncertain significance for reclassification using a rule-based algorithm in inherited retinal dystrophies

“…We chose to focus on IRDs due to the expertize and case mix of our laboratory, and thus the larger sub-cohort available for study; still proportion of unsolved cases pending on a VUS reclassification among diseases does not differ. Our diagnostic ratios (solved/unsolved) in IRDs are consistent with previous studies 36 , 37 , but differ from the non-IRD sub-cohort, illustrating both the general heterogeneity of rare diseases and the impact of expertize on diagnosis.…”

Epidemiology of Mutations in the 65-kDa Retinal Pigment Epithelium (RPE65) Gene-Mediated Inherited Retinal Dystrophies: A Systematic Literature Review

“…The proportions of RPE65 -IRD in the EU-5 counties and the European region ranged between 1.2% in the UK and 14% in Germany [ 83 , 91 ]. Lastly, RPE65 mutations were estimated at ~ 1% in the US, while for the Middle East region, it varied between 4.81% in Saudi Arabia and 8% in Iran’s clinically diagnosed cases with IRD [ 81 , 85 , 87 , 89 ]. Also, based on the available data, the proportions of RPE65 in molecularly diagnosed cases with IRD was reported at 1.78% in China, 2.82% in the United Arab Emirates (UAE) and 4% in Brazil [ 57 , 84 , 92 ] (Supplementary Table B).…”

“…RPE65 variants have been reported to be most prevalent cause of LCA in the Nordic country of Denmark (17.44%) [ 102 ]. In North America, mutations in RPE65 were reported to cause 3.0–15.55% of clinically diagnosed LCA cases in the US, 0.81–1.85% of RP in the US, 3.28% for RPE65 -RP in Mexico and ~ 1% for clinically diagnosed IRD cases in the US [ 45 , 49 , 56 , 79 , 81 , 85 ]. In South America, the proportion were high due to the identification in the molecularly diagnosed Brazilian patients with only RPE65 -LCA at 20.51% and RPE65 -IRD at 4% [ 57 , 58 ].…”

“…This proportion increased to 6.32% for the molecularly diagnosed 95 patients with RP and LCA [56]. 85,87,89]. Also, based on the available data, the proportions of RPE65 in molecularly diagnosed cases with IRD was reported at 1.78% in China, 2.82% in the United Arab Emirates (UAE) and 4% in Brazil [57,84,92] (Supplementary Table B).…”

Voretigene Neparvovec for the Treatment of RPE65-associated Retinal Dystrophy: Consensus and Recommendations from the Korea RPE65-IRD Consensus Paper Committee

“…In a survey conducted by our group in Korea in May 2022, biallelic RPE65 mutations were found in 6 out of 2,140 patients (0.28%), including 109 LCA and 2,031 RP patients (unpublished data). In other countries, the proportion of RPE65-associated retinal dystrophy ranged from 0.60% to 20% [2,6,7,12,13,25,43,48,49,67,[78][79][80][81][82][83][84][85][86][87][88][89][90][91][92][93]. The results of those studies are summarized in Table 1.…”