Searching the second hit in patients with inherited retinal dystrophies and monoallelic variants in ABCA4, USH2A and CEP290 by whole-gene targeted sequencing

Abstract

Inherited Retinal Dystrophies are clinically and genetically heterogeneous disorders affecting the photoreceptors. Although NGS has shown to be helpful for the molecular diagnosis of these conditions, some cases remain unsolved. Among these, several individuals harboured monoallelic variants in a recessive gene, suggesting that a comprehensive screening could improve the overall diagnosis. In order to assess the contribution of non-coding variations in a cohort of 29 patients, 25 of them with monoallelic mutations, we performed targeted NGS. The design comprised the entire genomic sequence of three genes (USH2A, ABCA4 and CEP290), the coding exons of 76 genes and two disease-associated intronic regions in OFD1 and PRPF31. As a result, likely causative mutations (8 novel) were identified in 17 probands (diagnostic rate: 58.62%), including two copy-number variations in USH2A (one deletion of exons 22–55 and one duplication of exons 46–47). Possibly damaging deep-intronic mutations were identified in one family, and another with a monoallelic variant harboured causal mutations in a different locus. In conclusion, due to the high prevalence of carriers of IRD mutations and the results obtained here, sequencing entire genes do not seem to be the approach of choice for detecting the second hit in IRD patients with monoallelic variants.

Genotype–Phenotype Correlations in a Spanish Cohort of 506 Families With Biallelic ABCA4 Pathogenic Variants

“…33 The new variant identified was a coding variant filtered out on the whole-exome sequencing analysis because of extremely low coverage. According to these data and findings from recent studies, 34 c.6148G>C should therefore be considered a likely benign variant. On the other hand, the frequent variant c.5603A>T, recently considered a low-penetrant variant, 18,19 was identified in 9 cases, allowing us to consider them solved, and only 2 carried an additional intronic variant in cis.…”

Molecular diagnosis based on comprehensive genetic testing in 800 Chinese families with non‐syndromic inherited retinal dystrophies

“…The shortage of some MLPA kits limited the detection of some CNVs, however the employment of QF‐PCR 38 or droplet digital PCR 39,40 may increase the detection rate of CNVs. Sanger sequencing, and the majority of NGS platforms are limited in their ability to detect CNVs, nevertheless, some genes involving PRPF31 and USH2A do have a high percentage of CNV variants 41‐43 . In our study, HEDEP achieved a sequencing depth of up to ×800.…”

Updating the Genetic Landscape of Inherited Retinal Dystrophies

“…A specific panel of genes will allow the study of known IRD at a high time/effectiveness ratio regardless of the clinical diagnosis, which is an advantage in those cases in which the clinical diagnosis is not well defined or overlaps between different clinical entities. Furthermore, it allows not only the possibility of including a greater number of probes in repetitive regions such as ORF15 of the RPGR gene, which is highly involved in X-linked IRD cases ( Vervoort et al, 2000 ; Megaw et al, 2015 ; Chiang et al, 2018 ; Charng et al, 2019 ), but also reported deep-intronic pathogenic variants ( González-del Pozo et al, 2018 ; Di Scipio et al, 2020 ). On the other hand, WES made it possible to find mutations in novel IRD-related genes without updating the panel in low prevalent genes not included for the sake of increasing the depth of coverage or to identify novel IRD genes.…”