Large scale smMIPs-based sequencing of 908 probands to provide a genetic diagnosis for patients with inherited macular diseases.

Abstract

Purpose : Inherited macular diseases (iMDs), including cone dystrophies (CD), cone-rod dystrophies (CRD) and Stargardt disease (STGD), are relatively rare and typically present with an early to mid- age of onset. Late onset iMDs also occur, which can display shared clinical features to age-related MD (AMD). The clinical and genetic heterogeneity in MDs makes a clear diagnosis challenging, therefore it is prudent to identify genetic variants associated with iMDs in order to differentiate between late-onset iMDs, AMDs, and other MD-phenocopies. We sought to identify pathogenic variants associated with iMDs and increase the diagnostic yield for this IRD subgroup.

Methods : We previously established a high-throughput, cost-effective sequencing strategy using single molecule Molecular Inversion Probes (smMIPs) to sequence genes and loci associated with MDs, and demonstrated that this method is effective in variant identification for MD patients (Hitti-Malin et al., Human Mutation, 2022, PMID: 36259723). We used this tailored smMIPs panel to sequence the coding regions of 105 iMD and AMD-associated genes, known pseudo-exons, and the mitochondrial genome of 908 patients in series of 360 samples using the Illumina NovaSeq 6000 platform. Single nucleotide variants (SNVs) were annotated with an in-house pipeline, filtering for autosomal recessive variants with an allele frequency of ≤0.5% in population databases, and autosomal dominant variants with an allele frequency of ≤0.1%. Variants with a Franklin-ACMG classification of class 3, 4 or 5 (VUS, likely pathogenic and pathogenic) were selected. Copy number variant (CNVs) analysis was performed using an in-house Excel script.

Results : Sequencing data for 908 iMD probands were analysed, achieving a diagnostic yield of 32% for this partially prescreened cohort. At least 60 novel pathogenic variants were identified. The five most frequently mutated genes were ABCA4 (14%), CDHR1 (8%), RP1L1 (6%), PROM1 (6%) and PRPH2 (5%).

Conclusions : We showed that this smMIPs panel can be utilised on a large scale to cost-effectively sequence MD genes and loci and provide patients and their families with a genetic diagnosis. Further analysis will be performed to reveal new genetic associations for MD and potential genetic overlaps between iMD and AMD.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.