Diagnostic genetic analysis of variants in the highly complex OPN1LW/OPN1MW gene cluster causal for mild to severe color vision deficiencies

Abstract

Purpose : Variants in the OPN1LW/OPN1MW gene cluster cause X-linked color vision deficiencies, such as the frequently occurring protanopia and deuteranopia or the more rare Bornholm eye disease and blue cone monochromacy. Genetic analysis of the cluster is extremely difficult, first due to the ~98% homology of the OPN1LW and OPN1MW genes, second because only the first two opsin genes in the cluster are clinically relevant, while the cluster can contain zero to as many as six gene copies and third because of the broad spectrum of causal variants.

Methods : A genetic assay was developed for the OPN1LW/OPN1MW gene cluster combining copy number analysis using multiplex ligation-dependent probe amplification (MLPA) and sequencing analysis using long-read circular consensus sequencing. The assay was tested on 50 clinical cases analyzed to confirm clinical diagnosis (n=43) or determine carrier status (n=7).

Results : Using the developed assay a broad range of pathogenic variants were detected, such as deletions of the LCR, hybrid genes, single variants (including 7 novel variants) and combinations of variants. In all 43 male cases in which a genetic confirmation of diagnosis was requested the genetic composition of the clinically relevant first and second gene in the OPN1LW/OPN1MW gene cluster could be determined. In 39 of these 43 cases the clinical diagnosis was genetically confirmed, in two cases it was unclear whether the detected variants were causal and in two causes no causal variants were detected. In the latter two cases the causal variants are most likely outside the OPN1LW/OPN1MW gene cluster. Indeed, in one of these cases variants in the ZNF644 gene were detected. In seven female samples carrier status could be determined, although in two samples parental samples were requested and analyzed to definitely conclude carrier status. Interestingly, in 4 of the 7 cases the causal allele arose de novo in the affected child.

Conclusions : The developed genetic assay for the OPN1LW/OPN1MW gene cluster is the first available diagnostic assay that can detect both structural and nucleotide variants with a straightforward analysis, as MLPA is a fast and efficient method for copy number analysis and the long read sequencing approach makes it possible to determine the genetic composition of the entire OPN1LW or OPN1MW gene in single sequencing reads.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.