Digital droplet PCR enables genotyping the red-green opsin array for clinical diagnostics

Abstract

Purpose : An accurate and efficient red-green opsin array genotyping strategy is required for molecular confirmation of X-linked color vision deficiencies, Bornholm eye disease (BED), X-linked cone dystrophy (XLCD), and blue cone monochromacy (BCM). Genotyping is challenging due to the highly homologous OPN1LW and OPN1MW (L and M) genes sharing ~98% identical DNA sequences and the presence of multiple, tandem-arranged copies. To overcome this, we designed a tiered approach to assess copy number, structural, and sequence variants for comprehensive genotyping of the complex opsin array.

Methods : A diagnostic test was designed using digital droplet PCR (ddPCR) copy number analysis of the locus control region (LCR), L and M genes, c.607T>C p.Cys203Arg (C203R) variant known to cause protein misfolding, and exon-3 haplotypes in affected males from 29 families with X-opsin array disorders (n=31) and unaffected males (n=45). Proximal and distal gene(s) were genotyped separately by long-range PCR, exon-specific nested PCR, and Sanger sequencing. ddPCR molecular milepost assays were employed to determine gene-variant concordance using reference points and linkage to infer gene copy order.

Results : Pathogenic genotypes of 100% (29/29) probands with X-opsin array disorders were successfully resolved. Of these, 7 included uncertainty of altered L or M gene copy position and required milepost assays. Of 17 males with color discrimination disorder, 14 deutans and 3 protans were molecularly confirmed by haplotype and spectrally sensitive residues. Three probands with BED or XLCD had splice-altering exon-3 haplotypes. Of 9 BCM probands, 6 had LCR deletions, and 3 had p.C203R in expressed gene copy(s). Molecular milepost assays delineated gene arrangements in multiple participants, including one individual with BCM due to p.C203R in 3 of 4 gene copies.

Conclusions : Determination of opsin gene-variant concordance by ddPCR was crucial for molecular diagnosis in ~24% (7/29) of probands with X-linked opsin-array disorders. Amino acids at positions 116, 180 and 230 in L and M genes can also be used to successfully predict X-linked color discrimination phenotypes. The ddPCR molecular milepost assay enables an accurate and efficient genotyping strategy for the X-opsin array, elucidating genotype-phenotype correlations and informing future gene-directed therapies.

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