Interrogating the M/L Opsin Cluster using Exome Sequencing

Abstract

Purpose : The M/L opsin cluster is a difficult region of the genome to sequence and interpret because it consists of a tandem array of nearly identical genes. Accurate sequence evaluation of this cluster is of value for detecting variations that underlie mild to moderate variations in human color vision as well as for the molecular diagnosis of a more severe condition known as Blue Cone Monochromacy (BCM).

Methods : We performed augmented, whole exome sequencing on a cohort of 1080 individuals with inherited retinal disease. Sequence was aligned using BWA to a modified reference genome containing only a single copy of OPN1MW. Coverage-based techniques were used to assess copy number variants in the cluster. Coverage was calculated over a portion of the locus control region (LCR) that was captured by an augmenting bait, as well as for the portion of exon 5 of each gene (amino acids 277 to 285) that confers the primary spectral difference between the opsins. Patients completely lacking coverage of the LCR would be expected to exhibit the clinical findings of BCM, while patients completely lacking coverage of either the LW or MW spectral signature fragment would be expected to exhibit the clinical findings of dichromacy or anomalous trichromacy.

Results : Coverage-based analysis of the locus control region revealed four patients with hemizygous deletions, indicating previously undiagnosed BCM. Twenty-one patients completely lacked coverage of OPN1LW exon 5, and 14 patients lacked coverage of OPN1MW exon 5, indicating at a minimum a severe color vision deficit in these patients. Of these 35 probable dichromats, four had a clinical suspicion of BCM noted in their records.

Conclusions : The two most common mutational mechanisms of BCM are 1) deletions of the LCR, and 2) missense changes in a cluster consisting of a single opsin. The coverage-based techniques described here can detect both major types of mutations. In addition, these techniques can reveal the genetic basis for milder forms of X-linked color vision loss using data that is incidentally collected as part of exome-based genetic testing of patients with inherited eye disease. The co-occurrence of common X-linked color vision deficits with photoreceptor disease can make patients appear to have a more cone-selective disease than they actually do, thus confounding the diagnosis. The ability to accurately interpret variants in the opsin cluster will improve the diagnosis of these patients.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.