Abstract
Purpose: :
The genetics underlying red-green color vision deficiencies are well-understood. The causative mutations are in the long (L) and middle (M) wavelength sensitive cone opsin genes, which lie in tandem on the X-chromosome. L and M opsin genes vary in copy number as well as in nucleotides that specify amino acids responsible for variability in spectral tuning of photopigments. The number and identity of genes in the array provides complete information about the color vision potential of an individual. We have developed a high-throughput genetic screen capable of providing such information for the purpose of cheaply and efficiently diagnosing red-green color vision defects.
Methods: :
The Sequenom MassArray uses PCR amplification of regions surrounding known SNPs followed by single nucleotide extension of primers across SNP sites, creating different products depending upon the alleles present. The products differ in mass and thus can be detected by mass spectrometry to provide a ratio of alleles. The color vision test is comprised of five multiplexed assays that can be carried out simultaneously in a single well: one that determines total array length, one that finds the percentage of L genes, and three that probe SNPs known to affect spectral tuning. One individual suspected to be XXY was excluded. Selective amplification of the last gene in the array was done on all subjects classified as deuteranomalous to prevent misdiagnosis of normal subjects with an extra L gene at the 5’ end of the array.
Results: :
A group of 409 males unselected for color vision status was classified using the MassArray. Frequencies of deuteranopia (1.0%), protanopia (0.2%), deuteranomaly (7.1%) and protanomaly (1.0%) agreed closely with previously reported frequencies based on behavioral tests. Additionally, the MassArray genetic screen correctly characterized 58 out of 58 color normals and 22/23 behaviorally tested colorblind subjects, thus showing perfect specificity and 96% sensitivity. The sole misdiagnosis was a deuteranope who we suspect has a loss-of-function mutation (e.g. Cis203Arg). In future iterations, such errors can be avoided by adding detection of SNPs for missense mutations to the screen.
Conclusions: :
The MassArray assay promises to provide nearly perfect diagnosis of inherited color vision deficiency including presence vs. absence, type, and severity. Compared to traditional behavioral tests, genetic analysis does not require lengthy subject testing and removes inconsistencies from variability in spectral content and intensity of illumination, viewing distance, and cheating that can decrease the reliability.
Keywords: color vision • genetics • opsins