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Novel mutations in the L visual pigment gene found in Japanese men with protan color-vision defect having a normal order L/M gene array

Among congenital color-vision defects, protan-(L [long wavelength-sensitive]cones are affected) and deutan-(M [medium wavelength-sensitive]cones are affected) defects are common. Both defects have phenotypes with dichromacy (protanopia and deuteranopia, respectively) and anomalous trichromacy (protanomaly and deuteranomaly, respectively). The genes for L pigment (expressed in L-cones) and M pigment (expressed in M-cones) are arranged in tandem to form an L/M gene array on the human X chromosome, with the L gene first and M gene (s) downstream in color-normal men. In this study, we call such an array an “L-M array.” Since the L and M genes are similar to each other in structure as well as at nucleotide level, non-homologous recombination tends to occur between these genes, leading to an abnormal single-M or M-M array, causing a protan defect, or single-L or L-L array, causing a deutan defect. With regard to arrays consisting of more than two genes, it is widely accepted that when products from the first two genes of an array are spectrally identical, the phenotype is dichromacy (protanopia or deuteranopia) and when they are spectrally different to each other, the phenotype is anomalous trichromacy (protanomaly or deuteranomaly). We have been analyzing L/M gene arrays in Japanese men with protan or deutan defect to study correlations of the array genotype with a subject’s color-vision phenotype. Analysis of gene arrays in a total of 236 Japanese men with protan defect until now revealed that more than a few men had an L gene at the first position (8 in the 135 men with protanopia and 6 in the 101 men with protanomaly [Table 1]), which does not agree with the non-homologous recombination mechanism as a cause of the color-vision defect. Such a high frequency (14 in a total of 236, 5.9%) has never been reported in Caucasians with protan defect, being 0 in 23 protans, 0 in 23 and 1 in 37. The only one Caucasian man with protan defect in reference 8 had a rare LIAVA haplotype, which consists of Leu-Ile-AlaVal-Ala, in exon 3 of the L gene. We have also found this haplotype in two Japanese men, as shown in Table 1. Since we have already reported abnormalities of the L genes in 11 of the 14 men, we analyzed the L genes of the other three men in the present study, and mutations such as Trp177STOP, −99T>G, and +3A>C in intron 2 were newly identified (Table 1). The nonsense mutation is the second identified one in our study (Table 1). The −99T>G substitution was at the PCE-1 (photoreceptor conserved element-1) site. Although a −71A>C substitution was reported in the M gene, abnormalities in the L gene promoter have never been documented. Since the PCE-1 sequence was frequently found in the promoter of photoreceptor-specific genes such as arrestin, interphotoreceptor retinoid-binding protein, rhodopsin, and cone opsin genes, the sequence would be necessary for the development of photoreceptor cells. Its consensus sequence is CAATTAR. The corresponding sequence in the wild-type L gene promoter is CAATTAA (perfect match). The −99T>G substitution causes one mismatch which may avoid binding of a specific protein for transcription to the element, resulting in a decrease of transcription activity. However, we found instead a slight increase in activity in the mutant promoter when compared to the wild-type promoter in the luciferase reporter assay (Supplemental Figure 1A – online only). In the gel retardation assay, the shifted band disappeared for the wild-type competitor but not for the mutant (−99T>G) competitor at 100-fold excess (Supplemental Figure 1B – online only). The mutant sequence did not seem to bind to the PCE-1specific binding protein. The mutation at intron 2 (+3A>C) was within the consensus sequence for the 5′ splice site, MAG|GTRAGT (the vertical bar represents the boundary of exon and intron). We analyzed this mutation by using minigenes consisting of L opsin cDNA and the genomic region of intron 2 exon 3 intron 3. Figure 1A shows that the mutant minigene yielded a slightly smaller product than the wild-type minigene. By sequencing the product of the mutant minigene, we found that the splicing had occurred just behind codon 114 in exon 2 (Figure 1B). The aberrant splicing eliminated 67 nucleotides from the mature mRNA and is supposed to cause premature termination of translation at codons 162-163 in exon 3. The RT-PCR product from the mutant minigene was less in amount than that from the wild-type minigene (Figure 1A). This was probably due to the nonsense-mediated mRNA decay mechanism which was activated by the resultant stop codon in exon 3. The