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We have analyzed L/M visual pigment gene arrays in 119 Japanese men with protanopia color vision defect and found that five had a normal gene order of L-M. Among the five men, two (identified as A376 and A642) had apparently normal L genes. To clarify their L gene defect, the whole L or M gene from A376 and control subjects was cloned in an expression vector. Total RNA extracted from the transfected HEK293 cells was analyzed by Northern blot and reverse transcription-polymerase chain reaction. The product from the cloned L gene of A376 was smaller than the normal control due to the absence of exon 3. To investigate such exon-skipping at splicing, minigenes of exon 3 accompanying introns 2 and 3 were prepared from A376, A642, and control subjects. The minigenes of A376 (L) and A642 (L) showed the product lacking exon 3 only, while the minigene of normal control N44 (L) showed the product retaining exon 3 only. Exchanging of introns 2 and 3 between the A376 (L) and N44 (L) minigenes showed that the skipping of exon 3 was caused by the exon itself. Seven differences in exon 3 between A376 (L) and N44 (L) were all within already-known polymorphisms as follows: G(151-3), C(153-1), G(155-3), A(171-1), T(171-3), G(178-1) and G(180-1) in A376 (L) and A642 (L), and A(151-3), A(153-1), C(155-3), G(171-1), G(171-3), A(178-1) and T(180-1) in N44 (L). An in vitro mutagenesis experiment with these nucleotides in the minigenes showed that exon 3 was completely skipped at splicing only in the haplotype observed in A376 (L) and A642 (L). These results suggest that complete skipping of exon 3 at splicing, due to the unique haplotype of the exon, causes loss of expression of L-opsin in these men.

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

Normal visual pigment gene arrays on the human X chromosome have a red gene at the first and a green gene at the second positions. More than half of the arrays have additional green genes downstream, but only the first two genes of the array are likely to be expressed in the retina. An array consisting of four genes in two Japanese participants, A121 and A447, was detected either by pulsed field gel electrophoresis and subsequent Southern hybridization or by single nucleotide primer extension reaction. In both participants, the first gene of the array was green, downstream genes were red and green, and the fourth gene was green. The red gene was determined to be at the second position by comparison of polymorphic sites among the intergenic regions that had been amplified by long-range PCR. Such an array with a reverse normal order of pigment genes, green-red as the first two, has never been reported before. They were expected to have normal color vision but showed protan deficiency (protanomaly), a phenotype lacking the red pigment. The red gene had no mutations in the exons and exon/intron boundaries, but had an A-71C substitution in the promoter in both participants.

TEX28 gene (fTEX) is present immediately downstream of the red/green visual pigment gene array on the human X chromosome. Its pseudogene (pTEX) that lacks exon 1 is present within the array between pigment genes. We found that both fTEX and pTEX genes had a 697 bp insertion/deletion polymorphism in their introns 3. In color-normal male subjects, the frequency of the 697 bp region was 43% (40/94) in pTEX and 97% (91/94) in fTEX in the array of Red-pTEX-Green-fTEX and 10% (9/94) in pTEX and 87% (41/47) in fTEX in the array of Red-pTEX-Green-pTEX-Green-fTEX. These results suggest that normal arrays with multiple green genes may have arisen through gene duplication rather than unequal homologous crossover. In color-vision-deficient male subjects with a single-gene array, the frequency of the 697 bp region was 83% (25/30) in the array of Green-fTEX and 66% (74/112) in the array of Red-fTEX. In color-vision-deficient male subjects with a 2-gene array, the frequency of the region was 44% (16/36) in pTEX and 97% (35/36) in fTEX in the array of Green-pTEX-Green-fTEX and 75% (18/24) in pTEX and 92% (22/24) in fTEX in the array of Red-pTEX-Red-fTEX. These results suggest that 2-green-gene arrays have arisen through unequal homologous crossover between a normal 2-gene array and a single-green-gene array. With data from a long-range PCR method using the insertion/deletion polymorphism, we proposed a structure of the second gene of 3-gene arrays, Green-pTEX-Green-pTEX-Green-fTEX and Red-pTEX-Red-pTEX-Red-fTEX, in color-vision-deficient subjects.

The L-cone/M-cone visual pigment gene arrays were analyzed in a group of 63 Japanese females consisting of 7 applicants for examination of their carrier status, 14 color-deficient females, 6 obligate carriers with no genotypic data available for affected father or sons, and 36 color-normals. The first and the downstream genes, the entire region from the promoter to exon 6, were each amplified very efficiently by the long-range PCR to give products of 15.8 and 14.4 kb, respectively. The products were gel-purified and used as the template in the second PCR for exon 5. The region from intron 4 of the last genes, to the nearest neighbor gene, TEX28, was also efficiently amplified by the long-range PCR and the gel-purified products (27.5 kb) were used as the template in the second PCR for exon 5. The status of the 7 applicants was thought to be 3 non-carriers, 2 protan carriers and 2 deutan carriers. All of the 14 color-deficient females had unusual arrays in which an M gene was present as the first gene, an L gene(s) was present downstream, or a single L gene constituted both of the two arrays. One protanopic subject, A348, had an L gene as one of the first genes. The 6 obligate carriers also had unusual arrays with the exception of the mother of the A187, a male subject with pigment color defect. In the 36 color-normal individuals, 4 had downstream L genes. The long-range PCR method is useful for analysis of the L/M visual pigment genes.

In 524 Japanese individuals with deutan colour vision defect, 76 had a normal-order pigment gene array, where the L gene is at the first position and the M gene(s) is located downstream. Of these 76 individuals, 69 had a -71A>C substitution in the M gene without any other mutation. Because the expression of L/M genes is up-regulated by thyroid hormone (T3) in human retinoblastoma WERI cells, we examined the effects of T3 on promoter activity; T3 increased the activity of the -71A promoter 2-fold, but it had no effect on the -71C promoter. Similarly, the -71C promoter was much less activated by T3 than the -71A promoter in HEK293 cells expressing thyroid hormone receptor isoform β2. Such a weak response of the -71C promoter to T3 may cause a decrease in the number of M cones and/or the density of M pigment during the differentiation of M cones. The average Rayleigh match midpoint was 18.9 ± 4.1 in 162 ordinary deuteranomaly individuals, but was 37.3 ± 9.1 in 63 deuteranomaly individuals with -71C. The -71A>C substitution was found to be specific to eastern Asia. These results suggest that there may be a new subset of deuteranomaly associated with -71C in the Japanese (and probably eastern Asian) population(s).

Purpose: We analyzed the red/green visual pigment genes in color-normal Japanese men to understand the relationship between color anomalies and genetic defects. Methods: DNA from 120 color-normal Japanese men was subjected to polymerase chain reaction (PCR)-amplification for exons 2–5 of the red/green visual pigment genes and the PCR products were sequenced. The red:green gene ratios were estimated from the sequencing electropherograms of exon 5 and also from MvaI-restriction fragment analysis of the same exon. The first gene and the downstream genes in the pigment gene array were separately analyzed by PCR, direct sequencing, and/or single-strand conformation polymorphisms. Results: The red:green gene ratios estimated from the ratios of peak heights of nucleotides on the sequencing electropherograms coincided with those estimated from the MvaI-restriction fragment analysis. Among the subjects analyzed, they were 1:1 in 43% (n = 52), 1:2 in 41% (n = 49), 1:3 in 6% (n = 7), and 1:>3 in 9% (n = 11). The first gene in the pigment gene arrays was red in all subjects. Only 1 subject (N22) had a green-red hybrid gene. Exons 2 and 4 had 2 haplotypes each, but exon 3 was highly polymorphic. Exon 5 of the green genes had one polymorphism at codon 283 with a frequency of 32%. Conclusions: The features of visual pigment genes in color-normal Japanese men were revealed. The data and establishing techniques may be useful for analyzing these genes in color-deficient subjects in the Japanese population.

Congenital color-vision deficiencies are frequent among males, 4.7-8.0%, suggesting that female carriers are present at a frequency of 9-15%. The purpose of this study was to determine whether carriers could be detected by analysis of the visual pigment genes.DNA from 29 males with congenital color-vision deficiencies, from their mothers, and from 117 randomly-selected females was analyzed. The most upstream genes, the downstream genes, and the most downstream genes in the red/green pigment gene arrays were amplified separately by PCR. Exon 5 of each gene was analyzed by single-strand conformation polymorphisms (SSCP).Analysis of the visual pigment genes suggests that one of the 29 mothers examined is a female protan and two others are carriers of both protan and deutan defects. The remaining 26 mothers were confirmed to be carriers of congenital color-vision deficiencies. Unusual patterns were observed in 15 (13%) of the randomly-selected females; among them, 5 appeared to be protan carriers and at least 4 to be deutan carriers.Female carriers of congenital color-vision deficiencies can be detected by analysis of the visual pigment genes. Since the proportion of females showing unusual patterns was slightly higher than expected, some must be false-positives and require more detailed examination.

Recent advancements in molecular biology have revealed genetic aspects of congenital color vision deficiencies (CVD). In many cases of CVD, the genotypes and phenotypes coincide with each other. But it is also known that there are cases in which the genotype alone cannot explain the clinical diagnosis of CVD. So it is still unclear whether routine gene analysis is useful as a clinical diagnostic tool for this anomaly. We evaluated the clinical usefulness of genetic analysis for congenital red-green color-vision deficiencies. The nucleotide sequences of red-green pigment genes of 62 CVD cases and 113 color-normal males were determined using PCR and a DNA sequencer (ABI PRISM 310 Genetic Analyzer). The CVD cases were diagnosed using clinical color-vision tests including an anomaloscope. The color-normal subjects were tested with Ishihara plates and, if needed, with an anomaloscope. All normals had two types of exon 5, i.e., both red and green pigment genes. Forty-seven (76%) of 62 CVD cases had genotypes that coincided with their phenotypes. Three dichromats (one protanope and two deuteranopes) had genetic potential to be anomalous trichromats. Twelve deutans (one deuteranope and 11 deuteranomals) could not be differentiated from the normals: they had both red and green types of genes, as did the normals. All in all, 15 cases (24%) had some inconsistencies between the phenotype and the genotype. Pigment gene analysis alone could not fully differentiate CVD from normals. © 2000 John Wiley & Sons, Inc. Col Res Appl, 26, S89–S92, 2001

Deuteranomaly, and to a lesser extent deuteranopia, are associated with a 5′ green-red 3′ (G-R) visual pigment hybrid gene. Such G-R hybrid genes may also exist in males with normal color vision. This discrepancy can be explained if the position of the G-R hybrid gene in the array determines the probability of its expression in the retina. In accord with our previous findings, we confirm that when a G-R hybrid gene occupies the second position in the array, a deutan defect results (N = 2); whereas when a normal green pigment gene does, normal color vision ensues (N = 3). Regarding gene expression as a function of position in the array, in 5 postmortem retinae from individuals whose arrays contained one red and two green pigment genes that differed at a polymorphic site in exon 5, only the green pigment gene that occupied the second position in the array was expressed into retinal mRNA. In conclusion, our data suggest that only the first two genes in the X-chromosome linked visual gene array are expressed in the retina and, therefore, influence the color vision phenotype. Green-red hybrid genes cause deuteranomaly only if they occupy the second position in the array. © 2000 John Wiley & Sons, Inc. Col Res Appl, 26, S79–S83, 2001

Among the 447 Japanese men with deutan color-vision deficiency that we analyzed, 61 had a normal order array of L/M pigment genes. Three of the 61 men had an exonic mutation, but the other 58 had no mutations even in the flanking introns of their M genes. In these 58 men, 55 had a -71A --> C substitution in the M gene. Two hypotheses were built up for the substitution: it is in linkage disequilibrium with a genuine cause of deficiency in the introns, or itself is the cause of the deficiency. For the first hypothesis, we sequenced entire regions of both the L and M genes in 30 color-normal Japanese men who had one each of the L and M genes to understand normal variations of the introns. Fifty-two already known and 15 newly identified polymorphic sites could be classified into three categories: those with no polymorphisms in the Japanese group, those essentially different between the L and the M genes, and the others. We then sequenced the entire region of the M genes in 12 representative deutan individuals with a normal gene-order array but found no significant mutations. For the second hypothesis, we performed a reporter assay and found that the M gene promoter with -71C had a 60-70% reduction in activity when compared to that with -71A. These results suggest that the -71A --> C substitution is not in linkage disequilibrium with an intronic mutation, but the substitution itself may affect the transcription of the M gene, leading to deutan deficiency.

Thirty-nine missense mutations, which had been identified in rod monochromacy or related disorders, in the CNGA3 subunit of cone photoreceptor cGMP-gated channels were analyzed. HEK293 cells were transfected with cDNA of the human CNGA3 subunit harboring each of these mutations in an expression vector. Patch-clamp recordings demonstrated that 32 of the 39 mutants did not show cGMP-activated current, suggesting that these 32 mutations cause a loss of function of the channels. From the remaining 7 mutants that showed cGMP-activated current, two mutations in the cyclic nucleotide-binding domain, T565M or E593K, were further studied. The half-maximal activating concentration (K(1/2)) for cGMP in the homomeric CNGA3-T565M channels (160microM) was 17.8-fold higher than that of the homomeric wild-type CNGA3 channels (9.0microM). Conversely, the K(1/2) for cGMP in the homomeric CNGA3-E593K channels (3.0microM) was 3-fold lower than that of the homomeric wild-type CNGA3 channels. These results suggest that the T565M and E593K mutations alter the apparent affinity for cGMP of the channels to cause cone dysfunction, resulting in rod monochromacy.

The L-cone/M-cone visual pigment gene arrays were analyzed in 125 Japanese males with protan color-vision deficiency. Arrays were successfully determined in 62/65 subjects with protanopia and 57/60 protanomaly subjects. Among the 62 protanopia subjects, 48 (77%) had an array consisting of a single 5′ L–M hybrid gene (PS-array) or a 5′ L–M hybrid gene followed by an M gene(s) that was structurally identical to the hybrid gene (PI-array). In the remaining 14 subjects, 11 had an array consisting of a 5′ L–M hybrid gene followed by an M gene(s) that was structurally different from the hybrid gene (PD-array) and 3 subjects had an apparently normal array consisting of a single L gene followed by an M gene(s) (PN-array). In the 11 subjects with the PD-array, subject A67 had an 11 bp-deletion in exon 3 of the downstream genes and 6 had an A-71C substitution in the second gene of the array. In the 3 subjects with the PN-array, subject A289 had a missense mutation (Pro231Leu) in exon 4 of the L gene. When the function of the missense mutation was studied by in vitro reconstitution of visual pigments, it was found to be deleterious to both cone opsin and rhodopsin. Among the 57 protanomaly subjects, 49 (86%) had the PD-array, but 25 subjects had a difference only in exon 2 between the first and downstream genes that suggested a contribution of exon 2-encoded difference in the M pigment to color-discrimination. In the remaining 8 subjects, 2 had the PS-array, 2 had the PI-array and the other 4, including subject A89 with a missense mutation (Glu338Gly) in the L gene, had the PN-array. Genotype–phenotype relationships in protan color-vision deficiency are discussed.

We studied 247 Japanese males with congenital deutan color-vision deficiency and found that 37 subjects (15.0%) had a normal genotype of a single red gene followed by a green gene(s). Two of them had missense mutations in the green gene(s), but the other 35 subjects had no mutations in either the exons or their flanking introns. However, 32 of the 35 subjects, including all 8 subjects with pigment-color defect, a special category of deuteranomaly, had a nucleotide substitution, A−71C, in the promoter of a green gene at the second position in the red/green visual-pigment gene array. Although the −71C substitution was also present in color-normal Japanese males at a frequency of 24.3%, it was never at the second position but always found further downstream. The substitution was found in 19.4% of Chinese males and 7.7% of Thai males but rarely in Caucasians or African Americans. These results suggest that the A−71C substitution in the green gene at the second position is closely associated with deutan color-vision deficiency. In Japanese and presumably other Asian populations further downstream genes with −71C comprise a reservoir of the visual-pigment genes that cause deutan color-vision deficiency by unequal crossing over between the intergenic regions.

The DNAs from 217 Japanese males with congenital red/green color-vision deficiencies were analyzed. Twenty-three subjects had the normal genotype of a single red gene, followed by a green gene. Four of the 23 were from the 69 protan subject group and 19 of the 23 were from the 148 deutan subject group. Three of the 23 subjects had missense mutations. The mutation Asn94Lys (AAC--AAA) occurred in the single green gene of a deutan subject (A155). The Arg330Gln (CGA--CAA) mutation was detected in both green genes of another deutan subject (A164). The Gly338Glu (GGG--GAG) mutation occurred in the single red gene of a protan subject (A89). Both normal and mutant opsins were expressed in cultured COS-7 cells and visual pigments were regenerated with 11-cis-retinal. The normal red and green opsins showed absorbance spectra with lambda(max) of 560 and 530 nm, respectively, but the three mutant opsins had altered spectra. The mutations in Asn94Lys and Gly338Glu resulted in no absorbance and the Arg330Gln mutation gave a low absorbance spectrum with a lambda(max) of 530 nm. Therefore these three mutant opsins are likely to be affected in the folding process, resulting in a loss of function as a visual pigment.

PURPOSE The human cone photoreceptor cyclic nucleotide-gated (CNG) channel comprises alpha- and beta-subunits, which are respectively encoded by hCNGA3 and hCNGB3. The purpose was to examine the functional role of hCNGB3 in modulation of human cone CNG channels and to characterize functional consequences of rod monochromacy-associated mutations in hCNGB3 (S435F and D633G). METHODS Macroscopic patch currents were recorded from human embryonic kidney (HEK) 293 cells expressing homomeric (hCNGA3 and hCNGB3) and heteromeric (hCNGA3/hCNGB3, hCNGA3/hCNGB3-S435F, and hCNGA3/hCNGB3-D633G) channels using inside-out patch-clamp technique. RESULTS Both hCNGA3 homomeric and hCNGA3/hCNGB3 heteromeric channels were activated by cGMP, with half-maximally activating concentration (K(1/2)) of 11.1 +/- 1.0 and 26.2 +/- 1.9 micro M, respectively. The hCNGA3 channels appeared to be more sensitive to inhibition by extracellular Ca(2+) compared with hCNGA3/hCNGB3 channels, when assessed by the degree of outward rectification. Coexpression of either of rod monochromacy-associated mutants of hCNGB3 with hCNGA3 significantly reduced K(1/2) value for cGMP but little affected the sensitivity to extracellular Ca(2+), compared with wild-type heteromeric channels. The selectivity of hCNGA3, hCNGA3/hCNGB3, hCNGA3/hCNGB3-S435F, and hCNGA3/hCNGB3-D633G channels for monovalent cations were largely similar. Immunoprecipitation experiments showed association of hCNGA3 subunit with both of wild-type and mutant hCNGB3 subunits. CONCLUSIONS The hCNGB3 plays an important modulatory role in the function of human cone CNG channels with respect to cGMP and extracellular Ca(2+) sensitivities. The rod monochromacy-associated S435F and D633G mutations in hCNGB3 evokes a significant increase in the apparent affinity for cGMP, which should alter cone function and thereby contribute at least partly to pathogenesis of the disease.