Shoko Tanabe

Web-page color modification for barrier-free color vision with genetic algorithm

In this paper, we propose a color modification scheme for web-pages described by HTML markup language in order to realize barrier-free color vision on the internet. First, we present an abstracted image model, which describes a color image as a combination of several regions divided with color information, and define some mutual color relations between regions. Next, based on fundamental research on the anomalous color vision, we design some fitness functions to modify colors in a web-page properly and effectively. Then we solve the color modification problem, which contains complex mutual color relations, by using Genetic Algorithm. Experimental results verify that the proposed scheme can make the colors in a web-page more recognizable for anomalous vision users through not only computer simulation but also psychological experiments with them.

Preliminary study on color modification for still images to realize barrier-free color vision

We propose a color modification scheme for still images in order to realize barrier-free color vision in the IT society. Based on the knowledge of Kondos anomalous color vision model, we quantify the degree of color discrimination among colors in a given image by the anomalous vision people, and modify the pixel colors to improve the discrimination by them while keeping naturalness of the image.

Novel missense mutations in red/green opsin genes in congenital color-vision deficiencies

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.

An A−71C substitution in a green gene at the second position in the red/green visual-pigment gene array is associated with deutan color-vision deficiency

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.

Unique haplotype in exon 3 of cone opsin mRNA affects splicing of its precursor, leading to congenital color vision defect.

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.

Number and variations of the red and green visual pigment genes in Japanese men with normal color vision

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.

Analysis of L-cone/M-cone visual pigment gene arrays in females by long-range PCR.

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.

Functional analysis of rod monochromacy-associated missense mutations in the CNGA3 subunit of the cone photoreceptor cGMP-gated channel

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.

An insertion/deletion TEX28 polymorphism and its application to analysis of red/green visual pigment gene arrays

AbstractTEX28 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.

Detection of female carriers of congenital color-vision deficiencies by visual pigment gene analysis.

Purpose. 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. Methods. 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). Results. 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. Conclusions. 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.