Genotype-phenotype relationships in human red/green color-vision defects: molecular and psychophysical studies.

The relationship between the molecular structure of the X-linked red and green visual pigment genes and color-vision phenotype as ascertained by anomaloscopy was studied in 64 color-defective males. The great majority of red-green defects were associated with either the deletion of the green-pigment gene or the formation of 5' red-green hybrid genes or 5' green-red hybrid genes. A rapid PCR-based method allowed detection of hybrid genes, including those undetectable by Southern blot analysis, as well as more precise localization of the fusion points in hybrid genes. Protan color-vision defects appeared always associated with 5' red-green hybrid genes. Carriers of single red-green hybrid genes with fusion in introns 1-4 were protanopes. However, carriers of hybrid genes with red-green fusions in introns 2, 3, or 4 in the presence of additional normal green genes manifested as either protanopes or protanomalous trichromats, with the majority being protanomalous. Deutan defects were associated with green-pigment gene deletions, with 5' green-red hybrid genes, or, rarely, with 5' green-red-green hybrid genes. Complete green-pigment gene deletions or green-red fusions in intron 1 were usually associated with deuteranopia, although we unexpectedly found three carriers of a single red-pigment gene without any green-pigment genes to be deuteranomalous trichromats. All but one of the other deuteranomalous subjects had green-red hybrid genes with intron 1, 2, 3, or 4 fusions, as well as several normal green-pigment genes. The one exception had a grossly normal gene array, presumably with a more subtle mutation. Amino acid differences in exon 5 largely determine whether a hybrid gene will be more redlike or more greenlike in phenotype. Various discrepancies as to severity (dichromacy or trichromacy) remain unexplained but may arise because of variability of expression, postreceptoral variation, or both. When phenotypic color-vision defects exist, the kind of defect (protan or deutan) can be predicted by molecular analysis. Red-green hybrid genes are probably always associated with protan color-vision defects, while the presence of green-red hybrid genes may not always manifest phenotypically with color-vision defects. Four subjects who were found to have 5' green-red hybrid genes in addition to normal red- and green-pigment genes had normal color vision as determined by anomaloscopy. These were discovered among a group of 129 Caucasian males who had been recruited as volunteers for a vision study.(ABSTRACT TRUNCATED AT 400 WORDS)     

A model of color vision in trichromats and protans

A model which explains the human vision protanopic deficiency and its biologic prototype with the absence of red-absorbing pigment (rabbit) was constructed from neuron-like elements. In behavioral experiments and by means of evoked potential technique it was shown that the rabbit's color space is characterized by a spherical four-dimensional with a reduction of red-coding area. Similar spherical four-dimensional structure of color space is characteristic for a group of protanopic human subjects. The perceptive space of another group of protanopic subjects (protanomals) is characterized by a reduction of both parts of the red-green opponent axis. These disorders are reproduced in the model either by a loss of some color-coding elements (the absence of the red-absorbing pigment as in protanops) or a shift of the spectral characteristics of the red pigment towards those of the green one (protanomals).     

A 195-kb cosmid walk encompassing the human Xq28 color vision pigment genes

By using cosmid walking, we have cloned a 195-kb region from chromosome band Xq28 that encompasses the red and green color pigment genes and 85 kb of flanking sequences. This has allowed us to confirm that the color pigment genes are within very homologous units arranged in tandem array. Each unit contains two BssHII sites and one NruI site that are frequently methylated in male leukocyte DNA. A NotI and an EagI site are present 6 kb upstream from the red pigment gene promoter; the NotI site was shown to be unmethylated in the active X chromosome in leukocytes and may represent a CpG island for the whole cluster. We have identified another CpG island, 61 kb 3' from the last green pigment gene, that is unmethylated in leukocytes on the active X chromosome, but methylated on the inactive X. This island is flanked by sequences conserved in evolution and may thus correspond to an expressed gene. We also describe an informative three-allele restriction fragment length polymorphism within the pigment gene cluster.     

THE DARK ADAPTATION OF THE COLOR ANOMALOUS MEASURED WITH LIGHTS OF DIFFERENT HUES

Determinations of minimum light thresholds as a function of time in the dark have been made for four color normal, three deuteranopic (or deuteranomalous), and four protanopic (or protanomalous) subjects. Measurements were made with red, reddish orange, yellow, green, violet, and white test lights. Dark adaptation curves for the deuteranopes and deuteranomalous are essentially identical with those of the color normal for all colors. The cone portions of the protanopic dark adaptation curves measured with the red, reddish orange, yellow, and white lights are higher than the corresponding data for the color normal, the discrepancy between the two sets of data decreasing from the long to short wave lengths. Dark adaptation curves for the protanopes and protanomalous measured with green and violet light are essentially normal in appearance. A theoretical explanation is advanced to account for these findings in terms of the known sensitivity characteristics of the normal and color-anomalous eye.     

Gene conversion and natural selection in the evolution of X-linked color vision genes in higher primates

During higher primate evolution, gene conversion seems to have occurred often between the red and green photo-pigment genes, which are tandemly linked on the X chromosome. To understand this phenomenon better, intron 4 sequences of the red and green pigment genes of a male human (an Asian Indian), a male chimpanzee, and a male baboon were amplified by PCR and sequenced. The data show that the intron 4 sequences between the two genes have been strongly or completely homogenized in the three species studied. Apparently recent gene conversion events have occurred in introns 4 of the red and green pigment genes in humans and chimpanzees. Two or more conversion events may have occurred at different times in introns 4 of the two pigment genes in baboons. The divergence between the two genes is significantly lower in intron 4 than in exons 4 and 5 in each species, contrary to the usual situation that introns evolve faster than exons. It is most likely that strong natural selection for maintaining the distinct functions of exons 4 and 5 of the red and green pigment genes has acted against sequence homogenization of these exons.      

Intronic gene conversion in the evolution of human X-linked color vision genes

Human red and green visual pigment genes are X-linked duplicate genes. To study their evolutionary history, introns 2 and 4 (1,987 and 1,552 bp, respectively) of human red and green pigment genes were sequenced. Surprisingly, we found that intron 4 sequences of these two genes are identical and that the intron 2 sequences differ by only 0.3%. The low divergences are unexpected because the duplication event producing the two genes is believed to have occurred before the separation of the human and Old World monkey (OWM) lineages. Indeed, the divergences in the two introns are significantly lower than both the synonymous divergence (3.2% +/- 1.1%) and the nonsynonymous divergence (2.0% +/- 0.5%) in the coding sequences (exons 1-6). A comparison of partial sequences of exons 4 and 5 of human and OWM red and green pigment genes supports the hypothesis that the gene duplication occurred before the human-OWM split. In conclusion, the high similarities in the two intron sequences might be due to very recent gene conversion, probably during evolution of the human lineage.      

Different patterns of X inactivation in MZ twins discordant for red-green color-vision deficiency.

Two female identical twins who were clinically normal were obligatory heterozygotes for X-linked deuteranomaly associated with a green-red fusion gene derived from their deuteranomalous father. On anomaloscopy, one of the twins was phenotypically deuteranomalous while the other had normal color vision. The color vision-defective twin had two sons with normal color vision and one deuteranomalous son. X-inactivation analysis was done with the highly informative probe M27 beta. This probe detects a locus (DXS255) which contains a VNTR and which is somewhat differentially methylated on the active and inactive X chromosomes. In skin cells of the color vision-defective twin, almost all paternal X chromosomes with the abnormal color-vision genes were active, thereby explaining her color-vision defect. In contrast, a different pattern was observed in skin cells from the woman with normal color vision; her maternal X chromosome was mostly active. However, in blood lymphocytes, both twins showed identical patterns with mixtures of inactivated maternal and paternal X chromosomes. Deuteranomaly in one of the twins is explained by extremely skewed X inactivation, as shown in skin cells. Failure to find this skewed pattern in blood cells is explained by the sharing of fetal circulation and exchange of hematopoietic precursor cells between twins. These data give evidence for X inactivation of the color-vision locus and add another MZ twin pair with markedly different X-inactivation patterns for X-linked traits.     

Molecular patterns and sequence polymorphisms in the red and green visual pigment genes of Japanese men

The red-green pigment gene arrays of 203 (101 from a previous study and 102 from this study) randomly selected men of Japanese ancestry from the Seattle area were screened for the abnormal molecular patterns (deletions and red/green or green/red hybrid genes) that are usually associated with defective color vision. Such molecular patterns were found in approximately 5% of these individuals, which is equivalent to the frequency of phenotypic color vision defects in Japanese males in Japan. Thus, the majority of hybrid genes carried by Japanese males appear to be associated with defective color vision. In contrast, the frequency of hybrid genes among Caucasians and African-Americans is approximately two and five times the frequency of color vision defects in these two ethnic groups, respectively. The coding sequences of 50 males of Japanese ancestry were determined. All the polymorphisms in the red and green pigment genes that were detected in the Japanese sample had been observed in Caucasians and African-Americans. The same polymorphisms of the red pigment gene were present in the green pigment gene, suggesting that gene conversion contributes to sequence homogenization between these pigment genes. As is the case for Caucasians, exon 3 of the red and green pigment genes was observed to be a hot spot for recombination and gene conversion. Fewer polymorphic sites (4 vs 11) and haplotypes (5 vs 14) of the red pigment gene were observed in Japanese than in Caucasians. The Japanese population was more uniform with respect to the red pigment gene, with 70% of individuals having the same haplotype, as compared with the 43% for the Caucasian population. This difference was largely due to the lower degree of polymorphism at position 180 of the red pigment gene in Japanese (84% Ser and 16% Ala vs 62% Ser and 38% Ala.) The number of polymorphic sites and haplotypes in the green pigment gene was similar in the two populations. Nevertheless, the Japanese population was more uniform with 65% having the same haplotype. The difference in the frequency of alleles at position 283 accounted for this difference in haplotype distribution.     

Spectrum of color gene deletions and phenotype in patients with blue cone monochromacy

Blue cone monochromacy (BCM) is an X-linked ocular disease characterized by poor visual acuity, nystagmus, and photodysphoria in males with severely reduced color discrimination. Deletions, rearrangements and point mutations in the red and green pigment genes have been implicated in causing BCM. We assessed the spectrum of genetic alterations in ten families with BCM by Southern blot, polymerase chain reaction, and sequencing analysis, and the phenotype was characterized by ophthalmoscopy, fluorescein angiography, and a battery of tests to assess color vision in addition to routine ophthalmological examination. All families showed clinical features associated with BCM. Acuities were reduced in all affected males, and photopic b-wave was reduced by more than 90% in seven families. In three families, however, the photopic b-wave response showed uncharacteristic relative preservation of 30-80% (of the clinical low-normal value). The color vision was unusually preserved in two affected males, but this was not correlated with photopic electroretinography retention. Progressive macular atrophy was observed in affected members of two BCM families while the rest of the families presented with normal fundus. In nine families deletions were identified in the gene encoding the red-sensitive photopigment and/or in the region up to 17.8 kb upstream of the red gene which contains the locus control region and other regulatory sequences. In the same nine families the red pigment gene showed a range of deletions from the loss of a single exon to loss of the complete red gene. In one family no mutation was found in the exons of the red gene or the locus control region but showed loss of the complete green gene. No association was observed between the phenotypes and genotypes in these families.     

Molecular evolution of human visual pigment genes

By comparing the published DNA sequences for (a) the genes encoding the human visual color pigments (red, green, and blue) with (b) the genes encoding human, bovine, and Drosophila rhodopsins, a phylogenetic tree for the mammalian pigment genes has been constructed. This evolutionary tree shows that the common ancestor of the visual color pigment genes diverged first from that of the rhodopsin genes; then the common ancestor of the red and green pigment genes and the ancestor of the blue pigment gene diverged; and finally the red and green pigment genes diverged from each other much more recently. Nucleotide substitutions in the rhodopsin genes are best explained by the neutral theory of molecular evolution. However, important functional adaptations seem to have occurred twice during the evolution of the color pigment genes in humans: first, to the common ancestor of the three color pigment genes after its divergence from the ancestor of the rhodopsin gene and, second, to the ancestor of the red pigment gene after its divergence from that of the green pigment gene.     

Design, chemical synthesis, and expression of genes for the three human color vision pigments.

Color vision in humans is mediated by three pigments from retinal cone photoreceptor cells: blue, green, and red. We have designed and chemically synthesized genes for each of these three pigments. The genes were expressed in COS cells, reconstituted with 11-cis-retinal chromophore, and purified to homogeneity using an immunoaffinity procedure. To facilitate the immunoaffinity purification, each pigment was modified at the carboxy terminus to contain an additional eight amino acid epitope for a monoclonal antibody previously used to purify bovine rhodopsin. The spectra for the isolated pigments had maxima of 424, 530, and 560 nm, respectively, for the blue, green, and red pigments. These maxima are in excellent agreement with the maxima previously observed by microspectrophotometry of individual human cone cells. The spectra are the first to be obtained from isolated human color vision pigments. They confirm the original identification of the three color vision genes, which was based on genetic evidence [Nathans, J., Thomas, D., & Hogness, D.S. (1986) Science 232, 193].     

The Luminosity Curve of the Protanomalous Fovea

Threshold spectral sensitivities (in the dark, or against bright colored backgrounds) are identical in the red-green range for both protanopes (dichromats) and protanomalous trichromatic color defectives. The latter, however, must have an additional photolabile cone pigment in the red-green range, and its presence is revealed by heterochromatic brightness matching through the spectrum (i.e. luminosity curves). The absorption spectrum of the anomalous cone pigment can be inferred from the protanomalous and protanopic luminosity curve, given reasonable assumptions as to how the different cone mechanisms pool their responses. Depending upon these assumptions, the pigment inferred is either (a) dilute solution of the normal red pigment (assumed density 1.0 for the deuteranope) or (b) similar in its absorption spectrum to the normal green pigment but shifted slightly toward the long wave end of the spectrum. Experimental attempts to choose between these alternatives have so far proved equivocal though (b) seems more likely on the basis of indirect evidence.     

A new mechanism in blue cone monochromatism

Blue cone monochromatism (BCM) is a rare X-linked colour vision disorder characterized by the absence of both red and green cone sensitivity. Most mutations leading to BCM fall into two classes of alterations in the red and green pigment gene array at Xq28. In one class the red and green pigment genes are inactivated by deletion in the locus control region. In the second class genetic rearrangements have created an isolated pigment gene that carries an inactivating point mutation. Here we describe a clinical case of BCM caused by a new mutation where exon 4 of an isolated red pigment gene has been deleted. The finding represents the first intragenic deletion yet described among red and green pigment genes. Received: 29 December 1995 / Revised: 30 May 1996     

Allele-specific PCR genotyping of the HSP70 gene polymorphism discriminating the green and red color variants sea cucumber （Apostichopus japonicus）

Color variation is a well-known feature of sea cucumbers (Apostichopus japonicus),which are classified into three groups based on their colors of red,green and black.It is also one of the most important traits related to how they taste,and it thereby affects their market price.Attempts were made to identify single-nucleotide polymorphisms (SNPs) and to analyze differences associated with SNP genotypes between green and red color variants using HSP70 as the target gene.The HSP70 gene,which is found universally in organisms from bacteria to humans,is one of the most evolutionarily conserved genes and the most widely studied biomarker of stress response.DNA fragments of 1074 bp covering a partial sequence of the sea cucumber HSP70 gene,were amplified from both red and green variants,and subsequently analyzed for the presence of SNPs.Twenty-seven polymorphic sites in total,including heterozygous sites,were observed.Of these,six sites were found to be significantly different SNP genotypes between green and red variants.Furthermore,PCR with an internal primer designed to include an allelespecific SNP at the 3' end (site 443) showed differentiation between the two variants,100％ and 4.2％ amplification in green and red variants,respectively.The validated SNPs may serve as informative genetic markers that can be used to distinguish variants at the early developmental stage,prior to color differentiation.     

Demonstration of a genotype-phenotype correlation in the polymorphic color vision of a non-callitrichine New World monkey, capuchin (Cebus apella)

Color-vision polymorphism in New World monkeys occurs because of an allelic polymorphism of the single-copy red-green middle-to-long-wavelength-sensitive (M/LWS) opsin gene on the X chromosome. Because color-vision types can readily be estimated from allelic types of the M/LWS opsin gene, this polymorphic system offers researchers an excellent opportunity to study the association between vision and behavior. As a prerequisite for such studies, genetically determined color-vision types must be concordant with phenotypes determined directly by behavioral criteria (e.g., by a color discrimination test). However, such correlations between genotypes and phenotypes have been studied only for callitrichine species. Using genetic, electrophysiological, and behavioral approaches, we evaluated the color vision of brown capuchin monkeys (Cebus apella), a representative non-callitrichine model animal for physiology and behavior. Two allelic M/LWS opsins-P545 and P530-were identified in the studied captive population. Females had one or both of the alleles, and males had either one. The retinal sensitivity in P530 dichromats was short-wave shifted relative to that in P545 dichromats, whereas that in P530/P545 trichromats was between the two groups. In a discrimination task using Ishihara pseudo-isochromatic plates, P530/P545 trichromats were successful in discriminating stimuli that P530 and P545 dichromats were unable to discriminate. In a food-search task, P530/P545 trichromats were able to locate red targets among green distracters as quickly as among white distracters, whereas both types of dichromats took longer. These results demonstrate the mutual consistency between genotypes and phenotypes of color vision, and provide a solid genetic basis on which the ecology and evolution of color vision can be investigated.     

Recent gene conversion between genes encoding human red and green visual pigments

Nucleotide sequences of the human X-linked red and green pigment genes were compared, and the number of silent substitutions per site (KSc) between these genes was analysed in comparison with the corresponding values of primate genes. Taking the retarded mutation rate of X-linked genes into consideration (Miyata et al., 1987), the red and green pigment genes were shown to have undergone gene conversion at around the time of separation of African apes and orangutan. Thus the recent gene conversion and retarded mutation rate in these X-linked genes are probably responsible for the strong sequence similarity between these genes, which is likely to facilitate the occurrence of red-green color blindness in the human population. It was also shown that the red pigment gene evolved about five times more rapidly than the green pigment gene since the latest gene conversion.     

Signatures of selection and gene conversion associated with human color vision variation

Trichromatic color vision in humans results from the combination of red, green, and blue photopigment opsins. Although color vision genes have been the targets of active molecular and psychophysical research on color vision abnormalities, little is known about patterns of normal genetic variation in these genes among global human populations. The current study presents nucleotide sequence analyses and tests of neutrality for a 5.5-kb region of the X-linked long-wave "red" opsin gene (OPN1LW) in 236 individuals from ethnically diverse human populations. Our analysis of the recombination landscape across OPN1LW reveals an unusual haplotype structure associated with amino acid replacement variation in exon 3 that is consistent with gene conversion. Compared with the absence of OPN1LW amino acid replacement fixation since divergence from chimpanzee, the human population exhibits a significant excess of high-frequency OPN1LW replacements. Our results suggest that subtle changes in L-cone opsin wavelength absorption may have been adaptive during human evolution.     

Frequent alterations of visual pigment genes in adrenoleukodystrophy.

Both adrenoleukodystrophy (ALD) and red/green color blindness have been mapped to the distal long arm of the human X chromosome (Xq28). Color-vision defects are frequently associated with ALD, and study of the red and green visual pigment genes in eight ALD kindreds has shown frequent structural changes including deletions and possible intragenic recombinations. Such changes may reflect chromosomal events underlying both ALD and the associated visual defects and should help define both the structural gene responsible for ALD and physical genetic relationships in the Xq28 region.