Shinichi Yamade

Magnetic resonance imaging measurements of extraocular muscle path shift and posterior eyeball prolapse from the muscle cone in acquired esotropia with high myopia

PURPOSE To investigate extraocular muscle (EOM) path shift and prolapse of posterior eyeball from muscle cone in acquired esotropia with high myopia (AEHM), using magnetic resonance imaging. DESIGN A case-control study. METHODS There were 16 eyes with AEHM, 11 with high myopia (HM), 12 with moderate myopia (MM), and 11 control eyes. Extraocular muscle shift was evaluated by measuring angles formed by the line connecting orbital centroids and the line connecting each orbital centroid and each EOM centroid. The ratio of the prolapse in the posterior eyeball from the muscle cone was also measured. RESULTS Both inferior shift of lateral rectus (LR) and nasal shift of superior rectus (SR) muscle were observed in the AEHM group, compared with HM, MM, and control groups. Neither shifted significantly in the HM group compared with control group. The prolapse ratio in AEHM group was higher than in the HM, MM, and control groups. Greater EOM shifts and eyeball prolapse were observed when the AEHM was more severe, as in esotropia fixus. CONCLUSIONS In AEHM, a prolapsing eyeball shifts LR inferiorly and SR nasally; these findings were not observed in high myopia with neither ocular deviation nor restriction. These shifts reduce abduction and supraduction and increase infraduction and adduction in AEHM. The shifts would be predicted to create a hypoesodeviation, which is a common finding in AEHM. Both EOM shifts and superotemporal eyeball prolapse tend to be greater in esotropia fixus.

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.

The importance of gene order in expression of the red and green visual pigment genes and in color vision

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.

Contrast (Modulation) Sensitivity Functions measured in patients with high refractive error with emphasis on aphakia: II. Determinations on patients

Measurements of Contrast Sensitivity Functions (CSF) were made on normal observers made artificially highly ametropic with spectacle lenses (with high back vertex) distance in order to determine the effect of retinal image size alterations upon CSF measures. While not an exact model for high ametropia per se, this experiment served to familiarize the experimenters with problems associated with the task.Image size alterations occur normally in aphakic patients and highly myopic patients. As a clinical trial, a series of aphakic observers were tested using an interferometric acuity device. CSF measures were made with the patients spectacle corrections in place and again with correcting contact lenses substituted. The contact lenses reduce induced image size alterations in these cases. The use of contact lenses in such measures allows differentiation between artifactual low frequency fall off in aphakia due to lens effects and possible low frequency fall-off due to other causes.

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.