Takuro Fujimaki

Japanese juvenile retinoschisis is caused by mutations of the XLRS1 gene.

Abstract We investigated the XLRS1 gene in Japanese patients with retinoschisis (RS). All exons of the XLRS1 gene were sequenced in 14 males, including a pair of monozygotic twins, from 11 individual families with RS and five of their mothers who are asymptomatic but diagnosed as carriers. Six kinds of missense mutations and a nonsense mutation, including six novel mutations, were detected in all 14 patients and carriers. Mutations in the XLRS1 gene are also responsible for RS in non-Caucasian patients. Most Japanese RS cases are caused by an XLRS1 gene defect. A novel mutation, Glu72Lys, was found in four families, suggesting a common mutation in the Japanese population. Clinical features of RS patients with both the Glu72Lys and Pro193Leu mutations indicate that a genotype–phenotype correlation is not recognized in RS.

Arg124Cys Mutation of the βig-h3 Gene in a Japanese Family With Lattice Corneal Dystrophy Type I

To characterize severe lattice corneal dystrophy, we analyzed the betaig-h3 gene, clinical features, histological findings, and genotype-phenotype correlation in an affected Japanese family. Deoxyribonucleic acid was extracted from leukocytes in 16 members (12 affected and 4 unaffected) of a Japanese family with lattice corneal dystrophy type I. Exon 4 of the betaig-h3 gene was amplified and analyzed using molecular biological methods. Clinical and pathological data were also collected. We found a heterozygous point mutation that causes the disease phenotype. It was a single base-pair transition leading to an amino acid substitution (CGC-->TGC, Arg124Cys). The phenotypic variation within families was not recognized. The affected members in the pedigree demonstrated severe visual disturbance in the third decade and required keratoplasty. Histopathological examination revealed amyloid deposits consisting of short and thin amyloid fibers and lattice corneal dystrophy type I. The heterozygous Arg124Cys mutation reported in Caucasian lattice corneal dystrophy caused severe lattice corneal dystrophy consisting of short and thin amyloid fibers in a Japanese family. Based on our study of many members of the family, we are able to construct the natural course of this disorder from its earliest clinical findings through its late manifestations.

The level of erythrocyte aldose reductase is associated with the severity of diabetic retinopathy

To examine whether the level of erythrocyte aldose reductase is a risk factor for the severity of diabetic retinopathy, the enzyme level in 97 non-insulin-dependent diabetes mellitus (NIDDM) patients was measured by the two-site enzyme-linked immunosorbent assay. Based on the results of fundus photography and biomicroscopy, the severity of retinopathy was classified among NIDDM patients of more than 10 years. The level of erythrocyte aldose reductase was significantly higher in the patients with active proliferative retinopathy than in those with nonproliferative or quiescent proliferative retinopathy. Multivariate logistic regression analysis demonstrated that the level of erythrocyte aldose reductase was an independent risk factor for active proliferative retinopathy (odds ratio, 1.28; 95% confidence interval, 1.01-1.61). The results suggest that a high level of erythrocyte aldose reductase in NIDDM patients may affect the prognosis of diabetic retinopathy. Patients with high enzyme levels would need to be closely followed up in the management of the retinal complication.

Novel myoc Gene Mutation, Phe369leu, in Japanese Patients with Primary Open-angle Glaucoma Detected by Denaturing High-performance Liquid Chromatography

Purpose:To screen for mutations in the MYOC gene in Japanese patients with primary open-angle glaucoma (POAG) using denaturing high-performance liquid chromatography (DHPLC). Patients and Methods:Blood samples were collected from 171 patients with POAG and 100 controls from seven institutions in Japan. For high-throughput analysis, seven exonic regions were amplified by polymerase chain reaction using DNA pooled from three patients; each DNA pool was then analyzed chromatographically. For analysis of a small number of samples, 7 exonic regions were amplified separately but simultaneously with annealing at 58°C in each patient and then chromatographed, using 7 wells of the same 96-well plate per sample. When chromatographic patterns were abnormal by either method, the PCR products of the individual samples were sequenced. Results:Four glaucoma-causing mutations were identified in five POAG patients (2.9%). One missense mutation, Phe369Leu, is new; and three others, Ile360Asn, Ala363Thr, and Thr448Pro, have been reported in Japanese patients. Phe369Leu was associated with adult onset POAG. Conclusions:Mutations in the MYOC gene were demonstrated chromatographically in 2.9% of our Japanese POAG patients. The use of pooled DNAs with DHPLC analysis is a time- and labor-saving technique. All mutations detected appear to be specific to Japanese patients.

A Case of Oculodentodigital Dysplasia Syndrome with Novel GJA1 Gene Mutation

PurposeTo report a case of oculodentodigital dysplasia syndrome (ODDD) with a heterozygous mutation in GJA1 (connexin 43) gene.MethodsA 9-year-old girl visited our hospital complaining of visual disturbances. The patient had microphthalmia, a small nose with hypoplastic alae nasi, and syndactyly. Visual acuity with prescribed glasses improved to 0.5 (1.2) OU 2 months after the first visit. She was satisfied with the new glasses and the improvement in visual acuity. Genomic DNA was extracted from leukocytes of the patient’s peripheral blood in accordance with standard procedures, after obtaining parental informed consent. We amplified GJA1 exon 2 from her genomic DNA by the PCR method, and sequenced the product using the dye terminator method.ResultsS5C (c. 13A > T), a novel mutation in exon 2 of GJA1, was found in the patient. The parents had no mutation of GJAI, nor was there any sign of abnormality in other family members. No similar mutation could be found in the 50 genotyped normal subjects in the control group.ConclusionsA novel GJA1 mutation was detected in a Japanese ODDD patient. Glaucoma complications associated with ODDD have already been reported. Careful long-term monitoring and treatment are also necessary.

Mitochondrial DNA mutations with Leber's hereditary optic neuropathy in Japanese patients with open-angle glaucoma.

PurposeAbnormal optic disc excavations are found in patients with Lebers hereditary optic neuropathy (LHON). The purpose of this study was to determine whether heteroplasmy for the major three LHON mutations or for the rare LHON mutations are risk factors for open-angle glaucoma.MethodsBlood samples from 835 Japanese subjects were screened with the Invader assay for ten LHON-associated mutations: three major mutations (G3460A, G11778A, T14484C) and seven rare mutations (T9101C, G9804A, C14482A, C14482G, G14459A, T14498C, and A14510G). Of the 835 subjects, 241 were patients with primary open-angle glaucoma (POAG), 310 were patients with normal-tension glaucoma (NTG), and 284 were healthy controls.ResultsFive POAG patients and three NTG patients had one of five mutations, C9099A, T9101G, T9101C, G9804A, or G11778A, but none of these patients had LHON. The C9099A (Ile191Met) and T9101G (Ile192Ser) mutations were novel and identified within the probes by lack of signal in the assay. Two patients with the G11778A mutation showed heteroplasmy, with 15% mutant mtDNA in the male patient and 80% in the female patient. The remaining LHON-associated mutations were not detected in any of the subjects. A case-control study did not show a significant difference (P = 0.099): eight potentially disease-associated variants in 551 patients versus zero variants in the 284 controls.ConclusionsRare LHON-associated mitochondrial DNA mutations were found in Japanese patients with open-angle glaucoma (OAG). However, whether mitochondrial DNA mutations are risk factors for OAG is still open to question. Jpn J Ophthalmol 2006;50:128–134

A novel nonsense mutation in rhodopsin gene in two Indonesian families with autosomal recessive retinitis pigmentosa.

Purpose: To report a novel, identical nonsense mutation in the rhodopsin (RHO) gene in two Indonesian families with autosomal recessive retinitis pigmentosa (arRP). Methods: Mutation screening for the RHO gene was performed in 38 unrelated patients with retinitis pigmentosa (RP) by direct sequencing. Clinical features were also characterized, through complete ophthalmologic examination. Family members of RP patients testing positive for the RHO gene were subjected to genetic and clinical examination. To assess the founder effect in the two families, haplotype analysis also was performed. Results: A novel homozygous nonsense mutation was detected in two patients by a G to A transition at nucleotide position 482 in exon 2 of the RHO gene, resulting in substitution of a tryptophan-to-stop at codon 161 (c.482G>A, p.W161X). Examination of family members of these 2 patients showed that the affected members were homozygous and unaffected carriers were heterozygous for the p.W161X mutation. Haplotype analysis revealed that members of the two families carried the same disease-associated variants in markers (IVS1 RHO and D3S2322). No p.W161X mutations were detected in 45 normal Indonesian subjects, nor were any mutations detected in exons 1–5 of the RHO gene in the remaining 36 RP patients. Conclusion: We detected a novel, recessive nonsense mutation (p.W161X) in the RHO gene of two families through mutation screening of RHO in 38 Indonesian RP patients. Haplotype analysis suggested that p.W161X was the founder mutation.

Analysis of peripherin/RDS gene for Japanese retinal dystrophies

We studied 133 Japanese patients with retinal dystrophies to detect peripherin/RDS (retinal degeneration slow) gene defects. The patients analyzed included 52 with autosomal dominant retinitis pigmentosa, 36 with autosomal recessive retinitis pigmentosa, 3 with simplex retinitis pigmentosa, 12 with cone-rod dystrophy, 5 with rod-cone dystrophy, 3 with vitelliform macular dystrophy (Bests disease), 4 with macular dystrophy, 2 with cone dystrophy, 2 with fundus flavimaculatus, 2 with fundus albipunctatus, and 12 with retinitis pigmentosa with macular degeneration as well as 40 unrelated normal persons. Three exons of the peripherin/RDS gene cut into 150-200 base-pair fragments were amplified by polymerase chain reaction and screened by single-strand conformation polymorphism. The DNA fragments with any suspected variations were directly sequenced. Eight point mutations were detected. Among them, two missense mutations at codons 304 and 338 result in an amino acid substitution of glutamine for glutamic acid and aspartic acid for glycine, respectively. However, they were not cosegregated with the diseases, and these mutations were also commonly found in normal controls. For these controls, the proportion of transversion from G to C at codon 304 (GAG-->CAG) and transition from G to A at codon 338 (GGC-->GAC) were 0.192 +/- 0.045 and 0.173 +/- 0.053, respectively. Our results suggest that a peripherin/RDS gene mutation might be rare in Japanese patients.

Mutations in the Membrane Component, Chromosome 1, Surface Marker 1 (M1S1) Gene in Gelatinous Drop-like Corneal Dystrophy

PurposeTo report mutations in the membrane component, chromosome 1, surface marker 1 (M1S1) gene in two members of the same family who showed symptoms of gelatinous drop-like corneal dystrophy (GDLD).MethodsDNA was extracted from leukocytes of peripheral blood of the two affected members of the family and from controls, and the coding region of M1S1 was amplified by polymerase chain reaction (PCR). The PCR products were analyzed by direct sequencing. Normal and mutant M1S1 expression vectors were constructed and transfected into CHO cells to identify the cellular location of the gene products.ResultsThe affected members had compound heterozygous mutations consisting of a nonsense change at codon 84 (K84X) and a missense mutation resulting in a substitution of arginine for cysteine at codon 108 (C108R). Neither of these mutations was found in the 50 controls. Protein expression analysis showed that the C108R product was distributed diffusely in the cytoplasm, whereas the normal gene product accumulated at cell-to-cell adhesion borders.ConclusionThese data indicate that the K84X and C108R mutations in M1S1 cause GDLD.

Hidden Genetic Variation in LCA9-Associated Congenital Blindness Explained by 5′UTR Mutations and Copy-Number Variations of NMNAT1

Leber congenital amaurosis (LCA) is a severe autosomal‐recessive retinal dystrophy leading to congenital blindness. A recently identified LCA gene is NMNAT1, located in the LCA9 locus. Although most mutations in blindness genes are coding variations, there is accumulating evidence for hidden noncoding defects or structural variations (SVs). The starting point of this study was an LCA9‐associated consanguineous family in which no coding mutations were found in the LCA9 region. Exploring the untranslated regions of NMNAT1 revealed a novel homozygous 5′UTR variant, c.‐70A>T. Moreover, an adjacent 5′UTR variant, c.‐69C>T, was identified in a second consanguineous family displaying a similar phenotype. Both 5′UTR variants resulted in decreased NMNAT1 mRNA abundance in patients’ lymphocytes, and caused decreased luciferase activity in human retinal pigment epithelial RPE‐1 cells. Second, we unraveled pseudohomozygosity of a coding NMNAT1 mutation in two unrelated LCA patients by the identification of two distinct heterozygous partial NMNAT1 deletions. Molecular characterization of the breakpoint junctions revealed a complex Alu‐rich genomic architecture. Our study uncovered hidden genetic variation in NMNAT1‐associated LCA and emphasized a shift from coding to noncoding regulatory mutations and repeat‐mediated SVs in the molecular pathogenesis of heterogeneous recessive disorders such as hereditary blindness.