Association of HK2 and NCK2 with normal tension glaucoma in the Japanese population.

Although family studies and genome-wide association studies have shown that genetic factors play a role in glaucoma, it has been difficult to identify the specific genetic variants involved. We tested 669 single nucleotide polymorphisms (SNPs) from the region of chromosome 2 that includes the GLC1B glaucoma locus for association with primary open-angle glaucoma (POAG) and normal tension glaucoma (NTG) in the Japanese population. We performed a two-stage case-control study. The first cohort consisted of 123 POAG cases, 121 NTG cases and 120 controls: the second cohort consisted of 187 POAG cases, 286 NTG cases, and 271 controls. Out of six SNPs showing significant association with POAG in the first round screening, seven SNPs were tested in the second round. Rs678350 in the HK2 gene coding sequence showed significant allelic (p=0.0027 in Stage Two, 2.7XE-4 in meta-analysis) association with POAG, and significant allelic (p=4.7XE-4 in Stage Two, 1.0XE-5 in meta-analysis) association with NTG. Although alleles in the TMEM182 gene did not show significant association with glaucoma in the second round, subjects with the A/A allele in TMEM182 rs869833 showed worse visual field mean deviation (p=0.01). Even though rs2033008 in the NCK2 gene coding sequence did not show significant association in the first round, it had previously shown association with NTG so it was tested for association with NTG in round 2 (p=0.0053 in Stage Two). Immunohistochemistry showed that both HK2 and NCK2 are expressed in the retinal ganglion cell layer. Once multi-testing was taken into account, only HK2 showed significant association with POAG and NTG in Stage Two. Our data also support previous reports of NCK2 association with NTG, and raise questions about what role TMEM182 might play in phenotypic variability. Our data suggest that HK2 may play an important role in NTG in the Japanese population.

Association of Toll-like receptor 4 gene polymorphisms in Japanese subjects with primary open-angle, normal-tension, and exfoliation glaucoma.

PURPOSE: To determine whether polymorphisms in the Toll-like receptor 4 (TLR4) gene are associated with primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG), and exfoliation glaucoma (XFG) in Japanese individuals. DESIGN: Genetic association study. SETTING: Multicenter study. STUDY POPULATION: One hundred eighty-four unrelated Japanese patients with POAG, 365 unrelated patients with NTG, and 109 unrelated patients with XFG from 5 hospitals. PROCEDURES: Genomic DNA was extracted from leukocytes of the peripheral blood, and 8 polymorphisms in the TLR4 genes were amplified by polymerase chain reaction (PCR) and directly sequenced. Allele and genotype frequencies and the inferred haplotypes were estimated. MAIN OUTCOME MEASURES: Differences in allele and genotype frequencies and haplotypes between subjects with POAG, NTG, and XFG. RESULTS: The allele frequency of rs2149356 of the TLR4 gene in the POAG, NTG, and XFG groups was the most significantly different from that of the control group (minor allele frequency 0.446, 0.395, 0.404, vs 0.308; P = .000058, P = .0030, and P = .015). The allele frequencies of the 5 TLR4 SNPs were higher in all of the glaucoma groups than that in the control group. The statistics of genotypes of TLR4 were approximately the same for all allele frequencies. The haplotypic frequencies with Tag SNPs studied earlier showed that only POAG was statistically significant. Other haplotypes, such as rs10759930, rs1927914, rs1927911, and rs2149356, had higher statistical significance (overall P = .00078 in POAG, overall P = .018 in NTG, and overall P = .014 in XFG). CONCLUSIONS: This study demonstrated that TLR4 polymorphisms are associated with NTG in the Japanese, and they also play a role in the pathogenesis of POAG and XFG.

Association between open-angle glaucoma and gene polymorphism for heat-shock protein 70-1.

PURPOSE: Heat-shock proteins (HSPs) or antibodies against them may contribute to glaucomatous optic neuropathy. We investigated the associations of HSP70-1 polymorphisms with open-angle glaucoma (OAG) in a Japanese population. METHODS: In 241 normal Japanese controls and 501 Japanese OAG patients, including 211 with primary open-angle glaucoma (POAG) and 290 with normal-tension glaucoma (NTG), two single-nucleotide polymorphisms, A-110C and G+190C, of HSP70-1 were identified by using an Invader assay and polymerase chain reaction-restriction fragment length polymorphism, respectively. Genotype distributions were compared between controls and OAG patients. Age at diagnosis, untreated maximum intraocular pressure, and visual field defects at diagnosis were examined for associations with the polymorphisms. RESULTS: Distribution of the A-110C genotype (AA versus AC+CC) differed significantly between controls and OAG patients (P = 0.007), POAG patients (P = 0.007), or NTG patients (P = 0.032). The genotype distribution of the G+190C polymorphism did not differ significantly between the controls and any patient group. No significant differences in the clinical characteristics of the patients were detected between genotype-defined groups by logistic regression analysis. CONCLUSION: The A-110C polymorphism of HSP70-1 may be associated with OAG pathogenesis in Japanese patients.

SNPs and interaction analyses of noelin 2, myocilin, and optineurin genes in Japanese patients with open-angle glaucoma.

PURPOSE: To evaluate the noelin 2 gene as a disease-causing factor for open-angle glaucoma (OAG) and the interactions between the noelin 2 (OLFM2), optineurin (OPTN), and myocilin (MYOC) genes. METHODS: OLFM2 was analyzed in 770 Japanese subjects including 215 patients with elevated intraocular pressure (IOP), 277 with normal IOP, 38 with juvenile open-angle glaucoma, and 240 control subjects. Two single-nucleotide polymorphisms (SNPs) in OPTN (c.412G-->A and c.603T-->A) and one SNP in MYOC (c.227G-->A) were examined. Single genes were investigated by univariate analysis and the gene-gene interactions by logistic regression analysis. Associations between genotypes and clinical characteristics at the time of diagnosis were examined. RESULTS: In OLFM2, 12 sequence variants were identified in 770 Japanese subjects. Arg144Gln (exon 4) was identified in two (0.3%) of the patients and in none of the control subjects. Combinations of OLFM2/317A and OPTN/412A or OLFM2/1281T and OPTN/412A were associated with patients with elevated IOP (P = 0.018 or P = 0.012, respectively). The combination of OLFM2/317G and OPTN/603A was significantly associated with elevated IOP (P = 0.018). No significant association was detected between SNPs in OLFM2 and in MYOC. Patients with normal IOP and with OLFM2/678A+OPTN/412G or OLFM2/1281C+OPTN/412G had significantly worse visual field scores (P = 0.022 or 0.030, respectively). CONCLUSIONS: The Arg144Gln mutation in OLFM2 is a possible disease-causing mutation in Japanese patients with OAG. Common polymorphisms in OLFM2 and OPTN may interactively contribute to the development of OAG, indicating a polygenic etiology.

[Low penetrance of His26Asp mutation in the optineurin gene in a Japanese family with normal-tension glaucoma].

PURPOSE: Three glaucoma genes have been identified in open-angle glaucoma(OAG). In this study, two of these genes were analyzed in Japanese patients with OAG. SUBJECTS AND METHODS: After informed consent was obtained, the myocilin gene and the optineurin gene were analyzed in 99 Japanese patients with OAG, including 49 cases of primary open-angle glaucoma(POAG) and 50 cases of normal-tension glaucoma(NTG). The patients were outpatients at the Tokyo Metropolitan Police Hospital. Family members were examined for the genes and clinical features if the proband had a mutation. RESULTS: One of the 99 patients had His26Asp mutation in the optineurin gene. None of the 240 subjects serving as controls had this mutation. The proband was a 37-year-old man diagnosed as having NTG. His father had the same mutation, but had normal clinical findings. His mother did not have the mutation, but had ocular hypertension. Polymorphism of Thr34Thr was observed in a wild type for the proband, and heterozygous change was found in his parents. For the myocilin gene, only the Asp208Glu mutation was found in his mother. CONCLUSION: His26Asp mutation in the optineurin gene showed low penetrance in a Japanese family with NTG.

Polymorphism of beta-adrenergic receptors and susceptibility to open-angle glaucoma.

PURPOSE: The human trabecular meshwork and ciliary body, which express beta-adrenergic receptors (ADRB1 and ADRB2), control aqueous humor dynamics. We investigated associations of ADRB polymorphisms with open-angle glaucoma (OAG), because ADRB gene polymorphisms alter receptor function. METHODS: We studied 240 Japanese controls and 505 Japanese OAG patients including 211 with primary open-angle glaucoma (POAG), and 294 with normal-tension glaucoma (NTG). Associations of four polymorphisms (Ser49Gly and Arg389Gly in the ADRB1 gene; Arg16Gly and Gln27Glu in the ADRB2 gene) were compared between patients and controls. Age, intraocular pressure (IOP), and visual field defects at diagnosis were examined for associations with polymorphisms. RESULTS: The Arg389Gly polymorphism in the ADRB1 gene showed significantly different allele and genotype frequencies in patients with NTG than in controls (p = 0.004 and 0.006, respectively). Other polymorphisms did not show a significant frequency difference. In POAG patients, carriers of Gly16 in the ADRB2 gene were significantly younger at diagnosis than noncarriers (p<0.001). The IOP at diagnosis was significantly higher in OAG patients carrying 27Glu in the ADRB2 gene than in patients without this allele (p<0.001). Clinical characteristics of OAG patients did not differ significantly in relation to other polymorphisms. CONCLUSIONS: Certain polymorphisms of the ADRB1 and ADRB2 genes influence the pathophysiology of OAG in Japanese patients.

Paraoxonase 1 gene polymorphisms influence clinical features of open-angle glaucoma.

BACKGROUND: The purpose of this study was to determine whether genetic polymorphisms affecting high-density lipoprotein (HDL)-associated antioxidant enzymes were associated with open-angle glaucoma (OAG). The rationale for this study was that the modification of low-density lipoprotein (LDL) by HDL prevents oxidative modification which can then cause dysfunction of endothelial cells. METHODS: We studied 284 normal Japanese controls and 555 Japanese patients with OAG, including primary open-angle glaucoma (POAG) and normal-tension glaucoma (NTG). The possible associations of polymorphisms of PON1/L55M, PON1/Q192R, PON2/S311C, and PAF-AH/V279F with OAG were investigated. We compared the genotype distributions and allele frequency in controls and patient groups. The age at diagnosis, intraocular pressure (IOP) at diagnosis, and visual field score at diagnosis were examined for association with polymorphisms. RESULTS: The distributions of genotypes and allele frequency for the four polymorphisms were not significantly different between any patient group and controls. In NTG patients, 55M carriers of the PON1 gene were significantly older at diagnosis than 55M non-carriers (P=0.001). The IOP at diagnosis was significantly higher in glaucoma patients carrying 192R in the PON1 gene than in patients not carrying 192R (P=0.006). No significant differences were seen in clinical characteristics of OAG patients in relation to other polymorphisms. CONCLUSION: PON1 gene polymorphisms may influence the features of Japanese patients with OAG.

Association between glaucoma and gene polymorphism of endothelin type A receptor.

PURPOSE: Endothelin 1 (ET-1), a potent vasoconstrictor, may affect regulation of intraocular pressure and ocular vessel tone. Thus, ET-1 and its receptors may contribute to development of glaucoma. We investigated whether gene polymorphisms of ET-1 (EDN1) and its receptors ETA (EDNRA) and ETB (EDNRB) were associated with glaucoma phenotypes and clinical features. METHODS: We studied 224 normal Japanese controls and 426 open angle glaucoma (OAG) patients including 176 with primary open angle glaucoma (POAG) and 250 with normal tension glaucoma (NTG). Nine single nucleotide polymorphisms were detected among the participants using the Invader assay; four for EDN1 (T-1370G, +138/ex1 del/ins, G8002A, K198N), four for EDNRA (G-231A, H323H, C+70G, C+1222T), and one for EDNRB (L277L). Genotype distributions were compared between normal controls and OAG. Age at diagnosis, untreated maximum intraocular pressure (IOP), and visual field defects at diagnosis were examined for association with polymorphisms. RESULTS: Of the 9 polymorphisms, genotype distributions showed no significant differences between OAG patients and controls adjusted by age. The GG genotype of EDNRA/C+70G was associated with worse visual field defects in NTG patients (p=0.014; Mann-Whitney U test, and p=0.027; logistic regression analysis). CONCLUSIONS: The polymorphism of EDNRA/C+70G may be related to NTG risk factors.

Genetic variants of TP53 and EPHX1 in Leber's hereditary optic neuropathy and their relationship to age at onset.

PURPOSE: To determine whether genetic polymorphisms of the genes for oxidative stress and apoptosis cause the clinical variability in patients with Leber's hereditary optic neuropathy (LHON). METHODS: Eighty-seven unrelated Japanese LHON patients carrying the 11778 mitochondrial mutation were studied at the Keio University Hospital. Their mean age (+/-SD) was 25.0 +/- 13.0 years with a range 3 to 65 years. Eleven polymorphisms in nine genes were studied: seven genes related to oxidative stress (SOD2, GSTT1, GSTM1, EPHX1, NQO1, p22 PHOX, and NOS3), and two genes related to apoptosis (TP53 and CD95). Each genetic polymorphism was analyzed in relation to the age at onset and the final visual acuity. RESULTS: Among the oxidative stress-related polymorphisms, a significant association between Tyr113His in the EPHX1 gene and the age at onset of the disease was identified (P = 0.026). LHON patients who were homozygous for His113 developed the disease earlier than those without this genotype (21.9 vs. 27.9 years). Among the apoptosis-related polymorphisms, a significant association between Arg72Pro in the TP53 gene and the age at onset was identified (P = 0.007). LHON patients who were homozygous for Arg72 developed the disease earlier than those without this genotype (20.5 vs. 28.1 years). In addition, LHON patients with both genotypes developed the disease significantly earlier (17.5 years, P = 0.011). No associations were found between the final visual acuity and the genetic polymorphisms examined. CONCLUSION: Nuclear genetic polymorphisms related to oxidative stress or apoptosis may modify the age at onset of LHON.

[Six cases of late-onset lattice corneal dystrophy associated with gene mutations induced by the transforming growth factor-beta].

PURPOSE: To investigate mutations in the transforming growth factor beta induced (TGFBI) gene and clinical features in 6 Japanese patients who were clinically diagnosed as having late-onset lattice corneal dystrophy (LCD). METHODS: The six patients were all male, and their ages at diagnosis were 56-82 years (average +/- standard deviation, 71.8 +/- 9.8 years). Molecular genetic analysis in the TGFBI gene was performed after informed consent was obtained. Exons 11, 12, and 14 were amplified by polymerase chain reaction (PCR), and the PCR products were directly sequenced. RESULTS: One of the 6 patients had a family history of corneal problems. Thick lattice lines in the middle to deep stroma (Cases 1, 2, and 3, right eye) and whitish nodular opaque lines in the middle stroma (Cases 4, 5, and 3, left eye) were observed. One patient showed tiny nodular deposits with thin lattice lines in the middle stroma (Case 6). A heterozygous Leu527Arg mutation in the TGFBI gene was detected in 5 patients (Cases 1 to 5); and there was Asn544Ser mutation in one patient (Case 6). CONCLUSIONS: Patients with Leu527Arg mutation in the TGFBI gene showed the late-onset form of LCD with low penetration, and varied corneal appearance.

Variants in optineurin gene and their association with tumor necrosis factor-alpha polymorphisms in Japanese patients with glaucoma.

PURPOSE: To investigate sequence variations in the optineurin (OPTN) gene and their association with TNF-alpha polymorphisms in Japanese patients with glaucoma. METHODS: The OPTN gene was analyzed in blood samples from 629 Japanese subjects. There were 194 patients with primary open-angle glaucoma (POAG), 217 with normal-tension glaucoma (NTG), and 218 with no eye disease (control subjects). The gene was screened for mutations by denaturing high-performance liquid chromatography. Genotyping of three polymorphisms of -308G-->A, -857C-->T, and -863C-->A in the TNF-alpha promoter region was performed. The associations between the genotypes and age, intraocular pressure (IOP), and visual field defects at the time of diagnosis were examined. RESULTS: A possible glaucoma-causing mutation, His26Asp, was identified in 1 of the 411 Japanese patients with glaucoma. A c.412G-->A (Thr34Thr) polymorphism in the OPTN gene was significantly associated with POAG (genotype frequency, P = 0.011; allele frequency, P = 0.003). The frequency of TNF-alpha/-857T and optineurin/412A carriers was significantly higher (P = 0.006) in patients with POAG than in control subjects. Among the patients with POAG who were carriers of TNF-alpha/-857T, the optineurin/412A carriers had significantly worse (P = 0.020) visual field scores than the non-optineurin/412A ones. The frequency of TNF-alpha/-863A and optineurin/603A (or Lys98) carriers was significantly higher in patients with POAG (P = 0.008) or NTG (P = 0.027) than in control subjects. Among the patients with POAG who were carriers of TNF-alpha/-863A, the ones with optineurin/603A (or Lys98) had significantly worse (P = 0.026) visual field scores than did those with non-optineurin/603A (or Lys98). CONCLUSIONS: These findings demonstrated that the OPTN gene is associated with POAG rather than NTG in the Japanese. Statistical analysis showed a possible interaction between polymorphisms in the OPTN and the TNF-alpha genes that would increase the risk for glaucoma.

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 degrees 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.

Rapid quantification of the heteroplasmy of mutant mitochondrial DNAs in Leber's hereditary optic neuropathy using the Invader technology.

PURPOSE: To quantify the degree of heteroplasmy of a mitochondrial DNA (mtDNA) mutation in Leber's hereditary optic neuropathy (LHON) a biplex Invader assay was applied. METHODS: To determine the optimum condition for the Invader assay, mtDNAs were assayed in various amounts of total DNA in 1-4-h incubations at 63 degrees C. To evaluate the suitability of the Invader assay to detect the three mutations, G3460A, G11778A, and T14484C, 10 ng of DNAs from 224 patients with bilateral optic atrophy was assayed. To quantify mtDNA heteroplasmy, a standard curve of known mixture ratios of mutation against calculation by the Invader assay was constructed. Seventy-two of the 224 patients had one of the three mutations, which corresponded with the mutation detected earlier by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. The percentages of mutant mtDNAs were calculated by the Invader assay in five heteroplasmic families, including 30 individuals with the G11778A mutation. The results were compared with those calculated earlier by labeled polymerase chain reaction followed by single-strand conformation polymorphism (PCR-SSCP) analysis. RESULTS: In 1-8 ng of DNA, the fluorescence intensity increased near linearly during a 4-h assay. With more than 16 ng of DNA, the intensities were saturated even at the 2-h assay. A linear relationship was observed between the results obtained from separate mixtures and from the Invader assay analysis. Because two fluorescent intensities are not always the same, one of the two intensities was modified to adjust to that of the other. Complete concordance was observed between PCR-RFLP analysis and Invader assay genotyping for the 224 patients. Results of percentage of heteroplasmy in five LHON families obtained by the Invader assay were consistent with those by the PCR-SSCP analysis. CONCLUSIONS: Invader assay is a simple, rapid, and reliable method of genotyping mtDNA mutations as well as quantifying heteroplasmy simultaneously under optimum conditions.