Novel MIP gene mutation causes autosomal-dominant congenital cataract.

AIM: To identify disease-causative mutations in families with congenital cataract. METHODS: Two Chinese families with autosomal-dominant congenital cataract (ADCC) were recruited and underwent comprehensive eye examinations. Gene panel next-generation sequencing of common pathogenic genes of congenital cataract was performed in the proband of each family. Sanger sequencing was used to valid the candidate gene mutations and sequence the other family members for co-segregation analysis. The effect of sequence changes on protein structure and function was predicted through bioinformatics analysis. Major intrinsic protein (MIP)-wildtype and MIP-G29R plasmids were constructed and microinjected into zebrafish single-cell stage embryos. Zebrafish embryonic lens phenotypes were screened using confocal microscopy. RESULTS: A novel heterozygous mutation (c.85G>A; p.G29R) in the MIP gene was identified in the proband of one family. A known heterozygous mutation (c.97C>T; p.R33C; rs864309693) in MIP was found in the proband of another family. In-silico prediction indicated that the novel mutation might affect the MIP protein function. Zebrafish embryonic lens was uniformly transparent in both wild-type PCS2+MIP and mutant PCS2+MIP. CONCLUSION: Two missense mutations in the MIP gene in Chinese cataract families are identified, and one of which is novel. These findings expand the genetic spectrum of MIP mutations associated with cataracts. The functional studies suggest that the novel MIP mutation might not be a gain-of-function but a loss-of-function mutation.

Exome-based investigation of the genetic basis of human pigmentary glaucoma.

BACKGROUND: Glaucoma is a leading cause of visual disability and blindness. Release of iris pigment within the eye, pigment dispersion syndrome (PDS), can lead to one type of glaucoma known as pigmentary glaucoma. PDS has a genetic component, however, the genes involved with this condition are largely unknown. We sought to discover genes that cause PDS by testing cohorts of patients and controls for mutations using a tiered analysis of exome data. RESULTS: Our primary analysis evaluated melanosome-related genes that cause dispersion of iris pigment in mice (TYRP1, GPNMB, LYST, DCT, and MITF). We identified rare mutations, but they were not statistically enriched in PDS patients. Our secondary analyses examined PMEL (previously linked with PDS), MRAP, and 19 other genes. Four MRAP mutations were identified in PDS cases but not in controls (p = 0.016). Immunohistochemical analysis of human donor eyes revealed abundant MRAP protein in the iris, the source of pigment in PDS. However, analysis of MRAP in additional cohorts (415 cases and 1645 controls) did not support an association with PDS. We also did not confirm a link between PMEL and PDS in our cohorts due to lack of reported mutations and similar frequency of the variants in PDS patients as in control subjects. CONCLUSIONS: We did not detect a statistical enrichment of mutations in melanosome-related genes in human PDS patients and we found conflicting data about the likely pathogenicity of MRAP mutations. PDS may have a complex genetic basis that is not easily unraveled with exome analyses.

Association of a Primary Open-Angle Glaucoma Genetic Risk Score With Earlier Age at Diagnosis.

IMPORTANCE: Genetic variants associated with primary open-angle glaucoma (POAG) are known to influence disease risk. However, the clinical effect of associated variants individually or in aggregate is not known. Genetic risk scores (GRS) examine the cumulative genetic load by combining individual genetic variants into a single measure, which is assumed to have a larger effect and increased power to detect relevant disease-related associations. OBJECTIVE: To investigate if a GRS that comprised 12 POAG genetic risk variants is associated with age at disease diagnosis. DESIGN, SETTING, AND PARTICIPANTS: A cross-sectional study included individuals with POAG and controls from the Glaucoma Genes and Environment (GLAUGEN) study and the National Eye Institute Glaucoma Human Genetics Collaboration (NEIGHBOR) study. A GRS was formulated using 12 variants known to be associated with POAG, and the alleles associated with increasing risk of POAG were aligned in the case-control sets. In case-only analyses, the association of the GRS with age at diagnosis was analyzed as an estimate of disease onset. Results from cohort-specific analyses were combined with meta-analysis. Data collection started in August 2012 for the NEIGHBOR cohort and in July 2008 for the GLAUGEN cohort and were analyzed starting in March 2018. MAIN OUTCOMES AND MEASURES: Association of a 12 single-nucleotide polymorphism POAG GRS with age at diagnosis in individuals with POAG using linear regression. RESULTS: The GLAUGEN study included 976 individuals with POAG and 1140 controls. The NEIGHBOR study included 2132 individuals with POAG and 2290 controls. For individuals with POAG, the mean (SD) age at diagnosis was 63.6 (9.8) years in the GLAUGEN cohort and 66.0 (13.7) years in the NEIGHBOR cohort. For controls, the mean (SD) age at enrollment was 65.5 (9.2) years in the GLAUGEN cohort and 68.9 (11.4) years in the NEIGHBOR cohort. All study participants were European white. The GRS was strongly associated with POAG risk in case-control analysis (odds ratio per 1-point increase in score = 1.24; 95% CI, 1.21-1.27; P = 3.4 × 10-66). In case-only analyses, each higher GRS unit was associated with a 0.36-year earlier age at diagnosis (ß = -0.36; 95% CI, -0.56 to -0.16; P = 4.0 × 10-4). Individuals in the top 5% of the GRS had a mean (SD) age at diagnosis of 5.2 (12.8) years earlier than those in the bottom 5% GRS (61.4 [12.7] vs 66.6 [12.9] years; P = 5.0 × 10-4). CONCLUSIONS AND RELEVANCE: A higher dose of POAG risk alleles was associated with an earlier age at glaucoma diagnosis. On average, individuals with POAG with the highest GRS had 5.2-year earlier age at diagnosis of disease. These results suggest that a GRS that comprised genetic variants associated with POAG could help identify patients with risk of earlier disease onset impacting screening and therapeutic strategies.

Family-Based Genome-Wide Association Study of South Indian Pedigrees Supports WNT7B as a Central Corneal Thickness Locus.

Purpose: To identify genetic risk factors contributing to central corneal thickness (CCT) in individuals from South India, a population with a high prevalence of ocular disorders. Methods: One hundred ninety-five individuals from 15 large South Indian pedigrees were genotyped using the Omni2.5 bead array. Family-based association for CCT was conducted using the score test in MERLIN. Results: Genome-wide association study (GWAS) identified strongest association for single nucleotide polymorphisms (SNPs) in the first intron of WNT7B and CCT (top SNP rs9330813; ß = -0.57, 95% confidence interval [CI]: -0.78 to -0.36; P = 1.7 × 10-7). We further investigated rs9330813 in a Latino cohort and four independent European cohorts. A meta-analysis of these data sets demonstrated statistically significant association between rs9330813 and CCT (ß = -3.94, 95% CI: -5.23 to -2.66; P = 1.7 × 10-9). WNT7B SNPs located in the same genomic region that includes rs9330813 have previously been associated with CCT in Latinos but with other ocular quantitative traits related to myopia (corneal curvature and axial length) in a Japanese population (rs10453441 and rs200329677). To evaluate the specificity of the observed WNT7B association with CCT in the South Indian families, we completed an ocular phenome-wide association study (PheWAS) for the top WNT7B SNPs using 45 ocular traits measured in these same families including corneal curvature and axial length. The ocular PheWAS results indicate that in the South Indian families WNT7B SNPs are primarily associated with CCT. Conclusions: The results indicate robust evidence for association between WNT7B SNPs and CCT in South Indian pedigrees, and suggest that WNT7B SNPs can have population-specific effects on ocular quantitative traits.

Serum Th1 and Th17 related cytokines and autoantibodies in patients with Posner-Schlossman syndrome.

Posner-Schlossman syndrome (PSS) shares some clinical features with uveitis and open angle glaucoma. Cytokines and autoantibodies have been associated with uveitis and open angle glaucoma. However, the role of serum cytokines and autoantibodies in the pathogenesis of PSS remains unknown. This study aimed to evaluate the associations of type 1 T helper (Th1) and Th17 related cytokines and autoantibodies with PSS. Peripheral blood serum samples were collected from 81 patients with PSS and 97 gender- and age-matched healthy blood donors. Th1 and Th17 related cytokines, including interleukin-1ß (IL-1ß), IL-12, tumor necrosis factor-α (TNF-α), interferon- γ (IFN-γ), IL-6 and IL-17, and glucose-6-phosphate isomerase (GPI) were determined by double antibody sandwich ELISA. Anti-nuclear antibody (ANA), anti-keratin antibody (AKA) and anti-neutrophil cytoplasmic antibody (ANCA) were detected by indirect immunofluorescence assay. Anti-cardiolipin antibody (ACA)-IgG, ACA-IgM, ACA-IgA, anti-double stranded DNA (anti-dsDNA) and anti-cyclic citrullinated peptide antibody (anti-CCP) were detected by indirect ELISA. Serum levels of IL-1ß, IL-12 and IL-6 in PSS patients were significantly lower than those in controls (P < 0.003), and these associations survived the Bonferroni correction (Pc < 0.018). There was no significant difference in serum levels of TNF-α, IFN-γ and IL-17 between the PSS and control groups (Pc > 0.12). Positive rate of serum anti-dsDNA in PSS patients was significantly higher than that in the control group (P = 0.002, Pc = 0.018), while positive rates of serum ANA, AKA, ANCA, ACA-IgG, ACA-IgM, ACA-IgA, GPI and anti-CCP in the PSS group were not significantly different from those in the control group (Pc > 0.09). These results suggest that anti-dsDNA may contribute to the pathogenesis of PSS, while Th1 and Th17 related cytokines and other autoantibodies may not be major contributors to PSS.

Screening of the LTBP2 gene in 214 Chinese sporadic CYP1B1-negative patients with primary congenital glaucoma.

PURPOSE: To identify deleterious mutations in the latent transforming growth factor-ß-binding protein 2 (LTBP2) gene in sporadic patients with primary congenital glaucoma (PCG) from a Han Chinese population, which had been excluded for mutations in the CYP1B1 gene. METHODS: In this retrospective case-control study, 36 coding exons and adjacent exon-intron boundaries of LTBP2 were amplified with PCR and screened for mutations with Sanger sequencing in DNA samples of 214 sporadic patients with PCG. Sequence variants identified in the patients with PCG were subsequently screened in 100 unaffected control subjects and the unaffected parents of the patients with PCG who had sequence changes in LTBP2. RESULTS: Eight heterozygous single nucleotide polymorphisms (SNPs) in coding regions of LTBP2 were identified in the patients with PCG. Four of these SNPs were missense changes that resulted in the replacement of amino acids (rs2304707, rs116914994, rs45468895, and rs763035721), two of which (rs2304707 and rs116914994) were also present in the control subjects. No significant differences in the frequencies of the missense SNPs were found between the patients with PCG and the controls. The two missense SNPs, rs45468895 and rs763035721, which were each found in one patient also existed in their unaffected parents, suggesting that these two SNPs were not segregated in these families and are unlikely to be a disease-causative variant. In addition, four synonymous SNPs were detected in the patients with PCG (rs61738025, rs862031, rs199805158, and rs12586758). CONCLUSIONS: The results showed that no deleterious mutations were found in coding regions of LTBP2 in patients with PCG, suggesting that it is not a causal gene for PCG in the Han Chinese population.

Association of Matrix Metalloproteinase-9 (MMP9) Variants with Primary Angle Closure and Primary Angle Closure Glaucoma.

Shorter axial length observed in patients with primary angle closure glaucoma (PACG) might be due to altered matrix metalloproteinase-9 (MMP9) activity resulting in ECM remodeling during eye growth and development. This study aimed to evaluate common variants in MMP9 for association with PACG. Six tag SNPs of MMP9 were genotyped in a Chinese sample of 1,030 cases, including 572 PACG and 458 primary angle closure (PAC), and 499 controls. None of 6 SNPs were significantly associated with overall PAC/PACG (P > 0.07) or with PAC/PACG subgroups (Pc > 0.18). Meta-analysis of two non-Chinese studies revealed significant association between rs17576 and PACG (ORs = 0.56, P < 0.0001); however, meta-analysis of our dataset with 4 Chinese datasets did not replicate this association (ORs = 1.23, P = 0.29). Prior significant association for rs3918249 in one Caucasian study (OR = 0.63, P = 0.006) was not replicated in meta-analysis of 3 Chinese studies including this study (ORs = 0.91, P = 0.13). Significant heterogeneity between non-Chinese and Chinese datasets precluded overall meta-analysis for rs17576 and rs3918249 (Q = 0.001 and 0.04 respectively). rs17577 was nominally associated with PACG in one Caucasian study (OR = 1.71, P = 0.02), but not in 3 Chinese studies including our study (ORs = 1.20, P = 0.07). Overall meta-analysis revealed nominal association for rs17577 and PAC/PACG (ORs = 1.26, Pc = 0.05). Meta-analysis did not show significant association between the other SNPs and PAC/PACG (P > 0.47). The largest association study to date did not find significant association between MMP9 and PAC/PACG in Chinese; meta-analysis with other Chinese datasets did not produce significant association. In most instances combination with non-Chinese datasets was not possible except for one variant showing nominally significant association. More work is needed to define the role of MMP9 variants in PACG.

Human Leukocyte Antigens-B and -C Loci Associated with Posner-Schlossman Syndrome in a Southern Chinese Population.

The etiology of Posner-Schlossman syndrome (PSS) remains unknown. The association of human leukocyte antigens (HLA) allelic diversity with PSS has been poorly investigated. To evaluate the association of allelic polymorphisms of class I HLA-A, -B and -C and class II HLA-DRB1 and -DQB1 with PSS, 100 unrelated patients with PSS and 128 age- and ethnically matched control subjects were recruited from a southern Chinese Han population. Polymorphisms in exons 2-4 for HLA-A, -B, -C loci, exon 2 for HLA-DRB1 and exons 2,3 for HLA-DQB1 were analyzed for association with PSS at allele and haplotype levels. The allele frequency of HLA-C*1402 in PSS patients was significantly higher than that in controls (P = 0.002, OR = 4.12). This association survived the Bonferroni correction (Pc = 0.04). The allele frequency of HLA-B*1301 in PSS patients was lower than that in the control group (P = 0.003, OR = 0.21), although this association did not survive the Bonferroni correction (Pc = 0.16). In PSS patients, the haplotype frequencies of HLA-A*1101~C*1402 and B*5101~C*1402 were higher than that in controls (P = 0.03, OR = 4.44; P = 0.02, OR = 3.20; respectively), while the HLA-B*1301~C*0304 was lower than that in controls (P = 0.007, OR = 0.23), although these associations did not survive the Bonferroni correction (Pc > 0.16). This study for the first time demonstrated that polymorphisms at the HLA-B and HLA-C loci were nominally associated with PSS in the southern Chinese Han population. Our results suggest that HLA-C*1402, A*1101~C*1402 and B*5101~C*1402 might be risk factors for PSS, whereas HLA-B*1301 plus B*1301~C*0304 might be protective factors against PSS, but even larger datasets are required to confirm these findings. Findings from this study provide valuable new clues for investigating the mechanisms and development of new diagnosis and treatment for PSS.

The p53 codon 72 PRO/PRO genotype may be associated with initial central visual field defects in caucasians with primary open angle glaucoma.

BACKGROUND: Loss of vision in glaucoma is due to apoptotic retinal ganglion cell loss. While p53 modulates apoptosis, gene association studies between p53 variants and glaucoma have been inconsistent. In this study we evaluate the association between a p53 variant functionally known to influence apoptosis (codon 72 Pro/Arg) and the subset of primary open angle glaucoma (POAG) patients with early loss of central visual field. METHODS: Genotypes for the p53 codon 72 polymorphism (Pro/Arg) were obtained for 264 POAG patients and 400 controls from the U.S. and in replication studies for 308 POAG patients and 178 controls from Australia (GIST). The glaucoma patients were divided into two groups according to location of initial visual field defect (either paracentral or peripheral). All cases and controls were Caucasian with European ancestry. RESULTS: The p53-PRO/PRO genotype was more frequent in the U.S. POAG patients with early visual field defects in the paracentral regions compared with those in the peripheral regions or control group (p=2.7 × 10(-5)). We replicated this finding in the GIST cohort (p  =7.3 × 10(-3), and in the pooled sample (p=6.6 × 10(-7)) and in a meta-analysis of both the US and GIST datasets (1.3 × 10(-6), OR 2.17 (1.58-2.98 for the PRO allele). CONCLUSIONS: These results suggest that the p53 codon 72 PRO/PRO genotype is potentially associated with early paracentral visual field defects in primary open-angle glaucoma patients.

SNP rs1533428 at 2p16.3 as a marker for late-onset primary open-angle glaucoma.

PURPOSE: To investigate the associations between gene variants in cholesterol 24S-hydroxylase (CYP46A1), LIM homeobox transcription factor 1-beta (LMX1B), plexin domain containing 2 (PLXDC2), toll-like receptor 4 (TLR4), transmembrane and tetratricopeptide repeat containing 2 (TMTC2), zona pellucida glycoprotein 4 (ZP4), chromosome 2p16.3, and primary open-angle glaucoma (POAG). METHODS: We studied 462 POAG patients and 577 controls from three cohorts (Hong Kong, Shantou, and Beijing, China). Twelve single-nucleotide polymorphisms (SNPs) were genotyped in the Hong Kong cohort using TaqMan genotyping assay. Significant associations were validated in the Shantou and Beijing cohorts. RESULTS: Association of POAG with TLR4 rs7037117, in a recessive model, was identified in the Hong Kong and Shantou cohorts (both southern Chinese, p(rec)=0.0019) but not the Beijing cohort (northern Chinese). rs1533428 at chromosome 2p16.3 showed a consistent trend of age-specific association in all three cohorts. Genotypes TT + CT conferred a 2.16 fold of significantly increased risk to late-onset POAG (p(dom)=0.00025), but no significant risk to POAG of younger ages of onset in the combined cohort. A joint effect was found between rs7037117 and rs1533428, with carriers of both higher-risk genotypes having a 4.53 fold of increased disease risk (p=0.00028). CONCLUSIONS: Our study reveals discrepant association patterns of 12 candidate SNPs in 7 genes/loci with POAG in Chinese, provides positive replications for POAG markers rs1533428 at 2p16.3 and TLR4 rs7037117, and suggests that rs1533428 is a putative risk variant for late-onset POAG. The identification of an age-specific association between rs1533428 and late-onset POAG highlights a new genotype-phenotype association in POAG. Further studies are warranted to confirm the age-specific association.

Genetic variants associated with optic nerve vertical cup-to-disc ratio are risk factors for primary open angle glaucoma in a US Caucasian population.

PURPOSE: Genetically complex disorders, such as primary open angle glaucoma (POAG), may include highly heritable quantitative traits as part of the overall phenotype, and mapping genes influencing the related quantitative traits may effectively identify genetic risk factors predisposing to the complex disease. Recent studies have identified SNPs associated with optic nerve area and vertical cup-to-disc ratio (VCDR). The purpose of this study was to evaluate the association between these SNPs and POAG in a US Caucasian case-control sample. METHODS: Five SNPs previously associated with optic disc area, or VCDR, were genotyped in 539 POAG cases and 336 controls. Genotype data were analyzed for single SNP associations and SNP interactions with VCDR and POAG. RESULTS: SNPs associated with VCDR rs1063192 (CDKN2B) and rs10483727 (SIX1/SIX6) were also associated with POAG (P = 0.0006 and P = 0.0043 for rs1063192 and rs10483727, respectively). rs1063192, associated with smaller VCDR, had a protective effect (odds ratio [OR] = 0.73; 95% confidence interval [CI], 0.58-0.90), whereas rs10483727, associated with larger VCDR, increased POAG risk (OR = 1.33; 95% CI, 1.08-1.65). POAG risk associated with increased VCDR was significantly influenced by the C allele of rs1900004 (ATOH7), associated with increased optic nerve area (P-interaction = 0.025; OR = 1.89; 95% CI, 1.22-2.94). CONCLUSIONS: Genetic variants influencing VCDR are associated with POAG in a US Caucasian population. Variants associated with optic nerve area are not independently associated with disease but can influence the effects of VCDR variants suggesting that increased optic disc area can significantly contribute to POAG risk when coupled with risk factors controlling VCDR.

LOXL1 promoter haplotypes are associated with exfoliation syndrome in a U.S. Caucasian population.

PURPOSE: LOXL1 is a major genetic risk factor for exfoliation syndrome (ES) and exfoliation glaucoma (EG). Recent evidence documenting reversal of risk alleles for the disease-associated missense variants R141L and G153D suggests that these variants are not causative and that they may be proxies for other unknown functional LOXL1 variants. The purpose of this study was to investigate the disease association of LOXL1 variants spanning the gene region, including the 5' and 3' regulatory regions, in a U.S. Caucasian case-control sample. METHODS: Twenty-five LOXL1 single-nucleotide polymorphisms (SNPs), distributed throughout the gene, were genotyped in 196 Caucasian patients with ES/EG and 201 matched controls. Genotype data were analyzed for single SNP associations, SNP interactions, and haplotype associations. RESULTS: Promoter region haplotypes that included the risk alleles for rs12914489, a SNP located in the distal promoter region and independently associated with ES, and rs16958477, a SNP previously shown to affect gene transcription, were associated with increased disease risk (P=0.0008; odds ratio [OR], 2.34; 95% confidence interval [CI], 1.42-3.85) and with protective effects (P=2.3 × 10(-6); OR, 0.38; 95% CI, 0.25-0.57). Haplotypes containing rs12914489 and rs16958477 risk and protective alleles also significantly influenced the disease risk associated with missense alleles R141L and G153D. CONCLUSIONS: LOXL1 promoter haplotypes were identified that are significantly associated with ES/EG in a U.S. Caucasian population. These results suggest that promoter region SNPs can influence LOXL1 gene expression, potentially causing a reduction of enzyme activity that may predispose to disease.

Glaucoma: genes, phenotypes, and new directions for therapy.

Glaucoma, a leading cause of blindness worldwide, is characterized by progressive optic nerve damage, usually associated with intraocular pressure. Although the clinical progression of the disease is well defined, the molecular events responsible for glaucoma are currently poorly understood and current therapeutic strategies are not curative. This review summarizes the human genetics and genomic approaches that have shed light on the complex inheritance of glaucoma genes and the potential for gene-based and cellular therapies that this research makes possible.

Association of polymorphisms of tumor necrosis factor and tumor protein p53 with primary open-angle glaucoma.

PURPOSE: To evaluate the variants of 10 genes for association with primary open-angle glaucoma (POAG) in a Chinese population. METHODS: A total of 405 unrelated patients with POAG (255 high-tension glaucoma [HTG], 100 normal-tension glaucoma [NTG], and 50 juvenile-onset open-angle glaucoma [JOAG]) and 201 control subjects were recruited. Seventeen variants in 10 genes with reported association with POAG were genotyped for analysis of allele and haplotype frequencies between cases and control subjects. These genes included CDH1 (cadherin 1, type 1, E-cadherin), CDKN1A (cyclin-dependent kinase inhibitor 1A), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), GSTM1 (glutathione S-transferase mu 1), GSTT1 (glutathione S-transferase theta 1), MTHFR (5,10-methylenetetrahydrofolate reductase), NOS3 (nitric oxide synthase 3), OPA1 (optic atrophy 1), TNF (tumor necrosis factor), and TP53 (tumor protein p53). RESULTS: One SNP (-308G>A; rs1800629) in TNF demonstrated a significant association with HTG (P = 0.012). The allele G frequency was higher in HTG patients than in control subjects (94.6% vs. 90.3%; OR = 1.89). One haplotype consisting of rs1799724 and rs1800629 was significantly associated with HTG (P = 0.015, corrected P = 0.045). One SNP (R72P; rs1042522) in TP53 was significantly associated with NTG (P = 0.018). The allele G frequency was higher in NTG patients than in control subjects (56.1% vs. 45.8%; OR = 1.52). The significance of these associations survived the Bonferroni correction (corrected P < 0.024). Other gene variants were not significantly associated with HTG (P > 0.063) or NTG (P > 0.13). None of the studied variants was significantly associated with JOAG (P > 0.17). CONCLUSIONS: The findings suggest that variants in TNF and TP53 are risk factors for POAG, whereas variants in other studied genes are not major risk factors for POAG, at least in the Chinese population.

Lack of association of polymorphisms in elastin with pseudoexfoliation syndrome and glaucoma.

PURPOSE: To evaluate the elastin gene (ELN) as a secondary risk factor for pseudoexfoliation syndrome (PXFS) and the associated glaucoma pseudoexfoliation glaucoma (PXFG). METHODS: One hundred seventy-eight unrelated patients with PXFS, including 132 patients with PXFG, and 113 unrelated controls were recruited from the Massachusetts Eye and Ear Infirmary. All the patients and controls were white of European ancestry. Three tag SNPs (rs2071307, rs3823879, and rs3757587) that capture the majority of alleles in ELN were genotyped. Single-SNP association was analyzed using Fisher exact test. Haplotype analysis and the set-based test were used to assess the association for the whole gene. Interaction analysis was done between the ELN SNP rs2071307 and LOXL1 SNP rs2165241 using logistic regression. Multiple comparisons were corrected using the Bonferroni method. RESULTS: All 3 ELN tag SNPs were not significantly associated with PXFS and PXFG (P>0.20). The minor allele frequencies in PXFS, PXFG, and controls were 40.7%, 39.8%, and 45.6%, respectively for rs2071307, 6.7%, 6.3%, and 5.4% for rs3823879, and 14.8%, 16.2%, and 13.6% for rs3757587. Haplotype analysis and the set-based test did not find significant association of ELN with PXFS (P=0.94 and 0.99, respectively). No significant interaction effects on PXFS were identified between the ELN and LOXL1 SNPs (P=0.55). CONCLUSIONS: Our results suggest that common polymorphisms of ELN are not associated with PXFS and PXFG in white populations. Further studies are required to identify secondary genetic factors that contribute to PXFS.

Endothelial nitric oxide synthase gene variants and primary open-angle glaucoma: interactions with sex and postmenopausal hormone use.

PURPOSE: To evaluate the association between the nitric oxide synthase gene (NOS3) variants and primary open-angle glaucoma (POAG). METHODS: Two functional single-nucleotide polymorphisms (SNPs) (T-786C: rs2070744; Glu298Asp: rs1799983) and three tagging SNPs (rs7830, rs3918188, and rs1800779) were evaluated in a nested case-control study from the Nurses' Health Study (1980-2002) and the Health Professionals' Follow-up Study (1986-2002). Participants were aged >or=40 years and Caucasian. Included were 527 incident cases and 1543 controls, matched by cohort, age, and eye examination at the matched cases' diagnosis dates. Cohort-specific relative risks (RR) were estimated by using multivariable conditional logistic regression and were pooled with meta-analysis. RESULTS: No NOS3 polymorphism was significantly associated with overall POAG. For high-tension POAG (HTPOAG), rs3918188 was significantly inversely associated among the women (AA versus CC genotype: RR = 0.48; 95% CI, 0.28-0.82) but not among the men (P-heterogeneity by sex = 0.02). The minor alleles of T -786C and rs1800779 showed positive association with high-tension POAG (P-trend < 0.02) in the women only, but P-heterogeneity was not significant. In the women, four of the five NOS3 SNPs showed significant interactions with postmenopausal hormone (PMH) use in relation to HTPOAG: for example, among the women with the TT genotype in T -786C, PMH use was inversely associated (RR = 0.41; 95% CI, 0.22-0.76), but among carriers of the minor allele, use of PMH was not associated. CONCLUSIONS: Interactions were observed between NOS3 SNPs and female sex and postmenopausal hormone use in the women in relation to HTPOAG. These findings should be confirmed in different racial/ethnic groups.

AC and AG dinucleotide repeats in the PAX6 P1 promoter are associated with high myopia.

PURPOSE: The PAX6 gene, located at the reported myopia locus MYP7 on chromosome 11p13, was postulated to be associated with myopia development. This study investigated the association of PAX6 with high myopia in 379 high myopia patients and 349 controls. METHODS: High myopia patients had refractive errors of -6.00 diopters or greater and axial length longer than 26 mm. Control subjects had refractive errors less than -1.00 diopter and axial length shorter than 24 mm. The P1 promoter, all coding sequences, and adjacent splice-site regions of the PAX6 gene were screened in all study subjects by polymerase chain reaction and direct sequencing. PAX6 P1 promoter-luciferase constructs with variable AC and AG repeat lengths were prepared and transfected into human ARPE-19 cells prior to assaying for their transcriptional activities. RESULTS: No sequence alterations in the coding or splicing regions showed an association with high myopia. Two dinucleotide repeats, (AC)(m) and (AG)(n), in the P1 promoter region were found to be highly polymorphic and significantly associated with high myopia. Higher repeat numbers were observed in high myopia patients for both (AC)(m) (empirical p = 0.013) and (AG)(n) (empirical p = 0.012) dinucleotide polymorphisms, with a 1.327-fold increased risk associated with the (AG)(n) repeat (empirical p = 0.016; 95% confidence interval: 1.059-1.663). Luciferase-reporter analysis showed elevated transcription activity with increasing individual (AC)(m) and (AG)(n) and combined (AC)(m)(AG)(n) repeat lengths. CONCLUSIONS: Our results revealed an association between high myopia and AC and AG dinucleotide repeat lengths in the PAX6 P1 promoter, indicating the involvement of PAX6 in the pathogenesis of high myopia.

Different WDR36 mutation pattern in Chinese patients with primary open-angle glaucoma.

PURPOSE: To determine the distribution of WD repeat domain 36 (WDR36) sequence variants in Chinese patients with primary open-angle glaucoma (POAG). METHODS: One hundred and thirty-five unrelated POAG patients (82 high tension glaucoma [HTG], 42 normal tension glaucoma [NTG], and 11 juvenile-onset POAG [JOAG] patients) and 77 unrelated controls were recruited. All 23 coding exons and splicing junctions of WDR36 were sequenced using BigDye Terminator v3.1 cycle sequencing kit. Single nucleotide polymorphism (SNP) and haplotype associations were analyzed using PLINK (version 1.04). RESULTS: Nineteen sequence alterations were identified, and eight of them were novel including two novel nonsynonymous SNPs (L240V and I713V). Except the common I264V polymorphism, no other previously reported disease-causing or disease-susceptibility mutations were found. The novel I713V mutation was observed in three (3.7%) patients with HTG. One intronic SNP, IVS5+30C>T (rs10038177), showed significantly higher frequency of minor allele T in HTG patients (16.5%) than in controls (1.3%; Odds ratio [OR]=15.0, p=7.9 x 10(-7), Bonferroni corrected p=1.5 x 10(-5)). Haplotype GTA, which is composed of rs13153937, rs10038177, and rs11241095, was significantly associated with HTG (OR=22.5, p=0.002, Bonferroni corrected p=0.013). Neither the individual SNPs nor haplotypes of WDR36 were associated with NTG or JOAG (Bonferroni corrected p>0.05). CONCLUSIONS: Findings in this study suggest WDR36 to be associated with sporadic HTG but not with NTG or JOAG. Our results also suggest a different mutation pattern of WDR36 in the Chinese population from other ethnic populations.

Lack of association of polymorphisms in homocysteine metabolism genes with pseudoexfoliation syndrome and glaucoma.

PURPOSE: To evaluate genes involved in homocysteine metabolism as secondary risk factors for pseudoexfoliation syndrome (PXFS) and the associated glaucoma (PXFG). METHODS: One hundred eighty-six unrelated patients with PXFS, including 140 patients with PXFG and 127 unrelated control subjects were recruited from the Massachusetts Eye and Ear Infirmary. All the patients and controls were Caucasian of European ancestry. Seventeen tag SNPs from 5 genes (methylenetetrahydrofolate reductase [MTHFR], methionine synthase [MTR], methionine synthase reductase [MTRR], methylenetetrahydrofolate dehydrogenase [MTHFD1], and cystathionine beta-synthase [CBS]) were genotyped. Single-SNP association was analyzed using Fisher's exact test (unconditional) or logistic regression after conditioning on the effects of age and three LOXL1 SNPs (rs1048661, rs3825942, and rs2165241). Interaction analysis was performed between the homocysteine and LOXL1 SNPs using logistic regression. Haplotype analysis and the set-based test were used to test for association of individual genes. Multiple comparisons were corrected using the Bonferroni method. RESULTS: One SNP (rs8006686) in MTHFD1 showed a nominally significant association with PXFG (p=0.015, OR=2.23). None of the seventeen SNPs tested were significantly associated with PXFS or PXFG after correcting for multiple comparisons (Bonferroni corrected p>0.25). After controlling for the effects of age and three associated LOXL1 SNPs, none of the seventeen tested SNPs were associated with PXFS (p>0.12). No significant interaction effects on PXFS were identified between the homocysteine and LOXL1 SNPs (p>0.06). Haplotype analysis and the set-based test did not find significant association of individual genes with PXFS (p>0.23 and 0.20, respectively). CONCLUSIONS: Five genes that are critical components of the homocysteine metabolism pathway were evaluated as secondary factors for PXFS and PXFG. Our results suggest that these genes are not significant risk factors for the development of these conditions.

EDN1 Lys198Asn is associated with diabetic retinopathy in type 2 diabetes.

PURPOSE: We tested the hypothesis that genetic variants in vasoactive and angiogenic factors regulating the retina vasculature contribute to the development of diabetic retinopathy (DR). METHODS: A case-control study was performed to study the genetic association between DR and polymorphic variants of EDN1 (Lys198Asn), LTA (IVS1-80C>A, IVS1-206G>C, IVS1-252A>G), eNOS (Glu298Asp), and ITGA2 (BgI II) in a Chinese population with type 2 diabetes mellitus. A well defined population with type 2 diabetes, consisting of 127 controls and 216 DR patients, was recruited. RESULTS: A higher frequency of the Asn/Asn genotype of EDN1 was found in individuals with at least 10 years of diabetes and no retinopathy (controls) compared with DR patients with any duration of diabetes (DR: 2.3%; control: 11.0%; p=0.0002). The Asn allele was also more frequent in controls than DR patients (DR: 16.4%; control: 29.5%; p=0.007). Multiple logistic regression analysis showed that the Asn/Asn genotype was the factor most significantly associated with reduced risk of DR (odds ratio=0.19; 95% CI: 0.07-0.53; p=0.002) and with late onset of diabetes (Asn/Asn: 59 years; Lys/Lys + Lys/Asn: 53 years; p=0.02). Moreover, the Lys/Lys genotype was more common among patients with nonproliferative (75.7%) than proliferative DR (56.9%; p=0.008). The distributions of Lys198Asn alleles in hypertension did not differ from normotensive subjects. No associations between DR and polymorphisms of LTA, eNOS, or ITGA2 were detected, and there were no detectable gene-gene or gene-environmental interactions among the polymorphisms. CONCLUSIONS: The Asn/Asn genotype of EDN1 was associated with a reduced risk of DR and with delayed onset of type 2 diabetes.