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Purpose: Despite the different etiology of primary open angle glaucoma (POAG), primary angle closure glaucoma (PACG), and pseudoexfoliative glaucoma (PEXG), several studies have suggested that these forms of glaucoma have overlapping genetic risk factors. Therefore, the aim of this study was to evaluate the role of genetic variants recently associated with POAG in different types of glaucoma in Pakistani POAG, PACG, and PEXG patient cohorts., Methods: Six variants in CDKN2B-AS1 (rs4977756), CDKN2B (rs1063192), ATOH7 (rs1900004), CAV1 (rs4236601), TMCO1 (rs4656461), and SIX1 (rs10483727) were genotyped using TaqMan assays. A total of 513 unrelated patients with glaucoma (268 with POAG, 125 with PACG, and 120 with PEXG) and 233 healthy controls were included in the study. Genotypic and allelic associations were analyzed with a chi-square test., Results: The frequency of the G allele of TMCO1 rs4656461 was significantly lower in the patients with POAG (p=0.003; OR [odds ratio]=0.57), PACG (p=0.009; OR=0.52), and PEXG (p=0.01; OR=0.54) compared to the control individuals. The T allele of ATOH7 rs1900004 was observed less frequently in the patients with PACG (p=0.03; OR=0.69) compared to the control individuals. The A allele of CAV1 rs4236601 was found more frequently in the patients with POAG (p=0.008; OR=1.49) compared to the control individuals. This study demonstrates that the TMCO1 rs4656461 variant is associated with POAG, PACG and PEXG in the Pakistani population. Our study was unable to confirm previous associations reported for variants in CDKN2B-AS1, CDKN2B, and SIX1 with any type of glaucoma., Conclusions: In conclusion, we found consistent evidence of the significant association of three common variants in TMCO1, ATOH7, and CAV1.

Purpose: Matrix metalloproteinases (MMPs) play an important role in remodeling of the extracellular matrix during development and growth of various tissues including the eye. Various functional polymorphisms in MMPs have been implicated in the pathogenesis of different types of glaucoma. The aim of the present study was to investigate the role of various polymorphisms in Pakistani patients with glaucoma., Methods: The present case-control study included 112 patients with primary open-angle glaucoma (POAG), 82 patients with primary angle closure glaucoma (PACG), and 118 control subjects. Genotyping of polymorphisms was done using PCR followed by restriction fragment length polymorphism analysis., Results: A significant difference in the genotype frequencies of MMP1 rs1799750 (-1607 1G/2G) was observed between the patients with POAG and the control subjects (p = 0.001). This was attributed to the female subjects (p < 0.001), while the association was not significant in male subjects (p > 0.47). In addition, a significant difference was observed in genotype frequencies of MMP9 rs17576 (c.836A>G) in patients with PACG compared to the control subjects (p < 0.001), which after gender stratification remained significant in men (p = 0.009) but not in women (p = 0.14). No significant associations were found for MMP7 (c.-181T>C) and MMP9 (c.-1562C>T) polymorphisms., Conclusions: Our data suggest that the MMP1 rs1799750 (-1607 1G/2G) and MMP9 rs17576 polymorphisms might be of value for further study as potential gender-dependent risk factors for developing POAG and PACG, respectively, in Pakistan.

Purpose: Single nucleotide polymorphisms (SNPs) rs1048661 (p.R141L) and rs3825942 (p.G153D) in the lysyl oxidase-like 1 (LOXL1) gene have been previously reported to be associated with pseudoexfoliation glaucoma (PEXG) in various Asian and European populations, but these SNPs have not yet been studied in the Pakistani population. Therefore the aim of the present study was to investigate the association of these two coding LOXL1 SNPs in Pakistani PEXG patients., Methods: One hundred twenty-eight Pakistani patients diagnosed with PEXG and 180 healthy controls were recruited for the study. Genomic DNA was extracted and both SNPs were genotyped by direct sequencing. Association of genotype and allele frequencies with PEXG were analyzed using the Chi-square (χ(2)) test., Results: Genotype and allele frequencies of both rs1048661 and rs3825942 were found to be significantly associated with PEXG. The GG genotypes of both LOXL1 SNPs were associated with an increased risk of developing PEXG. In addition the G alleles of rs1048661 and rs3825942 confer an increased risk for PEXG with an odds ratio (OR) of 2.98 (95% CI 1.94-4.57) and OR 6.83 (95% CI 2.94-16.67), respectively., Conclusions: A significant association was found for the G allele of rs1048661 and rs3825942 in PEXG patients of Pakistani origin.

Purpose: To describe a novel mutation in the fibrillin-1 (FBN1) gene in a large Pakistani family with autosomal dominant Marfan syndrome (MFS)., Methods: Blood samples were collected of 11 family members affected with Marfan syndrome, and DNA was isolated by phenol-extraction. The coding exons of FBN1 were analyzed by polymerase chain reaction (PCR) and direct sequencing. One hundred-thirty controls were screened for a mutation in the FBN1 gene that was identified in this family by restriction fragment length polymorphism (RFLP) analysis., Results: A novel heterozygous missense mutation c.2368T>A; p.Cys790Ser was observed in exon 19. This mutation substitutes a highly conserved cysteine residue by serine in a calcium binding epidermal growth factor-like domain (cbEGF) of FBN1. This mutation was present in all affected members and absent from unaffected individuals of the family in addition to 130 healthy Pakistani controls. Interestingly all affected family members presented with ectopia lentis, myopia and glaucoma, but lacked the cardinal cardiovascular features of MFS., Conclusions: This is a first report of a mutation in FBN1 in MFS patients of Pakistani origin. The identification of a FBN1 mutation in this family confirms the diagnosis of MFS patients and expands the worldwide spectrum of FBN1 mutations.

Purpose: The present study was designed to determine the association of polymorphisms of the DNA repair genes X-ray cross-complementing group 1 (XRCC1) (c.1316G>A [rs25487]) and xeroderma pigmentosum complementation group D (XPD) (c.2298A>C [rs13181]) with primary open-angle glaucoma (POAG) and primary closed-angle glaucoma (PCAG)., Methods: In this prospective case-control study, polymerase chain reaction-restriction fragment length polymorphism analysis was used to study the association of XRCC1 and XPD with 160 POAG patients, 163 PCAG patients, and 193 unaffected controls., Results: XRCC1 rs25487 was found to be significantly associated specifically with male POAG patients (χ(2) = 13.2 [p = 0.001]), only for the dominant model (odds ratio [OR] = 2.65 [95% confidence interval [CI] = 1.44-4.85], p < 0.005). In addition XPD rs13181 was also found to be associated with male POAG patients (χ(2) = 12.1 [p < 0.005]), for both dominant (OR = 2.44 [95% CI = 1.33-4.47], p < 0.005) as well as recessive model (OR = 3.62 [95% CI = 1.45-9.01], p < 0.01). Combined genotypes of both the genes revealed that the heterozygote AC/GA was significantly associated with the male POAG patients (z = 3.00 [p < 0.001]). The AA/GG genotype was present at a higher frequency in the male controls and the AA/GA in the female controls and could thus have a protective role in males and females, respectively., Conclusions: We postulate that defects in the DNA repair genes XRCC1 and XPD may possibly be associated with the progression of POAG in male patients of Pakistani origin.

Purpose: The aim of the present study was to investigate the association of glutathione S-transferase GSTT1 and GSTM1 genotypes with pseudoexfoliative glaucoma (PEXG) in a group of Pakistani patients., Methods: Multiplex polymerase chain reaction was used to study the GSTT1 and GSTM1 polymorphisms in 165 PEXG patients and 162 unaffected controls., Results: In the current study we describe a significant gender-specific association of GSTT1 and GSTM1 null genotypes with PEXG. The three null genotype combinations (i.e., T1M0, T0M1, and T0M0) were found at significantly higher frequencies in the PEXG patients as compared to the controls (χ(2)=21.82, p<0.001). This association was specifically related to the female patients (χ(2)=35.63, p<0.001); no such association was seen in the male patients (χ(2)=2.28, p>0.05)., Conclusions: The results suggest that there is a significant involvement of the GSTT1 and GSTM1 polymorphisms in female Pakistani patients having PEXG, which suggests a possible gender-specific impairment of detoxification in this group.

Purpose: To investigate the involvement of stress-regulating genes, endothelial nitric oxide synthase (eNOS) and heat shock protein 70 (HSP70) with primary open angle glaucoma (POAG) and primary closed angle glaucoma (PCAG)., Methods: POAG and PCAG patients recruited from different areas of Pakistan were diagnosed on the basis of clinical history, raised intraocular pressure (IOP), cup-to-disc ratio (CDR) and visual field defects. Their blood was collected and genomic DNA was extracted from it, followed by PCR amplification and VNTR typing of the eNOS gene, while the HSP70 SNP was analyzed with PCR-RFLP. For both of the polymorphisms, the genotype distribution of the POAG and PCAG patients was compared with unaffected controls., Results: HSP70 polymorphism was found to be significantly associated with PCAG (chi(2)=15.29 [p<0.001], OR=2.63 [95% CI=1.55-4.48]), with p<0.001 for the dominant model and OR=2.09 (95% CI=1.10-3.96) , with p<0.01 for the recessive model, but not with POAG (chi(2)=2.96 [p>0.05]). As opposed to this significant eNOS association, was seen with PCAG (chi(2)=6.33 [p<0.05], OR=2.09 [95% CI=1.12-3.89]), with p<0.01 for the dominant model, as well as with POAG (chi(2)=8.89 [p<0.05], OR=2.23 [95% CI=1.26-3.39]), with p<0.01 for dominant model. For the eNOS case, we found a significant association with the risk allele "a" for POAG patients (chi(2)=9.29 [p<0.01], OR=2.02 [95% CI=1.25-3.28, p=0.001]) and PCAG patients (chi(2)=7.59 [p<0.01], OR=1.99 [95% CI=1.18-3.37, p<0.01]). Similarly, in the HSP70 case, there was a significant association with the risk allele "C" for POAG patients (chi(2)=3.57 [p=0.05], OR=1.38 [95% CI=0.97-1.94, p<0.05]) and PCAG patients (chi(2)=18.32 (p<0.001), OR=2.16 [95% CI=1.49-3.13, p<0.001])., Conclusions: The intron 4 polymorphism of eNOS is associated with POAG, as well as PCAG, while the G+190C polymorphism in HSP70 is associated with PCAG, but not with POAG in the Pakistani population.

Purpose: The purpose of the present study was to determine the role of the tumor necrosis factor alpha (TNF-alpha) gene polymorphism G-308A and total serum immunoglobulin E (TsIgE) levels in the onset of pseudoexfoliation glaucoma (PEXG) in Pakistani patients., Methods: The TNF-alpha polymorphism G-308A was analyzed in 122 patients with PEXG and 126 healthy unrelated controls by using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). TsIgE levels were determined by solid-phase enzyme-linked immunosorbent assay (ELISA)., Results: The AA and GA genotypes were strongly associated with PEXG (p<0.001), with an odds ratio (OR) of 0.07 (95% confidence interval [CI]=0.02-0.27) and 0.24 (95% CI=0.12-0.51), respectively, while the GG genotype was found at a higher frequency in controls as compared to patients (p<0.001) OR=8.95 (95% CI=4.55-17.81). No significant difference was found in TsIgE levels of both patients and controls (p=0.86)., Conclusion: The present study concludes that the TNF-alpha polymorphism G-308A is strongly associated with PEXG. To our knowledge this is the first study in southeast Asia which demonstrates a strong association of a TNF-alpha polymorphism with PEXG.

Purpose: To investigate the methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C genotypes and plasma concentrations of total homocysteine (tHcy) in Pakistani patients with primary open angle glaucoma (POAG) and primary closed angle glaucoma (PCAG)., Methods: This was a prospective case-control study. A total of 295 patients (173 POAG, 122 PCAG) and 143 age- and sex-matched controls were subdivided into two ethnic groups, Punjabis (Punjab province, central Pakistan) and Pathans (North-West Frontier Province, northern Pakistan). Genotypes of the MTHFR C677T and A1298C polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). An enzyme-linked immunosorbent assay was used to determine the total serum homocysteine (tHcy) levels. Associations were determined by logistic regression analysis., Results: Frequency distributions of genotypes and combined genotypes as well as homocysteine levels were obtained. The overall distribution of the C677T genotype was found to be significantly associated with PCAG (CC 69%, CT 21%, TT 10%; p=0.001, chi(2)=12.6), but not with POAG (CC 71%, CT 28%, TT 1%; p=0.98, chi(2)=0.02) as compared to the controls (CC 71%, CT 29%, TT 1%). The Pathan cohorts revealed no association with the disease; however, the Punjabis demonstrated a significant association with PCAG (CC 75%, CT 11%, TT 13%; p<0.001, chi(2)=17.2). PCAG in the Punjabi subjects was also significantly associated with the A1298C polymorphism (AA 43%, AC 54%, CC 3%; p<0.001, chi(2)=33.9) as compared to the controls. Combined genotype data showed no association with POAG; however, a significant association with all combined genotypes was observed in the overall PCAG subjects (p<0.05, chi(2)=20.1). This difference was particularly apparent in the TTAA and TTAC combinations that were completely absent in the control groups (p<0.05. chi(2)=49.6). Mean serum tHcy levels were found to be significantly increased in the POAG (15.2+/-1.28 micromol/l, p<0.001) and PCAG (20.8+/-4.8 micromol/l) groups as compared to the controls (10.0+/-0.97 micromol/l). The tHcy levels in the TT and AC genotype were significantly elevated in the PCAG group (67+/-12.39 micromol/l, p<0.001; 23+/-5.94 micromol/l, p=0.027) as compared to the controls., Conclusion: The TT and AC genotypes of MTHFR C677T and A1298C polymorphisms and the combined genotype TTAC were associated with PCAG in Punjabi subjects of Pakistani origin and correlated with the high serum tHcy levels seen in these patients.

Purpose: To determine whether or not there is an association of the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism with disease in cohorts of primary open-angle glaucoma (POAG) and primary closed-angle glaucoma (PCAG) from Pakistan., Methods: This was a prospective study consisting of 150 patients (90 POAG and 60 PCAG) and 70 control subjects. Genomic DNA was extracted from leukocytes of the peripheral blood. MTHFR C677T polymorphism analysis was performed by the polymerase chain reaction-restriction fragment length polymorphism (RFLP) technique., Results: The prevalence of the MTHFR C/T genotype was 22.2% in POAG, 13.3% in PACG, and 18.6% in controls whereas the MTHFR T/T genotype was present solely in the PACG group (6.9%). The difference regarding the T/T genotype between PACG and controls was statistically significant (p<0.01)., Conclusions: The MTHFR C677T polymorphism was found to be associated with PCAG but not POAG in patients of Pakistani origin.