Association of eNOS and HSP70 gene polymorphisms with glaucoma in Pakistani cohorts

Abstract

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.

Figure 1

Amplification product of the 27-bp insertional variable number of tandem repeat polymorphism in intron 4 of eNOS. The amplified products were separated by electrophoresis on 4% agarose gel. Lane L, 100-bp DNA ladder; lane NC, negative control (no template DNA); lane PC, positive control; lane 1, 2 and 5, homozygous eNOSb/b genotype (420 bp fragment); lane 3, heterozygous, eNOSa/b genotype (420-bp and 393-bp fragments); and lane 4, homozygous a/a genotype (393-bp fragment).

Figure 2

BrsB1 restriction enzyme-digested, PCR-amplified product of HSP70 G+190C polymorphism (rs1043618). The digested products were separated by electrophoresis on 4% agarose gel. Lane L, 100-bp DNA ladder; lanes 1, 2 and 4, homozygous wild-type G allele (461 bp and 27 bp [not shown]); and lane 3 and 5, homozygous C risk allele (488 bp).