In this nested case-control, we observed that the associations with NOS3 SNPs in relation to POAG depended on age at menarche and parity, where those associations were most evident among those with greater estrogen exposure (age at menarche <13 years and parity of 3 or more children versus 1–2 children). Interactions were not observed with oral contraceptives in relation to POAG overall. Because this is the first study to examine the interaction between NOS3 SNPS and female reproductive factors, these results need to be confirmed in other studies.
These findings should be interpreted in the context of previous cohort studies describing the relation between reproductive factors and POAG [24
]. For age at menarche, in the NHS, we previously found that menarche >13 years was associated with increased risk of POAG with IOP≤21 mmHg at diagnosis [31
], which was consistent with the finding from the Blue Mountain Eye Study that reported that later age at menarche was associated with an increased risk of OAG [32
]. These data suggest that increased lifetime estrogen exposure during the reproductive years, as reflected in earlier menarche, is associated with POAG. Among the NOS3
SNPs, the functional consequences of the rs3918188
tagging SNP are unknown; however, we previously found that the minor allele (A) was associated with a reduced risk of POAG in women (p-trend=0.04) but not men (p-trend=0.58). We observed a significant interaction between the minor allelic variant of the rs3918188
tagging SNP and age at menarche in POAG: women with homozygosity for the minor allele and age at menarche <13 years had a 69% reduction in POAG risk compared to those with homozygosity for the wild-type allele and older age at menarche. Biologically, this is plausible as increased age at menarche may be related to reduced NOS3 activity [33
], which may also reduce endothelial cell relaxation in the trabecular meshwork cells and in the vascular endothelium that supply retinal ganglion cells. In addition, we previously observed significant interactions between this same SNP and current PMH use in POAG with IOP>21 mmHg at diagnosis: postmenopausal women with homozygosity for the minor allele and current users of PMH had a 67% reduced risk of high tension POAG.
We also observed that the association between the functional Glu298Asp SNP and POAG was significantly modified by parity. For parity, in the NHS, we reported a null association with POAG [31
], although the Blue Mountain Eye Study observed that increasing parity was associated with an increased risk of OAG [33
]. For the functional Glu298Asp SNP, which is known to reduce intercellular nitric oxide levels [34
], we previously did not find an association with POAG in women or in men, nor any interactions with age at menopause or PMH use. Here, we observed a “cross-over” interaction where the wild type genotype was associated with increased POAG risk for women with parity <3, while the dysfunctional genotype was associated with increased risk for women with parity 3+. Multiparity is known to produce endothelial dysfunction through reduced nitric oxide bioavailability [36
], thus, it is plausible that multiparity-induced endothelial dysfunction may be exacerbated in homozygotes of the dysfunctional NOS3 variant. The adverse association with the common SNP among those with lower parity is relative to women of the same genotype who had more children. Perhaps in the latter instance lower circulating estrogen milieu in women with low parity may be more important than multiparity-induced endothelial dysfunction in dictating POAG risk. We have previously reported a “cross-over” type interaction for the functional T-786C SNP and hypertension and POAG [37
]. It remains to be determined whether crossover interactions have real pathophysiological significance.
The associations between the NOS3
SNPs and reproductive factors that reflect exposure to estrogen, which potently modulates NOS3
activity, provides further support for the important role of endothelial cell dysfunction in POAG etiology. Such dysfunction in the trabecular meshwork and ocular vascular endothelium may lead to increased IOP and retinal ganglion cell vulnerability, resulting in glaucomatous visual loss. Recent emerging data from animal models, studies of POAG genetics and clinical trials of agents related to vascular tone also support this hypothesis. In a mouse model, there is evidence that knocking out soluble guanylate cyclase, the downstream mediator of nitric oxide, produces modest increases in IOP and considerable loss of retinal ganglion cells and nerve fiber layer dropout [38
]. In an Icelandic genome-wide association study of POAG, gene variants in the intergenic region between caveolin 1 and caveolin 2, which code for caveolins that reside near NOS3 in endothelial cell membranes and serve to mediate endothelial tone were associated with POAG [39
]. A randomized clinical trial found that topical brimonidine, an alpha 2 agonist with vasomotor activity at the level of the retinal vascular endothelial cells, was superior to timolol, in preserving vision in the normal tension variant of POAG [40
]. Other studies of statins, which also improve endothelial cell function, have found that they may favorably alter the course of POAG [44
]. Thus, our data, in combination with emerging data from laboratory as well as human studies, coherently implicate endothelial cell dysfunction in POAG pathogenesis.
There are limitations to this work that should be considered. First, our glaucoma definition was based on self-report with confirmation with medical records and visual fields. This definition has very high specificity, as we required documentation of reproducible defect on at least 2 reliable visual field tests and we have demonstrated strong associations with established risk factors, such as African-American heritage and family history [28
]. Given the insidiousness of glaucoma, some controls might have had undiagnosed glaucoma. However, it is unlikely to have had a major influence on our results, as the prevalence of glaucoma in adults over age 40 is 1.3% in Caucasians [46
]. Furthermore, our controls had an eye exam as of the matched cases’ diagnosis dates. In fact, the average number of eye exams reported as of their selection as controls were three exams, implying that manifest glaucoma, if present, would likely have been detected. Any misclassifications of the disease would have biased the results toward the null. Third, our participants were generally healthy Caucasians and thus we are unable to make inferences to less healthy populations or minorities. Finally, it is possible that our results may be due to chance, despite the fact we showed statistical significance after accounting for multiple comparisons. Therefore, these findings should be interpreted with caution and confirmed in future studies, particularly with different racial/ethnic groups.
In conclusion, the associations between NOS3 SNPs and POAG depended on parity and age at menarche. Previously we showed the associations between NOS3 SNPs and POAG depended on PMH use. These data indicate that NOS3 gene variants interact with sex hormone levels in ways to influence the risk of POAG. In the future we hope to validate these interactions in animal model systems.