clinically , primary glaucoma presents two major subtypes : primary open - angle glaucoma ( poag ) and primary angle closure glaucoma ( pacg ) . the classification relies mainly on the anterior segment anatomy , particularly that of the anterior chamber angle . pacg is characterized by obstruction of aqueous fluid drainage through the trabecular meshwork from the anterior chamber of the eye . the anterior chamber depth ( acd ) is a main factor affecting the drainage of aqueous humor . it has been reported that asian populations are at higher risk of developing pacg than other ethnic groups . eyes with pacg usually display characteristic anatomical features such as a shorter corneal diameter , a steeper corneal curvature , a shallower anterior chamber , a thicker and more anteriorly positioned lens , and a shortened eyeball , often accompanied by hyperopic refraction error . the risk factors for developing pacg include age , family history , and being female . first - degree relatives were found to have a 6- to 9-fold increased risk of developing pacg . siblings of chinese patients with primary angle closure ( pac ) or pacg have almost a 50% probability of having narrow angles and are more than 7 times more likely to have narrow angles than the general population . there is also a higher prevalence among inuits and asians compared to caucasians , suggesting a genetic predisposition for the disorder . because the ocular anatomic features are predisposing factors for pacg , genes involved in regulation of axial length and structural remodeling of connective tissues may contribute to development of pacg . some genes related to eye development or tissue remodeling including membrane frizzled - related protein ( mfrp ) [ 7 , 8 ] , extracellular matrix metalloprotease-9 ( mmp-9 ) [ 911 ] , and methylenetetrahydrofolate reductase ( mthfr )   have been reported to be associated with pacg . endothelial nitric oxide synthase ( enos ) is a nitric oxide synthase that generates nitric oxide ( no ) in blood vessels and is involved with regulating vascular tone by inhibiting smooth muscle contraction and platelet aggregation . studies also found that enos is a pressure - dependent regulator of intraocular pressure and a well - known predisposition gene for poag [ 14 , 15 ] . enos was recently reported to be associated with pacg assumed by regulating the expression of extracellular matrix metalloproteases [ 16 , 17 ] . a sequence variation in the intron 4 of enos was associated with both poag and pacg in the pakistani population . a c - t haplotype established by enosrs3793342 and enosrs11771443 may be a genetic marker for poag but not for pacg in the han chinese population . recently , enosrs3793342 was reported to be associated with pacg in the australian population but not in the nepal population . in short , the relationship of common variations in enos and pacg was inconsistent in different populations and the mechanism of enos in pacg pathogenesis is unclear . considering that both pacg and poag are characterized by apoptotic cell death of the retinal ganglion cells in the optic disc or retinal nerve fiber , we attempted to focus on pac instead of pacg for its possible association with enos variants . we also sought to investigate whether the snps of these loci are associated with ocular biometry . the study was a part of the jiangsu eye study and was conducted according to the declaration of helsinki and approved by the ethics committee of the affiliated hospital of nantong university . each participant was fully informed of the purpose and procedures involved in the study and signed the informed consent form . all participants were recruited from a population - based prevalence survey on eye diseases using a cluster random sampling strategy in funing county of jiangsu , china . of the 6032 people screened , 232 people with pac and 306 controls were enrolled in the study . pac subjects and controls were matched in groups for sex and age and were ethnically homogenous . there was no difference between the control group and the pac group in gender , age , or systemic disease distribution . all study participants were residents of funing county of jiangsu , china , aged 50 years and above . each participant received a thorough ophthalmic examination , included best - corrected visual acuity , anterior segment photography , goldmann applanation tonometry , fundus examination , optic disc photography , visual field , objective refraction , and subjective refraction . the subjects with a peripheral chamber depth less than one - third of corneal thickness were invited for gonioscope , a - scan ultrasonography , and ultrasound biomicroscopy ( ubm , sw-3200s , suoer , china ) examinations . the detailed protocol for gonioscopy and ubm was reported previously by barkana et al . . acd and axial length ( al ) were measured 3 times by a - scan to obtain mean values , and mean values of binoculus were used for statistical analyses . pac was defined according to the international society of geographical and epidemiologic ophthalmology ( isgeo ) classification by foster et al . ; it includes the following conditions : ( 1 ) either eye has the presence of an occluded angle ( at least 180 degrees of closed angle in which the trabecular meshwork is not visible on gonioscopy or iris apposition to the trabecular meshwork is more than 180 degrees on ubm ) ; ( 2 ) at least one of the following features was detected : peripheral anterior synechiae ; intraocular pressure > 21  mmhg ; excessive pigment deposition on the superior trabecular meshwork ; iris whorling ; history of symptoms ; or intraocular pressure elevated 8  mmhg after ubm examination in dark conditions ; ( 3 ) no signs of secondary angle closure were present ; ( 4 ) no signs of glaucomatous optic neuropathy and peripheral visual loss were present ; ( 5 ) no previous ocular surgery or laser therapy was present . the criteria for enrollment of the control group were ( 1 ) peripheral chamber depth more than one - third of corneal thickness ; ( 2 ) intraocular pressure less than 21  mmhg ; ( 3 ) normal optic nerve heads with cup - to - cup ratio less than 0.5 ; ( 4 ) normal visual field ; ( 5 ) no family history of glaucoma ; ( 6 ) no ophthalmic diseases except slight cataract ; and ( 7 ) refractive error of less than three diopters . genomic dna was extracted from the peripheral blood of each individual using the qiagen blood dna mini kit ( qiagen , valencia , ca ) according to the manufacturer 's instructions and stored at 20c . the samples were genotyped for rs3793342 ( in intron 4 ) and rs11771443 ( in 5-utr region ) by taqman genotyping assay ( applied biosystems , foster city , ca , usa ) using the real - time pcr 7500 system ( applied biosystems , foster city , ca , usa ) . pcrs were performed in a total volume of 10  l containing 1  l ( 10  ng ) dna , 5  l taqman universal master mixture , and 0.20  l taqman snp genotyping probes ( 40x ) . amplification was carried out with an initial denaturation at 95c for 5  min , followed by 40 cycles of denaturation at 95c for 30  s , and annealing at 60c for 30  s. statistical analysis was performed with spss version 15.0 software . differences in age and gender between pac subjects and controls were assessed using t - test and chi - square test , respectively . hardy - weinberg equilibrium was tested using chi - square test . to analyze the association of these two snps with pac and controls , the frequency of genotypes and alleles was evaluated using chi - square test . logistic regression analysis was performed to calculate the odds ratio ( or ) value and the 95% confidence interval ( 95% ci ) and to adjust the confounding effects of age and gender . three genetic models were analyzed : the additive model defined as minor allele homozygotes versus heterozygotes versus common allele homozygotes , the dominant model as heterozygotes plus minor allele homozygotes versus common allele homozygotes , and the recessive model as minor allele homozygotes versus common allele homozygotes plus heterozygotes . the association of these two snps with al , acd , and spherical power ( ds ) was also assessed under the additive genetic model , dominant model using f - test , and t - test , respectively . the call rates of the snps genotyped were 100% and the call accuracy was 100% in randomly selected 10% of samples . both snps conformed to hardy - weinberg equilibrium ( p > 0.05 ) in the pac group and in the control group . the two snps did not show differences in the distribution of allele frequency ( table 3 ) and genotypes ( table 4 ) between the cases and controls . the acd of rs11771443 aa and ga carriers is significantly deeper than that of rs11771443 gg carriers ( p = 0.0025 and p = 0.0005 for the additive model and dominant model , resp . ) . rs3793342 was not significantly associated with biometric parameters including acd , al , and ds ( table 5 ) . this study , to the best of our knowledge , is the first population - based study to investigate the association of rs11771443 and rs3793342 with pac and pac relevant biometric parameters such as acd , al , and ds in a han chinese population . the design of a population - based study can minimize sample selection bias often present in hospital - based case - control study . however , the variation of rs11771443 was associated with deeper acd that is an anatomical feature against pacg . enos gene is a stress - regulating gene and its expression is triggered when organisms are exposed to stress , hypoxia , or injury . under these unfavorable conditions , low no level may impair blood flow and related to neurodegenerative disorders , such as optic neuropathy . studies have found a decrease of nitric oxide in the plasma and aqueous humour of glaucoma patients , and a weak association of nonsynonymous snp with glaucoma patient with a history of migraine . nevertheless , an abundance of no has been found in the optic nerve head vessels of primary glaucoma patients , supporting that optic neuropathy in glaucoma may be related to enos overexpression . demonstrated that enosrs3793342 was associated with pacg and suggested dysregulation of the no system in this multifactorial optic neuropathy disease . we excluded patients with optic nerve neuropathy from this study to verify the relationship between these snps and ocular anatomic features . taking into account the result that the variation of rs11771443 was associated with deeper acd , we appraise that the influence of enos on pacg may be owing to another mechanism . the enos gene has been shown to play an important role in controlling the activity of matrix metalloproteinases . dysregulation of the no system may downregulate mmp9 expression , which has been shown to be associated with pacg . postulated that the downregulation in mmp9 activity causes deficiency in extracellular matrix remodeling during eye development and thus leads to hyperopic refractive error and shorter axial length . however , although awadalla et al . found that enosrs3793342   and mmp9rs17576   were associated with pacg , they did not investigate the relationship between these snps and al or refractive status . interestingly , mmp9rs17576 was also found to be associated with susceptibility to pacg in a taiwanese population , but there were no differences in al between the genotypes . found that mmp9rs2250889 was associated with pacg in southern china , but the patients in their study have regular al and no obvious microphthalmia . in our present study , two snps in enos were not associated with either pac or al and ds . the possible mechanism in which mmp9 and enos might contribute to pacg needs to be further studied . nathanson and mckee demonstrated an extensive no - containing cells in the human ciliary muscle ( cm ) and outflow pathway . production of no in cm could cause a relaxation of the muscle that would counteract the contractile effect of the neuronally released acetylcholine . contraction of the cm , induced by cholinergic agonists such as pilocarpine , caused forward movement of lens that resulted in shallower acd [ 30 , 31 ] . in our present study , we found that the variation of rs11771443 was associated with deeper acd but not associated with al and ds . we speculate that the variation of rs11771443 might increase no production in anterior segment endothelia , result in relaxation of ciliary muscle , and thus increase the depth of anterior chamber . a singapore study reported that lens vault and posterior corneal arc length were responsible for approximately 75% of the variation of acd , while axial length was a poor determinant of acd . in summary , our data indicates that enos rs11771443 is associated with acd and its role in the pathogenesis of pacg warranted further study .
recently , a study reported that single nucleotide polymorphisms ( snp ) in endothelial nitric oxide synthase ( enos ) were associated with primary angle closure glaucoma ( pacg ) in australian cohort . in this study , we aimed to investigate whether those enos snps are associated with primary angle closure ( pac ) or ocular biometric characteristics such as axial length ( al ) , anterior chamber depth ( acd ) , and diopter of spherical power ( ds ) in han chinese . 
 the samples consisted of 232 pac subjects and 306 controls collected from a population - based prevalence survey conducted in funing county of jiangsu , china . 
 the rs3793342 and rs11771443 in enos were genotyped by taqman - mgb probe using the rt - pcr system . 
 our data did not identify any association of the enos snps with pac . 
 however , the analysis on the quantitative traits of ocular biometrics showed that the acd of rs11771443 aa and ga carriers is significantly deeper than that of rs11771443 gg carriers ( p = 0.0025 ) , even though the al and ds are not associated with rs11771443 genotypes . 
 rs3793342 was not associated with any biometric parameters including acd , al and ds . in summary , 
 our data indicates that enos rs11771443 is associated with acd and its role in the pathogenesis of pacg warranted further study .