Endothelial Nitric Oxide Synthase Gene Variants and
Primary Open-Angle Glaucoma: Interactions with Sex
and Postmenopausal Hormone Use
Jae Hee Kang,1,2Janey L. Wiggs,2,3Bernard A. Rosner,1,4Susan E. Hankinson,1,5
Wael Abdrabou,3Bao Jian Fan,3Jonathan Haines,6and Louis R. Pasquale3
PURPOSE. To evaluate the association between the nitric oxide
synthase gene (NOS3) variants and primary open-angle glau-
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’ Fol-
low-up Study (1986–2002). Participants were aged ?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 inter-
actions 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. (Invest Ophthal-
mol Vis Sci. 2010;51:971–979) DOI:10.1167/iovs.09-4266
q24.3),1macrophage-derived isoforms encoded by genes on
chromosome 17 (NOS2A, -2B, and -2C),2and an endothelium-
derived isoform encoded by NOS3 (7q36).3Endothelial NOS is
constitutively expressed in the human outflow pathway and
ciliary muscle where it may modulate outflow facility.4It is also
expressed on all vascular endothelial cells, including those in
the optic nerve pedicle.5Nitric oxide (NO), formed by NOS in
endothelial cells, mediates vasodilation in response to acetyl-
choline and thus is important in mediating vascular tone and
modulating blood flow to the optic nerve.6
Endothelial dysfunction may play a key role in POAG patho-
genesis, and it can be detected in patients with early disease.7,8
Patients with POAG demonstrate abnormal vascular regulation
in various ocular tissue beds7–14and abnormal brachial artery
vasodilation in response to acetylcholine.8,15Also, an interven-
tional study using an NOS inhibitor found differences in ocular
blood flow response between patients with POAG and con-
trols, implicating the L-arginine/nitric oxide system.16Thus,
alterations in NOS3 activity produced either by genetic varia-
tion or by environmental influences could play a role in the
pathogenesis of glaucoma.
Recent genetic studies of NOS3 also support the involvement
of endothelial NOS expression and NO synthesis. Polymorphisms
in NOS3 or in the 5? upstream untranslated region have been
associated with high-tension POAG17and POAG with migraine.18
Studies to date have been relatively small (?200 cases) and their
results have been inconsistent. In addition, researchers have not
examined interactions between NOS3 and environment factors in
POAG. For example, estrogen is known to upregulate NOS3,19
and later age at menopause20,21and the use of postmenopausal
hormones (PMHs) is inversely associated with the risk of POAG.22
However, no investigators have evaluated the interaction be-
tween the individual’s sex, reproductive aging, and NOS3 in
relation to POAG.
Therefore, we evaluated the association between five single-
nucleotide polymorphisms (SNPs) of NOS3 and POAG in a
matched, nested case–control study with 527 cases and 1543
control subjects selected from participants in the Nurses’ Health
Study and Health Professionals’ Follow-up Study. We also exam-
ined whether there were interactions with age at menopause and
PMH use and POAG among the postmenopausal women.
itric oxide synthase (NOS) has three genetically distinct
isoforms: neuronal NOS encoded by NOS1 (12q24-
We conducted a nested case–control study within the Nurses’ Health
Study (NHS) and Health Professionals Follow-up Study (HPFS) cohorts.
From the1Channing Laboratory, Department of Medicine, Har-
vard Medical School and Brigham & Women’s Hospital, Boston, Mas-
School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts;
the Departments of4Biostatistics and5Epidemiology, Harvard School
of Public Health, Boston, Massachusetts; and the6Center for Human
Genetics Research, Vanderbilt University School of Medicine, Nash-
Presented at the annual meeting of the Association for Research in
Vision and Ophthalmology, Fort Lauderdale, Florida, May 2009.
2Contributed equally to the work and therefore should be consid-
ered equivalent authors.
Submitted for publication July 3, 2009; revised August 27, 2009;
accepted September 14, 2009.
Disclosure: J.H. Kang, None; J.L. Wiggs, None; B.A. Rosner,
None; S.E. Hankinson, None; W. Abdrabou, None; B.J. Fan, None;
J. Haines, None; L.R. Pasquale, None
Corresponding author: Louis R. Pasquale, Massachusetts Eye
and Ear Infirmary, 243 Charles Street, Boston, MA 02114;
3Department of Ophthalmology, Harvard Medical
Investigative Ophthalmology & Visual Science, February 2010, Vol. 51, No. 2
Copyright © Association for Research in Vision and Ophthalmology
The NHS was established in the United States in 1976, when 121,700
registered nurses between the ages of 30 and 55 years returned a
questionnaire on health-related exposures.23The HPFS started in 1986
with 51,529 U.S. male health professionals aged 40 to 75 years who
responded to a similar mailed health questionnaire. Participants were
followed up with biennial questionnaires to update information on
lifestyle factors and newly diagnosed illnesses, such as glaucoma.24
Follow-up rates were high (?95% of the total possible person time
through 2002). The Human Research Committees of Brigham & Wom-
en’s Hospital, Massachusetts Eye and Ear Infirmary, and the Harvard
School of Public Health approved the study. Our research adhered to
the tenets of the Declaration of Helsinki.
Blood and Buccal Sample Collection
Blood samples were collected from 32,826 (27%) women between
1989 and 1990 and from 18,225 (35%) men between 1993 and 1995.
Buccal cell samples were collected between 2001 and 2004 from
29,684 of the women who had not provided a blood sample. Follow-up
was ?95% in both of these subcohorts.
Blood samples, collected with heparin sodium used as an antico-
agulant, were returned within 26 hours of being obtained, immediately
centrifuged; aliquoted into plasma, red blood cells, and buffy coat
components; and stored in liquid nitrogen freezers. All buccal cell
samples were collected in a single “swish-and-spit” procedure in which
subjects were provided a small bottle of mouthwash and a small cup
with a screw-on cap and were asked to swish the mouthwash and then
spit into the cup.25Samples were processed within a week of receipt.
Case and Control Ascertainment
We ascertained POAG cases biennially, in a three-step process. First, in
each questionnaire, we asked whether eye examinations had been
performed and whether a diagnosis of glaucoma had been given.
Second, we sought permission to retrieve the ophthalmic records of
participants who reported glaucoma. We contacted the diagnosing
physician for copies of all visual field (VF) tests to date and for the
completion of a glaucoma questionnaire regarding maximum IOP, the
status of the filtration apparatus, optic nerve structural information,
prior ophthalmic surgery, and any VF loss. We allowed physicians to
send relevant medical records in lieu of completing the questionnaire.
To determine case status, a glaucoma specialist (LRP) evaluated all the
ophthalmic information from questionnaires/medical records and VF
results in a standardized manner.
Only participants with “definite” or “probable” POAG were
included. From participants with definite POAG, we required doc-
umentation of gonioscopy showing that angles were not occludable
in either eye, slit lamp biomicroscopy showing no indication in
either eye of pigment dispersion syndrome, uveitis, exfoliation
syndrome, trauma, or rubeosis, and two or more reliable VF results
showing reproducible defects that were consistent with glaucoma.
For probable POAG cases, the slit lamp examination and VF criteria
were also required, but documentation of pupil dilation without
subsequent adverse events was accepted instead of gonioscopy.
Among cases, ?70% met the criteria for definite POAG. For VF
results, there was no requirement regarding the type of perimetry
performed; however, in 95% of cases, full static threshold testing
was completed, and in ?1% of cases, kinetic VF tests were used. For
static threshold or suprathreshold testing, we considered the VF
result to be reliable if the fixation loss rate was ?33%, the false-
positive rate was ?20%, and the false-negative rate was ?20%. For
kinetic VFs, we consider the results reliable unless there was nota-
tion by the examiner to the contrary.
We included 527 glaucoma cases and 1543 control subjects (373
NHS cases and 1082 controls; 154 HPFS cases and 461 controls), aged
?40 years and Caucasian (?20 cases were Latino). The control sub-
jects were matched on sex, type of DNA sample (blood or cheek cell),
year of birth and ethnicity (Latino or not), and they were required to
have had an eye examination at the same period as the diagnosis date
of the matched case. Approximately three control subjects were
matched to each case, by using incidence density sampling.
Two functional single-nucleotide polymorphisms (SNPs) (T ?786C:
rs2070744, and Glu298Asp: rs1799983) and three tagging SNPs
(rs1800779, rs3918188, and rs7830) were genotyped. The tagging
SNPs corresponded to the three NOS3 linkage disequilibrium (LD)
blocks (Fig. 1) and were selected by using Haploview (ver. 4.1) ac-
cording to the HapMap data (release 22) from the CEU population.26
The minimum minor allele frequency for checking markers was set to
0.01. Three tagging SNPs (rs1800779, rs3918188, and rs7830) were
selected to capture the majority (88%) of alleles at r2? 0.8 across the
whole gene, including the 5? and 3? untranslated regions.
For DNA extraction, 50 ?L of buffy coat or 20 ?L of buccal cells
were diluted with 150 ?L of PBS and processed (QIAmp 96 spin
blood kit protocol; Qiagen, Inc., Chatsworth, CA). A quantitative
PCR approach (TaqMan Assay; Applied Biosystems, Inc. [ABI], Fos-
ter City, CA) was used for genotyping, according to the manufac-
turer’s instructions. The RT-PCR amplification of genomic DNA was
performed in 96-well plates with a sequence-detection system
(Prism 7000; ABI). The thermal cycler (model 2720; ABI) was set at
the following parameters: 50°C for 2 minutes, 95°C for 10 minutes,
92°C for 15 seconds, and 58°C for 1 minute, for 60 cycles. The
genotyping success rate was ?90% for all five SNPs included in the
study. Plates that passed quality control measures (including Hardy-
Weinberg equilibrium tests) were included, and in 5% of samples
that underwent repeat genotyping, there was ?95% concordance
on genotyping calls.
Assessment of Menopausal Status, Age at
Menopause, and Postmenopausal Hormone Use
Starting in 1976, we asked NHS participants to update the information
on their reproductive histories. We asked participants if they had
entered menopause, the age of onset, and the type of menopause
experienced (natural, radiation-induced, or surgical). Beginning in
1980, for surgically induced menopause, we inquired about the nature
of the surgery (whether one or both ovaries were removed and
whether the uterus was also removed). We also asked participants
whether they had taken PMHs, and if so, for how long and what type
of hormone was used (unopposed oral-conjugated estrogen, estrogen
with progesterone, and other estrogens). In validation studies, the
self-reported information on reproductive history given by the NHS
participants has been found to be highly accurate.27–30
We analyzed the cohort-specific data separately with conditional logis-
tic regression, adjusting for potential confounders. Then, we pooled
the results using meta-analytic methods, incorporating random ef-
fects.31P ? 0.05 was considered statistically significant (SAS, ver.
9.1.3; SAS, Cary, NC).
Information on potential confounders was obtained from the bien-
nial questionnaires and was updated through the questionnaire com-
pleted immediately before the date of the diagnosis of the index case.
Potential confounders were family history of glaucoma, body mass
index (?22, 22–23.9, 24–25.9, 26–27.9, 28–29.9, or ?30 kg/m2),
smoking status (current, past, or never), physical activity (quartiles of
activity intensity/day), self-reported history (yes/no) of hypertension
and diabetes, cumulatively updated alcohol intake (0, 1–9, 10–19,
20–20, or ?30 g/d), and cumulatively updated caffeine intake (0–149,
150–299, 300–449, 450–600, or ?600 g/d).
In secondary analyses, we separately analyzed the risk of high-tension
POAG, defined as maximum IOP ? 22 mm Hg before visual field loss
972Kang et al.
IOVS, February 2010, Vol. 51, No. 2
(67.5% of all POAG cases), and normal-tension POAG, defined as those
with maximum IOP ? 22 mm Hg before visual field loss. Polytomous
logistic regression was used to test whether an individual SNP was related
differently to high-tension compared with normal-tension glaucoma.32
We also conducted haplotype analyses using genotyping data from
all five SNPs. Haplotype frequencies of the NOS3 gene were estimated
(PROC Haplotype; SAS ver. 9.1.3; SAS).
Effect Modification with Age at Menopause and
Among the postmenopausal women only, we evaluated interactions
between the NOS3 SNPs and age at menopause (?54 years vs. ?54
years; 54 years was the median age at menopause) and between NOS3
SNPs and the use of PMHs (current use versus nonuse).
1 A T G A** C 30.3 32.2
2 G C T C C 23.6 23.6
3 G C G C A 15.7 15.4
4 A T G C A 13.1 13.8
5 A T T C C 9.0 7.4
6 A T G C C 8.3 7.6
* Likelihood ratio test of haplotype effect for overall POAG: NHS, p=0.28; HPFS, p=0.80; for high tension POAG: NHS, p=0.004;
HPFS, p=0.68; for normal tension POAG: NHS, p=0.28; HPFS, p=0.02.
** Bold denotes variant
† P-heterogeneity between men and women was significant: p=0.007. In the NHS, the RR = 2.10 (95% CI=1.28, 3.46), and in the
HPFS, the RR = 0.55 (95% CI, 0.24, 1.26)
analyses of NOS3 gene polymorphisms.
Linkage disequilibrium between NOS3 SNPs using the Haploview software (version 4.1)26expressed in terms of r2and haplotype
IOVS, February 2010, Vol. 51, No. 2
Nitric Oxide Synthase Gene Variants and Glaucoma 973
To evaluate effect modification, we tested the significance of the
pooled estimates of the interaction terms from the multivariate condi-
tional logistic regression models.
The POAG cases and matched control subjects were similar in
their characteristics as of the index case’s date of diagnosis
(Table 1). All cases and controls were Caucasian and matched
for age. As expected, cases had a higher frequency of a family
history of glaucoma. In addition, they had a higher frequency
of diabetes; however, they were somewhat less likely to be
obese, drink alcohol, or smoke. Among the women, the cases
were less likely to be current PMH users.
Association with NOS3 SNPs
The five NOS3 polymorphisms were not associated with over-
all POAG (Table 2), and the associations were not significantly
different between the men and the women. For example, for
the T ?786C polymorphism, compared with the TT genotype,
the pooled RR was 1.05 (95% CI, 0.83–1.34) for the CT geno-
type and 1.00 (95% CI, 0.64–1.55) for the CC genotype. Also,
for the Glu298Asp polymorphism, compared with the GG
genotype, the pooled RR was 1.04 (95% CI, 0.82–1.32) for GT
and 1.33 (95% CI, 0.94–1.86) for TT.
In secondary analyses in which we evaluated subtypes of
POAG defined by highest IOP at diagnosis, we found one
SNP that was associated with high-tension glaucoma (Table
3): for the tagging SNP rs1800779, the RR was 1.13 (95% CI,
0.85–1.52) for the AG genotype and 1.58 (95% CI, 0.97–
2.55) for the GG genotype, with a significant trend (P ?
0.02). For this SNP, although the P-heterogeneity for sex
effect was not significant, among the women, the associa-
tion was stronger (RR ? 1.87, 95% CI, 1.19–2.94 for the
comparison between the GG versus AA genotype, P-trend ?
0.01). Similarly, the associations of other SNPs appeared to
be stronger in the women, although the P-heterogeneity for
sex was not significant for most of the SNPs. In the one SNP
(rs3918188) that showed significantly different association
by sex (P-heterogeneity ? 0.02), the RR for the AA com-
pared with the CC genotype was 0.48 (95% CI, 0.28–0.82)
among the women (P-trend ? 0.0008) but 1.48 (95% CI,
0.77–2.84) among the men (P-trend ? 0.61).
In relation to normal-tension glaucoma, associations were
observed with the functional promoter SNP (Table 4). The
pooled RR for the CC homozygote was 0.44 (95% CI, 0.22–
0.87) and P-trend ? 0.03. The T minor allele for the Glu298Asp
polymorphism was adversely associated (TT versus GG: RR ?
1.81; 95% CI, 0.93–3.52) but the association was not signifi-
cant. Other SNPs were not associated with normal-tension
For the SNP rs3918188, that showed significantly differ-
ent associations between the men and the women, the
associations with high- and normal-tension glaucoma were
also significantly different (P ? 0.02) among the women.
Similarly, the associations with the two subtypes of POAG
were also different for the tagging SNP rs1800779 as well as
the T ?786C polymorphism (P ? 0.04 and P ? 0.02,
respectively) among women.
There were no haplotype effects observed for overall POAG
(P ? 0.20 in both the men and the women; Fig. 1). However,
in the women, we observed significant haplotype effects in
relation to high-tension glaucoma (likelihood ratio test P ?
0.004), whereas in the men we did not (likelihood ratio test
P ? 0.68). In the women, the significant increase in risk with
haplotype 6 (RR ? 2.10; 95% CI, 1.28–3.46) compared with
haplotype 1 in relation to high-tension glaucoma confirmed the
main effect of the tagging SNP rs3918188.
Effect Modification with Age of
Menopause/Postmenopausal Hormone Therapy
Because associations were observed between NOS3 SNPs in
relation to high-tension glaucoma, particularly in the women,
we examined the interaction between these SNPs and at-
tributes of reproductive aging. There were no significant inter-
actions between any of the NOS3 polymorphisms and age at
menopause (?54 vs. ?54 years; data not shown). However, in
relation to high-tension POAG, we observed significant inter-
actions between current PMH use and four of the five NOS3
SNPs we evaluated (Table 5). For example, among the women
who were homozygous for the common allele of the T ?786C
SNP, current PMH use was significantly inversely associated
with high-tension POAG risk (multivariate RR ? 0.41; 95% CI,
0.22–0.76); however, among carriers of the minor allele, cur-
rent PMH use was not associated with POAG (P-interaction ?
0.04). Similar trends were observed with the tagging SNPs
rs1800779 and rs7830. For the rs3918188 SNP, current PMH
use was inversely associated with high-tension POAG risk only
among the carriers of the minor allele (e.g., the multivariate RR
of high-tension POAG among the current PMH users with the
AA genotype was 0.37 [95% CI, 0.15–0.94]; P-interaction ?
In this large, population-based, case-control study, we found
no relation between NOS3 gene variants and POAG overall.
TABLE 1. Characteristics of Cases of POAG and Their Matched
Controls as of the Diagnosis Date
Age, mean, y
Family history of glaucoma, %
Obesity (Body mass index ? 30 kg/m2, %)
30? pack years of smoking, %
Caffeine (mean, mg/day)
Alcohol intake (mean, g/day)
Reported eye examinations, n*
Current postmenopausal hormone use
* Eye examinations have been asked every 2 years, seven times
from 1990 to 2002; the number represents the number of eye exami-
nations reported as of the diagnosis date of the index case in matched
974 Kang et al.
IOVS, February 2010, Vol. 51, No. 2
However, several SNPs, including the functional T –786C
SNP in the promoter region, were associated with high-
tension glaucoma, particularly in the women. The interac-
tions between NOS3 and their sex and NOS3 and current
PMH use among the postmenopausal women for high-ten-
sion POAG suggests that the biology of the sexes and gene–
environment interactions play a role in POAG pathogenesis.
As this is the first study to evaluate these gene–environment
interactions, these findings should be interpreted with cau-
tion and confirmed in future studies in different racial/
The functional SNP Glu298Asp, which has been linked to
ischemic heart disease33and ischemic stroke,34was not
associated with POAG overall or with the POAG subtypes.
Furthermore, polymorphisms in this SNP were not associ-
ated with POAG of either sex, and this SNP did not interact
with attributes of female reproductive aging. A relation
between the promoter region functional SNP (T ?786C)
polymorphism has been reported with coronary vasospasm
in a Japanese study35; however, this finding has not been
In our study, we observed associations between NOS3
gene variants and high-tension POAG, particularly among
the women. Similar associations have been found in other
studies. In a study of 56 cases of familial high-tension POAG
and 100 controls, Tunny et al.17found that a NOS3 variant
close to the functional T ?786C variant was positively
associated with familial high-tension POAG; the associations
by sex were not provided. In another study that included 58
patients with high-tension POAG, 76 with normal-tension
glaucoma, and 38 control subjects, Logan et al.18failed to
find an association between NOS3 allelic variants and POAG
overall, but did find an association between NOS3 allelic
haplotypes, including the T ?786C and POAG with mi-
graine.18The associations by sex were also not presented,
but it is known that migraine, which is characterized by a
dysfunction in the vasodilatory response, is a female-pre-
Our results suggest that reproductive hormones play a
role in modulating IOP and the risk of POAG in women. An
Australian study demonstrated that the incidence of POAG
in women was lower than that in men up to the sixth decade
of life, implying that before menopause, women are less
likely to develop POAG.37PMH use among postmenopausal
women produces modest reductions of IOP and may en-
hance optic nerve blood flow.38–44In the present study, the
percentage of POAG cases with elevated IOP at diagnosis
was lower in current PMH users than in non-PMH users (56%
among the users versus 71% among the nonusers), support-
ing the role of PMH in IOP modulation. The Rotterdam Eye
Study found that early menopause was associated with an
increased risk of POAG,20which was confirmed in the
NHS.21Furthermore, in the NHS, current use of estrogen
with progestin was associated with a reduced risk of high-
Circulating estrogen may influence NOS3, and current
PMH use in postmenopausal women may systemically up-
TABLE 2. Cohort-Specific and Pooled Relative Risks of POAG
NHS HPFS Pooled
(n ? 373)
(n ? 1082) RR (95% CI)
(n ? 154)
(n ? 461) RR (95% CI)RR (95% CI)
P-trend ? 0.35
P-trend ? 0.37
P-trend ? 0.94 0.21
P-trend ? 0.15
P-trend ? 0.70
P-trend ? 0.160.69
P-trend ? 0.19
P-trend ? 0.55
P-trend ? 0.620.23
P-trend ? 0.04
P-trend ? 0.58
P-trend ? 0.56 0.10
P-trend ? 0.70
P-trend ? 0.54
P-trend ? 0.990.47
Case and control data are the number of cases (% of total group). Results are based on conditional logistic regression, with family history of
glaucoma, body mass index (?22, 22–23.9, 24–25.9, 26–27.9, 28–29.9, or ?30 kg/m2), smoking status (current, past, or never smoker), physical
activity (quartiles of activity intensity/day), self-reported history (yes/no) of hypertension and diabetes, cumulatively updated alcohol intake (0, 1–9,
10–19, 20–20, or ?30 g/day) and cumulatively updated caffeine intake (0–149, 150–299, 300–449, 450–600, or ?600 g/day).
IOVS, February 2010, Vol. 51, No. 2
Nitric Oxide Synthase Gene Variants and Glaucoma 975
regulate NOS3. Yang et al.45found that estrogen enhances
the release of NO from cultured human coronary artery
endothelial cells. Furthermore, brachial artery vasoconstric-
tion was documented within 1 week of surgical menopause,
supporting the role of estrogen in modulating endothelial
NOS activity.46In postmenopausal women not treated with
PMHs, the endothelial NOS immunoreactivity in the uterine
wall was significantly lower when compared to tissue from
PMH users.47Perara et al.48found that small arteries from
patients with type 2 diabetes exhibited enhanced vascular
relaxation ex vivo after 6 months of PMH exposure. Presum-
ably, these changes are mimicked in other tissue beds,
including the eye, and these data support the interaction
between NOS3 variants and PMH in relation to high-tension
Limitations of our study should be considered. First, our
definition of glaucoma was based on self-report and confir-
mation with medical records and visual fields. This defini-
tion has very high specificity, as we required documentation
of reproducible defect on at least two reliable visual field
tests. It is possible that given the insidiousness of glaucoma,
some control subjects may 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.49Furthermore, our control subjects
had to have undergone an eye examination as of the
matched cases’ diagnosis dates, and the average number of
eye examinations reported as of their selection as controls
was three, implying that advanced glaucoma, if present,
would most likely have been detected. Any misclassifica-
tions of the disease in the control group or in the POAG
group 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 or minority
populations. Finally, it is possible that our results may be
due to chance, as several NOS3 polymorphisms as well as
several interactions were examined in relation to three out-
comes. Therefore, these findings should be confirmed in
future studies, particularly with different racial/ethnic
groups, and the results should be interpreted with caution.
To summarize the major findings of this study, we observed
● None of the NOS3 SNPs investigated was significantly
associated with overall POAG
● For high-tension POAG, the association with one tagging
SNP (rs3918188) was significantly inversely associated among
the women but not among the men (P-heterogeneity by sex ?
● In the women, four of the five NOS3 SNPs showed sig-
nificant interactions with current PMH use in relation to high-
tension POAG. For example, PMH use was inversely associated
(RR ? 0.41; 95% CI, 0.22–0.76) only among the women with
the TT genotype in T ?786C.
In conclusion, NOS3 gene polymorphisms may interact
with sex and PMH use in women in relation to high-tension
POAG, suggesting that sex-based biology and reproductive
hormones play a role in POAG pathogenesis.
TABLE 3. Cohort-Specific and Pooled RR of High-Tension Glaucoma (IOP ?22 mmHg)
(n ? 252)
(n ? 731)RR (95% CI)
(n ? 112)
(n ? 334)RR (95% CI) RR (95% CI)
P-trend ? 0.02*
P-trend ? 0.89
P-trend ? 0.12 0.24
P-trend ? 0.19
P-trend ? 0.87
P-trend ? 0.23 0.58
P-trend ? 0.01*
P-trend ? 0.68
P-trend ? 0.02 0.31
P-trend ? 0.0008*
P-trend ? 0.61
P-trend ? 0.48 0.02
P-trend ? 0.49
P-trend ? 0.37
P-trend ? 0.990.26
Results are based on conditional logistic regression, with an additional control for the covariates specified in the footnote of Table 2.
* P-heterogeneity was ?0.05 for the test between high-tension glaucoma and normal-tension glaucoma only among NHS participants (P ? 0.02
for the ?786 C/T SNP, P ? 0.02 for the rs3918188 SNP, and P ? 0.04 for the rs1800779 SNP).
976Kang et al.
IOVS, February 2010, Vol. 51, No. 2
TABLE 5. Effect Modification by Current PMH Use Status on the Associations of Selected NOS3 Polymorphisms and High-Tension Glaucoma
(IOP ?22 mmHg) among Women
Never or Past Users of PMHCurrent Users of PMH
(n ? 153)
(n ? 389)RR (95% CI)
(n ? 72)
(n ? 233) RR (95% CI)
Data are based on analysis of data from postmenopausal women only, with adjustment for age at menopause and type of menopause (natural,
surgery) in addition to those specified in the footnote of Table 2.
TABLE 4. Cohort-Specific and Pooled RR of Normal-Tension Glaucoma (IOP ?22 mm Hg)
NHS HPFS Pooled
(n ? 121)
(n ? 351)RR (95% CI)
(n ? 42)
(n ? 127) RR (95% CI) RR (95% CI)
P-trend ? 0.11*
P-trend ? 0.08
P-trend ? 0.030.32
P-trend ? 0.34
P-trend ? 0.21
P-trend ? 0.170.45
P-trend ? 0.24*
P-trend ? 0.11
P-trend ? 0.090.34
P-trend ? 0.26*
P-trend ? 0.54
P-trend ? 0.560.29
P-trend ? 0.98
P-trend ? 0.30
P-trend ? 0.730.33
Results based on conditional logistic regression, with additional control for covariates specified in the footnote of Table 2.
* P-heterogeneity was ?0.05 for the test between high-tension and normal-tension glaucoma, only among NHS participants. P ? 0.02 for the
?786 C/T SNP, P ? 0.02 for the rs3918188 SNP, and P ? 0.04 for the rs1800779 SNP).
IOVS, February 2010, Vol. 51, No. 2
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