The association of glutathione S-transferase GSTT1 and GSTM1 gene polymorphism with pseudoexfoliative glaucoma in a Pakistani population.

Abstract

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.
Multiplex polymerase chain reaction was used to study the GSTT1 and GSTM1 polymorphisms in 165 PEXG patients and 162 unaffected controls.
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).
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.

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The association of glutathione S-transferase GSTT1 and GSTM1
gene polymorphism with pseudoexfoliative glaucoma in a Pakistani
population
Muhammad Imran Khan,1 Shazia Micheal,1 Farah Akhtar,2 Waqar Ahmed,1 Bushra Ijaz,1 Asifa Ahmed,1
Raheel Qamar1,3
1Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan; 2Al-Shifa Trust Eye Hospital,
Rawalpindi, Pakistan; 3Shifa College of Medicine, Islamabad, Pakistan
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 (χ2=21.82, p<0.001). This association was specifically
related to the female patients (χ2=35.63, p<0.001); no such association was seen in the male patients (χ2=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.
Xenobiotic compounds of exogenous and endogenous
origin are a substantial threat to the human cells as they lead
to the production of highly reactive oxygen species (ROS).
The cells produce numerous antioxidants that counter the
effects of these compounds by reducing their accumulation.
Glutathione (GSH) is an important antioxidant that protects
against cellular damage caused by environmental toxins as
well as from ROS-mediated injury. GSH works by
neutralizing ROS and xenobiotics with the help of glutathione
S-transferase (GST); this enzyme catalyzes the conjugation of
these compounds with GSH, which being water soluble can
thus be easily eliminated from the body [1-3].
Mammalian GSTs are made up of a supergene family of
catalytic and binding proteins located on at least seven
different genes, which are divided into three major classes:
cytosolic, mitochondrial and microsomal GSTs. Tissue
expression studies have shown that most of the cytosolic GSTs
are expressed in the kidneys and the liver, where they play an
important role in the detoxification of various endogenous and
exogenous toxic chemicals in the body [4-6].
Of the cytosolic GSTs, the Mu (µ), Theta (σ), and Pi (π)
genes have been found at different frequencies in various
ethnic groups. In the Mu class of GSTs the M1 null genotype
(M0) is common in the Chinese, Japanese, French, and
Correspondence to: Raheel Qamar, Dean, Faculty of Science,
COMSATS Institute of Information Technology, Park Road,
Islamabad-45600, Pakistan; Phone: +92-51-9235033; FAX:
+92-51-8318499; email:raheelqamar@hotmail.com
English, with a frequency between 43% and 58% [7-11]. In
the Theta class of GSTs the T1 null genotype (T0) has been
found at varying frequencies in different ethnic groups: 64.4%
in Chinese, 60.2% in Koreans, and 20%–24% in African-
Americans [12]. Biochemical studies have associated these
allelic variations to intra-individual differences in the ability
to metabolize environmental and cellular toxins [13].
It has been shown that individuals carrying the null
genotypes of GST may have higher levels of intermediates of
oxidative metabolism because the detoxification pathways
have been disrupted, and this then directly or indirectly
exacerbates the pathological effects of ROS.
This has important implications in a range of diseases; for
example, various types of cancers, asthma and others, and has
also demonstrated involvement in causing neuronal cell death
in neurodegenerative diseases, such as Alzheimer, motor
neuron disease and Parkinson [14-17]. GSTs have also been
reported to be widely expressed in different ocular tissues.
Thus, for individuals carrying the null genotypes, the body’s
defense against oxidative damage may be impaired,
contributing to manifestation of the ocular diseases in
question [18-23].
Polymorphisms of GST have previously been shown to
be associated with glaucoma, cataract, exudative age-related
macular degeneration as well as various spontaneous optic
neuropathies [13,24-27].
Several studies have been conducted in different
populations to determine the association of GSTT1 and
Molecular Vision 2010; 16:2146-2152 <http://www.molvis.org/molvis/v16/a230>
Received 25 May 2010 | Accepted 13 October 2010 | Published 26 October 2010
© 2010 Molecular Vision
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GSTM1 polymorphisms with primary open-angle glaucoma
[13,26,28], but to date only three studies have been reported
on pseudo-exfoliative glaucoma (PEXG) in populations of
Arabs, Turks and Swedes [29-31]. There was no significant
association found between PEXG and the null genotypes of
GSTT1 and GSTM1 in the Turks and Swedes, whereas in the
Arab glaucoma patients (n=107), in a study that included
POAG, PCAG and PEXG, a significant association of all the
deletion genotypes was observed [29]. However, after
stratification of patients by glaucoma type the T0M0 genotype
was not found to be significantly associated with any type of
glaucoma.
The aim of the present study was to determine if there
was a significant association of the GSTT1 and GSTM1
polymorphisms with PEXG in a Pakistani cohort. The study
was approved by the Departmental Ethics Committee of
COMSATS Institute of Information Technology, Islamabad
and the relevant Hospitals’ Ethics Committee and conformed
to the principles of the Declaration of Helsinki. Informed
written consent was taken from all patients and unaffected
control individuals before sampling. PEXG patients were
recruited from the out-patients Department of the Al-Shifa
Trust Eye Hospital, Rawalpindi and Christian Eye Hospital,
Taxila.
METHODS
Criteria for patient selection, sample collection and DNA
extraction: Complete ophthalmic examinations were
performed on the PEXG patients, including measurement of
cup-to-disk ratio, tonometeric assessment of intra-ocular
pressure and slit lamp biomicroscopy was performed to detect
any presence of exfoliative material along the papillary border
and on the iris. Following this, the pupils of the patients were
dilated and the anterior of the lens surface was examined for
any deposits of white material. Angles were measured in all
the patients with the help of gonioscopy. All of the healthy
control individuals were also examined and they were found
to have normal visual fields, no exfoliation material in the eye
or any other evidence of glaucoma. Blood samples from all
the patients and controls were collected by venipuncture;
genomic DNA was extracted by a conventional phenol
chloroform method, as described previously [32].
Genotype analysis of GSTT1 and GSTM1 polymorphisms: To
determine the GSTT1 and GSTM1 genotypes of the subjects,
multiplex polymerase chain reaction (PCR) amplification was
performed using the following primers: GSTT1 forward
primer 5′-TTC CTT ACT GGT CCT CAC ATC TC-3′;
GSTT1 reverse primer 5′-TCA CCG GAT CAT GGC CAG
CA-3′; GSTM1 forward primer 5′-GAA CTC CCT GAA
AAG CTA AAG C-3′; and GSTM1 reverse primer 5′-GTT
GGG CTC AAA TAT ACG GTG G-3′. Beta globin gene
sequence amplification was used as an internal control in the
PCR reactions, for which the primers were: forward primer
5′-CAA CTT CAT CCA CGT TCA CC-3′; reverse primer 5′-
GAA GAG CCA AGG ACA GGT AC-3′. Each 25 μl PCR
reaction contained 1× Taq Buffer (10 mM Tris-HCl, pH 9.0,
50 mM KCl, 0.1% Triton X-100, 0.01% gelatine; Fermentas,
Burlington, Ontario), 30 pmol of each primer, 1.5 mM
MgCl2, 0.3 mM dNTP, 1.5 U of Taq DNA polymerase
(Fermentas) and 100 ng of genomic DNA.
Amplification was performed with initial denaturation at
95 °C for 5 min, followed by 30 cycles at 95 °C for 1 min,
65 °C for 1 min and 72 °C for 1 min, with a final extension at
72 °C for 7 min. PCR products were electrophoretically
separated on 2% agarose gels and the bands were visualized
by UV transillumination. For the T1M1 genotype three bands
were obtained: a 459 bp band of GSTT1, a 209 bp band of
GSTM1 and a 268 bp band of the internal control (β-globin
gene; Figure 1). The T1M0 genotype produced two bands of
459 bp and 268 bp; the T0M1 genotype produced two bands
of 209 bp and 268 bp. In the case of the T0M0 null genotype
only, the β-globin gene internal control band (268 bp) was
observed. To confirm the null results, confirmatory PCR tests
were performed separately for the GSTT1 as well as the
GSTM1 genotypes, using β-globin gene amplification as the
internal control, under identical conditions as those described
above, but using only the GSTT1 and β-globin or the
GSTM1 and β-globin primers, respectively.
Statistical analysis: Statistical analysis of the genotype
frequencies of both the PEXG patients and controls was
performed using the chi-square test (χ2). To prevent any false
positive inference, Bonferroni correction (p′b) was applied to
the genotype data. Correction involved multiplying the p
value obtained after each single test with the total number of
independent tests (k) performed during the study [33,34]. The
Sidak correction (p′s), an approximation of Bonferroni, was
also applied to correct the p value as some Bonferroni
corrected values were >1 [35,36]. Note that for the Bonferroni
corrected values, the level of significance remained 0.05; i.e.,
the association of any genotype group/variable with k×p<0.05
was considered as statistically significant [33]. The formulae
for the calculation of the Bonferroni and Sidak values are as
follows:
p′b
= k × p
p′s
= 1 −(1 − p)k
where k is the number of genotype groups tested and p is the
raw value obtained from the χ2 test.
All analyses were performed using SPSS v.16 statistical
analysis software (SPSS Inc., Chicago, IL) and StatCalc
EpiInfo package v.6 (Atlanta, GA).
RESULTS
The case-controlled study included 165 patients with
glaucoma (53% males, mean age 45.8±10.1 years and 47%
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females, mean age 46.31±11.6 years), as well as 162
unaffected controls (52% males, mean age 43.8±13.9 years
and 48% females, mean age 43.1±10.9 years); there was no
statistically significant difference (p>0.05) in the mean age of
male/female patients and controls.
There was a significant difference in the overall
distribution of the GSTT1 and GSTM1 genotypes in the PEXG
patients and controls (χ2=21.82, p<0.001; Table 1). The
difference between individual genotypes remained significant
even after the application of Bonferroni and Sidak corrections
(p’b & p’s<0.05). When the subjects were stratified according
to gender the overall genotype distribution of the female
patients was found to be significantly associated with the
disease (χ2=35.63, p<0.001; Table 1); this remained
significant even after applying the Bonferroni correction
(p’b & p’s<0.001). Interestingly, the T0M0 null genotype was
exclusively present in the PEXG female patients (χ2=20.12, p,
p’b & p’s<0.001). In the male patients 8.2% of the controls had
the T0M0 null genotype as compared to 6% of the PEXG male
patients (χ2=0.11, p, p’b & p’s>0.05, OR=0.81 [95% CI=0.20–
3.24]; Table 1).
The gender distribution of the null genotypes GSTT0 and
GSTM0 were also compared between patients and contols;
these genotypes were found at a significantly higher
frequency in the female patients than in the female controls
(χ2=19.90, and χ2=18.7, respectively, p<0.05; Table 2);
whereas in males the frequency of the null genotypes did not
differ significantly between the groups (χ2=0.43, χ2=0.01,
respectively, p>0.05). However, the male control samples as
compared to the female controls had a significantly higher
frequency of both the null genotypes GSTT0 and GSTM0
(χ2=12.13, χ2=14.41, respectively, p<0.001; Table 3), whereas
in patients in both these groups these genotypes were not
statistically different from each other (χ2=0.44, χ2=0.53,
respectively, p>0.05).
DISCUSSION
Oxidative stress along with cellular senescence is one of the
major factors affecting cellular processes. The inability of the
cells to cope with oxidative stress is due to a breakdown of
the body’s antioxidant defenses due to excessive production
of ROS; this in turn leads to damage to the cellular
macromolecules, including DNA, proteins and lipids. To
counteract the stress-induced damage, the cells upregulate
antioxidant enzymes, such as GST [37].
In the eye a system of trabecular meshwork (TM)
regulates the outflow of aqueous humor, and maintains normal
intraocular pressure (IOP); therefore, any challenge to the
structural and functional integrity of TM results in the
development of glaucoma. Several lines of evidence suggest
that one effect of the generation of oxidative free radicals in
the glaucomatous eye is the progressive loss of TM [38]. In
an experimental rat model of glaucoma the retinal ganglion
cells (RGC) and glial cells showed extensive protein and lipid
oxidation, which appears to lead to apoptosis of these and
other neuronal cells [37,39]. Thus, to maintain the
homeostatic balance between the production of ROS and their
clearance, the eye, under stress, is required to produce higher
levels of antioxidants. Indeed, the glaucomatous eye
upregulates several stress-related genes, including GST,
which are involved in the detoxification of ROS and other
related compounds [39,40].
Although, under stress, GST and other antioxidants are
demonstrably upregulated, the glaucomatous eye fails to
prevent oxidative damage, a fact that points to an underlying
molecular or genetic defect. One such genetic risk factor is the
presence of the GST null genotype in patients having
oxidative, stress-related diseases.
A potent antioxidant 8-hydroxy-2′-deoxyguanosine (8-
OH-dG) has been shown to be present at 3.6 fold higher levels
in the TM of POAG patients, as compared to controls [41]. In
these patients there was a positive correlation between
Figure 1. Multiplex PCR amplification
product of the GSTT1, GSTM1 and
internal control β-globin genes. The
amplified products were separated by
electrophoresis on 2% agarose gel. Lane
L, 100bp DNA ladder; lane 1, 4, 9, and
10, T1/M0 genotype (459 bp and 268 bp
fragments); lane 2, 3, and 8, T1/M1
genotype (459 bp, 268 bp, and 209 bp
fragments); lane 5, T0/M1 genotype
(268 bp and 209 bp fragments); lane 6,
7, and 11, T0/M0 genotype (268 bp
fragments).
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TABLE 1. OVERALL AND GENDER SEGREGATED DATA OF GST GENOTYPES IN UNAFFECTED CONTROLS AND PEXG PATIENTS.
Group
Genotype
Controls
Patients
p (χ2)
p (χ2)
pb
ps
OR (95% CI)
Total
T1M1
95 (59%)
57 (34.5%)
<0.001 (21.82)
Reference




T1M0
51 (31%)
69 (41%)

0.001 (10.79)
<0.05
<0.05
2.25 (1.34–3.79)

T0M1
9 (6%)
23 (14%)

<0.001 (12.71)
<0.001
<0.001
4.26 (1.73–10.74)

T0M0
7 (4%)
16 (9.5%)

<0.05 (8.45)
<0.05
<0.05
3.81 (1.37–10.96

Total
162
165





Females
Genotype
Controls
Patients
p (χ2)
p (χ2)
pb
ps
OR (95% CI)

T1M1
60 (80.5%)
26 (33.8%)
<0.001 (35.63)
Reference




T1M0
16 (20.8%)
31 (40.2%)

<0.001 (15.84)
<0.001
<0.001
4.47 (1.96–10.29)

T0M1
1 (1.2%)
9 (11.7%)

<0.001 (13.81)
<0.001
<0.001
20.77 (2.45–460.38)

T0M0
0 (0%)
11 (14.3%)

<0.001 (20.12)
<0.001
<0.001
N/A

Total
77
77





Males
Genotype
Controls
Patients
p (χ2)
p (χ2)
pb
ps
OR (95% CI)

T1M1
35 (41.2%)
31 (35%)
>0.05 (2.28)
Reference




T1M0
35 (41.2%)
38 (43%)

>0.05 (0.36)
>0.05
>0.05
1.23 (0.60–2.52)

T0M1
8(9.4)
14 (16%)

>0.05 (1.83)
>0.05
>0.05
1.98 (0.66–6.01)

T0M0
7 (8.2%)
5 (6%)

>0.05 (0.11)
>0.05
>0.05
0.81 (0.20–3.24)

Total
85
88






pb indicates Bonferroni corrected value and ps indicates Sidak corrected value. A p<0.05 was considered to be statistically significant.


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oxidative DNA damage and intraocular pressure as well as
visual field defects. The GSTT1 null genotype was found to
be more common in POAG cases, who also showed 2.2 fold
higher levels of 8-OH-dG as compared to the other genotypes
[41,42], resulting in oxidative DNA damage in the TM of
these patients. A higher level of oxidative damage to the
trabecular meshwork has also been seen in POAG patients
with the GSTM1 null genotype, indicating the possible
involvement of these genotypes in the manifestation of
disease [42].
In primary cultures of human optic nerve head astrocytes
from glaucomatous eyes it has been observed that the basal
levels of GSH antioxidants were well below those of primary
cultures from normal astrocytes; these data combined with all
the other evidence indicates that oxidative stress plays a
significant role in the manifestation and progression of
glaucoma [40].
Recently, using cDNA arrays, the involvement of several
stress-related candidate genes was studied in PEXG. The
authors found that a large set of cytoprotective gene products,
including antioxidant defense enzymes (e.g., GST) and stress-
inducible transcription factors, were consistently down-
regulated in PEXG at both the mRNA and protein levels; this
finding supports the conjecture that GSTs play an important
role in protecting the eye against the development of PEXG
[41].
The present study was based on the hypothesis that
inadequate expression of GST in PEXG correlates with the
null genotypes. This hypothesis has been tested in Swedes
[31] and Turks [28], but in those populations no significant
contribution of the polymorphic variants of GSTs with PEXG
was found. To the best of our knowledge, the investigators in
all of those studies did not stratify their data according to
gender. This may be significant considering our knowledge
that in mice there is a higher expression of some types of GSTs
in females as compared to males [6]. This indicates that any
defects in the relevant genetic pathways in females could
exacerbate the risk of disease to a significantly higher level
than for males.
The importance of the present study is that an association
between PEXG and all the GST null genotype combinations
was observed and this remained significant even after
Bonferroni correction. In addition, after stratifying the data
according to gender, a clear association of the null genotypes
with only the female PEXG patients was found; this occurred
because a large number of female PEXG patients were found
to carry the null genotypes, in different combinations, as
compared to the female controls (Table 1).
The GSTT1 and GSTM1 null genotypes in the total data
set of the patient cohort was also compared, as well as in the
data stratified according to gender. Although an overall
statistically significant difference in the null genotype
distribution between patients and controls was observed, this
was due to the significantly higher distribution of the null
genotypes in the female patients, as compared to female
controls (Table 2). In addition it must be pointed out that the
TABLE 2. COMPARISON OF GSTT1 AND GSTM1 NULL GENOTYPES ACCORDING TO GENDER DISTRIBUTION IN PATIENTS AND CONTROLS. A P<0.05 WAS
CONSIDERED STATISTICALLY SIGNIFICANT.
Genotype
T0
T1
M0
M1
Genotype
T0
T1
M0
M1
Genotype
T0
T1
M0
M1
Group
Total

Total


Females

Females


Males

Males

Controls (n=162)
16 (10%)
146 (90%)
58 (36%)
104 (64%)
Controls (n=77)
1 (1%)
76 (99%)
16(21%)
61(79%)
Controls (n=85)
15(18%)
70(82%)
42(49%)
43(51%)
Patients (n=165)
39 (24%)
126 (76%)
85 (52%)
80 (48%)
Patients (n=77)
20 (26%)
57 (74%)
42(55%)
35(45%)
Patients (n=88)
19(22%)
69(78%)
43(49%)
45(51%)
p (χ2)
<0.05 (11.06)

<0.05 (8.20)

p (χ2)
<0.05 (19.90)

<0.05 (18.70)

p (χ2)
>0.05(0.43)

>0.05(0.01)

TABLE 3. COMPARISON OF GSTT1 AND GSTM1 NULL GENOTYPES BETWEEN MALE AND FEMALE CONTROLS AND PATIENTS. A P<0.05 WAS CONSIDERED
STATISTICALLY SIGNIFICANT.

Genotypes
GSTT1 null
GSTM1 null
Controls

Patients

(Males) (n=85)
15 (18%)
42 (49%)
(Females) (n=77)
1 (1%)
16 (21%)
p (χ2)(Males) (n=88)
19 (22%)
43 (49%)
(Females) (n=77)
20 (26%)
42 (55%)
p (χ2)
<0.001 (12.13)
<0.001 (14.41)
>0.05 (0.44)
>0.05 (0.53)
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significantly higher frequency of the T1 and M1 in the female
unaffected controls as compared to the males (Table 3) could
be a result of a gender-specific protective effect of these
genotypes in the females only.
The association of the GSTM1 null genotype with female
PEXG patients in the current study is in accordance with the
data of a group of Greek patients with multiple sclerosis
[42]; using this data we calculated and compared the gender-
wise distribution of the null genotypes of GSTT1 and
GSTM1 between the Greek controls and patients. We found
that the female patients had a significantly higher frequency
of the GSTM1 null genotype (65.5% versus 41.7% in controls;
χ2=4.91, p=0.02) while in males there was no difference in the
distribution of any of the null genotypes.
Another interesting aspect of the present study is that, in
addition to the GSTM1 null genotype, the frequency of the
GSTT1 null genotype in the female PEXG patients occurred
also at a significantly higher level than in the control females
(Table 2), while in the Greeks there was no significant
difference in this group (χ2=0.05, p=0.82). Also, in contrast to
our data, in the Greeks the T0M0 combined null genotype was
not found to be associated with the females, which points to
the possible association of the GST T0M0 genotype with
PEXG only in the Pakistani female patients; the resultant
vulnerability to cellular and oxidative stress conditions may
therefore be a contributing factor in the disease pathology that
possibly has ethnic as well as gender associations.
In conclusion, we would like to emphasize the importance
of conducting further studies in different populations to
further ascertain the association of GST with PEXG; this will
allow the development of a consensus regarding the
involvement of GST in glaucoma. There is also a need to better
understand the mechanisms associated with the null
genotypes in female patients.
ACKNOWLEDGMENTS
We thank all the subjects in this study for donating blood
samples. This work was supported by grant no. PSF/RES/C-
COMSATS/MED(280) awarded to R.Q. by the Pakistan
Science Foundation and a core grant from the Shifa College
of Medicine.
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