Graefe's Archive for Clinical and Experimental Ophthalmology

, Volume 243, Issue 4, pp 327–333

May glutathione S-transferase M1 positive genotype afford protection against primary open-angle glaucoma?

Authors

    • Department of OphthalmologyMersin Üniversitesi Tip Fakültesi
  • Nurcan Aras Ateş
    • Department of Medical Biology and GeneticMersin Üniversitesi Tip Fakültesi
  • Lülüfer Tamer
    • Department of BiochemistryMersin Üniversitesi Tip Fakültesi
  • Özay Öz
    • Department of OphthalmologyMersin Üniversitesi Tip Fakültesi
  • Ayça Yilmaz
    • Department of OphthalmologyMersin Üniversitesi Tip Fakültesi
  • Uĝur Atik
    • Department of BiochemistryMersin Üniversitesi Tip Fakültesi
  • Handan Çamdeviren
    • Department of BiostatisticsMersin Üniversitesi Tip Fakültesi
Clinical Investigation

DOI: 10.1007/s00417-004-1013-9

Cite this article as:
Yildirim, Ö., Ateş, N.A., Tamer, L. et al. Graefe's Arch Clin Exp Ophthalmol (2005) 243: 327. doi:10.1007/s00417-004-1013-9

Abstract

Purpose

To find out whether the polymorphism at GSTM1, GSTT1 and GSTP1 loci is associated with increased susceptibility to glaucoma.

Methods

We genotyped 153 primary open angle patients and 159 healthy controls. Genomic DNA from peripheral blood was examined using polymerase chain reaction and defined for the genetic polymorphisms of glutathione S-transferase.

Results

The frequency of the GSTM1 null genotype individuals among the glaucoma patients was significanlty higher than in controls (54.9 vs 40.9%) with odds ratio of 1.64 (95% CI: 1.10–2.59). The frequency of the GSTT1 and GSTP1 in both groups were not statistically different.

Conclusion

The present study suggests that the GSTM1 null genotype may be a genetic risk factor for development of primary open angle glaucoma. Further associations studies in other polymorphic genes for xenobiotic–metabolizing enzymes are needed to elucidate the environmental-genetic interaction in the underlying cause of primary open angle glaucoma.

Keywords

Primary open angle glaucomaOxidative stressGlutathione S-transferasePolymorphism

Introduction

Glaucoma is a disease in which a progressive loss of retinal ganglion cells is characterized by a recognizable pattern of both visual functional loss and optic nerve head pallor and excavation. If untreated, the natural course is towards blindness, or at least significant visual disability [13]. Primary open-angle glaucoma (POAG), which affects almost 2% of the world’s population, accounts for most of the glaucoma cases [38]. Although the pathophysiology of POAG is not precisely known, its causes are clearly multifactorial. It is a result of multiple, interactive genetic and environmental effects. Although the most prominent known risk factor for developing POAG is elevated intraocular pressure, there are also other suspected risk factors such as positive family history, age, hypertension and diabetes [31, 35, 39].

Its prevalence increases with age [30]. It is well known that POAG is an age-related disorder [16]. There is a general consensus that cumulative oxidative damage is responsible for aging, and may, therefore, play an important role in the pathogenesis of an age-related disorder such as glaucoma [3]. Oxidant stress and antioxidant systems are potentially important for ocular tissues. Exposure to light by photosensitizing mechanisms may lead to the formation of reactive oxygen species. Many of the ocular tissues regenerate slowly, causing an increase in the risk for an accumulation of oxidant-inflicted damage in the tissue components [4]. The damage caused by xenobiotics and oxidants can result in a number of molecular changes that contribute to the development of glaucoma, cataract and other age-related eye diseases [2]. Therefore the eye must posses efficient reducing systems, as well as, detoxification enzymes such as catalase, superoxide dismutase, glutathione peroxidase and glutathione S-transferase (GST) for protection from oxidative damage [5]. The ocular ciliary epithelium expresses genes coding for GST and other enzymes involved in the glutathione cycle, such as glutathione peroxidase [8]. Moreover, in a bovine model, Nguyen et al. [28] have showed that trabecular meshwork cells have both glutathione peroxidase and glutathione reductase activities. Several epidemiological studies suggested that individual susceptibility to several disorders, including eye diseases might be connected with the GST system [19, 21, 34].

The GST supergene family, which encodes detoxification enzymes, is widely expressed in mammalian tissue cytosols and membranes. GSTs catalyse the conjugation of reduced glutathione with a wide variety of electrophiles those include known carcinogens and various compounds which are products of oxidative stress, including oxidized DNA and lipids. Indeed, several lines of evidence suggest that the level of expression of GST is a crucial factor in the determination of sensitivity of cells to a broad spectrum of toxic chemicals and oxidative stress [14, 15].

The GST isoenzymes expressed in human tissues comprise Alpha, Mu, Pi, Theta and Zeta gene families [1921]. Among these classes of GSTs, GSTM1, GSTM3, GSTT1, GSTP1 and GSTZ1 have been shown to be polymorphically distributed and are therefore of special interest in molecular epidemiological studies. Biochemical studies have shown interindividual variation in the ability to metabolize toxic and carcinogenic compounds and epidemiologic studies have revealed associations between specific alleles of GST genes and increased disease and cancer risk [9, 10, 23, 25]. The GSTM1 and GSTT1 genes both exhibit deletion polymorphisms. Homozygous deletions of those genes, called GSTM1 and GSTT1 null genotypes, result in a lack of enzyme activity [29, 33]. These polymorphisms are relevant for the general population, GSTM1 and GSTT1 are homozygously deleted in about 50% and 19% of caucasian individuals, respectively [12]. Individuals with GSTM1 deficiency show a greater level of DNA-damage following carcinogen exposure, this is determined by sister chromatid exchange (SCE) and formation of DNA-adducts [20]. Polymorphism in the GSTP1 gene consists of the presence of variant genotypes GSTP1 AB and GSTP1 BB next to the wild-type GSTP1 AA [27, 32, 37]. A transition of adenine (A) to guanine (G) at nucleotide 313 in exon 5 of the GSTP1 gene results in substitution of isoleucine (Ile) to valine (Val) at position 104 in the amino acid sequence of the protein [41]. The valine variants exhibit lower specific activities and affinities for the electrophilic substrates of the enzyme [37, 41].

To facilitate understanding the multifactorial causes of the disease, it is reasonable to study whether genetic polymorphisms of xenobiotic-metabolizing and antioxidant enzyme contribute to the development of POAG. In the present study, we investigated the distribution of GSTM1, GSTT1 and GSTP1 polymorphisms in patients with POAG and controls to explore the possible association between different GST variants and the occurrence of POAG.

Materials and methods

Patient selection

Patients with POAG were recruited from Ophthalmology Department of the University Hospital, Mersin. Diagnosis of POAG required all of the following: open angle; intraocular pressure higher than 21 mmHg; characteristic optic disc changes (e.g., vertical cup-to-disc ratio higher than 0.3); thin or notched neuroretinal rim or disc hemorrhage; and characteristic visual field changes. Patients were excluded if they had a history of eye surgery before the diagnosis of glaucoma or there was evidence of secondary glaucomas, such as exfoliation, pigment dispersion, traumatic or uveitic glaucoma. The patient group consisted of 153 patients with POAG. The mean age of the glaucoma group was 61.64±5.48 years (ranging from 44 to 72), 77 of them (50.3%) were women.

We recruited people who were over 40 years of age, had intraocular pressures below 20 mmHg, and had no family or personal history of glaucoma as control subjects. Also cases and controls were unrelated. The control group comprised 159 cases who were the volunteers living in the same region. The ones who had any type of malignancies or autoimmune diseases were not included in the study either in the patient or in the control group. The mean age of the control group was 62.74±6.58 years (ranging from 56 to 64); 69 of them (43.4%) were women. Informed consent was obtained from all subjects after explanation of the nature of the study.

Blood samples and DNA isolation

Venous blood was collected by venapuncture in sterile siliconized EDTA 2-ml vacutainer tubes. Immediately after collection, whole blood was stored at+4°C until it was used. Genomic DNA was extracted from whole blood using High Pure PCR Template Preparation kits (Roche Diagnostics, GmbH, Germany).

Analysis of GSTM1, GSTT1 and GSTP1 polymorphism

The detection of GSTT1, GSTM1 and GSTP1 gene polymorphisms were made by using Real-Time PCR (Roche diagnostics, GmbH, Mannheim, Germany). Appropriate fragments of the GST gene for GSTT1, GSTP1 and GSTM1 were amplified with specific primers from human genomic DNA. The polymerase chain reaction (PCR) primers were synthesized according to the Ko [22]. The sequences and the hybridization probes were shown in Table 1.
Table 1

Polymerase chain reaction (PCR) primer sequences and hybridization probes for glutathione S-transferase (GST) M1, T1 and P1

Gene

PCR primers

Hybridization probes

GSTM1

5′-GAACTCCCTGAAAAGCTAAAGC-3′

5′-LCR640-ATGGCCGCTTCCCAGAAACTCTG-3′

5′-GTTGGGCTCAAATATACGGTGG-3′

5′-TCACTCCTCCTTTACCTTGTTTCCTGCAAA-FL-3′

GSTT1

5′-TTCCTTACTGGTCCTCACATCTC-3′

5′-LCR640-TCDAAGGCCGACCCAAGCTGGC-3′

5′-TCCAGGTCAACCGGATCAT-3′

5′-CCGTGGGTGCTGGCTGCCAAGT-FL-3′

GSTP1

5′-ACCCCAGGGCTCTATGGGAA-3′

5′LCR640-TGTGAGCATCTGCACCAAGGGTTGGGG-3′

5′-TGAGGGCACAAGAAGCCCCT-3′

5′-TGCAAATACATCTCCCTCATCTACACAAC-FL-3′

β-globin

5′-CAACTTCATCCACGTTCACC-3′

 

5′-GAAGAGCCAAGGACAGGTAC-3′

 
PCR protocol of GST polymorphisms; 4 mmol/l MgCl2, 0.2 μmol/l of each hybridization probe, 10 pmol of each PCR primers, 2 μl of the Light Cycler DNA Master Hybridization Mix and 50 ng of genomic DNA in a final volume of 20 μl. These conditions were the same for the amplification of all three mutations (Table 2).
Table 2

PCR protocol of GST polymorphisms

Program

Target temperatures

Incubation time (s)

Temperatures transition rate

Amplification

  Denaturation

95°C

0a

20°C/s

  Annealing

66°C

10

20°C/s

  Extension

72°C

19/7b

20°C/s

Melting curve

  Preconditioning

45°C

10/15c

20°C/s

  Melting

95°C

0a

0.1°C/s

aZero second means no incubation at target temperature.

bIncubation times are 19 s for T1, 7 s for M1 and P1 polymorphisms.

cIncubation times are 10 s for T1, 15 s for M1 and P1 polymorphisms.

To test GSTM1 and GSTT1 false negative subjects, a simple PCR was used to identify nulled subjects. The PCR was performed in a Techne thermal cycler (Progene). β-globin gene was co-amplified as an internal positive control in the individual PCR reactions. Both the PCR primers and hybridization probes were synthesized by TIB MOLBIOL (Berlin, Germany). Amplification and melting curves which represent PCR results of GSTT1, M1, and P1 are shown in Figs. 1, 2, 3.
Fig. 1

The amplification curve graph for GSTT1. Lines with crosses express the GSTT1 negative subjects. Purple line implicates negative control and bold black line with dots implicates positive control. Subjects having GSTT1 gene show amplification in the exponential phase

Fig. 2

The amplification curve graph for GSTM1. Lines with crosses express the GSTM1 negative subjects. Line with dots implicates negative control and bold dark blue line implicates positive control. Subjects having GSTM1 gene show amplification in the exponential phase

Fig. 3

Melting curve graph for GSTP1. Red line implicates B allele, blue line implicates heterozygous genotypes, green line implicates A allele and the black line implicates negative control

Statistical analysis

Student’s t test was used for comparing the ages of two groups. Chi-Square test was used for determining the relationship between sex and allele frequencies with in groups. All values were represented as mean ± standard deviation (SD). p values of <0.05 were considered statistically significant. The association between age, sex, GSTM1, GSTT1 and GSTP1 polymorphisms, and glaucoma was modeled through multivariate logistic regression analysis [7]. The GST assays place individuals into distinct categories: those with present or null genotypes for GSTM1 and GSTT1 and homozygous 105 Ile or heterozygous or homozygous 105 Val genotypes for GSTP1. Odds ratio and confidence intervals were used to analyze the occurrence of frequencies of GSTM1, GSTT1 and GSTP1 genotypes in patients with glaucoma compared to the control groups. The reference group consisted of individuals with three putative low-risk genotypes, i.e., the presence of GSTM1 (non-deleted), GSTT1 (non-deleted) and GSTP1 (homozygous Ile-104) functional alleles.

Results

Table 3 describes the distribution of the glaucoma cases and the controls by sex and age status. The mean intraocular pressure of the glaucoma cases was 17.68±0.66 mmHg. The intraocular pressure of glaucoma patients were regulated by monotherapy in 48.34%, by two drugs in 39.96% and by three drugs in 11.70% of the cases.
Table 3

Descriptive statistics about age (p=0.11) and sex (p=0.22) in both groups

Variable

Cases (n=153) mean ± SD

Controls (n=159) mean ± SD

Age (years)

61.64±5.48

62.74±6.58

Sex

Cases (n=153) count (%)

Controls (n=159) count (%)

Female

77 (50.3)

69 (43.4)

Male

76 (49.7)

90 (56.6)

SD standard deviation.

The genotypes for each gene can be seen in Table 4. The frequency of the GSTM1 null genotype in patients (54.9%) was higher than in controls (40.9%). The GSTM1 null genotype had an increased risk of developing POAG (OR: 1.64, 95% CI: 1.10–2.59). The frequency of the GSTT1 null genotype was not significantly different between the glaucoma cases and the controls (OR: 0.94, 95% CI: 0.56–1.58). The GSTP1 val/val genotype was also not significantly different among cases and controls (OR: 1.35, 95% CI: 0.72–2.51).
Table 4

GST genotypes and the risk of developing POAG

Variable

Cases (n=153)

Controls (n=159)

n (%)

n (%)

ORa

95% CI

GSTM1b

 Present

69 (45.1)

94 (59.1)

1 (reference)

 Null

84 (54.9)

65 (40.9)

1.64

1.10–2.59

GSTT1b

 Present

115 (75.2)

118 (74.2)

1 (reference)

 Null

38 (24.8)

41 (25.8)

0.94

0.56–1.58

GSTP1

 Ile/Ile

75 (49.0)

76 (47.8)

1 (reference)

 Ile/Val

46 (30.1)

58 (36.5)

0.86

0.51–1.44

 Val/Val

32 (20.9)

25 (15.7)

1.35

0.72–2.51

aORs odds ratio; CI confidence interval from binary logistic regression.

bCarriers of at least one intact allele are used as reference.

Also, the frequency of the GSTP1 genotypes is shown in Table 4. The variant genotype, termed val/val, was seen in 20.9% of the cases and in 15.7% of the controls, which was not significantly different among groups.

The percents (%) of Val-105 allele were 34% for controls and 35.9% for cases. There was no significant difference between cases and controls about the percent of these alleles (p=0.60; Table 5).
Table 5

Allele counts and percents in each group

GSTP1 allelesa

Cases count (%)

Controls count (%)

Ile

196 (64.1)

210 (66.0)

Val

110 (35.9)

108 (34.0)

aNo statistically significant relation between allele frequencies and groups (p=0.60).

To investigate whether profiles of GST genotypes were associated with the risk of glaucoma or not, we examined the risk of glaucoma associated with combinations of genotypes. The reference group consisted of individuals with all three putative low-risk genotypes, i.e., the presence of GSTM1 and GSTT1 genotypes and the homozygous ile/ile genotype for GSTP1. Individuals heterozygous and homozygous for the Ile105val allele combined for this analysis. Table 6 displays the risk of glaucoma associated with each combination of genotypes as well as the trend in risk associated with one, two, or three putative high-risk genotypes. The data were suggestive of a trend of increasing risk with higher numbers of the combined GSTM1 null, GSTT1 null and GSTP1 105-val allele genotypes and the combined GSTM1 null, GSTT1 present and GSTP1 105-val allele genotypes; a 2.3-fold and 1.6-fold increased risk of glaucoma (95% CI: 0.75–7.08; 95% CI: 0.76–3.58; respectively) but this increase was not significant. These results indicate that GSTM1 influence glaucoma susceptibility in this study.
Table 6

Association between GST genotype profile and the development of POAG

S.no.

GSTM1

GSTT1

GSTP1

Cases n (%)

Control n (%)

OR (95% CI)

1

Present

Present

Ile/Ile genotype

25 (16.3)

29 (18.2)

1 (reference)

2

Null

Present

Ile/Ile

32 (20.9)

33 (20.8)

1.12 (0.54–2.31)

3

Present

Null

Ile/Ile

8 (5.2)

7 (4.4)

1.32 (0.42–4.17)

4

Present

Present

Ile/Val or Val/Val genotype

28 (18.3)

35 (22.0)

0.98 (0.47–2.05)

5

Null

Null

Ile/Ile

10 (6.5)

8 (5.0)

1.65 (0.54–4.99)

6

Null

Present

Ile/Val or Val/Val

30 (19.6)

21 (16.2)

1.65 (0.76–3.58)

7

Present

Null

Ile/Val or Val/Val

8 (5.2)

25 (13.2)

0.40 (0.15–1.06)

8

Null

Null

Ile/Val or Val/Val

12 (7.8)

3 (1.9)

2.32 (0.75–7.08)

Discussion

The basic cause of glaucoma is largely unknown. First degree relatives of glaucoma cases have 8–10 times increased risk of developing the disease, making genetic predisposition a strong risk factor [39]. Mutations in the TIGR/MYOC gene have been shown to be related with some forms of juvenile glaucoma, and they are also found in 1–4% of cases with adult onset POAG, but these can only explain a fraction of the genetics of the disease [1, 11, 36]. It is likely that other genes may contribute to the disease. Therefore, in recent years, the studies about the etiopathogenesis of POAG have focused on both gene mapping of different locuses of different chromosomes and GST, an antioxidant enzyme, polymorphisms.

Firstly, Juronen et al. [19] used an ELISA technique to show 60% of glaucoma patients were GSTM1 positive, as compared to 45% of the controls in the Estonian population (p=0.002). An association with a lower level of significance was found with the GSTM3 gene. The frequencies of the GSTT1 and GSTP1 genotypes in both groups were not statistically different. This study suggests that GSTM1 polymorphism may be associated with increased risk of development of POAG. Further evidence for involvement of GSTM in glaucoma comes from the studies of autoimmunity. GST antigen was found in 52% of cases with glaucoma and 20% of controls (p<0.05). The patients had significantly higher titers of anti-GST antibodies as compared to the controls (p=0.013 in normal tension glaucoma and p=0.0006 in POAG). Furthermore, in this study, it was shown that the related retinal antigen was GST class μ [40]. Thus, it is a reasonable hypothesis that the people who are expressing GSTM1 are at increased risk of developing auto-antibodies against this protein, which is related to an increased risk of developing glaucoma.

Then, a recent study from Sweden showed that the frequency of GSTM1 positive individuals was 44.0% in POAG and 44.5% in the controls [18]. There was no evidence of association between GSTM1 and glaucoma in the Swedish population. This study does not support the finding in Estonia, where 60% of the glaucoma cases were GSTM1 positive.

Contrary, Izzotti et al. [17] found that the GSTM1 null genotype was significantly more common in patients with POAG than the controls in Italian population. There were no differences in the frequencies of the GSTT1 null genotype, or the GSTT1 null and GSTM1 null genotypes, between controls and patients.

Our study also showed that the frequency of the GSTM1 null genotype in patients (54.9%) was higher than in controls (40.9%). The GSTM1 null genotype has been seem to have an increased risk of developing POAG. The frequency of the GSTT1 null genotype and the GSTP1 val/val genotype were not significantly different between the glaucoma cases and the controls. The results of our study supports the study of Izzotti et al. Interestingly, the patients and control groups studied in both studies belong to Mediterranean people.

The differences in the results of different populations may be due to the genetic and environmental background differences of these and/or the different techniques used in these studies. Finally, GSTM1 null genotype, which causes the absence of enzyme activity, may be associated with higher risk of developing glaucoma whereas GSTT1 and GSTP1 genotypes are not. Our results showed that GSTM1 was possibly associated with susceptibility to glaucoma. Although our results contradict earlier reports showing no association with GSTM1 genotype and open-angle glaucoma, the present data may reflect rassic differences between Scandinavian, Caucasian and Mediterranean populations. The results of this study are encouraging for further studies of genetic polymorphisms as the underlying causes of POAG.

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© Springer-Verlag 2004