Analysis of the Glutathione S-transferase M1 gene using pyrosequencing

and multiplex PCR–no evidence of association to glaucoma

Mattias Janssona, Alvaro Radaa, Lidija Tomicb, Lill-Inger Larssonb, Claes Wadeliusa,*

aRudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Uppsala SE-751 85, SwedenbDepartment of Ophthalmology, University Hospital, Uppsala, Sweden

Received 28 October 2002; accepted in revised form 2 April 2003

Abstract

The Glutathione S-transferase M1 (GSTM1) gene is reported to be involved in glaucoma, an eye disease with a largely unknown

mechanism. The gene is polymorphic and three alleles have been characterized. These are one complete deletion of the gene, GSTM1p0, and

two alleles differing only in a single amino acid substitution, GSTM1pA and pB. The two latter alleles seem to have equivalent function.

Approximately 45% of the European populations are GSTM1 positive. An Estonian study has found that 60% of the glaucoma patients are

GSTM1 positive as compared to 45% of controls ðP ¼ 0:002Þ: We genotyped 200 primary open angle glaucoma patients (POAG), 188

exfoliative glaucoma patients and 200 matched controls using multiplex PCR and pyrosequencing. Forty four per cent of the POAG patients

and exfoliative glaucoma patients, and 44.5% of the matched controls were GSTM1 positive. Using pyrosequencing we were able to

determine if the patients were homo- or hemizygous for the GSTM1 gene. Five per cent of the POAG patients, 7.4% of the exfoliative

glaucoma patients and 4.6% of the controls were homozygous for the presence of the GSTM1 gene. There is no evidence of association

between GSTM1 and glaucoma in the Swedish population.

q 2003 Elsevier Ltd. All rights reserved.

Keywords: glutathione S-transferase; GSTM1; primary open angle glaucoma; exfoliative glaucoma; pyrosequencing; genotyping

1. Introduction

The basic cause of glaucoma is largely unknown. First

degree relatives to glaucoma cases have 8–10 times

increased risk of developing the disease, making genetic

predisposition a strong risk factor (Wolfs et al., 1998).

Mutations in the TIGR/MYOC gene have been shown to

cause some forms of juvenile glaucoma, and they are also

found in 1–4% of cases with adult onset primary open angle

glaucoma (POAG), but they can only explain a fraction of

the genetics of the disease (Stone et al., 1997; Alward et al.,

1998; Wiggs et al., 1998; Fingert et al., 1999; Jansson et al.,

2003). By studying families with autosomal dominant forms

of glaucoma, other gene loci, presently called GLC1B, C, D,

E and F, have been mapped to chromosomes 2, 3, 7, 8 and

10, respectively (Stoilova et al., 1996; Raymond, 1997;

Wirtz et al., 1997; Sarfarazi et al., 1998; Trifan et al., 1998).

However, it is likely that still other genes contribute to the

disease. Recently, a gene in the GLC1F locus was identified

as encoding a protein named optineurin, which is mutated in

families mostly with normal tension glaucoma (NTG).

In addition, a predisposing allele, M98K, was found in

increased frequency in glaucoma patients with mostly

normal intraocular pressure (IOP) (Rezaie et al., 2002). At

present it is unknown whether this gene is involved in the

etiology of POAG as well.

Recently, evidence was presented which indicates that

the GSTM1 gene may be predisposing to glaucoma (Juronen

et al., 2000). The GSTM1 gene is polymorphic and presently

three alleles have been characterized, namely GSTM1pA,

GSTM1pB and GSTM1p0 (Seidegard et al., 1988; Widersten

et al., 1991). The latter allele contains a total deletion of the

gene. Approximately, half of the population are homo-

zygous for this deletion (GSTM1p0/0, or GSTM12/2 ) and do

not express any protein. The GSTM1pA and GSTM1pB alleles

differ only in the substitution of the amino acid K172N and

seem to have equivalent function. There are a number of

related genes, GSTM2, M3, M4 and M5, which are located in

0014-4835/03/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

DOI:10.1016/S0014-4835(03)00109-X

Experimental Eye Research 77 (2003) 239–243

www.elsevier.com/locate/yexer

* Corresponding author. Dr Claes Wadelius, Rudbeck Laboratory,

Department of Genetics and Pathology, Uppsala University, Uppsala SE-

751 85, Sweden.

E-mail address: claes.wadelius@genpat.uu.se (C. Wadelius).

a cluster on chromosome 1q13.3. GSTM1p0 is the result of

an unequal crossing over in this region resulting in

the deletion of the GSTM1 gene. Juronen et al. (2000)

used an ELISA technique to show that in the Estonian

population, 60% of glaucoma patients were GSTM1 positive

(GSTM1þ/2 or þ/þ ), as compared to 45% of the controls

ðp ¼ 0:002Þ: In a meta-analysis comprising 3500 individ-

uals from different European populations, it was found that

45.8% were GSTM1 positive (M. Wadelius, personal

communication), which is in agreement with the finding in

the controls from Estonia. 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 ðp , 0:05Þ: The patients

had significantly higher titers of anti-GST antibody as

compared to the controls (p ¼ 0:013 in NTG and p ¼

0:0006 in POAG). Furthermore, it was shown that the

related retinal antigen was GST class m (Yang et al., 2001).

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 connected to

an increased risk of developing glaucoma. We therefore

attempted to replicate the genetic study in the Swedish

population, using a clinically well-characterized material

of patients with POAG and exfoliative glaucoma, and

compare them to carefully matched controls in which

glaucoma was excluded by clinical examination. In

addition, we developed a method for genotyping GSTM1

using pyrosequencing.

Pyrosequencing is a method where one dNTP after the

other is added to the reaction and a light peak is generated

for every nucleotide that is incorporated. The height of the

peak is proportional to the number of bases integrated, so if

the sequence is, e.g. CC, then the peak has double height as

compared to the normal height of C. If a person is

heterozygous, e.g. for an A/C polymorphism, a ‘half height’

signal is generated for both the bases, one after the other

according to the dNTPs added. In this case, the GSTM4 gene

is always present in two copies whereas the GSTM1 gene can

be present in 0, 1 or 2 copies. Since the GSTM1 and GSTM4

genes differ at a number of individual bases in the

commonly amplified fragments, it should be possible to

distinguish the presence or absence of the GSTM1 gene by

analyzing the peak heights that identify the GSTM1 and M4

Fig. 1. Alignment of GSTM1 and M4. Primers P1, P2 and P3 were used for genotyping the GSTM1 gene. Primers P1 and biotinylated P2 was used for

amplification of product for pyrosequencing. The pyroprimer was used in the pyrosequencing reaction. Position denoted as N is a C/T polymorphism.

M. Jansson et al. / Experimental Eye Research 77 (2003) 239–243240

genes, respectively. Furthermore, it should also be possible

to quantify if a person has one or two copies of the GSTM1

gene.

2. Methods

2.1. Subjects

The patients were recruited at the out-patient clinic of the

Department of Ophthalmology, University Hospital,

Uppsala, Sweden and Tierp Hospital, and comprised 200

patients with POAG, 188 with exfoliative glaucoma and 200

age, sex and ethnically matched controls in which glaucoma

was excluded by measurement of the IOP and ophthal-

moscopy of the optic disc. DNA was extracted from white

blood cells by standard procedures (Miller et al., 1988). The

investigation was approved by the local Research Ethics

Committee, and an informed consent was obtained from all

subjects before inclusion into the study.

2.2. Genotyping

The frequency of GSTM1 positive and negative individ-

uals was determined by genotyping using a multiplex-PCR

protocol as described by Zhong et al. (1993) using primers

Fig. 2. Genotyping, using multiplex PCR, results where patients 1, 2, 5 and

9 have the GSTM1 and GSTM4 genes, whereas patients 3, 4, 6, 7, and 8 only

have the GSTM4 gene.

Table 1

Genotypes of the GSTM1 gene

GSTM12/2 GSTM1þ/2 or þ/þ

POAG 112 (56.0%) 88 (44.0%)

Exfoliative glaucoma 105 (56.0%) 83 (44.0%)

Controls 111 (55.5%) 89 (44.5%)

Fig. 3. Pyrosequencing pyrograms of the three possible outcomes of the GSTM1 assay. Arrows indicate some of the positions used to determine the number of

GSTM1 copies in the sample. Solid arrows indicate GSTM1 peaks and dotted arrows indicate reference peaks from GSTM4. Pyrogram A shows a sample with

the null phenotype, pyrogram B shows a sample containing one copy of the GSTM1 gene and pyrogram C shows a sample containing two copies.

M. Jansson et al. / Experimental Eye Research 77 (2003) 239–243 241

P1, P2 and P3. P1 and P2 amplify a 157 bp region from both

the GSTM1 and GSTM4 genes, while P1 and P3 amplify a

230 bp fragment from the GSTM1 gene only. In the

pyrosequencing protocol, DNA was amplified using primers

P1 and a 50 biotinylated P2 (Fig. 1). The amplification

products were then captured on streptavidin coated beads,

denatured and washed. The pyrosequencing primer was then

added and the mix was ready for analysis, as described

(Ronaghi et al., 1998). Pyrosequencing was performed using

the PSQe96 SQA Reagent Kit and Sample Preparation Kit

10 £ 96 with the pyrosequencing primer 50-CCT CCT TGG

CTG G-30. The pyrosequencing primer was designed using

the sequence from UCSC (NM_000850) and not the

sequence published by Zhong et al. (1993), which contained

errors. Statistical evaluation was made by x 2-analysis.

3. Results

Typical results of the genotyping are shown in Fig. 2.

Patients 1, 2, 5 and 9 have the GSTM1 gene (GSTM1þ/2

or þ/þ) and the others lack the gene. The result of the

analysis is shown in Table 1. The frequency of GSTM1

positive individuals was 44.0% in POAG and exfoliative

glaucoma and 44.5% in the controls. There is no

significant difference in the number of GSTM1 positive

individuals between the controls and the two patient

groups. This study does not support the findings in

Estonia, where 60% of the glaucoma cases were GSTM1

positive.

The significance of one or two copies of the GSTM1 gene

has not been investigated, mostly due to the difficulty in

assessing the number of copies. Using pyrosequencing, we

were able to distinguish between people who were hemi- or

homozygous for the presence of the GSTM1 gene (þ /2 or

þ /þ , respectively). Fig. 3 shows representative pyrograms

identifying people who have 0, 1 or 2 copies of the GSTM1

gene, respectively. Arrows indicate the distinguishing bases.

Five per cent of the POAG patients, 7.4% of the exfoliative

glaucoma patients and 4.6% of the controls were homo-

zygous, i.e. carried two copies of the GSTM1 gene (Table 2).

This is in accordance with the theoretical estimate using

Hardy–Weinberg equilibrium. Since 55% are GSTM1p0/0,

the allele frequency for GSTM1p0 is 0.74 and hence, 38%

are expected to be hemizygous and 7% homozygous for the

presence of a functional GSTM1 gene. No significant

difference in the number of homozygotes between the

patient groups and controls were found.

4. Conclusions

Some people lack the GSTM1 enzyme due to deletion of

the gene, and this is normally not associated with ocular

disease. A recent study from Estonia has indicated that there

may be an association between GSTM1 and glaucoma, since

60% of the glaucoma patients were GSTM1 positive as

compared to 45% of the controls. In the Swedish population,

44% of the patients affected by POAG and also exfoliative

glaucoma, and 44.5% of the matched unaffected controls

were GSTM1 positive as determined by multiplex amplifi-

cation genotyping. The frequency of GSTM1 positive

individuals in our study agrees well with a data from a

large meta-analysis of the European populations, where

46% were positive. Consequently, in the Swedish popu-

lation there is no indication that carrying a functional

GSTM1 gene is a risk factor for glaucoma. The higher

frequency found in glaucoma patients from Estonia could

represent a population specific effect, e.g. caused by

differences in genetic background between the Swedish

and Estonian populations. Such an explanation seems

unlikely. The positive association in the previous study

could also be a chance finding due to a statistical Type I

error. In our study, we used two methods for genotyping,

multiplex PCR and pyrosequencing, both of which gave the

same result. The Estonian analysis was performed using

ELISA, which should give the same result as genotyping

and could therefore not explain the different results.

Pyrosequencing is a recent method for genotyping and

reading short sequences. It has the sensitivity to determine

allele frequencies in pools of DNA with good precision

(Gruber et al., 2002). We have now shown that the method

can also be used for genotyping GSTM1, taking advantage of

differences in the sequence between GSTM1 and M4 in the

region of those genes amplified using the same primers. In

most of the individuals it was possible to unequivocally

determine the presence or absence of the GSTM1 gene

and also to establish if the person was GSTM1 hemizygous

( þ /2 ) or homozygous ( þ /þ ). In some people there were

sequence variants in the GSTM1 or GSTM4 genes, which

prevented determination of gene copy number. This is to be

expected, since rare alleles are present in all genes. In the

GSTM locus, there are several homologous genes, so

additional variation could in some cases be created by

gene conversion. However, such unusual alleles cannot

change the overall interpretation of the results.

Acknowledgements

We thank the patients for participating in the study.

Financial support came from the Swedish Research Council

(09747 and 12493), Synframjandet Research Foundation,

the 6th of December Foundation and the Makarna

Borgstrom Foundation. Pyrosequencing reagents were

supplied by Pyrosequencing AB.

Table 2

Number of homozygotes (þ/þ) of the GSTM1 gene

No. of patients typed GSTM1 þ/þ

POAG 199 10 (5.0%)

Exfoliative glaucoma 188 14 (7.4%)

Controls 194 9 (4.6%)

M. Jansson et al. / Experimental Eye Research 77 (2003) 239–243242

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