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Biochemical and Biophysical Research Communications 269, 676–680 (2000)

doi:10.1006/bbrc.2000.2338, available online at http://www.idealibrary.com on

0CA

STM1 and mEPHX Polymorphisms in Parkinson’sisease and Age of Onset

. Ahmadi,* M. Fredrikson,† H. Jerregård,* A. Åkerback,* P. A. Fall,‡

. Rannug,§ O. Axelson,† and P. Soderkvist,*Division of Cell Biology, Department of Biomedicine and Surgery, †Division of Occupational and Environmentaledicine, Department of Health and Environment, and ‡Division of Geriatrics, Department of Neuroscience andocomotion, Faculty of Health Sciences, University Hospital, S-581 85 Linkoping, Sweden; and §National Institute

or Working Life, Solna and Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden

eceived January 28, 2000

tron transport chain defects (2, 3). Impaired cellulardmpp

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Both environmental and genetic factors are in-olved in the development of PD and biotransforma-ion of exogenous and endogenous compounds anday play a role in inter-individual susceptibility.herefore, we investigated the presence of null geno-

ypes of GSTM1, GSTT1, and two polymorphisms ofEPHX in subjects with Parkinson’s disease and in a

eference population. The study included 35 male PDatients and a male control group including 283 sub-

ects. Homozygosity of the histidine (H) 113 isoform ofEPHX was significantly increased in PD patients

odds ratio 5 3.8 CI 95% 1.2–11.8) and analysis of allelerequencies displayed an increased frequency of the-allele among PD patients (odds ratio 5 1.9 CI 95%

.1–3.3). However, a significantly elevated median ageor the onset of PD was found among GSTM1 genearriers (median age 5 68 years) compared to PD pa-ients being GSTM1 null genotypes (median age 5 57ears). Our observations suggest that (H) 113 isoformf mEPHX, which has been suggested as a low activitysoform, is overrepresented in PD patients and thatnherited carriers of the GSTM1 gene postpone thenset of PD. These detoxification pathways may rep-esent important protective mechanisms against reac-ive intermediates modifying the susceptibility andnset of PD. © 2000 Academic Press

Key Words: GSTM1; mEPHX; polymorphism; Parkin-on’s disease; age of onset.

Parkinson’s disease (PD) is a common progressiveeurodegenerative disorder characterized by degener-tion of nigrostriatal dopaminergic neurons includinghe loss of cell bodies in the pars compacta of substan-ia nigra (SN) (1). The mechanism for neurodegenera-ion is unknown, but the pathogenesis is considered toe multifactorial involving exposure for toxins, geneticnheritance, oxidative stress and mitochondrial elec-

676006-291X/00 $35.00opyright © 2000 by Academic Pressll rights of reproduction in any form reserved.

efense against reactive intermediates in the oxidativeetabolism of endogenous as well as exogenous com-

ounds, oxidative stress or antioxidant deficiency mayromote the development of PD (4).Microsomal epoxide hydrolase (mEPHX) has a prom-

nent role in the xenobiotic metabolism and cleaves aange of reactive epoxides to form trans-dihydrodiols5). Two amino acid polymorphisms have been identi-ed in the coding region of exon three and four in theEPHX gene. In exon three, the tyrosine 113 histidine

Y113H) exchange, has been proposed to result in a lowctivity form of the enzyme, (6) which may influencepoxide deactivation in the cell. This low activity forms associated with susceptibility of aflatoxin B1 inducedepatocellular carcinoma (7) and decreased risk forvarian cancer (8). The polymorphism in exon four,istidine 139 arginine (H139R), has been suggested ashigh activity isoform of mEPHX (9, 10). Heterozy-

osity of this polymorphism (H139R) has been associ-ted to chronic obstructive pulmonary disease but notelated to lung cancer or emphysema (9, 10). Further-ore, epoxide hydrolase plays an important role in the

etoxication of procarcinogens activated by some cyto-hrome P450:s (10) and as a consequence, may alsolay an important role in adverse drug responses (11).Reduced glutathione (GSH) plays a major role in the

emoval of reactive metabolites in the cell and reducedevels of GSH in SN have been reported in PD andostulated to be important in the pathogenesis (12, 13).he glutathione S-transferases (GSTs) are critical foronjugation of electrophilic compounds to GSH, andultiple isozymes with overlapping substrate specific-

ties have been identified (14). The GSTM1 and GSTT1enes code for two isozymes that are polymorphic inumans and homozygous deletions of the genes, theull genotypes, have a frequency among Caucasians ofpproximately 50% and 10–20%, respectively (15, 16,

23). The GSTM1 null genotype has been associatedwbbtfmirnnvP

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Vol. 269, No. 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ith an increased risk of smoking induced lung andladder cancers (17) and the GSTT1 null genotype haseen linked to an about four-fold risk of myelodysplas-ic syndrome (18) and recently also as a predisposingactor for colorectal cancer (19, 20). Since the enzy-atic activities of GSTs and mEPHX may represent

mportant protection mechanisms against intracellulareactive intermediates both of exogenous and endoge-ous origin, we tested whether polymorphisms for theull alleles of GSTM1 and GSTT1 and the two geneticariants for mEPHX might affect the risk of developingD.

ATIENTS AND METHODS

Whole blood samples from 35 levedopa positive PD patients, age0–80 years old, visiting the Clinic for Geriatrics, University Hos-ital in Linkoping, Sweden, were collected and leukocyte DNA wassolated with Wizard Genome DNA purification kit (Promega Inc.).hese patients constitute a subgroup within an epidemiologicaltudy on risk factors for PD (21). The project was approved by thethical committee at the Faculty of Health Sciences in Linkopingniversity. As comparison group, a population-based control group

rom a lung cancer study in Sweden (n 5 283, age 30–80) washosen.Two amino acid polymorphisms in the mEPHX gene were deter-ined in genomic DNA in a PCR-RFLP assay (8, 9). These genetic

olymorphisms have been identified within the coding region of theEPHX gene at exon 3 and 4, and results in substitution of amino

cids Y to H at residue 113 and H to R at residue 139, respectively (8,). The GSTM1 and GSTT1 null genotypes were asesssed in a mul-iplex PCR-reaction with b-globin as an internal control gene for auccessful PCR-amplification (18).Neither the controls nor the PD patients were normally distrib-

ted with regard to age (Shapiro-Wilk test, SAS v. 6. 12, SAS Inst.nc. Cary, NC). Therefore, statistical analysis were performed usingnonparametric method; Wilcoxon rank sum test. Relative risk was

xpressed as Odds Ratio (OR) and 95% Confident Intervals (CI) werealculated for the ORs. Since the fraction of women and their ageistribution differed between PD and available women in controlroups, they were excluded in the analysis for homogeneity reasons.Isolation of DNA and RNA were performed by extracation with

RIzol (GibcoBRL, Grand Island, NY) from normal tissue of SN. Theissue was collected at the University Hospital, Linkoping, Swedenmmediately frozen within 24 h postmortem and kept at 270°C untilnalysis. Expression of GSTM1, GSTT1 and mEPHX genes werenalysed with exonic primers spanning an intervening intron byeverse transcriptase (RT)-PCR of isolated total RNA from SN (9,8). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was forhis reason used as an internal control instead of b-globin, in theT-PCR experiments.

ESULTS AND DISCUSSION

Null genotypes of drug metabolizing enzymes areost likely to influence disorders of a specific tissue or

ell type where the gene is expressed. Therefore, theene must be expressed in the tissue of study. Nonformation about GSTM1 or GSTT1 expression pat-erns in SN, the target tissue of PD, is reported earliern the literature. Therefore, a RT-PCR based expres-ion study was undertaken of these genes in SN (Fig.

677

). As demonstrated in Fig. 1, GSTM1 (1A) in contrasto GSTT1 (1B) is expressed in SN, from individualsho are GSTM1 and GSTT1 gene carriers. TheEPHX expression in SN was determined from exons

hree and four from cDNA (9). In Fig. 1C it is evidenthat mEPHX is expressed in this tissue.

The overall frequency of H 113 mEPHX variantmong PD cases and controls was 21.2% and 8.1%,espectively, and found to differ significantly (OR 5 3.8I 95% 1.2–11.8, p 5 0.008; Table 1). The number oflleles for Y and H were also found to be significantlyifferent among PD cases and controls (OR 5 1.9 CI5% 1.1–3.3 p 5 0.013; Table 1). Regarding the H139Rolymorphism, no difference between controls and PDases was found (Table 1). Our results reveal an asso-iation between a genetically defined polymorphism inEPHX, which may correspond to a low activity form

f the enzyme and susceptibility to PD.

FIG. 1. Analysis of GSTM1(A), GSTT1(B), and mEPHX(C)RNA expression in substantia nigra from normal individuals. CD

nd D are abbreviations for cDNA and genomic DNA, respectively.A) In lanes 1CD and 2CD, a PCR fragment of 136 bp indicates theresence of GSTM1 cDNA and in the corresponding genomic DNA;anes 1D and 2D, a 231 bp long PCR fragment is visible. Lanes 3CDnd 3D represent cDNA and genomic DNA from a GSTM1 nullndividual. The 356-bp GAPDH band serves as an internal control,or a successful PCR amplification for both cDNA and genomic DNA.B) Samples 2 and 3 display the presence of GSTT1 (459 bp) gene (2Dnd 3D) but absence of gene expression in SN cDNA (255 bp) (2CDnd 3CD) . In sample 1 expression of GSTT1 gene was not detectedither in cDNA or in genomic DNA. The 356-bp GAPDH band servess an internal control, for a successful PCR amplification for bothDNA and genomic DNA. Staining below 100-bp molecular markerepresents primer dimer formation. (C) PCR amplification ofEPHX with primers located in exon 3 and 4 spanning intron 3 (5–7

b long) with cDNA from SN, giving a 383-bp-long product.

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Vol. 269, No. 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The overall frequency of GSTM1 and GSTT1 nullenotypes among PD was 48.6% and 11.4%, respec-ively. In the control population for this study, therequency of GSTM1 null genotype was 54.4% (Table), which is not statistically different to the PD casesnd also consistent with frequencies found in otheraucasian populations (19). The GSTT1 gene displaysnull genotype in English, American and Swedish

opulations of approximately 10–20% (16, 22, 23). Inur study, the overall frequency of GSTT1 null geno-ype among the controls is 13.8%, and among Swedesn general is 13.1% (23). The frequency of GSTT1 null11.4%) in PD derives from only four PD individualsnd thus it is not possible to make any firm conclu-ions. On the other hand it is unlikely that GSTT1lays any major role in PD, since the enzyme is notxpressed in SN (Fig. 1B).Analysis of the age of onset of PD revealed a higheredian age for the onset of PD among GSTM1 gene

arriers (median age 5 68 years) compared to PD pa-

Distribution of GSTM1, GSTT1, and mEP

Genepolymorphism Genotype

Control,n (%)

1 129 (45.6)GSTM1

2 154 (54.4)1 244 (86.2)

GSTT12 39 (13.8)

YY 149 (52.7)mEPHX YH 111 (39.2)(Y113H) HH 23 (8.1)

Y allele 409

H allele 157HH 158 (56.6)

mEPHX HR 103 (36.9)(H139R) RR 18 (6.5)

H allele 419

R allele 139

a x2 test.b YY versus HH.c HH versus RR.

TAB

Age-Related Differences in GSTM1 Gen

Total n

GSTM1 null

n % Median

ontrol 283 154 54.4 49D 35 17 48.6 57

a Wilcoxon rank sum test.

678

ients being GSTM1 null genotypes (median age 5 57ears, p 5 0.013, Table 2). In the control population noifference in median age between GSTM1 null orSTM1 gene carriers was evident (Table 2). In order to

ule out any potential bias due to non-normal age dis-ribution among GSTM1 gene carriers and null geno-ypes in the PD patient group and the controls, the PDatients were age-matched with the control subjectsefore comparisons of genotype frequencies.There was no increased frequency of GSTM1 null

enotypes with increasing age in the general popula-ion, indicating that GSTM1 null genotype do not applyny selective pressure for survival. Some reports hy-othesize that oxidative stress and reactive intermedi-tes from biotransformation of exogenous as well asndogenous compounds may influence the develop-ent of PD (24, 25). Our results support this conten-

ion and the presence of GSTM1 genes thus might haveprotective function against reactive biomolecules in

N. A further result is that the GSTM1 null genotype

Genotypes in Controls and PD Patients

PD,n (%) ORa 95% CIa p value

18 (51.4)1.23 0.6–2.7 0.51

17 (48.6)31 (88.6)

1.24 0.4–4.4 0.704 (11.4)

12 (36.4)14 (42.4) 3.8b 1.2–11.8b 0.0087 (21.2)

381.9 1.1–3.3 0.013

2824 (68.6)10 (28.6) 0.4c 0.02–2.8c 0.321 (2.8)

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Vol. 269, No. 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

f onset is influenced by the GSTM1 genotype, thismplies that GSTM1 is a modifier and that some otherene(s) also are involved in the etiology of PD. Thisossibility have been indicated also by genetic linkagend cloning studies of familial PD (26) where causativeutations in familial PD were identified in the-synuclein gene (27). Gasser et al. have mapped an-ther susceptibility locus for PD to chromosome 2p13hich is involved in the development of both familialnd sporadic PD (28). It is interesting to note that theverall frequency of GSTM1 null genotype among PD isimilar to and not statistically different from the con-rol population and the general population (29). A sim-lar result is also reported in a phenotypic study for theresence of GSTM activity in the blood of PD patients,30) and also in a report where the slow acetylatorenotype for N-acetyltransferase 2, was found to bessociated with familial and sporadic PD (31).The age of onset for PD also differed with regard to

eing a GSTT1 gene carrier or not (median age 5 49mong GSTT1 null compared to median age 5 64mong GSTT1 gene carriers; p 5 0.18). The studyroup is too small to make any conclusions aboutSTT1 as a potentially predisposing factor. On thether hand, the GSTT1 gene exhibits a specific expres-ion distribution pattern involving lung, liver and co-on (32, 33) but not SN (Fig. 1B). Its role as a defenseystem in SN is therefore likely to be of limited impor-ance. In contrast, the GSTM1 enzyme activity is ex-ressed in the brain (32) and SN (Fig. 1A).The present study leads to the conclusion that a

uggested low activity isoform of mEPHX is over rep-esented in PD patients and there seems to be a sub-roup of PD patients carrying the GSTM1 gene thatostpone disease onset. The GSTM1 genotype mayherefore act as a modifier of the response to cellularxposure for endogenous and/or exogenous reactive in-ermediates, influencing the individual susceptibilityor PD. This also implies that other factors (i.e., geneticnd/or environmental) act in concert, resulting inarly-onset of PD among GSTM1 null individuals. In atudy on solvent induced chronic toxic encephalopathyepresenting a toxic neurodegenerative disorder, it wasound that individuals with GSTM1 null genotype weret increased risk after long-term exposure for solvents,34) revealing the interaction between genetic and en-ironmental factors for disease pathogenesis. Further-ore, the present study supports the potential role ofEPHX and GSTM1 as cellular protective mecha-isms in SN and modifiers for the onset of PD.

CKNOWLEDGMENTS

This study was partly supported by grants from the County Coun-il of Ostergotland, FORSS and the University Hospital in Linkop-ng. A. Ahmadi is grateful for a generous support from the Linkoping

679

trategic Research, Sweden. Thanks to Lisbeth Hjalle and Professoran Marcusson for human tissue samples of SN.

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