Determination of the GSTM1 gene deletion frequency in Parkinson's disease by allele specific PCR

Elsevier

PII: S1353-8020(96)0001&4

ELSEVIER

Parkinsonism 8 Related Disorders Vol. 2, No. 3, pp. 151-154,19!?6 Copyright 0 1996 Elswier Science Ltd. All rights reserved Printed in Great Britain 1353-6020/% $15.00 + 0.00

Determination of the GSTMl Gene Deletion Frequency in Parkinson’s Disease by Allele Specific PCR MAUDY C.M.J. STROOMBERGENV, ROSEMARY H. WARINGt, PHILIP BENNETTS AND ADRIAN C. WILLIAMS9 Received 3 October 1995; accepted 4 March 1996

We have examined the occurrence of GSTMl null, one of the glutathione S-transferase p genes, in a control and a Parkinson’s disease group. By using the polymerase chain reaction (PCR) we found 67% of non-expressors compared with 51% in a control group (xf = 5.535; p < 0.025). These results suggest that a deletion of the GSTMl gene may be associated with a susceptibility to Parkinson’s disease. Copyright 0 1996 Elsevier Science Ltd. Glutathione S-transferase

GSTMl

PCR

Parkinson’s disease

Gene deletion

more, the substantia nigra of patients with PD reveals evidence of oxidative stress as shown by increased levels of lipid peroxidation [9]. Glutathione also combines with many environmental toxins via the glutathione S-transferase family of enzymes to form Ssubstituted glutathione derivatives. The relevance of the glutathione S-transferase isoenzymes to neurodegenerative processes is not known.

INTRODUCTION Parkinson’s disease and oxiaUive stress Patients with a chronic neurological dysfunction [e.g. Parkinson’s disease (PD)] appear to have reduced activity for detoxification of certain environmental compounds [l-6]. This suggests that neurological diseases may be the result of interactions between exposure to toxins combined with increased susceptibility due to genetic faults in the enzyme systems which detoxify harmful chemicals. These toxins may be endogenous or exogenous. Oxidative stress has been suggested as a major factor in PD; this may be due to generation of reactive oxygen species (ROS) by a malfunction in the oxidative phosphorylation pathway [7]. Glutathione functions as a major antioxidant defence against reactive species derived from hydrogen peroxide (HZO,). The normal process for the decomposition of HzOZin the brain is the oxidation of reduced (GSH) to oxidized (GSSG) glutathione by glutathione peroxidase [8]. It has been shown that many patients with neurological problems have low levels of reduced glutathione in brain tissue. Further-

Glutathione S-transferuse The glutathione S-transferases (GST) are a family of dimeric enzymes that catalyse glutathione conjugation with electrophilic compounds. GST also mediates the repair of DNA and lipid oxidized by ROS [lo]. Therefore, the GST enzymes function as an intracellular detoxification system for mutagens, carcinogens and xenobiotic compounds. They also play an important role in the protection against oxidative stress, because they protect tissues from the endogenous organic hydroperoxides which are produced as a byproduct from the oxidative phosphorylation pathway 1111. Mammalian cytosolic GST can be grouped into a!, p, 0 and ?r gene families. The CX,p and ?r enzymes have basic, neutral and acidic iso-electric points respectively. In addition to these four cytosolic families, membrane bound forms of the enzyme exist. Ammo acid sequencing and cDNA cloning have demonstrated that the N-ethylmaleimide-activatable microsomal GST shares no homology with the cytosolic enzymes and therefore appears to be an example of convergent evolution [ 121.

tDepartment of Biochemistry, University of Birmingham, Birmingham, U.K. SUniversity Department of Medicine, Queen Elizabeth Hospital, Birmingham, U.K. SDepartment of Clinical Neurology, Selly Oak Hospital, Birmingham, U.K. *Address correspondence to: M.C.M.J. Stroombergen, Department of Biochemistry, University of Birmingham, Edgbaston, Birmingham, B15 2’IT, U.K. 151

M. C. M. J. STROOMBERGEN

152

The (Yand p GST isoforms are involved in removal of products of oxidative damage to DNA, such as DNA hydroperoxide [13, 141; the proteins themselves have antioxidant activity and their presence therefore reduces oxidative stress [15]. In humans, five ccGST genes have been identified so far. The most widely expressed gene is the GSTMl gene. It is polymorphic, due to a G-C534 exchange, leading to an amino acid exchange from the basic lysine to the near-neutral asparagine. Between 45 and 55% of a normal population does not have the GSTMl gene and therefore does not express the enzyme [17, 181. This results in homo- and heterozygous combinations of GSTMl null, GSTMl A and GSTMl B [16]. Much work has been published concerning GSTMl null homozygotes and possible susceptibility to several forms of malignacies [19-241. A characteristic feature of the enzyme encoded at this locus is its high activity towards certain mutagenic epoxides, including benzo[a]pyrene-4,5oxide and styrene-7,,8oxide [12]. This has led to the hypothesis that individuals with the null allele at this locus, and therefore deficient for p GST, are more susceptible to the toxic effects of various xenobiotics. Little research has been done on the GSTMl null variant and the susceptibility to PD. However, the pivotal role of these antioxidant enzymes and the protection against oxidative stress may provide an indicator of susceptibility to degenerative neurological disease. Other workers have found no significant differences in GST genotype in PD as compared with controls [25], but the numbers of PD patients were relatively small and all of the patients were on levodopa treatment.

MATERIALS

AND METHODS

Bloodsamples Blood samples (5 ml) from a group of non-neurological disease subjects (controls, 39 males and 45 females) and a PD group (71 males, 51 females) both in the same age group (mean age controls 59.8 + 13.3, mean age PD patients 66.3 f 10.7) were obtained from the Queen Elizabeth Hospital, Birmingham. Patients with clinically definite PD were recruited from the practice of two neurologists. None were taking, or had ever taken, L-dopa-containing preparations or any other medication. No patients with atypical features, such as ocular motility disorders, pyramidal or cerebellar signs, or autonomic failure were included. All patients exhibited at least two of the three cardinal features of the disease: tremor, rigidity and bradykinesia. Samples were collected in vacutainers containing EDTA and stored at -20°C until assayed. All subjects were Caucasian and had given written and informed consent. Ethical annroval II

et al.

TABLE 1. DNA regions and their primer sequences Region GSTMl Intron 6/7

Exon 415 j3-Globin

Primer sequence

5’-GCT TCA CGT GTT ATG AAG GTT C-3’ 5’-TTG GGA AGG CGT CCA AGC AG-3’ (A specific) 5’-TTG GGA AGG CGT CCA AGC GC-3’ (B specific) 5’-CGT CCC TAC ‘ITG ATT GAT GGG-3 5’-CTG GAT TGT AGC AGA TCA TGC-3 5’-ACA CAA CTG TGT TCA CTA GC-3’ 5’-CAA Cl-T CAT CCA CGT TCA CC-3

for this study had been obtained from the local committee. ldentifiuztion

ofpolymorphisms

at the GSTMl locus

DNA was isolated from leukocytes according to the method of Smith et al. [26]. Primers specific for exon 4/ 5 and intron 617 (GSTMI A or GSTMl B specific) of the GSTMl gene and primers for @lobin were used in a PCR reaction, allowing the amplification of DNA, depending on the GSTMl polymorphism [27] (Table 1). Primers for exon 4/ 5 are included in the reaction to determine presence of the GSTMl gene. The PCR reactions were performed in a solution containing 1 x reaction buffer (50 mM Tris-HCl (pH 9.0), 50 mM KCl, 7 mM MgClz, HT Biotechnology, Cambridge, U.K.), dNTPs (4 x 50 PM, Promega, Southampton, U.K.), Super Taq polymerase (0.25 U, HT Biotechnology, Cambridge, U.K.), 5 primers, A or B specific primer, (6 x 500 nM, Alta Bioscience, University of Birmingham, Birmingham, U.K.) and DNA isolated from leukocytes. The final volume of the solutions was 50 ~1. After the reaction (Table 2) the products were run on a 4% agarose gel containing ethidium bromide at 120 V for 40 min and the gel was photographed under U.V. light. Statistical methods Odds ratios and the corresponding x2 test were used, using one degree of freedom for the latter. Odds TABLE 2. PCR programme polymorphisms Temperature

(“C)

for the determination

Time (s)

of the GSTMl

No. of cycles

94

150

94 58 72

45 60 60

5

94 58 72

30 30 45

30

40

5

The temperatures 94”C, 58°C and 72°C are respectively DNA denaturation, primer annealing and elongation temperatures.

DETERMINATION

OF THE GSTMl GENE DELETION FREQUENCY

FIGURE 1. GSTMl polymorphisms on a 4% agarose gel. From left to right, lane 1: blank, lane 2: markers, lane 3,4: GSTMl A, lane 5,6: GSTMl B, lane 7,8: GSTMl AB, lane 9,lO: GSTMl null The I marks pglobin, II marks exon 4/5 and III marks intron 6/7.

ratios (OR) are odds of null phenotype in I’D patients divided by odds of null phenotype in controls. If OR-l, the GSTMl gene deletion is not related to the susceptibility to PD. RESULTS

The primers for @lobin, intron 6/ 7 and exon 4/5 of the GSTMl gene allowed amplification of DNA with base pair lengths of 110,132 and 273 respectively. The results of agarose-gel electrophoresis of the PCR amplification products for each of the possible GSTMl polymorphisms are shown in Fig. 1. DNA from those subjects heteroygous for GSTMl null and home- and heterozygous for GSTMl A or B allowed the amplification of the 132 and 273 bp fragments, whereas homozygotes for GSTMl null did not have these fragments. Table 3 shows the distribution of the GSTMl genotypes in both a control and I’D group. It shows that the PD group, in comparison with the controls, contains a significantly higher number of subjects (67%) who do not express the GSTMl gene (x? = 5.353; p < 0.025). The odds ratio was 1.955 (95% confidence interval = 1.104-3.459) The frequency of the deletion in the control group (51%) is in accordance with findings of previous research [17,18]. There was no significant difference in the incidence of the null variant in the control and PD group for both males (xf = 3.772) and females (XT = 1.777) and no significant difference between men and women in either the control (XT = 0.178) or the PD group (& = 0.012). DISCUSSION

The GST enzymes protect against oxidative stress as TABLE 3. GSTMl frequencies determined by PCR PD patients

Controls Total

Male

Female

Total

Male

Female

Subjects (n) GSTMl A (n)

84 7

39 2

45 5

122 11

71 7

51 4

GSTMl B (n) GSTMl AB (n)

29 5

15 3

14 2

26 3

15 1

11 2

GSTMl null (n) Nullfrequency

43 51

19 49

24 53

82 67

48 68

34 67

153

they remove many toxic substances by conjugation with glutathione while the proteins also act as antioxidants. A deletion of one of the genes for the GST ~1 enzyme, GSTMl has been related to the susceptibility of several different malignancies [19241. In this paper we have determined the frequency of the gene deletion in relation to Parkinson’s disease. We observed that in a Caucasian hospital control group 51% of the subjects have the GSTMl gene deletion and therefore do not express the GST p enzyme. This is comparable with results from normal control groups studied by other workers [17,18]. The I’D group however, shows a significant increase in the incidence of GSTMl null (67%). These results suggest that failure to detoxify environmental compounds adequately may be a risk factor for I’D, and that oxidative damage in the central nervous system (CNS) may lead to neurological dysfunction. It is not clear whether exogenous, environmental toxins are potentially involved. Such compounds could cross the blood-brain barrier and either exert direct effects on the CNS or down-regulate expression of the GST isoforms and thus indirectly reduce capacity to deal with oxidative stress. If, however, neurological damage in PD is largely due to ROS and endogenous toxins, then non-expression of the GSTMl genotype could be one of the factors in disease expression or progression. Furthermore, we found a 6% frequency in the control group for subjects who are heterozygous for GSTMl (GSTM AB) versus 3% in the PD group (xt = 1.626). Although the difference is not significant, previous work has shown that this heterozygous combination for GSTMl is related to a reduced incidence of coloredal and brain cancer [21]. Whether the GSTMl AB genotype is related to a reduced incidence of PD cannot be determined directly from these results as further studies are required with an increased number of subjects. As the PCR method used is not specific enough to determine whether the subjects are homo- (AA, BB) or heterozygous (A0, B0) for GSTMl A or B, it is not currently possible to discuss the relationship of these genotypes to possible susceptibility to or protection against Parkinson’s disease. The GSTMl gene deletion is autosomal and no differences were observed in frequencies of the different alleles in PD, although the x2 value for the differences for men in both the control and PD group are almost significant, whereas the differences for women in both groups are not significant. It has been shown that in female skin other GST isozymes (0~and ?r) are more abundant with a higher activity and protein content in comparison to male skin 1281. The female human colon also contains a GST 1~isozyme (p1 6.7) which is absent in male colon whereas the male colon has an approximately two-fold higher level of (Y class isozymes [29].

M. C. M. J. STROOMBERGEN et al.

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Although we have shown differences in the GSTMl genotype in DNA from white blood cells, this does not give information about the tissue expression of any particular p isozyme. GST p is known to be expressed in brain tissue [20] but the distribution is not known and nor are the effects of medication on enzyme activity. From the results presented here, it would be of interest to examine the expression and activity of the GSTMl forms in both PD and control brain, since the genotype differences found in leukocytes may reflect an altered capacity to metabolize both endogenous and exogenous toxins.

16.

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18.

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Acknozuledgements-We would Iike to thank the staff of the neurology ward E4A of the Queen Elizabeth Hospital and their patients for donating the blood, Dr Nuala Helsby for her help in writing this manuscript and Dr Andrew Birley for his statistical work.