Combined genotype analysis of GSTM1 and GSTT1 polymorphisms in a Polish population

Abstract This study describes the distribution of GSTT1 and GSTM1 polymorphisms in a normal population of central Poland. A homozygous inherited deletion of either gene leads to absence of enzyme activity in affected individuals, and those lacking more than one detoxifying gene are at the highest risk for diseases caused by environmental factors. The prevalence of the "null" genotype of the GSTM1 and GSTT1 genes was determined by using a multiplex polymerase chain reaction methodology in a group of 233 healthy individuals. We found the following frequencies of individuals with mutated alleles: 47.6% were homozygously deficient for GSTM1 (51.1% males, 42.4% females) and 16.3% for GSTT1 (17.7% males, 15.2% females). The combined analyses GSTM1/GSTT1 revealed the following genotypes: +/+, 44.2% (42.6% males, 46.7% females); "null"/+, 39.1% (39.7% males, 38.0% females); +/"null," 8.6% (7.1% males, 10.9% females); "null'/'null," 8.1% (10.6% males, 4.4% females). Of interest is the small number of women lacking both genes. Significant differences occurred between men and women in some age groups, which could suggest that sex differences in susceptibility to diseases may be caused by genotype differences in detoxifying enzymes such as glutathione S-transferase. The data obtained may prove to be useful for epidemiological studies.

KEY WORDS: GSTM1, GSTT1, GENOTYPE, POLYMORPHISM, CANCER EPIDEMlOLOGY

Cytosolic glutathione transferases (GSTs) catalyze the conjugation of toxic electrophils with glutathione-forming excretable hydrophilic metabolites (Mannervik and Danielson 1988). GSTs have also been implicated in a variety of resistance phenomena (McLellan and Wolf 1999; Walter et al. 1999; Walter and Kargas 2001). In the GSTM1 gene the "null" allele has been found to be present in high frequencies in the human population (depending on ethnicity). GSTM1 "null" may predispose affected individuals to greater risk from toxic xenobiotics (Strange et al. 2000; To-Figueras et al. 2000). GSTM1 "null" has been associated with increased susceptibility to inflammatory pathologies and increased risk of some cancers (Bell et al. 1993; Chenevix-Trench et al. 1995; Trizna et al. 1995). Clinical studies on GSTM1 "null" have suggested that lack of the gene might be associated with intermittent claudication and atherosclerosis (Pessah-Rasmussen et al. 1990; Evans et al. 1996) and with increased risk of recurrent dysmenorrhea (Wu et al. 2000). In contrast, it has been suggested that GSTM1 "null" is protective against myocardial infarction, especially in smokers (Wilson et al. 2000).

In the case of the GSTT1 gene the "null" allele occurs in 9% to 65% of various ethnic groups; its occurrence may possibly underlie an increased risk of toxicity in response to xenobiotics (Cotton et al. 2000; Hayes and Strange 2000; Landi 2000). Epidemiologic studies have indicated that GSTT1 "null" was associated with an increased risk of certain cancers among smokers (Chen et al. 1996; Brockmoller et al. 1996). Genotoxic effects such as induction of sister chromatid exchanges after exposure of human blood to methyl bromide and other agents in vitro have been found to be more pronounced among individuals lacking the GSTT1 gene (Schroder et al. 1995). The lack of a functional GSTT1*1 was estimated to increase the internal dose of ethylene oxide derived from cigarette smoke by 50% to 70% (Fennell et al. 2000).

Males with a deletion of the GSTM1 gene have been shown to be more susceptible to Parkinson's disease, and males with a deletion of the GSTT1 gene have been shown to be more susceptible to motor neuron disease and Alzheimer's disease (Stroombergen and Waring 1999).

This study was undertaken to determine the profile of the genetic polymorphisms in GSTM1 and GSTT1 genes separately and combined in a population from central Poland, with respect to sex and age differences.

Materials and Methods

To determine the GSTM1 and GSTT1 deletion, e used blood samples from 233 volunteers from the Regional Center of Blood Donation and Blood Therapy in Lodz, Poland. The volunteers consisted of 141 males and 92 females; they ranged in age from 18 to 60 years. Samples were obtained after meeting all ethical and legal requirements. DNA was extracted from human mononuclear blood cells. Genomic DNA was prepared using the guanidine isothiocyanate acid isolation method (Slomski et al. 1999). A multiplex polymerase chain reaction (PCR) method was used to detect the presence or absence of GSTM1and GSTT1 genes (Arand et al. 1996). PCR products were analyzed on 2.5% agarose gels that had been stained with ethidium bromide. As a DNA standard, a pUC19 (26-501 pb-MspI restricted) was used. The statistical significance of differences between groups was calculated by the [chi]^sup 2^ test. Confidence intervals (95% CI) for statistical evaluation of frequencies of studied genotypes were estimated. The expected number of all genotypes on the basis of Hardy-Weinberg proportions was deduced.

Results