Background: The variability in the distribution of the null phenotypes of GSTM1 and GSTT1, due to total or partial gene deletion resulting in the lack of the active enzyme, has been reported in different populations, especially in ethnically well-defined groups but not in Tabuk. This study investigated the variability in the distribution of the null phenotypes of GSTM1 and GSTT1 in the population of Tabuk (northwestern part of Saudi Arabia). Method: This study was conducted on 200 subjects of Tabuk—northwestern part of Saudi Arabia among which 100 were chronic smokers and 100 were nonsmokers. The subjects were reporting to hospital for routine checkup. All were without past history of any chronic disease and no significant abnormality. GST genotyping was done by multiplex PCR-based methods. The smoker and control groups were compared using a chi-square test with P < 0.05. Results: The distribution of the GSTM1 null genotype was more frequent among non smoker individuals (14%) than among smoker individuals (1%) and difference was statistically significant (P < 0.0001) whereas the distribution of the GSTT1 null genotype showed the highest frequency among non smokers (28%) than the smokers (6%) and the difference was statistically significant (P < 0.003). GSTM1 deletion homozygosity of 14% and 1% was reported among non smokers and smokers, respectively whereas GSTT1 deletion homozygosity of 28% and 6% was reported among non smokers and smokers, respectively. Our results indicate that there are major differences in allelic distribution of GSTM1 and GSTT1 genes between the two groups investigated. Combined analysis of both genes revealed that 15% of smokers and non smokers harbor the deleted genotype of GSTM1 and 34% of smokers and non smokers harbor the deleted genotype of GSTT1 with significant differences. Conclusion: This study enables selecting subgroups among the general population who are more susceptible to DNA damage and will help genetic studies on the association of GST polymorphisms with disease risks and drug effects in Arab population. Studies with a larger sample size are needed to evaluate and confirm the validity of our results.
GST family genes have a critical role in xenobiotic metabolism and drug resistance and have a major role in the carcinogen detoxification process. GSTs play a critical role by protecting against the ROS caused by the breakdown of the peroxidased lipids and they are capable of oxidizing DNA and generating damage [
A number of studies done so far in different populations have shown that polymorphisms within the GST genes play an important role in determining individual susceptibility to cancer; however, data obtained so far have been contradictory within the same or different populations. Molecular epidemiological studies have provided three pieces of information about the relationship of GSTM1 and GSTT1 with cancer susceptibility. First, the frequencies of homozygous GSTM1 and GSTT1 deletion carriers are very high (i.e., 20% - 50%) in most populations studied to date. Second, GSTM1 and, possibly, GSTT1 may be involved in the etiology of cancer at more than one site. Third, the risk conferred to individuals who carry homozygous deletions in GSTM1 and GSTT1 appears to be small in magnitude (e.g., odds ratio of <2). However, the magnitude of risk is larger (e.g., odds ratio of 3 - 5) when interactions of GSTM1 of GSTT1 with other factors (e.g., cigarette smoking) are considered. These findings have implications for studies of GSTM1 and GSTT1 in cancer susceptibility and for future applications of these biomarkers in cancer prevention of control strategies [
Several epidemiological studies suggest that individuals who are homozygous null at the GSTM1 and GSTT1 loci (i.e., lack both copies of the GSTM1 and/or GSTT1 variants of the GST gene family) have an increased risk of developing various types of neoplastic diseases, including cancers of the bladder, prostate, colon, skin, lung, head and neck and stomach [
The study was conducted in the Division of Cancer Molecular Genetics, Prince Fahd Bin Sultan Research chair, University of Tabuk, Saudi Arabia.
This study included 100 healthy individuals (smokers) and 100 nonsmokers of TABUK-northwestern Saudi Arabia. The subjects were reporting to hospital for routine checkup.
All the Subjects were interviewed using a structured questionnaire regarding epidemiological/demographic data, past history, history of addiction particularly smoking, family history of any significant diseases.
Venous blood was collected in EDTA tubes from all subjects and controls and the high molecular genomic DNA was isolated using DNA extraction QIAamp DNA Blood Mini Kit (51106) and samples were stored at −20˚C until PCR. The ratio of absorbance at 260 nm and 280 nm was used to assess the purity of DNA by Thermo Scientific NanoDrop™. A ratio of ~1.8 to 1.95 was generally obtained.
Briefly, 50 ng of DNA was amplified in a 25 μl multiplex reaction mixture containing 25 pmol of each of the following primers as depicted in
The distribution of the GSTM1 and GSTT1 genotypes was evaluated for Hardy-Weinberg equilibrium using chi-squared analysis, as described. Chi Square test or Fisher Exact Probability test was used for analysis of frequency distribution of subjects in various subgroups. Odds ratio and confidence interval were used to estimate risk of contracting disease in presence of various risk factors. Statistical significance was accepted as P < 0.05 (Two tailed). All analyses were done using EPI 6 software (Epi info 6 CDC).
Gene | Primer sequence | Annealing tempt | PCR products |
---|---|---|---|
GSTM1 | F-5’-GAACTCCCTGAAAA GCTAAAGC-3’ | 58˚C | 215 bp |
R-5’-GTTGGGCTCAAATA TACGGTGG-3’ | |||
GSTT1 | F-5’-TTCCTTACTGGTCCT CACATCTC-3’ | 480 bp | |
R-5’-TCACGGGATCATGGCC AGCA-3’ | |||
CYP1A1 | F-5’-GAACTGCCACTTCAGCTGTCT-3’ | 312 bp | |
R-5’-CAGCTGCATTTGGAAGTGCTC-3’ |
GSTM1 and GSTT1 genotyping were assessed by multiplex PCR in 100 smoker individuals and 100 non smoker individuals of Tabuk a Northwestern part of Saudi Arabia). While this method does not differentiate between wild-type and heterozygous states, it determines the percentages of the homozygous deletion of both GSTM1 and GSTT1 genes. GST genotypes were coded as positive (wild-type homozygotes and deletion heterozygotes), or as null (homozygous deletion). This made direct calculation of Hardy Weinberg Equilibrium impossible.
The distribution of the GSTM1 null genotype was more frequent among non smoker individuals (14%) than among smoker individuals (1%) as depicted in
The significant correlation was reported between non smoker individuals and smoker individuals. The difference was statistically significant (P < 0.0003) as depicted in the
The significant correlation was reported between non smoker individuals and smoker individuals as depicted in
The allele frequencies of GSTT1 (normal) and (null) genotypes in according to Chi-sq Hardy-Weinberg equilibrium test calculator for biallelic markers as depicted in
The allele frequencies of GSTM1 (normal) and (null) genotypes in according to Chi-sq Hardy-Weinberg equilibrium test calculator for biallelic markers as depicted in
The “worst” detoxifier genotype (the combined genotype of GSTM1 null and GSTT1 null) was found in 14 of
Group | N | GSTM1 normal genotype | GSTM1 null genotype | GSTT1 normal genotype | GSTT1 null genotype |
---|---|---|---|---|---|
Smokers | 100 | 99 (99%) | 01 (1%) | 94 (94%) | 6 (6%) |
Non smokers | 100 | 86 (86%) | 14 (14%) | 72 (72%) | 28 (28%) |
Group | GSTM1 normal genotype | GSTM1 null genotype | Chi-square | P value |
---|---|---|---|---|
Smokers | 99 | 01 | 10.38 | <0.0013 |
Non smokers | 86 | 14 |
GST = glutathione S-transferase; GSTM1 = GST mu; *P < 0.05 compared between smokers and non smokers (Fisher exact test).
Group | GSTT1 normal genotype | GSTT1 null genotype | Chi-square | P value |
---|---|---|---|---|
Smokers | 94 | 6 | 15.63 | <0.0001 |
Non smokers | 72 | 28 |
GST = glutathione S-transferase; GSTT1 = GST theta. *P < 0.05 compared between smokers and non smokers (Fisher exact test).
Group | GSTT1 normal genotype | GSTT1 null genotype | GSTT1 Allele freq | GSTT1 null freq |
---|---|---|---|---|
Smokers | 94 | 6 | 0.94 | 0.06 |
Non smokers | 72 | 28 | 0.72 | 0.28 |
Group | GSTM1 normal genotype | GSTM1 null genotype | GST M1 Allele freq | GST M1 null freq |
---|---|---|---|---|
Smokers | 99 | 01 | 0.99 | 0.01 |
Non smokers | 86 | 14 | 0.14 | 0.14 |
100 (GSTM1) and 28/100 (GSTT1) northwestern Saudi Arabia female populations and 1 of 100 (GSTM1) and 14/100 (GSTT1) northwestern Saudi Arabia male populations. The difference was statistically significant as depicted
This is the first study to evaluate the prevalence of the GSTT1 and GSTM1 gene polymorphisms in Tabuk Region (Northwestern Saudi Arabia), because no data are available for this region. This study provided us with an excellent opportunity to explore the frequency of GSTM1 and GSTT1 null genotypes, gene-gene and genotype- cigarette smoking interactions.
Tobacco smoke is a major cause of many diseases. Although its carcinogenic role via induction of DNA damage and mutation is well established, GSTM1-null was reported to be associated in some studies with increased susceptibility to inflammatory pathologies and increased risk of smoking-related cancers [
Tobacco smoking is a strong risk factor in the development of cancers. Tobacco is consumed both in smoking and smokeless forms. Tobacco smoke comprises nearly 60 carcinogenic compounds whereas its unburned form contains 16 identified carcinogens [
Group | N | Smokers | Non smokers | OR* (95% CI) | RR# (95% CI) | RD^ (95% CI) | P value |
---|---|---|---|---|---|---|---|
GSTT1 normal genotype | 100 | 94 (94%) | 72 (72%) | 1.00 | 1.00 | ||
GSTT1 null genotype | 6 (6%) | 28 (28%) | 5.2 (2.03 - 13.45) | 2.8 (1.37 - 5.86) | 36.6 (20.45 - 52.80) | <0.0005 | |
GSTM1 normal genotype | 100 | 99 (99%) | 86 (86%) | 1.00 | 1.00 | ||
GSTM1 null genotype | 01 (1%) | 14 (14%) | 16.1 (2.08 - 125.1) | 8.0 (1.2 - 53.58) | 46.8 (32.3 - 61.37) | <0.0013 |
*OR: Odd ratio #RR: Risk ratio ^RD: Risk difference.
polymorphisms on the risk of developing different disease. When the findings of them are examined in detail, it can be seen that different results exist. As the GSTs are important in the detoxification of toxic molecules in tobacco, their variant genotypes should be investigated together in susceptibility to cancer.
The underlying reasons of these differences were the sample size of study, ethnic differences, the smoking habits (heavy/mild/light smoker), the type of endogenous and exogenous genotoxicants and exposure time to them as well as genetic structures of enzymes having role for the detoxification. The genetic structure (in terms of mutation and polymorphisms) of other enzymes having a role for the detoxification, diet and the exposure time to the endogenous and exogenous genotoxicants may affect the age of onset and type of cancer among different populations.
The frequency of the GSTM1 null genotype was more frequent among non-smoker individuals (14%) than the smoker individuals (1%). Similarly the higher frequency of GSTT1 null genotype was reported among nonsmokers (Females) (28%) than the smokers (6%). While the distribution of GSTM1 and GSTT1 null genotypes were described previously for different population as depicted in the
We investigated the polymorphism at GST loci in the populations of Tabuk Region in northwestern Saudi Arabia. The frequency of the GSTT1 null genotype in the present study was significantly higher than those in the populations of North Indian, South India, England, Turkish-USA, Brazil, Italy, Caucasians and Africa whereas the frequency of null genotype in the present study was significantly lower than those in Japan and China as depicted in the
Comparing the frequencies of GSTM1 deletion in our study with other studies, we found homozygosity generally comparable with Gambia. Similarly comparing the frequencies of GSTT1 deletion in our study with other studies, we found homozygosity generally comparable with Tunisians (37%) Bahraini (29%) and Lebanese
Country/region | GSTM1 | GSTM1 null | GSTT1 null | References |
---|---|---|---|---|
Tabuk (Northwestern Saudi Arabia) | 100 | 15% | 34% | Our study |
Lebanese | 141 | 52.5% | 37.6% | [ |
Tunisians | 186 | 63.3% | 37.1% | [ |
Bahrainis | 167 | 49.7% | 28.7% | [ |
North India | 248 | 33% | 18.4% | [ |
England | 88 | 50.8% | 16.9% | [ |
Central Europe | 70 | 45% | 13% | [ |
Turkish | 64 | 51.9% | 16.3% | [ |
Japan | 39 | 55.7% | 44.3% | [ |
W-USA | 188 | 52% | 16% | [ |
Chinese | 234 | 51% | 46% | [ |
Brazilian Non White | 179 | 34.2% | 25.7% | [ |
Italians | 145 | 46.9% | 19% | [ |
Caucasian | 85 | 48.8% | 19.9% | [ |
South India | 361 | 30.1% | 16.8% | [ |
Egypt | 89 | 55.5% | 29.5% | [ |
African-American | 195 | 28% | 17% | [ |
Gambia | 269 | 20.2% | 37.1% | [ |
(37%) and Egypt (29%). Statistically significant differences were detected when we compared smokers and non smokers (P < 0.001), which may be due to gender and ethnic mixture in this population. The homozygosity for the GSTT1 deletion in the four populations Tunisian (37%) Bahraini (29%) and Lebanese (37%) and Egypt (29%) were comparable to those reported for Tabuk population of Saudi Arabia. GSTM1 null genotype are not uniformly distributed among diverse population, but an ethnic and geographic basis of distribution was suggested.
There are wide variations in the frequencies of GSTT1 deletion (GSTT1*0/0) among different ethnicities. Human phenotyping is facilitated by the GST activity towards methyl bromide or ethylene oxide in erythrocytes which is representative of the metabolic GSTT1 competence of the entire organism. Inter-individual variations in xenobiotic metabolism capacities may be due to polymorphisms of the genes coding for the enzymes themselves or of the genes coding for the receptors or transcription factors which regulate the expression of the enzymes.
The polymorphisms in several regions of genes may cause altered ligand affinity, transactivation activity or expression levels of the receptor subsequently influencing the expression of the downstream target genes. Studies of individual susceptibility to toxicants and gene-environment interaction are now emerging as an important component of molecular epidemiology.
Nevertheless the activity of these enzymes may differ due to polymorphisms which ultimately results in interindividual susceptibility to cancer development. Our results provided useful information in selecting target SNPs that are likely to have an impact on GST activity and contribute to an individual’s susceptibility to the disease. Furthermore, protective effect of GSTT1 was strongly associated with smokers only. It is suggested that GSTM1 and GSTT1 genotypes do not individually influence susceptibility to develop disease.
This is the first study that addresses GST gene polymorphism among the populations of Tabuk (northwestern part of Saudi Arabia) and will help genetic studies on the association of GSTM1 and GSTT1 polymorphisms with disease risks and drug effects in Arab population. However, further studies with a larger sample size are needed to provide more precise evidence.
We acknowledge the support of Dr. Fawzia Sharaf for providing Non smoker subjects and Dr. Jamsheed Javid for his technical support.
The authors declare that they have no competing interests.
RashidMir,AbdullahYahya Hamadi,Abu-DuhierF.M., (2016) Frequency of Null Phenotypes of Glutathione S-Transferase M1 and T1 among the Populations of Tabuk (Northwestern Part of Saudi Arabia). Open Journal of Genetics,06,9-18. doi: 10.4236/ojgen.2016.61002