Original Article

Human GSTs Polymorphisms in the Hakka Population of South China and Their Associations with Family History of Several Chronic Diseases*

PAN ShangXia¹, YANG XingFen^1,#, YANG LinQing² , WEI Qing², YANG Ying¹, XU GuangNing²,
LIN ZhongNing², and HUANG JunMing¹

1. Guangdong Provincial Center for Disease Control and Prevention, Guangzhou 510300, Guangdong, China;
2. School of Public Health, Sun Yat-Sen University, Guangzhou 510080, Guangdong, China

 

INTRODUCTION

he Hakka is a distinctive Han Chinese ethnic group speaking the unique Hakka dialect, and possessing its own civilization. The Hakka originated from ancient Han residents in middle China and migrated to the south because of war, starvation, and poor living conditions. After five long-distance migrations from the Eastern Jin Dynasty to the late Qing Dynasty, they settled in several global regions^[1]. In China, they live mainly in south Jiangxi, west Fujian, and east Guangdong provinces. These three areas are the primary residence of the Hakka, particularly northeast Guangdong. In Meizhou city, located in northeast Guangdong, over 95% of the 4 850 000 inhabitants are Hakka, and the dialect they speak is a particular Chinese dialect, which has been certified as the standardized Hakka dialect^[2].

The human glutathione S-transferases (GSTs) belong to a supergene family of detoxification enzymes. To date, GSTs have been grouped into eight classes: alpha, mu, theta, pi, zeta, sigma, kappa, and omega^[3], and most of them contain genetic polymorphisms. GSTs are involved in the metabolism of drugs and detoxification of a wide range of xenobiotic compounds, including carcinogens. Inherited genetic polymorphisms in the xenobiotic metabolizing enzymes play an important role in individual susceptibility to various diseases. Recent data have shown that GST genetic polymorphisms are closely correlated to drug effects, several cancers, and the outcomes of therapy for asthma, coronary heart disease, and atherosclerosis^[4-9].

A wealth of research on GST genetic polymorphisms has been carried out recently, showing that the distributions of GST polymorphisms vary among different ethnic, national, and regional populations^[10-14]. Although many studies on GST polymorphisms in north and middle China have been reported, polymorphisms in the Hakka population of south China have not yet been studied. Therefore, this study investigated the genetic polymorphisms of GSTT1, GSTM1, GSTM3, GSTP, and GSTA1 in the Hakka of Guangdong province and their possible associations with family histories of several chronic diseases were analyzed.

MATERIALS AND METHODS

Study Population

Five hundred and thirty-nine participants, 328 males and 211 females, were randomly selected by a multi-stage stratified random sampling method from five towns (Sanjiao, Chengjiang, Chengdong, Chengbei, and Yanyang) in Meizhou in northeast Guangong province from May to July of 2003, where the majority of residents are Hakka. All participants were healthy residents without ties of kindred to each other and most of them were married and had senior high schooling or above. The mean age of the participants was 38.4±13.5 years (ranging between 15 and 80 years). Each participant independently completed a structured questionnaire concerning drinking, smoking, dietary habits, and family history of special and chronic diseases, including esophageal cancer, nasopharyngeal cancer, favism, and hypertension. The involvement of these chronic diseases was determined by medical certification. Alcohol drinking was defined as drinking at least once a week and smoking was defined as smoking one cigarette a day for at least six months.

Genotyping

Genomic DNA was extracted from peripheral blood by a classic phenol/chloroform extraction method. All polymerase chain reactions (PCR) were in a volume of 20 μL containing target DNA, 0.2 mmoL/L d(NTP)₄, 10′ PCR buffer, 0.1-0.4 moL/L primers (Shanghai Shenggong Ltd. Co.), 1.5 mmoL/L MgCL₂, and 1U Taq DNA polymerase (American Promega Ltd. Co., Shanghai). Different genetic GST polymorphisms required different PCR conditions (Table 1), and gene-specific GST primers were designed as previously described^[15-17] (Table 2). GSTM1 and GSTT1 had a null-allele variant in which the entire gene was absent (null genotype), so the absence or presence of GSTM1 or GSTT1 was detected by co-amplifying GSTM1 or GSTT1 and the β-globin gene. The results were visualized on 2% agarose gels stained with ethidium bromide (Figure1 and Figure 2). For GSTP1, the wild-type GSTP1*A and variant GSTP1*G alleles were detected by PCR-RFLP (restriction fragment length polymorphism), with PCR products digested with 5U BsmA1 (Shanghai Shenggong Ltd. Co.) for 16 h at 37 °C in 20 l after PCR amplification. Fragments were visualized on 4% agarose gels stained with ethidium bromide (Figure 3 and Figure 4). For GSTA1, the same method was used to determine wild-type GSTA1*A and variant GSTA1*B alleles, but digestion was with Ear1 (New England Biolabs) for RFLP detection (Figure5). For GSTM3, the GSTM3*B allele had a 3 bp deletion in intron 6 that was absent in the GSTM3*A allele; therefore, PCR products were subjected to electrophoresis on a 12% polyacrylamide gel to distinguish the genotypes (Figure 6). DNA samples were kept at 4 °C for short-term storage and at -80 °C for longer storage.

Figure 1. Co-amplification products of GSTM1 (480 bp) and β-Globin (268 bp), Lanes 2, 3, 4, 5, 6, 8, 9, 13-15:GSTM1+ samples. lane 7, 10-12:GSTM1-null genotype samples. lane 1: 100 bp markers.

Table 1. PCR Conditions for GST Genes

Table 2. Primes Used to Identify Polymorphic Gene Frequencies of GSTs

Note. ^*β-Globin was a control that was co-amplified in GSTT1 or GSTM1 PCR reactions.

Figure 2. Co-amplification products of GSTT1 (215 bp) and β-Globin (268 bp), Lanes 2,3, and 7 :GSTT1-null genotype samples. Lanes 4, 5, 6, and 8: GSTT1+ samples. lane 1: 100 bp markers.

Figure 3. RFLP fragments of GSTP1 after digestion of PCR products. Amplified products (176 bp)   were cut into two fragments(91 bp and 85 bp) by BsmA1. Lane 2, 3, 4, 5, 6: homozygous GSTP1*A/*A genotype samples. Lane 7: homozygous GSTP1*G/*G genotype sample. lane 1: 100 bp markers.

Figure 4. RFLP fragments of GSTP1 after digestion of PCR products. Amplified products (176 bp) were cut into two fragments (91 bp and 85 bp) by BsmA1. Lanes 2, 3, 5,6,7, 10,11: homozygous GSTP1*A/*A genotype samples. Lanes 4, 8, 9: heterozygous GSTP1*A/*G genotype samples. lane 1: 100 bp markers.

Figure 5. RFLP fragments of GSTA1 after digestion of PCR products. Amplified products (480 bp) were cut into two fragments (380 bp and 100 bp) by Ear1. Lane 2: homozygous GSTA1*B/ *B genotype samples. Lane3:heterozygous GSTA1 *A/*B genotype sample. Lanes 4-6: homozygous GSTA1*B/*B genotype samples. lane 1: 100 bp markers.

Figure 6. PCR products of GSTM3 separated into 79 bp and 76 bp fragments on a 12% polyacrylamide gel.Lane 2: heterozygous GSTM3*A/*B genotype sample. Lanes 3, 5, 6: homozygous GSTM3*A/*A  ABLE 4-64.genotype samples. Lane 4: homozygous GSTA1*B/*B genotype sample. lane 1: 100 bp markers.

Statistical Analysis

Data were analyzed with SPSS software (ver. 10.0 SPSS In., Chicago, IL, USA). The statistical differences of GST polymorphism frequencies were determined by a Chi-squared test. Logistic regression analysis was used to determine the associations of GST genetic polymorphisms with family history of coronary heart disease, hypertension, stroke, lung cancer, esophageal cancer, nasopharyngeal cancer, favism, and thalassemia. A Chi-squared test for Hardy-Weinberg equilibrium was performed on GSTP1, GSTM3, and GSTA1 polymorphisms.

RESULTS

The allele frequencies of GSTP1A and GSTP1G were 83.2% and 16.8%, respectively. The allele frequencies of GSTM3A and GSTM3B were 80.2% and 19.8%, respectively. The allele frequency of GSTA1A and GSTA1B were 87.9% and 12.1%, respectively.

Polymorphisms of GSTT1, GSTM1, GSTP1, and GSTM3 were determined in our previous study on the Cantonese population in Guangdong province. Compared to this population, the Hakka had a significantly lower frequency of the GSTM3B*/*B genotype (3.1% vs. 11.9%, P<0.05). No statistical differences were observed between the Hakka and the Cantonese for GSTT1, GSTM1, or GSTP1 genetic polymorphisms (Table 3). According to the results of the Chi-square test for Hardy-Weinberg equilibrium, GSTP1, GSTM3, and GSTA1 polymorphisms in the Hakka population conformed to the Hardy-Weinberg equilibrium (Table 4, Table 5, and Table 6). To the best of our knowledge, this is the first report to determine genetic polymorphisms of GSTA1 in the Hakka in China. The distributions of GSTA1*A/*A, GSTA1*A/*B, and GSTA1*B/*B were 77.1%, 21.7%, and 1.2%, respectively.

 
Table 3. GST Genotype Frequencies in the Hakka versus
       the Cantonese populations in Guangdong

Note. ^*Some data are missing because of insufficient DNA or failure of PCR. ^**Blank: GSTA1 was not included in the previous study.

Table 4. Hardy-Weinberg Equilibrium Test on GSTP1

Note. ^*P＞0.05.

Table 5. Hardy-Weinberg Equilibrium Test on GSTA1

Note. ^*P＞0.05.

Table 6. Hardy-Weinberg Equilibrium Test on GSTM3

Note. ^*P＞0.05.

Comparisons of GST genetic polymorphisms in the Hakka population with those reported for Han Chinese in other areas of China, as well as Caucasian and Indian populations are shown (Table 7). All these populations came from healthy volunteers and had similar social characteristics. Compared to Han Chinese in other areas of China, the Hakka had similar distributions of genetic polymorphisms in GSTT1, GSTM1, GSTP1, and GSTM3, whereas the Hakka and the Han Chinese from other areas of China tend to lack the GSTT1 and GSTM1 genes, when compared with Caucasians and Indians. Compared to Caucasians, the Hakka had a lower frequency of the homozygous GSTP1^*G/^*G genotype, which was similar to the Indian population.

Logistic regression analysis results indicated a weak association of GSTM1 genetic polymorphisms with family history of hypertension (odds ratio (OR) = 1.868, 95% confidence intervals (95% CI) = 1.119 - 3.119). In addition, Chi-squared tests showed that people with a family history of hypertension had higher frequencies of the null-genotype for GSTM1 than those without such a family history (73.0% vs. 59.2%, P<0.05). No relationships were observed between other GST genotype distributions and family histories of coronary heart disease, stroke, lung cancer, esophageal cancer, nasopharyngeal cancer, favism, or thalassemia.

In addition, a significant difference was observed in GSTM1 genetic polymorphisms between alcohol drinkers and non-drinkers (70.5% vs. 59.8%, P<0.05). Significant differences were also observed in GSTP1 genetic polymorphisms between smoking and non-smoking groups (GSTP1*A/*A, GSTP1*A/*G, GSTP1*G/*G: 62.5%, 29.2%, 8.3% vs. 70.4%, 28.1%, 1.5%, P<0.05). However, no differences were seen in the frequency of the GSTP1 genotypes with regard to tobacco consumption, pickles consumption (Table 8), and differences in GSTs genetic polymorphisms were not observed among different age groups or genders (P>0.05).

Table 7. GST Genotype Frequencies in the Hakka Population Compared to other Populations

Note. ^aNA: not available; ^bA combination of GSTM3 A/B and GSTM3 B/B; ^creference 18; ^dreference 19; ^ereference 20; ^freference 21; ^greference 13.

DISCUSSION

Several studies have shown that GST genes are polymorphic, with variable regional, ethnic, and national distributions. In this study, genetic polymorphisms of the GSTT1, GSTM1, GSTP1, GSTM3, and GSTA1 genes in a Hakka population were compared to other populations. In the Hakka, the higher frequencies of null-GSTM1 and null-GSTT1 in comparison with both Caucasians and Indians, suggests that the Hakka is distinct from both these

Table 8. GST Genotype Frequencies in the Hakka Population and Life Style Factors

Note. ^*P<0.05 when non-smokers were compared with smokers for GSTP1 genotype frequencies. ^**P<0.05 when non-drinkers were compared with drinkers for GSTM1 genotype frequencies.

racial groups. However, when compared to the Cantonese population, the Hakka have statistically significant differences in GSTM3 genotype frequency distributions, a significantly lower frequency of the GSTM3B*/*B genotype, but have similar frequency distributions for the GSTT1, GSTM1, GSTP1, and GSTA1 genes. The Hakka population appears to be a special branch of the Han Chinese by origin, with some changes in genetic structure, possibly caused by exoteric factors from long-distance southward migrations more than one thousand years ago. This result is consistent with a genetic analysis of the origin of the Hakka by Hui, et al.^[22].

Tobacco smoking is one of the most important risk factors for lung cancer, with cigarettes producing many confirmed or suspected human chemical carcinogens and oxygen free radicals, such as polycyclic aromatic hydrocarbon amines, hydrogen peroxide free radicals, which might cause damage to DNA^[23-24]. However, not every smoker will develop lung cancer, possibly because of individual genetic susceptibility. GSTP1 is a candidate gene for lung cancer and is widely expressed in normal human epithelial tissue, particularly in the lung^[25]. GSTP1-catalysed glutathione (GSH) conjugation is an important enzymatic reaction for protecting respiratory cells against environmental pollutants. Individuals with decreased enzyme activity have lower detoxification ability and are predisposed to an increased risk of lung cancer. In the Hakka population, the smokers had higher distributions of variant GSTP1*A/*G and *G/*G compared to non-smokers (29.2% and 8.3% vs. 28.1%, 1.4%, respectively). This suggested that the smokers might be at a higher risk of lung cancer, but further case-control studies and cohort studies are needed for confirmation.

Considering the high incidence of esophageal cancer, nasopharyngeal cancer, favism, and thalassemia among the Hakka, logistic regression analysis was carried out to find possible associations between GST genotype distributions and family history of these diseases, together with lung cancer, coronary heart disease, hypertension and coronary heart disease. The results indicated a weak association of the GSTM1 genetic polymorphisms with family history of hypertension. Furthermore, Chi-squared tests showed that individuals with a family history of hypertension had a higher frequency of the null-GSTM1 allele compared to those without such family history (73.0% vs. 59.2%). GSTM1 has five subfamilies, GSTM1-5. Only the GSTM1 gene is abundantly expressed in hepatic tissue. The null-GSTM1 correlates with many diseases, including prostate cancer, colorectal cancer, gastric cancer, asthma, and heart rate variability (HRV)^[26-30]. In our study, the alcohol drinkers had higher frequencies of null-GSTM1 genotypes than the non-drinkers (70.5% vs. 59.8%). Drinking is known to be an important risk factor for hypertension. Oxidative metabolism of ethanol in alcohol produces acetaldehyde and various free radicals, causing hepatic damage^[31]. The null-GSTM1 might decrease detoxification ability and increase individual susceptibility to related diseases. As far as we know, the relationship between GSTM1 genetic polymorphisms and alcohol consumption are still unclear. A study showed that the correlation between the null-GSTM1 genotype and alcohol consumption was a low exposure-gene effect^[32]. Our results suggest that individuals with the null-GSTM1 genotype are at an increased risk for hypertension, but further studies are needed, based on the information presented here on the normal Hakka population.

As part of the research project, the family histories of several chronic diseases were selected as the research outcomes, which might be the limitation of the study. However, our results have provided basic genetic information on the Hakka population for further case-control study.

 In conclusion, we have reported the genotype distributions of GSTM1, GSTT1, GSTP1, GSTM3, and GSTA1 in the normal Hakka population of South China. We have also uncovered a possible primary relationship between smoking and GSTP1 genetic polymorphisms, as well as a relationship between drinking and GSTM1 genetic polymorphisms. These results might be useful in case-controlled, or cohort studies on associations between GST gene polymorphisms and disease risks, drug effects, and disease prognosis in the Hakka. 

ACKNOWLEDGEMENTS

We thank our partners (Center for Disease Control and Prevention of Meizhou City) for their help in sample collection. We also thank the staff (Toxicology Lab in GDCDC) for their experimental and technical assistances.

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