Glutathione-S-transferase M1 polymorphisms on the susceptibility to esophageal cancer among three Chinese minorities: Kazakh, Tajik and Uygur

Abstract

AIM: To investigate the glutathione-S-transferase M1 (GSTM1) polymorphisms in three Chinese minorities, Kazakh, Uygur, and Tajik; and the pathological significance of GSTM1 polymorphisms in esophageal carcinogenesis in Kazakh.

METHODS: A total of 1121 blood samples (442 males and 679 females) were obtained from healthy Kazakh (654), Uygur (412) and Tajik (55). Primary esophageal squamous cell cancer (ESCC) tissues from Kazakh were obtained from 116 patients who underwent surgery. GSTM1 polymorphisms were analyzed by a combined approach of PCR and electrophoresis techniques.

RESULTS: GSTM1 null genotype was found in 62.63% Uygur, 50.91% Tajik and 47.40% Kazakh. A significantly higher frequency of GSTM1 null genotype in Uygur was observed compared with Kazakh (OR: 1.859, 95% CI: 1.445 -2.391, χ² = 23.71, P = 0.000). In addition, GSTM1 null genotype was found in 23.53% of well-differentiated ESCC in Kazakh, in 49.23% of poorly differentiated ESCC, with a significant difference (OR: 3.152, 95% CI: 1.403-7.080, χ² = 8.018, P = 0.007).

CONCLUSION: There is a marked difference in the frequency of common GSTM1 null genotype between Uygur and Kazakh. GSTM1 null genotype is associated with differentiation of ESCC in Kazakh.

Key Words: Glutathione-S-transferase M1; Kazakh; Uygur; Tajik; Esophageal cancer

INTRODUCTION

Esophageal cancer (EC) is the sixth leading cause of cancer mortality worldwide[1]. The incidence of EC is highly variable in different populations, with more than a 50-fold difference between the high- and low-risk ethic groups[2,3]. For example, Turkomans in northeastern Iran are considered to be a very high-risk group, with age standardized prevalence (ASR) of over 100/100 000 for both men and women; whereas the prevalence of EC in pure Zoroastrian Persians in Iran and India are known to be considerably low, with ASRs of 3-7/100 000.

Epidemiological studies have identified several high EC incidence areas, such as the western and northern parts of China[4], certain areas of France and Brazil[5]. In Xinjiang Uygur Autonomous Region of China, there are thirteen minority ethnical groups (Uygur, Han, Hazakh, Tajik, Hui, Uzbek, Kerkez, Man, Mongolia, Tatar, Darur, Xibo, and Russian), who have lived there since ancient times. Uygur, Hazakh and Tajik are the major residents among those minorities with populations of 8 million, 2 million, and 40 thousand, respectively. Although they are all Muslims and have certain similarities in their life styles, the morbidities of EC among them are quite different. The incidence of EC in Kazakh is highest among all ethnics in Xinjiang, with an age-adjusted mortality of 90.7/100 000, significantly higher than that in Uygur (23.4/100 000) and almost 18-fold higher than that in Tajik (5.13/100 000)[4].

Glutathione S-transferases (GSTs) constitute a superfamily of ubiquitous multifunctional enzymes, which play a key role in cellular detoxification and protection of macromolecules from being attacked by reactive electrophiles[6]. GSTs catalyze the conjugation of tripeptide glutathione (GSH) to a wide variety of exogenous and endogenous chemicals with electrophilic functional groups (e.g. products of oxidative stress, environmental pollutants, and carcinogens), thereby neutralizing their electrophilic sites, and rendering the products more water-soluble[7]. Based on sequence homology and immunological cross-reactivity, human cytosolic GSTs have been grouped into seven families, designated GST Alpha, Mu, Pi, Sigma, Omega, Theta, and Zeta[8]. The GSTs presumably arise from a single common ancestor and their substrate specificity and diversity have been reshaped by gene duplication, recombination and mutation.

There are marked intra- and inter-ethnic differences in the frequencies of common GST mutations[9,10]. For example, the distribution of GSTM1 genotype frequencies in Indian is significantly different from that in Chinese[11]. GSTM1 polymorphisms have been considered as a risk factor for EC development in a number of studies; however the overall results of such studies are inconsistent[12,13]. Up to date, data on genetic analysis of GSTM1 in Uygur, Tajik are lacking[14], and the correlation between GSTM1 polymorphisms and high incidence of EC in Kazakh has not been clarified.

The present study aimed to investigate the GSTM1 polymorphisms in healthy Kazakh, Uygur, and Tajik; and to explore the pathological significance of GSTM1 polymorphisms in esophageal carcinogenesis.

MATERIALS AND METHODS

Blood and tissue

A total of 1121 blood samples was collected from healthy Kazakh (269 males and 385 females; age 35-65 years, mean 46.5 years), Uygur (146 males and 266 females; age 30-68 years, mean 45.5 years) and Tajik (27 males and 28 females; age 32-70 years, mean 47.5 years). All subjects from north-western of Xinjiang received clinical and biochemical assessments before entering this study and none of them has a clinical or family history of EC. Specimens of 116 primary EC tissues from Kazakh (84 males and 32 females; age 42-76 years, mean 55.5 years), with histological confirmation of primary ESCC, was recruited from two hospitals in Xinjiang from July 1999 to June 2004.

DNA extraction and GSTM1 genotyping

DNAs from healthy controls were extracted from peripheral leukocytes using the classical phenol-chloroform extraction method[15]. Genomic DNA in cancer tissue embedded by paraffin was prepared by proteinase K digestion and phenol/chloroform extraction, followed by ethanol precipitation, as described by Diffenbach[16]. The purity and concentration of DNA was examined by ultraviolet densitometry. GSTM1 genotyping for gene deletion was performed by PCR using primers 5’-GAACTCCCTGAAAAGCTAAAGC-3’ and 5’-GTTGGGCTCAAATATACGGTGG-3’[17], which produced a 219 bp product. At the same time, β-globin gene was amplified, resulting in a 350 bp product as an internal control. PCR was performed in a reaction mixture of 20 μL containing 100 ng sample DNA, 10 mmol/L Tris-HCl, 50 mmol/L KCl, 1.5 mmol/L MgCl₂ pH 8.4, 0.1 mmol/L of each dNTP and 1.25 U Taq polymerase. After initial denaturation for 5 min at 94°C, 35 cycles were performed at 94°C for 30 s (denaturation), at 63°C for 30 s (annealing) and at 72°C for 30 s (extension), followed by a final step for 5 min at 72°C. The amplified products were visualized by electrophoresis in ethidium-bromide-stained 1.5% agarose gel in TBE buffer. For genotype of GSTM1 deletion, no amplified product was observed except the band of β-globin gene.

Statistical analysis

Chi-square test was used to examine the correlation between the GSTM1 polymorphism among three healthy ethnics, and association of GSTM1 polymorphisms with differentiation of ESCC in Kazakh with SPSS software (11.0). Odds ratios (ORs) and 95% confidence intervals (CIs) of different variables among groups were calculated.

RESULTS

Histological types of primary ESCC

Histological confirmation of primary ESCC including well-differentiated and poorly-differentiated are shown in Figure 1.

Figure 1 Histological types of primary ESCC (HE × 400). A: Well-differentiated ESCC; B: Poorly-differentiated ESCC.

GSTM1 genetic polymorphisms in ESCC of Kazakh

Figure 2 shows the PCR-amplified fragment of GSTM1. Genotype data for GSTM1 in the three ethnics are summarized in Table 1. The frequency of GSTM1 null mutation in Kazakh was significantly lower than that in Uygur (OR:1.859, 95% CI: 1.445-2.391, χ² = 23.71, P = 0.000, P < 0.05). There was no significant difference in the frequency of the GSTM1 null genotype between Uygur (62.63%) and Tajik (50.91%) (χ² = 2.804, P > 0.05), and there was no significant difference between the Kazakh (47.40%) and Tajikic either (χ² = 0.250, P > 0.05). In addition, no significant difference of GSTM1 null polymorphisms between the two genders of each ethnical group was observed.

Table 1 Frequencies of GSTM1 polymorphisms in three ethnics.

Ethnic	GSTM1		OR (95% CI)	P
	Null [n (%)]	Present [n (%)]
Kazakh
Male	130 (48.33)	139 (51.67)
Female	180 (46.75)	205 (53.25)
Total	310 (47.40)	344 (52.60)
			11.859 (1.445-2.391)	0.000
Uygur
Male	99 (67.81)	47 (32.19)
Female	159 (59.77)	107 (40.23)
Total	258 (62.63)	154 (37.37)
			20.619 (0.352-1.809)	0.106
Tajik
Male	13 (50.00)	13 (50.00)
Female	15 (51.72)	14 (48.28)
Total	28 (50.91)	27 (49.09)
			31.151 (0.664-1.996)	0.674

OR: odds ratio; CI: confidence interval.

¹Kazakh vs Uygur;

²Uygur vs Tajik;

³Kazakh vs Tajik.

Figure 2 PCR of the GSTM1 genes. Lane M: DL 2000 DNA molecular weight marker; lane 1: GSTM1 genotype present; lane 2: homozygous deletion of GSTM1.

There was a significant difference in the frequency of the GSTM1 null genotype between well-differentiation (high grade) (76.47%) and poor-differentiation (low grade) group (50.77%) of EC of Kazakh (OR 3.152, 95% CI 1.403-7.080, χ² = 8.018, P < 0.05) (Table 2).

Table 2 Correlation of clinicopathological grade of EC with GSTM1 genotypes in Kazakh.

ESCC grade	GSTM1		OR (95% CI)	P
	Null [n (%)]	Present [n (%)]
High	12 (23.53)	39 (76.47 )
Low	32 (49.23)	33 (50.77)	3.152 (1.403-7.080)	0.007

The odds ratio of GSTM1 null genotype of Kazakh people with lowly differentiated ESCC was 3.152-fold higher than those people with highly differentiated ESCC.

DISCUSSION

In this study, we investigated differences in the prevalence of GSTM1 null genotypes in three ethnical groups, Kazakh, Tajik and Uygur, in Xinjiang. As far as we know, we are the first to report the frequency of GSTM1 null genotype in Tajik. GSTM1 null genotype in Uygur in Xinjiang has a similar frequency when compared with Zoroastrians Iranian[18] and Han Chinese[19,20].

The study showed the association of GSTM1 null genotype with ESCC differentiation in Kazakh, suggesting the involvement of GSTM1 null genotype in the development of ESCC. Differences in the risk of EC development between high- and low-risk populations may partly be attributed to the genetic make-up of the populations, reflected by their different susceptibility to EC. GSTM1 encoding metabolic enzymes, the alteration in expression and function of which may increase or decrease carcinogen activation/detoxication, expressed as different phenotypes with different cancer risk[21-23]. Homozygous deletions of such genes, called GSTM1 null genotypes, result in the phenotype of no enzyme activity[24]. Individuals with null genotypes of GSTM1 are reported at high risk for developing several types of cancers, e.g. breast, lung, cervix[25-27] and bladder cancers[28-32]. However, the frequency of GSTM1 null genotype was low in Kazakh with high risk to EC, suggesting that the lack of the null allele or the other genes may play roles in carcinogenesis of ESCC by different mechanisms or via different pathways, from that of the reported breast, lung, cervix and bladder cancers. This large sample study on 654 of healthy Kazakh individuals and our previous genotyping results[14] have confirmed this contradictory finding of low frequency GSTM1 null genotype among Kazakh with a high susceptibility to ESCC.

In conclusion, there are different frequencies of GSTM1 null genotype among Uygur, Tajik and Kazakh, however, a significant difference is only observed between Uygur and Kazakh. The GSTM1 null genotype may play a role in the carcinogenesis and progress of ESCC.

ACKNOWLEDGMENTS

We thank those people who provided the blood samples of Kazakh, Tajik and Uygur individuals.