Association of NAT2 gene substitution mutation T341C with increased risk for head and neck cancer in Tunisia.

Introduction

Squamous cell carcinoma of the head and neck, including the larynx, pharynx, and oral cavity is a relatively common human neoplasm. Increasing epidemiological studies indicate that these cancers are strongly linked to environmental factors such as chemical carcinogens present in tobacco and alcohol [1], [2]. The risk of head and neck cancer in smokers and alcohol users is more than twice that in non smokers and non alcohol users [3], [4]. Inter individual differences observed in the metabolism of carcinogens have been attributed to the genetic polymorphisms of genes encoding for enzymes involved in detoxification. This genetic variability seems to be associated with individual's susceptibility to some cancers, including head and neck cancer [5–8].

N acetyl transferase 2 (NAT2) metabolizes aromatic amines, and is involved in the phase II metabolism of different compounds. Several mutations in the NAT2 gene such as G191A, T341C, G590A and G857A reduce the activity of the protein [9], [10], disturbing the metabolism of the corresponding substrates.

Glutathione S-transferase (GST) M1 and T1 are two enzymes of GST family that catalyze the reduction of several electrophilic substrates, facilitating the excretion of these compounds. Both of them are polymorphic. The GSTM1*0 (GSTM1 deficiency) and GSTT1*0 (GSTT1 deficiency) allele represent a deletion of the GSTM1 and GSTT1 gene and result in a loss of enzymatic activity [11], [12].

The aim of this study was to investigate the influence of deletion polymorphisms at GSTM1, GSTT1 genes and substitution polymorphisms at NAT2 gene (T341C and G590A), on susceptibility to head and neck cancer in a Tunisian population.

Patients and methods

Study groups

We performed a case control study: The case group consisted of 64 newly diagnosed patients with confirmed primary squamous cell carcinoma of head and neck at histology (nasopharyngeal (n = 46), laryngeal (n = 11), lips (n = 3), tongue (n = 2) and oropharyngeal (n = 2)). All patients were recruited at Cancer Institute of Tunis. This group included 42 men and 22 women (mean age: 50.7±12.27 years; 17–80 years). The healthy group consisted of 160 individuals (72 men and 88 women) without any prior diagnosis of cancer, recruited among the professional surrounding (mean age: 53.6±8.89 years; 40–70 years).

Methods

Genotyping

DNA was isolated from leukocytes in 10 ml of peripheral blood. The presence of the GSTM1 null and GSTT1 null deletion was screened by using a multiplex PCR procedure using primers described in Table I. The amplification of albumin gene provides a positive control for each reaction. The 50 µl reaction mixture contained: 250 ng of genomic DNA, 22 pM for each primer, 200 µM of each dNTP, 2U of Taq DNA polymerase. Cycling conditions consisted of 5′at 94°C for initial denaturation and 2′at 64°C for initial hybridization followed by 30 cycles of extension for 30′′at 72°C, denaturating for 20′′at 94°C, hybridization for 20′′at 64°C and final extension at 72°C for 7′. Ten microliter of each PCR product was analyzed for efficient amplification on an agarose gel at 1%.

Table I.  Primer sequences used for PCR

Genes	Primer Sequences	Product Length
GSTM1	5′-GAACTCCCTGAAAAGCTAAAGC- 3′	215 bp
	5′-GTTGGGCTCAAATATACGGTGG -3′
GSTT1	5′-TTCCTTACTGGTCCTCACATCTC- 3′	480 bp
	5′-TCACCGGATCATGGCCAGCA- 3′
Albumin	5′-GCCCTCTGCTAACAAGTCCTAC- 3′	380 bp
	5′-GCCCTAAAAAGAAAATCGCCAATC-3′
NAT2	5′- CTTCTCCTGCAGGTGTCTA- 3′	243 bp
(T341C)	5′-TTTTTGGTGTTTCTTCTTTGGC- 3′
NAT2	5′ -GATCACATTGTAAGAAGAAACCG- 3′	835 bp
(G590A)	5′ -GGATGAAAGTATTTGATGTTTAGG- 3′

A PCR-RFLP method was used to detect substitution polymorphisms at NAT2 gene. Two fragments of 835 bp and 243 bp of NAT2 were amplified with a PCR procedure previously described [13]. The G590A mutation was detected by the loss of TaqI restriction site in the 835 bp PCR product. The T341C substitution was detected by AccI cleavage of the 243 bp product.

Statistical analysis

The χ2 test was used to assess the distribution of allele frequencies between groups. Odds ratio (OR) and 95% confidence intervals (CI) were calculated using epi_info 6 logiciel to estimate relative risk association with some genotypes.

Results

Frequencies of homozygous deletion of GSTM1 and GSTT1 genes did not differ significantly from patients and controls (Figure 1) (Table II).

Figure 1.  PCR multiplex for genotyping GSTM1 and GSTT1. Lane 1: GSTM1 null and GSTT1 null; lane 2: GSTM1 null; lane 3: GSTT1 null; lane 4: corresponds to an individual who is positive for both GSTM1 and GSTT1

Table II.  Frequencies of GSTT1 and GSTM1 null genotypes in patients and controls

	Frequency (%)
Genotypes	Patients	Normal controls
GSTM1 0/0	46,87	34,48
GSTT1 0/0	26,56	18,33

Frequencies of G590A and T341C mutations in NAT2 gene are listed in Table III.

Table III.  Frequencies of G590A and T341C mutations in patients and controls

	Frequency (%)
Mutations	Patients	Normal controls	p	OR
G590A	33,59	28,75	0,54
T341C	51,56	36,87	0,04	1,82 [2,86–12,26]

A significant difference was found between patients and controls for T341C mutation (p < 0.05) (Table III). Calculated OR showed an association between T341C mutation and increased risk of head and neck cancer.

When the patients were stratified by disease site, the T341C mutation's frequency for the patients with squamous cell carcinoma of nasopharynx, larynx, lip, tongue or oropharynx were not statistically different from that of total patient population (Table IV).

Table IV.  Frequency distribution of T341C mutation by disease site.

Disease site	T341C mutation's frequency (%)
nasopharynx	51.08
larynx	50
lip	25
tongue	66.6
oropharynx	75

Discussion

GST and NAT2 are important phase II detoxifying enzymes. Total or partial deficit of these enzymes lead to a reduced capacity of detoxifying chemical compounds whose accumulation may promote cancer development.

We found no association between GSTM1 and GSTT1 null genotypes and head and neck cancer risk. Several studies related to association between GST genotypes and head and neck cancer are rather conflicting. Some reported that polymorphic variants of the GSTM1 and GSTT1 genes result in no significant contribution to head and neck cancer development [3], [14–17]. While others suggested that GSTM1 and/ or GSTT1 genes deletion might be risk factors for developing head and neck carcinoma [6], [18]. Discrepancy between these studies may be ascribable to ethnic difference in allelic frequencies of the GSTM1 and GSTT1 polymorphism or to factors such as diet, alcohol consumption, smoking habit [19]. It is well known that variation in the geographic and ethnic distribution between cases and control individuals among studies may be a considerable bias, which might confound the results of pooling analysis [20], [21]. Z ye et al. [22] have observed such an imbalance in geographic and ethnic distribution. For GSTM1 status, the risk of head and neck cancer is higher in African-American and Asians than in whites, while the risk of head and neck cancer is higher in Asia than in America and Europe. Similarly, for GSTT1 status, the risk of head and neck cancer is higher in America than in Europe and Asia. However, the risk of head and neck cancer seems consistent in the different ethnic group. Thus, the large differences in prevalence of enzyme's detoxification's polymorphisms in different ethnic group may influence the susceptibility to cancer by modification of enzyme's activity on environmental carcinogens.

We also analyzed the link between NAT2 polymorphisms and susceptibility of head and neck cancer. T341C mutation emerged as a significant risk factor for such cancer. Our findings are in accordance to the reported association between squamous cell carcinoma and T341C mutation [23]. This is probably due to the great reduction in acetyltransferase 2 catalytic activity in relation with the T341C mutation in NAT2 gene [24].

Conclusion

Inter individual differences observed in the metabolism of carcinogens have been attributed to the genetic polymorphism of genes which encode for enzymes involved in detoxification. This genetic variability seems to be associated with the individual's susceptibility to head and neck cancer. Our results showed no association between GSTM1 and GSTT1 null genotypes and susceptibility to head and neck carcinoma. T341C mutation of NAT2 gene was found to be associated with an elevated risk for head and neck cancer in Tunisian population.