Study population

One hundred and seventy-six patients diagnosed with NPC were recruited at the general surgery outpatient clinics of the study hospital in Taichung, Taiwan, between 2003 and 2009. All patients participated voluntarily, completed a self-administered questionnaire, and provided peripheral blood samples. The questionnaire included questions on history and frequency of alcohol consumption, betel quid chewing, and smoking habits, and ‘ever’ was defined as more than twice a week for years. Self-reported alcohol consumption, betel quid chewing, and smoking habits were evaluated and classified as categorical variables.

For each case patient, two age- and gender-matched healthy controls, who had no NPC or other type of cancer, were selected from those attending the hospital for a health examination (age matching was done within less than 5 years of the case patient’s first diagnosis). These volunteers attended the hospital for regular health assessments by multidisciplinary team approach with registered health practitioners during the years 2002–2012; most of the volunteers underwent health examinations every 5–6 months. A total of 10,358 participants aged 1–104 years were recruited into this cohort, and those who were cancer-free by the age at diagnosis of the case patient, according to the International Classification of Diseases, Ninth Revision (ICD-9) codes, were chosen. Finally, 352 participants were included for analysis in the present study. For the convenience of the gene–environment interaction analysis, we preferentially selected those with alcoholism, betel quid chewing, and smoking habits when selecting the controls for genotyping and further analysis. Thus, the control group was not a general population control, but rather an alcohol-, betel quid-, and smoking-related control group. The overall agreement rate in this study was more than 85% in collection.

The study was approved by the institutional review board of the medical university hospital and written informed consent was obtained from all participants.

Genotyping protocol

The total genomic DNA of each subject was extracted from peripheral blood leucocytes using a QIAamp Blood Mini Kit (Qiagen, Taipei, Taiwan) and stored as reported previously. The primers used for XRCC6 were as follows: promoter T–991C, forward 5′-AACTCATGGACCCACGGTTGTGA-3′, and reverse 5′-CAACTTAAATACAGGAATGTCTTG-3′; promoter G–57C, forward 5′-AAACTTCAGACCACTCTCTTCT-3′, and reverse 5′-AAGCCGCTGCCGGGTGCCCGA-3′; promoter G–31A, forward 5′-TACAGTCCTGACGTAGAAG-3′, and reverse 5′-AAGCGACCAACTTGGACAGA-3′; intron 3, forward 5′-GTATACTTACTGCATTCTGG-3′, and reverse 5′-CATAAGTGCTCAGTACCTAT-3′. The following cycling conditions were used: one cycle at 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; and a final extension at 72 °C for 10 min.

Restriction fragment length polymorphism (RFLP) conditions

For the XRCC6 promoter T–991C, the resulting 301-base pair (bp) PCR product was mixed with 2 U Dpn II. The restriction site was located at −991, with a T/C polymorphism; the C form PCR products could be digested further, while the T form could not. Two fragments of 101 bp and 200 bp were present if the product was the digestible C form. The reaction was incubated for 2 h at 37 °C. Then, 10 μl of product was loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The polymorphism was categorized as (1) C/C homozygote (digested), (2) T/T homozygote (undigested), or (3) C/T heterozygote.

For the XRCC6 promoter G–57C, the resulting 298-bp PCR product was mixed with 2 U Hae II. The restriction site was located at −57, with a G/C polymorphism; the G form PCR products could be digested further, while the C form could not. Two fractions of 103 bp and 195 bp were present if the product was the digestible G form. The reaction was incubated for 2 h at 37 °C. Then, 10 μl of product was loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The polymorphism was categorized as (1) G/G homozygote (digested), (2) C/C homozygote (undigested), or (3) C/G heterozygote.

For the XRCC6 promoter G–31A, the resulting 226-bp PCR product was mixed with 2 U Mnl I. The restriction site was located at −31, with a G/A polymorphism; the A form PCR products could be digested further, while the G form could not. Two fractions of 80 bp and 146 bp were present if the product was the digestible A form. The reaction was incubated for 2 h at 37 °C. Then, 10 μl of product was loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The polymorphism was categorized as (1) A/A homozygote (digested), (2) G/G homozygote (undigested), or (3) A/G heterozygote.

For the XRCC6 intron 3, the resulting 160-bp PCR product was mixed with 2 U Msc I. The restriction site was located at intron 3, with a GG/GC polymorphism; the GG form PCR products could be digested further, while the GC form could not. Two fractions of 46 bp and 114 bp were present if the product was the digestible GG form. The reaction was incubated for 2 h at 37 °C. Then, 10 μl of product was loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The polymorphism was categorized as (1) GG/GG homozygote (digested), (2) GC/GC homozygote (undigested), or (3) GG/GC heterozygote.

mRNA XRCC6 expression pattern

To evaluate the correlation between XRCC6 mRNA expression and XRCC6 polymorphism, 20 surgically removed NPC tissue samples obtained from sites adjacent to tumours with different genotypes were subjected to extraction of the total RNA using Trizol Reagent (Invitrogen, Carlsbad, CA, USA); the manufacturer’s protocol was followed. Total RNA was measured by real-time quantitative RT-PCR using an FTC-3000 real-time quantitative PCR instrument (Funglyn Biotech Inc., Canada). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal quantitative control. The primers used for amplification of XRCC6 mRNA were forward 5′-CGATAATGAAGGTTCTGGAAG-3′ and reverse 5′-CTGGAAGTGCTTGGTGAG-3′, while for GAPDH the primers were forward 5′-GAAATCCCATCACCATCTTCCAGG-3′ and reverse 5′-GAGCCCCAGCCTTCTCCATG-3′. Fold changes were normalized using the levels of GAPDH expression, and each assay was done at least in triplicate.

Western blotting analysis

The NPC specimens were homogenized in radio immunoprecipitation assay (RIPA) lysis buffer (Upstate Biotechnology Inc., Lake Placid, NY, USA), the homogenates were centrifuged at 10,000 × g for 30 min at 4 °C, and the supernatants were used for Western blotting. Samples were denatured by heating at 95 °C for 10 min, were separated on a 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel, and were then transferred to a nitrocellulose membrane (BioRad Laboratories, Hercules, CA, USA). The membrane was blocked with 5% non-fat milk and incubated overnight at 4 °C with mouse monoclonal anti-human XRCC6 antibody (1:1000; BD Transduction Laboratories; BD Biosciences, Franklin Lakes, NJ, USA), and then with the corresponding horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Chemicon, Temecula, CA, USA) for 1 h at room temperature. After reaction with enhanced chemiluminescence (ECL) solution (Amersham, Arlington Heights, IL, USA), bound antibody was visualized using a chemiluminescence imaging system (Syngene, Cambridge, UK). Finally, the blots were incubated at 56 °C for 18 min in stripping buffer (0.0626 M Tris–HCl, pH 6.7, 2% SDS, 0.1 M mercaptoethanol) and re-probed with a monoclonal mouse anti-β-actin antibody (Sigma, St. Louis, MO, USA) as the loading control. The optical density of each specific band was measured using a computer-assisted imaging analysis system (GeneTools Match software; Syngene).

Statistical analyses

The results only for those with successful genotyping and completed questionnaires are presented and analyzed in the tables. To ensure that the control subjects were representative of the general population and to exclude the possibility of genotyping error, the deviation of the genotype frequencies of XRCC6 SNPs in the control subjects from those expected under Hardy–Weinberg equilibrium was assessed using the goodness-of-fit test. Pearson’s Chi-square test or Fisher’s exact test (when the expected number in any cell was less than five) was used to compare the distribution of the XRCC6 genotypes between cases and controls. The associations between the XRCC6 polymorphisms and NPC risk were estimated by computing odds ratios (ORs) and their 95% confidence intervals (CIs) from unconditional logistic regression analysis, with adjustment for possible confounders. P < 0.05 was considered statistically significant, and statistical analyses were carried out using SPSS version 16.0 software (SPSS Inc., Chicago, IL, USA); tests were two-sided.

Basic comparisons between the case and control groups

Selected characteristics of the control and case subjects are summarized in Table 1 . All characteristics of the patients and controls were matched and no frequency distribution was statistically different between the two groups ( P > 0.05). In this selected population, personal habits including smoking, alcohol consumption, and betel quid chewing appeared not to be direct risk factors for NPC.

Association of XRCC6 genotypes and NPC risk

The genotype distributions of the XRCC6 polymorphisms in the cases and controls are presented in Table 2 . Among the polymorphic sites, the most significant findings were found for the XRCC6 promoter T–991C genotyping. The ORs after adjusting for confounding factors (age, gender, and smoking, alcohol drinking and betel quid chewing status) for those subjects carrying TC and CC genotypes were 2.02 (95% CI 1.21–3.32) and 3.42 (95% CI 1.28–8.94), respectively, compared to those carrying the TT wild-type genotype. The P -value for trend was significant ( P = 0.0014). In the dominant (TC+CC vs. TT) and recessive (CC vs. TT+TC) models, the association between XRCC6 promoter T–991C polymorphism and the risk of NPC was also statistically significant (adjusted OR 2.15 and 2.45, 95% CI 1.40–3.19 and 1.23–7.74, respectively).

Table 2

Distributions of XRCC6 genotypic and allelic frequencies among the nasopharyngeal carcinoma patients and controls.

	NPC cases (%)	Controls (%)	aOR a (95% CI)	P -value b
Promoter T–991C (rs5751129)
TT	131 (74.4)	305 (86.6)	1.00 (Ref.)
TC	35 (19.9)	40 (11.4)	2.02 c (1.21–3.32)	0.0072
CC	10 (5.7)	7 (2.0)	3.42 c (1.28–8.94)	0.0165
P for trend				0.0014
(TC+CC) vs. TT			2.15 c (1.40–3.19)	0.0006
CC vs. (TT+TC)			2.45 c (1.23–7.74)	0.0341
Promoter G–57C (rs2267437)
CC	113 (64.2)	229 (65.1)	1.00 (Ref.)
CG	58 (33.0)	109 (31.0)	1.08 (0.77–1.58)	0.7644
GG	5 (2.8)	14 (3.9)	0.77 (0.35–1.84)	0.6239
P for trend				0.7479
(CG+GG) vs. CC			1.02 (0.78–1.49)	0.8474
GG vs. (CC+CG)			0.88 (0.39–1.98)	0.8140
Promoter G–31A (rs132770)
GG	144 (81.8)	298 (84.7)	1.00 (Ref.)
GA	25 (14.2)	43 (12.2)	1.14 (0.76–1.93)	0.4921
AA	7 (4.0)	11 (3.1)	1.26 (0.60–2.96)	0.6122
P for trend				0.6965
(GA+AA) vs. GG			1.18 (0.83–1.87)	0.4533
AA vs. (GG+GA)			1.22 (0.58–3.00)	0.6170
Intron 3 (rs132774)
GG	144 (81.8)	279 (79.3)	1.00 (Ref.)
GC	32 (18.2)	73 (20.7)	0.85 (0.58–1.29)	0.5633

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