The present study was a population-based case-control study. Between January 2008 and December 2011, 400 patients with ESCC (case group) whose diagnoses were confirmed by pathological biopsy of tissues removed by endoscopic or surgical excision were recruited. Healthy individuals (control group) were confirmed by endoscopic census to be free of esophageal cancer and precancerous lesions over the same period in the same geographical area. Information on gender, age, smoking status and alcohol consumption of case and control group individuals was obtained. The 400 healthy individuals were gender- and age-matched with the case group. Smoking was defined as ≥1 cigarette/day continuously for >6 months. Alcohol consumption was defined as at least once/week continuously for >6 months. The study was approved by the First People’s Hospital of Yancheng City Ethics Committee (Jiangsu, China) and all subjects provided written informed consent.

Peripheral venous blood samples were obtained from all participants in a fasting state and stored at −20°C for subsequent use.

Wizard genomic DNA extraction kits (Promega Corporation, Madison, WI, USA) were used to extract genomic DNA according to the manufacturer’s instructions.

ND-1000 UV/VIS spectrophotometer (Nanodrop Corporation, Wilmington, DE, USA) was used to perform quantitative determination of the DNA samples. Template DNA concentration was adjusted to 25–50 ng/μl.

Genotypes at the 312 and 751 loci of XPD were determined using PCR-restriction fragment length polymorphism (RFLP). Primers were synthesized by Sangon Biological Engineering Technology Co., Ltd. (Shanghai, China). For codon 312, the primer sequences used were: upstream, 5′-CTGTTGGTGGGTGCCCGTATCTGTT GGTCT-3′; downstream, 5′-TAATATCGGGGCTCACCCT GCAGCACTTCCT-3′. PCR mix (20 μl) containing 100 ng DNA template, 2.0 μl 10X PCR buffer solution (with 15 mM MgCl₂), 0.4 μl dNTPs (10 mM), 1.5 μl each primer (150 mM), 1 unit Taq DNA polymerase and 16.5 μl ddH₂O. Reaction conditions were as follows: 94°C for 5 min; 35 cycles of 94°C for 45 sec, 69°C for 45 sec and 72°C for 45 sec; and 72°C for 7 min. PCR products were digested using the StyI restriction enzyme (New England Biolabs, Ipswich, MA, USA) at 37°C for 16 h. For codon 751, primer sequences used were: upstream, 5′-GCCCGCTCTGGATTATACG-3′ downstream, 5′-CTATC ATCTCCTGGCCCCC-3′. PCR mix (20 μl) containing 100 ng DNA template, 2.0 μl 10Χ PCR buffer solution (Mg²⁺-free), 1.2 μl Mg²⁺ solution (25 mM MgCl₂), 0.5 μl dNTPs (10 mM), 1.5 μl each primer (150 mM) and 25 units Taq DNA polymerase. Reaction conditions were as follows: 94°C for 5 min; 35 cycles of 94°C for 45 sec, 63°C for 45 sec and 72°C for 45 sec; and 72°C for 7 min. PCR products were digested using the PstI restriction enzyme (New England Biolabs) at 37°C for 16 h. Codon products were visualized on 2% agarose gels using a gel imaging system. Restriction fragments were sent to Beijing Genomics Institute (Beijing, China) for direct gene sequencing.

SPSS 17.0 statistical software was used for statistical analysis. The two-sample t-test was used to compare differences between the two groups and the χ² test was used to compare age, gender, smoking status, alcohol consumption and genotype and allele frequencies between the two groups. The relationship between gene polymorphism and ESCC was analyzed using logistic regression to obtain odds ratios (OR) and 95% confidence intervals (CI). Analyses were performed with two-sided tests. P<0.05 was considered to indicate a statistically significant difference.

Age, gender, smoking status and alcohol consumption of the 400 ESCC cases and 400 healthy controls included in this study are presented in Table I. Controls were age- and gender-matched to cases. Age ranges and gender distributions of the two groups were not identified as significantly different, although ESCC occurred twice as often in males. However, statistically significant increases in cigarette smoking and alcohol consumption were observed in the ESCC patients (44.0 and 34.3%, respectively) compared with the controls (33.5 and 26%, respectively; P<0.05).

The expected PCR product size for the 312 locus was 751 bp. StyI restriction digestion generated various products based on the genotype: GG resulted in 2 fragments, 507 and 244 bp; GA resulted in 4 fragments, 507, 474, 244 and 33 bp; and AA resulted in 3 fragments, 474, 244 and 33 bp (Fig. 1). For the 751 locus, the expected PCR product size was 436 bp. PstI restriction digestion generated the following: 2 fragments for the AA genotype, 290 and 146 bp; 4 fragments for AC, 290, 227, 146 and 63 bp; and 3 fragments for CC, 227, 146 and 63 bp (Fig. 2). Sequencing of these restriction products was consistent with the expected sequence (Figs. 3 and 4).

The distributions of genotypes for XPD codons 312 and 751 were consistent with Hardy-Weinberg equilibrium for cases and controls (P>0.05). No statistically significant differences in genotype or allele frequencies for codon 312 were observed between cases and controls (Table II). However, statistically significant differences in genotype and allele frequencies for codon 751 were observed between cases and controls (P<0.05; Table III).

Results of the logistic regression analysis indicated that gender was a confounding factor for ESCC (P=0.039), while age, smoking status and alcohol consumption had no correlation with the occurrence of ESCC (P>0.05). Excluding the effect of gender resulted in no correlation between SNP in codon 312 and occurrence of ESCC (P>0.05). However, a correlation was observed between codon 751 genotype and ESCC occurrence. No significant increase in ESCC risk was observed for cases with the AC genotype compared with those with the AA genotype (P=0.137). ESCC risk in cases with the CC genotype was increased by 1.6-fold (OR, 1.600; 95% CI; 1.137–2.253; P=0.007; Table IV).