Association between novel PLCE1variants identified in published esophageal cancer genome-wide association studies and risk of squamous cell carcinoma of the head and neck

Participant recruitment has been described elsewhere [20, 21]. In brief, newly-diagnosed SCCHN cancer patients were consecutively recruited from The University of Texas M.D. Anderson Cancer Center between October 1999 and October 2007. All cases were diagnosed with histologically confirmed SCCHN, and there were no age, sex or stage restrictions. Exclusion criteria included second SCCHN primary tumors, primary tumors of the nasopharynx or sinonasal tract, metastasized cancer from other organs, or any histopathologic diagnosis other than SCCHN. Additionally, patients who had received prior surgery (other than diagnostic biopsies), chemotherapy or radiation therapy before recruitment, and any blood transfusion during the preceding 6 months were also excluded.

Cancer-free controls were recruited from those visitors in outpatient clinics at M.D. Anderson Cancer Center, who were not genetically related to the patients or to each other, and were frequency-matched to the cases on age (±5 years), sex and ethnicity. The inclusion criterion for controls was defined as the self-reported absence of prior history of cancer. After the first survey using a short questionnaire to find out whether potential subjects were willing to participate and qualified in this study, a written informed consent was obtained, and a structured questionnaire was administered by trained interviewers to collect demographic data and environmental exposure history, such as age, sex, ethnicity, smoking and alcohol consumption. After interview, approximately 30-ml venous blood sample was collected from each participant. The response rates for cases and controls were approximately 90% and 85%, respectively. In the analysis, a total of 1,098 cases and 1,090 controls that completed the interview and donated a one-time blood sample were included. All subjects were non-Hispanic whites. This study was approved by the Institutional Review Board of M. D. Anderson.

Besides the non-synonymous SNP (rs2274223) identified in the published GWAS, we also searched the NCBI dbSNP database (http://​www.​ncbi.​nlm.​nih.​gov/​ build 131) for other common [minor allele frequency (MAF) ≥ 0.05] and potentially functional SNPs located in the 5' near gene, 5'- and 3'-untranslated regions of PLCE1 by using a set of tools at the website SNPinfo (http://​snpinfo.​niehs.​nih.​gov/​) [22]. In addition, the LD analyses were performed to optimize SNP selection. We selected additional two potentially functional SNPs in PLCE1: rs3203713, located in the miRNA binding site (3'UTR) and rs11599672, located in the transcription factor binding site (TFBS in 5' near gene). Taken together, we included three SNPs (rs2274223A/G, rs3203713A/G and rs11599672T/G) in PLCE1 for the final genotyping.

We extracted genomic DNA from the buffy-coat fraction of the blood samples using the Qiagen Blood DNA Mini Kit (Qiagen Inc., Valencia, Calif) according to the manufacturer's instruction and genotyped SNPs rs2274223 and rs3203713 using the TaqMan allelic discrimination assay on an ABI 7900 system (Applied Biosystems). Genotyping was performed without knowing the subjects' case or control status, and four negative controls (no DNA) and duplicated commercial positive controls (TaqMan Control Genomic DNA; Applied Biosystems) included in each 384-well plate were used for quality control. The accordance achieved 100% for the duplicates of 5% of samples. Polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method was also used to identify genotypes of rs11599672, because the Taqman assay was not applicable to this SNP. The antisense primer was introduced a mismatched A to replace T at +2bp from the polymorphic site to create an SspI restriction site (sense: 5'- GGAGAGACATTCTGTTGGGTGA-3'; antisense: 5'- CCTTCAAACCACCGCTGTAAT- 3'). A 155-bp fragment containing this T/G site was amplified in the PCR mixture consisted with approximately 20 ng of genomic DNA, 2 pmol of each primer, 0.1 mM each dNTP, 1 × PCR buffer (50 mM KCl, 10 mM Tris HCl, and 0.1% Triton X-100), 1.5 mM MgCl ₂ , and 0.5 unit of Taq polymerase. The PCR profile consisted of an initial melting step of 95°C for 5 min; 35 cycles of 95°C for 30 s, 59°C for 45 s, and 72°C for 1 min; and final extension step of 72°C for 10 min. The PCR product was then digested with the restriction enzyme SspI (New England BioLabs, Beverly, MA) overnight at 37°C and separated on 3% agarose gel. The T allele has the restriction site and produces two bands of 134- and 21-bp, whereas the G allele lacks the SspI restriction site, resulting in one band of 155-bp (Figure 1). Finally, 10% of the samples were randomly selected to perform the repeated assays, and the results were 100% concordant. Direct sequencing was also conducted to confirm the genotypes of rs11599672 (Figure 2).

We evaluated differences in selected demographic variables, risk factors, and frequencies of the PLCE1 genotypes between cases and controls by using the χ² test and examined Hardy-Weinberg equilibrium by a goodness-of-fit χ² test to compare the observed genotype frequencies with the expected ones among the controls. We estimated associations between PLCE1 variants and SCCHN risk by computing odds ratios (ORs) and 95% confidence intervals (CIs) from both univariate and multivariate logistic regression analyses with adjustment for the known risk factors for SCCHN, such as age, sex, smoking status and alcohol use. Haplotype frequencies and individual haplotypes were generated by using SAS PROC HAPLOTYPE based on the observed genotypes. All statistical analyses were two sided, and P < 0.05 was considered statistically significant. All the statistical analyses were performed with Statistical Analysis System software (v.9.1 SAS Institute, Cary, NC).

The characteristics of cases and controls included in this study are summarized in Table 1. The mean age was 57.1 years (±11.1) for cases and 56.6 years (±11.0) for controls. There were no significant differences in the distributions of age and sex between cases and controls (P = 0.676 and 0.581, respectively), suggesting the frequency-matching on age and sex was adequate. However, 72.2% and 72.9% of the SCCHN cases were smokers and drinkers, respectively, which were significantly higher than that of the controls (50.9% and 56.6%, respectively) (P < 0.001 for the both). Of the 1,098 cases, 559 (50.9%) had tumors of the oropharynx and 539 (49.1%) had tumors arising at non-oropharynx sites including oral cavity, hypopharynx and larynx. Furthermore, 272 cases (24.8%) presented with stage I-II and 826 cases (75.2%) presented with III-IV stage.

The genotype and allele distributions of three SNPs (rs2274223, rs3203713 and rs11599672) in cases and controls are shown in Table 2. The observed genotype frequencies for these three polymorphisms were all in Hardy-Weinberg equilibrium in the controls (P = 0.07, 0.98 and 0.72, respectively). The single locus analyses revealed that genotype distributions of these three polymorphisms were not significantly different between overall cases and controls (P = 0.554 for rs2274223, P = 0.860 for rs3203713 and P = 0.265 for rs11599672, respectively). However, we found that the frequencies of variant rs2274223G and rs3203713G alleles (0.335 and 0.170, respectively) among the cases were slightly higher than those in the controls (0.320 and 0.162, respectively), while the frequency of the variant rs11599672G allele among the cases was slightly lower than that among the controls (0.277 vs. 0.297), suggesting rs2274223G, rs3203713G and rs11599672T alleles may be risk alleles to be considered in further analyses.

The LD analysis showed that two PLCE1 polymorphisms rs2274223 and rs3203713 were in incomplete LD in our study population (D' = 0.99, r² = 0.40), but these two SNPs were not in LD with rs11599672. To estimate possible joint effects and potential locus-locus interactions of PLCE1 polymorphisms on risk of SCCHN, we then examined the combined effects of these three variants by the number of putative risk alleles (i.e. rs2274223G, rs3203713G and rs11599672T). As shown in Table 2, we trichotomized the subjects into three groups with "0-1", "2-3" and "4-6" risk alleles. Compared with the group of "0-1" risk allele, there was a locus-dose effect as the risk allele number increased (P _trend = 0.046). After adjustment for age, sex, smoking and alcohol status, the group of " 2-3" risk alleles was borderline associated with risk of SCCHN (adjusted OR = 1.20, 95% CI = 0.97-1.50, adjusted P = 0.096) and the "4-6" risk allele group was significantly associated with risk of SCCHN (adjusted OR = 1.31, 95% CI = 1.00-1.73, adjusted P = 0.049). When "2-3" and "4-6" groups were combined for a larger number in the same stratum, the association between a larger number of risk alleles and risk of SCCHN remained statistically significant (adjusted OR = 1.23, 95% CI = 1.00-1.52, adjusted P = 0.048).

To investigate the modifying effects of PLCE1 variants on risk of SCCHN with different tumor sites, we conducted the stratification analysis by oropharyngeal and non-oropharyngeal cancers. As shown in Table 2, rs2274223 variant genotypes were associated with a significantly increased risk of SCCHN only for non-oropharyngeal sites (AG vs. AA: adjusted OR = 1.29, 95% CI = 1.01-1.64, adjusted P = 0.042; AG/GG vs. AA: adjusted OR = 1.30, 95% CI = 1.03-1.64; adjusted P = 0.025), while rs11599672 variant genotypes were associated with a significantly decreased risk of SCCHN for this group of patients (GG vs. TT: adjusted OR = 0.54, 95% CI = 0.34-0.86, adjusted P = 0.009; TG/GG vs. TT: adjusted OR = 0.76, 95% CI = 0.61-0.95, adjusted P = 0.018). No association was observed for rs3203713 variant genotypes and risk of SCCHN in both subgroups. In addition, the locus-dose effect of combined risk alleles was also seen in SCCHN arising at non-oropharyngeal sites (P _trend = 0.017). When the group of "0-1" risk allele was used as the reference, the group with "2-6" risk alleles had a significantly higher risk of SCCHN arising at non- oropharyngeal sites (adjusted OR = 1.35, 95% CI = 1.03-1.78; adjusted P = 0.033). However, these associations were not found for oropharyngeal cancer.

We further evaluated the combined effect of the three polymorphisms on risk of SCCHN arising at non-oropharyngeal sites by using the haplotype analysis (Table 3). A total of six haplotypes were derived from the observed genotypes, of which T_rs11599672A_rs2274223A_rs3203713 was the most common haplotype in cases and controls with the frequencies of 53.6% and 53.0%, respectively. Compared with the common haplotype, the G _rs11599672A _rs2274223A _rs3203713 haplotype was associated with a 28% decreased risk of SCCHN arising at non- oropharyngeal sites (adjusted OR = 0.72, 95% CI = 0.56-0.92); in contract, the T _rs11599672G _rs2274223A _rs3203713 haplotype was associated with a 31% increased risk of SCCHN arising at non-oropharyngeal sites (adjusted OR = 1.31, 95% CI = 1.00-1.72). These associations were not observed for other haplotypes.

We then performed stratification analyses to evaluate the effects of variant genotypes on risk of SCCHN arising at non-oropharyngeal sites by age, sex, smoking status, alcohol use and disease stage (Table 4). The results showed that the risk effect of rs2274223 was more evident in the younger (adjusted OR = 1.44, 95% CI = 1.02-2.02), male (adjusted OR = 1.51, 95% CI = 1.14-2.00), smokers (adjusted OR = 1.33, 95% CI = 1.02-1.72), drinkers (adjusted OR = 1.69, 95% CI = 1.25-2.28) and subjects with early stage (adjusted OR = 1.40, 95% CI = 1.03-1.91), whereas the protective effect of rs11599672 was more prominent in the younger (adjusted OR = 0.71, 95% CI = 0.50-1.00), smokers (adjusted OR = 0.73, 95% CI = 0.56-0.94) and non-drinkers (adjusted OR = 0.67, 95% CI = 0.46-0.99). Additionally, the increased risk associated with the combined effect of risk alleles was also more pronounced among the younger (adjusted OR = 1.68, 95% CI = 1.09-2.59), male (adjusted OR = 1.41, 95% CI = 1.00-1.98), smokers (adjusted OR = 1.65, 95% CI = 1.21-2.26), drinkers (adjusted OR = 1.42, 95% CI = 1.00-2.03) and subjects with early-stage tumors (adjusted OR = 1.46, 95% CI = 1.01-2.13). However, no significant associations were found for rs3203713 variant genotypes and risk of SCCHN arising at non-oropharyngeal sites in every stratum (data not shown). Heterogeneity test showed that there was no significant heterogeneity (P > 0.05) in every two stratums except for that of rs2274223 stratified by alcohol status (P = 0.005). Interestingly, we detected a significant interaction between alcohol status (never and ever) and rs2274223 variant genotypes (AA and AG/GG) for risk of SCCHN arising at non-oropharyngeal sites, even after the Bonferroni correction (P _int = 0.004).