CLPTM1L Genetic Polymorphisms and Interaction With Smoking and Alcohol Drinking in Lung Cancer Risk

Supplemental Digital Content is available in the text


INTRODUCTION
A s one of the most common malignant tumors, lung cancer causes the greatest number of cancer-related deaths 1 . The incidence of lung cancer appears to result from a combination of factors, including genetic susceptibility of the individual and risk factors in the environment. Lung cancer has the highest mortality rate, and it is the leading cause of cancer-related deaths in urban China. 2,3 Several genes at chromosomal locus 5p15.33, including cleft lip and palate trans-membrane 1-like (CLPTM1L), were found to be associated with lung cancer risk in recent genomewide association studies. [4][5][6][7][8] CLPTM1L, also known as CRR9, encodes an enzyme that may be associated with apoptosis. 9 This gene is expressed in cells of various tumor types, including lung tumor tissue, and is overexpressed in cisplatin-resistant cell lines.
In modern China, an increasing number of people have smoking and drinking habits. In the northwest China, drinking and smoking is very common. Previous studies have shown that there are interactions between certain genes and environmental factors that increase the risk of lung cancer. But there are few studies reporting an interaction between CLPTM1L and environmental factors like drinking and smoking habits. Therefore, in this study, we aimed to determine the relationship between CLPTM1L and lung cancer risk, as well as to explore the interaction between CLPTM1L and environmental factors. Nine SNPs of CLPTM1L were genotyped to perform an association analysis in a case-control study of the northwest Chinese Han population.

Study Participants
All patients and controls were members of the Han population living in Xi'an city and nearby. The patients were recruited between January 2011 and February 2014 at the First Affiliated Hospital of the Medical College of Xi'an Jiaotong University. All patients were newly diagnosed with lung cancer and were characterized histologically. None of the patients had previous history of other cancers, chemotherapy, or radiotherapy. Patients who had comorbidity, such as diabetes mellitus, hypertension, or any endocrine disorders were excluded. The 301 controls were randomly selected from the individuals who received physical examination at First Affiliated Hospital during the same time period as the cases were recruited. The control group comprised unrelated healthy individuals who had no known medical illness or hereditary disorders and who were not taking any medications. Control subjects were excluded if they had <12 months of data before their index date or had miss information. Participants were chosen without restrictions of age, gender, or disease stage. A total of 228 cases and 301 controls were included in the study. The basic characteristics of the participants (eg, gender, age, and pathology) are listed in Table 1.

Clinical Data and Demographic Information
We used a standard epidemiological questionnaire to collect personal data through an in-person interview, including residential region, age, gender, smoking status, alcohol use, education status, and family history of cancer. Patients were classified as alcohol drinkers if they consumed one or more drinks (Drinking alcohol intake calculated using standard units: one standard drink units ¼ 10 g of pure alcohol ¼ 0.5 two high spirits ¼ 1 two low spirits ¼ 355 mL beer ¼ 1.5 two wine ¼ 3 two wines.) per week, and as nondrinkers if they consumed less than 1 drink per week or abstained from alcohol. All of the smokers had at least 1 or more per day smoking and a period or periods aggregated more than 6 months were defined as eversmokers; Never-smokers were defined as those who smoked less than 100 cigarettes in their lifetime (or before diagnosis for cases) and former smokers as those who quit smoking at least 1 year before the time of the survey. The case information was collected through consultation with treating physicians or by review of medical charts. All of the participants signed informed consent. The Human Research Committee of the First Affiliated Hospital of Xi'an Jiaotong University and Northwest University for Approval of Research Involving Human Subjects approved the use of human tissue in this study.

SNP Selection and Genotyping
All 9 SNPs were previously published to be associated with lung cancer, with minor allele frequencies >5% in the HapMap Chinese Han Beijing (CHB) population. The blood samples were collected into tubes containing ethylenediaminetetraacetic acid. After centrifugation, the samples were stored at À808C until analysis. DNA was extracted from whole blood samples by GoldMag-Mini Whole Blood Genomic DNA Purification Kit (GoldMag Co. Ltd. Xi'an City, China) and DNA concentration was measured by NanoDrop 2000. The Multiplexed SNP MassEXTENDED assay was designed by Sequenom MassAR-RAY Assay Design 3.0 Software. 10 SNP genotyping was performed by Sequenom MassARRAY RS1000 using the standard protocol. 10

Statistical Analysis
The SPSS17.0 statistical software and Microsoft Excel were used for statistical analysis. All P values presented in this study are two-sided, and we used P 0.05 as the cutoff value for statistical significance. We used a Fisher's exact test to assess the departure of each SNP frequency from Hardy-Weinberg equilibrium (HWE) in the control subjects. We tested the difference of SNP genotype distribution between cases and controls by using a x 2 test. 11 We tested odds ratios (ORs) and constructed 95% confidence intervals (CIs) using unconditional logistic regression analysis with adjustments for age, gender, and smoking and drinking status. 12 The associations between the CLPTM1L gene and the risk of lung cancer were tested using genetic models (co-dominant, dominant, recessive, over-dominant and log-additive) analysis by SNP-tats, website software from http://bioinfo.iconcologia.net. We calculated ORs and 95% CIs by unconditional logistic regression analysis adjusted for age and gender. 12 Akaike's Information Criterion and Bayesian Information Criterion were applied to estimate the best-fit model for each SNP.
Haploview software version 4.2 was used to analyze the association between haplotypes and the lung cancer. Linkage disequilibrium (LD) analysis was performed using genotype data from all the subjects. The pattern of LD was analyzed using two parameters, r 2 and D 0 . Statistical significance was established when P < 0.05.

RESULTS
A total of 529 participants, including 228 lung cancer cases and 301 controls were successfully genotyped for further analysis (Table 1). Males were 78.1% among cases compared with 62.5% among controls. The mean age was 58.7 (AE10) years for cases and 50.2 (AE8.1) years for controls. There was significant difference between cases and controls as a function of age ( 50 and >50; 60 and >60; 70 and >70 years old, P < 0.001). Overall, there was an increased risk of lung cancer observed with increasing age. More smokers were observed in cases compared with subjects in the control group (P < 0.001). This result was expected because most lung cancers can be attributed to smoking. There was no significant difference between groups based upon drinking status (P ¼ 0.101). Of the 228 lung cancer cases, 37 (16.2%) were small-cell lung cancer (SCLC), 73 (32%) lung adenocarcinoma (LAC), 80 (35.1%) lung squamous cell carcinoma (LSCC), 11 (4.8%) lung adenosquamous carcinoma (LASC) and 27 (32%) large cell, mixed cell, or undifferentiated carcinomas.
Nine SNPs in the CLPTM1L gene were genotyped in lung cancer patients and the healthy controls. Each SNP/sample call rate was 98.5% in both cases and controls. Table 2 summarizes the basic characteristics of CLPTM1L SNPs in the study population. rs421629 and rs467095 were excluded at 5% HWE P level. Differences in frequency distributions of alleles between cases and controls we determined using the x 2 test or Fisher's exact tests. Three SNPs, rs451360, rs402710, and rs31484 were significantly associated with decreased lung cancer risk in the study population, and they respectively presented a 0.52-fold (95% CI, 0.33-0.81, P ¼ 0.004), 0.76-fold (95% CI, 0.58-0.99, P ¼ 0.045), and 0.70-fold (95% CI, 0.49-0.99, P ¼ 0.045) lung cancer risk. After adjustment for age, gender, and smoking and drinking status, the three SNPs respectively presented a 0.59fold, 0.75-fold and 0.74-fold lung cancer risk.
In addition to the allelic model analysis, the association between CLPTM1L SNPs and lung cancer risks was assessed using genetic models ( Table 3). The results showed that the genotype ''T/T'' of rs402710 significantly decreased the risk of lung cancer in co-dominant (OR ¼ 0.44; 95% CI, 0.19-0.99, The association of CLPTM1L genetic polymorphisms and lung cancer risk was analyzed among different populations (Supplemental, http://links.lww.com/MD/A103). In the allelic model analysis, rs402710 (OR ¼ 0.55; 95% CI, 0.37-0.81, P ¼ 0.002) and rs451360 (OR ¼ 0.50; 95% CI, 0.25-0.969, P ¼ 0.04) demonstrated a significantly decreased risk for lung cancer among smokers. After adjustment for age, gender, and drinking status, only rs402710 presented a 0.54-fold decreased risk of lung cancer risk (Table 4). Additionally, rs451360 were not significant anymore (Table 5). CLPTM1L polymorphisms were further characterized using linkage disequilibrium (LD) and haplotype analyses. LD was determined pairwise among all 9 SNPs and the haplotype structure of CLPTM1L gene was analyzed (D 0 and r 2 ).
Haplotype block divided by D' confidence interval method, D' value of 95% CI 0.70 $ 0.98 in adjacent SNPs were classified as the same haplotype block. In the control group, 2 LD blocks were detected. Block 1 consisted of 3 closely linked SNPs, rs421629, rs380286, and rs402710 ( Figure 1). The other SNPs in block 1 showed strong linkage. Block 2 included 2 completely linked SNPs, rs467093, and rs31484. Additionally, weak linkage between rs1007334 and rs45136 was observed.

DISCUSSION
In the present case-control study, we investigated the associations between the 9 SNPs of CLPTM1L and risk of lung cancer. We demonstrated that certain CLPTM1L genetic polymorphisms, rs402710, rs451360, and rs31484, are associated with the decreased risk of lung cancer in the northwest Chinese Han population. We found that alcohol drinking and tobacco smoking may interact with CLPTM1L polymorphisms to affect the development of lung cancer.
CLPTM1L is named for its similarity to a gene encoding cleft lip and palate transmembrane protein 1, which was identified as being disrupted in a family with cleft lip and palate. 13 However, for a long time, the functions of the CLPTM1L gene were poorly understood. It was observed as a cisplatin resistance factor in ovarian cancer cell lines, which cannot be interpreted as a function of the CLPTM1L gene during oncogenesis. 9,14 A recent study demonstrated that CLPTM1L, as an over expressed protein in lung tumor cells, protected cells from genotoxic stress induced apoptosis through regulation of Bcl-xL, which implicated an anti-apoptotic CLPTM1L function as a potential mechanism of susceptibility to lung tumorigenesis. 15 Of the 3 SNPs associated with decreased lung cancer risk, 2 of these SNPs show high consistency with previous studies in other populations. rs402710, located in intron 4 of the CLPTM1L gene, was found to be associated with higher DNA adduct formation in tumor adjacent lung tissue 16 and may enhance the formation and persistence of DNA adducts. However, the association between this variant and the CLPTM1L gene is not clear. The protective effect of rs402710 on lung cancer risk has been identified in epidemiologic studies of the Japanese population, 17 the Korean population 18,19 and in a mixed Asian population 20 ; however, 1 study of the Chinese population failed to demonstrate a similar finding. 7 Consistent with most of the previous studies, our study in northwest Chinese Han population confirmed that rs402710 was associated with decreased lung cancer risk in the allelic model. The association between CLPTM1L intronic SNP rs451360 and lung cancer has been evaluated in Caucasian 6,21 and Chinese populations, 4 which all demonstrated a statistically significant protective effect of rs451360. Consistently, we also found the same effect of rs451360 on lung cancer in the northwest Chinese Han population.
In contrast, rs31484, located in an intron region of CLPTM1L, displayed a totally opposite effect on lung cancer risk compared to results of 2 previous studies. 4,21 One study Linkage disequilibrium of polymorphic sites in the CLPTM1L gene. A standard color scheme is used to display LD with bright red for very strong LD (LOD ¼ 2, D 0 ¼ 1), white for no LD (LOD < 2, D 0 < 1), pink red (LOD ¼ 2, D 0 < 1), and blue (LOD < 2, D 0 ¼ 1) for intermediate LD.
found rs31484 was associated with an increased lung cancer risk in Caucasians, but the other study showed a decreased risk in Asians. In agreement with the Asian study, we revealed a protective effect of rs31484 in Han Chinese. These conflicting findings might be attributed to the different ethnicity of the subjects enrolled in each study, as the minor allele frequency (MAF) is very different between Caucasians (12%) and Asians (47%). When subjects were categorized by smoking status, we found that rs402710 and rs451360 were only significant in the smoking group. This result is very interesting because the general belief is that smoking increases the risk of lung cancer. It could be hypothesized that CLPTM1L may play a role in apoptotic response of lung cells that are exposed to genotoxic stress caused by tobacco-related carcinogens. However, this hypothesis needs to be investigated in future studies.
It is noteworthy that rs380286 was not associated with lung cancer risk in the general population, but displayed significantly decreased risk for lung cancer among non-drinking population in our study, suggesting it may present a protective effect for non-drinkers to the susceptibility to lung cancer. In one previous study, heavy alcohol consumption was found to be a risk factor for lung cancer. [22][23][24][25] Our data indicated that there may be a potential interaction between CLPTM1L polymorphisms and alcohol consumption. However, the role alcohol plays between the CLPTM1L polymorphisms and lung cancer risk remains unknown.
Besides the allelic model analysis, we also performed genotypic model analysis to investigate the role of CLPTM1L variants on lung cancer risk. We found that in the northwest Chinese Han population, rs401681 and rs402710 had a protective effect on lung cancer in recessive and co-dominant models. Our findings are corroborated by previous studies performed in Asians and Caucasians. 4,18,21,26,27 Despite the current study possessing enough statistical power, some limitations should be considered. Firstly, the sample size of our study was relatively small. Secondly, the association between CLPTM1L polymorphism and clinicopathological type was not evaluated in this study. For example, a meta-analysis showed that rs402710 conferred protective effects in both adenocarcinoma and squamous cell carcinoma. 28 In conclusion, our study provides new evidence regarding the relationship between CLPTM1L and risk of lung cancer in the Han individuals of northwestern China. Our findings suggest that the interaction of genetic variants of CLPTM1L and environmental factors, especially tobacco smoking and alcohol consumption, may play an important role in occurrence of lung cancer in this population. The biological functions of the SNPs in CLPTM1L are of great interest and warrant further research.