Abstract

   Cancer-testis (CT) genes played important roles in the progression of
   malignant tumors and were recognized as promising therapeutic targets.
   However, the roles of genetic variants in CT genes in lung cancer
   susceptibility have not been well depicted. This study aimed to
   evaluate the associations between genetic variants in CT genes and lung
   cancer risk in Chinese population. A total of 22,556 qualified SNPs
   from 268 lung cancer associated CT genes were initially evaluated based
   on our previous lung cancer GWAS (Genome-wide association studies) with
   2,331 cases and 3,077 controls. As a result, 17 candidate SNPs were
   further genotyped in 1,056 cases and 1,053 controls using Sequenom
   platform. Two variants (rs6941653, OPRM1, T > C, screening: OR = 1.24,
   95%CI: 1.12-1.38, P = 2.40×10^-5; validation: OR = 1.18, 95%CI:
   1.01-1.37, P = 0.039 and rs402969, NLRP8, C > T, screening: OR = 1.15,
   95%CI: 1.04-1.26, P = 0.006; validation: OR = 1.16, 95%CI: 1.02-1.33, P
   = 0.028) were identified as novel lung cancer susceptibility variants.
   Stratification analysis indicated that the effect of rs6941653 was
   stronger in lung squamous cell carcinoma (OR = 1.36) than that in lung
   adenocarcinoma (OR = 1.15, I^2 = 77%, P = 0.04). Finally, functional
   annotations, differential gene expression analysis, pathway and gene
   ontology analyses were performed to suggest the potential functions of
   our identified variants and genes. In conclusion, this study identified
   two novel lung cancer risk variants in Chinese population and provided
   deeper insight into the roles of CT genes in lung tumorigenesis.

   Keywords: cancer-testis genes, lung cancer susceptibility, single
   nucleotide polymorphisms, Chinese population, Sequenom platform

Introduction

   Lung cancer has been the most frequently diagnosed caner type and the
   leading cause of cancer-related deaths for decades in China [59]^1. The
   tumorigenesis of lung cancer was a multiple-stage process, and both
   environmental and genetic factors were involved. It was estimated that
   the heritability of lung cancer was about 15.2% in Chinese population
   [60]^2. However, up till now, GWAS (Genome-Wide Association
   Study)-reported lung cancer associated single nucleotide polymorphisms
   (SNPs) could only account for limited lung cancer heritability (less
   than 1%) [61]^2^, [62]^3. Therefore, more effective strategies were
   wanted to identify novel lung cancer risk loci based on GWAS data.

   Epigenetic alterations have been recognized as an important feature of
   tumorigenesis [63]^4^, [64]^5. Notably, cancer-testis (CT) genes, which
   were restrictedly expressed in germ cells and malignant tumor cells,
   were usually activated through epigenetic mechanisms [65]^6. The
   activation of CT genes in cancer samples made them oncogene candidates
   and their remained high immunogenicity made them perfect
   immunotherapeutic targets for cancer treatment [66]^7^, [67]^8. In
   addition, associations between genetic variants in CT genes and the
   susceptibility of cancers have been described in previous studies. For
   example, genetic variants in HORMAD2 and GPATCH2 were reported
   associated with lung cancer risk [68]^9^, [69]^10, and variants in
   CTNNA2, CCDC33 and SPAG17 showed significant association with the
   susceptibility of breast cancer [70]^11^, [71]^12. All these studies
   suggested that genetic variants in CT genes could also contribute to
   the development of cancers.

   Therefore, systematic analysis of the associations between genetic
   variants in CT genes and lung cancer risk could help identify more
   novel lung cancer susceptibility loci. In our previous study, we
   performed a systematic identification of CT genes in 19 cancer types
   based on multiple public-available databases [72]^13. As a result, 876
   novel CT genes in 19 cancer types were recognized. In lung cancer, we
   identified 268 CT genes (including 61 known CT genes) that were
   activated in at least 2% of cancer samples [73]^13. This finding
   provided us an unprecedented opportunity to explore the associations
   between genetic variants in CT genes and the susceptibility of lung
   cancer.

   In this study, a two-stage case control study was performed. The NJMU
   GWAS, which has been established in our previous study, was used to
   screen candidate lung cancer risk variants [74]^9. These promising
   variants were further validated in an independent Chinese population
   with a total of 1,056 lung cancer cases and 1,053 controls based on the
   Sequenom MassARRAY iPLEX platform. This study would identify novel lung
   cancer susceptibility loci in Chinese population and help reveal the
   roles of genetic variants in CT genes in the development of lung
   cancer.

Materials and Methods

Study subjects

   Two independent datasets were used in this study. The NJMU GWAS
   contained 2,331 lung cancer cases and 3,077 controls, and was used as
   screening dataset. The detailed information about the study subjects in
   NJMU GWAS was described previously [75]^9. Genotype imputation for NJMU
   GWAS was performed using IMPUTE2 and Shapeit v2 with the 1000 Genomes
   Project (the Phase III integrated variant set release, across 2504
   samples) as the reference [76]^14. The validation dataset consisted of
   1,056 lung cancer cases and 1,053 controls. All the cases were
   histopathologically confirmed patients and were recruited from the
   First Affiliated Hospital of Nanjing Medical University and Jiangsu
   Cancer Hospital. Controls were obtained from a screening program for
   non-infectious diseases conducted in Jiangsu Province and matched to
   the cases for age and gender. All the participants have signed the
   informed consent acknowledgement and this study was approved by the