Transcriptome‐wide association study reveals candidate causal genes for lung cancer

Abstract

We have recently completed the largest GWAS on lung cancer including 29,266 cases and 56,450 controls of European descent. The goal of our study has been to integrate the complete GWAS results with a large‐scale expression quantitative trait loci (eQTL) mapping study in human lung tissues (n = 1,038) to identify candidate causal genes for lung cancer. We performed transcriptome‐wide association study (TWAS) for lung cancer overall, by histology (adenocarcinoma, squamous cell carcinoma and small cell lung cancer) and smoking subgroups (never‐ and ever‐smokers). We performed replication analysis using lung data from the Genotype‐Tissue Expression (GTEx) project. DNA damage assays were performed in human lung fibroblasts for selected TWAS genes. As expected, the main TWAS signal for all histological subtypes and ever‐smokers was on chromosome 15q25. The gene most strongly associated with lung cancer at this locus using the TWAS approach was IREB2 (pTWAS = 1.09E−99), where lower predicted expression increased lung cancer risk. A new lung adenocarcinoma susceptibility locus was revealed on 9p13.3 and associated with higher predicted expression of AQP3 (pTWAS = 3.72E−6). Among the 45 previously described lung cancer GWAS loci, we mapped candidate target gene for 17 of them. The association AQP3‐adenocarcinoma on 9p13.3 was replicated using GTEx (pTWAS = 6.55E−5). Consistent with the effect of risk alleles on gene expression levels, IREB2 knockdown and AQP3 overproduction promote endogenous DNA damage. These findings indicate genes whose expression in lung tissue directly influences lung cancer risk.

Rare deleterious germline variants and risk of lung cancer

“…These networks are known as DNA damageome proteins (DDPs) 37 . The DNA damageome also includes LoF variants that show DNA damage-up phenotypes 38 , most of which are not directly related to DNA repair but rather participate in the DNA damage production. We selected six prioritized genes for the assay: CHEK2 , ATM, MPZL2 , MLNR , POMC , and MME .…”

“…Endogenous DNA damage is proposed to drive cancers by genome instability — a hallmark of cancer 37 , 38 . To test whether knockdown or overexpression of the candidate genes or variants induces endogenous DNA damage, we performed flow cytometric assays to measure γH2AX levels, a DNA double-strand-break marker 107 , following siRNA knockdown and overproduction of GFP fusions of proteins of interest.…”

“… Human cell lines and reagents . MRC5-SV40, a human lung fibroblasts derived cell line was maintained in standard Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, 2 mM L-glutamine, 100 μg/mL penicillin, and 100 μg/mL streptomycin 37 , 38 . The cell line was authenticated by ATCC STR analysis and routinely check to be mycoplasma-free.…”

“…Primers used included GAPDH (housekeeping gene) forward: CAA TGA CCC CTT CAT TGA CC; GAPDH reverse: GAT CTC GCT CCT GGA AGA TG; POMC forward: GCC AGT GTC AGG ACC TCA C; POMC reverse: GGG AAC ATG GGA GTC TCG G; CHEK2 forward: TCT CGG GAG TCG GAT GTT GAG; CHEK2 reverse: CCT GAG TGG ACA CTG TCT CTA A; ATM forward: GGC TAT TCA GTG TGC GAG ACA; ATM reverse: TGG CTC CTT TCG GAT GAT GGA; MPZL2 f orward: TTA ATG GGA CAG ATG CTC GGT; MPZL2 reverse: AAG ACA CCC GGT CCT TAA ACC; MME forward: AGA AGA AAC AGC GAT GGA CTC C; MME reverse: CAT AGA GTG CGA TCA TTG TCA CA; MLNR forward (siRNA): CTG AGC GCA TCT ATC AAC CCA; MLNR reverse (siRNA): TCC CAT CGT CTT CAC GTT AGC; MLNR forward (overexpression): GTG GTG ACC GTG ATG CTG AT; MLNR reverse (overexpression): AGC AGG ATG AGT AGG TCG GA. Flow-cytometric DNA damage assays . Sensitive DNA damage assays by flow cytometry were performed as previously described 37 , 38 . γH2AX primary antibody (Sigma, Catalog #05-636) and goat anti-mouse secondary antibody, Alexa Fluor 647 (Thermo Fisher, Catalog #A21236) were used to stain cells.…”

Implications of the Adiponectin System in Non-Small Cell Lung Cancer Patients: A Case-Control Study

“…Despite substantial advances in our understanding of the molecular basis of lung cancer [ 4 , 5 , 6 , 7 , 8 , 9 ], ongoing research on driver genes, mechanisms of immune evasion, and the tumor microenvironment, which also triggers crosstalk phenomena between organs/tissues, is expected to improve both early disease detection and survival [ 10 ]. Data from genome-wide (GWAS) and transcriptome-wide association studies (TWAS) in large lung cancer cohorts have documented prominent heterogeneity in the genetic susceptibility across lung cancer histological subtypes, possibly reflecting different underlying oncogenic molecular drivers [ 11 , 12 , 13 ]. Recently, obesity was recognized as a major risk factor linked to both the incidence and progression of several cancer types [ 14 ].…”