Rare deleterious germline variants and risk of lung cancer

Abstract

Recent studies suggest that rare variants exhibit stronger effect sizes and might play a crucial role in the etiology of lung cancers (LC). Whole exome plus targeted sequencing of germline DNA was performed on 1045 LC cases and 885 controls in the discovery set. To unveil the inherited causal variants, we focused on rare and predicted deleterious variants and small indels enriched in cases or controls. Promising candidates were further validated in a series of 26,803 LCs and 555,107 controls. During discovery, we identified 25 rare deleterious variants associated with LC susceptibility, including 13 reported in ClinVar. Of the five validated candidates, we discovered two pathogenic variants in known LC susceptibility loci, ATM p.V2716A (Odds Ratio [OR] 19.55, 95%CI 5.04-75.6) and MPZL2 p.I24M frameshift deletion (OR 3.88, 95%CI 1.71-8.8); and three in novel LC susceptibility genes, POMC c.*28delT at 3' UTR (OR 4.33, 95%CI 2.03-9.24), STAU2 p.N364M frameshift deletion (OR 4.48, 95%CI 1.73-11.55), and MLNR p.Q334V frameshift deletion (OR 2.69, 95%CI 1.33-5.43). The potential cancer-promoting role of selected candidate genes and variants was further supported by endogenous DNA damage assays. Our analyses led to the identification of new rare deleterious variants with LC susceptibility. However, in-depth mechanistic studies are still needed to evaluate the pathogenic effects of these specific alleles.

Fig. 1. Gene exons, protein domains, and rare deleterious variants of the candidate genes.

The top five candidate variants (red arrows): 1) POMC c.*28 deletion (del) located at target sites of several miRNAs in 3′ UTR; 2) STAU2 p.N364M fs*67del located in the double-stranded RNA-binding motif (dsrm), and next to a phosphorylation site p.S363; 3) ATM V2716A located in the PI3-kinase (PI3K) catalytic domain; 4) MPZL2 p.I24M fs*22del was close to the antibody variable domain of immunoglobulins (Ig-V); 5) MLNR p.Q334V fs*3del located in the transmembrane receptor domain (TM), and close to a phosphorylation site p.S327. The color vertical bars represent different types of variants: ClinVar pathogenic variants (bold blue: POMC W84* stop-gain, ATM Q414* stop-gain, and MPZL2 M1T* start-loss), previous reported LC-associated variants (blue: ATM P1054R and L2307F, and MPZL2 deletion rs13915729), and ClinVar variants of uncertain significance (black). Gene exons (green blocks), introns (horizontal green lines), untranslated regions (UTRs, orange blocks), and protein domain/motif (framed rectangles) are shown. The length of the gene (kb) and protein (number of amino acids, AA) are shown to the right.

Fig. 2. Discovery of DNA damageome genes/proteins and variants.

a siRNA knockdown endogenous DNA damage assay scheme. b Increased DNA damage (γH2AX) levels in five out of the six genes knockdowns (mean ± SEM, n = 2~4), MLNR, CHEK2, POMC, ATM, and MME, compared with non-targeting (NT) siRNA control. There is no increasing DNA damage in MPZL2 knockdown cells. c Representative flow histograms showing higher γH2AX levels in gene knockdowns. d–f MLNR, MME, and POMC knockdown by two individual siRNAs confirmed the DNA damage-up phenotypes by pooled siRNAs in b. DNA damage quantified by d median fluorescence intensity or e DNA-damage positive subpopulation. f Examples of flow cytometry dot plots showing DNA-damage positive subpopulation. g Overproduction endogenous DNA damage assay scheme. h Wildtype POMC and mutant MLNR p.Q334V fs*3del overproduction promote DNA damage. GFP-Tubulin as a control. i Representative histograms of (g). *P-value < 0.05, **P-value < 0.01, n.s not significant (P-value > 0.05).