br Materials and Methods br Results br

Materials and Methods
Results
Discussion In this study, we examined the cancer genomes of HPR NSCLCs to identify the genomic mutation profile associated with prolonged exposure to smoky coal pollutants. Of the 14 WGS patients, there was a mean of 12.75somaticgenomicmutations/Mb, 8.16exonicmutations/Mb, and 289 exonic mutations/tumor. Among the 2010 genes sequenced by targeted exome sequencing, the HPR patients had 68 mutated genes/tumor, 3 times higher than that in CR cases. Previous studies demonstrated that smoker NSCLCs bear more somatic mutations than never-smokers (Imielinski et al., 2012; Govindan et al., 2012). However, in HPR, the smokers and non-smokers harbored equal numbers of mutations and gene rearrangements in their genome. In CR, stages III–IV cancers had more mutations in 6 genes (KRAS, MYH13, TNR, ADAMTS20, PXDNL and SEZ6L) than stages I–II tumors, while patients ≥65years harbored more mutations in 4 genes (RYR2, COL22A1, ADAMTS12 and ZFPM2) than patients <65years; in HPR, only ACVR2A had more mutations in stages III–IV cancers than stages I–II tumors, and ADCY7 had a higher mutation rate in patients ≥65years than those <65years (Table S7 sheet 4). These results demonstrate the genotoxic effect of air pollution and the urgent need to attenuate pollution. PAHs are important carcinogens in PM2.5 and PM10 (Zielinska et al., 2010; Mumford et al., 1987). A variety of enzymes metabolize PAHs to more polar and water-soluble metabolites to be excreted from the body. However, during the course of metabolism, some unstable and reactive intermediates are formed, which can bind to DNA to form bulky DNA adducts (Hecht, 2012; DeMarini et al., 2001). At the same time, the PR619 constantly deal with the formation of DNA adducts by DNA repair processes to eliminate these alterations so that mutation does not occur (Irigaray and Belpomme, 2010). We showed that in the WGS NSCLCs, genes responsible for PAH detoxification (GSTM1, GSTP1, GSTT1) were mainly copy loss (Fig. S5B) or down-regulated (Fig. S5C), while genes involved in PAH activation (CYP1B1 in particular; Fig. S5C) were mainly up-regulated. DNA repair genes were mainly copy loss or mutated (Table S3). Mutations in DNA repair pathways have also been implicated in the production of chromosomal translocations (Aplan, 2006). Therefore, the events in PAH metabolism and DNA repair genes may pave the way to genomic mutations and chromosomal translocations, and may represent an essential step to allow accumulation of significant mutations to initiate malignant transformation. PAHs are associated with the C:G→A:T transversions in nucleotides (Ruggeri et al., 1993; Eisenstadt et al., 1982), and recent studies in cell lines showed that BaP can induce this type of nucleotide substitutions (Olivier et al., 2014). We found that the C:G→A:T substitutions were the most frequent nucleotide substitutions in 12/14 patients (Fig. S8), and exome sequencing confirmed the prevalent of C:G→A:T transversions in HPR NSCLCs (Table 2), indicating that PAHs were the main carcinogens for these patients. However, in 2/14 cases the most frequent nucleotide changes were A:T→T:A transitions (Fig. S8), suggesting that there might be other pollutants that caused this signature in the genomes. Some genes, e.g., TP53, EGFR, and KRAS, have high frequency of mutations in lung cancer (The Cancer Genome Atlas Research Network, 2012; Imielinski et al., 2012), and ethnic and sex-related differences in mutation spectrum are noted (Dearden et al., 2013; Kosaka et al., 2004). We showed that the mutation pattern of TP53, EGFR, and KRAS in CR NSCLCs (Figs. 2D and 3) was in consistence with previous report in Asian patients (Dearden et al., 2013), and HPR patients also had high mutation rates in these genes (Fig. 2D). Some genes, e.g., COL22A1, PAPPA2, TNR, TMEM132C, ADAMTS20, BAI3, CPS1, and OTOA, had high mutation frequencies in both regions (Table S7). Mutations in most genes, e.g., TP53, COL22A1, PAPP2A, CACNA1E, MYH3, NRXN2, RYR2, XIRP2, and TMEM132C, distributed throughout the entire genes were either missense or nonsense in nature; on the contrary, some genes, e.g., EGFR and KRAS, had mutation hot spots (Fig. 3). The results indicated that although NSCLCs from HPR and CR had distinct mutation patterns in many genes, they did show some similar points in some somatic mutations.