3.1 Mutation spectrum of CRC patients from TCGA
Mutation spectrum of CRC patients from TCGA was first explored which uncovered the predominance of missense mutation variant class and C to T transition as shown in Figure S1. Mutational signature (MS) is a combination of multiple mutational processes which could reflect to some extent the incentives through which those mutational processes occurred, such as defective DNA repair, mutagen exposures and so on. In this study, we summarized a total of eight MSs which named W1-8 as shown in Figure S2A. Cosine similarities between our MSs and those deposited in COSMIC database were calculated and provided as a heatmap in Figure S2B which shows high similarities between our MSs and signature1, signature6, signature10, signature28 and signature30. Signature1 might be a cell division/mitotic clock and correlated with individual age. Signature6 is associated with defective DNA mismatch repair and microsatellite instability which could contribute genome instability. Percentages of mutations attributed to each signature at individual and whole level were shown in Figure S3A and Figure S3B, respectively.
Mutations located in the top 30 most frequently mutated genes could cover all the 399 CRC samples and TTN represented the most frequently one whose mutations were found in about 74% samples (Figure 1A). Genes belong to NEB gene family that with mutation rate greater than 1% along with several well-known DNA damage repair (DDR)-related genes including BRCA1, BRCA2, POLE and MLH3 were analyzed for their mutation state across CRC samples. As a result, NEB was mutated in 16% CRC samples that was distinctly higher than PEG3, the second frequently mutated genes in Figure 1B which mutated in 9% CRC samples. Besides, CRC samples harbored NEB mutations also tend to have mutations located in BRCA1, BRCA2, POLE and MLH3 and shows higher mutation number than those without NEB mutation, which should indicate potential roles of NEB in genome stability.
3.2 NEB mutation is closely associated with CRC patients' tumor mutation burden
Tumor mutation burden (TMB) that represents somatic mutation number within a million bases is closely related with genome integrity and immunotherapy sensitivity of malignant tumor patients. In this study, TMB of CRC patients with at least one NEB mutation was significantly higher than those without NEB mutation (Figure 2A). What's more, NEB mutation was recognized as an independent high CRC TMB signature after excluded influences of confounding factors including patients' age, gender, tumor stage and mutation state of BRCA1, BRCA2 and POLE as shown in Figure 2B.
3.3 NEB mutation is closely associated with CRC patients' overall survival
Kaplan-Meier curves of samples stratified by NEB mutation state were plotted and log-rank test indicated that NEB mutation was significantly associated with inferior CRC overall survival (Figure 3A). Association between NEB mutation and CRC overall survival was still significant after controlling for confounding factors including patients' age, gender, tumor stage and mutation states of BRCA1, BRCA2 and POLE (Figure 3B).
3.4 Significantly mutated genes
We analyzed the SMGs in samples with and without NEB mutations, and Figure 4A and Figure 4B illustrated the mutation spectrum of SMGs NEB mutated and wild-type CRC samples, respectively. APC, TP53 and FBXW7 were identified simultaneously as SMGs in the two sample groups. Mutation frequency of SMGs in the two groups of samples were detailed shown in Figure 5, and several genes such as SETD1B, RNF43, MBD6 and etc were only significantly mutated in NEB mutated samples, whose mutation state might be profoundly influenced by NEB.