NRAS germline variant G138R and multiple rare somatic mutations on APC in colorectal cancer patients in Taiwan by next generation sequencing

Abstract

Colorectal cancer (CRC) arises from mutations in a subset of genes. We investigated the germline and somatic mutation spectrum of patients with CRC in Taiwan by using the AmpliSeq Cancer Hotspot Panel V2. Fifty paired freshly frozen stage 0-IV CRC tumors and adjacent normal tissue were collected. Blood DNA from 20 healthy donors were used for comparison of germline mutations. Variants were identified using an ion-torrent personal genomic machine and subsequently confirmed by Sanger sequencing or pyrosequencing. Five nonsynonymous germline variants on 4 cancer susceptible genes, CDH1, APC, MLH1, and NRAS, were observed in 6 patients with CRC (12%). Among them, oncogene NRAS G138R variant was identified as having a predicted damaging effect on protein function, which has never been reported by other laboratories. CDH1 T340A variants were presented in 3 patients. The germline variants in the cancer patients differed completely from those found in asymptomatic controls. Furthermore, a total of 56 COSMIC and 21 novel somatic variants distributed in 20 genes were detected in 44 (88%) of the CRC samples. High inter- and intra-tumor heterogeneity levels were observed. Nine rare variants located in the β-catenin binding region of the APC gene were discovered, 7 of which could cause amino acid frameshift and might have a pathogenic effect. In conclusion, panel-based mutation detection by using a high-throughput sequencing platform can elucidate race-dependent cancer genomes. This approach facilitates identifying individuals at high risk and aiding the recognition of novel mutations as targets for drug development.

Keywords: APC; NRAS; ampliSeq cancer hotspot panel; colorectal cancer; next-generation sequencing.

Figure 1. Five unique independent nonsynonymous variants were identified in the 50 normal tissue samples from the CRC patients (left half) and 20 PBMC DNA from the asymptomatic controls (right half)

The frequency of each mutant is shown. The red asterisk indicates that the APC V1125A germline variant is present in one patient (Sample ID 1461) with diagnosed HNPCC.

Figure 2. Distribution of 77 somatic variants on 20 genes in the 50 CRC patients

The number on the uppermost layer represents the number of somatic variants per sample. The size and color of the circle in the cell represents the individual variant frequency. For interpreting the meaning of each symbol, please refer to the scale bar. The number labeled in the cell indicates the designated variant serial number of each gene, which are listed in Table 3. More than one number in one cell indicates multiple mutations in the same gene. The gray line at the right part of the figure indicates a missense mutation, and the black bar denotes an indel or nonsense mutation. Columns a and b denote the number and percentage of samples altered per gene; Column c denotes the accumulated percentage of samples with mutated genes. The patient information denoted at the bottom of the figure includes the tumor ID, AJCC stage, location, and metastasis condition. “Re” = rectum; “R” = right site; “L” = left site; 1 = metastasis; 0 = no evidence of metastasis; * = follow-up observation of metastasis events 1 year after surgery in 2 stage III patients.

Figure 3. Spatial distribution of somatic variants on the protein function domains of the top-4 mutated genes

One triangle represents one variant found in one tumor (green = COSMIC mutation; red = novel variant). Color blocks illustrate the different functional domains of each protein. For comparison, the variants of each gene with a frequency of ≥1% recorded in the COSMIC database are marked with a black line below the domain bar, and the mutation rate is indicated in parentheses and indicated by the length of the line. A. TP53; B. KRAS; C. APC; D. PIK3CA.