Identifying Novel Susceptibility Genes for Colorectal Cancer Risk From a Transcriptome-Wide Association Study of 125,478 Subjects.

BACKGROUND AND AIMS: Susceptibility genes and the underlying mechanisms for the majority of risk loci identified by genome-wide association studies (GWAS) for colorectal cancer (CRC) risk remain largely unknown. We conducted a transcriptome-wide association study (TWAS) to identify putative susceptibility genes. METHODS: Gene-expression prediction models were built using transcriptome and genetic data from the 284 normal transverse colon tissues of European descendants from the Genotype-Tissue Expression (GTEx), and model performance was evaluated using data from The Cancer Genome Atlas (n = 355). We applied the gene-expression prediction models and GWAS data to evaluate associations of genetically predicted gene-expression with CRC risk in 58,131 CRC cases and 67,347 controls of European ancestry. Dual-luciferase reporter assays and knockdown experiments in CRC cells and tumor xenografts were conducted. RESULTS: We identified 25 genes associated with CRC risk at a Bonferroni-corrected threshold of P < 9.1 × 10-6, including genes in 4 novel loci, PYGL (14q22.1), RPL28 (19q13.42), CAPN12 (19q13.2), MYH7B (20q11.22), and MAP1L3CA (20q11.22). In 9 known GWAS-identified loci, we uncovered 9 genes that have not been reported previously, whereas 4 genes remained statistically significant after adjusting for the lead risk variant of the locus. Through colocalization analysis in GWAS loci, we additionally identified 12 putative susceptibility genes that were supported by TWAS analysis at P < .01. We showed that risk allele of the lead risk variant rs1741640 affected the promoter activity of CABLES2. Knockdown experiments confirmed that CABLES2 plays a vital role in colorectal carcinogenesis. CONCLUSIONS: Our study reveals new putative susceptibility genes and provides new insight into the biological mechanisms underlying CRC development.

Hypermethylation of the MLH1 promoter with concomitant absence of transcript and protein occurs in small patches of crypt cells in unaffected mucosa from sporadic colorectal carcinoma.

Hypermethylation of the MLH1 promoter is present in most sporadic colorectal cancers and leads to the abrogation of MLH1 transcription. The significance of the hypermethylation of the promoter that can be detected in rare foci of cells in the normal colonic epithelium adjacent to the carcinoma is unclear. The purpose of this study was to determine the distribution of hypermethylation of the MLH1 promoter DNA with MLH1 RNA expression by RT in situ PCR and MLH1 protein expression by immunohistochemistry in normal colonic tissues. Hypermethylation of the MLH1 promoter DNA was not evident in sections of normal colon from 10 people with no history of colon cancer. MLH1 RNA and protein were detected diffusely throughout the colonic epithelium in these 10 controls. In comparison, hypermethylation of either of 2 regions of the promoter was evident in the normal mucosa from cases adjacent to adenocarcinoma of the colon. In situ analysis of the DNA methylation showed that there were rare foci that involved small groups of cells, typically toward the middle and surface area of a given crypt. In these areas, small foci of colonic epithelial cells, typically in groups of 5 to 10, were found to lack MLH1 RNA and protein in adjacent serial sections, consistent with silencing of the gene by hypermethylation that may play a role in the origin of these neoplasms. It is concluded that the rare foci of hypermethylation of the MLH1 promoter in normal colon epithelium from people with colorectal cancer is associated with silencing of the gene that may play a role in the early evolution of these neoplasms. A combination of methylation-specific in situ PCR with in situ RNA and protein analysis can assist in documenting rare foci of promoter hypermethylation-induced gene silencing.

Identification of a novel noncoding RNA gene, NAMA, that is downregulated in papillary thyroid carcinoma with BRAF mutation and associated with growth arrest.

In search of tumor suppressor genes in papillary thyroid carcinoma (PTC), we previously used gene expression profiling to identify genes underexpressed in tumor compared with paired unaffected tissue. While searching for loss of heterozygosity (LOH) in genomic regions harboring candidate tumor suppressor genes, we detected LOH in a approximately 20 kb region around marker D9S176. Several ESTs flanking D9S176 were underexpressed in PTC tumors, and for one of the ESTs, downregulation was highly associated with the activating BRAF mutation V600E, the most common genetic lesion in PTC. A novel gene, NAMA, (noncoding RNA associated with MAP kinase pathway and growth arrest) containing the affected EST was cloned and characterized. NAMA is weakly expressed in several human tissues, and the spliced forms are primarily detected in testis. Several characteristics of NAMA suggest that it is a nonprotein coding but functional RNA; it has no long open reading frames (ORFs); the exons exhibit low sequence identity in the evolutionarily conserved regions; it is inducible by knockdown of BRAF, inhibition of the MAP kinase pathway, growth arrest and DNA damage in cancer cell lines. We suggest that NAMA is a noncoding RNA associated with growth arrest.