Functional annotation with expression validation identifies novel metastasis-relevant genes from post-GWAS risk loci in sporadic colorectal carcinomas.

BACKGROUND: Colorectal cancer (CRC) is the third highest incidence cancer and is the leading cause of cancer mortality worldwide. Metastasis to distal organ is the major cause of cancer mortality. However, the underlying genetic factors are unclear. This study aimed to identify metastasis-relevant genes and pathways for better management of metastasis-prone patients. METHODS: A case-case genome-wide association study comprising 2677 sporadic Chinese CRC cases (1282 metastasis-positive vs 1395 metastasis-negative) was performed using the Human SNP6 microarray platform and analysed with the correlation/trend test based on the additive model. SNP variants with association testing -log10 p value ≥5 were imported into Functional Mapping and Annotation (FUMA) for functional annotation. RESULTS: Glycolysis was uncovered as the top hallmark gene set. Transcripts from two of the five genes profiled, hematopoietic substrate 1 associated protein X 1 (HAX1) and hyaluronan-mediatedmotility receptor (HMMR), were significantly upregulated in the metastasis-positive tumours. In contrast to disease-risk variants, HAX1 appeared to act synergistically with HMMR in significantly impacting metastasis-free survival. Examining the subtype datasets with FUMA and Ingenuity Pathway Analysis (IPA) identified distinct pathways demonstrating sexual dimorphism in CRC metastasis. CONCLUSIONS: Combining genome-wide association testing with in silico functional annotation and wet-bench validation identified metastasis-relevant genes that could serve as features to develop subtype-specific metastasis-risk signatures for tailored management of patients with stage I-III CRC.

Genome-wide association study identified copy number variants associated with sporadic colorectal cancer risk.

BACKGROUND: Multiple single nucleotide polymorphisms (SNPs) have been associated with colorectal cancer (CRC) risk. The role of structural or copy number variants (CNV) in CRC, however, remained unclear. We investigated the role of CNVs in patients with sporadic CRC. METHODS: A genome-wide association study (GWAS) was performed on 1000 Singapore Chinese patients aged 50 years or more with no family history of CRC and 1000 ethnicity-matched, age-matched and gender-matched healthy controls using the Affymetrix SNP 6 platform. After 16 principal component corrections, univariate and multivariate segmentations followed by association testing were performed on 1830 samples that passed quality assurance tests. RESULTS: A rare CNV region (CNVR) at chromosome 14q11 (OR=1.92 (95% CI 1.59 to 2.32), p=2.7e-12) encompassing CHD8, and common CNVR at chromosomes 3q13.12 (OR=1.54 (95% CI 1.33 to 1.77), p=2.9e-9) and 12p12.3 (OR=1.69 (95% CI 1.41 to 2.01), p=2.8e-9) encompassing CD47 and RERG/ARHGDIB, respectively, were significantly associated with CRC risk. CNV loci were validated in an independent replication panel using an optimised copy number assay. Whole-genome expression data in matched tumours of a subset of cases demonstrated that copy number loss at CHD8 was significantly associated with dysregulation of several genes that perturb the Wnt, TP53 and inflammatory pathways. CONCLUSIONS: A rare CNVR at 14q11 encompassing the chromatin modifier CHD8 was significantly associated with sporadic CRC risk. Copy number loss at CHD8 altered expressions of genes implicated in colorectal tumourigenesis.

Genome-wide association study identifies a new SMAD7 risk variant associated with colorectal cancer risk in East Asians.

Genome-wide association studies (GWAS) of colorectal cancer (CRC) have been conducted primarily in European descendants. In a GWAS conducted in East Asians, we first analyzed approximately 1.7 million single-nucleotide polymorphisms (SNPs) in four studies with 1,773 CRC cases and 2,642 controls. We then selected 66 promising SNPs for replication and genotyped them in three independent studies with 3,612 cases and 3,523 controls. Five SNPs were further evaluated using data from four additional studies including up to 3,290 cases and 4,339 controls. SNP rs7229639 in the SMAD7 gene was found to be associated with CRC risk with an odds ratio (95% confidence interval) associated with the minor allele (A) of 1.22 (1.15-1.29) in the combined analysis of all 11 studies (p = 2.93 × 10(-11) ). SNP rs7229639 is 2,487 bp upstream from rs4939827, a risk variant identified previously in a European-ancestry GWAS in relation to CRC risk. However, these two SNPs are not correlated in East Asians (r(2) = 0.008) nor in Europeans (r(2) = 0.146). The CRC association with rs7229639 remained statistically significant after adjusting for rs4939827 as well as three additional CRC risk variants (rs58920878, rs12953717 and rs4464148) reported previously in this region. SNPs rs7229639 and rs4939827 explained approximately 1% of the familial relative risk of CRC in East Asians. This study identifies a new CRC risk variant in the SMAD7 gene, further highlighting the significant role of this gene in the etiology of CRC.

Advances in colorectal cancer genomics and transcriptomics drive early detection and prevention.

Colorectal carcinoma (CRC) is a high incidence cancer and leading cause of cancer mortality worldwide. The advances in genomics and transcriptomics in the past decades have improved the detection and prevention of CRC in familial CRC syndromes. Nevertheless, the ultimate goal of personalized medicine for sporadic CRC is still not within reach due no less to the difficulty in integrating population disparity and clinical data to combat what essentially is a very heterogenous disease. This minireview highlights the achievement of the past decades and present possible direction in the hope of early detection and metastasis prevention for reducing CRC-associated morbidity and mortality.

Enhancer-derived long non-coding RNAs CCAT1 and CCAT2 at rs6983267 has limited predictability for early stage colorectal carcinoma metastasis.

Up-regulation of long non-coding RNAs (lncRNAs), colon-cancer associated transcript (CCAT) 1 and 2, was associated with worse prognosis in colorectal cancer (CRC). Nevertheless, their role in predicting metastasis in early-stage CRC is unclear. We measured the expression of CCAT1, CCAT2 and their oncotarget, c-Myc, in 150 matched mucosa-tumour samples of early-stage microsatellite-stable Chinese CRC patients with definitive metastasis status by multiplex real-time RT-PCR assay. Expression of CCAT1, CCAT2 and c-Myc were significantly up-regulated in the tumours compared to matched mucosa (p < 0.0001). The expression of c-Myc in the tumours was significantly correlated to time to metastasis [hazard ratio = 1.47 (1.10-1.97)] and the risk genotype (GG) of rs6983267, located within CCAT2. Expression of c-Myc and CCAT2 in the tumour were also significantly up-regulated in metastasis-positive compared to metastasis-negative patients (p = 0.009 and p = 0.04 respectively). Nevertheless, integrating the expression of CCAT1 and CCAT2 by the Random Forest classifier did not improve the predictive values of ColoMet19, the mRNA-based predictor for metastasis previously developed on the same series of tumours. The role of these two lncRNAs is probably mitigated via their oncotarget, c-Myc, which was not ranked high enough previously to be included in ColoMet19.

Human colorectal cancer initiation is bidirectional, and cell growth, metabolic genes and transporter genes are early drivers of tumorigenesis.

The role of stem cells in the development of solid tumors remains controversial. In colorectal cancers (CRC), this is complicated by the conflicting "top-down" or "bottom-up" hypotheses of cancer initiation. We profiled the expressions of genes from the top (T) and bottom (B) crypt fractions of normal-appearing human colonic mucosa (M) at least 20 cm away from the tumor as a baseline and compared this to the genes of matched mucosa adjacent to tumors (MT) in twenty-three sporadic CRC patients. In thirteen patients, the genetic distance (M-MT) between the B fractions is smaller than the distance between the T fractions, indicating that the expressions diverge further in the top fractions (B < T). In the remaining patients, the reverse effect is observed (B > T). Assuming that a greater genetic divergence in the top or bottom fractions indicates that position as the initiation site, it is thus equally likely that human CRC initiates from 'top-down' via de-differentiated colonocytes or 'bottom-up' via dysregulated intestinal stem cells. Dysregulated genes that persist until tumor stage are not limited to tumor suppressors or oncogenes but include metabolic and transporter genes such as CA7, PHLPP2, and AQP8.

Chromosome 19q13 disruption alters expressions of CYP2A7, MIA and MIA-RAB4B lncRNA and contributes to FAP-like phenotype in APC mutation-negative familial colorectal cancer patients.

Familial adenomatous polyposis (FAP) is an autosomal-dominantly inherited form of colorectal cancer (CRC) caused by mutation in the adenomatous polyposis coli (APC) gene. Our ability to exhaustively screen for APC mutations identify microsatellite-stable and APC-mutation negative familial CRC patients, enabling us to search for novel genes. We performed genome-wide scan on two affected siblings of one family and 88 ethnicity- and gender-matched healthy controls to identify deletions shared by the siblings. Combined loss of heterozygosity, copy number and allelic-specific copy number analysis uncovered 5 shared deletions. Long-range polymerase chain reaction (PCR) confirmed chromosome 19q13 deletion, which was subsequently found in one other family. The 32 kb deleted region harbors the CYP2A7 gene and was enriched with enhancer, repressor and insulator sites. The wildtype allele was lost in the polyps of the proband. Further, real-time RT-PCR assays showed that expressions of MIA and MIA-RAB4B located 35 kb upstream of the deletion, were up-regulated in the polyps compared to the matched mucosa of the proband. MIA-RAB4B, the read-through long non-coding RNA (lncRNA), RAB4B, PIM2 and TAOK1 share common binding site of a microRNA, miR-24, in their 3'UTRs. PIM2 and TAOK1, two target oncogenes of miR-24, were co-ordinately up-regulated with MIA-RAB4B in the polyps, suggesting that MIA-RAB4B could function as competitive endogenous RNA to titrate miR-24 away from its other targets. The data suggest that the 19.13 deletion disrupted chromatin boundary, leading to altered expression of several genes and lncRNA, could contribute to colorectal cancer via novel genetic and epigenetic mechanisms.

Association of Caucasian-identified variants with colorectal cancer risk in Singapore Chinese.

BACKGROUND: Genome-wide association studies (GWAS) in Caucasians have identified fourteen index single nucleotide polymorphisms (iSNPs) that influence colorectal cancer (CRC) risk. METHODS: We investigated the role of eleven iSNPs or surrogate SNPs (sSNPs), in high linkage disequilibrium (LD, r(2)≥ 0.8) and within 100 kb vicinity of iSNPs, in 2,000 age- and gender-matched Singapore Chinese (SCH) cases and controls. RESULTS: Only iSNP rs6983267 at 8q24.21 and sSNPs rs6695584, rs11986063, rs3087967, rs2059254, and rs7226855 at 1q41, 8q23.3, 11q23.1, 16q22.1 and 18q21.1 respectively showed evidence of association with CRC risk, with odds ratios (OR) ranging from 1.13 to 1.40. sSNP rs827401 at 10p14 was associated with rectal cancer risk (OR = 0.74, 95% CI 0.63-0.88) but not disease prognosis (OR = 0.91, 95% CI 0.69-1.20). Interestingly, sSNP rs3087967 at 11q23.1 was associated with CRC risk in men (OR = 1.34, 95% CI 1.14-1.58) but not women (OR = 1.07, 95% CI: 0.88-1.29), suggesting a gender-specific role. Half of the Caucasian-identified variants, including the recently fine-mapped BMP pathway loci, BMP4, GREM1, BMP2 and LAMA 5, did not show any evidence for association with CRC in SCH (OR ~1; p-value >0.1). Comparing the results of this study with that of the Northern and Hong Kong Chinese, only variants at chromosomes 8q24.21, 10p14, 11q23.1 and 18q21.1 were replicated in at least two out of the three Chinese studies. CONCLUSIONS: The contrasting results between Caucasians and Chinese could be due to different LD patterns and allelic frequencies or genetic heterogeneity. The results suggest that additional common variants contributing to CRC predisposition remained to be identified.