Lai Fun Thean

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 (r2 = 0.008) nor in Europeans (r2 = 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.

Genome‐wide scan identifies a copy number variable region at 3q26 that regulates PPM1L 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. However, APC mutations are not detected in 10–50% of FAP patients. We searched for a new cancer gene by performing genome‐wide genotyping on members of an APC mutation‐negative FAP variant family and ethnicity‐matched healthy controls. No common copy number change was found in all affected members using the unaffected members and healthy controls as baseline. A 111 kb copy number variable (CNV) region at 3q26.1 was shown to have copy number loss in all eight polyps compared to matched lymphocytes of two affected members. A common region of loss in all polyps, which are precursors to CRC, is likely to harbor disease‐causing gene in accordance to Knudsens “two‐hit” hypothesis. There is, however, no gene within the deleted region. A 2‐Mb scan of the genomic region encompassing the deleted region identified PPM1L, coding for a novel serine‐threonine phosphatase in the TGF‐β and BMP signaling pathways. Real‐time PCR analyses indicate that the 3′UTR of PPM1L transcript was down‐regulated more than two‐folds in all six polyps and tumors compared to matched mucosa of the affected member. This down‐regulation was not observed in APC mutation‐positive FAP patients. Our results suggest that the CNV region at 3q26 harbors an element that regulates the expression of an upstream candidate tumor suppressor, PPM1L, thus providing a novel mechanism for colorectal tumorigenesis in APC mutation‐negative familial CRC patients.

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, r2≥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.

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.

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.

Abstract 2559: Genome-wide haplotype association tests identified three candidate loci associated with sporadic colorectal cancer risk in Singapore Chinese

Colorectal cancer (CRC) is one of the most frequent cancers and leading cause of cancer mortality in the developed world. Twin study has attributed about 35% of the etiology of sporadic CRC to genes. Hitherto, more than 20 single nucleotide polymorphism (SNP) loci from genome-wide association studies (GWAS) were associated with CRC risk. Most of these index SNPs however have small effect sizes and often located in gene deserts. Thus they are unlikely to be causal. Haplotype-based methods may offer a more powerful approach to disease gene mapping. We previously performed a GWAS on 2,000 ethnicity-, age-, and gender-matched Singapore Chinese CRC patients and healthy controls using the Affymetrix SNP 6 platform. Sporadic patients were defined as aged 50 or more at the time of surgery and with no dominant family history of CRC. Haplotype block detection was performed in Golden Helix SVS after this SNP6 dataset was filtered for genotype call rate (>95%), Hardy-Weinberg equilibrium in the controls and principal component analysis. Block detection was performed requiring strong linkage disequilibrium between block pairs, minor allelic frequencies (MAF) of SNPs more than 0.01, the maximum number of markers and length of the block at 30 and 160kb respectively. Genome-wide, 73,333 blocks were computed using 336, 466 markers on 22 autosomes. Haplotype frequencies were estimated using the EM algorithm. Chi-Squared and logistic regression with odds ratio tests of haplotype association were performed using these pre-computed blocks on a per-haplotype basis. Three candidate loci at chromosomes 3, 15 and 20, each with 6 to 9 SNPs and sizes ranging from 4 to 15 kb achieved genome-wide significance and encompass potential novel disease genes. These loci are currently being validated in an independent replication panel using the Fluidgm SNPtype assays and pooled analyses will be performed. Citation Format: Peh Yean Cheah, Lai Fun Thean, Yik Ying Teo, Woon-Puay Koh, Jian-Min Yuan, Min Hoe Chew, Choong Leong Tang. Genome-wide haplotype association tests identified three candidate loci associated with sporadic colorectal cancer risk in Singapore Chinese. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2559.

Abstract 2189: A rare copy number variant at chromosome 14q11 was associated with sporadic colorectal cancer risk in Singapore Chinese

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Colorectal cancer (CRC) is one of the most frequent cancers and leading cause of cancer mortality in the developed world. Twin study has attributed about 35% of the etiology of sporadic CRC to genes. Structural variations in the human genome have recently been associated with complex neurological diseases. Their role in CRC, however, is unclear. We performed genome-wide association study (GWAS) on 2,000 ethnicity-, age-, and gender-matched Singapore Chinese CRC patients (aged 50 or more and with no dominant family history of CRC) and healthy controls. Genome-wide genotyping was performed using Affymetrix SNP 6 platform, and rare copy number variants (CNV) were interrogated in 1830 samples that passed the quality assurance tests. A 400 kb region at chromosome 14q11 was identified to be significantly associated (-log10 p-value = 11) with sporadic CRC risk. A chromatin modifier implicated in the β-catenin/Wnt signalling pathway is one of the candidate genes found in the region. Primers unique for this gene applied to an optimal real-time copy number assay verified the CNV region in an independent replication panel comprising another 1,000 sporadic CRC cases. About 6% of the cases exhibit copy number alterations at this region compared to 1% in the healthy controls. A second gene, a small guanosine triphosphatase (GTPase) involved in protein trafficking and preferentially up-regulated in colonic tumors , was shown by long-range polymerase chain reaction to have more structural variations in the cases compared to the controls. Furthermore, expression of the H1 binding domain of the chromatin modifier was positively correlated to the expressions of Cyclin D1 and C-myc. The data suggest that the rare CNV in 14q11 was dynamically associated with the activation of genes implicated in sporadic CRC in the Singapore Chinese. Citation Format: Peh Yean Cheah, Lai Fun Thean, Yik-Ying Teo, Woon-Puay Koh, Jian-Min Yuan, Poh Koon Koh, Min Hoe Chew, Choong Leong Tang. A rare copy number variant at chromosome 14q11 was associated with sporadic colorectal cancer risk in Singapore Chinese. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2189. doi:10.1158/1538-7445.AM2014-2189

Abstract 3764: Genome-wide association study identifies potential susceptibility loci associated with differential response to environmental insults in sporadic Chinese CRC patients

To date, genome-wide association studies (GWAS) have identified fourteen SNPs that influence risk of developing colorectal cancer (CRC) in Caucasians. None of these tagging SNPs is within coding region of a candidate gene. Only five of these SNPs were associated with CRC risk in Chinese population. We aimed to perform GWAS to identify new susceptibility loci associated with differential response to environmental insults in sporadic Chinese CRC patients. A total of 1,000 cases (aged 50 years or more) and 1,000 age- and gender-matched healthy controls were genotyped with Affymetrix SNP 6 array, a newer microarray platform which includes the ability to interrogate structural variants. Over 90% of the arrays have call rate of 99% or more. Thirty five arrays with call rate less than 97% and either null QC or negative QC (enzyme) values were replicated. Principal component analysis performed together with 270 HapMap and 92 Singapore Genome Variant Project (SGVP) samples indicate that there is no population substructure. Sixteen outliers were removed. SNPs with call rate less than 99% and minor allelic frequency less than 0.1, and control SNPs not in Hardy-Weinburg equilibrium were filtered. Subsequent quantile-quantile plot shows no evidence of allelic test statistic inflation. Twelve new chromosomal regions with SNPs having -log10 p-values greater than four and good clustering patterns were identified via Manhattan plot. Linkage disequilibrium (LD) analysis was performed to ascertain the LD blocks around these SNPs. SNPs in three chromosomal regions within the vicinity of promising CRC candidate genes were prioritized and currently being validated in a replication panel consisting of another 2,000 cases and controls. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3764. doi:10.1158/1538-7445.AM2011-3764