Wei-Yu Lin

Meta association of colorectal cancer confirms risk alleles at 8q24 and 18q21

Background: Genome-wide association studies of colorectal cancer (CRC) have identified genetic variants that reproducibly associate with CRC. Associations of 12 single nucleotide polymorphisms at 8q24, 9p24, and 18q21 (SMAD7) and CRC were investigated in a three-center collaborative study including two U.K. case-control cohorts (Sheffield and Leeds) and a U.S. case-control study of CRC cases from high-risk Utah pedigrees. Methods: Our combined resource included 1,092 CRC case subjects and 1,060 age- and sex-matched controls. Meta statistics and Monte Carlo significance testing using Genie software provided a valid combined analysis of our mixed independent and related case-control resource. We also evaluated whether these associations differed by sex, age at diagnosis, family history, or tumor site. Results: At 8q24, we observed two independent significant associations at single nucleotide polymorphisms located in two different risk regions of 8q24: rs6983267 in region 3 [Ptrend = 0.01; per allele odds ratio (OR), 1.17; 95% confidence intervals (95% CI), 1.03-1.32] and rs10090154 in region 5 (Ptrend = 0.05; per allele OR, 1.24; 95% CI, 1.01-1.51). At 18q21, associations were observed in distal colon tumors but not in proximal or rectal cancers: rs4939827 (Ptrend = 0.007; per allele OR, 0.77; 95% CI, 0.64-0.93; case-case pdiff = 0.03) and rs12953717 (Ptrend = 0.01; per allele OR, 1.27; 95% CI, 1.06-1.52). We were unable to detect any associations at 9p24 with CRC. Conclusions: Our investigation confirms that variants across multiple risk regions of 8q24 are associated with CRC, and that associations at 18q21 differ by tumor site. (Cancer Epidemiol Biomarkers Prev 2009;18(2):616–21)

Genetic Variants in XRCC2: New Insights Into Colorectal Cancer Tumorigenesis

Polymorphisms in DNA double-strand break repair gene XRCC2 may play an important role in colorectal cancer etiology, specifically in disease subtypes. Associations of XRCC2 variants and colorectal cancer were investigated by tumor site and tumor instability status in a four-center collaboration including three U.K. case-control studies (Sheffield, Leeds, and Dundee) and a U.S. case-control study of cases from high-risk Utah pedigrees (total: 1,252 cases and 1,422 controls). The 14 variants studied were tagging single nucleotide polymorphisms (SNP) selected from National Institute of Environmental Health Sciences/HapMap data supplemented with SNPs identified from sequencing of 125 cases chosen to represent multiple colorectal cancer groups (familial, metastatic disease, and tumor subsite). Monte Carlo significance testing using Genie software provided valid meta-analyses of the total resource that includes family-based data. Similar to reports of colorectal cancer and other cancer sites, the rs3218536 R188H allele was not associated with increased risk. However, we observed a novel, highly significant association of a common SNP, rs3218499G>C, with increased risk of rectal tumors (odds ratio, 2.1; 95% confidence interval, 1.3-3.3; Pχ2 = 0.0006) versus controls, with the largest risk found for female rectal cases (odds ratio, 3.1; 95% confidence interval, 1.6-6.1; Pχ2 = 0.0006). This difference was significantly different to that for proximal and distal colon cancers (Pχ2 = 0.02). Our investigation supports a role for XRCC2 in colorectal cancer tumorigenesis, conferring susceptibility to rectal tumors. (Cancer Epidemiol Biomarkers Prev 2009;18(9):2476–84)

A role for XRCC2 gene polymorphisms in breast cancer risk and survival

Background The XRCC2 gene is a key mediator in the homologous recombination repair of DNA double strand breaks. It is hypothesised that inherited variants in the XRCC2 gene might also affect susceptibility to, and survival from, breast cancer. Methods The study genotyped 12 XRCC2 tagging single nucleotide polymorphisms (SNPs) in 1131 breast cancer cases and 1148 controls from the Sheffield Breast Cancer Study (SBCS), and examined their associations with breast cancer risk and survival by estimating ORs and HRs, and their corresponding 95% CIs. Positive findings were further investigated in 860 cases and 869 controls from the Utah Breast Cancer Study (UBCS) and jointly analysed together with available published data for breast cancer risk. The survival findings were further confirmed in studies (8074 cases) from the Breast Cancer Association Consortium (BCAC). Results The most significant association with breast cancer risk in the SBCS dataset was the XRCC2 rs3218408 SNP (recessive model p=2.3×10−4, minor allele frequency (MAF)=0.23). This SNP yielded an ORrec of 1.64 (95% CI 1.25 to 2.16) in a two-site analysis of SBCS and UBCS, and a meta-ORrec of 1.33 (95% CI 1.12 to 1.57) when all published data were included. This SNP may mark a rare risk haplotype carried by two in 1000 of the control population. Furthermore, the XRCC2 coding R188H SNP (rs3218536, MAF=0.08) was significantly associated with poor survival, with an increased per-allele HR of 1.58 (95% CI 1.01 to 2.49) in a multivariate analysis. This effect was still evident in a pooled meta-analysis of 8781 breast cancer patients from the BCAC (HR 1.19, 95% CI 1.05 to 1.36; p=0.01). Conclusions These findings suggest that XRCC2 SNPs may influence breast cancer risk and survival.

A breast cancer risk haplotype in the caspase-8 gene

Recent large-scale studies have been successful in identifying common, low-penetrance variants associated with common cancers. One such variant in the caspase-8 (CASP8) gene, D302H (rs1045485), has been confirmed to be associated with breast cancer risk, although the functional effect of this polymorphism (if any) is not yet clear. In order to further map the CASP8 gene with respect to breast cancer susceptibility, we performed extensive haplotype analyses using single nucleotide polymorphisms (SNP) chosen to tag all common variations in the gene (tSNP). We used a staged study design based on 3,200 breast cancer and 3,324 control subjects from the United Kingdom, Utah, and Germany. Using a haplotype-mining algorithm in the UK cohort, we identified a four-SNP haplotype that was significantly associated with breast cancer and that was superior to any other single or multi-locus combination (P=8.0 x 10(-5)), with a per allele odds ratio and 95% confidence interval of 1.30 (1.12-1.49). The result remained significant after adjustment for the multiple testing inherent in mining techniques (false discovery rate, q=0.044). As expected, this haplotype includes the D302H locus. Multicenter analyses on a subset of the tSNPs yielded consistent results. This risk haplotype is likely to carry one or more underlying breast cancer susceptibility alleles, making it an excellent candidate for resequencing in homozygous individuals. An understanding of the mode of action of these alleles will aid risk assessment and may lead to the identification of novel treatment targets in breast cancer.

Identification of candidate driver genes in common focal chromosomal aberrations of microsatellite stable colorectal cancer.

Colorectal cancer (CRC) is a leading cause of cancer deaths worldwide. Chromosomal instability (CIN) is a major driving force of microsatellite stable (MSS) sporadic CRC. CIN tumours are characterised by a large number of somatic chromosomal copy number aberrations (SCNA) that frequently affect oncogenes and tumour suppressor genes. The main aim of this work was to identify novel candidate CRC driver genes affected by recurrent and focal SCNA. High resolution genome-wide comparative genome hybridisation (CGH) arrays were used to compare tumour and normal DNA for 53 sporadic CRC cases. Context corrected common aberration (COCA) analysis and custom algorithms identified 64 deletions and 32 gains of focal minimal common regions (FMCR) at high frequency (>10%). Comparison of these FMCR with published genomic profiles from CRC revealed common overlap (42.2% of deletions and 34.4% of copy gains). Pathway analysis showed that apoptosis and p53 signalling pathways were commonly affected by deleted FMCR, and MAPK and potassium channel pathways by gains of FMCR. Candidate tumour suppressor genes in deleted FMCR included RASSF3, IFNAR1, IFNAR2 and NFKBIA and candidate oncogenes in gained FMCR included PRDM16, TNS1, RPA3 and KCNMA1. In conclusion, this study confirms some previously identified aberrations in MSS CRC and provides in silico evidence for some novel candidate driver genes.

Associations of ATR and CHEK1 Single Nucleotide Polymorphisms with Breast Cancer

DNA damage and replication checkpoints mediated by the ATR-CHEK1 pathway are key to the maintenance of genome stability, and both ATR and CHEK1 have been proposed as potential breast cancer susceptibility genes. Many novel variants recently identified by the large resequencing projects have not yet been thoroughly tested in genome-wide association studies for breast cancer susceptibility. We therefore used a tagging SNP (tagSNP) approach based on recent SNP data available from the 1000 genomes projects, to investigate the roles of ATR and CHEK1 in breast cancer risk and survival. ATR and CHEK1 tagSNPs were genotyped in the Sheffield Breast Cancer Study (SBCS; 1011 cases and 1024 controls) using Illumina GoldenGate assays. Untyped SNPs were imputed using IMPUTE2, and associations between genotype and breast cancer risk and survival were evaluated using logistic and Cox proportional hazard regression models respectively on a per allele basis. Significant associations were further examined in a meta-analysis of published data or confirmed in the Utah Breast Cancer Study (UBCS). The most significant associations for breast cancer risk in SBCS came from rs6805118 in ATR (p=7.6x10-5) and rs2155388 in CHEK1 (p=3.1x10-6), but neither remained significant after meta-analysis with other studies. However, meta-analysis of published data revealed a weak association between the ATR SNP rs1802904 (minor allele frequency is 12%) and breast cancer risk, with a summary odds ratio (confidence interval) of 0.90 (0.83-0.98) [p=0.0185] for the minor allele. Further replication of this SNP in larger studies is warranted since it is located in the target region of 2 microRNAs. No evidence of any survival effects of ATR or CHEK1 SNPs were identified. We conclude that common alleles of ATR and CHEK1 are not implicated in breast cancer risk or survival, but we cannot exclude effects of rare alleles and of common alleles with very small effect sizes.

Discordant Haplotype Sequencing Identifies Functional Variants at the 2q33 Breast Cancer Risk Locus

The findings from genome-wide association studies hold enormous potential for novel insight into disease mechanisms. A major challenge in the field is to map these low-risk association signals to their underlying functional sequence variants (FSV). Simple sequence study designs are insufficient, as the vast numbers of statistically comparable variants and a limited knowledge of noncoding regulatory elements complicate prioritization. Furthermore, large sample sizes are typically required for adequate power to identify the initial association signals. One important question is whether similar sample sizes need to be sequenced to identify the FSVs. Here, we present a proof-of-principle example of an extreme discordant design to map FSVs within the 2q33 low-risk breast cancer locus. Our approach employed DNA sequencing of a small number of discordant haplotypes to efficiently identify candidate FSVs. Our results were consistent with those from a 2,000-fold larger, traditional imputation-based fine-mapping study. To prioritize further, we used expression-quantitative trait locus analysis of RNA sequencing from breast tissues, gene regulation annotations from the ENCODE consortium, and functional assays for differential enhancer activities. Notably, we implicate three regulatory variants at 2q33 that target CASP8 (rs3769823, rs3769821 in CASP8, and rs10197246 in ALS2CR12) as functionally relevant. We conclude that nested discordant haplotype sequencing is a promising approach to aid mapping of low-risk association loci. The ability to include more efficient sequencing designs into mapping efforts presents an opportunity for the field to capitalize on the potential of association loci and accelerate translation of association signals to their underlying FSVs. Cancer Res; 76(7); 1916-25. ©2016 AACR.

Novel Bayes Factors That Capture Expert Uncertainty in Prior Density Specification in Genetic Association Studies.

Bayes factors (BFs) are becoming increasingly important tools in genetic association studies, partly because they provide a natural framework for including prior information. The Wakefield BF (WBF) approximation is easy to calculate and assumes a normal prior on the log odds ratio (logOR) with a mean of zero. However, the prior variance (W) must be specified. Because of the potentially high sensitivity of the WBF to the choice of W, we propose several new BF approximations with logOR ∼N(0,W) , but allow W to take a probability distribution rather than a fixed value. We provide several prior distributions for W which lead to BFs that can be calculated easily in freely available software packages. These priors allow a wide range of densities for W and provide considerable flexibility. We examine some properties of the priors and BFs and show how to determine the most appropriate prior based on elicited quantiles of the prior odds ratio (OR). We show by simulation that our novel BFs have superior true‐positive rates at low false‐positive rates compared to those from both P‐value and WBF analyses across a range of sample sizes and ORs. We give an example of utilizing our BFs to fine‐map the CASP8 region using genotype data on approximately 46,000 breast cancer case and 43,000 healthy control samples from the Collaborative Oncological Gene‐environment Study (COGS) Consortium, and compare the single‐nucleotide polymorphism ranks to those obtained using WBFs and P‐values from univariate logistic regression.

Abstract 3267: Targeted DNA and RNA sequencing identifies breast cancer risk variants associated with differential expression of CASP8 and CFLAR/CASP10

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA CASP8 at 2q33 is an established common, low risk susceptibility locus for breast cancer but functional variants are yet to be elucidated. Previously we used haplotype mining of 45 tSNPs in 3778 breast cancer cases and controls to identify a risk haplotype maximizing association evidence. A protective haplotype was also suggested. Here, we perform a focused sequencing study to identify the critical sequence variant/s. To not exclude potential regulatory variants, we designed a custom Agilent SureSelect capture array to target all non-repetitive sequence in the 1 Mb region surrounding CASP8. The resulting 41 libraries for germline DNA (24 breast cancer cases homozygous for the risk haplotype, 17 cancer-free women carrying the protective haplotype) were sequenced on the Life Technologies SOLID system. A total of 1464 sequence variants were prioritized by their propensity to lie on the risk or protective haplotypes, resulting in the selection of 42 variants to follow-up with expression-QTL analysis (MAFs 0.1-0.4). Our RNA sequencing panel comprised 156 tissue samples from 88 women (68 breast tumor, 88 normal breast). Illumina TruSeq stranded mRNA sequencing was performed on an Illumina HiSeq 2000. Genotyping for the 42 selected variants was performed using Illumina BeadXpress. RNAseq eQTL analysis focused on 4 genes: CFLAR, CASP10, CASP8, ALS2CR12 using counts standardized to fragments per kilobase per million bases (FPKM). Differences in FPKM between carriers and non-carriers of the 42 DNA sequence variants were assessed in both normal and tumor tissues. Replication analyses were carried out on available TCGA data and published blood eQTL microarray data. In normal tissue, an intronic variant in ALS2CR12 on the risk haplotype was best associated with CASP8 expression (19% decreased FPKM, p=9x10-5). Eight other variants (all in ALS2CR12 or CASP8) showed similar CASP8 decreases (p<10-3). Increased ALS2CR12 expression was also observed for the same variants, and decreased CASP8 expression was seen in tumor. Despite the difference in technologies, lower coverage and sparser genotyping, the decreased CASP8 and increased ALS2CR12 levels were replicated in both in the TCGA normal tissue (p=3x10-4) and blood eQTL data (p=4x10-14). In tumor, our strongest eQTL results were for decreased expression of CFLAR (p=1x10-4) and CASP10 (p=4x10-5), identified for variants on the protective haplotype (including rs1045485). Examination of TCGA breast tumors did not replicate these findings; however read depths were substantially lower for these genes in the TCGA data. We find evidence for two separate paths to 2q33 breast cancer risk. Reduction of CASP8 expression, which may lead to reduced apoptosis and an environment conducive to tumor development. Reduction of CFLAR-CASP10 expression in tumor may implicate autophagy leading to a challenging environment for tumors, but requires more replication. Citation Format: Nicola J. Camp, Wei-Yu Lin, Alex Bigelow, Marina A. Parry, Tim Mosbruger, George Burghel, Venkatesh Rajamanickam, Sushilaben H. Rigas, Rachel Cosby, Dan Connley, Guoying Wang, Tresa George, Rosalie Waller, Lisa A. Cannon-Albright, Brandt Jones, Rob Sargent, Malcolm W.R. Reed, Angela Cox. Targeted DNA and RNA sequencing identifies breast cancer risk variants associated with differential expression of CASP8 and CFLAR/CASP10. [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 3267. doi:10.1158/1538-7445.AM2014-3267

Abstract 3055: Identification of potential colorectal cancer driver genes in focal chromosomal aberrations

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Microsatellite stable colorectal cancer (MSS-CRC) is characterized by frequent chromosomal amplifications and deletions. Even though most of these aberrations are random and case specific, some of them are common indicating a potentially important role in cancer development. However, most of the common aberrations affect large chromosomal segments which provide a challenge for the identification of cancer driver genes within these areas. But, some of these common aberrations are focal in size (<3Mb) and harbor small number of genes which facilitates the detection of candidate driver genes. Array comparative genome hybridization (aCGH) allows the analysis of chromosomal abnormalities across the whole cancer genome with high spatial resolution. When applied to a large number of cancer cases, high resolution aCGH helps in the identification of common focal chromosomal aberrations that play an important role in carcinogenesis. The aim of this study is to identify novel and common focal aberrations in CRC. Hypothesis: Common focal deletions and amplifications play an important role in MSS-CRC development through affecting key genes and microRNAs. Methods: Thirty eight CRC samples were investigated for MSI status using quantitative fluorescent PCR and a set of 5 mono-nucleotide microsatellites (BAT25, BAT26, NR21, NR24 and MONO27). High resolution Agilent 44K and 180K CGH arrays were applied on 6 and 32 CRC DNA samples respectively. The arrays contained 60-mer oligonucleotides probes for 42494 (44K) and 170334 (180K) distinct chromosomal locations distributed over the genome. The resolutions of the arrays were 43kb (44K) and 13kb (180K). All the tumor DNA samples were referenced against peripheral blood DNA extracted from the same patients. aCGH analysis was performed using the Agilent genomic workbench software (v5.0) and the aberrations were called using the aberration detection method 2 (ADM2) algorithm. Common aberration analysis was performed using the context corrected common aberration algorithm with a chromosomal scope. Results: All the samples were shown to be MSS and to carry mutations in APC (50%), TP53 (50%), KRAS (39.4%) and BRAF (5.2%). A Large number of common and focal deletions and amplifications were identified, some of which are novel. Some of the identified focal aberrations contained only single genes such as the deletion of PARK2 (∼18.4%), RGS12 (∼18.4%), FHIT (15.8%), NAALADL2 (10.5%) and the amplification of KCNMA1 (15.8%) or microRNAs such as the deletion of miR661 (13.2%) and the amplification of miR885 (15.8%). A list of novel candidate CRC-related genes and microRNAs was established. Conclusion: A number of novel candidate CRC-related genes have been identified. These results will be followed up by gene over-expression and knock-down studies in CRC cell lines. 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 3055. doi:10.1158/1538-7445.AM2011-3055