Graham Casey

Principles for the post-GWAS functional characterization of cancer risk loci

Genome wide association studies (GWAS) have identified more than 200 mostly new common low-penetrance susceptibility loci for cancers. The predicted risk associated with each locus is generally modest (with a per-allele odds ratio typically less than 2) and so, presumably, are the functional effects of individual genetic variants conferring disease susceptibility. Perhaps the greatest challenge in the ‘post-GWAS’ era is to understand the functional consequences of these loci. Biological insights can then be translated to clinical benefits, including reliable biomarkers and effective strategies for screening and disease prevention. The purpose of this article is to propose principles for the initial functional characterization of cancer risk loci, with a focus on non-coding variants, and to define ‘post-GWAS’ functional characterization. By December 2010, there were 1,212 published GWAS studies1 reporting significant (P < 5 × 10−8) associations for 210 traits (Table 1), and the Catalog of Published GWAS states that by March 2011, 812 publications reported 3,977 SNP associations1. This is likely a small fraction of the common susceptibility loci of low penetrance that will eventually be identified. Despite these successes in identifying risk loci, the causal variant and/or the molecular basis of risk etiology has been determined for only a small fraction of these associations2–4. Plausible candidate genes can be based on proximity to risk loci, but few have so far been defined in a more systematic manner (Supplementary Table 1). Table 1 The genomic context in which a variant is found can be used as preliminary functional analysis Increased investment in post-GWAS functional characterization of risk loci5 has now been advocated across diseases and for cardiovascular disease and diabetes6. For cancer biology, the complex interplay between genetics and the environment in many cancers poses a particularly exciting challenge for post-GWAS research. Here we suggest a systematic strategy for understanding how cancer-associated variants exert their effects. We mostly refer to SNPs throughout the paper, but we recognize that other types of common genetic (for example, copy number variants) or epigenetic variation may influence risk. Our understanding of the way in which a risk variant initiates disease pathogenesis progresses from statistical association between genetic variation and trait or disease variation to functionality and causality. The functional consequences of variants in protein-coding regions causing most monogenic disorders are more readily interpreted because we know the genetic code. For non-Mendelian or multifactorial traits, most of the common DNA variants have so far mapped to non-protein–coding regions2, where our understanding of functional consequences and causality is more rudimentary. Our hypothesis is that the trait-associated alleles exert their effects by influencing transcriptional output (such as transcript levels and splicing) through multiple mechanisms. We emphasize appropriate assays and models to test the functional effects of both SNPs and genes mapping to cancer predisposition loci. Although much of what is written is applicable to alleles discovered for any trait, the section on modeling gene effects will emphasize measuring cancer-related phenotypes. At some loci, multiple, independently associated risk alleles rather than single risk alleles may be functionally responsible for the occurrence of disease. Genotyping susceptibility loci (and their correlated variants) in multiple populations with different linkage disequilibrium (LD) structures may prove effective in substantially reducing the number of potentially causative variants (that is, the same causal variant may segregate in multiple populations), as shown for the FGFR2 locus in breast cancer7, but for most loci there will remain a set of potentially causative variants that cannot be separated at the statistical level from case-control genotype data. A susceptibility locus should be re-sequenced to ascertain all genetic variation, identifying candidate functional or causal variants and identifying candidate causal genes. Ideally, the identification of a causal SNP would be the next step to reveal the molecular mechanisms of risk modification. Practically, however, it is unclear what the criteria for causality should be, particularly in non-protein–coding regions. Thus, although we propose a framework set of analyses (Box 1), we acknowledge that the techniques and methods will continue to evolve with the field. Box 1 Strategies to progress from tag SNP to mechanism Target resequencing efforts using linkage disequilibrium (LD) structure. Use other populations to refine LD regions (for example African ancestry with shorter LD and more heterogeneity). Determine expression levels of nearby genes as a function of genotype at each locus (eQTL). Characterize gene regulatory regions by multiple empirical techniques bearing in mind that these are tissue and context specific. Combine regulatory regions with risk loci using coordinates from multiple reference genomes to capture all variation within the shorter regulatory regions that correlates with the tag SNP at each locus. Multiple experimental manipulations in model systems are needed to progressively implicate transcription units (genes) in mechanisms relevant to the associated loci: Knockouts of regulatory regions in animal (difficult and may be limited by functional redundancy, but new targeting methods in rat are promising) models followed by genome-wide expression analysis. Use chromatin association methods (3C, CHIA-PET) of regulatory regions to determine the identity of target genes (compare with eQTL data). Targeted gene perturbations in somatic cell models. Explore fully genome-wide eQTL and miRNA quantitative variation correlation in relevant tissues and cells. Explore epigenetic mechanisms in the context of genome-wide genetic polymorphism. Employ cell models and tissue reconstructions to evaluate mechanisms using gene perturbations and polymorphic variants. The human cancer cell xenograft has re-emerged as a minimal in vivo validation of these models. Above all, resist the temptation to equate any partial functional evidence as sufficient. Published claims of functional relevance should be fully evaluated using the steps detailed above.

RNASEL Arg462Gln variant is implicated in up to 13% of prostate cancer cases.

RNASEL (encoding ribonuclease L) has recently been proposed as a candidate for the hereditary prostate cancer (HPC1) gene. We determined that the RNASEL variant Arg462Gln has three times less enzymatic activity than the wildtype and is significantly associated with prostate cancer risk (P = 0.007). At least one copy of the mutated allele that causes this substitution is carried by nearly 60% of the men in our study. Men that are heterozygous with respect to the mutated allele have 50% greater risk of prostate cancer than non-carriers, and homozygotes have more than double the risk.

SLC5A8, a sodium transporter, is a tumor suppressor gene silenced by methylation in human colon aberrant crypt foci and cancers

We identify a gene, SLC5A8, and show it is a candidate tumor suppressor gene whose silencing by aberrant methylation is a common and early event in human colon neoplasia. Aberrant DNA methylation has been implicated as a component of an epigenetic mechanism that silences genes in human cancers. Using restriction landmark genome scanning, we performed a global search to identify genes that would be aberrantly methylated at high frequency in human colon cancer. From among 1,231 genomic NotI sites assayed, site 3D41 was identified as methylated in 11 of 12 colon cancers profiled. Site 3D41 mapped to exon 1 of SLC5A8, a transcript that we assembled. In normal colon mucosa we found that SLC5A8 exon 1 is unmethylated and SLC5A8 transcript is expressed. In contrast, SLC5A8 exon 1 proved to be aberrantly methylated in 59% of primary colon cancers and 52% of colon cancer cell lines. SLC5A8 exon 1 methylated cells were uniformly silenced for SLC5A8 expression, but reactivated expression on treatment with a demethylating drug, 5-azacytidine. Transfection of SLC5A8 suppressed colony growth in each of three SLC5A8-deficient cell lines, but showed no suppressive effect in any of three SLC5A8-proficient cell lines. SLC5A8 exon 1 methylation is an early event, detectable in colon adenomas, and in even earlier microscopic colonic aberrant crypt foci. Structural homology and functional testing demonstrated that SLC5A8 is a member of the family of sodium solute symporters, which are now added as a class of candidate colon cancer suppressor genes.

CCAT2, a novel noncoding RNA mapping to 8q24, underlies metastatic progression and chromosomal instability in colon cancer

The functional roles of SNPs within the 8q24 gene desert in the cancer phenotype are not yet well understood. Here, we report that CCAT2, a novel long noncoding RNA transcript (lncRNA) encompassing the rs6983267 SNP, is highly overexpressed in microsatellite-stable colorectal cancer and promotes tumor growth, metastasis, and chromosomal instability. We demonstrate that MYC, miR-17-5p, and miR-20a are up-regulated by CCAT2 through TCF7L2-mediated transcriptional regulation. We further identify the physical interaction between CCAT2 and TCF7L2 resulting in an enhancement of WNT signaling activity. We show that CCAT2 is itself a WNT downstream target, which suggests the existence of a feedback loop. Finally, we demonstrate that the SNP status affects CCAT2 expression and the risk allele G produces more CCAT2 transcript. Our results support a new mechanism of MYC and WNT regulation by the novel lncRNA CCAT2 in colorectal cancer pathogenesis, and provide an alternative explanation of the SNP-conferred cancer risk.

Breast cancers with brain metastases are more likely to be estrogen receptor negative, express the basal cytokeratin CK5/6, and overexpress HER2 or EGFR

Brain metastases (BM) from breast cancer are associated with significant morbidity and mortality. In the current study, we have examined a cohort of breast cancer patients who went on to develop BM for clinical-pathologic features and predictive markers that identify this high-risk subgroup of patients at the time of diagnosis. The primary tumors from 55 patients who developed BM were used to construct a tissue microarray. The clinical and pathologic features were recorded and the tissue microarray was stained for estrogen receptor, human epidermal growth factor receptor 2, cytokeratin 5/6, and epidermal growth factor receptor by immunohistochemistry . This cohort of patients was compared against a group of 254 patients who remain free of metastases (67 mo mean follow-up), and another cohort of 40 patients who developed mixed visceral and bone metastatic disease without brain recurrence over a similar period of time. Breast cancer patients who went on to develop BM were more likely to be <50 years old (P<0.001), and the primary tumors were more likely to be estrogen receptor negative (P<0.001) and high grade (P=0.002). The primary tumors were also more likely to express cytokeratin 5/6 (P<0.001) and epidermal growth factor receptor (P=0.001), and to overexpress human epidermal growth factor receptor 2 (P=0.001). The data presented above suggest a profile for breast cancer patients at increased risk for developing BM. Predictive factors to help identify patients with metastatic breast cancer who are at an increased risk for developing central nervous system recurrence might allow for screening of this population for early detection and treatment or for the development of targeted strategies for prevention.

The landscape of recombination in African Americans

Recombination, together with mutation, gives rise to genetic variation in populations. Here we leverage the recent mixture of people of African and European ancestry in the Americas to build a genetic map measuring the probability of crossing over at each position in the genome, based on about 2.1 million crossovers in 30,000 unrelated African Americans. At intervals of more than three megabases it is nearly identical to a map built in Europeans. At finer scales it differs significantly, and we identify about 2,500 recombination hotspots that are active in people of West African ancestry but nearly inactive in Europeans. The probability of a crossover at these hotspots is almost fully controlled by the alleles an individual carries at PRDM9 (P value < 10−245). We identify a 17-base-pair DNA sequence motif that is enriched in these hotspots, and is an excellent match to the predicted binding target of PRDM9 alleles common in West Africans and rare in Europeans. Sites of this motif are predicted to be risk loci for disease-causing genomic rearrangements in individuals carrying these alleles. More generally, this map provides a resource for research in human genetic variation and evolution.

Colorectal and Other Cancer Risks for Carriers and Noncarriers From Families With a DNA Mismatch Repair Gene Mutation: A Prospective Cohort Study

PURPOSE To determine whether cancer risks for carriers and noncarriers from families with a mismatch repair (MMR) gene mutation are increased above the risks of the general population. PATIENTS AND METHODS We prospectively followed a cohort of 446 unaffected carriers of an MMR gene mutation (MLH1, n = 161; MSH2, n = 222; MSH6, n = 47; and PMS2, n = 16) and 1,029 their unaffected relatives who did not carry a mutation every 5 years at recruitment centers of the Colon Cancer Family Registry. For comparison of cancer risk with the general population, we estimated country-, age-, and sex-specific standardized incidence ratios (SIRs) of cancer for carriers and noncarriers. RESULTS Over a median follow-up of 5 years, mutation carriers had an increased risk of colorectal cancer (CRC; SIR, 20.48; 95% CI, 11.71 to 33.27; P < .001), endometrial cancer (SIR, 30.62; 95% CI, 11.24 to 66.64; P < .001), ovarian cancer (SIR, 18.81; 95% CI, 3.88 to 54.95; P < .001), renal cancer (SIR, 11.22; 95% CI, 2.31 to 32.79; P < .001), pancreatic cancer (SIR, 10.68; 95% CI, 2.68 to 47.70; P = .001), gastric cancer (SIR, 9.78; 95% CI, 1.18 to 35.30; P = .009), urinary bladder cancer (SIR, 9.51; 95% CI, 1.15 to 34.37; P = .009), and female breast cancer (SIR, 3.95; 95% CI, 1.59 to 8.13; P = .001). We found no evidence of their noncarrier relatives having an increased risk of any cancer, including CRC (SIR, 1.02; 95% CI, 0.33 to 2.39; P = .97). CONCLUSION We confirmed that carriers of an MMR gene mutation were at increased risk of a wide variety of cancers, including some cancers not previously recognized as being a result of MMR mutations, and found no evidence of an increased risk of cancer for their noncarrier relatives.

Identification of genetic susceptibility loci for colorectal tumors in a genome-wide meta-analysis

BACKGROUND & AIMS Heritable factors contribute to the development of colorectal cancer. Identifying the genetic loci associated with colorectal tumor formation could elucidate the mechanisms of pathogenesis. METHODS We conducted a genome-wide association study that included 14 studies, 12,696 cases of colorectal tumors (11,870 cancer, 826 adenoma), and 15,113 controls of European descent. The 10 most statistically significant, previously unreported findings were followed up in 6 studies; these included 3056 colorectal tumor cases (2098 cancer, 958 adenoma) and 6658 controls of European and Asian descent. RESULTS Based on the combined analysis, we identified a locus that reached the conventional genome-wide significance level at less than 5.0 × 10(-8): an intergenic region on chromosome 2q32.3, close to nucleic acid binding protein 1 (most significant single nucleotide polymorphism: rs11903757; odds ratio [OR], 1.15 per risk allele; P = 3.7 × 10(-8)). We also found evidence for 3 additional loci with P values less than 5.0 × 10(-7): a locus within the laminin gamma 1 gene on chromosome 1q25.3 (rs10911251; OR, 1.10 per risk allele; P = 9.5 × 10(-8)), a locus within the cyclin D2 gene on chromosome 12p13.32 (rs3217810 per risk allele; OR, 0.84; P = 5.9 × 10(-8)), and a locus in the T-box 3 gene on chromosome 12q24.21 (rs59336; OR, 0.91 per risk allele; P = 3.7 × 10(-7)). CONCLUSIONS In a large genome-wide association study, we associated polymorphisms close to nucleic acid binding protein 1 (which encodes a DNA-binding protein involved in DNA repair) with colorectal tumor risk. We also provided evidence for an association between colorectal tumor risk and polymorphisms in laminin gamma 1 (this is the second gene in the laminin family to be associated with colorectal cancers), cyclin D2 (which encodes for cyclin D2), and T-box 3 (which encodes a T-box transcription factor and is a target of Wnt signaling to β-catenin). The roles of these genes and their products in cancer pathogenesis warrant further investigation.

Common variation near CDKN1A , POLD3 and SHROOM2 influences colorectal cancer risk

We performed a meta-analysis of five genome-wide association studies to identify common variants influencing colorectal cancer (CRC) risk comprising 8,682 cases and 9,649 controls. Replication analysis was performed in case-control sets totaling 21,096 cases and 19,555 controls. We identified three new CRC risk loci at 6p21 (rs1321311, near CDKN1A; P = 1.14 × 10−10), 11q13.4 (rs3824999, intronic to POLD3; P = 3.65 × 10−10) and Xp22.2 (rs5934683, near SHROOM2; P = 7.30 × 10−10) This brings the number of independent loci associated with CRC risk to 20 and provides further insight into the genetic architecture of inherited susceptibility to CRC.

p53 mutations are common in pancreatic cancer and are absent in chronic pancreatitis.

Pancreatic expression of the p53 tumor suppressor gene was studied in pancreatic adenocarcinomas and chronic pancreatitis. By immunohistochemistry, 16 of 34 (47%) cancers and none of the 24 chronic pancreatitis samples revealed nuclear staining. Sequence analysis indicated that 8 of 24 (33%) cancers were mutated for the p53 gene. Point substitutions occurred at codons 35, 105, 133, 213, 213, 258, and 299. A three base-pair in-frame insertion was identified between codons 261 and 262. None of 8 chronic pancreatitis samples exhibited p53 gene mutations. These data support a role for p53 gene alterations in human pancreatic cancer, and suggest that loss of its regulatory functions may constitute one of the differences between pancreatic cancer and chronic pancreatitis.