Alexandra E. Gylfe

CTCF/cohesin-binding sites are frequently mutated in cancer

Cohesin is present in almost all active enhancer regions, where it is associated with transcription factors. Cohesin frequently colocalizes with CTCF (CCCTC-binding factor), affecting genomic stability, expression and epigenetic homeostasis. Cohesin subunits are mutated in cancer, but CTCF/cohesin-binding sites (CBSs) in DNA have not been examined for mutations. Here we report frequent mutations at CBSs in cancers displaying a mutational signature where mutations in A•T base pairs predominate. Integration of whole-genome sequencing data from 213 colorectal cancer (CRC) samples and chromatin immunoprecipitation sequencing (ChIP-exo) data identified frequent point mutations at CBSs. In contrast, CRCs showing an ultramutator phenotype caused by defects in the exonuclease domain of DNA polymerase ɛ (POLE) displayed significantly fewer mutations at and adjacent to CBSs. Analysis of public data showed that multiple cancer types accumulate CBS mutations. CBSs are a major mutational hotspot in the noncoding cancer genome.

Exome sequencing reveals frequent inactivating mutations in ARID1A, ARID1B, ARID2 and ARID4A in microsatellite unstable colorectal cancer

ARID1A has been identified as a novel tumor suppressor gene in ovarian cancer and subsequently in various other tumor types. ARID1A belongs to the ARID domain containing gene family, which comprises of 15 genes involved, for example, in transcriptional regulation, proliferation and chromatin remodeling. In this study, we used exome sequencing data to analyze the mutation frequency of all the ARID domain containing genes in 25 microsatellite unstable (MSI) colorectal cancers (CRCs) as a first systematic effort to characterize the mutation pattern of the whole ARID gene family. Genes which fulfilled the selection criteria in this discovery set (mutations in at least 4/25 [16%] samples, including at least one nonsense or splice site mutation) were chosen for further analysis in an independent validation set of 21 MSI CRCs. We found that in addition to ARID1A, which was mutated in 39% of the tumors (18/46), also ARID1B (13%, 6/46), ARID2 (13%, 6/46) and ARID4A (20%, 9/46) were frequently mutated. In all these genes, the mutations were distributed along the entire length of the gene, thus distinguishing them from typical MSI target genes previously described. Our results indicate that in addition to ARID1A, other members of the ARID gene family may play a role in MSI CRC.

Eleven Candidate Susceptibility Genes for Common Familial Colorectal Cancer

Hereditary factors are presumed to play a role in one third of colorectal cancer (CRC) cases. However, in the majority of familial CRC cases the genetic basis of predisposition remains unexplained. This is particularly true for families with few affected individuals. To identify susceptibility genes for this common phenotype, we examined familial cases derived from a consecutive series of 1514 Finnish CRC patients. Ninety-six familial CRC patients with no previous diagnosis of a hereditary CRC syndrome were included in the analysis. Eighty-six patients had one affected first-degree relative, and ten patients had two or more. Exome sequencing was utilized to search for genes harboring putative loss-of-function variants, because such alterations are likely candidates for disease-causing mutations. Eleven genes with rare truncating variants in two or three familial CRC cases were identified: UACA, SFXN4, TWSG1, PSPH, NUDT7, ZNF490, PRSS37, CCDC18, PRADC1, MRPL3, and AKR1C4. Loss of heterozygosity was examined in all respective cancer samples, and was detected in seven occasions involving four of the candidate genes. In all seven occasions the wild-type allele was lost (P = 0.0078) providing additional evidence that these eleven genes are likely to include true culprits. The study provides a set of candidate predisposition genes which may explain a subset of common familial CRC. Additional genetic validation in other populations is required to provide firm evidence for causality, as well as to characterize the natural history of the respective phenotypes.

A Finnish founder mutation in RAD51D: analysis in breast, ovarian, prostate, and colorectal cancer

Background RAD51D and RAD54L are involved in homologous recombination, and rare mutations in RAD51D were recently found in breast-ovarian cancer families. This study investigated RAD51D and RAD54L for mutations in breast and ovarian cancer patients in the Finnish population. Methods The study sequenced the RAD51D and RAD54L genes in 95 breast and/or ovarian cancer families and genotyped the identified mutation in an additional 2200 breast and 553 ovarian cancer patients and 2102 population controls. To investigate the role of the mutation in other common cancers, 1094 prostate and 980 colorectal cancer patients were genotyped. Results In the screening of RAD51D, one deleterious founder mutation c.576+1G>A was identified in two breast-ovarian cancer families. No mutations were found in RAD54L. Altogether, the c.576+1G>A mutation was detected in 5/707 patients with a personal or family history of ovarian cancer (OR 9.16, 95% CI 1.07 to 78.56; p=0.024), with the highest frequency among breast-ovarian cancer families (3/105 vs 1/1287 controls, OR 37.82, 95% CI 3.90 to 366.91; p=0.0016), but no elevated frequency among breast cancer patients/families (2/2105, p=1). The mutation was not found among prostate or colorectal cancer patients. Conclusions The results of this study on familial and unselected breast, ovarian, colorectal, and prostate cancer patients suggest that RAD51D is primarily a moderate penetrance susceptibility gene for ovarian cancer, with clinical significance for the carriers.

Somatic mutations and germline sequence variants in patients with familial colorectal cancer.

It is estimated that up to 35% of colorectal cancers (CRC) can be explained by hereditary factors. However, genes predisposing to highly penetrant CRC syndromes account for only a small fraction of all cases. Thus, most CRCs still remain molecularly unexplained. A recent systematic sequencing study on well‐annotated human protein coding genes identified 280 somatically mutated candidate cancer genes (CAN genes) in breast and colorectal cancer. It is estimated that 8% of all reported cancer genes show both somatic and germline mutations. Therefore, the identified CAN genes serve as a distinct set of candidates for being involved in hereditary susceptibility. The aim of this study was to evaluate the role of colorectal CAN genes in familial CRC. Samples from 45 familial CRCs without known cancer predisposing mutations were screened for somatic and germline variants in 15 top‐ranked CAN genes. Six of the genes were found to be somatically mutated in our tumor series. We identified 22 nonsynonymous somatic mutations of which the majority was of missense type. In germline, three novel nonsynonymous variants were identified in the following genes: CSMD3, EPHB6 and C10orf137, and none of the variants were present in 890 population‐matched healthy controls. It is possible that the identified germline variants modulate predisposition to CRC. Functional validation and larger sample sets, however, will be required to clarify the role of the identified germline variants in CRC susceptibility.

Mendelian randomisation analysis strongly implicates adiposity with risk of developing colorectal cancer

Background:Observational studies have associated adiposity with an increased risk of colorectal cancer (CRC). However, such studies do not establish a causal relationship. To minimise bias from confounding we performed a Mendelian randomisation (MR) analysis to examine the relationship between adiposity and CRC.Methods:We used SNPs associated with adult body mass index (BMI), waist-hip ratio (WHR), childhood obesity and birth weight as instrumental variables in a MR analysis of 9254 CRC cases and 18 386 controls.Results:In the MR analysis, the odds ratios (ORs) of CRC risk per unit increase in BMI, WHR and childhood obesity were 1.23 (95% CI: 1.02–1.49, P=0.033), 1.59 (95% CI: 1.08–2.34, P=0.019) and 1.07 (95% CI: 1.03–1.13, P=0.018), respectively. There was no evidence for association between birth weight and CRC (OR=1.22, 95% CI: 0.89–1.67, P=0.22). Combining these data with a concurrent MR-based analysis for BMI and WHR with CRC risk (totalling to 18 190 cases, 27 617 controls) provided increased support, ORs for BMI and WHR were 1.26 (95% CI: 1.10–1.44, P=7.7 × 10−4) and 1.40 (95% CI: 1.14–1.72, P=1.2 × 10−3), respectively.Conclusions:These data provide further evidence for a strong causal relationship between adiposity and the risk of developing CRC highlighting the urgent need for prevention and treatment of adiposity.

Characterization of the colorectal cancer-associated enhancer MYC-335 at 8q24: The role of rs67491583

Recent genome-wide association studies have identified multiple regions at 8q24 that confer susceptibility to many cancers. In our previous work, we showed that the colorectal cancer (CRC) risk variant rs6983267 at 8q24 resides within a TCF4 binding site at the MYC-335 enhancer, with the risk allele G having a stronger binding capacity and Wnt responsiveness. Here, we searched for other potential functional variants within MYC-335. Genetic variation within MYC-335 was determined in samples from individuals of European, African, and Asian descent, with emphasis on variants in putative transcription factor binding sites. A 2-bp GA deletion rs67491583 was found to affect a growth factor independent (GFI) binding site and was present only in individuals with African ancestry. Chromatin immunoprecipitation performed in heterozygous cells showed that the GA deletion had an ability to reduce binding of the transcriptional repressors GFI1 and GFI1b. Screening of 1,027 African American colorectal cancer cases and 1,773 healthy controls did not reveal evidence for association (odds ratio: 1.17, 95% confidence interval: 0.97-1.41, P = 0.095). In this study, rs67491583 was identified as another functional variant in the CRC-associated enhancer MYC-335, but further studies are needed to establish the role of rs67491583 in the colorectal cancer predisposition of African Americans.

Identification of 33 candidate oncogenes by screening for base-specific mutations.

Background:Genes with recurrent codon-specific somatic mutations are likely drivers of tumorigenesis and potential therapeutic targets. Hypermutable cancers may represent a sensitive system for generation and selection of oncogenic mutations.Methods:We utilised exome-sequencing data on 25 sporadic microsatellite-instable (MSI) colorectal cancers (CRCs) and searched for base-specific somatic mutation hotspots.Results:We identified novel mutation hotspots in 33 genes. Fourteen genes displayed mutations in the validation set of 254 MSI CRCs: ANTXR1, MORC2, CEP135, CRYBB1, GALNT9, KRT82, PI15, SLC36A1, CNTF, GLDC, MBTPS1, OR9Q2, R3HDM1 and TTPAL. A database search found examples of the hotspot mutations in multiple cancer types.Conclusions:This work reveals a variety of new recurrent candidate oncogene mutations to be further scrutinised as potential therapeutic targets.

Pro-inflammatory fatty acid profile and colorectal cancer risk: A Mendelian randomisation analysis.

Background While dietary fat has been established as a risk factor for colorectal cancer (CRC), associations between fatty acids (FAs) and CRC have been inconsistent. Using Mendelian randomisation (MR), we sought to evaluate associations between polyunsaturated (PUFA), monounsaturated (MUFA) and saturated FAs (SFAs) and CRC risk. Methods We analysed genotype data on 9254 CRC cases and 18,386 controls of European ancestry. Externally weighted polygenic risk scores were generated and used to evaluate associations with CRC per one standard deviation increase in genetically defined plasma FA levels. Results Risk reduction was observed for oleic and palmitoleic MUFAs (OROA = 0.77, 95% CI: 0.65–0.92, P = 3.9 × 10−3; ORPOA = 0.36, 95% CI: 0.15–0.84, P = 0.018). PUFAs linoleic and arachidonic acid had negative and positive associations with CRC respectively (ORLA = 0.95, 95% CI: 0.93–0.98, P = 3.7 × 10−4; ORAA = 1.05, 95% CI: 1.02–1.07, P = 1.7 × 10−4). The SFA stearic acid was associated with increased CRC risk (ORSA = 1.17, 95% CI: 1.01–1.35, P = 0.041). Conclusion Results from our analysis are broadly consistent with a pro-inflammatory FA profile having a detrimental effect in terms of CRC risk.

Mendelian randomisation implicates hyperlipidaemia as a risk factor for colorectal cancer.

While elevated blood cholesterol has been associated with an increased risk of colorectal cancer (CRC) in observational studies, causality is uncertain. Here we apply a Mendelian randomisation (MR) analysis to examine the potential causal relationship between lipid traits and CRC risk. We used single nucleotide polymorphisms (SNPs) associated with blood levels of total cholesterol (TC), triglyceride (TG), low‐density lipoprotein (LDL), and high‐density lipoprotein (HDL) as instrumental variables (IV). We calculated MR estimates for each risk factor with CRC using SNP‐CRC associations from 9,254 cases and 18,386 controls. Genetically predicted higher TC was associated with an elevated risk of CRC (odds ratios (OR) per unit SD increase = 1.46, 95% confidence interval [CI]: 1.20–1.79, p = 1.68 × 10−4). The pooled ORs for LDL, HDL, and TG were 1.05 (95% CI: 0.92–1.18, p = 0.49), 0.94 (95% CI: 0.84–1.05, p = 0.27), and 0.98 (95% CI: 0.85–1.12, p = 0.75) respectively. A genetic risk score for 3‐hydoxy‐3‐methylglutaryl‐coenzyme A reductase (HMGCR) to mimic the effects of statin therapy was associated with a reduced CRC risk (OR = 0.69, 95% CI: 0.49–0.99, p = 0.046). This study supports a causal relationship between higher levels of TC with CRC risk, and a further rationale for implementing public health strategies to reduce the prevalence of hyperlipidaemia.