Investigating the Potential Role of Genetic and Epigenetic Variation of DNA Methyltransferase Genes in Hyperplastic Polyposis Syndrome

BACKGROUND: Hyperplastic Polyposis Syndrome (HPS) is a condition associated with multiple serrated polyps, and an increased risk of colorectal cancer (CRC). At least half of CRCs arising in HPS show a CpG island methylator phenotype (CIMP), potentially linked to aberrant DNA methyltransferase (DNMT) activity. CIMP is associated with methylation of tumor suppressor genes including regulators of DNA mismatch repair (such as MLH1, MGMT), and negative regulators of Wnt signaling (such as WIF1). In this study, we investigated the potential for interaction of genetic and epigenetic variation in DNMT genes, in the aetiology of HPS. METHODS: We utilized high resolution melting (HRM) analysis to screen 45 cases with HPS for novel sequence variants in DNMT1, DNMT3A, DNMT3B, and DNMT3L. 21 polyps from 13 patients were screened for BRAF and KRAS mutations, with assessment of promoter methylation in the DNMT1, DNMT3A, DNMT3B, DNMT3L MLH1, MGMT, and WIF1 gene promoters. RESULTS: No pathologic germline mutations were observed in any DNA-methyltransferase gene. However, the T allele of rs62106244 (intron 10 of DNMT1 gene) was over-represented in cases with HPS (p<0.01) compared with population controls. The DNMT1, DNMT3A and DNMT3B promoters were unmethylated in all instances. Interestingly, the DNMT3L promoter showed low levels of methylation in polyps and normal colonic mucosa relative to matched disease free cells with methylation level negatively correlated to expression level in normal colonic tissue. DNMT3L promoter hypomethylation was more often found in polyps harbouring KRAS mutations (p = 0.0053). BRAF mutations were common (11 out of 21 polyps), whilst KRAS mutations were identified in 4 of 21 polyps. CONCLUSIONS: Genetic or epigenetic alterations in DNMT genes do not appear to be associated with HPS, but further investigation of genetic variation at rs62106244 is justified given the high frequency of the minor allele in this case series.Musa Drini, Nicholas C. Wong, Hamish S. Scott, Jeffrey M. Craig, Alexander Dobrovic, Chelsee A. Hewitt, Christofer Dow, Joanne P. Young, Mark A. Jenkins, Richard Saffery and Finlay A. Macra

Linkage to chromosome 2q32.2-q33.3 in familial serrated neoplasia (Jass syndrome)

Causative genetic variants have to date been identified for only a small proportion of familial colorectal cancer (CRC). While conditions such as Familial Adenomatous Polyposis and Lynch syndrome have well defined genetic causes, the search for variants underlying the remainder of familial CRC is plagued by genetic heterogeneity. The recent identification of families with a heritable predisposition to malignancies arising through the serrated pathway (familial serrated neoplasia or Jass syndrome) provides an opportunity to study a subset of familial CRC in which heterogeneity may be greatly reduced. A genome-wide linkage screen was performed on a large family displaying a dominantly-inherited predisposition to serrated neoplasia genotyped using the Affymetrix GeneChip Human Mapping 10 K SNP Array. Parametric and nonparametric analyses were performed and resulting regions of interest, as well as previously reported CRC susceptibility loci at 3q22, 7q31 and 9q22, were followed up by finemapping in 10 serrated neoplasia families. Genome-wide linkage analysis revealed regions of interest at 2p25.2-p25.1, 2q24.3-q37.1 and 8p21.2-q12.1. Finemapping linkage and haplotype analyses identified 2q32.2-q33.3 as the region most likely to harbour linkage, with heterogeneity logarithm of the odds (HLOD) 2.09 and nonparametric linkage (NPL) score 2.36 (P = 0.004). Five primary candidate genes (CFLAR, CASP10, CASP8, FZD7 and BMPR2) were sequenced and no segregating variants identified. There was no evidence of linkage to previously reported loci on chromosomes 3, 7 and 9

Risk Factors for Colorectal Cancer in Patients with Multiple Serrated Polyps: A Cross-Sectional Case Series from Genetics Clinics

Patients with multiple serrated polyps are at an increased risk for developing colorectal cancer (CRC). Recent reports have linked cigarette smoking with the subset of CRC that develops from serrated polyps. The aim of this work therefore was to investigate the association between smoking and the risk of CRC in high-risk genetics clinic patients presenting with multiple serrated polyps. Methods and Findings We identified 151 Caucasian individuals with multiple serrated polyps including at least 5 outside the rectum, and classified patients into non-smokers, current or former smokers at the time of initial diagnosis of polyposis. Cases were individuals with multiple serrated polyps who presented with CRC. Controls were individuals with multiple serrated polyps and no CRC. Multivariate logistic regression was performed to estimate associations between smoking and CRC with adjustment for age at first presentation, sex and co-existing traditional adenomas, a feature that has been consistently linked with CRC risk in patients with multiple serrated polyps. CRC was present in 56 (37%) individuals at presentation. Patients with at least one adenoma were 4 times more likely to present with CRC compared with patients without adenomas (OR = 4.09; 95%CI 1.27 to 13.14; P = 0.02). For females, the odds of CRC decreased by 90% in current smokers as compared to never smokers (OR = 0.10; 95%CI 0.02 to 0.47; P = 0.004) after adjusting for age and adenomas. For males, there was no relationship between current smoking and CRC. There was no statistical evidence of an association between former smoking and CRC for both sexes. Conclusion A decreased odds for CRC was identified in females with multiple serrated polyps who currently smoke, independent of age and the presence of a traditional adenoma. Investigations into the biological basis for these observations could lead to non-smoking-related therapies being developed to decrease the risk of CRC and colectomy in these patients.Daniel D. Buchanan, Kevin Sweet, Musa Drini, Mark A. Jenkins, Aung Ko Win, Dallas R. English, Michael D. Walsh, Mark Clendenning, Diane M. McKeone, Rhiannon J. Walters, Aedan Roberts, Sally-Ann Pearson, Erika Pavluk, John L. Hopper, Michael R. Gattas, Jack Goldblatt, Jill George, Graeme K. Suthers, Kerry D. Phillips, Sonja Woodal, Julie Arnold, Kathy Tucker, Amanda Muir, Michael Field, Sian Greening, Steven Gallinger, Renee Perrier, John A. Baron, John D. Potter, Robert Haile, Wendy Franke, Albert de la Chapelle, Finlay Macrae, Christophe Rosty, Neal I. Walker, Susan Parry and Joanne P. Youn

Peptic ulcer disease and non-steroidal anti-inflammatory drugs

Non-steroidal anti-inflammatory drugs including low-dose aspirin are some of the most commonly used medicines. They are associated with gastrointestinal mucosal injury. Before prescribing, it is important to assess the patient’s gastrointestinal risk factors such as age and history of peptic ulcers. Patients at high risk may require co-prescription to reduce the risk of peptic ulcers. A daily dose of a proton pump inhibitor is the most effective method of reducing the risk of ulcers induced by non-steroidal anti-inflammatory drugs

Genotypic and phenotypic studies in hyperplastic polyposis syndrome

Deposited with the permission of the author. © 2010 Dr. Musa DriniHyperplastic Polyposis Syndrome (HPS) is a condition associated with multiple serrated polyps and colorectal cancer (CRC) risk, and is related to a third pathway to human CRC known as “serrated neoplasia pathway”. Several genetic aberrations have been linked to HPS, including frequent BRAF and KRAS mutations and aberrant methyation known as CIMP. At least half of CRCs arising in HPS also show a CpG island methylator phenotype (CIMP). CIMP usually involves a wide panel of tumor suppressor genes including regulators of DNA mismatch repair (such as MLH1, MGMT), and negative regulators of Wnt signaling (such as WIF1). DNA methylation is one of many epigenetic factors that are important for cellular differentiation, gene regulation and genomic imprinting. The aberrant DNA methylation found in CIMP phenotype may be a consequence of dysfunctional machinery involved in establishing and maintaining normal DNA methylation. DNA methyltransferases DNMTs (DNMT1, -3A, -3B and -3L) are a family of proteins responsible for transfer of methyl groups specifically to cytosine in CpG dinucleotides of DNA. Given the role of DNMT’s in DNA methylation, we investigated the potential for interaction of genetic and epigenetic variation in DNMT genes, together with variation in BRAF and KRAS genes, in the molecular pathogenesis of HPS. A candidate gene approach identified no novel sequence variants that showed significant association with HPS. However, the T allele of rs62106244 (intron 10 of DNMT1 gene) was over-represented in cases with HPS (p<0.01), a finding deserving further study. The DNMT1, DNMT3A and DNMT3B promoters were un-methylated in all instances. Interestingly, the DNMT3L promoter showed low levels of methylation in polyps and normal colonic mucosa relative to matched disease free cells, and this may be important in expression of the gene in vivo. Expression levels of DNMT3L in polyps and normal terminal ileum tissue varied inversely with methylation may be a functionally important defect in gut. BRAF mutations were common (11 out of 21 polyps), whilst KRAS mutations were identified in 4 of 21 polyps. In addition, DNMT3L promoter hypermethylation was more often found in polyps harbouring KRAS mutations (p=0.0053). Attempts with candidate gene DNA methylation approaches were unsuccessful or unconvincing in assigning a single responsible gene responsible for HPS. We therefore applied a hypothesis-free approach by studying serrated polyps with Infinium HumanMethylation27 BeadChip Illumina® genome-wide promoter methylation. This approach identified three pathways: homedomain and basic-helix-loop transcription factors, homeobox genes and the ERK pathway, demonstrating co-ordinate methylation of genes in the polyps compared to normal controls; we speculated that the functional consequences, could explain HPS pathogenesis. This study has found that DNMT3L may be of a functional importance in gut functioning and in particular is associated with polyps harbouring KRAS mutation. A non-candidate gene approach also identified important pathways that may explain the complex aetiology of HPS