title: The dynamic DNA methylation landscape of themutL homolog 1shore is altered byMLH1-93G>A polymorphism in normal tissues and colorectal cancer.
abstract: Colorectal cancers (CRCs) undergo distinct genetic and epigenetic alterations. Expression ofmutL homolog 1(MLH1), a mismatch repair gene that corrects DNA replication errors, is lost in up to 15% of sporadic tumours due to mutation or, more commonly, due to DNA methylation of its promoter CpG island. A single nucleotide polymorphism (SNP) in the CpG island ofMLH1(MLH1-93G>A or rs1800734) is associated with CpG island hypermethylation and decreasedMLH1expression in CRC tumours. Further, in peripheral blood mononuclear cell (PBMC) DNA of both CRC cases and non-cancer controls, the variant allele of rs1800734 is associated with hypomethylation at theMLH1shore, a region upstream of its CpG island that is less dense in CpG sites.
  
  
  
  To determine whether this genotype-epigenotype association is present in other tissue types, including colorectal tumours, we assessed DNA methylation in matched normal colorectal tissue, tumour, and PBMC DNA from 349 population-based CRC cases recruited from the Ontario Familial Colorectal Cancer Registry. Using the semi-quantitative real-time PCR-based MethyLight assay,MLH1shore methylation was significantly higher in tumour tissue than normal colon or PBMCs (P< 0.01). When shore methylation levels were stratified by SNP genotype, normal colorectal DNA and PBMC DNA were significantly hypomethylated in association with variant SNP genotype (P< 0.05). However, this association was lost in tumour DNA. Among distinct stages of CRC, metastatic stage IV CRC tumours incurred significant hypomethylation compared to stage I-III cases, irrespective of genotype status. Shore methylation ofMLH1was not associated with MSI status or promoter CpG island hypermethylation, regardless of genotype. To confirm these results, bisulfite sequencing was performed in matched tumour and normal colorectal specimens from six CRC cases, including two cases per genotype (wildtype, heterozygous, and homozygous variant). Bisulfite sequencing results corroborated the methylation patterns found by MethyLight, with significant hypomethylation in normal colorectal tissue of variant SNP allele carriers.
  
  
  
  These results indicate that the normal tissue types tested (colorectum and PBMC) experience dynamic genotype-associated epigenetic alterations at theMLH1shore, whereas tumour DNA incurs aberrant hypermethylation compared to normal DNA.
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Sinai Health System, 60 Murray St., Toronto, Ontario M5T 3L9 Canada.|Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, 27 King's College Circle, Toronto, Ontario M5S 1A1 Canada.
authors.forename: Andrea J
authors.initials: AJ
authors.lastname: Savio
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Sinai Health System, 60 Murray St., Toronto, Ontario M5T 3L9 Canada.|Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, 27 King's College Circle, Toronto, Ontario M5S 1A1 Canada.
authors.forename: Miralem
authors.initials: M
authors.lastname: Mrkonjic
authors.affiliation: Ontario Institute for Cancer Research, 661 University Avenue, Toronto, Ontario M5G 0A3 Canada.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Lemire
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Sinai Health System, 60 Murray St., Toronto, Ontario M5T 3L9 Canada.|Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, 27 King's College Circle, Toronto, Ontario M5S 1A1 Canada.|Ontario Institute for Cancer Research, 661 University Avenue, Toronto, Ontario M5G 0A3 Canada.|Ontario Familial Colorectal Cancer Registry, Cancer Care Ontario, 60 Murray St., Toronto, Ontario M5T 3L9 Canada.|Department of Surgery, University Health Network, 200 Elizabeth St., Toronto, ON M5G 2C4 Canada.
authors.forename: Steven
authors.initials: S
authors.lastname: Gallinger
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Sinai Health System, 60 Murray St., Toronto, Ontario M5T 3L9 Canada.|Dalla Lana School of Public Health, University of Toronto, 155 College St., Toronto, ON M5T 3M7 Canada.
authors.forename: Julia A
authors.initials: JA
authors.lastname: Knight
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Sinai Health System, 60 Murray St., Toronto, Ontario M5T 3L9 Canada.|Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, 27 King's College Circle, Toronto, Ontario M5S 1A1 Canada.|Department of Pathology, University Health Network, 200 Elizabeth St., Toronto, ON M5G 2C4 Canada.
authors.forename: Bharat
authors.initials: B
authors.lastname: Bapat
mesh_terms: D000328:Adult; D000368:Aged; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology*; D018899:CpG Islands; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D008297:Male; D008875:Middle Aged; D000070957:MutL Protein Homolog 1 / Q000097:blood / Q000235:genetics*; D009367:Neoplasm Staging; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D055815:Young Adult
chemical_list: C501486:MLH1 protein, human; D000070957:MutL Protein Homolog 1
keywords: Colorectal cancer; CpG island; CpG shore; DNA methylation; Epigenetics; MethyLight; Microsatellite instability; Mismatch repair; Single nucleotide polymorphism; mutL homolog 1