title: Tumour MLH1 promoter region methylation testing is an effective prescreen for Lynch Syndrome (HNPCC).
abstract: OBJECTIVE
  Lynch syndrome (LS) patients have DNA mismatch repair deficiency and up to 80% lifetime risk of colorectal cancer (CRC). Screening of mutation carriers reduces CRC incidence and mortality. Selection for constitutional mutation testing relies on family history (Amsterdam and Bethesda Guidelines) and tumour-derived biomarkers. Initial biomarker analysis uses mismatch repair protein immunohistochemistry and microsatellite instability. Abnormalities in either identify mismatch repair deficiency but do not differentiate sporadic epigenetic defects, due to MLH1 promoter region methylation (13% of CRCs) from LS (4% of CRCs). A diagnostic biomarker capable of making this distinction would be valuable. This study compared two biomarkers in tumours with mismatch repair deficiency; quantification of methylation of the MLH1 promoter region using a novel assay and BRAF c.1799T>A, p.(Val600Glu) mutation status in the identification of constitutional mutations.
  
  
  METHODS
  Tumour DNA was extracted (formalin fixed, paraffin embedded, FFPE tissue) and pyrosequencing used to test for MLH1 promoter methylation and presence of the BRAF c.1799T>A, p.(Val600Glu) mutation 71 CRCs from individuals with pathogenic MLH1 mutations and 73 CRCs with sporadic MLH1 loss. Specificity and sensitivity was compared.
  
  
  RESULTS
  Unmethylated MLH1 promoter: sensitivity 94.4% (95% CI 86.2% to 98.4%), specificity 87.7% (95% CI 77.9% to 94.2%), Wild-type BRAF (codon 600): sensitivity 65.8% (95% CI 53.7% to 76.5%), specificity 98.6% (95% CI 92.4% to 100.0%) for the identification of those with pathogenic MLH1 mutations.
  
  
  CONCLUSIONS
  Quantitative MLH1 promoter region methylation using pyrosequencing is superior to BRAF codon 600 mutation status in identifying constitutional mutations in mismatch repair deficient tumours.
authors.affiliation: Department of General Surgery, Manchester Royal Infirmary, Central Manchester University Hospitals NHS Trust, Manchester, UK.
authors.forename: K
authors.initials: K
authors.lastname: Newton
authors.affiliation: Genomic Diagnostics Laboratory, Manchester Centre for Genomic Medicine, Central Manchester University Hospitals NHS Foundation Trust, Saint Mary's Hospital, Manchester, UK.
authors.forename: N M
authors.initials: NM
authors.lastname: Jorgensen
authors.affiliation: Genomic Diagnostics Laboratory, Manchester Centre for Genomic Medicine, Central Manchester University Hospitals NHS Foundation Trust, Saint Mary's Hospital, Manchester, UK.
authors.forename: A J
authors.initials: AJ
authors.lastname: Wallace
authors.affiliation: Cancer and Population Studies Group, Queensland Institute of Medical Research, Herston, Queensland, Australia Oncogenomics Group, Genetic Epidemiology Laboratory, Department of Pathology, The University of Melbourne, Parkville, Victoria, Australia Centre for Epidemiology and Biostatistics, Melbourne School of Population and Global Health, The University of Melbourne, Parkville, Victoria, Australia.
authors.forename: D D
authors.initials: DD
authors.lastname: Buchanan
authors.affiliation: Manchester Centre for Genomic Medicine, Central Manchester University Hospitals NHS Foundation Trust, Saint Mary's Hospital, Manchester, UK.
authors.forename: F
authors.initials: F
authors.lastname: Lalloo
authors.affiliation: Department of Histopathology, Manchester Royal Infirmary, Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK Manchester Medical School, University of Manchester, Manchester, UK.
authors.forename: R F T
authors.initials: RF
authors.lastname: McMahon
authors.affiliation: Department of General Surgery, Manchester Royal Infirmary, Central Manchester University Hospitals NHS Trust, Manchester, UK.
authors.forename: J
authors.initials: J
authors.lastname: Hill
authors.affiliation: Manchester Centre for Genomic Medicine, Central Manchester University Hospitals NHS Foundation Trust, Saint Mary's Hospital, Manchester, UK.
authors.forename: D G
authors.initials: DG
authors.lastname: Evans
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics*; D000328:Adult; D000483:Alleles; D001932:Brain Neoplasms / Q000235:genetics; D015179:Colorectal Neoplasms / Q000235:genetics; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000175:diagnosis* / Q000235:genetics*; D018899:CpG Islands; D019175:DNA Methylation; D005820:Genetic Testing / Q000379:methods / Q000592:standards; D006579:Heterozygote; D006801:Humans; D008875:Middle Aged; D000070957:MutL Protein Homolog 1; D009154:Mutation; D009369:Neoplasms / Q000235:genetics*; D009386:Neoplastic Syndromes, Hereditary / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D048493:Proto-Oncogene Proteins B-raf; D000070957:MutL Protein Homolog 1
keywords: Cancer: colon; Genetics