title: Reduced mRNA expression in paraffin-embedded tissue identifies MLH1- and MSH2-deficient colorectal tumours and potential mutation carriers.
abstract: Based on the principle of nonsense-mediated mRNA decay, we sought to identify MLH1 or MSH2-deficient colorectal tumours through relative quantification of mRNA expression with real-time PCR (RT-PCR) analysis. MLH1 and MSH2 mRNAs were almost equally expressed as defined by MLH1 to MSH2 transcript ratio (mean 1.41) in microsatellite stable, mismatch repair (MMR) proficient tumours (n = 16). A close correlation between loss of protein expression and MMR-mRNA levels was found in highly microsatellite instable (MSI-H) tumours deficient of MLH1 or MSH2. MLH1/MSH2 ratio was low in 11 sporadic and nine hereditary MLH1-deficient carcinomas (mean 0.51), whereas the ratio was high in 17 MSH2-deficient hereditary non-polyposis colorectal cancer (HNPCC) associated carcinomas (mean 6.8). Notably, in the normal tissues of HNPCC patients with MSH2 mutations, the MLH1/MSH2 transcript ratios were significantly elevated (ratio > 2.0) as compared to the ratios of normal mucosa in patients with MMR-proficient tumours (27 of 32 ratio < 2.0; p = 0.00113). Analysis of B-lymphocytes of HNPCC patients with proven MMR gene mutation confirmed these findings. In conclusion, RT-PCR allows relative quantification of MMR gene mRNA expression in formalin-fixed and paraffin-embedded tissue. Furthermore, this approach enables quantification of haploinsufficiency due to nonsense-mediated mRNA decay in normal tissue and B-lymphocytes from patients carrying MSH2 germline mutations and may be useful for identification of asymptomatic carriers of pathogenic germline mutations.
authors.affiliation: Department of General Surgery, University of Göttingen, Göttingen, Germany.
authors.forename: Annegret
authors.initials: A
authors.lastname: Müller
authors.forename: Dirk
authors.initials: D
authors.lastname: Zielinski
authors.forename: Nicolaus
authors.initials: N
authors.lastname: Friedrichs
authors.forename: Barbara
authors.initials: B
authors.lastname: Oberschmid
authors.forename: Sabine
authors.initials: S
authors.lastname: Merkelbach-Bruse
authors.forename: Hans K
authors.initials: HK
authors.lastname: Schackert
authors.forename: Markus
authors.initials: M
authors.lastname: Linnebacher
authors.forename: Magnus
authors.initials: M
authors.lastname: von Knebel Doeberitz
authors.forename: Reinhard
authors.initials: R
authors.lastname: Büttner
authors.forename: Josef
authors.initials: J
authors.lastname: Rüschoff
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000172:deficiency* / Q000235:genetics; D000236:Adenoma / Q000235:genetics / Q000378:metabolism*; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001402:B-Lymphocytes / Q000378:metabolism; D016022:Case-Control Studies; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism*; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D020022:Genetic Predisposition to Disease / Q000235:genetics; D005820:Genetic Testing; D018095:Germ-Line Mutation / Q000235:genetics*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D008875:Middle Aged; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein / Q000172:deficiency* / Q000235:genetics; D009687:Nuclear Proteins / Q000172:deficiency* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012334:RNA, Neoplasm / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein