title: Functional testing strategy for coding genetic variants of unclear significance in MLH1 in Lynch syndrome diagnosis.
abstract: Lynch syndrome is caused by inactivating mutations in the MLH1 gene, but genetic variants of unclear significance frequently preclude diagnosis. Functional testing can reveal variant-conferred defects in gene or protein function. Based on functional defect frequencies and clinical applicability of test systems, we developed a functional testing strategy aimed at efficiently detecting pathogenic defects in coding MLH1 variants. In this strategy, tests of repair activity and expression are prioritized over analyses of subcellular protein localization and messenger RNA (mRNA) formation. This strategy was used for four unclear coding MLH1 variants (p.Asp41His, p.Leu507Phe, p.Gln689Arg, p.Glu605del + p.Val716Met). Expression was analyzed using a transfection system, mismatch repair (MMR) activity by complementation in vitro, mRNA formation by reverse transcriptase-PCR in carrier lymphocyte mRNA, and subcellular localization with dye-labeled fusion constructs. All tests included clinically meaningful controls. The strategy enabled efficient identification of defects in two unclear variants: the p.Asp41His variant showed loss of MMR activity, whereas the compound variant p.Glu605del + p.Val716Met had a defect of expression. This expression defect was significantly stronger than the pathogenic expression reference variant analyzed in parallel, therefore the defect of the compound variant is also pathogenic. Interestingly, the expression defect was caused additively by both of the compound variants, at least one of which is non-pathogenic when occurring by itself. Tests were neutral for p.Leu507Phe and p.Gln689Arg, and the results were consistent with available clinical data. We finally discuss the improved sensitivity and efficiency of the applied strategy and its limitations in analyzing unclear coding MLH1 variants.
authors.affiliation: Biomedical Research Laboratory, Department of Internal Medicine 1 and Department of Human Genetics, Universitätsklinikum Frankfurt, Frankfurt D-60590, Germany, Institute of Human Genetics, University of Bonn, Bonn D-53127, Germany and Department of Internal Medicine 1, Universitätsklinikum Frankfurt D-60590, Frankfurt, Germany.
authors.forename: Inga
authors.initials: I
authors.lastname: Hinrichsen
authors.affiliation: Department of Human Genetics, Universitätsklinikum Frankfurt, Frankfurt D-60590, Germany.
authors.forename: Dieter
authors.initials: D
authors.lastname: Schäfer
authors.affiliation: Biomedical Research Laboratory, Department of Internal Medicine 1 and Department of Human Genetics, Universitätsklinikum Frankfurt, Frankfurt D-60590, Germany, Institute of Human Genetics, University of Bonn, Bonn D-53127, Germany and Department of Internal Medicine 1, Universitätsklinikum Frankfurt D-60590, Frankfurt, Germany.
authors.forename: Deborah
authors.initials: D
authors.lastname: Langer
authors.affiliation: Biomedical Research Laboratory, Department of Internal Medicine 1 and Department of Human Genetics, Universitätsklinikum Frankfurt, Frankfurt D-60590, Germany, Institute of Human Genetics, University of Bonn, Bonn D-53127, Germany and Department of Internal Medicine 1, Universitätsklinikum Frankfurt D-60590, Frankfurt, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Köger
authors.affiliation: Biomedical Research Laboratory, Department of Internal Medicine 1 and Department of Human Genetics, Universitätsklinikum Frankfurt, Frankfurt D-60590, Germany, Institute of Human Genetics, University of Bonn, Bonn D-53127, Germany and Department of Internal Medicine 1, Universitätsklinikum Frankfurt D-60590, Frankfurt, Germany.
authors.forename: Margarethe
authors.initials: M
authors.lastname: Wittmann
authors.affiliation: Institute of Human Genetics, University of Bonn, Bonn D-53127, Germany and.
authors.forename: Stefan
authors.initials: S
authors.lastname: Aretz
authors.affiliation: Institute of Human Genetics, University of Bonn, Bonn D-53127, Germany and.
authors.forename: Verena
authors.initials: V
authors.lastname: Steinke
authors.affiliation: Institute of Human Genetics, University of Bonn, Bonn D-53127, Germany and.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Holzapfel
authors.affiliation: Department of Internal Medicine 1, Universitätsklinikum Frankfurt D-60590, Frankfurt, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Trojan
authors.affiliation: Department of Human Genetics, Universitätsklinikum Frankfurt, Frankfurt D-60590, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: König
authors.affiliation: Department of Internal Medicine 1, Universitätsklinikum Frankfurt D-60590, Frankfurt, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Zeuzem
authors.affiliation: Biomedical Research Laboratory, Department of Internal Medicine 1 and Department of Human Genetics, Universitätsklinikum Frankfurt, Frankfurt D-60590, Germany, Institute of Human Genetics, University of Bonn, Bonn D-53127, Germany and Department of Internal Medicine 1, Universitätsklinikum Frankfurt D-60590, Frankfurt, Germany.
authors.forename: Angela
authors.initials: A
authors.lastname: Brieger
authors.affiliation: Biomedical Research Laboratory, Department of Internal Medicine 1 and Department of Human Genetics, Universitätsklinikum Frankfurt, Frankfurt D-60590, Germany, Institute of Human Genetics, University of Bonn, Bonn D-53127, Germany and Department of Internal Medicine 1, Universitätsklinikum Frankfurt D-60590, Frankfurt, Germany plotz@med.uni-frankfurt.de.
authors.forename: Guido
authors.initials: G
authors.lastname: Plotz
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000096:biosynthesis / Q000235:genetics*; D000251:Adenosine Triphosphatases / Q000235:genetics*; D000328:Adult; D000483:Alleles; D045744:Cell Line, Tumor; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000175:diagnosis* / Q000235:genetics*; D053843:DNA Mismatch Repair / Q000235:genetics*; D045643:DNA Repair Enzymes / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D005260:Female; D015870:Gene Expression / Q000235:genetics; D005820:Genetic Testing; D014644:Genetic Variation; D057809:HEK293 Cells; D006801:Humans; D008214:Lymphocytes / Q000166:cytology; D008297:Male; D008875:Middle Aged; D000070976:Mismatch Repair Endonuclease PMS2; D000070957:MutL Protein Homolog 1; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D012333:RNA, Messenger; D000251:Adenosine Triphosphatases; C094934:PMS2 protein, human; D000070976:Mismatch Repair Endonuclease PMS2; D000070957:MutL Protein Homolog 1; D045643:DNA Repair Enzymes