title: Expression defect size among unclassified MLH1 variants determines pathogenicity in Lynch syndrome diagnosis.
abstract: OBJECTIVE
  Lynch syndrome is caused by a germline mutation in a mismatch repair gene, most commonly the MLH1 gene. However, one third of the identified alterations are missense variants with unclear clinical significance. The functionality of these variants can be tested in the laboratory, but the results cannot be used for clinical diagnosis. We therefore aimed to establish a laboratory test that can be applied clinically.
  
  
  METHODS
  We assessed the expression, stability, and mismatch repair activity of 38 MLH1 missense variants and determined the pathogenicity status of recurrent variants using clinical data.
  
  
  RESULTS
  Four recurrent variants were classified as neutral (K618A, H718Y, E578G, V716M) and three as pathogenic (A681T, L622H, P654L). All seven variants were proficient in mismatch repair but showed defects in expression. Quantitative PCR, pulse-chase, and thermal stability experiments confirmed decreases in protein stability, which were stronger in the pathogenic variants. The minimal cellular MLH1 concentration for mismatch repair was determined, which corroborated that strongly destabilized variants can cause repair deficiency. Loss of MLH1 tumor immunostaining is consistently reported in carriers of the pathogenic variants, showing the impact of this protein instability on these tumors.
  
  
  CONCLUSIONS
  Expression defects are frequent among MLH1 missense variants, but only severe defects cause Lynch syndrome. The data obtained here enabled us to establish a threshold for distinguishing tolerable (clinically neutral) from pathogenic expression defects. This threshold allows the translation of laboratory results for uncertain MLH1 variants into pathogenicity statements for diagnosis, thereby improving the targeting of cancer prevention measures in affected families.
authors: Medizinische Klinik 1, Biomedical Research Laboratory, Johann Wolfgang Goethe-Universität, Frankfurt, Germany.
authors: Inga
authors: I
authors: Hinrichsen
authors: Angela
authors: A
authors: Brieger
authors: Jörg
authors: J
authors: Trojan
authors: Stefan
authors: S
authors: Zeuzem
authors: Mef
authors: M
authors: Nilbert
authors: Guido
authors: G
authors: Plotz
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D017124:Conserved Sequence; D053843:DNA Mismatch Repair; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques; D000070957:MutL Protein Homolog 1; D020125:Mutation, Missense; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D055550:Protein Stability
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D000070957:MutL Protein Homolog 1