title: A new strategy to screen MMR genes in Lynch Syndrome: HA-CAE, MLPA and RT-PCR.
abstract: OBJECTIVE
  Hereditary non-polyposis colon cancer (HNPCC) is an autosomal dominant disorder that is genetically heterogeneous because of underlying mutations in mismatch repair (MMR) genes, primarily MLH1, MSH2 and MSH6. One challenge to correctly diagnose HNPCC is that the large size of the causative genes makes identification of mutations both labour intensive and expensive.
  
  
  METHODS
  Our heteroduplex analysis by capillary array electrophoresis (HA-CAE) method, previously developed to increase the throughput and allow other multi-exon genes to be scanned, has been adapted for MMR genes. The altered peak patterns were then sequenced. Furthermore, the mutational scanning was completed using the Multiplex Ligation-Dependent Probe Amplification (MLPA) test in all negative HA-CAE cases, and these results were confirmed by RT-PCR.
  
  
  RESULTS
  We studied 216 individuals belonging to 100 unrelated families that met the Amsterdam I/II criteria for HNPCC. We detected 40 different variants that are classified as follows: 8 (20%) deleterious mutation, 8 (20%) unknown pathogenic significance variants and 24 (60%) coding and intronic sequence variants. Pathogenic mutations were detected in 12% of the families and about 42% of these had a deletion variant. Unknown pathogenic significance variants (UVs) affected 13% of the families. We also found 12.5% of novel polymorphisms in the rest of the variants. CONCLUDING: In short, using a combined method that includes HA-CAE, MLPA and RT-PCR, it is possible to detect the entire mutational spectrum of MMR genes. Twenty percent of the mutations found in the three genes have not been reported before. Relatives at risk will be offered predictive molecular analysis with potential exclusion of non-carriers of mutations.
authors.affiliation: Laboratorio Cáncer Hereditario, IBGM-CSIC, Universidad de Valladolid, C/ Sanz y Forés s/n, 47003 Valladolid, Spain.
authors.forename: Lucia
authors.initials: L
authors.lastname: Perez-Cabornero
authors.forename: Eladio
authors.initials: E
authors.lastname: Velasco
authors.forename: Mar
authors.initials: M
authors.lastname: Infante
authors.forename: David
authors.initials: D
authors.lastname: Sanz
authors.forename: Enrique
authors.initials: E
authors.lastname: Lastra
authors.forename: Lara
authors.initials: L
authors.lastname: Hernández
authors.forename: Cristina
authors.initials: C
authors.lastname: Miner
authors.forename: Mercedes
authors.initials: M
authors.lastname: Duran
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics*; D053843:DNA Mismatch Repair / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D004268:DNA-Binding Proteins / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D015321:Gene Rearrangement; D020022:Genetic Predisposition to Disease; D016678:Genome; D018095:Germ-Line Mutation; D006801:Humans; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C094443:G-T mismatch-binding protein; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein