title: Combined use of MLPA and nonfluorescent multiplex PCR analysis by high performance liquid chromatography for the detection of genomic rearrangements.
abstract: Large genomic rearrangements are recognized as playing a pathogenic role in an increasing number of human genetic diseases. It is important to develop efficient methods for the routine detection and confirmation of these germline defects. Multiplex ligation-dependent probe amplification (MLPA) is considered an early step for molecular diagnosis of several genetic disorders. However, artifacts might hamper the interpretation of MLPA analysis, especially when rearrangements involve a single exon. Therefore, rearrangements must be verified by two independent methods. In this study, we developed nonfluorescent multiplex-PCR coupled to high-performance liquid chromatography (NFMP-HPLC) and analyzed whether the use of this method combined with MLPA could be helpful in the detection and confirmation of gross MSH2 and MLH1 genomic rearrangements. A total of nine nonfluorescent multiplex-PCRs were developed to analyze the 16 MSH2 and 19 MLH1 exons. Reliable multiplex amplifications and nonfluorescent peak quantitation were obtained with a limited number of cycles (< or = 25) using a denaturing HPLC (DHPLC) instrument under nondenaturing conditions. The results obtained by NFMP-HPLC were highly reproducible. The combined use of MLPA and NFMP-HPLC identified and independently confirmed putative MLH1 and MSH2 deletions in eight out of 50 unrelated patients with hereditary nonpolyposis colorectal cancer (HNPCC). In five cases, the deletions affected a single exon and in three cases multiple contiguous exons. These results were in agreement with breakpoint and complementary DNA (cDNA) analyses. Considering that MLPA and NFMP-HPLC are unlikely to be affected by the same artifacts, their combined use could also provide a robust and cost-effective strategy for routine screening and confirmation of putative rearrangements in other genes, especially when a single exon is involved or a precise characterization of breakpoints is not achieved.
authors.affiliation: Department of Oncology and Neurosciences, University G. D'Annunzio, Chieti, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: De Lellis
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Curia
authors.forename: Teresa
authors.initials: T
authors.lastname: Catalano
authors.forename: Simona
authors.initials: S
authors.lastname: De Toffol
authors.forename: Chiara
authors.initials: C
authors.lastname: Bassi
authors.forename: Cristina
authors.initials: C
authors.lastname: Mareni
authors.forename: Lucio
authors.initials: L
authors.lastname: Bertario
authors.forename: Pasquale
authors.initials: P
authors.lastname: Battista
authors.forename: Renato
authors.initials: R
authors.lastname: Mariani-Costantini
authors.forename: Paolo
authors.initials: P
authors.lastname: Radice
authors.forename: Alessandro
authors.initials: A
authors.lastname: Cama
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins / Q000235:genetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004252:DNA Mutational Analysis / Q000379:methods; D005091:Exons / Q000235:genetics; D017353:Gene Deletion; D015321:Gene Rearrangement / Q000235:genetics*; D006801:Humans; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C501486:MLH1 protein, human; D009687:Nuclear Proteins; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein