title: Validation of a quantitative PCR-high-resolution melting protocol for simultaneous screening of COL1A1 and COL1A2 point mutations and large rearrangements: application for diagnosis of osteogenesis imperfecta.
abstract: Osteogenesis imperfecta (OI) is a connective tissue disorder mostly characterized by autosomal dominant inheritance. Over 1,100 causal mutations have been identified scattered along all exons of genes encoding type I collagen precursors, COL1A1 and COL1A2. Because of the absence of mutational hotspots, Sanger sequencing is considered the gold standard for molecular analysis even if the workload is very laborious and expensive. To overcome this issue, different prescreening methods have been proposed, including DHPLC and biochemical studies on cultured dermal fibroblasts; however, both approaches present different drawbacks. Moreover, in case of patients who screen negative for point mutations, an additional screening step for complex rearrangements is required; the added causative variants expected from this approach are about 1-2%. The aim of this study was to optimize and validate a new protocol that combines quantitative PCR (qPCR) and high-resolution melting (HRM) curve analysis to reduce time and costs for molecular diagnosis. Results of qPCR-HRM screening on 57 OI patients, validated by DHPLC-direct sequencing and multiplex ligation-dependent probe amplification (MLPA), indicate that all alterations identified with the mentioned methodologies are successfully detected by qPCR-HRM. Moreover, HRM was able to discriminate complex genotypes and homozygous variants. Finally, qPCR-HRM outperformed direct sequencing and DHPLC-MLPA in terms of rapidity and costs.
authors: Department of Medical Genetics and Skeletal Rare Diseases, Rizzoli Orthopedic Institute, Bologna, Italy.
authors: Filomena Valentina
authors: FV
authors: Gentile
authors: Monia
authors: M
authors: Zuntini
authors: Alessandro
authors: A
authors: Parra
authors: Luca
authors: L
authors: Battistelli
authors: Martina
authors: M
authors: Pandolfi
authors: Gerard
authors: G
authors: Pals
authors: Luca
authors: L
authors: Sangiorgi
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000191:economics / Q000592:standards; D024042:Collagen Type I / Q000235:genetics*; D000090602:Collagen Type I, alpha 1 Chain; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004252:DNA Mutational Analysis / Q000191:economics / Q000592:standards*; D018628:Gene Dosage; D060005:Genotyping Techniques / Q000191:economics; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000191:economics / Q000592:standards*; D010013:Osteogenesis Imperfecta / Q000175:diagnosis* / Q000235:genetics; D017354:Point Mutation; D060888:Real-Time Polymerase Chain Reaction / Q000191:economics / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results; D044366:Transition Temperature; D014178:Translocation, Genetic
chemical_list: D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D004247:DNA