title: The usefulness of single-strand DNA conformation polymorphism analysis to detect mutations in protein C deficiency.
abstract: The standard approach for the molecular genetic analysis of protein C deficiency, polymerase chain reaction (PCR) amplification followed by direct sequencing, although very accurate, is time-consuming. The aim of this study is to investigate the usefulness of a simplified, time-saving screening method for the detection of protein C mutations consisting of the combination of multiplex PCR amplifications using the same primers that were designed for sequencing, followed by single-strand DNA conformation polymorphism (SSCP) electrophoresis analysis performed with one set of conditions. The study was designed in two phases. First, we tested six known point mutations located in different exons of the protein C gene by SSCP. Second, we prospectively studied nine patients with protein C deficiency type I using SSCP as the first screening technique. All the exons were amplified with a common PCR protocol, either as single fragments or as multiplex combinations of several of them. In the retrospective study, three out of the six point mutations were visible as a band shift: 40 T-->G (exon 2), 1432 C-->T (exon 3) and 7253 C-->T (exon 8). In the prospective analysis SSCP detected three different mutations. These mutations were: 6128 T-->C (exon 7), 6216 C-->T (exon 7) and in two probands 8631 C-->T (exon 9). In the five remaining patients we identified only two different mutations by direct sequencing: 6246 G-->A (exon 7) in two patients and 8589 G-->A (exon 9) in four patients. In summary, the results from both studies show that only 60% of all mutations can be detected using this simplified method. It also suggests that a multiple set of conditions, smaller PCR fragments, or both, should be used in order to achieve a sensitivity comparable to sequencing.
authors.affiliation: Haemostasis and Thrombosis Research Center, Leiden University Medical Center, The Netherlands.
authors.forename: E
authors.initials: E
authors.lastname: Gómez
authors.forename: S R
authors.initials: SR
authors.lastname: Poort
authors.forename: R M
authors.initials: RM
authors.lastname: Bertina
authors.forename: P H
authors.initials: PH
authors.lastname: Reitsma
mesh_terms: D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D011446:Prospective Studies; D020151:Protein C Deficiency; D012189:Retrospective Studies; D012680:Sensitivity and Specificity