THE USEFULNESS OF SINGLE-STRAND DNA CONFORMATION POLYMORPHISM ANALYSIS TO DETECT MUTATIONS IN PROTEIN-C DEFICIENCY

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-s aving screening method for the detection of protein C mutations consis ting 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 performe d with one set of conditions. The study was designed in two phases. Fi rst, we tested six known point mutations located in different exons of the protein C gene by SSCP. Second, we prospectively studied nine pat ients with protein C deficiency type I using SSCP as the first screeni ng 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 mutation s were visible as a band shift: 40 T --> G (exon 2), 1432 C --> T (exo n 3) and 7253 C --> T (exon 8). In the prospective analysis SSCP detec ted three different mutations. These mutations were: 6128 --> C (exon 7), 6216 C --> T (exon 7) and in two probands 8631 C --> T (exon 9). I n the five remaining patients we identified only two different mutatio ns by direct sequencing: 6246 G --> A (exon 7) in two patients and 858 9 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 thi s simplified method. It also suggests that a multiple set of condition s, smaller PCR fragments, or both, should be used in order to achieve a sensitivity comparable to sequencing.