Scanning method to establish the molecular basis of protein C deficiencies

We describe a scanning procedure for the detection of protein C gene mutations and polymorphisms. The method is based on a combination of polymerase chain reaction (PCR) and denaturant gradient gel electrophoresis (DGGE) of 13 amplified fragments that cover exon I and most of the protein C coding regions. Exons IV and V are studied by routine direct sequencing. To validate our experimental conditions, and to verify that we were able to detect any point mutation in the fragments studied by DGGE, we tested a series of selected DNAs in which mutations had already been identified by another method. In addition, we studied the protein C gene of patients with qualitative deficiencies of protein C. In both instances, we detected all the causal mutations. We also present data on the detection of the three frequent neutral Caucasian polymorphisms in the protein C gene and on five novel mutations identified using the strategy described. These results show that DGGE is an efficient tool for establishing the molecular basis of hereditary protein C deficiencies.