Genotype establishments for protein C deficiency by use of a DNA polymorphism in the gene.

During the course of structural gene analyses for protein C deficiency, we have confirmed that a T or G nucleotide variation is present at exon 6 of the protein C gene. This single-base substitution was located at the third nucleotide coding for Ser (TCT) at 99 residue, and neither produces an amino acid substitution nor creates a new restriction enzyme site. By using mutagenic primers that could introduce A instead of G at the third nucleotide 3' to the de novo polymorphic site, we have created the polymorphic Xba I site (T/CTAGA) in a more-frequent allele. Polymerase chain reaction using these mutagenic primers and subsequent Xba I digestion of 20 normal Japanese genomic samples showed that the frequency of this new sequence polymorphism designated as PC-493 was 0.18 and that the estimated heterozygosity rate was 28.9%. In Caucasians, the frequency of this polymorphism was 0.25, and a significant difference did not exist between Japanese and Caucasian populations. The examination of the haplotype inter-relationships with PC-493 and the Msp I polymorphism 5' to the protein C gene established that PC-493 gave a 16.7% chance of new information per individual for people who were previously homozygous for the Msp I polymorphism. We have performed a family study of the protein C-deficient pedigree using this sequence polymorphism, and found that the PC-493 DNA polymorphism was a useful marker for tracing the affected gene in protein C-deficient family members.