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

Abstract

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.

Protein C Deficiency: A Database of Mutations

“…Three single base-pair substitutions have been found in the putative promoter region of the protein C gene. Two (nt-1533 and nt-1528) occur within an AAATA sequence located between 34 and 29 bp upstream of the transcriptionai initiation site at -L50L.…”

A Quantitative Protein S Deficiency Associated with a Novel Nonsense Mutation and Markedly Reduced Levels of Mutated mRNA

“…A mutagenic primer PSI4R (.V-AATACTGTAATATATCGGGT-3') was used to introduce a new Rsal site al the position of the base substitution (Fig. 2) (13,26).…”

A case of purpura fulminans is caused by homozygous Δ8857 mutation (protein C‐Nagoya) and successfully treated with activated protein C concentrate

“…Protein C Plasma protein C activity was determined using protein C‐deficient plasma as a substrate and Protac (Boehringer Mannheim) as an activator of protein C. Total protein C antigen level was measured using an enzyme‐linked immunosorbent assay (ELISA) kit using a polyclonal rabbit antibody for human protein C (Boehringer Mannheim). For the initial laboratory tests of samples from the patient and parents, protein C activity and antigen levels were expressed as a percentage of those in pooled plasma obtained from 20 normal individuals containing 100% of protein C ( Yamamoto et al , 1991 ). The APC concentrate was affinity‐purified from human plasma, as described elsewhere ( Katsuura et al , 1994 ) .…”

“…DNA analysis DNA was isolated from peripheral blood leucocytes. The PCR reaction for the protein C gene was performed as described ( Yamamoto et al, 1991). The PCR products were cloned using the TA‐cloning method and were subjected to the sequence analysis by ABI PRISM 310.…”

“…The PCR products were cloned using the TA‐cloning method and were subjected to the sequence analysis by ABI PRISM 310. A PCR method using the mutagenic primer pairs has been described earlier ( Yamamoto et al , 1991 ).…”