Homozygous protein C deficiency: identification of a novel missense mutation that causes impaired secretion of the mutant protein C.

We analyzed the promoter region and all the coding exons and exon-intron boundaries of the protein C gene in a Japanese patient with recurrent thromboembolism and complete protein C deficiency. By sequencing these fragments we identified a previously undescribed mutation. A guanine residue was replaced by an adenine residue converting Gly-292 (GGC) to Ser (AGC) in the last exon coding for the catalytic domain. Substitution of this key amino acid, invariably conserved in the serine protease superfamily to which protein C belongs, probably leads to destabilization of the tertiary structure. In a transient expression assay with COS 7 cells, the protein C level was extremely low in the culture medium of the cells transfected with the mutated protein C expression vector, as compared with the normal vector. In contrast, the cell extracts contained similar amounts of mutant and normal protein C, suggesting impaired secretion of the mutant protein C. Using mutagenic primers to introduce a new PvuII site into the mutant allele, we made a study of the family members in this patient's pedigree, revealing that the mutant allele had been inherited in the affected individuals in this pedigree.