GENETIC-CHARACTERIZATION OF PROTEIN-C DEFICIENCY IN JAPANESE SUBJECTSUSING A RAPID AND NONRADIOACTIVE METHOD FOR SINGLE-STRAND CONFORMATIONAL POLYMORPHISM ANALYSIS AND A MODEL-BUILDING

We studied the molecular basis of protein C deficiency in 25 Japanese families including 4 asymptomatic families. Two showed a decreased lev el of function with a normal antigen concentration consistent with typ e II protein C deficiency and the remaining 26 showed type I deficienc y with decreases in both function and antigen level. All the exons and intron/exon junctions of the protein C gene were studied using a stra tegy combining polymerase chain reaction (PCR) amplification and rapid nonradioactive single-strand conformational polymorphism (SSCP) analy sis, The PCR-amplified fragments with aberrant migration on SSCP analy sis were sequenced. We identified II missense mutations, I nonsense mu tation, 2 neutral polymorphisms, 1 frameshift deletion. I inframe dele tion, and 1 splice site mutation. We also identified two different rar e mutations in the 5'-untranslated region in the protein C gene that m ay be responsible for the phenotype. Of these molecular defects, ten w ere novel. From the results of genetic analysis of 47 Japanese familie s with protein C deficiency reported in this and previous studies. Phe 139Val and Met364IIe substitutions and a G8857 deletion were only foun d in Japanese subjects and seem to be a founder effect. In contrast, A rg169Trp and Val297Met substitutions, both occurring at CG dinucleotid es, were commonly observed in not only Japanese but also Western popul ations, indicating that these ara hot spots for mutation in the protei n C gene. These molecular defects were found in 22 families in total, accounting for 47% of Japanese families with protein C deficiency, The structural models of the second EGF and protease domains of activated wild-type and mutant human protein C suggest a possible substrate bin ding exosite on two loops; one from amino acid position 349 to 357 and the other from position 385 to 388, both of which are close to each o ther in the three-dimensional model.