6 MISSENSE MUTATIONS ASSOCIATED WITH TYPE-I AND TYPE-II PROTEIN-C DEFICIENCY AND IMPLICATIONS OBTAINED FROM MOLECULAR MODELING

The molecular basis of protein C deficiency was studied in three type I and three type II heterozygotes. Three probands showed thrombotic co mplications. All the exons and intron/exon junctions of the protein C gene were studied using a strategy combining by the polymerase chain r eaction (PCR) amplification, single-strand conformational polymorphism (SSCP) analysis, and DNA sequencing of the PCR-amplified fragments. S ix missense mutations were identified, including three novel ones. One was located in exon II, in which the initiating translation codon (AT G) encoding for Met at position -42 was replaced by ACG encoding for T hr. The other five were located in exon IX, and included TAC(Tyr399)-- >CAC(His), CCG(Pro327)-->CTG(Leu), GAC(Asp359)-->AAC(Asn) in two cases , and GGG(Gly350)-->AGG(Arg). Four of the six missense mutations occur red in CG dinucleotide. Sequence analysis of the other exons excluded additional mutations. By restriction enzyme analysis, co-segregation o f the mutation with protein C deficiency was observed in four families . The other two mutations at amino acid positions -42 and 350 were als o considered to be associated with protein C deficiency due to the abs ence of these mutations in 50 normal individuals. A structural model o f the protease domain of mutant activated protein C was constructed by the chimeric modelling method, and the resultant model suggested conf ormational changes due to each missense mutation identified in protein C deficiency. The present data also provide some evidence regarding t he genetic heterogeneity,of protein C deficiency.