Six missense mutations associated with type I and type II protein C deficiency and implications obtained from molecular modelling

• Published in: Blood coagulation & fibrinolysis Vol. 5; no. 5; p. 687
• Main Authors: Zheng, Y Z, Sakata, T, Matsusue, T, Umeyama, H, Kato, H, Miyata, T
• Format: Journal Article
• Language: English
• Published: England 01-10-1994
• Subjects: Adult; Aged; Amino Acid Sequence; Base Sequence; Codon; DNA Restriction Enzymes; Exons; Female; Humans; Introns; Male; Middle Aged; Models, Molecular; Molecular Sequence Data; Mutation; Pedigree; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Protein C - chemistry; Protein C - genetics; Protein C Deficiency; Sequence Analysis, DNA; Thrombosis - enzymology; Thrombosis - genetics
• ISSN: 0957-5235
• DOI: 10.1097/00001721-199410000-00003

The molecular basis of protein C deficiency was studied in three type I and three type II heterozygotes. Three probands showed thrombotic complications. All the exons and intron/exon junctions of the protein C gene were studied using a strategy combining by the polymerase chain reaction (PCR) amplification, single-strand conformational polymorphism (SSCP) analysis, and DNA sequencing of the PCR-amplified fragments. Six missense mutations were identified, including three novel ones. One was located in exon II, in which the initiating translation codon (ATG) encoding for Met at position -42 was replaced by ACG encoding for Thr. 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 occurred in CG dinucleotide. Sequence analysis of the other exons excluded additional mutations. By restriction enzyme analysis, co-segregation of the mutation with protein C deficiency was observed in four families. The other two mutations at amino acid positions -42 and 350 were also considered to be associated with protein C deficiency due to the absence of these mutations in 50 normal individuals. A structural model of the protease domain of mutant activated protein C was constructed by the chimeric modelling method, and the resultant model suggested conformational changes due to each missense mutation identified in protein C deficiency. The present data also provide some evidence regarding the genetic heterogeneity of protein C deficiency.