Genetic Characterization of Protein C Deficiency in Japanese Subjects Using a Rapid and Nonradioactive Method for Single-Strand Conformational Polymorphism Analysis and a Model Building

Abstract

SummaryWe studied the molecular basis of protein C deficiency in 28 Japanese families including 4 asymptomatic families. Two showed a decreased level of function with a normal antigen concentration consistent with type II protein C deficiency and the remaining 26 showed type I deficiency with decreases in both function and antigen level. All the exons and intron/exon junctions of the protein C gene were studied using a strategy combining polymerase chain reaction (PCR) amplification and rapid nonradioactive single-strand conformational polymorphism (SSCP) analysis. The PCR-amplified fragments with aberrant migration on SSCP analysis were sequenced. We identified 11 missense mutations, 1 nonsense mutation, 2 neutral polymorphisms, 1 frameshift deletion, 1 inframe deletion, and 1 splice site mutation. We also identified two different rare mutations in the 5-untranslated region in the protein C gene that may be responsible for the phenotype. Of these molecular defects, ten were novel. From the results of genetic analysis of 47 Japanese families with protein C deficiency reported in this and previous studies, Phel39Val and Met364Ile substitutions and a G8857 deletion were only found in Japanese subjects and seem to be a founder effect. In contrast, Argl69Trp and Val297Met substitutions, both occurring at CG dinucleotides, were commonly observed in not only Japanese but also Western populations, indicating that these are hot spots for mutation in the protein 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 binding 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 other in the three-dimensional model.

Different impact of two mutations of a novel compound heterozygous protein C deficiency with late onset thrombosis

“…However, the PC level of the neonate was only 15% compared to the adults, the first observed onset of thrombosis of the proband with CHPD was at 30 years old, and no symptoms of thromboembotic events at birth had been observed. This condition indicated that the compound heterozygous mutations caused mild PC deficiency, inconsistent with most previous reports of CHPD (Manabe and Matsuda, 1985;Deguchi et al, 1992;Miyata et al, 1996).…”

“…At present, the database of PROC mutations (www.hgmd.cf.ac.uk/ac/gene.php?gene=PROC) has registered 310 mutations or polymorphisms. F139V has been found in the PC deficiency pedigrees of Chinese and Japanese (Miyata et al, 1996;Zhou et al, 2007). D255H has also been reported in patients with PC deficiency and venous thrombosis (Tsay and Shen, 2004).…”

Defective Sorting to Secretory Vesicles in Trans-Golgi Network Is Partly Responsible for Protein C Deficiency

“…[11][12][13][14][15] On the basis of analyses of synthesis rates, posttranslational modifications, and secretion of wild-type PC and mutant PC with single amino acid substitution, we demonstrated that conformational defects resulted in the impaired sorting of abnormal molecules to secretory vesicles in the trans-Golgi network. This impairment may be at least partly responsible for PC deficiency caused by single amino acid substitution.…”

“…In the human PC, Asp71 is modified to a ␤-hydroxylated residue and is thought to be involved in high-affinity calcium ion binding. 10 Many clinical reports have shown that patients with decreased circulating levels of PC develop thrombophilia, [11][12][13][14][15] with venous thrombosis in heterozygotes and purpura fulminans in homozygotes. A wide variety of genetic mutations can lead to PC deficiency and PC molecular abnormality.…”

“…A wide variety of genetic mutations can lead to PC deficiency and PC molecular abnormality. [11][12][13][14][15] We found genetic mutations in three patients with thrombotic episodes and decreased levels of plasma PC. In the present study, we expressed wild-type PC and PC mutants corresponding to the respective genetic abnormalities of each patient to elucidate the molecular mechanisms of the secretion of wild-type PC and impaired secretion of abnormal PC.…”