Protein CVermont: symptomatic type II protein C deficiency associated with two GLA domain mutations

Abstract

This study investigates type II protein C deficiency in a family with manifestations of both arterial and venous thrombosis. Of 64 members of the kindred, 14 have been tested and 7 have PC deficiency. Among affected individuals (n = 7), mean protein C levels by different assays were as follows: enzyme-linked immunosorbent assay (ELISA), 3.8 micrograms/mL (2.1 to 4.3 micrograms/mL); amidolytic with venom activator, 115% (60% to 140%); clotting with venom activator, 42% (23% to 59%). The mean ratio of clotting to amidolytic assays for the affected individuals was 0.37 compared with a normal range of 0.8 to 1.2. Thus, the affected individuals have normal total protein C and their activated protein C has a normal active site assessed by chromogenic substrate; however, they have markedly diminished clotting activity. Immunoassay and chromatography data suggested an abnormality of carboxylation in the gamma carboxyglutamic acid (Gla) domain. Polymerase chain reaction amplification and direct DNA sequencing of exon 2 from genomic DNA of affected individuals showed two nucleotide substitutions. One of the mutations (A----C) results in Glu20----Ala, thereby eliminating a site for vitamin K-dependent gamma-carboxylation. The other substitution (G----A) results in a Val34----Met mutation. DNA sequencing of the other exons from affected individuals has shown no further difference from that of the wild-type gene. The former mutation also removes a Bgl II restriction endonuclease site, which has allowed us to confirm the mutation in affected individuals by direct digestion and Southern hybridization of genomic DNA from family members. This is the first reported family with documented Gla domain mutations in the protein C gene.

Computational analysis of the functional and structural impact of the most deleterious missense mutations in the human Protein C

“…Therefore, any change in these amino acids can affect the regulation of the biological function of the protein. These data are consistent with previous experimental studies that found that the mutants R42S [82], R57W [83,84], E62A [85], V420L [86], and W444C [87] are associated with thrombophilia disorders due to PC deficiency.…”

Laboratory Limitations of Excluding Hereditary Protein C Deficiency by Chromogenic Assay: Discrepancies of Phenotype and Genotype

“…For membrane-bound thrombin–thrombomodulin, activation of PC Gla domain is essential. 46 Thus, Gla-domainless PC could be one explanation for the overestimation of measured PC activity by chromogenic assay, which could be demonstrated in patients receiving vitamin K antagonist (VKA). 47 We found 5 patients (cases 6, 9, 17, 19, and 20) with heterozygous nucleotide exchange affecting the Gla domain (Figure 1).…”

Thrombophilia Screening: Not So Straightforward

“…Consequently, chromogenic assays cannot detect type IIb defects, which are due to mutations affecting the PC Gla domain. 79,[87][88][89] Clotting-based assays can detect all PC deficiency types, although variable sensitivity to the Asn2Ile substitution, causing a type IIb deficiency, has been described. 90 The rarity of type IIb defects helps to justify use of chromogenic assays as first-line tests, although the preponderance of chromogenic assays in routine diagnostic use could explain why these deficiencies are not encountered as often as they might be.…”