[Analysis of phenotypes and genetic mutations in two pedigrees affected with hereditary protein C deficiency]

To explore the pathogenesis of protein C deficiency in two pedigrees through mutation detection and model analysis.

Chromogenic substrate method and enzyme linked immunosorbent assay (ELISA) were used to determine the plasma protein C activity (PC: A) and protein C antigen (PC: Ag) in the two probands and their family members. All of the 9 exons and intron-exon boundaries of the PROC gene were amplified by PCR and analyzed with Sanger sequencing after purification. Corresponding mutate sites of the family members were also amplified and sequenced. The PolyPhen-2 software was used to analyze the perniciousness of the mutations and Clustal X was to analyze the conservatism. The protein model and amino acids interaction of the mutations were analyzed by Swiss-PdbViewer software.

The PC: A and PC: Ag of proband 1 was 30% and 35%, while PC:A of his father, mother and aunt were all slightly under the reference range. Two heterozygous missense mutations were found in exons 7 and 5 of the PROC gene, namely c.565 C>T (p.Arg147Trp) and c.383 G>A (p.Gly86Asp). His father and aunt were carriers for c.565 C>T, while his mother had carried c.383 G>A. The PC: A of proband 2 and his son were 50% and 64%, respectively. And they were both positive for p.Arg147Trp. Analysis of PolyPhen-2 indicated that p.Arg147Trp was benign, while p.Gly86Asp was damaging. Clustal X analysis indicated that the p.Arg147Trp was non-conservative, while the p.Gly86Asp was highly conservative. Modeling for the mutant proteins revealed that the simple aromatic ring of Trp147 in p.Arg147Trp destroyed the two hydrogen bonds between Arg147-Lys146 and Arg147-Lys151, and steric hindranted with Arg178. The side chain of Asp86 extended and generated steric clash with Gln90 with the occurrence of p.Gly86Asp. The change of hydrogen bonds and steric effects has altered the spatial configuration of amino acids, which led to unstable mutate proteins and interfered with the secretion.

Both probands had hereditary protein C deficiencies, for which their parents were all carriers. The heterozygous mutations p.Arg147Trp and p.Gly86Asp were the main cause for PC: A activity decrease. Among these, p.Gly86Asp was discovered for the first time.