Functional and Structural Characterization of a Novel PROC Missense Mutation (p.Ser312Trp) in Pediatric Protein C Deficiency
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Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease
Published on: April 4, 2018
Mengxin Yang1, Libing Chen2, Huaxia Xiang1
1Department of Pediatrics, The Second Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China.
Background And Objective:
Hereditary protein C deficiency (HPCD) is an autosomal dominant thrombophilia caused by PROC gene mutations. The genotype-phenotype correlation is complex, and functional characterization of novel variants, especially in pediatric populations, remains limited. This study aimed to characterize the genetic profiles of Chinese pediatric patients with HPCD and to functionally validate the molecular mechanisms of a newly identified PROC missense mutation.
Methods:
Four Chinese pediatric patients with HPCD were enrolled. PROC mutations were identified by high-throughput sequencing. Wild-type (WT) and the p.Ser312Trp mutant expression plasmid were constructed and transfected into HEK293T cells. Intracellular protein expression was confirmed by Western blotting, amidolytic activity was measured by a chromogenic substrate assay, and secreted PC levels were quantified by ELISA. Protein structural modeling was performed using AlphaFold2 and Schrödinger software.
Results:
Six distinct PROC mutations were identified in four patients, including two novel variants: p.Ser312Trp and p.Ser312Ter. Functional characterization was focused on p.Ser312Trp, a novel missense variant. The mutant protein was secreted at levels comparable to WT but exhibited significantly reduced amidolytic activity (79.2% ± 3.1% vs. 100.5% ± 3.9%, p = 0.0017), consistent with a Type II (qualitative) deficiency phenotype. Structural modeling revealed that the bulky tryptophan substitution creates a new π-π stacking interaction with Trp276 and reorganizes the local hydrogen-bond network, causing minimal to mild destabilization (ΔΔG = -0.16 kcal/mol) that may be one of the factors contributing to the observed functional impairment.
Conclusion:
This study provides the first functional and structural characterization of p.Ser312Trp, a novel PROC mutation causing Type II protein C deficiency in a Chinese pediatric patient. Our findings expand the mutational spectrum of HPCD and provide mechanistic insights into how this substitution selectively impairs catalytic function without affecting protein secretion, offering valuable evidence for molecular diagnosis and genetic counseling.
