Functional and Structural Characterization of a Novel PROC Missense Mutation (p.Ser312Trp) in Pediatric Protein C Deficiency
Mengxin Yang, Libing Chen, Huaxia Xiang, Jiali Jiang, Yuping Li, Zhihao Lin, Changjin He, Tao Xie, Hanying Nong, Jialiang Liao, Hongying WeiABSTRACT
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 (ΔΔ G = −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.