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Clinical and functional characterization of rare compound heterozygous mutations in the SERPINC1 gene causing severe
Ke Zhang1, Haiyue Zhang2, Dandan Yu1
1Department of Clinical Laboratory, Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.
Insights
Hereditary antithrombin (AT) deficiency in a patient resulted from compound heterozygous mutations in the SERPINC1 gene. One mutation caused AT protein degradation, while the other led to reduced mRNA, both causing type I AT deficiency.
Area of Science:
- Genetics
- Molecular Biology
- Hematology
Background:
- Hereditary antithrombin (AT) deficiency is a rare, heterogeneous autosomal dominant disorder.
- Identifying the genetic basis of AT deficiency is crucial for understanding thrombosis risk.
Observation:
- A patient with recurrent venous thrombosis presented with type I deficiency (33% AT activity and antigen).
- Genetic analysis revealed compound heterozygous mutations: c.318_319insT (p.Asn107*) and c.922G>T (p.Gly308Cys) in the SERPINC1 gene.
Findings:
- The p.Gly308Cys mutation altered local hydrophobicity, leading to proteasome-dependent AT protein degradation.
- The c.318_319insT mutation triggered nonsense-mediated mRNA decay, significantly reducing AT mRNA levels.
- Both mutations contributed to type I AT deficiency and were conserved across species, impacting thrombin generation.
Implications:
- These findings elucidate the molecular mechanisms underlying AT deficiency in this patient.
- Understanding mutation-specific effects on AT protein and mRNA levels aids in predicting clinical phenotypes.
- This study highlights the importance of comprehensive genetic and functional analysis in diagnosing rare bleeding disorders.
Introduction:
Hereditary antithrombin (AT) deficiency is a rare autosomal dominant disorder with significant clinical heterogeneity. In the study, we identified a patient with AT deficiency caused by compound heterozygous mutations in the SERPINC1 gene.
Methods:
A total of 9 individuals from three generations were investigated. The mutations were identified by direct sequencing of SERPINC1. Multiple in silico tools were programmed to predict the conservation of mutations and the effect on the AT structure. The coagulation state was evaluated by the thrombin generation assay. Recombinant AT was overexpressed in HEK293T cells; the mRNA level was determined using RT-qPCR. Western blotting, ELISA, and immunocytofluorescence were applied to characterize the recombinant AT protein.
Results:
The proband was a 26-year-old male who experienced recurrent venous thrombosis. He presented the type I deficiency with 33 % AT activity and a synchronized decrease in AT antigen. Genetic screening revealed that he carried a heterozygous c.318_319insT (p.Asn107*) in exon 2 and a heterozygous c.922G > T (p.Gly308Cys) in exon 5, both of which were completely conserved in homologous species and resulted in enhanced thrombin generation capability. Hydrophobicity analysis suggested that the p.Gly308Cys mutation may interfere with the hydrophobic state of residues 307-313. In vitro expression studies indicated that the levels of the recombinant protein AT-G308C decreased to 46.98 % ± 2.94 % and 41.35 % ± 1.48 % in transfected cell lysates and media, respectively. After treatment with a proteasome inhibitor (MG132), the quantity of AT-G308C protein in the cytoplasm was replenished to a level comparable to that of the wild type. The mRNA level of AT-N107* was significantly reduced and the recombinant protein AT-N107* was not detected in either the lysate or the culture media.
Conclusion:
These two mutations were responsible for the AT defects and clinical phenotypes of the proband. The p.Gly308Cys mutation could lead to proteasome-dependent degradation of the AT protein in the cytoplasm by altering local residue hydrophobicity. The c.318_319insT could eliminate aberrant transcripts by triggering nonsense-mediated mRNA degradation. Both mutations resulted in type I AT deficiency.
