Molecular insights into the comorbidity of vitamin K-dependent clotting factor deficiency and chondrodysplasia punctata.

Background: Autosomal recessive mutations in genes encoding vitamin K cycle enzymes cause hereditary vitamin K-dependent clotting factor deficiency, a disorder characterized by excessive bleeding and a spectrum of nonbleeding phenotypes. While high-dose vitamin K therapy can partially or fully correct coagulopathy, its effect on nonbleeding symptoms is limited.

Objectives: To investigate the molecular basis underlying the differential response to vitamin K therapy, we characterized novel gamma-glutamyl carboxylase (GGCX) mutations identified in a patient with vitamin K-dependent clotting factor deficiency.

Methods: We employed bioluminescent immunoassays, fluorescence confocal imaging, split-nanoluciferase complementation assays, and structural modeling to investigate how GGCX mutations affect its interaction with, and carboxylation of, various vitamin K-dependent proteins (VKDPs) in live cells.

Results: The patient harbored 2 novel compound heterozygous GGCX mutations: c.1760A>G (p.H587R) and c.1787del (p.P596fs). While oral vitamin K improved coagulation deficiency, it failed to correct defects associated with calcification abnormalities. Functional analysis revealed that the P596fs variant abolished enzymatic activity, whereas H587R impaired extrahepatic VKDPs more profoundly than hepatic VKDPs, thereby explaining the distinct clinical responses to vitamin K therapy. The H587R mutation significantly altered GGCX binding to extrahepatic VKDPs, such as the calcification inhibitor matrix Gla protein, while having a lesser effect on hepatic VKDPs. Structural modeling and biochemical characterization further revealed that conserved residues H587 and Y601 form an internal hydrogen bond critical for stabilizing the GGCX molecule.

Conclusion: These findings show how rare patient mutations can provide new insights into the biochemistry of GGCX and how its unique interactions with different VKDPs lead to distinct disease phenotypes.