Jin Da-Yun, Chen Xuejie, Wang Mengying, Qi Xiaofeng, Stafford Darrel W, Lewis Sara, Weiss Mitchell J, Reiss Ulrike M, Tie Jian-Ke
Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
J Thromb Haemost. 2025 Aug 26. doi: 10.1016/j.jtha.2025.08.011.
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.
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.
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.
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.
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.
编码维生素K循环酶的基因中的常染色体隐性突变会导致遗传性维生素K依赖凝血因子缺乏症，这是一种以出血过多和一系列非出血表型为特征的疾病。虽然高剂量维生素K疗法可以部分或完全纠正凝血病，但其对非出血症状的影响有限。
为了研究对维生素K疗法产生不同反应的分子基础，我们对在一名维生素K依赖凝血因子缺乏症患者中鉴定出的新型γ-谷氨酰羧化酶（GGCX）突变进行了特征分析。
我们采用生物发光免疫测定、荧光共聚焦成像、分裂纳米荧光素酶互补测定和结构建模，以研究GGCX突变如何影响其在活细胞中与各种维生素K依赖蛋白（VKDPs）的相互作用及其羧化作用。
该患者携带2种新型复合杂合GGCX突变：c.1760A>G（p.H587R）和c.1787del（p.P596fs）。虽然口服维生素K改善了凝血缺陷，但未能纠正与钙化异常相关的缺陷。功能分析表明，P596fs变体消除了酶活性，而H587R对肝外VKDPs的损害比对肝内VKDPs更严重，从而解释了对维生素K疗法的不同临床反应。H587R突变显著改变了GGCX与肝外VKDPs（如钙化抑制剂基质Gla蛋白）的结合，而对肝内VKDPs的影响较小。结构建模和生化特征进一步表明，保守残基H587和Y601形成了一个对稳定GGCX分子至关重要的内部氢键。
这些发现表明罕见的患者突变如何能够为GGCX的生物化学提供新的见解，以及其与不同VKDPs的独特相互作用如何导致不同的疾病表型。
