GGCX mutations show different responses to vitamin K thereby determining the severity of the hemorrhagic phenotype in VKCFD1 patients

Ghosh, Suvoshree; Kraus, Katrin; Biswas, Arijit; Müller, Jens; Buhl, A; Forin, Francesco; Singer, Heike; Höning, Klara; Hornung, Veit; Watzka, Matthias; Czogalla‐Nitsche, Katrin J.; Oldenburg, Johannes

doi:10.1111/jth.15238

Cited by 14 publications

(8 citation statements)

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“…Moreover, RSVs in ABCC6 and GGCX have been reported in a family with PXE without coagulation disorder (6). Blood coagulation tests performed on our patient did not reveal decreased levels of vitamin K-dependent clotting factors, consistently with the observation that a single RSV on GGCX gene is not sufficient to reduce the clotting factor activity (18). Our data support the occurrence of a GGCX and ABCC6 PXE digenic inheritance (6).…”

Section: Discussionsupporting

confidence: 87%

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confidence: 87%

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“…These findings suggests that only the biallelic deficiency to γ-carboxylate GRP lead to a PXE-like phenotype, and brings us to the conclusion that the two patients being heterozygous for either GGCX:p.(R476C) or GGCX:p.(R476H) must have an additional defect because of the unusual early age of onset. Since the reported genotype is also not fitting to the haemorrhagic phenotype as already observed in a previous study (Ghosh et al, 2021), we recommend performing next generation sequencing to potentially identify additional variants in GGCX or other genes.…”

Section: Discussionmentioning

confidence: 97%

“…The GGCX wt/mut together with VKD proteins in a bicistronic vector were expressed in a GGCX -/-HEK293T cell line as previously described (Ghosh et al, 2021). Four hours post transfection cells were treated with K 1 .…”

Section: μεασυρεμεντ οφ γ-ςαρβοξψλατιον οφ νον-ηαεμοστατις ῞κδ προτεινςmentioning

confidence: 99%

GGCX variants leading to biallelic deficiency to γ-carboxylate GRP cause skin laxity in VKCFD1 patients

Ghosh¹,

Kraus²,

Biswas

et al. 2021

Preprint

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γ-Glutamyl carboxylase (GGCX) catalyses γ-carboxylation of 15 different vitamin K dependent (VKD) proteins. Pathogenic variants in GGCX cause a rare hereditary bleeding disorder called Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1). In addition to bleedings, some VKCFD1 patients develop skin laxity and skeletal dysmorphologies. However, the pathophysiological mechanisms underlying these non-haemorrhagic phenotypes remain elusive. Therefore, we analyzed the effect of 22 GGCX pathogenic variants on γ-carboxylation of six non-haemostatic VKD proteins (UCMA/GRP, MGP, BGLAP, GAS6, PRGP1, TMG4) in a GGCX-/- HEK293T cell line by a functional ELISA. We observed that biallelic deficiency to γ-carboxylate Gla-rich protein lead to the development of skin laxity. Markedly reduced level of γ-carboxylated MGP is crucial but not exclusive for causing facial dysmorphologies. Moreover, we identified the vitamin K hydroquinone binding site in GGCX in an in silico model by docking studies, which was further validated by functional assays. Variants affecting this site result into loss-of-function or severely diminished ability to γ-carboxylate VKD proteins and hence are involved in the most severe phenotypes. This genotype-phenotype analysis will help to develop new treatment options for VKCFD1 patients, where individualized therapy with γ-carboxylated VKD proteins may represent a promising strategy.

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“…CRISPR/CAS9 gene‐editing technology was used to generate a GGCX −/− HEK293T cell line to eliminate endogenous GGCX activity as previously described (Ghosh et al, 2021). In this GGCX −/− HEK293T cell line, pIRES vectors harboring cDNAs of GGCX wt or variants were transfected together with one myc‐tagged VKD protein.…”

Section: Methodsmentioning

confidence: 99%

GGCX variants leading to biallelic deficiency to γ‐carboxylate GRP cause skin laxity in VKCFD1 patients

Ghosh¹,

Kraus²,

Biswas³

et al. 2021

Human Mutation

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γ‐Glutamyl carboxylase (GGCX) catalyzes the γ‐carboxylation of 15 different vitamin K dependent (VKD) proteins. Pathogenic variants in GGCX cause a rare hereditary bleeding disorder called Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1). In addition to bleedings, some VKCFD1 patients develop skin laxity and skeletal dysmorphologies. However, the pathophysiological mechanisms underlying these non‐hemorrhagic phenotypes remain elusive. Therefore, we have analyzed 20 pathogenic GGCX variants on their ability to γ‐carboxylate six non‐hemostatic VKD proteins in an in vitro assay, where GGCX variants were expressed in GGCX‐/‐ cells and levels of γ‐carboxylated co‐expressed VKD proteins were detected by a functional ELISA. We observed that GGCX variants causing markedly reduced γ‐carboxylation of Gla rich protein (GRP) in vitro were reported in patients with skin laxity. Reduced levels of γ‐carboxylated Matrix gla protein (MGP) are not exclusive for causing skeletal dysmorphologies in VKCFD1 patients. In silico docking of vitamin K hydroquinone on a GGCX model revealed a binding site, which was validated by in vitro assays. GGCX variants affecting this site result in disability to γ‐carboxylate VKD proteins and hence are involved in the most severe phenotypes. This genotype‐phenotype analysis will help to understand the development of non‐hemorrhagic phenotypes and hence improve treatment in VKCFD1 patients.

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GGCX mutations show different responses to vitamin K thereby determining the severity of the hemorrhagic phenotype in VKCFD1 patients

Abstract: This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Cited by 14 publications

References 46 publications

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GGCX variants leading to biallelic deficiency to γ-carboxylate GRP cause skin laxity in VKCFD1 patients

GGCX variants leading to biallelic deficiency to γ‐carboxylate GRP cause skin laxity in VKCFD1 patients

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