The interplay of differentially γ-carboxylated vitamin K dependent proteins constitute variable VKCFD1 phenotypes
The interplay of differentially γ-carboxylated vitamin K dependent proteins constitute variable VKCFD1 phenotypes
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DissertationPrüfungsdatum
27.10.2021Datum der Veröffentlichung
18.11.2021Erstgutachter
Oldenburg, JohannesZweitgutachter
Imhof, DianaMetadaten
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Inhalt
Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1) is a rare hereditary bleeding disorder caused by mutations in γ-Glutamyl carboxylase (GGCX). VKCFD1 patients are treated with high dose of vitamin K, which corrects the haemostatic phenotype in most cases. In addition, these patients often exhibit non-haemostatic phenotypes such as skin hyperlaxity, skeletal and cardiac abnormalities. These phenotypes are most likely developed due to the undercarboxylation of one or more vitamin K dependent proteins. However, it remained elusive how GGCX mutations affect γ-carboxylation of VKD proteins and which undercarboxylated protein leads to the development of a specific phenotype.
In this PhD thesis, I have used CRISPR/Cas9 engineered GGCX knockout HEK293T cells to evaluate the effect of 22 GGCX missense mutations on γ-carboxylation of 9 different vitamin K dependent proteins with respect to various concentrations of vitamin K. For this evaluation a specific ELISA assay was established, where only functional, γ-carboxylated VKD proteins were detected. Furthermore, a GGCX in silico model was generated, where molecular docking was performed to identify the vitamin K hydroquinone binding site.
All GGCX mutations were categorized into responder and low responder mutations, thereby determining the efficiency of vitamin K supplementation. It was observed that all VKCFD1 patients have at least one vitamin K responsive GGCX allele that is able to γ-carboxylate clotting factors sufficient for a viable phenotype. For non-haemostatic phenotypes, the findings highlight that the deficiency to γ-carboxylate Gla-rich protein leads to skin hyper-laxity whereas facial dysmorphologies are caused by reduced γ-carboxylated MGP. Moreover, the vitamin K hydroquinone binding site in GGCX was identified, where mutations within this site severely affect γ-carboxylation efficiency.
With these new structural and functional data, the clinical outcome of each VKCFD1 genotype can be predicted thus enabling optimized treatment strategies.
In this PhD thesis, I have used CRISPR/Cas9 engineered GGCX knockout HEK293T cells to evaluate the effect of 22 GGCX missense mutations on γ-carboxylation of 9 different vitamin K dependent proteins with respect to various concentrations of vitamin K. For this evaluation a specific ELISA assay was established, where only functional, γ-carboxylated VKD proteins were detected. Furthermore, a GGCX in silico model was generated, where molecular docking was performed to identify the vitamin K hydroquinone binding site.
All GGCX mutations were categorized into responder and low responder mutations, thereby determining the efficiency of vitamin K supplementation. It was observed that all VKCFD1 patients have at least one vitamin K responsive GGCX allele that is able to γ-carboxylate clotting factors sufficient for a viable phenotype. For non-haemostatic phenotypes, the findings highlight that the deficiency to γ-carboxylate Gla-rich protein leads to skin hyper-laxity whereas facial dysmorphologies are caused by reduced γ-carboxylated MGP. Moreover, the vitamin K hydroquinone binding site in GGCX was identified, where mutations within this site severely affect γ-carboxylation efficiency.
With these new structural and functional data, the clinical outcome of each VKCFD1 genotype can be predicted thus enabling optimized treatment strategies.
Klassifikation (DDC)
570 Biowissenschaften, BiologieGhosh, Suvoshree: The interplay of differentially γ-carboxylated vitamin K dependent proteins constitute variable VKCFD1 phenotypes. - Bonn, 2021. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-64490
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-64490
@phdthesis{handle:20.500.11811/9413,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-64490,
author = {{Suvoshree Ghosh}},
title = {The interplay of differentially γ-carboxylated vitamin K dependent proteins constitute variable VKCFD1 phenotypes},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2021,
month = nov,
note = {Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1) is a rare hereditary bleeding disorder caused by mutations in γ-Glutamyl carboxylase (GGCX). VKCFD1 patients are treated with high dose of vitamin K, which corrects the haemostatic phenotype in most cases. In addition, these patients often exhibit non-haemostatic phenotypes such as skin hyperlaxity, skeletal and cardiac abnormalities. These phenotypes are most likely developed due to the undercarboxylation of one or more vitamin K dependent proteins. However, it remained elusive how GGCX mutations affect γ-carboxylation of VKD proteins and which undercarboxylated protein leads to the development of a specific phenotype.
In this PhD thesis, I have used CRISPR/Cas9 engineered GGCX knockout HEK293T cells to evaluate the effect of 22 GGCX missense mutations on γ-carboxylation of 9 different vitamin K dependent proteins with respect to various concentrations of vitamin K. For this evaluation a specific ELISA assay was established, where only functional, γ-carboxylated VKD proteins were detected. Furthermore, a GGCX in silico model was generated, where molecular docking was performed to identify the vitamin K hydroquinone binding site.
All GGCX mutations were categorized into responder and low responder mutations, thereby determining the efficiency of vitamin K supplementation. It was observed that all VKCFD1 patients have at least one vitamin K responsive GGCX allele that is able to γ-carboxylate clotting factors sufficient for a viable phenotype. For non-haemostatic phenotypes, the findings highlight that the deficiency to γ-carboxylate Gla-rich protein leads to skin hyper-laxity whereas facial dysmorphologies are caused by reduced γ-carboxylated MGP. Moreover, the vitamin K hydroquinone binding site in GGCX was identified, where mutations within this site severely affect γ-carboxylation efficiency.
With these new structural and functional data, the clinical outcome of each VKCFD1 genotype can be predicted thus enabling optimized treatment strategies.},
url = {https://hdl.handle.net/20.500.11811/9413}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-64490,
author = {{Suvoshree Ghosh}},
title = {The interplay of differentially γ-carboxylated vitamin K dependent proteins constitute variable VKCFD1 phenotypes},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2021,
month = nov,
note = {Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1) is a rare hereditary bleeding disorder caused by mutations in γ-Glutamyl carboxylase (GGCX). VKCFD1 patients are treated with high dose of vitamin K, which corrects the haemostatic phenotype in most cases. In addition, these patients often exhibit non-haemostatic phenotypes such as skin hyperlaxity, skeletal and cardiac abnormalities. These phenotypes are most likely developed due to the undercarboxylation of one or more vitamin K dependent proteins. However, it remained elusive how GGCX mutations affect γ-carboxylation of VKD proteins and which undercarboxylated protein leads to the development of a specific phenotype.
In this PhD thesis, I have used CRISPR/Cas9 engineered GGCX knockout HEK293T cells to evaluate the effect of 22 GGCX missense mutations on γ-carboxylation of 9 different vitamin K dependent proteins with respect to various concentrations of vitamin K. For this evaluation a specific ELISA assay was established, where only functional, γ-carboxylated VKD proteins were detected. Furthermore, a GGCX in silico model was generated, where molecular docking was performed to identify the vitamin K hydroquinone binding site.
All GGCX mutations were categorized into responder and low responder mutations, thereby determining the efficiency of vitamin K supplementation. It was observed that all VKCFD1 patients have at least one vitamin K responsive GGCX allele that is able to γ-carboxylate clotting factors sufficient for a viable phenotype. For non-haemostatic phenotypes, the findings highlight that the deficiency to γ-carboxylate Gla-rich protein leads to skin hyper-laxity whereas facial dysmorphologies are caused by reduced γ-carboxylated MGP. Moreover, the vitamin K hydroquinone binding site in GGCX was identified, where mutations within this site severely affect γ-carboxylation efficiency.
With these new structural and functional data, the clinical outcome of each VKCFD1 genotype can be predicted thus enabling optimized treatment strategies.},
url = {https://hdl.handle.net/20.500.11811/9413}
}
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