Vaskulopathiespezifische Manifestationen des Cobalamin C Defekts. Proteinbiochemische Untersuchungen der Auswirkungen einer Deletion des 2. Exons im MMACHC-Gen.
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Philipps-Universität Marburg
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Abstract
Introduction
Cobalamin deficiency type C (CblC) is the most common inborn intracellular cobalamin metabolism error. It is inherited in an autosomal-recessive manner, caused by mutations in the MMACHC gene. Biochemically the multi-systemic disease is characterized by a combined occurrence of methylmalonic aciduria and homocystinuria. Specific MMACHC gene mutations result in erroneous splicing with in-frame exon 2 deletion. Patients with this truncated ΔEx2-MMACHC protein show a phenotypically notable combination of renal thrombotic microangiopathy (rTMA) or an atypical hemolytic uremic syndrome (aHUS) and pulmonary-arterial hypertension (PAH). The objective of this study was 1) to provide a detailed analysis of the relationship between ΔEx2-splice site mutations and the occurance of renal and pulmonary vasculopathies, 2) to characterize the truncated protein in order to learn more about its pathophysiology 3) to test new therapeutical approaches.
Methods
Literature research was conducted using PubMed to identify the genotype- phenotype correlation between ΔEx2-splice site mutations and renal and pulmonary vasculopathies. The truncated protein was characterized on the basis of protein biochemical examinations using Western Blot techniques and immuncytological stainings. The half-life periods of the truncated protein were determined by way of the cycloheximide chase method and a proximity ligation assay was used to identify interaction partners. Effects of cobalamin and homocysteine supplementation were also analysed in HEK293 and HepG2 cell lines.
Results
Eighty-four CblC patients with aHUS/ rTMA and/or PAH and the reported genotype were identified, with more than 65 % of the 37 patients carrying ΔEx2-splice site mutations showing a combined renal and pulmonary phenotype. This was contrasted by five CblC patients who showed no ΔEx2-splice site mutations and renal abnormalities and PAH.
In a comparison between the ΔEx2-MMACHC protein and the wild-type protein, laboratory tests revealed reduced stability, increased proteasomal degradation and a reduced interaction with the partner proteins MMADHC and MSR. The truncated protein was furthermore characterized by the formation of disulfide bonds.
The truncated protein half-life period was increased from 6.4 h to 9.2 h (monomers) or 7.4 h to 10.6 h (dimers) with cyanocobalamine (CN-Cbl) treatment. Adding 100 μM of homocysteine to the cell culture medium resulted in a significant increase in expressed ΔEx2-MMACHC protein, while the wt-MMACHC protein reacted with a significant decrease in protein expression.
Discussion
Literature research clearly revealed a strong genotype-phenotype correlation between ΔEx2-splice site mutations and renal or pulmonary vasculopathies. The combined phenotype in particular seems to be almost pathognomonic for CblC patients with ΔEx2-splice site mutations.
The reduced stability of the truncated protein is most probable of pathophysiological relevance. This was considerably increased by an excessive supply of CN-Cbl. The experimental findings reflect the observed efficacy of treatment with CN-Cbl in six CblC patients with ΔEx2-splice site mutations, thus opening up new treatment options for this group of patients.
The strong tendency towards a formation of disulfide bonds between two ΔEx2- MMACHC proteins might be responsible for the disturbed interaction of the truncated protein with MMADHC and MSR. Furthermore, this distinct disulfide bond formation and the stabilizing effects of homocysteine are an indication that protection against oxidation of Cbl by the truncated protein is impaired. The resulting increase in oxidative stress levels provides a conclusive explanation towards understanding the genesis of vasculopathies in patients with ΔEx2-splice site mutations.
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Dates
Created: 2024Issued: 2024-06-20Updated: 2024-06-20
Faculty
Medizin
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
genetische Erkrankung des intrazellulären Vitamin B 12 StoffwechselsCobalamin C Defektkombiniertes Auftreten einer renalen thrombotischen Mikroangiopathie und pulmonal arteriellen Hypertension bei Patienten mit einer Deletion des 2. Exons im MM
DFG-subjects
pulmonale veno-okklusive ErkrankungCobalamin C Defekt (CblC)MMACHC mit Deletion des 2. Exonspulmonal arterielle Hypertonie (PAH)MMACHC
DDC-Numbers
610
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Hofmann, Jana Katrin: Vaskulopathiespezifische Manifestationen des Cobalamin C Defekts. Proteinbiochemische Untersuchungen der Auswirkungen einer Deletion des 2. Exons im MMACHC-Gen.. : Philipps-Universität Marburg 2024-06-20. DOI: https://doi.org/10.17192/z2024.0210.