Mevalonate kinase assay using DEAE‐cellulose column chromatography for first‐trimester prenatal diagnosis and complementation analysis in mevalonic aciduria

Hoffmann, Georg F.; Brendel, Susanne; Scharfschwerdt, S. R.; Shin, Yoon S.; Speidel, I. M.; Gibson, K. Michael

doi:10.1007/bf01800016

Cited by 44 publications

(29 citation statements)

References 14 publications

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“…10 Preparation of the lymphoblasts lysates was carried out as described earlier. 11 Mevalonate kinase assay in lymphoblast lysates, including separation of substrate and products by thin layer chromatography, was performed according to Gibson et al 10 Results…”

Section: Mk Enzyme Analysismentioning

confidence: 99%

Molecular analysis of MVK mutations and enzymatic activity in hyper-IgD and periodic fever syndrome

et al. 2001

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Hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS) is an autosomal recessive inflammatory disorder characterised by recurrent episode of fever associated with lymphadenopathy, abdominal distress, joint involvement and skin lesions. We recently demonstrated that mutations in the mevalonate kinase gene (MVK) are associated with HIDS. Direct DNA sequencing was done to screen the entire coding region of MVK in 25 unrelated patients with HIDS. Mutations were detected in the coding region of the gene including 11 missense mutations, one deletion, the absence of expression of one allele, as well as three novel polymorphisms. Seven of these mutations are novel. The large majority of the patients were compound heterozygotes for two mutations. Of these, V377I (G?A) is the most common mutation occurring in 20 unrelated patients and was found to be associated with I268T in six patients. Mutations were associated with a decrease of mevalonate kinase (MK) (ATP:mevalonate 5-phosphotransferase, EC 2.7.I.36) enzymatic activity but not as profound as in mevalonic aciduria, a syndrome also caused by a deficient activity of MK. In HIDS the mutations are located all along the protein which is different from mevalonic aciduria where MK mutations are mainly clustered to a same region of the protein. On the basis of this study, we propose that the diagnostic screen of MVK in HIDS should be first directed on V377I and I268T mutations. Three patients are also described to illustrate the genotypic and phenotypic overlap with mevalonic aciduria. European Journal of Human Genetics (2001) 9, 260 ± 266.

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Section: Mk Enzyme Analysismentioning

confidence: 99%

Molecular analysis of MVK mutations and enzymatic activity in hyper-IgD and periodic fever syndrome

et al. 2001

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“…Phosphomevalonate Kinase Assay-The incubation conditions were the same as for mevalonate kinase except that (R)- [2][3][4][5][6][7][8][9][10][11][12][13][14] C]mevalonic acid-5-phosphate was added at a specific activity of 6,000 dpm/nmol and 50,000 dpm per assay was used. The products were loaded on AG1-X8 formate resin column (3 ml) and washed with 50 ml of 4 N formic acid to elute first the substrate and subsequently with 50 ml of 0.8 M ammonium formate in 4 N formic acid to elute the phosphorylated products (9,14).…”

Section: Materials-biochemicalsmentioning

confidence: 99%

“…Reactions were terminated by scraping cells into Eppendorf tubes and boiling the sample for 3 min. [1-14 C]Isopentenyl diphosphate was added as an internal standard to each sample, and the products were separated on DEAE-cellulose (Cl Ϫ form) columns (8,13).…”

Section: Materials-biochemicalsmentioning

confidence: 99%

Compartmentalization of Cholesterol Biosynthesis

Biardi

Krisans

1996

Journal of Biological Chemistry

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We have recently demonstrated that mevalonate kinase and farnesyl diphosphate (FPP) synthase are localized predominantly in peroxisomes. This observation raises the question regarding the subcellular localization of the enzymes that catalyze the individual steps in the pathway between mevalonate kinase and FPP synthase (phosphomevalonate kinase, mevalonate diphosphate decarboxylase, and isopentenyl diphosphate isomerase). These enzyme are found in the 100,000 ؋ g supernatant fraction of cells or tissues and have been considered to be cytoplasmic proteins. In the current studies, we show that the activities of mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase are equal in extracts prepared from intact cells and selectively permeabilized cells, which lack cytosolic enzymes. We also demonstrate structure-linked latency of phosphomevalonate kinase and mevalonate diphosphate decarboxylase that is consistent with a peroxisomal localization of these enzymes. Finally, we show that cholesterol biosynthesis from mevalonate can occur in selectively permeabilized cells lacking cytosolic components. These results suggest that the peroxisome is the major site of the synthesis of FPP from mevalonate, since all of the cholestrogenic enzymes involved in this conversion are localized in the peroxisome.Recently, it has been demonstrated by our group and others that peroxisomes contain a number of enzymes involved in cholesterol biosynthesis that previously were considered to be cytosolic or located exclusively in the endoplasmic reticulum. Peroxisomes have been shown to contain acetoacetyl-CoA thiolase (1, 2), 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) 1 synthase (3), HMG-CoA reductase (4 -6), mevalonate kinase (7,8), and most recently farnesyl diphosphate (FPP) synthase (9). Both mevalonate kinase and FPP synthase seem to be localized predominantly, if not exclusively, to peroxisomes (8, 9).The demonstration that mevalonate kinase and FPP synthase are localized predominantly in peroxisomes (8, 9) raises the question regarding the localization of the enzymes that catalyze the individual steps in the pathway between mevalonate kinase and FPP synthase (phosphomevalonate kinase, mevalonate diphosphate decarboxylase, and isopentenyl diphosphate isomerase). Based on results obtained from fractionation studies, these enzymes are believed to be localized in the cytosol. However, recent data have shown that the activities of these enzymes as well as mevalonate kinase and FPP synthase are significantly reduced in liver tissue obtained from patients with peroxisome-deficient diseases (Zellweger syndrome and neonatal adrenoleukodystrophy), thus indicating a peroxisomal localization (9).We have routinely employed three different methods to study subcellular localization of proteins: (i) analytical subcellular fractionation and measurements of enzyme activities, (ii) immunoblotting of the protein in the isolated fractions with a monospecific antibody, and (iii) immunoelectron and immunofluorescence micro...

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“…MK activity was measured in leukocytes prepared from 2-3 ml of peripheral blood treated with EDTA. The assay was performed as previously described (14). 14 C-mevalonate-5-phosphate was separated from the substrate 14 C-mevalonic acid on a mini DEAE-cellulose column (15).…”

Section: Methodsmentioning

confidence: 99%

Molecular analysis of the MVK and TNFRSF1A genes in patients with a clinical presentation typical of the hyperimmunoglobulinemia D with periodic fever syndrome: A low‐penetrance TNFRSF1A variant in a heterozygous MVK carrier possibly influences the phenotype of hyperimmunoglobulinemia D with periodic fever syndrome or vice versa

Stojanov¹,

Lohse²,

Lohse³

et al. 2004

Arthritis & Rheumatism

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Objective. To describe biochemical findings and the spectrum of mevalonate kinase (MVK) gene mutations as well as an associated TNFRSF1A lowpenetrance variant in a series of patients with clinical features of the hyperimmunoglobulinemia D with periodic fever syndrome (HIDS).Methods. The MVK gene was sequenced in 8 children and 1 adult (including 2 siblings) fulfilling the clinical criteria for HIDS. In addition, sequencing of exons 2, 3, 4, and 6 of the TNFRSF1A gene was performed in patients with only one or no MVK mutation. Mevalonate kinase (MK) enzyme activity in leukocytes and renal excretion of mevalonic acid were also measured.Results. Mutations in the coding region of the MVK gene were detected in 6 patients, and the most common mutation was V377I. Among these patients were 2 novel mutations, both of which were located in exon 6. These novel mutations resulted in the substitution of tryptophan (TGG) by a stop codon (TGA) at amino acid position 188 (W188X) and in the exchange of valine (GTG) for alanine (GCG) at amino acid position 203 (V203A). In 1 patient, a combination of one MVK (V377I) mutation and one TNFRSF1A (R92Q) mutation was present. The patient's clinical phenotype resembled a mixture of variant-type HIDS and tumor necrosis factor receptor-associated periodic syndrome (TRAPS). Her IgD values varied between normal and slightly increased, and the MK activity was in the low-normal range, while urinary mevalonate concentrations were always normal.Conclusion. The genotype findings indicate that a relatively small number of genes may be involved in the clinical manifestation of HIDS, with low-penetrance TNFRSF1A variants possibly influencing the HIDS phenotype or MVK mutations contributing to TRAPS.The hyperimmunoglobulinemia D with periodic fever syndrome (HIDS; MIM no. 260920) is an autosomal recessively inherited autoinflammatory disease. It is characterized by febrile episodes of 3-7 days' duration recurring every 4-8 weeks and by a persistently high serum level of IgD (Ͼ100 IU/ml). Symptoms accompanying a typical attack comprise cervical lymphadeno-

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Mevalonate kinase assay using DEAE‐cellulose column chromatography for first‐trimester prenatal diagnosis and complementation analysis in mevalonic aciduria

Cited by 44 publications

References 14 publications

Molecular analysis of MVK mutations and enzymatic activity in hyper-IgD and periodic fever syndrome

Molecular analysis of MVK mutations and enzymatic activity in hyper-IgD and periodic fever syndrome

Compartmentalization of Cholesterol Biosynthesis

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