Gerrit Jan Romeijn

Mutations in MVK, encoding mevalonate kinase, cause hyperimmunoglobulinaemia D and periodic fever syndrome

Hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS; MIM 260920) is an autosomal recessive disorder characterized by recurrent episodes of fever associated with lymphadenopathy, arthralgia, gastrointestinal dismay and skin rash. Diagnostic hallmark of HIDS is a constitutively elevated level of serum immunoglobulin D (IgD), although patients have been reported with normal IgD levels. To determine the underlying defect in HIDS, we analysed urine of several patients and discovered increased concentrations of mevalonic acid during severe episodes of fever, but not between crises. Subsequent analysis of cells from four unrelated HIDS patients revealed reduced activities of mevalonate kinase (MK; encoded by the gene MVK), a key enzyme of isoprenoid biosynthesis. Sequence analysis of MVK cDNA from the patients identified three different mutations, one of which was common to all patients. Expression of the mutant cDNAs in Escherichia coli showed that all three mutations affect the activity of the encoded proteins. Moreover, immunoblot analysis demonstrated a deficiency of MK protein in patient fibroblasts, indicating a protein-destabilizing effect of the mutations.

Mutations in the 3β-hydroxysterol Δ24-reductase gene cause desmosterolosis, an autosomal recessive disorder of cholesterol biosynthesis

Desmosterolosis is a rare autosomal recessive disorder characterized by multiple congenital anomalies. Patients with desmosterolosis have elevated levels of the cholesterol precursor desmosterol, in plasma, tissue, and cultured cells; this abnormality suggests a deficiency of the enzyme 3β-hydroxysterol Δ24-reductase (DHCR24), which, in cholesterol biosynthesis, catalyzes the reduction of the Δ24 double bond of sterol intermediates. We identified the human DHCR24 cDNA, by the similarity between the encoded protein and a recently characterized plant enzyme—DWF1/DIM, from Arabidopsis thaliana—catalyzing a different but partially similar reaction in steroid/sterol biosynthesis in plants. Heterologous expression, in the yeast Saccharomyces cerevisiae, of the DHCR24 cDNA, followed by enzyme-activity measurements, confirmed that it encodes DHCR24. The encoded DHCR24 protein has a calculated molecular weight of 60.1 kD, contains a potential N-terminal secretory-signal sequence as well as at least one putative transmembrane helix, and is a member of a recently defined family of flavin adenine dinucleotide (FAD)–dependent oxidoreductases. Conversion of desmosterol to cholesterol by DHCR24 in vitro is strictly dependent on reduced nicotinamide adenine dinucleotide phosphate and is increased twofold by the addition of FAD to the assay. The corresponding gene, DHCR24, was identified by database searching, spans ∼46.4 kb, is localized to chromosome 1p31.1-p33, and comprises nine exons and eight introns. Sequence analysis of DHCR24 in two patients with desmosterolosis revealed four different missense mutations, which were shown, by functional expression, in yeast, of the patient alleles, to be disease causing. Our data demonstrate that desmosterolosis is a cholesterol-biosynthesis disorder caused by mutations in DHCR24.

Organization of the mevalonate kinase ( MVK ) gene and identification of novel mutations causing mevalonic aciduria and hyperimmunoglobulinaemia D and periodic fever syndrome

Mevalonic aciduria (MA) and hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS) are two autosomal recessive inherited disorders both caused by a deficient activity of the enzyme mevalonate kinase (MK) resulting from mutations in the encoding MVK gene. Thus far, disease-causing mutations only could be detected by analysis of MVK cDNA. We now describe the genomic organization of the human MVK gene. It is 22 kb long and contains 11 exons of 46 to 837 bp and 10 introns of 379 bp to 4.2 kb. Three intron-exon boundaries were confirmed from natural splice variants, indicating the occurrence of exon skipping. Sequence analysis of 27 HIDS and MA patients confirmed all previously reported genotypes based on cDNA analysis and identified six novel nucleotide substitutions resulting in missense or nonsense mutations, providing new insights in the genotype/phenotype relation between HIDS and MA.

Mevalonic aciduria in 12 unrelated patients with hyperimmunoglobulinaemia D and periodic fever syndrome

Mevalonic aciduria is an inborn error of cholesterol and nonsterol isoprene biosynthesis due to mevalonate kinase deficiency (MKD; McKusick 251170). Urinary excretion of mevalonate is massively increased. Clinical manifestations include psychomotor retardation, hypotonia, dysmorphic features, failure to thrive, cataracts and hepatosplenomegaly. In addition to the multisystemic symptoms, patients present recurrent febrile attacks accompanied by adenopathy, arthralgias, rash and diarrhoea. These recurrent episodes of unexplained high fever with the associated features are also a characteristic finding of hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS), a relatively benign condition in which elevated immunoglobulin D (IgD) itself appears not to be causative. The febrile episodes in HIDS have an average duration of 24 h to 7 days, varying frequency, and a tendency to decrease in both numbers and severity of the attacks with age. We report 12 HIDS patients in whom a minimally increased mevalonate excretion during febrile episodes appeared to be related to mevalonate kinase (MK) deficiency resulting from mutations in the gene encoding mevalonate kinase. (Less)

Peroxisome Deficiency Does Not Result in Deficiency of Enzymes involved in Cholesterol Biosynthesis

To unravel conflicting literature data on the dependence of cholesterol biosynthesis on functional peroxisomes, we have determined activities and levels of selected cholesterol biosynthetic enzymes in livers of a PEX5 knock-out mouse, a well-characterized animal model for human Zellweger syndrome. All enzymes measured, including putative peroxisomal enzymes, were normally active in the peroxisome-deficient mice indicating that mislocalization of these enzymes to the cytosol does not lead to decreased activity or degradation. Since these findings were in apparent contrast to previously reported data in human livers, we re-examined cholesterol biosynthetic enzymes in livers of Zellweger patients and control subjects. We found that the previously reported deficient enzymes mevalonate kinase and phosphomevalonate kinase are inactivated rapidly in a time-and temperature-dependent manner. This inactivation is not due to degradation as a result of mislocalization since normal levels of protein can be detected. Therefore, the deficiencies of these enzymes in livers of Zellweger patients reflect the bad condition of the livers, rather than mislocalization to the cytosol. Our data explain the conflicting findings in literature and demonstrate that great care should be taken in the interpretation of data obtained in postmortem material (1). To exclude that genetic differences, resulting in different defects in peroxisomal biogenesis, might influence the activity of selected cholesterol biosynthetic enzymes, we also have determined activities and levels of these enzymes in human fibroblasts of patients belonging to various different complementation groups. We included fibroblasts of patients with mutations in genes encoding PEX1, PEX5, PEX7, PEX16, and PEX19, resulting in selective deficiencies in import of PTS1 or PTS2 containing proteins or in a more general defect in peroxisome biogenesis resulting in the complete absence of peroxisomes or peroxisomal ghosts. We measured normal protein levels and activities of enzymes involved in cholesterol biosynthesis in all cell lines. We conclude that peroxisomes are not a prerequisite for cholesterol biosynthesis in humans.

Erratum: Organization of the mevalonate kinase (MVK) gene and identification of novel mutations causing mevalonic aciduria and hyperimmunoglobulinaemia D and periodic fever syndrome (European Journal of Human Genetics (2001) vol. 9 (253-259))

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