Mutations in the 3beta-hydroxysterol Delta24-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 3beta-hydroxysterol Delta24-reductase (DHCR24), which, in cholesterol biosynthesis, catalyzes the reduction of the Delta24 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 approximately 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.

Identification and characterization of three novel missense mutations in mevalonate kinase cDNA causing mevalonic aciduria, a disorder of isoprene biosynthesis.

Mevalonic aciduria is a rare autosomal recessive metabolic disorder, characterized by psychomotor retardation, failure to thrive, hepatosplenomegaly, anemia and recurrent febrile crises. The disorder is caused by a deficient activity of mevalonate kinase due to mutations in the encoding gene. Thus far, only two disease-causing mutations have been identified. We now report four different missense mutations including three novel ones, which were identified by sequence analysis of mevalonate kinase cDNA from three mevalonic aciduria patients. All mutations affect conserved amino acids. Heterologous expression of the corresponding mutant mevalonate kinases as fusion proteins with glutathione S -transferase in Escherichia coli showed a profound effect of each of the mutations on enzyme activity. In addition, immunoblot analysis of fibroblast lysates from patients using specific antibodies against mevalonate kinase identified virtually no protein. These results demonstrate that the mutations affect not only the activity but also the stability of the mutant proteins.

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.

Differential deficiency of mevalonate kinase and phosphomevalonate kinase in patients with distinct defects in peroxisome biogenesis: evidence for a major role of peroxisomes in cholesterol biosynthesis.

Peroxisomes catalyze a number of essential metabolic functions especially related to lipid metabolism. There is increasing evidence suggesting that peroxisomes are also involved in the synthesis of isoprenoids via the mevalonate pathway at least in rat liver. In order to obtain independent evidence for a role of peroxisomes in isoprenoid synthesis in man, we have measured the activity of two key enzymes of the mevalonate pathway in patients suffering from certain defined defects in peroxisome biogenesis. We now report that mevalonate kinase is not only deficient in livers from Zellweger patients in which peroxisome biogenesis is defective, but also in livers from rhizomelic chondrodysplasia punctata (RCDP) Type 1 patients. In the latter group of patients there is a selective defect in peroxisome biogenesis due to a genetic defect in the PTS2-receptor, a mobile receptor-protein guiding peroxisomal proteins with a certain peroxisomal targeting signal (PTS2) to the peroxisome. Phosphomevalonate kinase was found to be strongly deficient in Zellweger patients thus suggesting that this enzyme is also peroxisomal. Taken together, our data indicate that in human liver mevalonate kinase and phosphomevalonate kinase are truly peroxisomal enzymes which strongly suggests that peroxisomes play a major role in cholesterol biosynthesis.

Measurement of dihydroxyacetone-phosphate acyltransferase (DHAPAT) in chorionic villous samples, blood cells and cultured cells.

Dihydroxyacetone-phosphate acyltransferase (DHAPAT) is a peroxisomal enzyme catalysing the first step in ether-phospholipid biosynthesis. DHAPAT is deficient in cells from patients suffering from a variety of peroxisomal disorders. Accurate measurement of the activity of this enzyme is of great importance, especially since it is a central parameter in the prenatal diagnosis of the disorders of peroxisome biogenesis, rhizomelic chondrodysplasia punctata and DHAPAT-deficiency. We describe a straightforward and accurate assay allowing the activity of DHAPAT to be measured reliably in chorionic villus samples, blood cells, cultured skin fibroblasts, cultured chorionic villus fibroblasts and cultured amniocytes.