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.

Decreased oxidative phosphorylation and PGAM deficiency in horses suffering from atypical myopathy associated with acquired MADD.

Earlier research on ten horses suffering from the frequently fatal disorder atypical myopathy showed that MADD (multiple acyl-CoA dehydrogenase deficiency) is the biochemical derangement behind atypical myopathy. From five horses that died as a result of this disease and seven healthy control horses, urine and plasma were collected ante mortem and muscle biopsies were obtained immediately post-mortem (2 patients and 7 control horses), to analyse creatine, purine and carbohydrate metabolism as well as oxidative phosphorylation. In patients, the mean creatine concentration in urine was increased 17-fold and the concentration of uric acid approximately 4-fold, compared to controls. The highest degree of depletion of glycogen was observed in the patient with the most severe myopathy clinically. In this patient, glycolysis was more active than in the other patients and controls, which may explain this depletion. One patient demonstrated very low phosphoglycerate mutase (PGAM) activity, less than 10% of reference values. Most respiratory chain complex activity in patients was 20-30% lower than in control horses, complex II activity was 42% lower than normal, and one patient had severely decrease ATP-synthase activity, more than 60% lower than in control horses. General markers for myopathic damage are creatine kinase (CK) and lactic acid in plasma, and creatine and uric acid in urine. To obtain more information about the cause of the myopathy analysis of carbohydrate, lipid and protein metabolism as well as oxidative phosphorylation is advised. This study expands the diagnostic possibilities of equine myopathies.

Expanding the clinical spectrum of 3-phosphoglycerate dehydrogenase deficiency.

UNLABELLED: 3-Phosphoglycerate dehydrogenase (3-PGDH) deficiency is considered to be a rare cause of congenital microcephaly, infantile onset of intractable seizures and severe psychomotor retardation. Here, we report for the first time a very mild form of genetically confirmed 3-PGDH deficiency in two siblings with juvenile onset of absence seizures and mild developmental delay. Amino acid analysis showed serine values in CSF and plasma identical to what is observed in the severe infantile form. Both patients responded favourably to relatively low dosages of serine supplementation with cessation of seizures, normalisation of their EEG abnormalities and improvement of well-being and behaviour. These cases illustrate that 3-PGDH deficiency can present with mild symptoms and should be considered as a treatable disorder in the differential diagnosis of mild developmental delay and seizures. SYNOPSIS: we present a novel mild phenotype in patients with 3-PGDH deficiency.

Equine acquired multiple acyl-CoA dehydrogenase deficiency (MADD) in 14 horses associated with ingestion of Maple leaves (Acer pseudoplatanus) covered with European tar spot (Rhytisma acerinum).

This case-series describes fourteen horses suspected of equine acquired multiple acyl-CoA dehydrogenase deficiency (MADD) also known as atypical myopathy of which seven cases were confirmed biochemically with all horses having had access to leaves of the Maple tree (Acer pseudoplatanus) covered with European tar spot (Rhytisma acerinum). Assessment of organic acids, glycine conjugates, and acylcarnitines in urine was regarded as gold standard in the biochemical diagnosis of equine acquired multiple acyl-CoA dehydrogenase deficiency.

Acquired multiple Acyl-CoA dehydrogenase deficiency in 10 horses with atypical myopathy.

The aim of the current study was to assess lipid metabolism in horses with atypical myopathy. Urine samples from 10 cases were subjected to analysis of organic acids, glycine conjugates, and acylcarnitines revealing increased mean excretion of lactic acid, ethylmalonic acid, 2-methylsuccinic acid, butyrylglycine, (iso)valerylglycine, hexanoylglycine, free carnitine, C2-, C3-, C4-, C5-, C6-, C8-, C8:1-, C10:1-, and C10:2-carnitine as compared with 15 control horses (12 healthy and three with acute myopathy due to other causes). Analysis of plasma revealed similar results for these predominantly short-chain acylcarnitines. Furthermore, measurement of dehydrogenase activities in lateral vastus muscle from one horse with atypical myopathy indeed showed deficiencies of short-chain acyl-CoA dehydrogenase (0.66 as compared with 2.27 and 2.48 in two controls), medium-chain acyl-CoA dehydrogenase (0.36 as compared with 4.31 and 4.82 in two controls) and isovaleryl-CoA dehydrogenase (0.74 as compared with 1.43 and 1.61 nmol min(-1) mg(-1) in two controls). A deficiency of several mitochondrial dehydrogenases that utilize flavin adenine dinucleotide as cofactor including the acyl-CoA dehydrogenases of fatty acid beta-oxidation, and enzymes that degrade the CoA-esters of glutaric acid, isovaleric acid, 2-methylbutyric acid, isobutyric acid, and sarcosine was suspected in 10 out of 10 cases as the possible etiology for a highly fatal and prevalent toxic equine muscle disease similar to the combined metabolic derangements seen in human multiple acyl-CoA dehydrogenase deficiency also known as glutaric acidemia type II.

Equine metabolic myopathies with emphasis on the diagnostic approach. Comparison with human myopathies. A review.

This review gives an overview of the presently known human and equine metabolic myopathies with emphasis on the diagnostic approach. Metabolic myopathies are muscle disorders caused by a biochemical defect of the skeletal muscle energy system, which results in inefficient muscle performance. Myopathies can arise in different levels of the metabolic system. In this review the metabolic myopathies are categorized in disorders of the carbohydrate metabolism, lipid metabolism, mitochondrial myopathies (other than those described in lipid metabolism), disorders of purine metabolism, primary disorders involving ion channels and electrolyte flux and secondary or acquired metabolic myopathies.

Removal of chloroform from biodegradable therapeutic microspheres by radiolysis.

Radioactive holmium-166 loaded poly(l-lactic acid) microspheres are promising systems for the treatment of liver malignancies. These microspheres are loaded with holmium acetylacetonate (HoAcAc) and prepared by a solvent evaporation method using chloroform. After preparation the microspheres (Ho-PLLA-MS) are activated by neutron irradiation in a nuclear reactor. It was observed that relatively large amounts of residual chloroform (1000-6000 ppm) remained in the microspheres before neutron irradiation. Since it is known that chloroform is susceptible for high-energy radiation, we investigated whether neutron and gamma irradiation could result in the removal of residual chloroform in HoAcAc-loaded and placebo PLLA-MS by radiolysis. To investigate this, microspheres with relatively high and low amounts of residual chloroform were subjected to irradiation. The effect of irradiation on the residual chloroform levels as well as other microsphere characteristics (morphology, size, crystallinity, molecular weight of PLLA and degradation products) were evaluated. No chloroform in the microspheres could be detected after neutron irradiation. This was also seen for gamma irradiation at a dose of 200 kGy phosgene, which can be formed as the result of radiolysis of chloroform, was not detected with gas chromatography-mass spectrometry (GC-MS). A precipitation titration showed that radiolysis of chloroform resulted in the formation of chloride. Gel permeation chromatography and differential scanning calorimetry showed a decrease in molecular weight of PLLA and crystallinity, respectively. However, no differences were observed between irradiated microsphere samples with high and low initial amounts of chloroform. In conclusion, this study demonstrates that neutron and gamma irradiation results in the removal of residual chloroform in PLLA-microspheres.

Structure determination of two oligomannoside-type glycopeptides obtained from bovine lactotransferrin, by 500 MHz 1H-NMR spectroscopy.

The elucidation of the structures of two carbohydrate units, N-glycosidically linked to an asparagine residue of bovine lactotransferrin, is described. These carbohydrate structures are of the oligomannoside type and contain eight or nine mannose residues, respectively. The potency of 500 MHz 1H-NMR spectroscopy in primary structure determination of two closely related carbohydrate chains present in a mixture is demonstrated. This implies that 500 MHz 1H-NMR spectroscopy can disclose microheterogeneity which is almost untraceable using other approaches.

A 360-MHz 1H-NMR study of three oligosaccharides isolated from cow kappa-casein.

360-MHz 1H-NMR spectra were recorded of NeuAc alpha(2 leads to 3)Gal beta (1 leads to 3)GalNAc-ol (I), Gal beta(1 leads to 3)[NeuAc alpha(2 leads to 6)] GalNAc-ol (II) and NeuAc alpha (2 leads to 3)-Gal beta(1 leads to 3) [NeuAc alpha(2 leads to 6)]GalNAc-ol (III). The chemical shifts and coupling constants of the anomeric protons, the H-3ax and H-3eq of NeuAc, the GalNAc-ol skeleton protons, the H-3 of Gal and the N-acetyl protons of GalNAc-ol and NeuAc provide conclusive evidence for the identification of the primary structures. Compound II represents a novel carbohydrate chain of kappa-casein.

2-Acetamidoglucal, a new metabolite isolated from the urine of a patient with sialuria.

A new metabolite, namely 2-acetamidoglucal, has been found in the urine of a patient with sialuria in addition to the metabolites N-acetylneuraminic acid, N-acetylmannosamine, N-acetylglucosamine and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid reported earlier. the structure has been identified by mass spectrometry and 360 MHz proton nuclear magnetic resonance spectroscopy and verified by synthesis. All accumulated compounds fit into the metabolic pathway for the biosynthesis of CMP-N-acetylneuraminic acid. Sialuria is discussed in terms of a failure of regulation of UDP-N-acetylglucosamine 2-epimerase.

The primary structure of the asialo-carbohydrate units of the first glycosylation site of human plasma alpha 1-acid glycoprotein.

The elucidation of the structures of the carbohydrate units linked to glycosylation site I of human plasma alpha 1-acid glycoprotein is described. These carbohydrate units can be grouped into compounds with bi- (class A) and triantennary (class B) structures and the triantennary structure with a fucose residue (class BF) (Fig. 1). The structural variability of the carbohydrate units of glycosylation site I and also of glycosylation sites II to V (Fournet, B., Montreuil, J., Strecker, G., Dorland, L., Haverkamp, J., Vliegenthart, J.F.G., Binette, J.P. and Schmid, K. (1978) Biochemistry 17, 5206--5214) accounts largely for the microheterogeneity of alpha 1-acid glycoprotein.

Prenatal and early postnatal treatment in 3-phosphoglycerate-dehydrogenase deficiency.

3-phosphoglycerate-dehydrogenase (3-PGDH) deficiency is an L-serine biosynthesis disorder, characterised by congenital microcephaly, severe psychomotor retardation, and intractable seizures. We report prenatal diagnosis of an affected fetus by DNA mutation analysis. Ultrasound assessment showed a reduction in fetal head circumference from the 75th percentile at 20 weeks' gestation to the 29th percentile at 26 weeks. L-serine was then given to the mother, which resulted in an enlarged fetal head circumference to the 76th percentile at 31 weeks. At birth, the girl's head circumference was normal, and at 48 months' follow-up, her psychomotor development has been unremarkable. 3-PGDH deficiency is an inborn metabolic error that can be successfully treated antenatally.

Two Novel Mutations in the SLC25A4 Gene in a Patient with Mitochondrial Myopathy.

In a 28-year-old male with a mild mitochondrial myopathy manifesting as exercise intolerance and early signs of cardiomyopathy without muscle weakness or ophthalmoplegia, we identified two novel mutations in the SLC25A4 gene: c.707G>C in exon 3 (p.(R236P)) and c.116_137del in exon 2 (p.(Q39Lfs*14)). Serum lactate levels at rest were elevated (12.7 mM). Both the patient's father and brother were heterozygous carriers of the c.707G>C mutation and were asymptomatic. The second mutation causes a 22 bp deletion leading to a frame shift likely giving rise to a premature stop codon and nonsense-mediated decay (NMD). The segregation of the mutations could not be tested directly as the mother had died before. However, indirect evidence from NMD experiments showed that the two mutations were situated on two different alleles in the patient. This case is unique compared to other previously reported patients with either progressive external ophthalmoplegia (PEO) or clear hypertrophic cardiomyopathy with exercise intolerance and/or muscle weakness carrying recessive mutations leading to a complete absence of the SLC25A4 protein. Most likely in our patient, although severely reduced, SLC25A4 is still partially present and functional.

Genomic organization of the human phosphomannose isomerase (MPI) gene and mutation analysis in patients with congenital disorders of glycosylation type Ib (CDG-Ib).

CDG-Ib is the "gastro-intestinal" type of the congenital disorders of glycosylation (CDG) and a potentially treatable disorder. It has been described in patients presenting with congenital hepatic fibrosis and protein losing enteropathy. The symptoms result from hypoglycosylation of serum- and other glycoproteins. CDG-Ib is caused by a deficiency of mannose-6-phosphate isomerase (synonym: phosphomannose isomerase, EC 5.3.1.8), due to mutations in the MPI gene. We determined the genomic structure of the MPI gene in order to simplify mutation detection. The gene is composed of 8 exons and spans only 5 kb. Eight (7 novel) different mutations were found in seven patients with a confirmed phosphomannose isomerase deficiency, analyzed in the context of this study: six missense mutations, a splice mutation and one insertion. In the last, the mutation resulted in an unstable transcript, and was hardly detectable at the mRNA level. This emphasizes the importance of mutation analysis at the genomic DNA level.

Primary structure of a novel N-glycosidic carbohydrate unit, derived from hen ovomucoid. A 500-MHz 1H-NMR study.

The N-glycosidic carbohydrate chains of hen beta-ovomucoid were released from the protein by hydrazinolysis, and separated by HPLC. Primary structural analysis of 3 major fractions was conducted by applying 500-MHz 1H-NMR spectroscopy in combination with methylation analysis. One of the fractions investigated appeared to consist of an intersected penta-antennary structure extended with one Gal residue. The location of the latter in a certain branch could be established unambiguously by NMR. This structure is a novel member of the family of N-glycosidic carbohydrates of glycoproteins.

Asymptomatic and late-onset ornithine transcarbamylase deficiency caused by a A208T mutation: clinical, biochemical and DNA analyses in a four-generation family.

We describe a 4-generation family in which a previously healthy 10-year-old boy died of late-onset ornithine transcarbamylase (OTC) deficiency. Pedigree analysis and allopurinol loading tests in female relatives were not informative. A missense mutation (A208T) in the OTC gene was detected in the deceased patient and in several clinically healthy male and female relatives, the oldest male being 97 years old. OTC deficiency was established in autopsy liver tissue of the propositus and liver biopsy samples of his sister, mother, and a maternal uncle. The males had 4% and 6% residual activity, respectively, the females 58% and 67%, respectively. The observed relation between the mutation and the decreased OTC activity in liver tissue of these subjects suggests that the mutation is a deleterious one. Late-onset, "mild" OTC deficiency can have a fatal or a favorable outcome. The disease can segregate undetected in families.

Studies on the interaction of Clostridium perfringens sialidase with sialic acid linked to the internal galactose in monosialogangliotetraosyl ceramide.

Investigation of the action of highly purified Clostridium perfringens sialidase on ganglioside II3Neu5Ac-Gg4Cer and its oligosaccharide II3Neu5Ac-Gg4, in the presence and absence of sodium cholate, extend earlier results obtained with impure enzyme fractions. Sialidase labeled with 125I was found to bind to various ganglioside substrate micelles, including II3Neu5Ac-Gg4Cer, and to mixed ganglioside-sodium cholate micelles. No binding occurred between the enzyme and the ganglioside-derived oligosaccharide II3Neu5Ac-Gg4, even when radioactive II3Neu5Ac-Gg4-[3H]ol was used. The binding of sialidase to micellar substrate is a condition for enzymic hydrolysis. Correspondingly, II3Neu5Ac-Gg4Cer and II3Neu5Ac-Gg4Cer-sodium cholate micelles were hydrolyzed by the enzyme but II3Neu5Ac-Gg4 was not. Ganglioside oligosaccharide analogues containing an amino function at the reducing terminus or between two oligosaccharide chains, II3Neu5Ac-Gg4-NH2 and (II3Neu5Ac-Gg4)2NH, were hydrolyzed in the absence of cholate. A synthetic analogue of II3Neu5Ac-Gg4Cer containing only the fatty acid moiety and not the sphingosine residue (I1-deoxy-I1-stearamido-II3-monosialo-gangliotetraitol ) behaved as the ganglioside in the presence and absence of sodium cholate.

Congenital hepatic fibrosis in 3 siblings with phosphomannose isomerase deficiency.

Congenital hepatic fibrosis is a rare disorder of intrahepatic bile ducts with the persistence of embryological bile duct structures in ductal plate configuration. Three siblings aged 18, 17 and 14 years old were found to have congenital hepatic fibrosis associated with a deficiency of the enzyme phosphomannose isomerase. The clinical symptoms were recurrent attacks of persistent vomiting with diarrhea and mild hepatomegaly. The biochemical abnormalities included elevated serum transferases during attacks, clotting factor deficiencies and persistent hypoalbuminemia. In the youngest patient protein-losing enteropathy was present. Liver biopsies of the three patients taken when they were 1, 3 and 14 years old showed an excess of bile duct structures in ductal plate configuration with mild fibrosis in the portal triads. In one patient the liver biopsy was repeated after 18 years and showed only a mild progression of fibrosis in the portal triads. Duodenal biopsies taken in infancy in two of the three patients did not show any abnormalities. Recognition of phosphomannose isomerase deficiency in association with congenital hepatic fibrosis and protein-losing enteropathy is important, because some of the clinical symptoms are potentially treatable by oral mannose therapy.

3-, 6- and 7-hydroxyoctanoic acids are metabolites of medium-chain triglycerides and excreted in urine as glucuronides.

Three new metabolites of medium-chain fatty acid oxidation, 3-, 6- and 7-hydroxyoctanoyl beta-D-glucuronide, were identified in the urine of six infants who were fed a diet enriched in medium-chain triglycerides (MCT). Glucuronides were extracted from the urine by organic solvent extraction with ethyl acetate and by solid-phase extraction on Sep-Pak C18 cartridges. The compounds of interest were also purified from the organic solvent extract by preparative one-dimensional thin-layer chromatography. Cleavage of the glucuronides was achieved by either alkaline hydrolysis or enzymatic hydrolysis with beta-D-glucuronidase. The analyses of the trimethylsilylated derivatives were performed both by gas chromatography with flame ionization detection (GC/FID) and by gas chromatography/mass spectrometry (GC/MS). The structure of the hydroxyoctanoic acids was proved by comparison of their mass spectra with those of reference substances. Authentic 6-hydroxyoctanoic acid was synthesized. The presence of 6-hydroxyoctanoyl glucuronide shows that in addition to beta-oxidation, omega-oxidation and (omega-1)-hydroxylation, medium-chain fatty acids can be oxidized at the omega-2 position. The conjugation of medium-chain hydroxy-monocarboxylic acids with glucuronic acid has not been described in humans before.

1,6-Anhydro-beta-D-glucopyranose (beta-glucosan), a constituent of human urine.

When screening for abnormal urinary saccharides with one-dimensional thin-layer chromatography, an unknown component was observed in a position just above that of xylose. This compound was studied by gas chromatography-mass spectrometry and identified as the anhydro sugar beta-glucosan. It was observed in approximately 20% of all urine samples investigated by thin-layer chromatography. Excretory levels varied widely from zero up to 5.3 mmol/l. No correlation with age or disease could be established. The compound was thought to be of exogenous origin.