Dorothea Haas

Mevalonate kinase deficiencies: from mevalonic aciduria to hyperimmunoglobulinemia D syndrome

Mevalonic aciduria (MVA) and hyperimmunoglobulinemia D syndrome (HIDS) represent the two ends of a clinical spectrum of disease caused by deficiency of mevalonate kinase (MVK), the first committed enzyme of cholesterol biosynthesis. At least 30 patients with MVA and 180 patients with HIDS have been reported worldwide. MVA is characterized by psychomotor retardation, failure to thrive, progressive cerebellar ataxia, dysmorphic features, progressive visual impairment and recurrent febrile crises. The febrile episodes are commonly accompanied by hepatosplenomegaly, lymphadenopathy, abdominal symptoms, arthralgia and skin rashes. Life expectancy is often compromised. In HIDS, only febrile attacks are present, but a subgroup of patients may also develop neurological abnormalities of varying degree such as mental retardation, ataxia, ocular symptoms and epilepsy. A reduced activity of MVK and pathogenic mutations in the MVK gene have been demonstrated as the common genetic basis in both disorders. In MVA, the diagnosis is established by detection of highly elevated levels of mevalonic acid excreted in urine. Increased levels of immunoglobulin D (IgD) and, in most patients of immunoglobulin A (IgA), in combination with enhanced excretion of mevalonic acid provide strong evidence for HIDS. The diagnosis is confirmed by low activity of mevalonate kinase or by demonstration of disease-causing mutations. Genetic counseling should be offered to families at risk. There is no established successful treatment for MVA. Simvastatin, an inhibitor of HMG-CoA reductase, and anakinra have been shown to have beneficial effect in HIDS.

Mutations in the Gene Encoding Peroxisomal Sterol Carrier Protein X (SCPx) Cause Leukencephalopathy with Dystonia and Motor Neuropathy

In this report, we describe the first known patient with a deficiency of sterol carrier protein X (SCPx), a peroxisomal enzyme with thiolase activity, which is required for the breakdown of branched-chain fatty acids. The patient presented with torticollis and dystonic head tremor as well as slight cerebellar signs with intention tremor, nystagmus, hyposmia, and azoospermia. Magnetic resonance imaging showed leukencephalopathy and involvement of the thalamus and pons. Metabolite analyses of plasma revealed an accumulation of the branched-chain fatty acid pristanic acid, and abnormal bile alcohol glucuronides were excreted in urine. In cultured skin fibroblasts, the thiolytic activity of SCPx was deficient, and no SCPx protein could be detected by western blotting. Mutation analysis revealed a homozygous 1-nucleotide insertion, 545_546insA, leading to a frameshift and premature stop codon (I184fsX7).

Maternal apo E genotype is a modifier of the Smith-Lemli-Opitz syndrome

Background: Smith-Lemli-Opitz syndrome (MIM 270400) is an autosomal recessive malformation and mental retardation syndrome that ranges in clinical severity from minimal dysmorphism and mild mental retardation to severe congenital anomalies and intrauterine death. Smith-Lemli-Opitz syndrome is caused by mutations in the Δ7 sterol-reductase gene (DHCR7; EC 1.3.1.21), which impair endogenous cholesterol biosynthesis and make the growing embryo dependent on exogenous (maternal) sources of cholesterol. We have investigated whether apolipoprotein E, a major component of the cholesterol transport system in human beings, is a modifier of the clinical severity of Smith-Lemli-Opitz syndrome. Method: Common apo E, DHCR7, and LDLR genotypes were determined in 137 biochemically characterised patients with Smith-Lemli-Opitz syndrome and 59 of their parents. Results: There was a significant correlation between patients’ clinical severity scores and maternal apo E genotypes (p = 0.028) but not between severity scores and patients’ or paternal apo E genotypes. In line with their effects on serum cholesterol levels, the maternal apo ϵ2 genotypes were associated with a severe Smith-Lemli-Opitz syndrome phenotype, whereas apo E genotypes without the ϵ2 allele were associated with a milder phenotype. The correlation of maternal apo E genotype with disease severity persisted after stratification for DHCR7 genotype. There was no association of Smith-Lemli-Opitz syndrome severity with LDLR gene variation. Conclusions: These results suggest that the efficiency of cholesterol transport from the mother to the embryo is affected by the maternal apo E genotype and extend the role of apo E and its disease associations to modulation of embryonic development and malformations.

Ubiquinol-induced gene expression signatures are translated into altered parameters of erythropoiesis and reduced low density lipoprotein cholesterol levels in humans

Studies in vitro and in mice indicate a role for Coenzyme Q10 (CoQ10) in gene expression. To determine this function in relationship to physiological readouts, a 2‐week supplementation study with the reduced form of CoQ10 (ubiquinol, Q10H2, 150 mg/d) was performed in 53 healthy males. Mean CoQ10 plasma levels increased 4.8‐fold after supplementation. Transcriptomic and bioinformatic approaches identified a gene–gene interaction network in CD14‐positive monocytes, which functions in inflammation, cell differentiation, and peroxisome proliferator‐activated receptor‐signaling. These Q10H2‐induced gene expression signatures were also described previously in liver tissues of SAMP1 mice. Biochemical and NMR‐based analyses showed a reduction of low density lipoprotein (LDL) cholesterol plasma levels after Q10H2 supplementation. This effect was especially pronounced in atherogenic small dense LDL particles (19–21 nm, 1.045 g/L). In agreement with gene expression signatures, Q10H2 reduces the number of erythrocytes but increases the concentration of reticulocytes. In conclusion, Q10H2 induces characteristic gene expression patterns, which are translated into reduced LDL cholesterol levels and altered parameters of erythropoiesis in humans.

Assays of plasma dehydrocholesteryl esters and oxysterols from Smith-Lemli-Opitz syndrome patients.

Smith-Lemli-Opitz syndrome (SLOS) is caused by mutations in the gene encoding 3β-hydroxysterol-Δ7-reductase and as a result of this defect, 7-dehydrocholesterol (7-DHC) and 8-dehydrocholesterol (8-DHC) accumulate in the fluids and tissues of patients with this syndrome. Both 7- and 8-DHC are susceptible to peroxidation reactions, and several biologically active DHC oxysterols are found in cell and animal models of SLOS. Ex vivo oxidation of DHCs can be a confounding factor in the analysis of these sterols and their esters, and we developed HPLC/MS methods that permit the direct analysis of cholesterol, 7-DHC, 8-DHC, and their esters in human plasma, thus avoiding ex vivo oxidation. In addition, three oxysterols were classified as endogenously formed products by the use of an isotopically-labeled 7-DHC (d7-7-DHC) added to the sample before workup, followed by MS analysis of products formed. Analysis of 17 SLOS plasma samples shows that 8-DHC linoleate correlates better with the SLOS severity score of the patients than other sterols or metabolites, including cholesterol and 7-DHC. Levels of 7-ketocholesterol also correlate with the SLOS severity score. 8-DHC esters should have utility as surrogate markers of severity in SLOS for prognostication and as endpoints in clinical trials.

Differential diagnosis in patients with suspected bile acid synthesis defects

AIM To investigate the clinical presentations associated with bile acid synthesis defects and to describe identification of individual disorders and diagnostic pitfalls. METHODS Authors describe semiquantitative determination of 16 urinary bile acid metabolites by electrospray ionization-tandem mass spectrometry. Sample preparation was performed by solid-phase extraction. The total analysis time was 2 min per sample. Authors determined bile acid metabolites in 363 patients with suspected defects in bile acid metabolism. RESULTS Abnormal bile acid metabolites were found in 36 patients. Two patients had bile acid synthesis defects but presented with atypical presentations. In 2 other patients who were later shown to be affected by biliary atresia and cystic fibrosis the profile of bile acid metabolites was initially suggestive of a bile acid synthesis defect. Three adult patients suffered from cerebrotendinous xanthomatosis. Nineteen patients had peroxisomal disorders, and 10 patients had cholestatic hepatopathy of other cause. CONCLUSION Screening for urinary cholanoids should be done in every infant with cholestatic hepatopathy as well as in children with progressive neurological disease to provide specific therapy.

Abnormal sterol metabolism in holoprosencephaly.

Holoprosencephaly (HPE) is the most common structural malformation of the developing forebrain in humans. The HPE phenotype is extremely variable and the etiology is heterogeneous. Among a variety of embryological toxins that can induce HPE, inhibitors, and other pertubations of cholesterol biosynthesis have been shown to be important factors, most likely because cholesterol is required in the Sonic hedgehog signaling cascade. Decreased levels of maternal cholesterol during pregnancy increase the risk for preterm delivery, but they are not associated with congenital malformations. However, if the fetus is affected by an inborn error of endogenous cholesterol synthesis, a reduction of maternal cholesterol concentration and cholesterol transport over the placenta aggravates the phenotypic expression. Exposure to lipophilic statins in early pregnancy may be associated with a substantial risk for structural CNS defects.

Abnormal sterol metabolism in holoprosencephaly: studies in cultured lymphoblasts

Background: Holoprosencephaly (HPE) is the most common structural malformation of the developing forebrain in humans. The aetiology is heterogeneous and remains unexplained in approximately 75% of patients. Objective: To examine cholesterol biosynthesis in lymphoblastoid cell lines of 228 patients with HPE, since perturbations of cholesterol homeostasis are an important model system to study HPE pathogenesis in animals. Methods: An in vitro loading test that clearly identifies abnormal increase of C27 sterols in lymphoblast-derived cells was developed using [2-14C] acetate as substrate. Results: 22 (9.6%) HPE cell lines had abnormal sterol pattern in the in vitro loading test. In one previously reported patient, Smith–Lemli–Opitz syndrome was diagnosed, whereas others also had clearly reduced cholesterol biosynthesis of uncertain cause. The mean (SD) cholesterol levels were 57% (15.3%) and 82% (4.7%) of total sterols in these cell lines and controls, respectively. The pattern of accumulating sterols was different from known defects of cholesterol biosynthesis. In six patients with abnormal lymphoblast cholesterol metabolism, additional mutations in genes known to be associated with HPE or chromosomal abnormalities were observed. Conclusions: Impaired cholesterol biosynthesis may be a contributing factor in the cause of HPE and should be considered in the evaluation of causes of HPE, even if mutations in HPE-associated genes have already been found.

The reduced form of coenzyme Q10 mediates distinct effects on cholesterol metabolism at the transcriptional and metabolite level in SAMP1 mice

Studies in humans and mice indicate a role for coenzyme Q10 (CoQ10) in gene expression. To analyze this function in relation to metabolism, SAMP1 mice were supplemented with the reduced (ubiquinol) or oxidized (ubiquinone) form of CoQ10 (500 mg/kg BW/d) for 14 months. Microarray analyses in liver tissues of SAMP1 mice identified 946 genes as differentially expressed between ubiquinol‐treated and control animals (≥1.5‐fold, P < 0.05). Text mining analyses revealed for a part of the ubiquinol‐regulated genes, a functional connection in PPARα and LXR/RXR signalling pathways. Because these pathways are involved in cholesterol homeostasis, relevant metabolites were determined by gas chromatography/mass spectrometry (GC/MS). We found a significant increase of desmosterol (2.0‐fold, P < 0.001) in the liver of ubiquinol‐supplemented SAMP1 mice when related to control animals. In agreement, cholesterol concentrations were also distinctly increased (1.3‐fold, P = 0.057). The Q10H2‐induced PPARα and LXR/RXR gene expression signatures and effects on cholesterol metabolism were not apparent for the oxidized form of CoQ10. In conclusion, the reduced form of CoQ10 mediates distinct effects on cholesterol metabolism at the transcriptional and metabolite level in SAMP1 mice.

Coenzyme Q(10) is decreased in fibroblasts of patients with methylmalonic aciduria but not in mevalonic aciduria.

SummaryThe content of coenzyme Q10 (CoQ10) was examined in skin fibroblasts of 10 patients with mevalonic aciduria (MVA) and of 22 patients with methylmalonic aciduria (MMA). Patients with these inborn errors of metabolism are thought to be at risk for CoQ10 depletion either by direct inhibition of the proximal pathway of CoQ10 synthesis (MVA) or indirectly by inhibition of mitochondrial energy metabolism (MMA). We demonstrated that CoQ10 concentrations were not significantly different from controls in MVA patients, suggesting that there may be upregulatory effects. On the other hand the CoQ10 content in fibroblasts of patients with MMA was significantly reduced.