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Biomedical subjects

Wolfgang Sperl

Publications and source records attributed to Wolfgang Sperl.

At least 19 recordsLinked to original sources
PubMed2006

A novel sporadic mutation G14739A of the mitochondrial tRNA(Glu) in a girl with exercise intolerance.

We describe a 7-year-old girl who presented with loss of appetite, weakness and excercise intolerance. Enzyme investigation of the respiratory chain in muscle tissue revealed a combined complex I, III and IV deficiency. A novel heteroplasmic G-->A exchange at nucleotide position 14739 was found in the MTTE gene of the tRNA glutamic acid. The mutation load in muscle was 72%, urine sediment 38%, blood 31% and fibroblasts 29% and it correlated with COX-negative fibres. Our patient presented with a predominantly myopathic phenotype. The G14739A mutation is the third reported in the mitochondrial tRNA glutamic acid gene, and it occurred in a sporadic case.

Brain↗
PubMed2006

Prevalence of congenital adrenal hyperplasia among sudden infant death in the Czech Republic and Austria.

This study aimed to estimate the number of infants who died of unrecognized congenital adrenal hyperplasia (CAH) in Austria and the Czech Republic within the past 13 years, before the introduction of adequate neonatal screening. The study was based on retrospective analysis of neonatal screening cards of 242 infants who died suddenly between 7 days and 12 months of age and whose cause of death could not be identified. 17-hydroxyprogesterone (17-OHP) was measured by fluoroimmunoassay and positive samples were subsequently genotyped. Three infants out of 242 may have had unrecognized CAH due to CYP21 (steroid 21-hydroxylase) gene defect. Their newborn 17-OHP levels and CYP21 genotypes were 706 nmol/l and del/conv//del/conv, 53 nmol/l and I2//I2, and 811 nmol/l and I2//Gln318stop, respectively. CAH due to CYP21 defect can lead to sudden unexpected death without prior symptoms typical for the condition. Hence, newborn screening would have prevented these deaths had it been available. In addition, we have shown that the I2 point mutation that is expected to lead to simple virilizing form may lead to a fatal outcome.

Adrenal Hyperplasia, Congenital↗
PubMed2006

Influence of hematocrit and localisation of punch in dried blood spots on levels of amino acids and acylcarnitines measured by tandem mass spectrometry.

BACKGROUND: Detection of amino acids (AA), acylcarnitines (AC), and guanidinoacetate (GAA) in dried blood spots by tandem mass spectrometry has made it possible to detect different inborn errors of metabolism in neonatal screening programs. Despite its proven sensitivity many issues related to sample preparation remain unsolved. Hematocrit has a profound effect on blood viscosity, and may thereby influence flux and diffusion properties of the blood. As newborn infants show a considerable interindividual variability of hematocrit levels, we investigated its effect on levels of AA and AC in dried blood spots. METHODS: Blood samples with defined hematocrit levels (20%, 30%, 40%, 50%, 60%) were produced by diluting blood cells with plasma from a single donor. Forty dried blood spots were made for each hematocrit level and a central as well as a peripheral 3 mm disk was punched and analysed for AA, AC, and GAA, respectively. RESULTS: Levels of most AA and GAA increased significantly with increasing hematocrit (p<0.001), while the effect of hematocrit on some AA was less pronounced. Total AC, free carnitine, some long, medium and short chain AC correlated positively with hematocrit levels (p<0.001). In samples with low hematocrit, levels of most AA and free carnitine were higher in the peripheral than in the central disk (p<0.0001). CONCLUSIONS: Both hematocrit and position of the disk within the dried blood spot have a significant and sometimes additive effect on levels of AA, AC and GAA in dried blood spots. Theoretically, diagnoses may be missed depending on hematocrit and position of the disk.

Amino Acids↗
PubMed2006

Multiplex primer extension analysis for rapid detection of major European mitochondrial haplogroups.

The evolution of the human mitochondrial genome is reflected in the existence of ethnically distinct lineages or haplogroups. Alterations of mitochondrial DNA (mtDNA) have been instrumental in studies of human phylogeny, in population genetics, and in molecular medicine to link pathological mutations to a variety of human diseases of complex etiology. For each of these applications, rapid and cost effective assays for mtDNA haplogrouping are invaluable. Here we describe a hierarchical system for mtDNA haplogrouping that combines multiplex PCR amplifications, multiplex single-base primer extensions, and CE for analyzing ten haplogroup-diagnostic mitochondrial single nucleotide polymorphisms. Using this rapid and cost-effective mtDNA genotyping method, we were able to show that within a large, randomly selected cohort of healthy Austrians (n = 1172), mtDNAs could be assigned to all nine major European haplogroups. Forty-four percent belonged to haplogroup H, the most frequent haplogroup in European Caucasian populations. The other major haplogroups identified were U (15.4%), J (11.8%), T (8.2%) and K (5.1%). The frequencies of haplogroups in Austria is within the range observed for other European countries. Our method may be suitable for mitochondrial genotyping of samples from large-scale epidemiology studies and for identifying markers of genetic susceptibility.

Austria↗
PubMed2006

Gangliocytes in neuroblastic tumors express alarin, a novel peptide derived by differential splicing of the galanin-like peptide gene.

In neuroblastic tumors a relationship of differentiation of the tumor to galanin receptor expression and antiproliferative and apoptotic effects upon activation of galanin receptors in neuroblastoma cells was reported. To elucidate the expression of other components of the galanin peptide family in neuroblastic tumors, RT-PCR analysis of a variety of human neuroblastic tumor tissues was performed. Ganglioneuroma tissues revealed the presence of a splice variant of the galanin-like peptide (GALP) mRNA, which results in exclusion of exon 3 and a frame shift after the signal peptide sequence of GALP. This generates a peptide of 25 amino acids, which we have termed alarin because of the N-terminal alanine and the C-terminal serine. The novel neuropeptide alarin does not reveal significant homology to other peptides. Immunohistochemistry with antibodies directed against synthetic alarin peptide detected specific cytoplasmic granular staining in ganglia of human ganglioneuroma and ganglioneuroblastoma, as well as differentiated tumor cells of neuroblastoma tissues. Undifferentiated neuroblasts of these tumor tissues did not show alarin-like immunoreactivity and alarin-specific mRNA. Our findings indicate that alarin expression is a feature of ganglionic differentiation in neuroblastic tumor tissues.

Amino Acid Sequence↗
PubMed2005

Combined deficiency of factor V and factor VIII is due to mutations in either LMAN1 or MCFD2.

Mutations in LMAN1 (ERGIC-53) or MCFD2 cause combined deficiency of factor V and factor VIII (F5F8D). LMAN1 and MCFD2 form a protein complex that functions as a cargo receptor ferrying FV and FVIII from the endoplasmic reticulum to the Golgi. In this study, we analyzed 10 previously reported and 10 new F5F8D families. Mutations in the LMAN1 or MCFD2 genes accounted for 15 of these families, including 3 alleles resulting in no LMAN1 mRNA accumulation. Combined with our previous reports, we have identified LMAN1 or MCFD2 mutations as the causes of F5F8D in 71 of 76 families. Among the 5 families in which no mutations were identified, 3 were due to misdiagnosis, with the remaining 2 likely carrying LMAN1 or MCFD2 mutations that were missed by direct sequencing. Our results suggest that mutations in LMAN1 and MCFD2 may account for all cases of F5F8D. Immunoprecipitation and Western blot analysis detected a low level of LMAN1-MCFD2 complex in lymphoblasts derived from patients with missense mutations in LMAN1 (C475R) or MCFD2 (I136T), suggesting that complete loss of the complex may not be required for clinically significant reduction in FV and FVIII.

Alleles↗
PubMed2005

Galanin and galanin receptors in human cancers.

The increasing interest in peptides and peptide receptors in cancer is based on the possibility of receptor targeting, because peptide receptors are often expressed in different human tumors. The neuropeptide galanin has also been suggested to be involved in the development of neuroendocrine tumors based on the development of estrogen-induced tumors in estrogen-sensitive rats. This study summarizes our current knowledge on the expression of galanin peptide and galanin receptors in different human neuroendocrine tumors. The expression of both, peptide and corresponding receptor, seems to be a common feature of human gliomas, pheochromocytomas, pituitary and neuroblastic tumors. The co-expression of galanin and its receptors supports a role for galanin in tumor cell pathology via autocrine/paracrine mechanisms.

Galanin↗
PubMed2005

Rapid screening of the entire mitochondrial DNA for low-level heteroplasmic mutations.

Alterations of the mitochondrial DNA (mtDNA) are implicated in various pathological conditions. In this study, we used denaturing high performance liquid chromatography (DHPLC) as a method to rapidly screen the entire mtDNA for mutations. Overlapping DNA fragments, amplified by one single cycling protocol from frozen pre-formulated PCR mixes, were subjected to DHPLC analysis. Single DHPLC injections of fragments yielded straightforward interpretation of results with a detection limit down to 1% mtDNA heteroplasmy. Furthermore, collection and re-amplification of low degree heteroduplex peak-fractions allowed sequence analysis of mtDNA mutations down to the detection limit of the DHPLC method. In order to demonstrate that the method has diagnostic value, we analyzed and confirmed known mtDNA mutations in patient samples.

Chromatography, High Pressure Liquid↗
PubMed2005

Yeast mediates lactic acidosis suppression after antibiotic cocktail treatment in short small bowel?

During acidotic periods in a girl with a short small bowel, very high D-lactic acid concentrations were measured in blood and urine; the patient's characteristic faecal flora contained mainly lactobacilli, and during antibiotic cocktail treatment also many yeasts. In this case report we sought to understand the beneficial effect of the antibiotic cocktail. Microbiological analysis was performed in faecal samples. Total lactic acid in serum and urine was studied using capillary gas chromatography-mass spectrometry, and D- and L-lactic acid in serum and urine by enzymatic assay. The results were coupled to patient's condition. Antibiotic cocktail therapy reduced the acidosis-associated symptoms, faecal lactobacilli and D-lactic acid production, but simultaneously the antibiotic therapy strongly increased the percentage of yeast in the faecal flora. Four to six weeks after each course of treatment the percentage of yeast decreased, whereas the percentage of intestinal lactobacilli increased; D-lactic acid also simultaneously increased in blood and urine. The patient felt well and showed a high percentage of intestinal yeast, but she often suffered from acidosis owing to a high percentage of lactobacilli. The yeast was identified as the pathogenic Candida glabrata. From the mentioned data together with data from the literature it was concluded that during several weeks the selected pathogenic yeast, C. glabrata, acted as a microbiological and metabolic buffer. Shortly after the course of antibiotic treatment this intestinal yeast strongly competed with the intestinal lactobacilli and thus prevented renewed rapid growth, massive D-lactic acid production from glucose and consequently also D-lactic acid-associated acidosis. The emergence of this yeast led us to consider probiotic lactobacilli or yeast for therapeutic use. The lack of knowledge regarding bile acid-deconjugating activity in both lactobacilli and probiotic yeast means that a final recommendation is not yet possible.

Acidosis, Lactic↗
PubMed2004

The problem of interlab variation in methods for mitochondrial disease diagnosis: enzymatic measurement of respiratory chain complexes.

Due to the large importance of mitochondrial function for numerous diseases the detection of respiratory chain defects for diagnostic purposes is an important task of mitochondrial medicine. For comparing the methods, standard mitochondrial homogenate prepared from bovine skeletal muscle was sent on dry ice to 14 labs of 8 countries. Activities of complexes I, I + III, II, II + III, IV (cytochrome c oxidase) and V (F0F1ATPase) as well as of citrate synthase were measured. For all enzymes the results differed at more than one order of magnitude. From eight labs we were able to compare the results with their control values for human skeletal muscle. Four labs found normal activity of cytochrome-c-oxidase whereas three labs found higher and one lab found lower activities compared to the own controls. Since all labs used different temperatures (25, 30 and 37 degrees C) in one lab the temperature dependencies were measured experimentally. The temperature correction did not much reduce the divergence of the results. It is concluded that differences in the lab protocols are the reason for the large variation of results. Since the experimental results strongly depend on the used method a strict standardization is necessary.

Journal Article↗
PubMed2004

Decrease of mitochondrial DNA content and energy metabolism in renal cell carcinoma.

To elucidate the relationship between tumor genesis and the mitochondrial energy metabolism in renal neoplasms, we studied three individual enzyme activities of the oxidative phosphorylation, two components of the Krebs cycle and the mitochondrial DNA content of renal carcinomas including 29 conventional, five papillary, two unclassified carcinomas with sarcomatoid features and one collecting duct carcinoma. A significant reduction of all mitochondrial enzyme activities including complex V, as well as of the mitochondrial DNA content was detected in 34 of 37 renal carcinoma tissues as compared with control kidney. Mitochondrial enzyme activities and mitochondrial DNA levels were not statistically different between the conventional, papillary and unclassified sarcomatoid type of renal carcinoma and did not correlate with tumour grade, metastasis, ploidy and proliferative activity as determined by Ki-67 staining. Taken together, our data indicate that a co-ordinated down-regulation of all components necessary for mitochondrial energy metabolism occurs in most renal carcinomas as an early event in carcinoma formation, which does not change with progression of the disease.

Adult↗
PubMed2004

2-Methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency: impaired catabolism of isoleucine presenting as neurodegenerative disease.

We describe a further case of recently reported 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiency, a disorder of isoleucine metabolism. The development of pronounced brain atrophy and symmetrical alterations of the basal ganglia were observed and the importance of specific enzymatic tests is emphasized, which should be performed if urinary metabolites suggest impaired catabolism of isoleucine.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed2004

Glycerol kinase deficiency: residual activity explained by reduced transcription and enzyme conformation.

Four unrelated patients with glyceroluria ranging from 7 to 170 mmol/l were studied. The activity of glycerol kinase (GK) in cultured fibroblasts was determined with a specific enzyme assay and with two indirect methods, that is, incorporation into macromolecules of [(14)C] from [(14)C]glycerol and its oxidation to [(14)C]CO(2). Exon amplification and RT-PCR were used to identify mutations. In patient 1, with low activity in all three assays, we identified a c.1194A>C (E398D) missense mutation. In patient 2 with a considerable activity of the GK enzyme (22% of reference), oxidation to [(14)C]CO(2) (37%) and a high incorporation of [(14)C] into macromolecules (92%), we identified a c.182T>C (L61P) mutation that causes the enzyme to have a higher K(m) for glycerol ( approximately 300 microM) than normals (2-8 microM). In patient 3, the GK activity estimated by the three different methods ranged from 16 to 22% of reference. Analysis of mRNA from the GK gene revealed three alternatively spliced transcripts. A mutation in intron 3 (g.16835G>A) resulted in an insertion of a cryptic exon between exon 2 or 3 and exon 4. Patient 4 with minor glyceroluria (7 mmol/l) and normal plasma glycerol concentration had normal activity with all three assay methods, thus excluding GK deficiency (GKD) as a cause of slight glyceroluria. To evaluate fully patients with glyceroluria, one needs to measure the GK activity and relate this and the clinical data to genetic findings. Residual enzyme activities in cultured fibroblasts can be found in GKD patients with severe clinical symptoms.

Alternative Splicing↗
PubMed2004

Reduced respiratory control with ADP and changed pattern of respiratory chain enzymes as a result of selective deficiency of the mitochondrial ATP synthase.

The F(o)F(1)-ATPase, a multisubunit protein complex of the inner mitochondrial membrane, produces most of the ATP in mammalian cells. Mitochondrial diseases as a result of a dysfunction of ATPase can be caused by mutations in mitochondrial DNA-encoded ATPase subunit a or rarely by an ATPase defect of nuclear origin. Here we present a detailed functional and immunochemical analysis of a new case of selective and generalized ATPase deficiency found in an Austrian patient. The defect manifested with developmental delay, muscle hypotonia, failure to thrive, ptosis, and varying lactic acidemia (up to 12 mmol/L) beginning from the neonatal period. A low-degree dilated cardiomyopathy of the left ventricle developed between the age of 1 and 2 y. A >90% decrease in oligomycin-sensitive ATPase activity and an 86% decrease in the content of the ATPase complex was found in muscle mitochondria. It was associated with a significant decrease of ADP-stimulated respiration of succinate (1.5-fold) and respiratory control with ADP (1.7-fold) in permeabilized muscle fibers, and with a slight decrease of the respiratory chain complex I and compensatory increase in the content of complexes III and IV. The same ATPase deficiency without an increase in respiratory chain complexes was found in fibroblasts, suggesting a generalized defect with tissue-specific manifestation. Absence of any mutations in mitochondrial ATP6 and ATP8 genes indicates a nuclear origin of the defect.

Adenosine Diphosphate↗
PubMed2003

Galanin receptor subtype GalR2 mediates apoptosis in SH-SY5Y neuroblastoma cells.

Recently we have shown that galanin binding significantly correlates with survival in neuroblastoma patients, indicating a possible modulatory role of galanin receptors in neuroblastic tumor biology. However, the molecular mechanisms beyond this correlation have not been elucidated. Here, the cellular effects on activation of specific galanin receptor subtypes in human SH-SY5Y neuroblastoma cells were analyzed using a tetracycline-controlled expression system. Pharmacological studies confirmed the inducible expression of high affinity binding sites for galanin in SH-SY5Y cells transfected with the galanin receptors GalR1 (SY5Y/GalR1) and GalR2 (SY5Y/GalR2). Microphysiometry revealed that both receptor subtypes were able to mediate an intracellular signal upon galanin application. Interestingly, induction of receptor expression and treatment with 100 nm galanin resulted in a dramatic decrease in cell viability in SY5Y/GalR2 cells (93 +/- 3%) compared with a less pronounced effect in SY5Y/GalR1 cells (19 +/- 10%). The antiproliferative potency of galanin was 100-fold higher in SY5Y/GalR2 (50% effective concentration, 1.1 nm) than in SY5Y/GalR1 cells (50% effective concentration, 190 nm). Furthermore, activation of receptor expression and exposure to galanin resulted in apparent morphological changes indicative of apoptosis in SY5Y/GalR2 cells only. Induction of cell death by the apoptotic process was confirmed by poly-(ADP-ribose)-polymerase cleavage, caspase-3 activation, and the typical laddering of DNA. This study indicates that a high level of GalR2 expression is able to inhibit cell proliferation and induce apoptosis in neuroblastoma cells and therefore identifies GalR2 as a possible target for pharmacological intervention in neuroblastoma.

Apoptosis↗
PubMed2003

S-homocysteinylation of transthyretin is detected in plasma and serum of humans with different types of hyperhomocysteinemia.

While the association of homocystinuria with disease is known for more than four decades, mild hyperhomocysteinemia has been detected more recently as a risk factor for a number of diseases. However, the mechanism which apparently renders (even mild) hyperhomocystenemia harmful is not known. Following reports on N-homocysteinylation of proteins by the homocysteine derivative homocysteine thiolactone, it has been suggested that homocysteinylation of proteins may contribute to the induction of biological effects by homocysteine. This has prompted us to study by electrospray ionization mass spectrometry homocysteinylation of transthyretin (TTR) in plasma and serum of humans with different types of hyperhomocysteinemia. We did not detect any N-homocysteinylation, but found pronounced S-homocysteinylation of TTR, if the concentration of total homocysteine was high. Our findings support a possible role of S-homocysteinylation of proteins in the mediation of detrimental effects of hyperhomocysteinemia. Careful study of posttranslational modifications of individual proteins may contribute to a better understanding of diseases associated with hyperhomocysteinemia.

Homocysteine↗
PubMed2003

2-Methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency is caused by mutations in the HADH2 gene.

2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiency is a novel inborn error of isoleucine degradation. In this article, we report the elucidation of the molecular basis of MHBD deficiency. To this end, we purified the enzyme from bovine liver. MALDI-TOF mass spectrometry analysis revealed that the purified protein was identical to bovine 3-hydroxyacyl-CoA dehydrogenase type II. The human homolog of this bovine enzyme is a short-chain 3-hydroxyacyl-CoA dehydrogenase, also known as the "endoplasmic reticulum-associated amyloid-beta binding protein" (ERAB). This led to the identification of the X-chromosomal gene involved, which previously had been denoted "HADH2." Sequence analysis of the HADH2 gene from patients with MHBD deficiency revealed the presence of two missense mutations (R130C and L122V). Heterologous expression of the mutant cDNAs in Escherichia coli showed that both mutations almost completely abolish enzyme activity. This confirms that MHBD deficiency is caused by mutations in the HADH2 gene.

3-Hydroxyacyl CoA Dehydrogenases↗