Biomedical subjects

David Meierhofer

Publications and source records attributed to David Meierhofer.

4 recordsLinked to original sources

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↗

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↗

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↗

PubMed2002

Severe depletion of mitochondrial DNA in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disorder in childhood leading to a dramatic loss of muscle strength. Functional investigations with high-resolution polarography and enzyme measurements of the respiratory chain revealed lowered activities in muscle tissue of SMA patients. To gain a better understanding of this low energy supply we analyzed the amount of mitochondrial DNA (mtDNA) in skeletal muscle of 20 unrelated children with genetically proven SMA and 31 controls. Quantitative Southern blot analysis revealed a severe and homogeneous decrease in the content of muscle mtDNA in relation to nuclear DNA in SMA patients (90.3+/-7.8%), whereas by immunofluorescence no decrease in the number of mitochondria was detected. In addition, a two- to threefold reduction of the nuclear-encoded complex II (succinate dehydrogenase) activity was detected in SMA muscle tissue. Western blot analysis showed a significant reduction of both mitochondrial- and nuclear-encoded cytochrome c oxidase subunits. Our results indicate that mtDNA depletion in SMA is a consequence of severe atrophy, and has to be differentiated by measurement of complex II from an isolated reduction of mtDNA as found in patients with mitochondriocytopathies and the so-called mtDNA depletion syndrome.

Adolescent↗