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PubMed2006

Controlled clinical trial of dichloroacetate for treatment of congenital lactic acidosis in children.

OBJECTIVE: Open-label studies indicate that oral dichloroacetate (DCA) may be effective in treating patients with congenital lactic acidosis. We tested this hypothesis by conducting the first double-blind, randomized, control trial of DCA in this disease. METHODS: Forty-three patients who ranged in age from 0.9 to 19 years were enrolled. All patients had persistent or intermittent hyperlactatemia, and most had severe psychomotor delay. Eleven patients had pyruvate dehydrogenase deficiency, 25 patients had 1 or more defects in enzymes of the respiratory chain, and 7 patients had a mutation in mitochondrial DNA. Patients were preconditioned on placebo for 6 months and then were randomly assigned to receive an additional 6 months of placebo or DCA, at a dose of 12.5 mg/kg every 12 hours. The primary outcome results were (1) a Global Assessment of Treatment Efficacy, which incorporated tests of neuromuscular and behavioral function and quality of life; (2) linear growth; (3) blood lactate concentration in the fasted state and after a carbohydrate meal; (4) frequency and severity of intercurrent illnesses and hospitalizations; and (5) safety, including tests of liver and peripheral nerve function. OUTCOME: There were no significant differences in Global Assessment of Treatment Efficacy scores, linear growth, or the frequency or severity of intercurrent illnesses. DCA significantly decreased the rise in blood lactate caused by carbohydrate feeding. Chronic DCA administration was associated with a fall in plasma clearance of the drug and with a rise in the urinary excretion of the tyrosine catabolite maleylacetone and the heme precursor delta-aminolevulinate. CONCLUSIONS: In this highly heterogeneous population of children with congenital lactic acidosis, oral DCA for 6 months was well tolerated and blunted the postprandial increase in circulating lactate. However, it did not improve neurologic or other measures of clinical outcome.

Acidosis, Lactic↗
PubMed2005

Loss of editing activity during the evolution of mitochondrial phenylalanyl-tRNA synthetase.

Accurate selection of amino acids is essential for faithful translation of the genetic code. Errors during amino acid selection are usually corrected by the editing activity of aminoacyl-tRNA synthetases such as phenylalanyl-tRNA synthetases (PheRS), which edit misactivated tyrosine. Comparison of cytosolic and mitochondrial PheRS from the yeast Saccharomyces cerevisiae suggested that the organellar protein might lack the editing activity. Yeast cytosolic PheRS was found to contain an editing site, which upon disruption abolished both cis and trans editing of Tyr-tRNA(Phe). Wild-type mitochondrial PheRS lacked cis and trans editing and could synthesize Tyr-tRNA(Phe), an activity enhanced in active site variants with improved tyrosine recognition. Possible trans editing was investigated in isolated mitochondrial extracts, but no such activity was detected. These data indicate that the mitochondrial protein synthesis machinery lacks the tyrosine proofreading activity characteristic of cytosolic translation. This difference between the mitochondria and the cytosol suggests that either organellar protein synthesis quality control is focused on another step or that translation in this compartment is inherently less accurate than in the cytosol.

Adenosine Triphosphate↗
PubMed1996

Extent of heterogeneity in mitochondrial DNA of ethnic Asian populations.

Variation in the mitochondrial DNA (mtDNA) control region as detected by sequence-specific oligonucleotide (SSO) probes is described for 993 individuals in 11 ethnic Asian populations. Estimates of diversity for mtDNA types exceed 0.94 in all populations, while 53% of the 255 types that were observed occur only once. Of 96 shared types, four occur at frequencies of greater than 10% but less than 17% in any one population. There is statistically significant heterogeneity among these 11 populations, however, an analysis of variance incorporating genetic distances between types shows that at least 95% of the variation present in the total sample is attributable to within-population diversity, while only 5% is due to between-population differences. Overall, heterogeneity with respect to mtDNA SSO types is grossly correlated with geographic distance between populations; the most extreme heterogeneity was observed between populations from East Asia and populations from West Asia. With respect to population genetics, the control region of mtDNA exhibits satisfactory qualities as a DNA typing locus.

Analysis of Variance↗
PubMed2003

Assessment of precision and concordance of quantitative mitochondrial DNA assays: a collaborative international quality assurance study.

BACKGROUND: A number of international research groups have developed DNA quantitation assays in order to investigate the role of mitochondrial DNA depletion in anti-retroviral therapy-induced toxicities. OBJECTIVES: A collaborative study was undertaken to evaluate intra-assay precision and between laboratory concordance of measurements of mitochondrial DNA quantity, as a component of a comprehensive quality assurance project. STUDY DESIGN: Four laboratories were asked to measure and report mitochondrial DNA and nuclear DNA genome copy number, as well as mitochondrial DNA copy number/cell, for 17 coded aliquots of DNA derived from serial dilutions of pooled DNA from a lymphoblastoid cell line. Samples included masked replicates and five standards. All samples had similar mitochondrial DNA/nuclear DNA ratios. Precision within laboratories was assessed by determining the coefficient of variation of replicates. Concordance between laboratories was assessed by determining the average coefficient of variation of the mean replicate values for each sample. The effect of standardising the assay for these three measurements was also assessed for laboratories A, B and C. RESULTS: Measurements of mitochondrial DNA and nuclear DNA content for replicate samples varied by an average of less than 6% (based on log(10) values, 72% non-logged values), and measurements of mitochondrial DNA/cell for replicates varied by less than 12% (based on log(10) values, 32% non-logged values), with no improvement of precision after standardisation. Standardisation did significantly improve the concordance of results for measurements of mitochondrial DNA content and mitochondrial DNA/cell. Non-standardised measurements of mitochondrial DNA content for the same sample set varied by 19% between laboratories (based on log(10) values, 96% non-logged values), and after standardisation results varied by less than 3% (based on log(10) values, 54% non-logged values). There was no significant improvement for concordance of measures of nuclear DNA content after standardisation, with results varying by 4.56% between laboratories (based on log(10) values, 45% non-logged values) before standardisation, and by 2.49% (based on log(10) values, 50% non-logged values) after standardisation. Derived values of mitochondrial DNA/cell varied between laboratories by an average of 91% (non-logged, 56% log(10) values) before and by 56% (non-logged, 13% log(10) values) after standardisation. CONCLUSION: All assays demonstrated good precision. The use of common standards is an important step in improving the comparability of data between laboratories.

Cell Line↗
PubMed2006

Freezing complete polymerase chain reaction master mix reagents for routine molecular diagnostics.

The potential of storing complete frozen real-time polymerase chain reactions (PCRs) and real-time reverse transcription PCRs (RT-PCRs), which require only thawing and the addition of template nucleic acid before PCR cycling, was examined. Master mixes containing all necessary reagents at working concentration (except template nucleic acid) were aliquoted into single-reaction volumes and stored at -70 degrees C for periods of up to 8 months. Reactions were removed from storage and nucleic acid template was added and amplified using different real-time PCR instruments. Threshold cycle values were used to monitor changes in assay performance after storage. Results for hybridization probe and TaqMan probe assays showed that freezing complete real-time PCR and RT-PCR reaction mixes was possible without deterioration in assay performance. This approach has advantages for routine molecular diagnostics in areas such as convenience, test consistency, quality control, and ease of use by nonspecialist staff.

Animals↗
PubMed2004

Gene expression profiling in mitochondrial disease: assessment of microarray accuracy by high-throughput Q-PCR.

Mitochondrial diseases are a heterogeneous array of disorders with a complex etiology. Use of microarrays as a tool to investigate complex human disease is increasingly common, however, a principle drawback of microarrays is their limited dynamic range, due to the poor quantification of weak signals. Although it is generally understood that low-intensity microarray 'spots' may be unreliable, there exists little documentation of their accuracy. Quantitative PCR (Q-PCR) is frequently used to validate microarray data, yet few Q-PCR validation studies have focused on the accuracy of low-intensity microarray signals. Hence, we have used Q-PCR to systematically assess microarray accuracy as a function of signal strength in a mouse model of mitochondrial disease, the superoxide dismutase 2 (SOD2) nullizygous mouse. We have focused on a unique category of data--spots with only one weak signal in a two-dye comparative hybridization--and show that such 'high-low' signal intensities are common for differentially expressed genes. This category of differential expression may be more important in mitochondrial disease in which there are often mosaic expression patterns due to the idiosyncratic distribution of mutant mtDNA in heteroplasmic individuals. Using RNA from the SOD2 mouse, we found that when spotted cDNA microarray data are filtered for quality (low variance between many technical replicates) and spot intensity (above a negative control threshold in both channels), there is an excellent quantitative concordance with Q-PCR (R2 = 0.94). The accuracy of gene expression ratios from low-intensity spots (R2 = 0.27) and 'high-low' spots (R2 = 0.32) is considerably lower. Our results should serve as guidelines for microarray interpretation and the selection of genes for validation in mitochondrial disorders.

Journal Article↗
PubMed2003

Misfolding, degradation, and aggregation of variant proteins. The molecular pathogenesis of short chain acyl-CoA dehydrogenase (SCAD) deficiency.

Short chain acyl-CoA dehydrogenase (SCAD) deficiency is an inborn error of the mitochondrial fatty acid metabolism caused by rare variations as well as common susceptibility variations in the SCAD gene. Earlier studies have shown that a common variant SCAD protein (R147W) was impaired in folding, and preliminary experiments suggested that the variant protein displayed prolonged association with chaperonins and delayed formation of active enzyme. Accordingly, the molecular pathogenesis of SCAD deficiency may rely on intramitochondrial protein quality control mechanisms, including degradation and aggregation of variant SCAD proteins. In this study we investigated the processing of a set of disease-causing variant SCAD proteins (R22W, G68C, W153R, R359C, and Q341H) and two common variant proteins (R147W and G185S) that lead to reduced SCAD activity. All SCAD proteins, including the wild type, associate with mitochondrial hsp60 chaperonins; however, the variant SCAD proteins remained associated with hsp60 for prolonged periods of time. Biogenesis experiments at two temperatures revealed that some of the variant proteins (R22W, G68C, W153R, and R359C) caused severe misfolding, whereas others (R147W, G185S, and Q341H) exhibited a less severe temperature-sensitive folding defect. Based on the magnitude of in vitro defects, these SCAD proteins are characterized as folding-defective variants and mild folding variants, respectively. Pulse-chase experiments demonstrated that the variant SCAD proteins either triggered proteolytic degradation by mitochondrial proteases or, especially at elevated temperature, aggregation of non-native conformers. The latter finding may indicate that accumulation of aggregated SCAD proteins may play a role in the pathogenesis of SCAD deficiency.

Animals↗
PubMed2000

Aggregate formation and degradation of overexpressed wild-type and mutant urate oxidase proteins. Quality control of organelle-destined proteins by the endoplasmic reticulum.

To analyze the cellular response caused by the overexpression of proteins in subcellular compartments, we constructed four expression clones encoding wild-type peroxisomal urate oxidase (UO), truncated UO lacking the peroxisomal targeting signal (UOdC), and chimeric UOs with a mitochondrial targeting signal (MTS) at the N-terminus of UOdC (MUOdC) or UO (MUO). After transfection, we examined COS-1 and HEK293 cells by immunofluorescence and immunoelectron microscopy, transmission electron microscopy, and pulse-chase experiments. The overexpressed UO and UOdC formed large electron-dense aggregates with no limiting membrane in both the cytoplasm and the nucleus. The UO aggregates exhibited the crystalloid structure quite similar to that of rat liver peroxisomal cores, whereas the UOdC aggregates formed a loose mass consisting of small dense substructures. The overexpressed MUOdC and MUO, on the other hand, formed other types of aggregates which were distributed in the cytoplasm. They consisted of tubular and circular membrane structures, which were morphologically confirmed to be derived from the endoplasmic reticulum (ER). No immunolabeling signals for MUOdC and MUO were present free in the cytoplasm and most of them were associated with membrane structures, suggesting that overexpressed UO containing the MTS attached to the ER membranes soon after synthesis and segregated from the cytosolic compartment. All the UO aggregates were stained for ubiquitin antigen. Pulse-chase experiments in combination with proteasome inhibitors suggested that proteasomes did not contribute to the degradation of these products.

Animals↗
PubMed2002

Results of the 1999-2000 collaborative exercise and proficiency testing program on mitochondrial DNA of the GEP-ISFG: an inter-laboratory study of the observed variability in the heteroplasmy level of hair from the same donor.

The Spanish and Portuguese working group (GEP) of international society for forensic genetics (ISFG) 1999-2000 collaborative exercise on mitochondrial DNA (mtDNA) included the analysis of four bloodstain samples and one hair shaft sample by 19 participating laboratories from Spain, Portugal and several Latin-American countries. A wide range of sequence results at position 16,093 of the HV1 (from T or C homoplasmy to different levels of heteroplasmy) were submitted by the different participating laboratories from the hair shaft sample during the first phase of this exercise. During the discussion of these results in the Annual GEP-ISFG 2000 Conference a second phase of this exercise was established with two main objectives: (i) to evaluate the incidence of the HV1 sequence heteroplasmy detected in Phase I across different sample types from the same donor including blood, saliva, and hair shafts, (ii) to perform a technical review of the electropherograms to evaluate the relative levels of heteroplasmies obtained by the different laboratories and also to examine the source of possible errors detected in Phase I. Anonymous review of the raw sequence data permitted the detection of three transcription errors and three errors due to methodological problems. Highly variable levels of heteroplasmy were found in the hair shaft and more stability in blood and saliva. Three laboratories found variable levels of heteroplasmy at position 16,093 across adjacent fragments from the same hair shaft. Two laboratories also described more than one heteroplasmic position from a single hair. The relevance of these findings for the interpretation of mtDNA data in the forensic context is also discussed.

Blood Stains↗
PubMed2006

East Asian mtDNA haplogroup determination in Koreans: haplogroup-level coding region SNP analysis and subhaplogroup-level control region sequence analysis.

The present study analyzed 21 coding region SNP markers and one deletion motif for the determination of East Asian mitochondrial DNA (mtDNA) haplogroups by designing three multiplex systems which apply single base extension methods. Using two multiplex systems, all 593 Korean mtDNAs were allocated into 15 haplogroups: M, D, D4, D5, G, M7, M8, M9, M10, M11, R, R9, B, A, and N9. As the D4 haplotypes occurred most frequently in Koreans, the third multiplex system was used to further define D4 subhaplogroups: D4a, D4b, D4e, D4g, D4h, and D4j. This method allowed the complementation of coding region information with control region mutation motifs and the resultant findings also suggest reliable control region mutation motifs for the assignment of East Asian mtDNA haplogroups. These three multiplex systems produce good results in degraded samples as they contain small PCR products (101-154 bp) for single base extension reactions. SNP scoring was performed in 101 old skeletal remains using these three systems to prove their utility in degraded samples. The sequence analysis of mtDNA control region with high incidence of haplogroup-specific mutations and the selective scoring of highly informative coding region SNPs using the three multiplex systems are useful tools for most applications involving East Asian mtDNA haplogroup determination and haplogroup-directed stringent quality control.

Asian People↗
PubMed2002

Noninvasive molecular tracking of colonizing wolf (Canis lupus) packs in the western Italian Alps.

We used noninvasive methods to obtain genetic and demographic data on the wolf packs (Canis lupus), which are now recolonizing the Alps, a century after their eradication. DNA samples, extracted from presumed wolf scats collected in the western Italian Alps (Piemonte), were genotyped to determine species and sex by sequencing parts of the mitochondrial DNA (mtDNA) control-region and ZFX/ZFY genes. Individual genotypes were identified by multilocus microsatellite analyses using a multiple tubes polymerase chain reaction (PCR). The performance of the laboratory protocols was affected by the age of samples. The quality of excremental DNA extracts was higher in samples freshly collected on snow in winter than in samples that were older or collected during summer. Preliminary mtDNA screening of all samples allowed species identification and was a good predictor of further PCR performances. Wolf, and not prey, DNA targets were preferentially amplified. Allelic dropout occurred more frequently than false alleles, but the probability of false homozygote determinations was always < 0.001. A panel of six to nine microsatellites would allow identification of individual wolf genotypes, also whether related, with a probability of identity of < 0.015. Genealogical relationships among individuals could be determined reliably if the number of candidate parents was 6-8, and most of them had been sampled and correctly genotyped. Genetic data indicate that colonizing Alpine wolves originate exclusively from the Italian source population and retain a high proportion of its genetic diversity. Spatial and temporal locations of individual genotypes, and kinship analyses, suggest that two distinct packs of closely related wolves, plus some unrelated individuals, ranged in the study areas. This is in agreement with field observations.

Animals↗
PubMed2006

[Application of flow cytometry to the evaluation of semen quality].

OBJECTIVE: To evaluate the role of flow cytometry in semen assessment. METHODS: Semen samples from 104 infertile male patients (as the case group) and 10 fertilized donors (as the control group) were analyzed for the volume of ejaculate and sperm concentration, motility and atypical morphology by computer-assisted semen analysis (CASA), the viability, chromatin structure and mitochondrial membrane potential (MMP) of the sperm stained by SYBR-14/PI, AO and JC-1 respectively, and assessed with flow cytometry. The results were analyzed through SAS software. RESULTS: U tests indicated that the semen from the infertile patients had not only lower concentration (U = 2.51, P = 0.0143), lower motility (U = 3.44, P = 0.001) and higher rate of atypical morphology (U = -5.88, P < 0.0001), but also lower viability (U = 4.72, P < 0.0001), MMP (U = -2.53, P = 0.0309), and chromatin integrity (alpha t: U = -3.82, P = 0.0003; SD alpha t: U = -3.98, P = 0.0001; COMP alpha t: U = -3.57, P = 0.0005). The multiple stepwise regression analysis revealed that sperm motility was positively correlated with sperm membrane integrity (t = 1.66, P = 0.1016), sperm MMP (t = 3.33, P = 0.0014) and sperm acrosome integrity (t = 3.24, P = 0.0019), while sperm MMP was negatively correlated with the rates of sperm neck and tail defects (t = -3.44, P = 0.001). CONCLUSION: Flow cytometry plays a significant role in the evaluation of the quality of human sperm, and can be adopted as a useful tool in the diagnosis and treatment of male infertility.

Adult↗
PubMed2007

The role of mitochondrial function in the oocyte and embryo.

Mitochondria have long been known to be the powerhouses of the cell but they also contribute to redox and Ca2+ homeostasis, provide intermediary metabolites and store proapoptotic factors. Mitochondria have a unique behavior during development. They are maternally transmitted with little (if any) paternal contribution, and they originate from a restricted founder population, which is amplified during oogenesis. Then, having established the full complement of mitochondria in the fully grown oocyte, there is no further increase of the mitochondrial population during early development. The localization of mitochondria in the egg during maturation and their segregation to blastomeres in the cleaving embryo are strictly regulated. Gradients in the distribution of mitochondria present in the egg have the potential to give rise to blastomeres receiving different numbers of mitochondria. Such maternally inherited differences in mitochondrial distribution are thought to play roles in defining the long-term viability of the blastomere in some cases and embryonic axes and patterning in others. Mitochondria may also regulate development by a number of other means, including modulating Ca2+ signaling, and the production of ATP, reactive oxygen species, and intermediary metabolites. If the participation of mitochondria in the regulation of sperm-triggered Ca2+ oscillations is now well established, the role of other properties of mitochondrial function during development remain largely unexplored probably due to the difficulty of accessing the mitochondrial compartment in an embryo. Maintaining a functional complement of maternally derived mitochondria is vital for the early embryo. Mitochondrial dysfunction may not only compromise developmental processes but also trigger apoptosis in the embryo. This dual role for mitochondria (to maintain life or to commit to cell death) may well represent a quality control system in the early embryo that will determine whether the embryo proceeds further into development or is quickly eliminated.

Adenosine Triphosphate↗
PubMed2003

Oocyte competence in repeat-breeder heifers: effects of an optimized ovum pick-up schedule on expression of oestrus, follicular development and fertility.

Repeat-breeder heifers (RBH) have been shown to present reproductive perturbations during spontaneous cyclicity, which affects oestrus and ovulation. Some of these disturbances (e.g. deviating hormone patterns) are also present during and after cycles of twice-weekly ovum pick-up (o.p.u.), performed according to an optimized schedule allowing normal oestrous cyclicity. In the present study, the effects of o.p.u. on oocyte competence in in vitro maturation (i.v.m.) and in vitro fertilization (i.v.f.) have been evaluated, as were the effects on expression of oestrus and fertility in five RBH (> or =4 artificial inseminations) and five virgin heifers (VH controls). In total, 269 RBH and 174 VH oocytes were scored for quality prior to i.v.m. and i.v.f. The number of follicles available for puncture was higher in RBH, but the oocyte recovery rate after o.p.u. was lower in RBH compared with VH controls and the recovered RBH oocytes were of lower quality, as judged by their appearance at retrieval. Confocal laser scanning and transmission electron microscopy of immature oocytes did not reveal any differences between RBH and VH control oocytes with respect to nuclear and mitochondrial status. However, after i.v.m., the cytoplasmic spatial reorganization of mitochondria and cortical granules was less advanced in RBH, which could contribute to the subfertility that defines the syndrome. Cleavage rates after i.v.f. were similar in RBH and VH controls. Subsequent to the o.p.u. period, in vivo fertility after controlled artificial insemination was comparable with field fertility rates in both RBH and VH.

Animals↗
PubMed2025

Genome-wide phylogeny reshapes our understanding of the evolution of deep-sea dragonfishes, bristlemouths, viperfishes, and allies (Stomiiformes).

BACKGROUND: The evolutionary relationships within Stomiiformes, a diverse order of deep-sea fishes dominating the mesopelagic and bathypelagic zones, remain contentious due to conflicting morphological and molecular evidence. These fishes, comprising 464 species across four traditionally recognized families (Gonostomatidae, Sternoptychidae, Phosichthyidae, and Stomiidae), exhibit remarkable adaptations such as bioluminescence, ultra-black pigmentation, and extreme jaw morphologies. Their global abundance and ecological significance, including contributions to the biological carbon pump, underscores the need to resolve their phylogeny amid escalating threats from climate change and human activities. RESULTS: We conducted the most comprehensive phylogenomic analysis of Stomiiformes to date, integrating 936 nuclear loci from 60 species and an expanded dataset of 135 species with mitochondrial sequences from publicly available repositories such as the Barcode of Life Data Systems (BOLD) database. We used maximum likelihood and coalescent-based approaches to assess family monophyly and relationships, including extensive quality control to address contamination in public databases. Our analyses reveal unstable tree topologies and complex evolutionary histories that challenge traditional classifications, while our quality control analyses identified 29% of BOLD sequences as misidentified or contaminated, emphasizing rigorous curation for deep-sea taxa. Congruent with a recent taxonomic treatment of Stomiiformes, the families Phosichthyidae and Gonostomatidae exhibit polyphyly and paraphyly, respectively, while subfamilies within Stomiidae are extensively non-monophyletic, leading us to recommend their abandonment. We propose the recognition of eight monophyletic families: Vinciguerriidae, Diplophidae, Gonostomatidae, Yarrellidae, Ichthyococcidae, Phosichthyidae, Sternoptychidae, and Stomiidae, supported by robust molecular and morphological evidence. CONCLUSIONS: This revised classification reflects the morphological and ecological diversity of Stomiiformes, aligning with their evolutionary diversification in the deep sea. Our phylogenomic framework resolves longstanding systematic uncertainties and highlights the power of genome-wide data in tackling taxonomically challenging clades. These findings provide a foundation for understanding deep-sea fish diversification and assessing the potential ecological drivers for their evolutionary diversity.

Animals↗
PubMed2006

Application of a quasi-median network analysis for the visualization of character conflicts to a population sample of mitochondrial DNA control region sequences from southern Germany (Ulm).

Entire mtDNA control region sequences from 100 individuals in a west Eurasian population sample from southern Germany (around the city of Ulm) were generated and analyzed. The control region was amplified in one piece and sequenced with ten different sequencing primers. Sequence evaluation was performed independently. Phylogenetic analyses were used for quality assurance purposes and for the determination of the haplogroup affiliation of the samples. The sequences were scrutinized performing a quasi-median network analysis. To visualize character conflicts, frequent mutations were filtered, and the reduced data were represented by the torso of their quasi-median network. Character incompatibilities were found to be based on real biological patterns of homoplasy. The population data will be incorporated in the EMPOP database ( http://www.empop.org ).

DNA, Mitochondrial↗
PubMed2004

Simple and rapid detection of uncoupling protein-2 - 866G/A polymorphism by mutagenically separated polymerase chain reaction.

BACKGROUND: Uncoupling protein-2 (UCP2), a recently identified member of the mitochondrial transporter superfamily, is a candidate gene for obesity. A common G/A polymorphism in the UCP2 promoter region is associated with enhanced adipose tissue mRNA expression in vivo. METHODS: We developed a rapid and simple method, mutagenically separated polymerase chain reaction (MS-PCR) for genotyping UCP2 - 866G/A polymorphism. Two reverse mutagenic allele-specific primers of different lengths for the UCP2 - 866G/A polymorphic site were paired with the same forward primer in the same PCR reaction. RESULTS: Agarose gel electrophoresis (3.5%) showed at least one of the two allelic products and provided a within-assay quality control to exclude false-negative results. The 203-bp fragment of the PCR products was A allele-specific and the 183-bp fragment was G allele-specific. The frequencies of the UCP2 - 866G/A genotypes in 72 Japanese subjects were AA: 21 (29.2%), AG: 32 (44.4%), and GG: 19 (26.4%). The results were confirmed by the PCR-RFLP genotyping method, in which a 360-bp fragment of PCR products was cut into 290- and 70-bp fragments by the restriction enzyme MluI when the G allele was present. This Japanese group showed a higher frequency of the AA genotype, which is associated with a low prevalence of obesity, than Caucasian populations. CONCLUSIONS: The MS-PCR technique is a simple, rapid, and reliable method for genotyping UCP2 - 866G/A polymorphism.

Adolescent↗
PubMed2005

Exercise training in mitochondrial myopathy: a randomized controlled trial.

Patients with mitochondrial myopathies (MM) usually suffer from exercise intolerance due to their impaired oxidative capacity and physical deconditioning. We evaluated the effects of a 12-week supervised randomized rehabilitation program involving endurance training in patients with MM. Twenty MM patients were assigned to a training or control group. For three nonconsecutive days each week, patients combined cycle exercise at 70% of their peak work rate with three upper-body weight-lifting exercises performed at 50% of maximum capacity. Training increased maximal oxygen uptake (28.5%), work output (15.5%), and minute ventilation (40%), endurance performance (62%), walking distance in shuttle walking test (+95 m), and peripheral muscle strength (32%-62%), and improved Nottingham Health Profile scores (21.47%) and clinical symptoms. Control MM patients did not change from baseline. Results show that our exercise program is an adequate training strategy for patients with mitochondrial myopathy.

Adolescent↗