Mutation and single nucleotide polymorphism detection using temperature gradient capillary electrophoresis

Rapid, high-throughput mutation and single nucleotide polymorphism detection technologies are necessary to identify sequence alterations responsible for human disease. Several screening techniques have been developed as alternatives to the costly and time-consuming task of direct gene sequencing. Unfortunately, many of these techniques have relatively low mutation detection sensitivities and/or require significant up-front assay optimization. Temperature gradient capillary electrophoresis is a relatively new mutation screening technology that capitalizes on the denaturing effects of temperature and the high resolution capacity of capillary electrophoresis to detect heteroduplexes formed between mutant and wild type gene sequences. The utility of temperature gradient capillary electrophoresis for the detection of known sequence alterations and as a tool for mutation discovery is reviewed.     

Complete gene scanning by temperature gradient capillary electrophoresis using the cystic fibrosis transmembrane conductance regulator gene as a model

Many inherited diseases involve large genes with many different mutations. Identifying a wide spectrum of mutations requires an efficient gene-scanning method. By differentiating thermodynamic stability and mobility of heteroduplexes from heterozygous samples, temperature gradient capillary electrophoresis (TGCE) was used to scan the entire coding region of the cystic fibrosis transmembrane conductance regulator gene. An initial panel (29 different mutations) showed 100% agreement between TGCE scanning and previously genotyped results for heterozygous samples. Different peak patterns were observed for single base substitutions and base insertions/deletions. Subsequently, 12 deidentified clinical samples genotyped as wild type for 32 mutations were scanned for the entire 27 exons. Results were 100% concordance with the bidirectional sequence analysis. Ten samples had nucleotide variations including a reported base insertion in intron 14b (2789 + 2insA) resulting in a possible mRNA splicing defect, and an unreported missense mutation in exon 20 (3991 G/A) with unknown clinical significance. This methodology does not require labeled primers or probes for detection and separation through a temperature gradient eliminates laborious temperature optimization required for other technologies. TGCE automation and high-throughput capability can be implemented in a clinical environment for mutation scanning with high sensitivity, thus reducing sequencing cost and effort.     

Genotyping energy-transfer-cassette-labeled short-tandem-repeat amplicons with capillary array electrophoresis microchannel plates

BACKGROUND: Genetic analysis of microsatellite DNA is a powerful tool used in linkage analysis, gene mapping, and clinical diagnosis. To address the expanding needs of studies of short tandem repeats (STRs), we demonstrated high-performance STR analysis on a high-throughput microchannel plate-based platform. METHODS: Energy-transfer-cassette-labeled STR amplicons were separated and typed on a microfabricated 96-channel radial capillary array electrophoresis (CAE) microchannel plate system. Four-color detection was accomplished with a laser-excited confocal fluorescence rotary scanner. RESULTS: Multiplex STR analysis with single base-pair resolution was demonstrated on denaturing polyacrylamide gel media. The high-throughput multiplex capabilities of this genetic analysis platform were demonstrated by the simultaneous separation of STR amplicons representing 122 samples in ninety-six 5.5-cm-long channels in <8 min. Sizing values obtained for these amplicons on the CAE microchannel plate were comparable to those measured on a conventional commercial CAE instrument and exhibit <1% sizing variance. CONCLUSIONS: Energy-transfer-cassette labeling and microfabricated CAE microchannel plates allow high-performance multiplex STR analyses.     

吸烟肺癌患者口腔黏膜细胞全线粒体DNA获得性突变分析吸烟/口腔黏膜获得性突变与康复预防

目的了解肺癌患者口腔黏膜mtDNA获得性突变情况,探讨肺癌外细胞的线粒体基因突变与肺癌的关系和作为对肺癌预测的生物学指标的可能性。方法利用时相温度梯度凝胶电泳法对12例肺癌患者(吸烟者10例,非吸烟者2例)口腔黏膜细胞及匹配的血液细胞线粒体DNA进行突变筛选,然后进行测序分析。结果在大部分吸烟患者中(8/9),发现口腔黏膜具有两个以上的获得性突变,而在两个非吸烟的患者仅发现1个突变。在11例(11/12,92%)患者中共发现有26个获得性突变,15个突变发生在高变的D环区(58%),11个在mRNA区(42%)。除303～309位点具有长度不稳定外,未见其他微卫星不稳定和5kb的大范围缺失突变。结论吸烟的肺癌患者口腔黏膜细胞线粒体DNA具有高发生频率的获得性突变。     

Application of mitochondrial genome information in cancer epidemiology

Two genomes, nuclear and mitochondrial, exist in humans although information contained in the mitochondrial genome has not been fully utilized in cancer epidemiology. Over the last few years, a variety of approaches have been developed to improve results of conventional cancer screening by detecting molecular markers in different populations. Mitochondrial DNA alterations (mutations, deletions and instability) are emerging as new molecular markers for detecting a variety of cancers in tissue samples and biofluids which can be included in population screening studies. Since mitochondrial genome is small (16.6 kb) and high-throughput assays have been developed for sequencing whole mitochondrial genome, it can be adopted by most of the laboratories conducting epidemiological studies. Applications of mitochondrial DNA markers to identify high risk populations and future challenges are discussed in this article.     

Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detection

BACKGROUND: As we enter the post-genome sequencing era and begin to sift through the enormous amount of genetic information now available, the need for technologies that allow rapid, cost-effective, high-throughput detection of specific nucleic acid sequences becomes apparent. Multiplexing technologies, which allow for simultaneous detection of multiple nucleic acid sequences in a single reaction, can greatly reduce the time, cost and labor associated with single reaction detection technologies. METHODS: The Luminex xMAP system is a multiplexed microsphere-based suspension array platform capable of analyzing and reporting up to 100 different reactions in a single reaction vessel. This technology provides a new platform for high-throughput nucleic acid detection and is being utilized with increasing frequency. Here we review specific applications of xMAP technology for nucleic acid detection in the areas of single nucleotide polymorphism (SNP) genotyping, genetic disease screening, gene expression profiling, HLA DNA typing and microbial detection. CONCLUSIONS: These studies demonstrate the speed, efficiency and utility of xMAP technology for simultaneous, rapid, sensitive and specific nucleic acid detection, and its capability to meet the current and future requirements of the molecular laboratory for high-throughput nucleic acid detection.     

Genotyping Parkinson disease-associated mitochondrial polymorphisms

OBJECTIVE: The purpose of this study was to establish a system for rapidly detecting single nucleotide polymorphisms (SNPs) in mitochondrial DNA (mtDNA) using hybridization probes and melting temperature (T(m)) analysis. This technology should prove useful for population-based studies on the interaction between genetic factors and environmental exposures and the risk of Parkinson disease (PD). METHODS: Mitochondrial DNA (mtDNA) was extracted from whole blood. Rapid polymerase chain reaction (PCR) and melting curve analyses were performed with primers and fluorochrome-labeled probes on a LightCycler (Roche Molecular Biochemical, Mannheim, Germany). Genotyping of 10 SNPs in 15 subjects was based on the analysis of allele-specific T(m) of detection probes. The results of melting curve analyses were verified by sequencing all 150 PCR products. RESULTS: Real-time monitoring showed optimal PCR amplification of each mtDNA fragment. The nucleotide changes at positions 1719, 4580, 7028, 8251, 9055, 10398, 12308, 13368, 13708, and 16391 from wild-type to mutant genotype resulted in 6.51, 8.29, 3.26, 7.82, 4.79, 2.84, 2.73, 9.04, 8.53, and 9.52 degrees C declines in T(m) of the detection probes, respectively. Genotyping of all 150 samples was verified by 100% correspondence with the results of sequencing. Fourteen subjects were haplogrouped by combining results for all 10 SNPs. CONCLUSION: A rapid and reliable detection system for identifying mitochondrial polymorphisms and haplotypes was developed based on hybridization probe technology. This method may be suitable for mitochondrial genotyping of samples from large-scale epidemiology studies, and may prove useful for exploring the molecular etiopathogenesis of PD, identifying markers of genetic susceptibility, and protecting susceptible individuals from PD.     

A rapid and accurate approach to identify single nucleotide polymorphisms of mitochondrial DNA using MALDI-TOF mass spectrometry.

BACKGROUND: Single nucleotide polymorphisms (SNPs) of mitochondrial DNA (mtDNA) are involved in physiological and pathological conditions. We developed a rapid, accurate, highly sensitive and high-throughput approach with low cost to identify mtDNA SNPs. METHODS: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to detect 18 SNPs of mtDNA by uniplex and multiplex assays. The sensitivity and specificity of the MALDI-TOF MS were evaluated. The accuracy of the approach was validated by the comparison of using the robust sequencing analysis. RESULTS: The detection limit achieved with the assays corresponded to the identification of five-genome equivalence of mtDNA per reaction after first round PCR amplification. The testing system enabled the discrimination of as little as 5% of mtDNA polymorphism in the predominating background of mtDNA not containing the SNP. No false positive and false negative results were obtained using the uniplex and multiplex MALDI-TOF MS assays for the analysis of the 18 SNPs compared with those obtained by sequencing analysis. CONCLUSIONS: Possible fields which could benefit from this powerful and sensitive tool include forensic medicine, tracing of matrilineage, transplantation immunology, transfusion medicine, the diagnosis of mtDNA mutation related disorders, and the research regarding aging, apoptosis and carcinogenesis based on physiologic and pathogenic alterations of mtDNA for the analysis of large-scale samples, multiple SNPs or rare mtDNA.     

Real-time nucleic acid sequence-based amplification assay to quantify changes in mitochondrial DNA concentrations in cell cultures and blood cells from HIV-infected patients receiving antiviral therapy

BACKGROUND: To study the clinical relevance of changes in mitochondrial DNA (mtDNA) in peripheral blood mononuclear cells (PBMCs) attributable to HIV infection and/or combination antiretroviral therapy (cART), a high-throughput molecular assay to quantify mtDNA is required. METHODS: We developed a quantitative real-time duplex nucleic acid sequence-based amplification assay in which both mtDNA and nuclear DNA are simultaneously amplified in 1 tube. The assay could accurately quantify mtDNA in a range of 15-1500 copies of mtDNA per 2 genomic copies with an intrarun variation of 11% and an interrun variation of 16%. We compared this real-time assay with the lactate/pyruvate ratios in fibroblasts incubated with glucose and exposed to zalcitabine. Additionally, we studied the effects of platelet contamination and the in vivo effects of cART on mtDNA in PBMCs from a small group of patients. RESULTS: Decreases in mtDNA preceded the increase in lactate/pyruvate ratios and vice versa when zalcitabine was eliminated from the culture. Platelets affected the mtDNA in PBMCs if >5 platelets per PBMC were present. Within 12 weeks, mtDNA increased and remained increased in PBMCs from patients on continuous treatment with zidovudine/lamivudine/indinavir therapy (P = 0.03), but increased if patients were switched to stavudine/didanosine therapy (P = 0.008). CONCLUSION: After drug exposure, the mtDNA assay can detect changes in mtDNA concentrations in cell lines and PBMCs, when properly controlled for platelet effects, earlier than traditional assays.     

Single cell PCR from archival stained bone marrow slides: a method for molecular diagnosis and characterization

Molecular analysis of isolated single cells is a powerful tool for clarifying issues of cell origin and clonality. Previous reports have described PCR amplifications from total DNA and RNA extracted from archival bone marrow and peripheral blood smears and have also shown the feasibility of amplifications from single cells, microdissected from stained histological sections. In this study, a method is described for performing PCR from morphologically defined single cells isolated from archival May-Gruenwald-Giemsa-stained bone-marrow and blood smears. Using three DNA extraction procedures, the organic lysis showed reproducible high efficiencies of amplifications. With this method, we were able to amplify long range amplicons up to 14.5 kb from mitochondrial DNA as well as PCR products of conventional length. The usability of such products for molecular diagnosis is demonstrated by restriction fragment length polymorphism (RFLP)characterization of a mitochondrial disorder. In conclusion, this method has the power to perform molecular diagnosis and characterization of diseases on the single cell level, and should provide valuable information to aid disease treatment and prognosis of hematological disorders.     

A protocol for detection of mitochondrial DNA deletions: characterization of a novel deletion

Objectives: To develop a protocol capable of identifying deletions in mitochondrial DNA and use it to identify the breakpoints of a mtDNA deletion in a patient with chronic progressive external ophthalmoplegia (CPEO).

Design and Methods: Deletions in mtDNA were identified by a combination of long range PCR and Southern blotting. The precise breakpoints were determined by automated DNA sequencing.

Results: A series of DNA samples from patients with suspected mitochondrial disease was subjected to a protocol, which combines long range PCR and Southern blotting. We found a unique deletion in a patient with CPEO and we identified the precise location of this deletion through DNA sequencing.

Conclusions: Long range PCR has the advantages of speed, minimal sample requirements, and sensitivity. Southern blotting is better able to evaluate heteroplasmy and detect duplications. We suggest a protocol that enables us to identify precisely the breakpoints in a unique mutation of mtDNA in a patient with CPEO.     

Identifying allogeneic platelets by resolution of point mutations in mitochondrial DNA using single-stranded conformational polymorphism PCR

BACKGROUND: The purpose of this study was to evaluate single-stranded conformational polymorphism (SSCP)-PCR utilizing two different regions of mitochondrial DNA (mtDNA) as a method to discriminate between donor platelets and recipient cells. STUDY DESIGN AND METHODS: Twenty-eight mixtures of platelets (1:1 ratio) were prepared from eight randomly selected persons to simulate donor-recipient combinations after allogeneic platelet transfusion. The mtDNA was extracted from each donor and each prepared mixture. Four primer pairs were designed to amplify two regions of mtDNA, hypervariable region (HVR) 1 and 2. An SSCP-PCR method was developed to analyze the four different amplicons. In addition, the amplified DNA samples containing HVR1 and HVR2 mtDNA of the eight persons were sequenced by using dye-terminator cycle sequencing to determine mtDNA polymorphisms. RESULTS: With four different primer pairs and SSCP-PCR, it was possible to discriminate between donor and recipient DNA in all 28 combinations. DNA sequencing confirmed that the suspected differences were localized within the amplicons examined by SSCP-PCR. CONCLUSION: SSCP-PCR analysis targeting the HVR1 and HVR2 mtDNA is a promising new method to potentially identify donor cells on the basis of mtDNA polymorphisms. The method does not require prior knowledge of sequence differences between donor and recipient and can be optimized to quantify the amount of residual transfused allogeneic platelets.     

Design and use of a peptide nucleic acid for detection of the heteroplasmic low-frequency mitochondrial encephalomyopathy,lactic acidosis,and stroke-like episodes (MELAS) mutation in human mitochondrial DNA

BACKGROUND: Most pathogenic human mitochondrial DNA (mtDNA) mutations are heteroplasmic (i.e., mutant and wild-type mtDNA coexist in the same individual) and are difficult to detect when their concentration is a small proportion of that of wild-type mtDNA molecules. We describe a simple methodology to detect low proportions of the single base pair heteroplasmic mutation, A3243G, that has been associated with the disease mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) in total DNA extracted from blood. METHODS: Three peptide nucleic acids (PNAs) were designed to bind to the wild-type mtDNA in the region of nucleotide position 3243, thus blocking PCR amplification of the wild-type mtDNA while permitting the mutant DNA to become the dominant product and readily discernable. DNA was obtained from both apparently healthy and MELAS individuals. Optimum PCR temperatures were based on the measured ultraviolet thermal stability of the DNA/PNA duplexes. The presence or absence of the mutation was determined by sequencing. RESULTS: In the absence of PNAs, the heteroplasmic mutation was either difficult to detect or undetectable by PCR and sequencing. Only PNA 3 successfully inhibited amplification of the wild-type mtDNA while allowing the mutant mtDNA to amplify. In the presence of PNA 3, we were able to detect the heteroplasmic mutation when its concentration was as low as 0.1% of the concentration of the wild-type sequence. CONCLUSION: This methodology permits easy detection of low concentrations of the MELAS A3243G mutation in blood by standard PCR and sequencing methods.     

Analysis of potential cancer biomarkers in mitochondrial DNA

Understanding mitochondrial biology is a fundamental research goal in human genetics and medicine. The use of mitochondria to serve as a biomarker is rapidly expanding in disciplines ranging from cancer, rare metabolic diseases, aging, the tracing of human migration patterns in antiquity, population characterization using maternal markers, and human identification. Mitochondrial DNA (mtDNA) mutations occur frequently in cancer, and there is an important need for validating mtDNA mutations as cancer biomarkers for the detection of early-stage disease. Although a few studies have suggested tissue-specific mtDNA mutations, there is no single mutational hotspot associated with the wide spectrum of cancer patients; hence, sequencing the entire mitochondrial genome and further characterization of the multiple deletions associated with tumors is required to detect the mutation load on an individual basis. Microarray-based technology provides a reliable and rapid method to detect all mutations of the entire mitochondrial genome. In addition to microarray-based sequencing, real-time PCR is an important method for deletion analysis. Mutations throughout the mitochondrial genome are recurrent events in primary tumor tissues and in corresponding non-invasively collected body fluids. Thus, mtDNA mutation analysis may provide a molecular tool for the early detection and prognosis of cancer. Recent findings have verified that relatively simple diagnostic tests for detecting mtDNA mutations, involving mitochondrial microarray chips and/or real-time PCR bioassays, have exciting predictive potential for cancer detection and prognosis.     

Identification of the multiple beta-thalassemia mutations by denaturing gradient gel electrophoresis.

We used denaturing gradient gel electrophoresis to detect the beta-thalassemia mutations in the Chinese population. By amplifying the beta-globin gene in four separate fragments and electrophoresing the amplified DNA in two gels, we were able to distinguish all the 12 known mutations on the basis of the mobility of the homoduplexes and heteroduplexes. We conclude that denaturing gradient gel electrophoresis offers a nonradioactive means of detecting multiple mutations in genetic disorders.     

A sequence-specific polymerase chain reaction assay for mitochondrial DNA polymorphisms in human platelets and white cells

BACKGROUND: Because mitochondria are abundant in white cells and are also present in platelets, polymorphic sequences in mitochondrial DNA (mtDNA) represent a unique target for polymerase chain reaction (PCR)- based detection of donor material. STUDY DESIGN AND METHODS: A PCR assay was developed that uses sequence-specific primers (SSP) focused on two continent-specific mtDNA polymorphisms. Results were validated by the use of informative restriction endonucleases. Three commercially available methods to extract mtDNA from white cell-reduced human platelets was compared. In preparation for in vivo studies, in vitro mixing studies designed to mimic transfusion were conducted to investigate the performance of the SSP-PCR assay. RESULTS: The gene sequences of two representative examples of amplicons obtained with the new SSP-PCR matched the sequence expected from the published genetic code. Fifteen individuals were classified as either positive (n = 6) or negative (n = 9) for the Asian polymorphism by the use of published primers known to flank the polymorphic site followed by digestion with appropriate restriction enzymes. Results with SSP-PCR were nearly perfectly concordant with those of restriction enzyme analysis. Although the use of three DNA extraction methods allowed the preparation of mtDNA that was suitable for PCR, large and consistent differences (ranging from 10- to 1000-fold) in endpoint sensitivity were found. In vitro mixing studies reproducibly documented that the SSP-PCR assay could detect as little as 1 percent of donor platelets mixed with recipient blood. CONCLUSION: PCR-SSP can be reliably used to identify human mtDNA polymorphisms. By optimization of the method of mtDNA extraction, the sensitivity of PCR-SSP assay was greatly increased. This assay should prove useful in investigations of allogeneic platelet transfusions without cell labeling. It may also be applied to studies of the donor cell microchimerism that follows transfusion or transplantation.     

邯郸市特教学校耳聋患者线粒体12SrRNA基因突变分析

目的利用基因诊断的方法调查邯郸市聋哑学校非综合征型耳聋患者的常见分子病因,对线粒体12SrRNA基因编码区突变进行分析,以明确非综合征型耳聋患者发病的分子学基础,为耳聋基因的筛查和产前诊断提供实验和理论依据。方法用PCR法检查100名聋哑学生（年龄6岁-17岁,男性58例,女性42例）线粒体12SrRNA基因,直接测序法分别对线粒体12SrRNA基因的2个基因片段分析。结果①100例研究对象中,被测者聋前或其母亲孕期明确应用过氨基糖甙类抗生素者30例（30%）,30例中有4例（7.5%）存在线粒体基因A1555G位点突变。②PCR检测结果100例患者的DNA样本（线粒体12SrRNA基因的2个基因片段）进行PCR扩增,扩增产物片段大小与预期相符。③测序结果100名聋哑学校非综合征型耳聋患者中,携带线粒体12SrRNA基因A1555G点突变的10例（占10%）;携带线粒体12SrRNA基因C1494T点突变的2例（占2%）。结论河北省邯郸地区特教学校耳聋患者存在较高的线粒体12SrRNA基因A1555G突变发生率,线粒体12SrRNA基因A1555G突变发生率高于全国平均水平,通过其突变筛查可以有效避免高危人群出现耳聋。     

Assessing the relative incidence of mitochondrial DNA A3243G in migraine without aura with maternal inheritance

OBJECTIVE: To determine whether patients with migraine without aura with maternal "inheritance" are affected by a monosymptomatic form of the MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes) or carry the most common mitochondrial DNA (mtDNA) mutation associated with MELAS, namely the A3243G transition in the transfer RNA (tRNA)Leu(UUR) gene. BACKGROUND: The association between migraine and abnormal mitochondrial function has been suggested on clinical, biochemical, and neuroradiological grounds. Migraine attacks with vomiting and cerebral infarctions, most often in the posterior cerebral regions, which are reminiscent of complicated migraine, are typical features of MELAS. The observation that migrainous patients have affected mothers more often than affected fathers suggests a possible role for maternally transmitted genetic factors. METHODS: We studied 25 patients with migraine with aura whose mothers were also affected. A sensitive polymerase chain reaction restriction fragment length polymorphism analysis was used to detect mutated genomes. CONCLUSIONS: We failed to detect the MELAS mutation, but migraine may still be associated with point mutations of mtDNA other than A3243G or with as-yet-unidentified nuclear DNA factors related to mitochondrial function.     

Modulating mtDNA heteroplasmy by mitochondria-targeted restriction endonucleases in a 'differential multiple cleavage-site' model

The ability to manipulate mitochondrial DNA (mtDNA) heteroplasmy would provide a powerful tool to treat mitochondrial diseases. Recent studies showed that mitochondria-targeted restriction endonucleases can modify mtDNA heteroplasmy in a predictable and efficient manner if it recognizes a single site in the mutant mtDNA. However, the applicability of such model is limited to mutations that create a novel cleavage site, not present in the wild-type mtDNA. We attempted to extend this approach to a 'differential multiple cleavage site' model, where an mtDNA mutation creates an extra restriction site to the ones normally present in the wild-type mtDNA. Taking advantage of a heteroplasmic mouse model harboring two haplotypes of mtDNA (NZB/BALB) and using adenovirus as a gene vector, we delivered a mitochondria-targeted Scal restriction endonuclease to different mouse tissues. Scal recognizes five sites in the NZB mtDNA but only three in BALB mtDNA. Our results showed that changes in mtDNA heteroplasmy were obtained by the expression of mitochondria-targeted ScaI in both liver, after intravenous injection, and in skeletal muscle, after intramuscular injection. Although mtDNA depletion was an undesirable side effect, our data suggest that under a regulated expression system, mtDNA depletion could be minimized and restriction endonucleases recognizing multiple sites could have a potential for therapeutic use.     

Real-time detection and quantification of mitochondrial mutations with oligonucleotide primers containing locked nucleic acid

BACKGROUND: The phenotypic expression of disorders caused by point mutations, deletions or depletions within the mitochondrial genome (mtDNA) is heterogeneous. This relates to the phenomena of heteroplasmy, tissue threshold as well as the distribution of mutant DNA among tissues. Hence, the diagnostics of these disorders demands highly specific, sensitive and quantitative methods. METHODS: We have developed an allele-specific quantitative real-time PCR method for the detection of two of the most prevalent disease causing mitochondrial mutations, m.3243A>G (MELAS) and m.8993T>G (NARP). Locked Nucleic Acid (LNA) modified primers were used to obtain high allele specificity. In order to monitor mtDNA depletion a real-time method for mtDNA/nuclear DNA copy number ratio determination was developed. RESULTS: Rapid and sensitive detection and quantification of MELAS and NARP mtDNA alleles were achieved. Heteroplasmy levels as low as 0.01% could be detected, and the mtDNA/nuclear DNA ratio could be determined. CONCLUSIONS: The present method that allows simultaneous determination of heteroplasmy levels and mtDNA/nuclear DNA copy number ratio, will provide a useful tool in molecular diagnostics and in future epidemiological studies of mitochondrial diseases.