Pathogenic mtDNA Mutations Research Articles

Mitochondrial autophagy in cells with mtDNA mutations results from synergistic loss of transmembrane potential and mTORC1 inhibition

Autophagy has emerged as a key cellular process for organellar quality control, yet this pathway apparently fails to eliminate mitochondria containing pathogenic mutations in mitochondrial DNA (mtDNA) in patients with a variety of human diseases. In order to explore how mtDNA-mediated mitochondrial dysfunction interacts with endogenous autophagic pathways, we examined autophagic status in a panel of human cytoplasmic hybrid (cybrid) cell lines carrying a variety of pathogenic mtDNA mutations. We found that both genetic- and chemically induced loss of mitochondrial transmembrane potential (Δψ(m)) caused recruitment of the pro-mitophagic factor Parkin to mitochondria. Strikingly, however, the loss of Δψ(m) alone was insufficient to prompt delivery of mitochondria to the autophagosome (mitophagy). We found that mitophagy could be induced following treatment with the mTORC1 inhibitor rapamycin in cybrids carrying either large-scale partial deletions of mtDNA or complete depletion of mtDNA. Further, we found that the level of endogenous Parkin is a crucial determinant of mitophagy. These results suggest a two-hit model, in which the synergistic induction of both (i) mitochondrial recruitment of Parkin following the loss of Δψ(m) and (ii) mTORC1-controlled general macroautophagy is required for mitophagy. It appears that mitophagy can be accomplished by the endogenous autophagic machinery, but requires the full engagement of both of these pathways.

Large-Scale Screening of Mitochondrial DNA Mutations Among Iranian Patients with Prelingual Nonsyndromic Hearing Impairment

Hereditary hearing impairment (HI) is a genetically heterogeneous disorder caused by mutations either in nuclear DNA (nDNA) or in mitochondrial DNA (mtDNA). The nDNA mutations account for the majority of prelingual nonsyndromic HI (NSHI). The present survey was conducted to screen for known pathogenic mtDNA mutations including A1555G, A3243G, C1494T, and A7445G to provide an accurate estimate of their prevalence in prelingual NSHI for the first time in the Iranian subpopulations. One thousand unrelated probands with NSHI (including both GJB2-negative and GJB2 heterozygote cases) and 1000 healthy matched controls were investigated using the PCR/RFLP method followed by DNA sequencing to confirm the observed mtDNA mutations. Two of the studied mutations, namely A3243G and A7445G, were each found in a single family (a frequency of 0.1% for each). Mutation screening for A3243G followed by DNA sequencing led to the identification of G3316A substitution, with no prior link to HI. Surprisingly, screening for A3243G in the studied population identified 6 cases (0.6%) in probands and 10 (1%) in normal subjects. A1555G, the most common mtDNA mutation associated with deafness in other populations, was not found in the studied samples. To conclude, our findings indicate G3316A as a nonpathogenic variant in the prelingual NSHI subpopulations of Iran and suggest that mtDNA mutations do not play a major role in the etiology of NSHI in Iran.

Prevalence of Deafness in Children with Mitochondrial DNA Disease

Background: An estimated 840 children are born with sensori-neural hearing loss (SNHL) each year in the UK and in some a causative genetic defect is identified. Many children ‘acquire’ a hearing deficit during childhood, which may be a consequence of an inherited mitochondrial DNA (mtDNA) mutation. The association between SNHL and mitochondrial disease is well established in adults, although the correlation between genotype and phenotype remains unclear. Aims: To determine the prevalence of SNHL in children with mtDNA disease; and to assess correlation between mutation load and severity of SNHL. Methods: Retrospective analysis of a validated mitochondrial disease rating scale and audiology in 34 patients aged < 30yrs with disease presenting in childhood, attending a specialist clinic was performed. Heteroplasmy level in urine was determined for each patient. Results: 11 patients had SNHL (6 female, 5 male). Patients harbouring the m.3243A>G mutation formed the largest cohort (8/11 patients), with the highest prevalence in patients harbouring the same mutation (8/21 patients). The earliest age of onset of SNHL was in the m.3243A>G group (2 years) but this did not predict severity or progression of hearing loss. Heteroplasmy levels did not predict onset or severity of hearing loss. Conclusions: Several genotypes recognised in mitochondrial disease are associated with SNHL in childhood. Both onset in infancy, and a gradual progression to deafness in later childhood, is recognised. In those children with recognised SNHL, heteroplasmy level does not predict the severity. Formal audiology is advised in all children harbouring pathogenic mtDNA mutations.

Mutations in mitochondrial DNA associated with hypertension

Mutations in mitochondrial DNA (mtDNA) are one of the molecular bases of hypertension. Among these, the tRNAMet A4435G, tRNAMet/tRNAGln A4401G, tRNAIle A4263G, T4291C and A4295G mutations have been reported to be associated with essential hypertension. These mutations alter the structure of the corresponding mitochondrial tRNAs and cause failures in tRNA metabolism. These shortages of these tRNAs lead to an impairment of mitochondrial protein synthesis and a failure in the oxidative phosphorylation function. These result in a deficit in ATP synthesis and an increase of generation of reactive oxygen species. As a result, these mitochondrial dysfunctions may contribute to the development of hypertension. Furthermore, the tissue specificity of these pathogenic mtDNA mutations might be associated with tRNA metabolism and nuclear modifier genes. These mtDNA mutations should be considered as inherited risk factors for future molecular diagnosis. Thus, these findings provide new insights into the molecular mechanism, management and treatment of maternally inherited hypertension. This review summarized the association between mtDNA mutations and hypertension.

Maternally inherited mitochondrial DNA disease in consanguineous families

Mitochondrial respiratory chain disease represents one of the most common inborn errors of metabolism and is genetically heterogeneous, with biochemical defects arising from mutations in the mitochondrial genome (mtDNA) or the nuclear genome. As such, inheritance of mitochondrial respiratory chain disease can either follow dominant or recessive autosomal (Mendelian) inheritance patterns, the strictly matrilineal inheritance observed with mtDNA point mutations or X-linked inheritance. Parental consanguinity in respiratory chain disease is often assumed to infer an autosomal recessive inheritance pattern, and the analysis of mtDNA may be overlooked in the pursuit of a presumed nuclear genetic defect. We report the histochemical, biochemical and molecular genetic investigations of two patients with suspected mitochondrial disease who, despite being born to consanguineous first-cousin parents, were found to harbour well-characterised pathogenic mtDNA mutations, both of which were maternally transmitted. Our findings highlight that any diagnostic algorithm for the investigation of mitochondrial respiratory chain disease must include a full and complete analysis of the entire coding sequence of the mitochondrial genome in a clinically relevant tissue. An autosomal basis for respiratory chain disease should not be assumed in consanguineous families and that 'maternally inherited consanguineous' mitochondrial disease may thus be going undiagnosed.

Organ-specific shifts in mtDNA heteroplasmy following systemic delivery of a mitochondria-targeted restriction endonuclease

Most pathogenic mtDNA mutations are heteroplasmic and there is a clear correlation between high levels of mutated mtDNA in a tissue and pathology. We have found that in vivo double-strand breaks (DSBs) in mtDNA lead to digestion of cleaved mtDNA and replication of residual mtDNA. Therefore, if DSB could be targeted to mutations in mtDNA, mutant genomes could be eliminated and the wild-type mtDNA would repopulate the cells. This can be achieved by using mitochondria-targeted restriction endonucleases as a means to degrade specific mtDNA haplotypes in heteroplasmic cells or tissues. In this work, we investigated the potential of systemic delivery of mitochondria-targeted restriction endonucleases to reduce the proportion of mutant mtDNA in specific tissues. Using the asymptomatic NZB/BALB mtDNA heteroplasmic mouse as a model, we found that a mitochondria-targeted ApaLI (that cleaves BALB mtDNA at a single site and does not cleave NZB mtDNA) increased the proportion of NZB mtDNA in target tissues. This was observed in heart, using a cardiotropic adeno-associated virus type-6 (AAV6) and in liver, using the hepatotropic adenovirus type-5 (Ad5). No mtDNA depletion or loss of cytochrome c oxidase activity was observed in any of these tissues. These results show the potential of systemic delivery of viral vectors to specific organs for the therapeutic application of mitochondria-targeted restriction enzymes in mtDNA disorders.

Expanding the clinical phenotype of the mitochondrial m.13513G&gt;A mutation with the first report of a fatal neonatal presentation: importance of DNA collection for post-mortem diagnosis

The evolving symbiotic union of infectious prokaryotes and eukaryotic hosts has led to the presence of mitochondria within cells. The mitochondria are now recognized to be of central importance in the milieu of cellular metabolism and transport where they carry out a variety of functions, ranging from disposal of toxic substances to the storage of excess energy as fat. The ubiquity of mitochondria results in the potential for any tissue to dysfunction in the context of an inborn metabolic defect of the respiratory chain. The universal role of the mitochondria is in the process of oxidative phosphorylation (OXPHOS), which is the transformation of energy from the breakdown of nutrients in the presence of oxygen to the synthesis of adenosine triphotphate (ATP). After the discovery of the first human mitochondrial disorders and realization that mutations in mitochondrial and nuclear DNA are associated with these conditions, a new field was born.1 Mitochondrial diseases are difficult to identify because of the variable presentations involving multiple combinations of organ systems, age at onset, and range of severity from mild to severe.2 Mitochondrial disorders may begin at birth or not occur until adult life, often precipitated by illness or other significant metabolic stressors. Debilitating or fatal forms are more frequent in children than in adults, but adults often have chronic multi-systemic manifestations requiring symptomatic treatment and long-term surveillance to minimize life-threatening episodes of acute illness. The central and peripheral nervous systems engender some of the body’s most metabolically demanding cells, highly dependent upon ATP produced by OXPHOS. Thus, the nervous system is more often frequently involved in patients with mitochondrial disease. Symptoms differ depending on the part of the nervous system affected, although almost any neurological symptom can be due to mitochondrial disease. For clinicians to predict and ultimately prevent transmission of mitochondrial disorders knowledge about mitochondrial genetics and cell biology of mitochondrial DNA (mtDNA) inheritance is critical, in particular the genetic bottleneck which accounts for the segregation of mtDNA in the female germline. A key aspect of mitochondrial transmission genetics, whose importance has only begun to be recognized rather recently, concerns the highly dynamic nature of the mitochondria, which undergo fission, fusion, and intracellular movement in the majority of cell types. van Karnebeek et al.3 present a fatal presentation of the mitochondrial m.13513G>A mutation with the first report of a fatal neonatal presentation. This mutation was initially reported as causal of mitochondrial encephalopathy with lactic acidosis and stroke-like episodes, but subsequently has been described in patients with other phenotypes with hypotonia, ptosis, and ataxia being the recognizable symptoms. In the newborn, the repertoire of responses to illness are typically limited and include poor feeding, poor temperature regulation, tone, and central nervous system effects such as seizures and encephalopathy. If the infant dies prior to diagnosis, valuable information may be preserved if blood and tissues are obtained for post-mortem diagnosis. This information will be helpful to bring closure as well as to provide genetic counseling for other family members, either those currently suspected to have a mitochondrial disorder or those at risk. Given the advances in mitochondrial genetics, mutations are being recognized with increasing pace. What was once unknown a year ago, may be understood to be due to a defined sequence alteration in DNA.4, 5 Therefore, in acutely ill patient cases where the diagnosis is suspected, but not proven, it is valuable to collect blood for future studies. This blood can be used for DNA banking, as a resource for future mtDNA and nuclear gene testing. DNA from non-invasive sample types such as hair follicles, buccal cells, and urine epithelial cells can also be useful, as the sensitivity of testing for some mtDNA mutations is higher in these sample types than in blood and is also due to heteroplasmy. Since most pathogenic mtDNA mutations are heteroplasmic, quantification of mutation heteroplasmy is important to correlate with disease expression. Availability of lower-cost, higher-throughput technologies for DNA analysis will facilitate mitochondrial diagnostic investigations.

Placing mitochondrial DNA mutations within the progression model of type I endometrial carcinoma

Mitochondrial DNA (mtDNA) mutations have been described in almost all types of cancer. However, their exact role and timing of occurrence during tumor development and progression are still a matter of debate. A Vogelstein-like model of progression is well established for endometrial carcinoma (EC), however, mtDNA has been scarcely investigated in these tumors despite the fact that mitochondrial biogenesis increase has been shown to be a hallmark of type I EC. Here, we screened a panel of 23 type I EC tissues and matched typical hyperplasia for mutations in mtDNA and in four oncosupressors/oncogenes, namely PTEN, KRAS, CTNNB1 and TP53. Overall, mtDNA mutations were identified in 69% of cases, while mutational events in nuclear genes occurred in 56% of the cases, indicating that mtDNA mutations may precede the genetic instability of these genes canonically involved in progression from hyperplasia to tumor. Protein expression analysis revealed an increase in mitochondrial biogenesis and activation of oxidative stress response mechanisms in tumor tissues, but not in hyperplasia, in correlation with the occurrence of pathogenic mtDNA mutations. Our results point out an involvement of mtDNA mutations in EC progression and explain the increase in mitochondrial biogenesis of type I EC. Last, since mtDNA mutations occur after hyperplasia, their potential role in contributing to genetic instability may be envisioned.

Mitochondrial disease is a common cause of severe cardiomyopathy in children

AimsRespiratory chain abnormalities have been documented in children with idiopathic cardiomyopathy undergoing diagnostic endomyocardial biopsy.1 Mitochondrial disease can present with isolated cardiomyopathy or prominent cardiac involvement in multisystem disease, but...

Phenotypic spectrum of m.3243A>G mitochondrial DNA mutation in children

BackgroundMitochondrial disease are multi-system disorders affecting virtually any organ and cause significant morbidity. The prevalence of mitochondrial DNA (mtDNA) disease is estimated at 1 in 5000, but epidemiological studies suggest...

Respiratory chain complex I deficiency caused by mitochondrial DNA mutations

Defects of the mitochondrial respiratory chain are associated with a diverse spectrum of clinical phenotypes, and may be caused by mutations in either the nuclear or the mitochondrial genome (mitochondrial DNA (mtDNA)). Isolated complex I deficiency is the most common enzyme defect in mitochondrial disorders, particularly in children in whom family history is often consistent with sporadic or autosomal recessive inheritance, implicating a nuclear genetic cause. In contrast, although a number of recurrent, pathogenic mtDNA mutations have been described, historically, these have been perceived as rare causes of paediatric complex I deficiency. We reviewed the clinical and genetic findings in a large cohort of 109 paediatric patients with isolated complex I deficiency from 101 families. Pathogenic mtDNA mutations were found in 29 of 101 probands (29%), 21 in MTND subunit genes and 8 in mtDNA tRNA genes. Nuclear gene defects were inferred in 38 of 101 (38%) probands based on cell hybrid studies, mtDNA sequencing or mutation analysis (nuclear gene mutations were identified in 22 probands). Leigh or Leigh-like disease was the most common clinical presentation in both mtDNA and nuclear genetic defects. The median age at onset was higher in mtDNA patients (12 months) than in patients with a nuclear gene defect (3 months). However, considerable overlap existed, with onset varying from 0 to >60 months in both groups. Our findings confirm that pathogenic mtDNA mutations are a significant cause of complex I deficiency in children. In the absence of parental consanguinity, we recommend whole mitochondrial genome sequencing as a key approach to elucidate the underlying molecular genetic abnormality.

Mitochondrial DNA haplogroups and mutations in children with acquired central demyelination

We investigated mitochondrial DNA (mtDNA) variants in children with a first episode of acquired demyelinating syndromes (PD-ADS) of the CNS and their relationship to disease phenotype, including subsequent diagnosis of multiple sclerosis (MS). This exploratory analysis included the initial 213 children with PD-ADS in the prospective Canadian Pediatric Demyelinating Study and 166 matched healthy sibling controls from the Canadian Autism Genome Project. A total of 31 single nucleotide polymorphisms (SNPs) were analyzed, including haplogroup-defining SNPs and mtDNA variants previously reported to be associated with MS. Primary Leber hereditary optic neuropathy (LHON) mutations and other known pathogenic mtDNA mutations were absent in both patients with pediatric acquired demyelinating syndromes and controls. The 13708A haplogroup J-associated variant, previously linked to adult MS, was more frequent among subjects with PD-ADS (13.0%) compared to controls (6.2%; odds ratio [OR] 2.27; 95% confidence interval [CI] 1.06 to 4.83) and haplogroup M was associated with an earlier age at onset of PD-ADS (-1.74 years; 95% CI -3.33 to -0.07). In contrast, the haplogroup cluster UKJT, as well as 3 other SNPs, were each associated with a lower risk of PD-ADS. A total of 33 subjects with PD-ADS were diagnosed with MS during a mean follow-up period of 3.11 ± 1.14 (SD) years. No single SNP was associated with the risk of subsequent diagnosis of MS. However, haplogroup H was associated with an increased risk of MS (OR 2.60; 95% CI 1.21 to 5.55). These data suggest an association between mtDNA variants and the risk of PD-ADS and of a subsequent MS diagnosis. Replication of these findings in an independent population of subjects with PD-ADS is required.

Leber's Hereditary Optic Neuropathy-Gene Therapy: From Benchtop to Bedside

Leber's hereditary optic neuropathy (LHON) is a maternally transmitted disorder caused by point mutations in mitochondrial DNA (mtDNA). Most cases are due to mutations in genes encoding subunits of the NADH-ubiquinone oxidoreductase that is Complex I of the electron transport chain (ETC). These mutations are located at nucleotide positions 3460, 11778, or 14484 in the mitochondrial genome. The disease is characterized by apoplectic, bilateral, and severe visual loss. While the mutated mtDNA impairs generation of ATP by all mitochondria, there is only a selective loss of retinal ganglion cells and degeneration of optic nerve axons. Thus, blindness is typically permanent. Half of the men and 10% of females who harbor the pathogenic mtDNA mutation actually develop the phenotype. This incomplete penetrance and gender bias is not fully understood. Additional mitochondrial and/or nuclear genetic factors may modulate the phenotypic expression of LHON. In a population-based study, the mtDNA background of haplogroup J was associated with an inverse relationship of low-ATP generation and increased production of reactive oxygen species (ROS). Effective therapy for LHON has been elusive. In this paper, we describe the findings of pertinent published studies and discuss the controversies of potential strategies to ameliorate the disease.

MtDNA disease for the neurologist

Inherited and acquired mutations of mtDNA cause an extraordinary group of diseases that are associated with a diverse panoply of neurological and non-neurological features. These diseases are surprisingly common and are often severely debilitating and readily transmitted through families. Remarkable advances in understanding molecular mechanisms have been made since the first pathogenic mtDNA mutations were identified in 1988, and while widely available genetic techniques have facilitated diagnosis, the complexities of mitochondrial genetics leave the neurologist facing important challenges in recognizing, managing and counseling patients with mtDNA mutations. In this article, we will discuss the clinical phenotypes associated with mtDNA disease, current diagnostic strategies, disease management and genetic counseling, as well as presenting new developments in preventing disease transmission and secondary complications.

Gimap3 Regulates Tissue-Specific Mitochondrial DNA Segregation

Mitochondrial DNA (mtDNA) sequence variants segregate in mutation and tissue-specific manners, but the mechanisms remain unknown. The segregation pattern of pathogenic mtDNA mutations is a major determinant of the onset and severity of disease. Using a heteroplasmic mouse model, we demonstrate that Gimap3, an outer mitochondrial membrane GTPase, is a critical regulator of this process in leukocytes. Gimap3 is important for T cell development and survival, suggesting that leukocyte survival may be a key factor in the genetic regulation of mtDNA sequence variants and in modulating human mitochondrial diseases.

Multiplex analysis of mitochondrial DNA pathogenic and polymorphic sequence variants

The mitochondrial DNA (mtDNA) encompasses two classes of functionally important sequence variants: recent pathogenic mutations and ancient adaptive polymorphisms. To rapidly and cheaply evaluate both classes of single nucleotide variants (SNVs), we have developed an integrated system in which mtDNA SNVs are analyzed by multiplex primer extension using the SNaPshot system. A multiplex PCR amplification strategy was used to amplify the entire mtDNA, a computer program identifies optimal extension primers, and a complete global haplotyping system is also proposed. This system genotypes SNVs on multiplexed mtDNA PCR products or directly from enriched mtDNA samples and can quantify heteroplasmic variants down to 0.8% using a standard curve. With this system, we have developed assays for testing the common pathogenic mutations in four multiplex panels: two genotype the 13 most common pathogenic mtDNA mutations and two genotype the 10 most common Leber Hereditary Optic Neuropathy mutations along with haplogroups J and T. We use a hierarchal system of 140 SNVs to delineate the major global mtDNA haplogroups based on a global phylogenetic tree of coding region polymorphisms. This system should permit rapid and inexpensive genotyping of pathogenic and lineage-specific mtDNA SNVs by clinical and research laboratories.

A novel mitochondrial tRNA Glu ( MTTE) gene mutation causing chronic progressive external ophthalmoplegia at low levels of heteroplasmy in muscle

Mitochondrial respiratory chain defects are associated with diverse clinical phenotypes in both adults and children, and may be caused by mutations in either nuclear or mitochondrial DNA (mtDNA). We report the molecular genetic investigations of a patient with chronic progressive external ophthalmoplegia (CPEO) and myopathy where muscle biopsies taken 11 years apart revealed a progressive increase in the proportion of cytochrome c oxidase (COX)-deficient fibres. Mitochondrial genetic analysis of the early biopsy had seemingly excluded both mtDNA rearrangements and mtDNA point mutations. Sequencing mtDNA from individual COX-deficient muscle fibres in the second biopsy, however, identified an unreported m.14723 T > C substitution within the mitochondrial tRNA Glu ( MTTE) gene, which fulfilled all canonical criteria for pathogenicity. The m.14723 T > C mutation was absent from several tissues, including muscle, from maternal relatives suggesting a de novo event, whilst quantitative analysis of the first muscle biopsy confirmed a very low level of the mutation (7% mutated mtDNA), highlighting a potential problem whereby pathogenic mtDNA mutations may remain undetected using established screening methodologies.

Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease

Mitochondrial DNA (mtDNA) mutations are a common cause of genetic disease with pathogenic mtDNA mutations being detected in approximately 1 in 250 live births1-3 and at least 1 in 10,000 adults in the UK affected by mtDNA disease4. Treatment options for patients with mtDNA disease are extremely limited and are predominantly supportive in nature. MtDNA is transmitted maternally and it has been proposed that nuclear transfer techniques may be an approach to prevent the transmission of human mtDNA disease5,6. Here we show that transfer of pronuclei between abnormally fertilised human zygotes results in minimal carry-over of donor zygote mtDNA and is compatible with onward development to the blastocyst stage in vitro. By optimising the procedure we found the average level of carry-over following transfer of two pronuclei is <2.0%, with many of the embryos containing no detectable donor mtDNA. We believe that pronuclear transfer between zygotes, as well as the recently described metaphase II spindle transfer, has potential to prevent the transmission of mtDNA disease in humans.

Organ-specific shifts in mtDNA heteroplasmy following systemic delivery of a mitochondria-targeted restriction endonuclease

Most pathogenic mtDNA mutations are heteroplasmic and there is a clear correlation between high levels of mutated mtDNA in a tissue and pathology. We have found that in vivo double strand breaks (DSB) in mtDNA lead to digestion of cleaved mtDNA and replication of residual mtDNA. Therefore, if DSB could be targeted to mutations in mtDNA, mutant genomes could be eliminated and the wild-type mtDNA would repopulate the cells. This can be achieved by using mitochondria-targeted restriction endonucleases as a means to degrade specific mtDNA haplotypes in heteroplasmic cells or tissues. In the present work we investigated the potential of systemic delivery of mitochondria-targeted restriction endonucleases to reduce the proportion of mutant mtDNA in specific tissues. Using the asymptomatic NZB/BALB mtDNA heteroplasmic mouse as a model, we found that a mitochondria-targeted ApaLI (that cleaves BALB mtDNA at a single site and does not cleave NZB mtDNA) increased the proportion of NZB mtDNA in target tissues. This was observed in heart, using a cardiotropic adeno-associated virus type-6 (AAV6) and in liver, using the hepatotropic adenovirus type-5 (Ad5). No mtDNA depletion or loss of cytochrome c oxidase activity was observed in any of these tissues. These results demonstrate the potential of systemic delivery of viral vectors to specific organs for the therapeutic application of mitochondria-targeted restriction enzymes in mtDNA disorders.

Mutational analysis of whole mitochondrial DNA in patients with MELAS and MERRF diseases

Mitochondrial diseases are clinically and genetically heterogeneous disorders, which make the exact diagnosis and classification difficult. The purpose of this study was to identify pathogenic mtDNA mutations in 61 Korean unrelated families (or isolated patients) with MELAS or MERRF. In particular, the mtDNA sequences were completely determined for 49 patients. From the mutational analysis of mtDNA obtained from blood, 5 confirmed pathogenic mutations were identified in 17 families, and 4 unreported pathogenically suspected mutations were identified in 4 families. The m.3243A>G in the tRNA(Leu(UUR))was predominantly observed in 10 MELAS families, and followed by m.8344A>G in the tRNA(Lys) of 4 MERRF families. Most pathogenic mutations showed heteroplasmy, and the rates were considerably different within the familial members. Patients with a higher rate of mutations showed a tendency of having more severe clinical phenotypes, but not in all cases. This study will be helpful for the molecular diagnosis of mitochondrial diseases, as well as establishment of mtDNA database in Koreans.