A whole mitochondrial genome screening in a MELAS patient: a novel mitochondrial tRNA(Val) mutation

Mitochondrial encephalopathy, lactic acidosis and strokelike episodes (MELAS) syndrome is a mitochondrial disorder characterized by a wide variety of clinical presentations and a multisystemic organ involvement. In this study, we report a Tunisian girl with clinical features of MELAS syndrome who was negative for the common m.3243A>G mutation, but also for the reported mitochondrial DNA (mtDNA) mutations and deletions. Screening of the entire mtDNA genome showed several known mitochondrial variants besides to a novel transition m.1640A>G affecting a wobble adenine in the anticodon stem region of the tRNA(Val). This nucleotide was conserved and it was absent in 150 controls suggesting its pathogenicity. In addition, no mutations were found in the nuclear polymerase gamma-1 gene (POLG1). These results suggest further investigation nuclear genes encoding proteins responsible for stability and structural components of the mtDNA or to the oxidative phosphorylation machinery to explain the phenotypic variability in the studied family.     

The investigation and diagnosis of pathogenic mitochondrial DNA mutations in human urothelial cells

Patients with mitochondrial DNA disease are amongst the most challenging to diagnose and manage given the striking phenotypic and genetic heterogeneity, which characterise these conditions. Recently, we and others have demonstrated the m.3243A>G mutation, one of the most common mitochondrial DNA pathogenic mutations, is present at clinically relevant levels in urinary epithelium, thus providing a practical, non-invasive test for diagnosis and mutation screening. In this study we further evaluate the use of these cells in detecting the m.3243A>G mutation, other mtDNA tRNA gene point mutations including the m.8344A>G mutation and single large-scale mtDNA deletions. We observe a robust relationship between m.3243A>G levels in urothelial cells and clinically affected tissues that does not change with time. Conversely, single large-scale mtDNA deletions can be detected in urothelial cells, with higher levels present in younger patients with more severe disease, but generally mtDNA deletion levels are not representative of those seen in a clinically affected tissue. Our results have implications for the diagnosis, management and counselling of families with mtDNA disease.     

A specific point mutation in the mitochondrial genome of Caucasians with MELAS

Summary The mitochondrial DNA (mtDNA) of Japanese patients suffering from the syndrome of mitochondrial myopathy, encephalopathy, lactic acidosis and strokelike episodes (MELAS) exhibits a specific heteroplasmic AG transition in the tRNA^Leu at position 3243. In this study, we investigated mtDNA from skeletal muscle, cardiac muscle, brain, liver, diaphragm, fibroblasts and blood cells of four Caucasians with MELAS, one younger healthy sister of two MELAS patients, and eleven controls. We found that 1) the mutation was present in all investigated tissues of Caucasians with MELAS but not in controls, 2) within a single patient, the tissue-specific variation of the copy number of mutated mtDNA covered the same range as in the skeletal muscle of different patients, 3) the mutation was also present in the blood cells of the healthy sister of two MELAS siblings.     

Diseases caused by mutations in mitochondrial DNA

Mitochondrial diseases associated with mutations within mitochondrial genome are a subgroup of metabolic disorders since their common consequence is reduced metabolic efficiency caused by impaired oxidative phophorylation and shortage of ATP. Although the vast majority of mitochondrial proteins (approximately 1500) is encoded by nuclear genome, mtDNA encodes 11 subunits of respiratory chain complexes, 2 subunits of ATP synthase, 22 tRNAs and 2 rRNAs. Up to now, more than 250 pathogenic mutations have been described within mtDNA. The most common are point mutations in genes encoding mitochondrial tRNAs such as 3243A-->G and 8344T-->G that cause, respectively, MELAS (mitochondrial encephalopathy, lactic acidosis and stroke-like episodes) or MIDD (maternally-inherited diabetes and deafness) and MERRF (myoclonic epilepsy with ragged red fibres) syndromes. There have been also found mutations in genes encoding subunits of ATP synthase such as 8993T-->G substitution associated with NARP (neuropathy, ataxia and retinitis pigmentosa) syndrome. It is worth to note that mitochondrial dysfunction can also be caused by mutations within nuclear genes coding for mitochondrial proteins.     

The MELAS mutation m.3243A>G promotes reactivation of fetal cardiac genes and an epithelial-mesenchymal transition-like program via dysregulation of miRNAs

The pathomechanisms underlying oxidative phosphorylation (OXPHOS) diseases are not well-understood, but they involve maladaptive changes in mitochondria-nucleus communication. Many studies on the mitochondria-nucleus cross-talk triggered by mitochondrial dysfunction have focused on the role played by regulatory proteins, while the participation of miRNAs remains poorly explored. MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) is mostly caused by mutation m.3243A>G in mitochondrial tRNA^Leu(UUR) gene. Adverse cardiac and neurological events are the commonest causes of early death in m.3243A>G patients. Notably, the incidence of major clinical features associated with this mutation has been correlated to the level of m.3243A>G mutant mitochondrial DNA (heteroplasmy) in skeletal muscle. In this work, we used a transmitochondrial cybrid model of MELAS (100% m.3243A>G mutant mitochondrial DNA) to investigate the participation of miRNAs in the mitochondria-nucleus cross-talk associated with OXPHOS dysfunction. High-throughput analysis of small-RNA-Seq data indicated that expression of 246 miRNAs was significantly altered in MELAS cybrids. Validation of selected miRNAs, including miR-4775 and miR-218-5p, in patient muscle samples revealed miRNAs whose expression declined with high levels of mutant heteroplasmy. We show that miR-218-5p and miR-4775 are direct regulators of fetal cardiac genes such as NODAL, RHOA, ISL1 and RXRB, which are up-regulated in MELAS cybrids and in patient muscle samples with heteroplasmy above 60%. Our data clearly indicate that TGF-β superfamily signaling and an epithelial-mesenchymal transition-like program are activated in MELAS cybrids, and suggest that down-regulation of miRNAs regulating fetal cardiac genes is a risk marker of heart failure in patients with OXPHOS diseases.     

MELAS syndrome, cardiomyopathy, rhabdomyolysis, and autism associated with the A3260G mitochondrial DNA mutation

The A to G transition mutation at position 3260 of the mitochondrial genome is usually associated with cardiomyopathy and myopathy. One Japanese kindred reported the phenotype of mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS syndrome) in association with the A3260G mtDNA mutation. We describe the first Caucasian cases of MELAS syndrome associated with the A3260G mutation. Furthermore, this mutation was associated with exercise-induced rhabdomyolysis, hearing loss, seizures, cardiomyopathy, and autism in the large kindred. We conclude that the A3260G mtDNA mutation is associated with wide phenotypic heterogeneity with MELAS and other “classical” mitochondrial phenotypes being manifestations.     

The MELAS mutation m.3243A>G alters the expression of mitochondrial tRNA fragments

Recent evidences highlight the importance of mitochondria-nucleus communication for the clinical phenotype of oxidative phosphorylation (OXPHOS) diseases. However, the participation of small non-coding RNAs (sncRNAs) in this communication has been poorly explored. We asked whether OXPHOS dysfunction alters the production of a new class of sncRNAs, mitochondrial tRNA fragments (mt tRFs), and, if so, whether mt tRFs play a physiological role and their accumulation is controlled by the action of mt tRNA modification enzymes. To address these questions, we used a cybrid model of MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), an OXPHOS disease mostly caused by mutation m.3243A>G in the mitochondrial tRNA^Leu(UUR) gene. High-throughput analysis of small-RNA-Seq data indicated that m.3243A>G significantly changed the expression pattern of mt tRFs. A functional analysis of potential mt tRFs targets (performed under the assumption that these tRFs act as miRNAs) indicated an association with processes that involve the most common affected tissues in MELAS. We present evidences that mt tRFs may be biologically relevant, as one of them (mt i-tRF GluUUC), likely produced by the action of the nuclease Dicer and whose levels are Ago2 dependent, down-regulates the expression of mitochondrial pyruvate carrier 1 (MPC1), promoting the build-up of extracellular lactate. Therefore, our study underpins the idea that retrograde signaling from mitochondria is also mediated by mt tRFs. Finally, we show that accumulation of mt i-tRF GluUUC depends on the modification status of mt tRNAs, which is regulated by the action of stress-responsive miRNAs on mt tRNA modification enzymes.     

Screening of common mitochondrial mutations in Chinese patients with mitochondrial encephalomyopathies

To investigate the spectrum of common mitochondrial mutations in Northern China during the years of 2000-2005, 552 patients of mitochondrial encephalomyopathies clinically diagnosed as MELAS, MERRF or Leigh's syndrome, 14 cases of LHON and 46 cases of aminoglycoside induced deafness along with their family members, accepted routine point mutation tests at nucleotide positions 3243, 8344, 8993, 11778 or 1555 in mitochondrial genome. PCR-RFLP analysis, site-specific PCR and PCR-sequencing methods were used to identify the mutations. Fifty-seven cases with A3243G mutation, 4 cases with A8344G, 2 cases with T8993C and 1 case with T8993G were identified from the 552 encephalomyopathy patients. In addition, one case with G11778A was found from the 14 cases of LHON, and 5 cases with A1555G from the 46 cases of aminoglycoside ototoxicity patients. Additional screening for T8356G and T3271C merely had limited significance for the diagnosis of MERRF and MELAS. Differential diagnosis among mitochondrial encephalomyopathies was often complicated due to many similar clinical manifestations. For A3243G mutation, the proportion of mutant mtDNA was not related to severity of the disease but to the age of onset.     

Retinal ganglion cell neurodegeneration in mitochondrial inherited disorders

Since the early days of mitochondrial medicine, it has been clear that optic atrophy is a very common and sometimes the singular pathological feature in mitochondrial disorders. The first point mutation of mitochondrial DNA (mtDNA) associated with the maternally inherited blinding disorder, Leber's hereditary optic neuropathy (LHON), was recognized in 1988. In 2000, the other blinding disorder, dominant optic atrophy (DOA) Kjer type, was found associated with mutations in the nuclear gene OPA1 that encodes a mitochondrial protein. Besides these two non-syndromic optic neuropathies, optic atrophy is a prominent feature in many other neurodegenerative diseases that are now recognized as due to primary mitochondrial dysfunction.We will consider mtDNA based syndromes such as LHON/dystonia/Mitochondrial Encephalomyopahty Lactic Acidosis Stroke-like (MELAS)/Leigh overlapping syndrome, or nuclear based diseases such as Friedreich ataxia (mutations in FXN gene), deafness-dystonia-optic atrophy (Mohr-Tranebjerg) syndrome (mutations in TIMM8A), complicated hereditary spastic paraplegia (mutations in SPG7), DOA “plus” syndromes (mutations in OPA1), Charcot-Marie-Tooth type 2A (CMT2A) with optic atrophy or hereditary motor and sensory neuropathy type VI (HMSN VI) (mutations in MFN2), and Costeff syndrome and DOA with cataract (mutations in OPA3). Thus, genetic errors in both nuclear and mitochondrial genomes often lead to retinal ganglion cell death, a specific target for mitochondrial mediated neurodegeneration. Many mechanisms have been studied and proposed as the bases for the pathogenesis of mitochondrial optic neuropathies including bioenergetic failure, oxidative stress, glutamate toxicity, abnormal mitochondrial dynamics and axonal transport, and susceptibility to apoptosis.     

A novel m.3395A > G missense mutation in the mitochondrial ND1 gene associated with the new tRNA m.4316A > G mutation in a patient with hypertrophic cardiomyopathy and profound hearing loss

Mitochondria are essential for early cardiac development and impaired regulation of mitochondrial function was implicated in congenital heart diseases. We described a newborn girl with hypertrophic cardiomyopathy and profound hearing loss. The mtDNA mutational analysis revealed the presence of known polymorphisms associated to cardiomyopathy and/or hearing loss, and 2 novel heteroplasmic mutations: m.3395A > G (Y30C) occurring in a highly conserved aminoacid of the ND1 gene and the m.4316A > G located in the residue A54 of the tRNA^Ile gene. These 2 novel variations were absent in 150 controls. All these variants may act synergistically and exert a cumulative negative effect on heart function to generate the cardiomyopathy.     

Novel nucleotide changes in mutational analysis of mitochondrial 12SrRNA gene in patients with nonsyndromic and aminoglycoside-induced hearing loss

Mitochondria have essential role in cellular energy metabolism and defects in their function lead to many metabolic diseases. Mitochondrial DNA (mtDNA) mutations have been associated with number diseases such as nonsyndromic and aminoglycoside-induced hearing loss. Mutational screening of entire 12SrRNA and tRNA ^{ser (UCN)} genes in 107 unrelated Iranian patients with amino glycoside-induced and nonsyndromic bilateral hearing loss by direct sequencing analysis method were performed. Twenty different homoplasmic sequence variants were identified; including fifteen common polymorphisms, two putatively pathogenic variants: m.921T>C and m.1005T>C, one 12SrRNA sequence variant m.739C>T and two nucleotides substitution; m.1245T>C and m.1545T>C. Deafness-associated mutation, m.1555A>G, was not found. In our patients we found the mutation 1005 was associated with R haplogroup. These finding show that m.1555A>G mutation is not important in our population. Nucleotide change, m.739C>T, previously reported with very low frequency. We suggested the variation of two nucleotides 1245 and 1545 that localized at conserved site of 12SrRNA may be new candidate for amino glycoside-induced and nonsyndromic hearing impairment associated mutations. However, aminoglycoside exposure is a risk factor for clinical phenotype appearance of these mutations.     

Clinical phenotype, prognosis and mitochondrial DNA mutation load in mitochondrial encephalomyopathies

We studied 42 individuals, including 8 patients with either complete or partial syndrome of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), 8 patients with either complete or partial syndrome of myoclonic epilepsy with ragged-red fibers (MERRF) and 26 maternal family members who carried either the A3243G or A8344G mutation of mitochondrial DNA (mtDNA). Clinical manifestations and prognosis were followed up in the patients harboring the A3243G or A8344G mutation. The relationship between clinical features and proportions of mutant mtDNAs in muscle biopsies, blood cells and/or hair follicles was studied. In the 8 regularly followed patients with the A3243G mutation, 4 died within 1 month to 7 years due to status epilepticus and/or recurrent stroke-like episodes. Two patients developed marked mental deterioration and 2 remained stationary. All of the patients harboring the A8344G mutation were stable or deteriorated slightly, except for 1 patient who died due to brain herniation after putaminal hemorrhage. The A3243G and A8344G mtDNA mutations were heteroplasmic in the muscle biopsies, blood cells and hair follicles of both the probands and their maternal family members. The mean proportion of A3243G mutant mtDNA in the muscle biopsies of the patients with MELAS syndrome (68.5 ± 21.3%, range 33–92%) was significantly higher than that of the asymptomatic family members (37.1 ± 12.6%, range 0–51%). The average proportions of A8344G mutant mtDNA in the muscle biopsies (90.1 ± 3.9%, range 89–95%) and hair follicles (93.9 ± 6.4%, range 84–99%) of the patients with MERRF syndrome were also significantly higher than those of the asymptomatic family members (muscle: 40.3 ± 39.5%, range 1–80%; hair follicles: 51.0 ± 44.5%, range 0.1–82%). We concluded that measurement of the proportion of mutant mtDNA in muscle biopsies may provide useful information in the identification of symptomatic patients with mitochondrial encephalomyopathies. For patients with the A3243G mutation, the prognosis was related to status epilepticus and the number of recurrent stroke-like episodes and was much worse than for patients with the A8344G mutation of mtDNA, who had stable or slowly deteriorating clinical courses.     

Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid chromatography

In patients with mitochondrial disease a continuously increasing number of mitochondrial DNA (mtDNA) mutations and polymorphisms have been identified. Most pathogenic mtDNA mutations are heteroplasmic, resulting in heteroduplexes after PCR amplification of mtDNA. To detect these heteroduplexes, we used the technique of denaturing high performance liquid chromatography (DHPLC). The complete mitochondrial genome was amplified in 13 fragments of 1–2 kb, digested in fragments of 90–600 bp and resolved at their optimal melting temperature. The sensitivity of the DHPLC system was high with a lowest detection of 0.5% for the A8344G mutation. The muscle mtDNA from six patients with mitochondrial disease was screened and three mutations were identified. The first patient with a limb-girdle-type myopathy carried an A3302G substitution in the tRNA^Leu(UUR) gene (70% heteroplasmy), the second patient with mitochondrial myopathy and cardiomyopathy carried a T3271C mutation in the tRNA^Leu(UUR) gene (80% heteroplasmy) and the third patient with Leigh syndrome carried a T9176C mutation in the ATPase6 gene (93% heteroplasmy). We conclude that DHPLC analysis is a sensitive and specific method to detect heteroplasmic mtDNA mutations. The entire automatic procedure can be completed within 2 days and can also be applied to exclude mtDNA involvement, providing a basis for subsequent investigation of nuclear genes.     

β-Lapachone attenuates mitochondrial dysfunction in MELAS cybrid cells

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is a mitochondrial disease caused by mutations in the mitochondrial genome. This study investigated the efficacy of β-lapachone (β-lap), a natural quinone compound, in rescuing mitochondrial dysfunction in MELAS cybrid cells. β-Lap significantly restored energy production and mitochondrial membrane potential as well as normalized the elevated ROS level in MELAS cybrid cells. Additionally, β-lap reduced lactic acidosis and restored glucose uptake in the MELAS cybrid cells. Finally, β-lap activated Sirt1 by increasing the intracellular NAD⁺/NADH ratio, which was accompanied by increased mtDNA content. Two other quinone compounds (idebenone and CoQ10) that have rescued mitochondrial dysfunction in previous studies of MELAS cybrid cells had a minimal effect in the current study. Taken together, these results demonstrated that β-lap may provide a novel therapeutic modality for the treatment of MELAS.     

A Clinical,Neuropathological and Genetic Study of Homozygous A467T POLG-Related Mitochondrial Disease

Mutations in the nuclear gene POLG (encoding the catalytic subunit of DNA polymerase gamma) are an important cause of mitochondrial disease. The most common POLG mutation, A467T, appears to exhibit considerable phenotypic heterogeneity. The mechanism by which this single genetic defect results in such clinical diversity remains unclear. In this study we evaluate the clinical, neuropathological and mitochondrial genetic features of four unrelated patients with homozygous A467T mutations. One patient presented with the severe and lethal Alpers-Huttenlocher syndrome, which was confirmed on neuropathology, and was found to have a depletion of mitochondrial DNA (mtDNA). Of the remaining three patients, one presented with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), one with a phenotype in the Myoclonic Epilepsy, Myopathy and Sensory Ataxia (MEMSA) spectrum and one with Sensory Ataxic Neuropathy, Dysarthria and Ophthalmoplegia (SANDO). All three had secondary accumulation of multiple mtDNA deletions. Complete sequence analysis of muscle mtDNA using the MitoChip resequencing chip in all four cases demonstrated significant variation in mtDNA, including a pathogenic MT-ND5 mutation in one patient. These data highlight the variable and overlapping clinical and neuropathological phenotypes and downstream molecular defects caused by the A467T mutation, which may result from factors such as the mtDNA genetic background, nuclear genetic modifiers and environmental stressors.     

Mutation analysis of mitochondrial 12S rRNA gene in Polish patients with non-syndromic and aminoglycoside-induced hearing loss

Mutations in mitochondrial DNA have been reported as associated with non-syndromic and aminoglycoside-induced hearing loss. In the present study, we have performed mutational screening of entire 12S rRNA gene in 250 unrelated patients with non-syndromic and aminoglycoside-induced hearing loss. Twenty-one different homoplasmic sequence variants were identified, including eight common polymorphisms, one deafness-associated mutation m.1555 A>G and three putatively pathogenic variants: m.669 T>C, m.827 A>G, m.961 delT+C(n)ins. The incidence of m.1555 A>G was estimated for 3.6% (9/250); however, where aminoglycoside exposure was taken as a risk factor, the frequency was 5.5% (7/128). Substitution m.669 T>C was identified only in patients with hearing impairment and episode of aminoglycoside exposure, which may suggest that such additional risk factors must appear to induce clinical phenotype. Moreover, two 12S rRNA sequence variants: m.988 G>A and m.1453 A>G, localized at conserved sites and affected RNA secondary structure, may be new candidates for non-syndromic and aminoglycoside-induced hearing loss associated mutations.