A novel mutation in the mitochondrial tRNA(Ser(AGY)) gene associated with mitochondrial myopathy, encephalopathy, and complex I deficiency

Purpose

To identify molecular defects in a girl with clinical features of MELAS (mitochondrial encephalomyopathy and lactic acidosis) and MERRF (ragged‐red fibres) syndromes.

Methods

The enzyme complex activities of the mitochondrial respiratory chain were assayed. Temporal temperature gradient gel electrophoresis was used to scan the entire mitochondrial genome for unknown mitochondrial DNA (mtDNA) alterations, which were then identified by direct DNA sequencing.

Results

A novel heteroplasmic mtDNA mutation, G12207A, in the tRNA^Ser(AGY) gene was identified in the patient who had a history of developmental delay, feeding difficulty, lesions within her basal ganglia, cerebral atrophy, proximal muscle weakness, increased blood lactate, liver dysfunction, and fatty infiltration of her muscle. Muscle biopsy revealed ragged red fibres and pleomorphic mitochondria. Study of skeletal muscle mitochondria revealed complex I deficiency associated with mitochondrial proliferation. Real time quantitative PCR analysis showed elevated mtDNA content, 2.5 times higher than normal. The tRNA^Ser(AGY) mutation was found in heteroplasmic state (92%) in the patient''s skeletal muscle. It was not present in her unaffected mother''s blood or in 200 healthy controls. This mutation occurs at the first nucleotide of the 5′ end of tRNA, which is involved in the formation of the stem region of the amino acid acceptor arm. Mutation at this position may affect processing of the precursor RNA, the stability and amino acid charging efficiency of the tRNA, and overall efficiency of protein translation.

Conclusion

This case underscores the importance of comprehensive mutational analysis of the entire mitochondrial genome when a mtDNA defect is strongly suggested.     

Mitochondrial DNA Polymerase γ Mutations and Their Implications in mtDNA Alterations in Colorectal Cancer

Mitochondrial DNA was found to be highly mutated in colorectal cancer cells. One of the key molecules involved in the maintenance of the mitochondrial genome is the nuclear‐encoded polymerase gamma. The aim of our study was to determine if there is a link between polymorphisms within the polymerase gamma gene (POLG) and somatic mutations within the mitochondrial genome in cancer cells. We investigated POLG sequence variability in 50 colorectal cancer patients whose complete mitochondrial genome sequences were determined. Relative mtDNA copy number was also determined. We identified 251 sequence variants in the POLG gene. Most of them were germline‐specific (～92%). Twenty‐one somatic changes in POLG were found in 10 colorectal cancer patients. We have found no association between the occurrence of mtDNA somatic mutations and the somatically occurring variants in POLG. MtDNA content was reduced in patients carrying somatic variants in POLG or germline nucleotide variants located in the region encoding the POLG polymerase domain, but the difference did not reach statistical significance. Our findings suggest that somatic mtDNA mutations occurring in colorectal cancer are not a consequence of somatic mutations in POLG. Nevertheless, POLG nucleotide variants may lead to a decrease in mtDNA content, and consequently result in mitochondrial dysfunction.     

Mitochondrial genome architecture in non‐alcoholic fatty liver disease

Non‐alcoholic fatty liver disease (NAFLD) is associated with mitochondrial dysfunction, a decreased liver mitochondrial DNA (mtDNA) content, and impaired energy metabolism. To understand the clinical implications of mtDNA diversity in the biology of NAFLD, we applied deep‐coverage whole sequencing of the liver mitochondrial genomes. We used a multistage study design, including a discovery phase, a phenotype‐oriented study to assess the mutational burden in patients with steatohepatitis at different stages of liver fibrosis, and a replication study to validate findings in loci of interest. We also assessed the potential protein‐level impact of the observed mutations. To determine whether the observed changes are tissue‐specific, we compared the liver and the corresponding peripheral blood entire mitochondrial genomes. The nuclear genes POLG and POLG2 (mitochondrial DNA polymerase‐γ) were also sequenced. We observed that the liver mtDNA of patients with NAFLD harbours complex genomes with a significantly higher mutational (1.28‐fold) rate and degree of heteroplasmy than in controls. The analysis of liver mitochondrial genomes of patients with different degrees of fibrosis revealed that the disease severity is associated with an overall 1.4‐fold increase in mutation rate, including mutations in genes of the oxidative phosphorylation (OXPHOS) chain. Significant differences in gene and protein expression patterns were observed in association with the cumulative number of OXPHOS polymorphic sites. We observed a high degree of homology (～98%) between the blood and liver mitochondrial genomes. A missense POLG p.Gln1236His variant was associated with liver mtDNA copy number. In conclusion, we have demonstrated that OXPHOS genes contain the highest number of hotspot positions associated with a more severe phenotype. The variability of the mitochondrial genomes probably originates from a common germline source; hence, it may explain a fraction of the ‘missing heritability’ of NAFLD. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Metabolic consequences of a novel missense mutation of the mtDNA CO I gene

We have identified a novel heteroplasmic C6489A missense mutation in the mitochondrial DNA (mtDNA) CO I gene encoding the cytochrome c oxidase (COX) subunit I in a 17-year-old girl with epilepsia partialis continua. This point mutation leads to an exchange of the highly conserved Leu196 to Ileu196. Muscle biopsy showed in single fibers decreased COX activity and lowered binding of COX antibodies, indicating decreased stability of the mutated enzyme. The analysis of blood mtDNA revealed about 30% mutant mtDNA in the patients blood but about 90% mutant mtDNA in the blood of two non-affected family members. Quantitative analysis of the mutation gene dose effect on COX activity on single muscle fiber level revealed a very high threshold-a COX deficiency was observed only in fibers containing >95% mutant mtDNA. In apparent contrast to this high mutation gene dose threshold, in vivo investigations of mitochondrial function in saponin-permeabilized muscle fibers of the index patient containing approximately 90% mutated mtDNA showed decreased maximal rates of respiration and an increased sensitivity of fiber respiration to cyanide. This is due to a 2-fold increase of COX flux control on muscle fiber respiration and a 30% decrease of COX metabolic threshold, supporting the concept of tight COX control of oxidative phosphorylation in skeletal muscle.     

Nonsense mutation in pseudouridylate synthase 1 (PUS1) in two brothers affected by myopathy, lactic acidosis and sideroblastic anaemia (MLASA)

Introduction

Myopathy, lactic acidosis and sideroblastic anaemia (MLASA) is a rare condition that combines early‐onset myopathy with lactic acidosis and sideroblastic anaemia. MLASA has been associated with a missense mutation in pseudouridylate synthase 1 (PUS1), an enzyme located in both nucleus and mitochondria, which converts uridine into pseudouridine in several cytosolic and mitochondrial tRNA positions and increases the efficiency of protein synthesis in both compartments.

Subjects and methods

We have identified two Italian brothers, offspring of distantly related parents, both of whom are affected by MLASA. The six exons of the PUS1 gene were analysed by automated sequencing.

Results

We found combined defects in mitochondrial respiratory chain complexes in muscle and fibroblast homogenates of both patients, and low levels of mtDNA translation products in fibroblast mitochondria. A novel, homozygous stop mutation was present in PUS1 (E220X). We have investigated the structural and mechanistic aspects of the double localisation of PUS1, demonstrating that the isoform located in the nucleus contains an N‐terminal extension which is absent in the mature mitochondrial isoform.

Conclusions

The stop mutation in PUS1 is likely to determine the loss of function of the protein, since it predicts the synthesis of a protein missing 208/427 amino acid residues on the C terminus, and was associated with low mtDNA translation. The structural differences in nuclear versus mitochondrial isoforms of PUS1 may be implicated in the variability of the clinical presentations in MLASA.     

Identification of a novel heterozygous guanosine monophosphate reductase (GMPR) variant in a patient with a late-onset disorder of mitochondrial DNA maintenance

Autosomal dominant progressive external ophthalmoplegia (adPEO) is a late-onset, Mendelian mitochondrial disorder characterised by paresis of the extraocular muscles, ptosis, and skeletal-muscle restricted multiple mitochondrial DNA (mtDNA) deletions. Although dominantly inherited, pathogenic variants in POLG, TWNK and RRM2B are among the most common genetic defects of adPEO, identification of novel candidate genes and the underlying pathomechanisms remains challenging. We report the clinical, genetic and molecular investigations of a patient who presented in the seventh decade of life with PEO. Oxidative histochemistry revealed cytochrome c oxidase-deficient fibres and occasional ragged red fibres showing subsarcolemmal mitochondrial accumulation in skeletal muscle, while molecular studies identified the presence of multiple mtDNA deletions. Negative candidate screening of known nuclear genes associated with PEO prompted diagnostic exome sequencing, leading to the prioritisation of a novel heterozygous c.547G>C variant in GMPR (NM_006877.3) encoding guanosine monophosphate reductase, a cytosolic enzyme required for maintaining the cellular balance of adenine and guanine nucleotides. We show that the novel c.547G>C variant causes aberrant splicing, decreased GMPR protein levels in patient skeletal muscle, proliferating and quiescent cells, and is associated with subtle changes in nucleotide homeostasis protein levels and evidence of disturbed mtDNA maintenance in skeletal muscle. Despite confirmation of GMPR deficiency, demonstrating marked defects of mtDNA replication or nucleotide homeostasis in patient cells proved challenging. Our study proposes that GMPR is the 19th locus for PEO and highlights the complexities of uncovering disease mechanisms in late-onset PEO phenotypes.     

Single‐fiber PCR in MELAS3243 patients: Correlations between intratissue distribution and phenotypic expression of the mtDNAA3243G genotype

We performed morphological, biochemical, and genetic studies, including single‐fiber PCR (sf PCR), on muscle biopsies obtained from a mother and daughter with MELAS syndrome due to the A3243G transition of mitochondrial DNA (mtDNA). The severity of muscle involvement appeared quite distinct, in spite of the fact that both patients segregated similar mutant mtDNA levels on total muscle DNA. The daughter did not show any clinical muscle involvement: muscle biopsy revealed many ragged red fibers (RRFs) mostly positive for cytochrome‐c oxidase (COX) activity. In contrast, her mother had developed a generalized myopathy without progressive external ophthalmoplegia (PEO), morphologically characterized by many COX‐negative RRFs. Single‐muscle fiber PCR demonstrated in both patients significantly higher percentages of wild‐type mtDNA in normal fibers (daughter: 23.25 ± 15.22; mother: 43.13 ± 26.11) than in COX‐positive RRFs (daughter: 11.25 ± 5.22, P < 0.005; mother: 9.12 ± 5.9, P < 0.001) and in COX‐negative RRFs (daughter: 8.9 ± 4.2, P < 0.001 mother: 4.8 ± 2.8, P < 0.001). Wild‐type mtDNA levels resulted higher also in COX‐positive vs. COX‐negative RRFs (daughter: P < 0.05; mother: P < 0.001). Our data confirm a direct correlation between A3243G levels and impairment of COX function at the single‐muscle fiber level. Moreover, the evidence of a clinical myopathy in the patient with higher amounts of COX‐negative RRFs bolsters the concept that a differential distribution of mutant mtDNAs at the cellular level may have effects on the clinical involvement of individual tissues. However, the occurrence of a similar morphological and biochemical muscle phenotype also in PEO³²⁴³ patients suggests that other genetic factors involved in the interaction between mitochondrial and nuclear DNA, rather than the stochastic distribution of mtDNA genomes during embryogenesis, are primarily implicated in determining the various clinical expressions of the A3243G of mtDNA. Am. J. Med. Genet. 94:201–206, 2000. © 2000 Wiley‐Liss, Inc.     

Is 8860 variation a rare polymorphism or associated as a secondary effect in HCM disease?

Introduction

mtDNA defects, both deletions and point mutations, have been associated with hypertrophic cardiomyopathies. The aim of this study was to establish a spectrum for mtDNA mutations in Iranian hypertrophic cardiomyopathy (HCM) patients.

Material and methods

The control group was chosen among the special medical centre visitors who did not have hypertrophic cardiomyopathy or any related heart disease. Hypertrophic cardiomyopathy (HCM) is widely accepted as a pluricausal or multifactorial disease. Because of the linkage between energy metabolism in the mitochondria and cardiac muscle contraction, it is reasonable to assume that mitochondrial abnormalities may be responsible for some forms of HCM. Point mutations and deletions in the two hot spot regions of mtDNA were investigated by PCR and sequencing methods.

Results

Some unreported point mutations have been found in this study but no deletion was detected. Meanwhile some of these point mutations have been investigated among HCM patients for the first time.

Conclusions

A8860G transition was detected in a high proportion, raising the question whether this rare polymorphism is associated as a secondary effect in HCM disease.     

Microbiopsies versus Bergström needle for skeletal muscle sampling: impact on maximal mitochondrial respiration rate

Introduction

Microbiopsies are increasingly used as an alternative to the standard Bergström technique for skeletal muscle sampling. The potential impact of these two different procedures on mitochondrial respiration rate is unknown. The objective of this work was to compare microbiopsies versus Bergström procedure on mitochondrial respiration in skeletal muscle.

Methods

52 vastus lateralis muscle samples were obtained from 13 anesthetized pigs, either with a Bergström [6 gauges (G)] needle or with microbiopsy needles (12, 14, 18G). Maximal mitochondrial respiration (V _GM-ADP) was assessed using an oxygraphic method on permeabilized fibers.

Results

The weight of the muscle samples and V _GM-ADP decreased with the increasing gauge of the needles. A positive nonlinear relationship was observed between the weight of the muscle sample and the level of maximal mitochondrial respiration (r = 0.99, p < 0.05) and between needle size and maximal mitochondrial respiration (r = 0.99, p < 0.05).

Conclusion

Microbiopsies give lower muscle sample weight and maximal rate of mitochondrial respiration compared to the standard Bergström needle.Therefore, the higher the gauge (i.e. the smaller the size) of the microbiopsy needle, the lower is the maximal rate of respiration. Microbiopsies of skeletal muscle underestimate the maximal mitochondrial respiration rate, and this finding needs to be highlighted for adequate interpretation and comparison with literature data.     

A novel heteroplasmic tRNAleu(CUN) mtDNA point mutation in a sporadic patient with mitochondrial encephalomyopathy segregates rapidly in skeletal muscle and suggests an approach to therapy

A novel mtDNA point mutation was detected in the tRNAleu(CUN) gene (G to A at position 12315) in a sporadic patient with chronic progressive external ophthalmoplegia, ptosis, limb weakness, sensorineural hearing loss and a pigmentary retinopathy. The mutation disrupts base pairing in the T psi C stem at a site which has been conserved throughout evolution. Although the other mtDNA tRNAleu gene (UUR) is a hotspot for mutation, this is the first pathogenic mutation to be reported in the gene coding for tRNAleu(CUN). MtDNAs carrying the mutation constituted 94% of total mtDNAs in two separate muscle biopsies. Single fibre analysis showed that skeletal muscle fibres without detectable cytochrome c oxidase activity (COX-ve fibres) contained predominantly mutant mtDNAs (93-98%) while fibres with apparently normal COX activity had up to 90% mutant mtDNAs, demonstrating that the G12315A mutation is functionally recessive. Immunofluorescence studies with specific antibodies to mtDNA- or nuclear-encoded subunits of COX were consistent with a defect in mitochondrial protein translation. The mutation was not present in blood cells or cultured fibroblasts and surprisingly, it could not be detected in satellite cells cultured from the patient's muscle. This pattern, which may by typical of patients who have inherited new germline pathogenic mtDNA mutations, possibly reflects loss of the mutation by random genetic drift in mitotic tissues and proliferation of mitochondria containing the mutant mtDNA in post- mitotic cells. The absence of mtDNA carrying the mutation in satellite cells suggests that regeneration of skeletal muscle fibres from satellite cells could restore a wild-type mtDNA genotype and normal muscle function.      

The 13042G --> A/ND5 mutation in mtDNA is pathogenic and can be associated also with a prevalent ocular phenotype

Background

Overlapping phenotypes including LHON, MELAS, and Leigh syndrome have recently been associated with numerous mtDNA point mutations in the ND5 gene of complex I, now considered a mutational hot spot.

Objective

To identify the mtDNA defect in a family with a prevalent ocular phenotype, including LHON‐like optic neuropathy, retinopathy, and cataract, but characterised also by strokes, early deaths, and miscarriages on the maternal line.

Results

Sequencing of the entire mitochondrial genome from the proband''s muscle DNA identified the heteroplasmic 13042G→A transition, which was previously described only once in a patient with a different mitochondrial disease. This mutation fulfils the major pathogenic criteria, inducing an amino acid change (A236T) at an invariant position in a highly conserved domain of the ND5 gene. Phosphorus magnetic resonance spectroscopy in the proband disclosed an in vivo brain and skeletal muscle energy metabolism deficit.

Conclusions

These findings conclusively establish the pathogenic role of the 13042G→A mutation and underscore its variable clinical expression.     

Elevated serum fibroblast growth factor 21 levels correlate with immune recovery but not mitochondrial dysfunction in HIV infection

Background

Anti-retroviral treated HIV-infected patients are at risk of mitochondrial toxicity, but non-invasive markers are lacking. Serum FGF-21 (fibroblast growth factor 21) levels correlate strongly with muscle biopsy findings in inherited mitochondrial disorders. We therefore aimed to determine whether serum FGF-21 levels correlate with muscle mitochondrial dysfunction in HIV-infected patients.

Findings

We performed a cross-sectional study of anti-retroviral treated HIV-infected subjects (aged 29 – 71 years, n?=?32). Serum FGF-21 levels were determined by quantitative ELISA. Cellular mitochondrial dysfunction was assessed by COX (cytochrome c oxidase) histochemistry of lower limb skeletal muscle biopsy. Serum FGF-21 levels were elevated in 66% of subjects. Levels correlated significantly with current CD4 lymphocyte count (p?=?0.042) and with total CD4 count gain since initiation of anti-retroviral therapy (p?=?0.016), but not with the nature or duration of past or current anti-retroviral treatment. There was no correlation between serum FGF-21 levels and severity of the muscle mitochondrial (COX) defect.

Conclusions

Serum FGF-21 levels are a poor predictor of muscle mitochondrial dysfunction in contemporary anti-retroviral treated patients. Serum FGF-21 levels are nevertheless commonly elevated, in association with the degree of immune recovery, suggesting a non-mitochondrial metabolic disturbance with potential implications for future comorbidity.

     

Metabolic adaptations in skeletal muscle,adipose tissue,and whole-body oxidative capacity in response to resistance training

Purpose

The effects of resistance training on mitochondrial biogenesis and oxidative capacity in skeletal muscle are not fully characterized, and even less is known about alterations in adipose tissue. We aimed to investigate adaptations in oxidative metabolism in skeletal muscle and adipose tissue after 8 weeks of heavy resistance training in apparently healthy young men.

Methods

Expression of genes linked to oxidative metabolism in the skeletal muscle and adipose tissue was assessed before and after the training program. Body composition, peak oxygen uptake (VO_{2 peak}), fat oxidation, activity of mitochondrial enzyme in muscle, and serum adiponectin levels were also determined before and after resistance training.

Results

In muscle, the expression of the genes AdipoR1 and COX4 increased after resistance training (9 and 13 %, respectively), whereas the expression levels of the genes PGC-1α, SIRT1, TFAM, CPT1b, and FNDC5 did not change. In adipose tissue, the expression of the genes SIRT1 and CPT1b decreased after training (20 and 23 %, respectively). There was an increase in lean mass (from 59.7 ± 6.1 to 61.9 ± 6.2 kg), VO_{2 peak} (from 49.7 ± 5.5 to 56.3 ± 5.0 ml/kg/min), and fat oxidation (from 6.8 ± 2.1 to 9.1 ± 2.7 mg/kg fat-free mass/min) after training, whereas serum adiponectin levels decreased significantly and enzyme activity of citrate synthase and 3-hydroxyacyl-CoA dehydrogenase did not change.

Conclusion

Despite significant increases in VO_{2 peak}, fat oxidation, and lean mass following resistance training, the total effect on gene expression and enzyme activity linked to oxidative metabolism was moderate.     

Clinical,biochemical, cellular and molecular characterization of mitochondrial DNA depletion syndrome due to novel mutations in the MPV17 gene

Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are severe autosomal recessive disorders associated with decreased mtDNA copy number in clinically affected tissues. The hepatocerebral form (mtDNA depletion in liver and brain) has been associated with mutations in the POLG, PEO1 (Twinkle), DGUOK and MPV17 genes, the latter encoding a mitochondrial inner membrane protein of unknown function. The aims of this study were to clarify further the clinical, biochemical, cellular and molecular genetic features associated with MDS due to MPV17 gene mutations. We identified 12 pathogenic mutations in the MPV17 gene, of which 11 are novel, in 17 patients from 12 families. All patients manifested liver disease. Poor feeding, hypoglycaemia, raised serum lactate, hypotonia and faltering growth were common presenting features. mtDNA depletion in liver was demonstrated in all seven cases where liver tissue was available. Mosaic mtDNA depletion was found in primary fibroblasts by PicoGreen staining. These results confirm that MPV17 mutations are an important cause of hepatocerebral mtDNA depletion syndrome, and provide the first demonstration of mosaic mtDNA depletion in human MPV17 mutant fibroblast cultures. We found that a severe clinical phenotype was associated with profound tissue-specific mtDNA depletion in liver, and, in some cases, mosaic mtDNA depletion in fibroblasts.     

A novel heteroplasmic point mutation in the mitochondrial tRNA(Lys) gene in a sporadic case of mitochondrial encephalomyopathy: de novo mutation and no transmission to the offspring

We have identified a new mutation in the tRNA(Lys) gene of mtDNA, in a 49-year-old patient with mitochondrial encephalomyopathy. The mutation is a heteroplasmic G-->A transition at position 8328, which affects the anticodon stem loop at a conserved site. The mutation was neither found in 100 controls nor in the maternal relatives of the patient. The level of mutated mtDNA was 57% in muscle, 13% in fibroblasts, and 10% in lymphocytes. Histochemistry of muscle tissue revealed cytochrome c oxidase-deficient fibers with abnormal accumulation of mitochondria. Biochemistry of muscle mitochondria showed slight cytochrome c oxidase deficiency. The mean ratio of mutant mtDNA to normal mtDNA in cytochrome c oxidase-positive muscle fibers was 59%, whereas a mean ratio of 95% was found in cytochrome c oxidase-negative fibers. The difference between cytochrome c oxidase-positive and cytochrome c oxidase-negative fibers was highly significant (P < 0.001). The mutation was not found in muscle or lymphocytes of the mother and daughter of the proband. This is the first report of a de novo point mutation in the tRNA(Lys) gene in an individual expressing disease and the first report of lack of transmission of the mutation to the offspring of a patient expressing a mitochondrial encephalomyopathy caused by a point mutation in mtDNA.     

Tigecycline-induced inhibition of mitochondrial DNA translation may cause lethal mitochondrial dysfunction in humans

BackgroundA 65-year-old patient developed an unexplained and ultimately lethal metabolic acidosis under prolonged treatment with tigecycline. Tigecycline is known to have a selective inhibitory effect on eukaryotic mitochondrial translation. The underlying molecular mechanisms of the metabolic acidosis in this patient were explored.MethodsOxidative phosphorylation system (OXPHOS) analysis, blue native polyacrylamide gel electrophoresis followed by in-gel activity staining in mitochondria, molecular analysis of mitochondrial DNA (mtDNA) for genomic rearrangements and sequencing of the rRNA genes was performed on the subject's skeletal muscle.ResultsOXPHOS analysis revealed a combined deficiency of the complexes I, III, IV and V, with a preserved function of complex II (encoded by nuclear DNA), thus demonstrating a defective mtDNA translation. There were no known underlying mitochondrial genetic defects. The patient had a (m.1391T>A) variant within the 12SrRNA gene in heteroplasmy (50–60%).ConclusionsThis patient developed an ultimately lethal mitochondrial toxicity while receiving prolonged treatment with tigecycline, which was caused by a defective translation of the mtDNA. Tigecycline is known to suppress eukaryotic mitochondrial DNA translation, but until now this effect has been considered to be clinically insignificant. The observations in this patient suggest a clinically significant mitochondrial toxicity of tigecycline in this patient, and warrant further investigation.     

Mutations in the ND5 subunit of complex I of the mitochondrial DNA are a frequent cause of oxidative phosphorylation disease

Background

Detection of mutations in the mitochondrial DNA (mtDNA) is usually limited to common mutations and the transfer RNA genes. However, mutations in other mtDNA regions can be an important cause of oxidative phosphorylation (OXPHOS) disease as well.

Objective

To investigate whether regions in the mtDNA are preferentially mutated in patients with OXPHOS disease.

Methods

Screening of the mtDNA for heteroplasmic mutations was performed by denaturing high‐performance liquid chromatography analysis of 116 patients with OXPHOS disease but without the common mtDNA mutations.

Results

An mtDNA sequence variant was detected in 15 patients, 5 of which were present in the ND5 gene. One sequence variant was new and three were known, one of which was found twice. The novel sequence variant m.13511A→T occurred in a patient with a Leigh‐like syndrome. The known mutation m.13513G→A, associated with mitochondrial encephalomyopathy lactic acidosis and stroke‐like syndrome (MELAS) and MELAS/Leigh/Leber hereditary optic neuropathy overlap syndrome, was found in a relatively low percentage in two patients from two different families, one with a MELAS/Leigh phenotype and one with a MELAS/chronic progressive external ophthalmoplegia phenotype. The known mutation m.13042G→A, detected previously in a patient with a MELAS/myoclonic epilepsy, ragged red fibres phenotype and in a family with a prevalent ocular phenotype, was now found in a patient with a Leigh‐like phenotype. The sequence variant m.12622G→A was reported once in a control database as a polymorphism, but is reported in this paper as heteroplasmic in three brothers, all with infantile encephalopathy (Leigh syndrome) fatal within the first 15 days of life. Therefore, a causal relationship between the presence of this sequence variant and the onset of mitochondrial disease cannot be entirely excluded at this moment.

Conclusions

Mutation screening of the ND5 gene is advised for routine diagnostics of patients with OXPHOS disease, especially for those with MELAS‐ and Leigh‐like syndrome with a complex I deficiency.Mitochondria are key for many cellular processes. One of the most important mechanisms is oxidative phosphorylation (OXPHOS) resulting in the production of cellular energy in the form of ATP. The OXPHOS system consists of five multiprotein complexes (I–V) and two mobile electron carriers (coenzyme q and cytochrome c) embedded in the lipid bilayer of the mitochondrial inner membrane.^1,2 The mitochondrial genome encodes 13 essential polypeptides of the OXPHOS system and the necessary RNA machinery (two ribosomal RNAs and 22 transfer RNAs (tRNA)). The remaining structural proteins and proteins involved in import, assembly and mitochondrial DNA (mtDNA) replication are encoded by the nucleus and specifically targeted to the mitochondria. OXPHOS disease is characterised by a wide variety of clinical symptoms, in which one or more organs can be involved, and by genetic and clinical heterogeneity.^2,3 With an estimated total number of about 1500 nuclear mitochondrial genes of which 600 have been identified so far,⁴ this complicates the process of identification of the underlying genetic defect. Although mutations in the mtDNA tRNA genes have been reported far more often than other mutations in mtDNA protein‐coding genes,² this figure is highly biased by a preferential screening of these genes.In this study, the complete mtDNA was screened for heteroplasmic mutations using denaturing high‐performance liquid chromatography (DHPLC) analysis in a group of 116 unrelated patients suspected for OXPHOS disease but without the common mutations for mitochondrial encephalomyopathy, lactic acidosis and stroke‐like syndrome (MELAS) m.3243A→G, myoclonic epilepsy, ragged red fibres (MERRF) m.8344A→G, Leigh/neuropathy, ataxia and retinitis pigmentosa m.8993T→G/C or large deletions. For this group of patients, we report that the ND5 gene is a commonly mutated gene.