Normal Respiratory Chain Research Articles

The Role of Cytoglobin in Cancer

Cytoglobin (Cygb) is a new member of the globin protein family, following the discovery of other globin proteins such as hemoglobin (Hb), myoglobin (Mb), and neuroglobin (Ngb). In 2001, Kawada et al. identified Cygb in hepatic stellate cells (HSCs) that play an important role in the repair of damaged liver cell tissue, hence the name stellate cell activation-associated protein (STAP). Cygb has a more universal role than Hb, Mb, and Ngb, and is expressed throughout mammalian tissues. Cygb is a globin with 6 coordination bonds (hexacoordinate globin). Cygb has been shown to play an important role in the normal cell respiratory chain, including oxygen storage, destruction of reactive oxygen species (ROS), terminal oxidase activity, regulation of fibrogenesis, and regulation of apoptosis. The role of Cygb in the respiratory process has been studied because it is associated with the globin family, and because of the upregulation of Cygb during hypoxia, but its specific role has not been elaborated. Recent research reports that Cygb has several implications for cancer. In most cancer cells, Cygb expression is upregulated by hypermethylation, suggesting epigenetic control. In cancer cells, downregulation of Cygb occurs which indicates a possible role as a tumor suppressor gene. In some malignancies, on the contrary, Cygb upregulation may be associated with resistance to hypoxia which indicates Cygb has two sides or Janus faces related to its role in cancer cells.

Clinical, biochemical, and histopathological diagnosis of Egyptian paediatric patients with suspected mitochondrial diseases: A hospital‑based study

Background. Mitochondrial respiratory chain (RC) disorders are a growing group of disorders with a large variety of clinical presentations ranging from well-defined clinical syndromes to nonspecific manifestations, such as failure to thrive, exercise intolerance and seizures.Objective. To describe the clinical, biochemical, and histochemical spectrum of 38 Egyptian patients clinically suspected of havingmitochondrial RC disorders.Methods. A total of 38 patients (female, n=18 (47.4%); male, n=20 (52.6%)) clinically suspected of having mitochondrial diseases had been referred to the Inherited Metabolic Diseases Laboratory at Cairo University Children’s Hospital. Laboratory investigations and analyses included histochemical staining of cytochrome c oxidase and succinate dehydrogenase in muscle biopsies, as well as spectrophotometric assays of RC complexes in muscle homogenates.Results. Twenty-three patients (60.5%) were diagnosed with different RC enzyme deficiencies. Fifteen patients (65%) had complex Ideficiency and all of them also had lactic acidosis (mean (standard deviation)) plasma lactate concentration of 4 (1.65) mmol/L). Twopatients (9%) with marked complex IV deficiency both showed COX-negative ragged red fibers (RRFs) on histochemical staining.Combined complex I and complex II deficiency with scattered COX-stain deficiency and RRFs was diagnosed in 2 patients (5.25%), whilea further 2 patients (5.26%) had combined (complex I, II+III, complex IV) deficiencies. Isolated complex II deficiency was diagnosed in2 patients (5.26%) and 15 (39.5%) patients had normal RC enzyme activities.Conclusion. Biochemical assay of RC complexes is considered the cornerstone for diagnosis of RC complex mitochondrial disorders.These disorders are common among Egyptian paediatric patients.

Impact of measuring heteroplasmy of a pathogenic mitochondrial DNA variant at the single-cell level in individuals with mitochondrial disease.

Pathogenic mitochondrial DNA heteroplasmy has mainly been assessed with bulk sequencing in individuals with mitochondrial disease. However, the distribution of heteroplasmy at the single-cell level in skin fibroblasts obtained from individuals, together with detailed clinical and biochemical information, remains to be investigated. We used the mitochondrial DNA single-cell assay for the transposase-accessible chromatin sequencing method. Skin fibroblasts were obtained from six individuals with mitochondrial disease and pathogenic m.3243A>G variants of differing severity. Different distributions of heteroplasmy at the single-cell level were identified in skin fibroblasts from all six individuals. Four individuals with different outcomes showed similar averaged heteroplasmy rates with normal mitochondrial respiratory chain enzyme activity, while the distribution of single-cell heteroplasmy patterns differed among the individuals. This study showed different heteroplasmy distribution patterns at the single-cell level in individuals with the m.3243A>G variant, who had a similar averaged heteroplasmy rates with normal mitochondrial respiratory chain enzyme activity. Whether such different heteroplasmy distribution patterns explain the different clinical outcomes should be assessed further in future studies. Measuring heteroplasmy of pathogenic mitochondrial DNA variants at the single-cell level could be important in individuals with mitochondrial disease.

Visible-light-driven photocatalytic inactivation of S. aureus in aqueous environment by hydrophilic zinc oxide (ZnO) nanoparticles based on the interfacial electron transfer in S. aureus/ZnO composites

Waterborne diseases caused by pathogenic microorganisms pose severe threats to human health. ZnO nanoparticles (NPs) hold great potentials as an effective, economical and eco-friendly method for water disinfection, but the exact antimicrobial mechanism of ZnO NPs under visible-light illumination is still not clear. Herein, we investigate the visible-light-driven photocatalytic inactivation mechanism of amino-functionalized hydrophilic ZnO (AH-ZnO) NPs against Staphylococcus aureus (S. aureus) in aqueous environment from the perspective of electron transfer theory. The results show that the antibacterial effects of AH-ZnO NPs are dependent on the AH-ZnO NPs concentration and treatment time. The bulk ORP value and released Zn2+ concentration in AH-ZnO NPs solutions increase with AH-ZnO NPs concentration. The SEM and intracellular protein leakage results indicate that AH-ZnO NPs can adhere to S. aureus surface without causing obvious cell membrane disruption. The photoluminescence (PL) intensity and fluorescence lifetime of AH-ZnO NPs are remarkedly decreased after adding S. aureus, which confirms the electron transfer from S. aureus to AH-ZnO NPs. Moreover, the ΔPL intensity is closely correlated with the inactivation efficiency, demonstrating that the interfacial electron transfer in S. aureus/AH-ZnO NPs composites contributes to the antibacterial activity, which is speculated to disrupt the normal respiratory electron transfer chain of S. aureus, thereby causing intracellular ROS generation, cell membrane depolarization and eventually apoptosis-like death.

Pyruvate Kinase M2 and Cancer: The Role of PKM2 in Promoting Tumorigenesis.

Pyruvate kinase plays a pivotal role in regulating cell metabolism. The final and rate-limiting step of glycolysis is the conversion of Phosphoenolpyruvate (PEP) to Pyruvate, which is catalyzed by Pyruvate Kinase. There are four isomeric, tissue-specific forms of Pyruvate Kinase found in mammals: PKL, PKR, PKM1, and PKM2. PKM1 and PKM2 are formed bya single mRNA transcript of the PKM gene by alternative splicing. The oligomers of PKM2 exist in high activity tetramer and low activity dimer forms. The dimer PKM2 regulates the rate-limiting step of glycolysis that shifts the glucose metabolism from the normal respiratory chain to lactate production in tumor cells. Besides its role as a metabolic regulator, it also acts as protein kinase, which contributes to tumorigenesis. This review is focused on the metabolic role of pyruvate kinase M2 in normal cells vs. cancerous cells and its regulation at the transcriptional level. The review also highlights the role of PKM2 as a potential diagnostic marker and as a therapeutic target in cancer treatment.

Expanding the clinical phenotype of IARS2-related mitochondrial disease

BackgroundIARS2 encodes a mitochondrial isoleucyl-tRNA synthetase, a highly conserved nuclear-encoded enzyme required for the charging of tRNAs with their cognate amino acid for translation. Recently, pathogenic IARS2 variants have been identified in a number of patients presenting broad clinical phenotypes with autosomal recessive inheritance. These phenotypes range from Leigh and West syndrome to a new syndrome abbreviated CAGSSS that is characterised by cataracts, growth hormone deficiency, sensory neuropathy, sensorineural hearing loss, and skeletal dysplasia, as well as cataract with no additional anomalies.MethodsGenomic DNA from Iranian probands from two families with consanguineous parental background and overlapping CAGSSS features were subjected to exome sequencing and bioinformatics analysis.ResultsExome sequencing and data analysis revealed a novel homozygous missense variant (c.2625C > T, p.Pro909Ser, NM_018060.3) within a 14.3 Mb run of homozygosity in proband 1 and a novel homozygous missense variant (c.2282A > G, p.His761Arg) residing in an ~ 8 Mb region of homozygosity in a proband of the second family. Patient-derived fibroblasts from proband 1 showed normal respiratory chain enzyme activity, as well as unchanged oxidative phosphorylation protein subunits and IARS2 levels. Homology modelling of the known and novel amino acid residue substitutions in IARS2 provided insight into the possible consequence of these variants on function and structure of the protein.ConclusionsThis study further expands the phenotypic spectrum of IARS2 pathogenic variants to include two patients (patients 2 and 3) with cataract and skeletal dysplasia and no other features of CAGSSS to the possible presentation of the defects in IARS2. Additionally, this study suggests that adult patients with CAGSSS may manifest central adrenal insufficiency and type II esophageal achalasia and proposes that a variable sensorineural hearing loss onset, proportionate short stature, polyneuropathy, and mild dysmorphic features are possible, as seen in patient 1. Our findings support that even though biallelic IARS2 pathogenic variants can result in a distinctive, clinically recognisable phenotype in humans, it can also show a wide range of clinical presentation from severe pediatric neurological disorders of Leigh and West syndrome to both non-syndromic cataract and cataract accompanied by skeletal dysplasia.

The protective effects of melatonin on oxidative damage and the immune system of the Chinese mitten crab (Eriocheir sinensis) exposed to deltamethrin

Deltamethrin (Del), an important broad-spectrum insecticide, is widely used in agricultural activities. However, Del is an effective reactive oxygen species (ROS) inducer that induces oxidative stress damage in cells or tissues. Del is significantly more toxic to aquatic organisms, especially crustaceans, than to mammals and birds. This study was designed to evaluate the protective effect of melatonin (MT) on the toxicity-induced damage of Del after 6 h in Eriocheir sinensis. The results showed that Del exposure significantly induced oxidative damage in the hepatopancreas and mitochondria, with malondialdehyde (MDA) and glutathione (GSH) levels being significantly increased and superoxide dismutase (SOD) activity being significantly decreased. Moreover, Del exposure significantly induced functional damage of the hepatopancreas and mitochondria, with a significant increase in alanine aminotransferase (ALT), aspartate aminotransferase (AST), acid phosphatase (ACP) and alkaline phosphatase (AKP) activities in the hepatopancreas and the ratio of albumin/globulin (ALB/GLB) in serum, which indicated the permeability and integrity of the membranes were damaged and had caused cell damage. In addition, ATP content, Na+-K+-ATPase activity and cytochrome C (Cyt‑C) content in mitochondria decreased significantly, which indicated that Del exposure destroyed the normal respiratory chain of mitochondria. We also evaluated the hematological parameters. Although there were no significant differences in total hemocyte count (THC) levels, hemocyte apoptosis was significantly induced by Del exposure, and the hemocyte phagocytic activity and the hemocyanin levels decreased significantly with Del exposure. However, MT pretreatment not only prevented oxidative damage and functional damage caused by Del exposure to the hepatopancreas and mitochondria, but it also restored the hemocyte apoptotic rate and phagocytic activity to normal levels. In short, Del exposure caused significant oxidative and functional damage to the hepatopancreas, mitochondria and hemocytes of E. sinensis, whereas the use of MT almost completely eliminated the damage caused by Del exposure.

Control of Mitochondrial Function by Fusion and Fission

Several neurodegenerative diseases are associated with defects in mitochondrial fusion or fission, which are opposing processes that regulate mitochondrial function. This observation raises the issue of whether a defect in one process can be alleviated by simultaneously reducing the opposing process. We used mouse models to address this issue. Mff is a mitochondrial outer membrane protein important for mitochondrial fission. We generated Mff mutant mice and found that they die at 13 weeks due to heart failure caused by dilated cardiomyopathy. Mff cardiac tissue shows reduced mitochondrial density, reduced respiratory chain activity, and increased mitophagy. Mfn1 deficient mice die shortly after birth. However, mice with loss of both Mff and Mfn1 have normal heart function, lifespan and respiratory chain function. The reciprocal rescue with both mutations indicate that retuning mitochondrial dynamics can restore tissue integrity and mitochondrial physiology at the whole organ level.

Compound heterozygosity for severe and hypomorphic NDUFS2 mutations cause non-syndromic LHON-like optic neuropathy

BackgroundNon-syndromic hereditary optic neuropathy (HON) has been ascribed to mutations in mitochondrial fusion/fission dynamics genes, nuclear and mitochondrial DNA-encoded respiratory enzyme genes or nuclear genes of poorly known mitochondrial function....

Incidence of Primary Mitochondrial Disease in Children Younger Than 2 Years Presenting With Acute Liver Failure.

ABSTRACTBackground:Mitochondrial liver disease (MLD), and in particular mitochondrial DNA (mtDNA) depletion syndrome (MDS) is an important cause of acute liver failure (ALF) in infancy. Early and accurate diagnosis is important because liver transplantation (LT) is often contraindicated. It is unclear which methods are the best to diagnose MLD in the setting of ALF.Objective:The aim of the study was to determine the incidence of MLD in children younger than 2 years with ALF and the utility of routine investigations to detect MLD.Methods:Thirty-nine consecutive infants with ALF were admitted to a single unit from 2009 to 2011. All were extensively investigated using an established protocol. Genes implicated in mitochondrial DNA depletion syndrome were sequenced in all cases and tissue mtDNA copy number measured where available.Results:Five infants (17%) had genetically proven MLD: DGUOK (n = 2), POLG (n = 2), and MPV17 (1). Four of these died, whereas 1 recovered. Two had normal muscle mtDNA copy number and 3 had normal muscle respiratory chain enzymes. An additional 8 children had low hepatic mtDNA copy number but pathogenic mutations were not detected. One of these developed fatal multisystemic disease after LT, whereas 5 who survived remain well without evidence of multisystemic disease up to 6 years later. Magnetic resonance spectroscopy did not distinguish between those with and without MLD.Conclusions:Low liver mtDNA copy number may be a secondary phenomenon in ALF.Screening for mtDNA maintenance gene mutations may be the most efficient way to confirm MLD in ALF in the first 2 years of life.

Mitochondrial defects associated with β-alanine toxicity: relevance to hyper-beta-alaninemia.

Hyper-beta-alaninemia is a rare metabolic condition that results in elevated plasma and urinary β-alanine levels and is characterized by neurotoxicity, hypotonia, and respiratory distress. It has been proposed that at least some of the symptoms are caused by oxidative stress; however, only limited information is available on the mechanism of reactive oxygen species generation. The present study examines the hypothesis that β-alanine reduces cellular levels of taurine, which are required for normal respiratory chain function; cellular taurine depletion is known to reduce respiratory function and elevate mitochondrial superoxide generation. To test the taurine hypothesis, isolated neonatal rat cardiomyocytes and mouse embryonic fibroblasts were incubated with medium lacking or containing β-alanine. β-alanine treatment led to mitochondrial superoxide accumulation in conjunction with a decrease in oxygen consumption. The defect in β-alanine-mediated respiratory function was detected in permeabilized cells exposed to glutamate/malate but not in cells utilizing succinate, suggesting that β-alanine leads to impaired complex I activity. Taurine treatment limited mitochondrial superoxide generation, supporting a role for taurine in maintaining complex I activity. Also affected by taurine is mitochondrial morphology, as β-alanine-treated fibroblasts undergo fragmentation, a sign of unhealthy mitochondria that is reversed by taurine treatment. If left unaltered, β-alanine-treated fibroblasts also undergo mitochondrial apoptosis, as evidenced by activation of caspases 3 and 9 and the initiation of the mitochondrial permeability transition. Together, these data show that β-alanine mediates changes that reduce ATP generation and enhance oxidative stress, factors that contribute to heart failure.

Early Infantile Epileptic Encephalopathy in an STXBP1 Patient with Lactic Acidemia and Normal Mitochondrial Respiratory Chain Function.

A wide range of clinical findings have been associated with mutations in Syntaxin Binding Protein 1 (STXBP1), including multiple forms of epilepsy, nonsyndromic intellectual disability, and movement disorders. STXBP1 mutations have recently been associated with mitochondrial pathology, although it remains unclear if this phenotype is a part of the core feature for this gene disorder. We report a 7-year-old boy who presented for diagnostic evaluation of intractable epilepsy, episodic ataxia, resting tremor, and speech regression following a period of apparently normal early development. Mild lactic acidemia was detected on one occasion at the time of an intercurrent illness. Due to the concern for mitochondrial disease, ophthalmologic evaluation was performed that revealed bilateral midperiphery pigmentary mottling. Optical coherence tomography (OCT) testing demonstrated a bilaterally thickened ganglion cell layer in the perifovea. Skeletal muscle biopsy analysis showed no mitochondrial abnormalities or respiratory chain dysfunction. Exome sequencing identified a de novo c.1651C>T (p.R551C) mutation in STXBP1. Although mitochondrial dysfunction has been reported in some individuals, our proband had only mild lactic acidemia and no skeletal muscle tissue evidence of mitochondrial disease pathology. Thus, mitochondrial dysfunction is not an obligate feature of STXBP1 disease.

The Respiratory Chain Supercomplex Organization Is Independent of COX7a2l Isoforms

SummaryThe organization of individual respiratory chain complexes into supercomplexes or respirasomes has attracted great interest because of the implications for cellular energy conversion. Recently, it was reported that commonly used mouse strains harbor a short COX7a2l (SCAFI) gene isoform that supposedly precludes the formation of complex IV-containing supercomplexes. This claim potentially has serious implications for numerous mouse studies addressing important topics in metabolism, including adaptation to space flights. Using several complementary experimental approaches, we show that mice with the short COX7a2l isoform have normal biogenesis and steady-state levels of complex IV-containing supercomplexes and consequently have normal respiratory chain function. Furthermore, we use a mouse knockout of Lrpprc and show that loss of complex IV compromises respirasome formation. We conclude that the presence of the short COX7a2l isoform in the commonly used C57BL/6 mouse strains does not prevent their use in metabolism research.

Cerebellar Ataxia, Vertical Supranuclear Gaze Palsy, Sensorineural Deafness, Epilepsy, Dementia, and Hallucinations in an Adolescent Male

We encountered an adolescent male with cerebellar ataxia since age 11, difficulty in vertical gaze from age 2, leg weakness since age 10, and partial epilepsy since age 8. At age 14, he developed visual and auditory hallucinations, as well as mild sensorineural deafness. He was evaluated as having a mitochondrial disorder. No common mitochondrial DNA mutations were detected in the blood. Muscle biopsy revealed nonspecific changes and normal respiratory chain enzyme complexes. He developed progressive cognitive decline leading to diagnosis of dementia at age 15, and intractablepartial epilepsy persisted despite treatment with multiple anticonvulsants. He also had progressive dysphagia requiring gastrostomy tube for nutrition. He required many other diagnostic investigations for neurodegenerative disorders, but was eventually confirmed as having Niemann-Pick disease type C with excessive free cholesterol using filipin staining and zero activity for cholesterol esterification in fibroblast as well as two pathogenic mutations in the NPC1 gene.

G82(P) Hepato-cerebral mitochondrial DNA depletion: a rare and fatal cause of prolonged neonatal jaundice

Case presentationThe only child of consanguineous moroccan parents presented at 6 weeks of age with poor weight gain and prolonged jaundice. He was profoundly hypotonic and not yet smiling or...

Liver adapts mitochondrial function to insulin resistant and diabetic states in mice

To determine if diabetic and insulin-resistant states cause mitochondrial dysfunction in liver or if there is long term adaptation of mitochondrial function to these states, mice were (i) fed with a high-fat diet to induce obesity and T2D (HFD), (ii) had a genetic defect in insulin signaling causing whole body insulin resistance, but not full blown T2D (IR/IRS-1(+/-) mice), or (iii) were analyzed after treatment with streptozocin (STZ) to induce a T1D-like state. Hepatic lipid levels were measured by thin layer chromatography. Mitochondrial respiratory chain (RC) levels and function were determined by Western blot, spectrophotometric, oxygen consumption and proton motive force analysis. Gene expression was analyzed by real-time PCR and microarray. HFD caused insulin resistance and hepatic lipid accumulation, but RC was largely unchanged. Livers from insulin resistant IR/IRS-1(+/-) mice had normal lipid contents and a normal RC, but mitochondria were less well coupled. Livers from severely hyperglycemic and hypoinsulinemic STZ mice had massively depleted lipid levels, but RC abundance was unchanged. However, liver mitochondria isolated from these animals showed increased abundance and activity of the RC, which was better coupled. Insulin resistance, induced either by obesity or genetic manipulation and steatosis do not cause mitochondrial dysfunction in mouse liver. Also, mitochondrial dysfunction is not a prerequisite for liver steatosis. However, severe insulin deficiency and high blood glucose levels lead to an enhanced performance and better coupling of the RC. This may represent an adaptation to fuel overload and the high energy-requirement of an unsuppressed gluconeogenesis.

Marked phenotypic differences of endurance performance and exercise-induced oxygen consumption between AMPK and LKB1 deficiency in mouse skeletal muscle: changes occurring in the diaphragm

LKB1 phosphorylates members of the AMP-activated protein kinase (AMPK) family. LKB1 and AMPK in the skeletal muscle are believed to regulate not only fuel oxidation during exercise but also exercise capacity. LKB1 was also required to prevent diaphragm fatigue, which was shown to affect exercise performance. Using mice expressing dominant negative (DN) mutants of LKB1 and AMPK, specifically in the skeletal muscle but not in the heart, we investigated the roles of LKB1 and AMPK activity in exercise performance and the effects of these kinases on the characteristics of respiratory and locomotive muscles. In the diaphragm and gastrocnemius, both AMPK-DN and LKB1-DN mice showed complete loss of AMPKα2 activity, and LKB1-DN mice showed a reduction in LKB1 activity. Exercise capacity was significantly reduced in LKB1-DN mice, with a marked reduction in oxygen consumption and carbon dioxide production during exercise. The diaphragm from LKB1-DN mice showed an increase in myosin heavy chain IIB and glycolytic enzyme expression. Normal respiratory chain function and CPT I activity were shown in the isolated mitochondria from LKB1-DN locomotive muscle, and the expression of genes related to fiber type, mitochondria function, glucose and lipid metabolism, and capillarization in locomotive muscle was not different between LKB1-DN and AMPK-DN mice. We concluded that LKB1 in the skeletal muscle contributes significantly to exercise capacity and oxygen uptake during exercise. LKB1 mediated the change of fiber-type distribution in the diaphragm independently of AMPK and might be responsible for the phenotypes we observed.

Movement Disorders in Children with Acute Bilateral Lesions of the Basal Ganglia: A Case Series (IN10-1.009)

Objective: To review the clinical features of children with acute neurological symptoms associated with bilateral basal ganglia lesions. Background Acute bilateral lesions of the basal ganglia in childhood are a rare and heterogeneous group of disorders. Movement disorders are a prominent feature, but the full clinical spectrum remains incompletely characterized. Design/Methods: Retrospective case series. Results: We reviewed the charts of 6 patients who were evaluated by movement disorders specialists in our institution. The patients ranged in age from 10 months to 12 years. Neurological symptom onset was preceded by systemic infection in 4 patients, and surgery in 2 patients. Four of the patients were encephalopathic with altered consciousness at onset. Motor symptoms developed hyper-acutely in one patient, while the other 5 experienced worsening motor symptoms over the course of days. The movement disorder syndromes observed were generalized dystonia (n=3), dystonia-parkinsonism (n=2), and chorea (n=1). Brain MRI demonstrated lesions limited to the striatum in 3 patients, while 3 patients had involvement of the globus pallidus with other brain regions. Striatal lesions were associated with a variety of motor syndromes (chorea, dystonia, parkinsonism). Investigations revealed abnormal Mycoplasma pneumoniae serology in 1 patient with striatal necrosis, and elevated blood or CSF lactate in 3 patients, one of whom had a mutation in mitochondrial DNA. Two patients had normal mitochondrial respiratory chain function on muscle biopsy. None of the patients had evidence of infection or inflammation in the CSF. Four patients had follow-up >12 months at our center. All were non-ambulatory and had persistent residual symptoms responsive to symptomatic medical treatments including tetrabenazine, artane, clonazepam and levodopa-carbidopa. Conclusions: This case series illustrates the complex patterns of manifestations which may be associated with pallidal and striatal necrosis in children. Supported by: In part by the Parkinson9s Disease Foundation. Disclosure: Dr. Virmani has nothing to disclose. Dr. Pearson has nothing to disclose.

Pyruvate dehydrogenase deficiency: Novel PDHA1 mutation in a child with progressive encephalopathy

Pyruvate dehydrogenase complex (PDHc) deficiency is an inborn error of carbohydrate metabolism that is heterogeneous in its presentation and clinical course. Most cases of PDHc deficiency are attributable to mutations in the X-chromosomal PDHA1 gene which encodes the E1α subunit. We report the case of a girl with first symptoms at age 4 months including muscular hypotonia, lactic acidosis and microcephaly. Brain MRI revealed an incomplete corpus callosum and a pronounced generalized cortical atrophy. Muscle biopsy performed for clinically suspected mitochondriopathy revealed normal respiratory chain enzymes. Functional investigation of intact mitochondria from unfrozen muscle tissue showed a deficiency of pyruvate oxidation. Sequencing of PDHA1 gene revealed a heterogeneous mutation c.523G>A resulting in a predicted alanine to threonine substitution at amino acid 175. To the best of our knowledge, this mutation has not yet been reported. However, another mutation in the same codon has been described in a girl with PDHc-deficiency resulting in an amino acid substitution of alanine to proline. The fact that alanine 175 is highly conserved suggests that this substitution is the causative mutation.

Isoleucyl-tRNA synthetase levels modulate the penetrance of a homoplasmic m.4277T>C mitochondrial tRNAIle mutation causing hypertrophic cardiomyopathy

The genetic and epigenetic factors underlying the variable penetrance of homoplasmic mitochondrial DNA mutations are poorly understood. We investigated a 16-year-old patient with hypertrophic cardiomyopathy harboring a homoplasmic m.4277T>C mutation in the mt-tRNA(Ile) (MTTI) gene. Skeletal muscle showed multiple respiratory chain enzyme abnormalities and a decreased steady-state level of the mutated mt-tRNA(Ile). Transmitochondrial cybrids grown on galactose medium demonstrated a functional effect of this mutation on cell viability, confirming pathogenicity. These findings were reproduced in transmitochondrial cybrids, harboring a previously described homoplasmic m.4300A>G MTTI mutation. The pathogenic role of the m.4277T>C mutation may be ascribed to misfolding of the mt-tRNA molecule, as demonstrated by the altered electrophoretic migration of the mutated mt-tRNA. Indeed, structure and sequence analyses suggest that thymidine at position 4277 of mt-tRNA(Ile) is involved in a conserved tertiary interaction with thymidine at position 4306. Interestingly, the mutation showed variable penetrance within family members, with skeletal muscle from the patient's clinically unaffected mother demonstrating normal muscle respiratory chain activities and steady-state levels of mt-tRNA(Ile), while homoplasmic for the m.4277T>C mutation. Analysis of mitochondrial isoleucyl-tRNA synthetase revealed significantly higher expression levels in skeletal muscle and fibroblasts of the unaffected mother when compared with the proband, while the transient over-expression of the IARS2 gene in patient transmitochondrial cybrids improved cell viability. This is the first observation that constitutively high levels of aminoacyl-tRNA synthetases (aaRSs) in human tissues prevent the phenotypic expression of a homoplasmic mt-tRNA point mutation. These findings extend previous observations on aaRSs therapeutic effects in yeast and human.