De Novo DNM1L Mutation in a Patient with Encephalopathy, Cardiomyopathy and Fatal Non-Epileptic Paroxysmal Refractory Vomiting.

Mitochondrial fission and fusion are vital dynamic processes for mitochondrial quality control and for the maintenance of cellular respiration; they also play an important role in the formation and maintenance of cells with high energy demand including cardiomyocytes and neurons. The DNM1L (dynamin-1 like) gene encodes for the DRP1 protein, an evolutionary conserved member of the dynamin family that is responsible for the fission of mitochondria; it is ubiquitous but highly expressed in the developing neonatal heart. De novo heterozygous pathogenic variants in the DNM1L gene have been previously reported to be associated with neonatal or infantile-onset encephalopathy characterized by hypotonia, developmental delay and refractory epilepsy. However, cardiac involvement has been previously reported only in one case. Next-Generation Sequencing (NGS) was used to genetically assess a baby girl characterized by developmental delay with spastic-dystonic, tetraparesis and hypertrophic cardiomyopathy of the left ventricle. Histochemical analysis and spectrophotometric determination of electron transport chain were performed to characterize the muscle biopsy; moreover, the morphology of mitochondria and peroxisomes was evaluated in cultured fibroblasts as well. Herein, we expand the phenotype of DNM1L-related disorder, describing the case of a girl with a heterozygous mutation in DNM1L and affected by progressive infantile encephalopathy, with cardiomyopathy and fatal paroxysmal vomiting correlated with bulbar transitory abnormal T2 hyperintensities and diffusion-weighted imaging (DWI) restriction areas, but without epilepsy. In patients with DNM1L mutations, careful evaluation for cardiac involvement is recommended.

Evaluation of Mitochondrial Dysfunction and Idebenone Responsiveness in Fibroblasts from Leber's Hereditary Optic Neuropathy (LHON) Subjects.

Leber's hereditary optic neuropathy (LHON) is a disease that affects the optical nerve, causing visual loss. The diagnosis of LHON is mostly defined by the identification of three pathogenic variants in the mitochondrial DNA. Idebenone is widely used to treat LHON patients, but only some of them are responders to treatment. In our study, we assessed the maximal respiration rate (MRR) and other respiratory parameters in eight fibroblast lines from subjects carrying LHON pathogenic variants. We measured also the effects of idebenone treatment on cell growth and mtDNA amounts. Results showed that LHON fibroblasts had significantly reduced respiratory parameters in untreated conditions, but no significant gain in MRR after idebenone supplementation. No major toxicity toward mitochondrial function and no relevant compensatory effect in terms of mtDNA quantity were found for the treatment at the tested conditions. Our findings confirmed that fibroblasts from subjects harboring LHON pathogenic variants displayed impaired respiration, regardless of the disease penetrance and severity. Testing responsiveness to idebenone treatment in cultured cells did not fully recapitulate in vivo data. The in-depth evaluation of cellular respiration in fibroblasts is a good approach to evaluating novel mtDNA variants associated with LHON but needs further evaluation as a potential biomarker for disease prognosis and treatment responsiveness.

Homozygous mutations in C1QBP as cause of progressive external ophthalmoplegia (PEO) and mitochondrial myopathy with multiple mtDNA deletions.

Biallelic mutations in the C1QBP gene have been associated with mitochondrial cardiomyopathy and combined respiratory-chain deficiencies, with variable onset (including intrauterine or neonatal forms), phenotypes, and severity. We studied two unrelated adult patients from consanguineous families, presenting with progressive external ophthalmoplegia (PEO), mitochondrial myopathy, and without any heart involvement. Muscle biopsies from both patients showed typical mitochondrial alterations and the presence of multiple mitochondrial DNA deletions, whereas biochemical defects of the respiratory chain were present only in one subject. Using next-generation sequencing approaches, we identified homozygous mutations in C1QBP. Immunoblot analyses in patients' muscle samples revealed a strong reduction in the amount of the C1QBP protein and varied impairment of respiratory chain complexes, correlating with disease severity. Despite the original study indicated C1QBP mutations as causative for mitochondrial cardiomyopathy, our data indicate that mutations in C1QBP have to be considered in subjects with PEO phenotype or primary mitochondrial myopathy and without cardiomyopathy.

Clinical-genetic features and peculiar muscle histopathology in infantile DNM1L-related mitochondrial epileptic encephalopathy.

Mitochondria are highly dynamic organelles, undergoing continuous fission and fusion. The DNM1L (dynamin-1 like) gene encodes for the DRP1 protein, an evolutionary conserved member of the dynamin family, responsible for fission of mitochondria, and having a role in the division of peroxisomes, as well. DRP1 impairment is implicated in several neurological disorders and associated with either de novo dominant or compound heterozygous mutations. In five patients presenting with severe epileptic encephalopathy, we identified five de novo dominant DNM1L variants, the pathogenicity of which was validated in a yeast model. Fluorescence microscopy revealed abnormally elongated mitochondria and aberrant peroxisomes in mutant fibroblasts, indicating impaired fission of these organelles. Moreover, a very peculiar finding in our cohort of patients was the presence, in muscle biopsy, of core like areas with oxidative enzyme alterations, suggesting an abnormal distribution of mitochondria in the muscle tissue.

Homozygous variant in OTX2 and possible genetic modifiers identified in a patient with combined pituitary hormone deficiency, ocular involvement, myopathy, ataxia, and mitochondrial impairment.

Here we report on a singleton patient affected by a complicated congenital syndrome characterized by growth delay, retinal dystrophy, sensorineural deafness, myopathy, ataxia, combined pituitary hormone deficiency, associated with mitochondrial impairment. Targeted clinical exome sequencing led to the identification of a homozygous missense variant in OTX2. Since only dominant mutations within OTX2 have been associated with cases of syndromic microphthalmia, retinal dystrophy with or without pituitary dysfunctions, this represents the first report of an OTX2 recessive mutation. Part of the phenotype, including ataxia, myopathy and multiple mitochondrial respiratory chain defects, seemed not related to OTX2. Further analysis of next generation sequencing (NGS) data revealed additional candidate variants: a homozygous variant in LETM1, and heterozygous rare variants in AFG3L2 and POLG. All three genes encode mitochondrial proteins and the last two are known to be associated with ataxia, a neurological sign present also in the father of the proband. With our study, we aim to encourage the integration of NGS data with a detailed analysis of clinical description and family history in order to unravel composite genotypes sometimes associated with complicated phenotypes.

A novel de novo dominant mutation in ISCU associated with mitochondrial myopathy.

BACKGROUND: Hereditary myopathy with lactic acidosis and myopathy with deficiency of succinate dehydrogenase and aconitase are variants of a recessive disorder characterised by childhood-onset early fatigue, dyspnoea and palpitations on trivial exercise. The disease is non-progressive, but life-threatening episodes of widespread weakness, metabolic acidosis and rhabdomyolysis may occur. So far, this disease has been molecularly defined only in Swedish patients, all homozygous for a deep intronic splicing affecting mutation in ISCU encoding a scaffold protein for the assembly of iron-sulfur (Fe-S) clusters. A single Scandinavian family was identified with a different mutation, a missense change in compound heterozygosity with the common intronic mutation. The aim of the study was to identify the genetic defect in our proband. METHODS: A next-generation sequencing (NGS) approach was carried out on an Italian male who presented in childhood with ptosis, severe muscle weakness and exercise intolerance. His disease was slowly progressive, with partial recovery between episodes. Patient's specimens and yeast models were investigated. RESULTS: Histochemical and biochemical analyses on muscle biopsy showed multiple defects affecting mitochondrial respiratory chain complexes. We identified a single heterozygous mutation p.Gly96Val in ISCU, which was absent in DNA from his parents indicating a possible de novo dominant effect in the patient. Patient fibroblasts showed normal levels of ISCU protein and a few variably affected Fe-S cluster-dependent enzymes. Yeast studies confirmed both pathogenicity and dominance of the identified missense mutation. CONCLUSION: We describe the first heterozygous dominant mutation in ISCU which results in a phenotype reminiscent of the recessive disease previously reported.

Nitric oxide regulates homeoprotein OTX1 and OTX2 expression in the rat myenteric plexus after intestinal ischemia-reperfusion injury.

Neuronal and inducible nitric oxide synthase (nNOS and iNOS) play a protective and damaging role, respectively, on the intestinal neuromuscular function after ischemia-reperfusion (I/R) injury. To uncover the molecular pathways underlying this dichotomy we investigated their possible correlation with the orthodenticle homeobox proteins OTX1 and OTX2 in the rat small intestine myenteric plexus after in vivo I/R. Homeobox genes are fundamental for the regulation of the gut wall homeostasis both during development and in pathological conditions (inflammation, cancer). I/R injury was induced by temporary clamping the superior mesenteric artery under anesthesia, followed by 24 and 48 h of reperfusion. At 48 h after I/R intestinal transit decreased and was further reduced by Nω-propyl-l-arginine hydrochloride (NPLA), a nNOS-selective inhibitor. By contrast this parameter was restored to control values by 1400W, an iNOS-selective inhibitor. In longitudinal muscle myenteric plexus (LMMP) preparations, iNOS, OTX1, and OTX2 mRNA and protein levels increased at 24 and 48 h after I/R. At both time periods, the number of iNOS- and OTX-immunopositive myenteric neurons increased. nNOS mRNA, protein levels, and neurons were unchanged. In LMMPs, OTX1 and OTX2 mRNA and protein upregulation was reduced by 1400W and NPLA, respectively. In myenteric ganglia, OTX1 and OTX2 staining was superimposed with that of iNOS and nNOS, respectively. Thus in myenteric ganglia iNOS- and nNOS-derived NO may promote OTX1 and OTX2 upregulation, respectively. We hypothesize that the neurodamaging and neuroprotective roles of iNOS and nNOS during I/R injury in the gut may involve corresponding activation of molecular pathways downstream of OTX1 and OTX2.NEW & NOTEWORTHY Intestinal ischemia-reperfusion (I/R) injury induces relevant alterations in myenteric neurons leading to dismotility. Nitrergic neurons seem to be selectively involved. In the present study the inference that both neuronal and inducible nitric oxide synthase (nNOS and iNOS) expressing myenteric neurons may undergo important changes sustaining derangements of motor function is reinforced. In addition, we provide data to suggest that NO produced by iNOS and nNOS regulates the expression of the vital transcription factors orthodenticle homeobox protein 1 and 2 during an I/R damage.

Mutations in APOPT1, encoding a mitochondrial protein, cause cavitating leukoencephalopathy with cytochrome c oxidase deficiency.

Cytochrome c oxidase (COX) deficiency is a frequent biochemical abnormality in mitochondrial disorders, but a large fraction of cases remains genetically undetermined. Whole-exome sequencing led to the identification of APOPT1 mutations in two Italian sisters and in a third Turkish individual presenting severe COX deficiency. All three subjects presented a distinctive brain MRI pattern characterized by cavitating leukodystrophy, predominantly in the posterior region of the cerebral hemispheres. We then found APOPT1 mutations in three additional unrelated children, selected on the basis of these particular MRI features. All identified mutations predicted the synthesis of severely damaged protein variants. The clinical features of the six subjects varied widely from acute neurometabolic decompensation in late infancy to subtle neurological signs, which appeared in adolescence; all presented a chronic, long-surviving clinical course. We showed that APOPT1 is targeted to and localized within mitochondria by an N-terminal mitochondrial targeting sequence that is eventually cleaved off from the mature protein. We then showed that APOPT1 is virtually absent in fibroblasts cultured in standard conditions, but its levels increase by inhibiting the proteasome or after oxidative challenge. Mutant fibroblasts showed reduced amount of COX holocomplex and higher levels of reactive oxygen species, which both shifted toward control values by expressing a recombinant, wild-type APOPT1 cDNA. The shRNA-mediated knockdown of APOPT1 in myoblasts and fibroblasts caused dramatic decrease in cell viability. APOPT1 mutations are responsible for infantile or childhood-onset mitochondrial disease, hallmarked by the combination of profound COX deficiency with a distinctive neuroimaging presentation.

Pantethine treatment is effective in recovering the disease phenotype induced by ketogenic diet in a pantothenate kinase-associated neurodegeneration mouse model.

Pantothenate kinase-associated neurodegeneration, caused by mutations in the PANK2 gene, is an autosomal recessive disorder characterized by dystonia, dysarthria, rigidity, pigmentary retinal degeneration and brain iron accumulation. PANK2 encodes the mitochondrial enzyme pantothenate kinase type 2, responsible for the phosphorylation of pantothenate or vitamin B5 in the biosynthesis of co-enzyme A. A Pank2 knockout (Pank2(-/-)) mouse model did not recapitulate the human disease but showed azoospermia and mitochondrial dysfunctions. We challenged this mouse model with a low glucose and high lipid content diet (ketogenic diet) to stimulate lipid use by mitochondrial beta-oxidation. In the presence of a shortage of co-enzyme A, this diet could evoke a general impairment of bioenergetic metabolism. Only Pank2(-/-) mice fed with a ketogenic diet developed a pantothenate kinase-associated neurodegeneration-like syndrome characterized by severe motor dysfunction, neurodegeneration and severely altered mitochondria in the central and peripheral nervous systems. These mice also showed structural alteration of muscle morphology, which was comparable with that observed in a patient with pantothenate kinase-associated neurodegeneration. We here demonstrate that pantethine administration can prevent the onset of the neuromuscular phenotype in mice suggesting the possibility of experimental treatment in patients with pantothenate kinase-associated neurodegeneration.

AAV-mediated liver-specific MPV17 expression restores mtDNA levels and prevents diet-induced liver failure.

Mutations in human MPV17 cause a hepatocerebral form of mitochondrial DNA depletion syndrome (MDS) hallmarked by early-onset liver failure, leading to premature death. Liver transplantation and frequent feeding using slow-release carbohydrates are the only available therapies, although surviving patients eventually develop slowly progressive peripheral and central neuropathy. The physiological role of Mpv17, including its functional link to mitochondrial DNA (mtDNA) maintenance, is still unclear. We show here that Mpv17 is part of a high molecular weight complex of unknown composition, which is essential for mtDNA maintenance in critical tissues, i.e. liver, of a Mpv17 knockout mouse model. On a standard diet, Mpv17-/- mouse shows hardly any symptom of liver dysfunction, but a ketogenic diet (KD) leads these animals to liver cirrhosis and failure. However, when expression of human MPV17 is carried out by adeno-associated virus (AAV)-mediated gene replacement, the Mpv17 knockout mice are able to reconstitute the Mpv17-containing supramolecular complex, restore liver mtDNA copy number and oxidative phosphorylation (OXPHOS) proficiency, and prevent liver failure induced by the KD. These results open new therapeutic perspectives for the treatment of MPV17-related liver-specific MDS.

A novel MT-ATP6 variant associated with complicated ataxia in two unrelated Italian patients: case report and functional studies.

BACKGROUND: MT-ATP6 is a mitochondrial gene which encodes for the intramembrane subunit 6 (or A) of the mitochondrial ATP synthase, also known asl complex V, which is involved in the last step of oxidative phosphorylation to produce cellular ATP through aerobic metabolism. Although classically associated with the NARP syndrome, recent evidence highlights an important role of MT-ATP6 pathogenic variants in complicated adult-onset ataxias. METHODS: We describe two unrelated patients with adult-onset cerebellar ataxia associated with severe optic atrophy and mild cognitive impairment. Whole mitochondrial DNA sequencing was performed in both patients. We employed patients' primary fibroblasts and cytoplasmic hybrids (cybrids), generated from patients-derived cells, to assess the activity of respiratory chain complexes, oxygen consumption rate (OCR), ATP production and mitochondrial membrane potential. RESULTS: In both patients, we identified the same novel m.8777 T > C variant in MT-ATP6 with variable heteroplasmy level in different tissues. We identifed an additional heteroplasmic novel variant in MT-ATP6, m.8879G > T, in the patients with the most severe phenotype. A significant reduction in complex V activity, OCR and ATP production was observed in cybrid clones homoplasmic for the m.8777 T > C variant, while no functional defect was detected in m.8879G > T homoplasmic clones. In addition, fibroblasts with high heteroplasmic levelsof m.8777 T > C variant showed hyperpolarization of mitochondrial membranes. CONCLUSIONS: We describe a novel pathogenic mtDNA variant in MT-ATP6 associated with adult-onset ataxia, reinforcing the value of mtDNA screening within the diagnostic workflow of selected patients with late onset ataxias.

An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model.

Deficiency of the mitochondrial respiratory chain complexes that carry out oxidative phosphorylation (OXPHOS) is the biochemical marker of human mitochondrial disorders. From a genetic point of view, the OXPHOS represents a unique example because it results from the complementation of two distinct genetic systems: nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Therefore, OXPHOS defects can be due to mutations affecting nuclear and mitochondrial encoded genes. The groundbreaking work by King and Attardi, published in 1989, showed that human cell lines depleted of mtDNA (named rho0) could be repopulated by exogenous mitochondria to obtain the so-called "transmitochondrial cybrids." Thanks to these cybrids containing mitochondria derived from patients with mitochondrial disorders (MDs) and nuclei from rho0 cells, it is possible to verify whether a defect is mtDNA- or nDNA-related. These cybrids are also a powerful tool to validate the pathogenicity of a mutation and study its impact at a biochemical level. This paper presents a detailed protocol describing cybrid generation, selection, and characterization.

Leber's Hereditary Optic Neuropathy: A Report on Novel mtDNA Pathogenic Variants.

Leber's hereditary optic neuropathy (LHON) is due to missense point mutations affecting mitochondrial DNA (mtDNA); 90% of cases harbor the m.3460G>A, m.11778G>A, and m.14484T>C primary mutations. Here, we report and discuss five families with patients affected by symptomatic LHON, in which we found five novel mtDNA variants. Remarkably, these mtDNA variants are located in complex I genes, though without strong deleterious effect on respiration in cellular models: this finding is likely linked to the tissue specificity of LHON. This study observes that in the case of a strong clinical suspicion of LHON, it is recommended to analyze the whole mtDNA sequence, since new rare mtDNA pathogenic variants causing LHON are increasingly identified.

Movement Disorders in Children with a Mitochondrial Disease: A Cross-Sectional Survey from the Nationwide Italian Collaborative Network of Mitochondrial Diseases.

Movement disorders are increasingly being recognized as a manifestation of childhood-onset mitochondrial diseases (MDs). However, the spectrum and characteristics of these conditions have not been studied in detail in the context of a well-defined cohort of patients. We retrospectively explored a cohort of individuals with childhood-onset MDs querying the Nationwide Italian Collaborative Network of Mitochondrial Diseases database. Using a customized online questionnaire, we attempted to collect data from the subgroup of patients with movement disorders. Complete information was available for 102 patients. Movement disorder was the presenting feature of MD in 45 individuals, with a mean age at onset of 11 years. Ataxia was the most common movement disorder at onset, followed by dystonia, tremor, hypokinetic disorders, chorea, and myoclonus. During the disease course, most patients (67.7%) encountered a worsening of their movement disorder. Basal ganglia involvement, cerebral white matter changes, and cerebellar atrophy were the most commonly associated neuroradiological patterns. Forty-one patients harbored point mutations in the mitochondrial DNA, 10 carried mitochondrial DNA rearrangements, and 41 cases presented mutations in nuclear-DNA-encoded genes, the latter being associated with an earlier onset and a higher impairment in activities of daily living. Among our patients, 32 individuals received pharmacological treatment; clonazepam and oral baclofen were the most commonly used drugs, whereas levodopa and intrathecal baclofen administration were the most effective. A better delineation of the movement disorders phenotypes starting in childhood may improve our diagnostic workup in MDs, fine tuning management, and treatment of affected patients.

Targeting Multiple Mitochondrial Processes by a Metabolic Modulator Prevents Sarcopenia and Cognitive Decline in SAMP8 Mice.

The age-dependent declines of skeletal muscle and cognitive functions often coexist in elderly subjects. The underlying pathophysiological mechanisms share common features of mitochondrial dysfunction, which plays a central role in the development of overt sarcopenia and/or dementia. Dietary supplementation with formulations of essential and branched-chain amino acids (EAA-BCAA) is a promising preventive strategy because it can preserve mitochondrial biogenesis and function. The senescence-accelerated mouse prone 8 (SAMP8) is considered an accurate model of age-related muscular and cognitive alterations. Hence, we aimed to investigate the progression of mitochondrial dysfunctions during muscular and cognitive aging of SAMP8 mice and to study the effects of a novel EAA-BCAA-based metabolic modulator on these changes. We evaluated body condition, motor endurance, and working memory of SAMP8 mice at 5, 9, 12, and 15 months of age. Parallel changes in protein levels of mitochondrial respiratory chain subunits, regulators of mitochondrial biogenesis and dynamics, and the antioxidant response, as well as respiratory complex activities, were measured in the quadriceps femoris and the hippocampus. The same variables were assessed in 12-month-old SAMP8 mice that had received dietary supplementation with the novel EAA-BCAA formulation, containing tricarboxylic acid cycle intermediates and co-factors (PD-0E7, 1.5 mg/kg/body weight/day in drinking water) for 3 months. Contrary to untreated mice, which had a significant molecular and phenotypic impairment, PD-0E7-treated mice showed preserved healthy body condition, muscle weight to body weight ratio, motor endurance, and working memory at 12 months of age. The PD-0E7 mixture increased the protein levels and the enzymatic activities of mitochondrial complex I, II, and IV and the expression of proliferator-activated receptor γ coactivator-1α, optic atrophy protein 1, and nuclear factor, erythroid 2 like 2 in muscles and hippocampi. The mitochondrial amyloid-ß-degrading pitrilysin metallopeptidase 1 was upregulated, while amyloid precursor protein was reduced in the hippocampi of PD-0E7 treated mice. In conclusion, we show that a dietary supplement tailored to boost mitochondrial respiration preserves skeletal muscle and hippocampal mitochondrial quality control and health. When administered at the early onset of age-related physical and cognitive decline, this novel metabolic inducer counteracts the deleterious effects of precocious aging in both domains.

Primary mitochondrial myopathy: Clinical features and outcome measures in 118 cases from Italy.

OBJECTIVE: To determine whether a set of functional tests, clinical scales, patient-reported questionnaires, and specific biomarkers can be considered reliable outcome measures in patients with primary mitochondrial myopathy (PMM), we analyzed a cohort of Italian patients. METHODS: Baseline data were collected from 118 patients with PMM, followed by centers of the Italian network for mitochondrial diseases. We used the 6-Minute Walk Test (6MWT), Timed Up-and-Go Test (x3) (3TUG), Five-Times Sit-To-Stand Test (5XSST), Timed Water Swallow Test (TWST), and Test of Masticating and Swallowing Solids (TOMASS) as functional outcome measures; the Fatigue Severity Scale and West Haven-Yale Multidimensional Pain Inventory as patient-reported outcome measures; and FGF21, GDF15, lactate, and creatine kinase (CK) as biomarkers. RESULTS: A total of 118 PMM cases were included. Functional outcome measures (6MWT, 3TUG, 5XSST, TWST, and TOMASS) and biomarkers significantly differed from healthy reference values and controls. Moreover, functional measures correlated with patients' perceived fatigue and pain severity. Patients with either mitochondrial or nuclear DNA point mutations performed worse in functional measures than patients harboring single deletion, even if the latter had an earlier age at onset but similar disease duration. Both the biomarkers FGF21 and GDF15 were significantly higher in the patients compared with a matched control population; however, there was no relation with severity of disease. CONCLUSIONS: We characterized a large cohort of PMM by evaluating baseline mitochondrial biomarkers and functional scales that represent potential outcome measures to monitor the efficacy of treatment in clinical trials; these outcome measures will be further reinvestigated longitudinally to define the natural history of PMM.

Expanding the molecular and phenotypic spectrum of truncating MT-ATP6 mutations.

OBJECTIVE: To describe the clinical and functional consequences of 1 novel and 1 previously reported truncating MT-ATP6 mutation. METHODS: Three unrelated probands with mitochondrial encephalomyopathy harboring truncating MT-ATP6 mutations are reported. Transmitochondrial cybrid cell studies were used to confirm pathogenicity of 1 novel variant, and the effects of all 3 mutations on ATPase 6 and complex V structure and function were investigated. RESULTS: Patient 1 presented with adult-onset cerebellar ataxia, chronic kidney disease, and diabetes, whereas patient 2 had myoclonic epilepsy and cerebellar ataxia; both harbored the novel m.8782G>A; p.(Gly86*) mutation. Patient 3 exhibited cognitive decline, with posterior white matter abnormalities on brain MRI, and severely impaired renal function requiring transplantation. The m.8618dup; p.(Thr33Hisfs*32) mutation, previously associated with neurogenic muscle weakness, ataxia, and retinitis pigmentosa, was identified. All 3 probands demonstrated a broad range of heteroplasmy across different tissue types. Blue-native gel electrophoresis of cultured fibroblasts and skeletal muscle tissue confirmed multiple bands, suggestive of impaired complex V assembly. Microscale oxygraphy showed reduced basal respiration and adenosine triphosphate synthesis, while reactive oxygen species generation was increased. Transmitochondrial cybrid cell lines studies confirmed the deleterious effects of the novel m.8782 G>A; p.(Gly86*) mutation. CONCLUSIONS: We expand the clinical and molecular spectrum of MT-ATP6-related mitochondrial disorders to include leukodystrophy, renal disease, and myoclonic epilepsy with cerebellar ataxia. Truncating MT-ATP6 mutations may exhibit highly variable mutant levels across different tissue types, an important consideration during genetic counseling.

Alteration of mitochondrial membrane inner potential in three Italian patients with megaconial congenital muscular dystrophy carrying new mutations in CHKB gene.

Congenital Muscular Dystrophies (CMDs) are a heterogeneous group of autosomal recessive disorders presenting at birth with psychomotor delay, cognitive impairment, muscle weakness and hypotonia. Here we described an alteration of mitochondrial inner membrane potential and mitochondrial network in cells derived from Italian patients carrying three novel mutations in CHKB gene, recently associated with "megaconial CMD". On the bases of our findings, we hypothesize that the mitochondrial membrane potential alteration, presumably as a consequence of the altered biosynthesis of phosphatidylcholine, could be responsible for the peculiar morphological aspect of mitochondria in this disease and might be involved in the disease pathogenesis.

Characterization of c-kit (CD117) expression in human normal pituitary cells and pituitary adenomas.

c-kit (CD117) is a tyrosine kinase receptor involved in the proliferation, differentiation, and secretory functions of various cells. In experimental animal models, c-kit has been detected in the pars intermedia of the normal pituitary gland and in alpha-melanocyte-stimulating-hormone-positive adenomas and it has been suggested that it plays a role in regulating adrenocorticotropic hormone (ACTH) secretion. To the best of our knowledge, the expression of c-kit in normal human pituitary cells and in pituitary adenomas has never been reported, so the possible biological role of this receptor in the control of pituitary hormone secretion remains unclear. The aim of this study was to evaluate the immunohistochemical expression of c-kit in normal human pituitary glands and in a series of 62 well-characterized pituitary adenomas. In normal adenohypophyses, several cells, mainly located in the central mucoid wedge, showed a c-kit immunoreactivity (IR). Double label immunostaining procedures showed that the c-kit-IR cells corresponded to ACTH cells. Out of 62 adenomas, 15 (24%) were c-kit-IR, including 7/16 (44%) ACTH cell, 3/7 (42%) null cell, 4/11 (36%) alpha-subunit cell, and 1/11 (10%) follicle-stimulating hormone-luteinizing hormone cell adenomas. By contrast, all ten prolactin cell and seven growth hormone cell adenomas were c-kit negative. These data suggest that, in normal conditions, c-kit may be involved in the pituitary-adrenal axis regulation.