Valentina Emmanuele

Nuclear genome transfer in human oocytes eliminates mitochondrial DNA variants

Mitochondrial DNA mutations transmitted maternally within the oocyte cytoplasm often cause life-threatening disorders. Here we explore the use of nuclear genome transfer between unfertilized oocytes of two donors to prevent the transmission of mitochondrial mutations. Nuclear genome transfer did not reduce developmental efficiency to the blastocyst stage, and genome integrity was maintained provided that spontaneous oocyte activation was avoided through the transfer of incompletely assembled spindle–chromosome complexes. Mitochondrial DNA transferred with the nuclear genome was initially detected at levels below 1%, decreasing in blastocysts and stem-cell lines to undetectable levels, and remained undetectable after passaging for more than one year, clonal expansion, differentiation into neurons, cardiomyocytes or β-cells, and after cellular reprogramming. Stem cells and differentiated cells had mitochondrial respiratory chain enzyme activities and oxygen consumption rates indistinguishable from controls. These results demonstrate the potential of nuclear genome transfer to prevent the transmission of mitochondrial disorders in humans.

Heterogeneity of Coenzyme Q10 Deficiency: Patient Study and Literature Review

Coenzyme Q(10) (CoQ(10)) deficiency has been associated with 5 major clinical phenotypes: encephalomyopathy, severe infantile multisystemic disease, nephropathy, cerebellar ataxia, and isolated myopathy. Primary CoQ(10) deficiency is due to defects in CoQ(10) biosynthesis, while secondary forms are due to other causes. A review of 149 cases, including our cohort of 76 patients, confirms that CoQ(10) deficiency is a clinically and genetically heterogeneous syndrome that mainly begins in childhood and predominantly manifests as cerebellar ataxia. Coenzyme Q(10) measurement in muscle is the gold standard for diagnosis. Identification of CoQ(10) deficiency is important because the condition frequently responds to treatment. Causative mutations have been identified in a small proportion of patients.

Tissue-specific oxidative stress and loss of mitochondria in CoQ-deficient Pdss2 mutant mice

Primary human CoQ10 deficiencies are clinically heterogeneous diseases caused by mutations in PDSS2 and other genes required for CoQ10 biosynthesis. Our in vitro studies of PDSS2 mutant fibroblasts, with <20% CoQ10 of control cells, revealed reduced activity of CoQ10‐dependent complex II+III and ATP synthesis, without amplification of reactive oxygen species (ROS), markers of oxidative damage, or antioxidant defenses. In contrast, COQ2 and ADCK3 mutant fibroblasts, with 30–50% CoQ10 of controls, showed milder bioenergetic defects but significantly increased ROS and oxidation of lipids and proteins. We hypothesized that absence of oxidative stress markers and cell death in PDSS2 mutant fibroblasts were due to the extreme severity of CoQ10 deficiency. Here, we have investigated in vivo effects of Pdss2 deficiency in affected and unaffected organs of CBA/Pdss2kd/kd mice at presymptomatic, phenotypic‐onset, and end‐stages of the disease. Although Pdss2 mutant mice manifest widespread CoQ9 deficiency and mitochondrial respiratory chain abnormalities, only affected organs show increased ROS production, oxidative stress, mitochondrial DNA depletion, and reduced citrate synthase activity, an index of mitochondrial mass. Our data indicate that kidney‐specific loss of mitochondria triggered by oxidative stress may be the cause of renal failure in Pdss2kd/kd mice.—Quinzii, C. M., Garone, C., Emmanuele, V., Tadesse, S., Krishna, S., Dorado, B., Hirano, M. Tissue‐specific oxidative stress and loss of mitochondria in CoQ‐deficient Pdss2 mutant mice. FASEB J. 27, 612–621 (2013). www.fasebj.org

Deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency

Autosomal recessive mutations in the thymidine kinase 2 gene (TK2) cause mitochondrial DNA depletion, multiple deletions, or both due to loss of TK2 enzyme activity and ensuing unbalanced deoxynucleotide triphosphate (dNTP) pools. To bypass Tk2 deficiency, we administered deoxycytidine and deoxythymidine monophosphates (dCMP+dTMP) to the Tk2 H126N (Tk2−/−) knock‐in mouse model from postnatal day 4, when mutant mice are phenotypically normal, but biochemically affected. Assessment of 13‐day‐old Tk2−/− mice treated with dCMP+dTMP 200 mg/kg/day each (Tk2−/−200dCMP/dTMP) demonstrated that in mutant animals, the compounds raise dTTP concentrations, increase levels of mtDNA, ameliorate defects of mitochondrial respiratory chain enzymes, and significantly prolong their lifespan (34 days with treatment versus 13 days untreated). A second trial of dCMP+dTMP each at 400 mg/kg/day showed even greater phenotypic and biochemical improvements. In conclusion, dCMP/dTMP supplementation is the first effective pharmacologic treatment for Tk2 deficiency.

Clinical Presentations of Coenzyme Q10 Deficiency Syndrome

Coenzyme Q10 (CoQ10) deficiency is a clinically and genetically heterogeneous syndrome which has been associated with 5 major clinical phenotypes: (1) encephalomyopathy, (2) severe infantile multisystemic disease, (3) nephropathy, (4) cerebellar ataxia, and (5) isolated myopathy. Of these phenotypes, cerebellar ataxia and syndromic or isolated nephrotic syndrome are the most common. CoQ10 deficiency predominantly presents in childhood. To date, causative mutations have been identified in a small proportion of patients, making it difficult to identify a phenotype-genotype correlation. Identification of CoQ10 deficiency is important because the disease, in particular muscle symptoms and nephropathy, frequently responds to CoQ10 supplementation.

A Novel Mutation in the Mitochondrial DNA Cytochrome b Gene (MTCYB) in a Patient With Mitochondrial Encephalomyopathy, Lactic Acidosis, and Strokelike Episodes Syndrome

Mutations in the mitochondrial DNA cytochrome b gene (MTCYB) have been commonly associated with isolated mitochondrial myopathy and exercise intolerance, rarely with multisystem disorders, and only once with a parkinsonism/mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) overlap syndrome. Here, we describe a novel mutation (m.14864 T>C) in MTCYB in a 15-year-old girl with a clinical history of migraines, epilepsy, sensorimotor neuropathy, and strokelike episodes, a clinical picture reminiscent of MELAS. The mutation, which changes a highly conserved cysteine to arginine at amino acid position 40 of cytochrome b, was heteroplasmic in muscle, blood, fibroblasts, and urinary sediment from the patient but absent in accessible tissues from her asymptomatic mother. This case demonstrates that MTCYB must be included in the already long list of mitochondrial DNA genes that have been associated with the MELAS phenotype.

MERRF and Kearns–Sayre overlap syndrome due to the mitochondrial DNA m.3291T>C mutation

A 48‐year‐old man presented with a complex phenotype of myoclonus epilepsy with ragged‐red fibers (MERRF) syndrome and Kearns–Sayre syndrome (KSS), which included progressive myoclonus epilepsy, cerebellar ataxia, hearing loss, myopathic weakness, ophthalmoparesis, pigmentary retinopathy, bifascicular heart block, and ragged‐red fibers. The m.3291T>C mutation in the tRNALeu(UUR) gene was found with 92% heteroplasmy in muscle. This mutation has been reported with MELAS, myopathy, and deafness with cognitive impairment. This is the first description with a MERRF/KSS syndrome. Muscle Nerve 44: 448–451, 2011

Retinopathy screening in patients with type 1 diabetes diagnosed in young age using a non-mydriatic digital stereoscopic retinal imaging.

Background: Diabetic retinopathy seriously impairs patients’ quality of life, since it represents the first cause of blindness in industrialized countries. Aim: To estimate prevalence of retinopathy in young Type 1 diabetes patients using a non-mydriatic digital stereoscopic retinal imaging (NMDSRI), and to evaluate the impact of socio-demographic, clinical, and metabolic variables. Subjects and methods: In 247 young patients glycated hemoglobin (HbA1c), gender, age, pubertal stage, presence of diabetic ketoacidosis (DKA), HLA-DQ heterodimers of susceptibility for Type 1 diabetes, and β-cell autoimmunity at clinical onset were considered. At retinopathy screening, we evaluated age, disease duration, pubertal stage, body mass index (BMI-SDS), insulin requirement, HbA1c levels, other autoimmune diseases, diabetes-related complications, serum concentrations of cholesterol and triglycerides, systolic and diastolic blood pressure. Results: Retinopathy was found in 26/247patients: 25 showed background retinopathy, and 1 had a sight-threatening retinopathy. A significant relationship between retinopathy and female gender (p=0.01), duration of disease ≥15 yr (p<0.0001), serum triglycerides levels >65 mg/dl (p=0.012) and mean HbA1c ≥7.5% or >9% (p=0.0014) were found at the multivariate logistic analysis. Conclusions: Metabolic control is the most important modifiable factor and promotion of continuous educational process to reach a good metabolic control is a cornerstone to prevent microangiopathic complications. Symptoms appear when the complication is already established; a screening program with an early diagnosis is mandatory to prevent an irreversible damage.

Recurrent myoglobinuria in a sporadic patient with a novel mitochondrial DNA tRNAIle mutation

The differential diagnosis of myoglobinuria includes multiple etiologies, such as infection, inflammation, trauma, endocrinopathies, drugs toxicity, and primary metabolic disorders. Metabolic myopathies can be due to inherited disorders of glycogen metabolism or to defects of fatty acid oxidation. Primary respiratory chain dysfunction is a rare cause of myoglobinuria, but it has been described in sporadic cases with mutations in genes encoding cytochrome b or cytochrome c oxidase (COX) subunits and in four cases with tRNA mutations. We describe a 39-year-old woman with myalgia and exercise-related recurrent myoglobinuria, who harbored a novel mitochondrial DNA mutation at nucleotide 4281 (m.4281A>G) in the tRNA-isoleucine gene. Her muscle biopsy revealed ragged-red and COX-deficient fibers. No deletions or duplication were detected by Southern blot analysis. The m.4281A>G mutation was present in the patients muscle with a mutation load of 46% and was detected in trace amounts in urine and cheek mucosa. Single-fiber analysis revealed significantly higher levels of the mutation in COX-deficient (65%) than in normal fibers (45%). This novel mutation has to be added to the molecular causes of recurrent myoglobinuria.

Decreased hippocampal expression of Calbindin D28K and cognitive impairment in MELAS

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a maternally inherited mitochondrial syndrome characterized by seizures, migrainous headaches, lactic acidosis, vomiting, and recurrent stroke-like episodes. Patients often suffer from cognitive dysfunction of unclear pathogenesis. In this study, we explored a possible link between cognitive dysfunction and hippocampal expression of calbindin D(28KD) (CB), a high affinity calcium-binding protein, in four MELAS patients, using post mortem hippocampal tissues. We found significantly reduced CB levels in all patients by immunohistochemistry, Western blot, and quantitative real-time PCR. Reduction in CB expression has been associated with aging and with neurodegenerative disorders, including Alzheimers disease. We postulate that the reduced CB expression may play a role in the cognitive abnormalities associated with MELAS.