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1.  A POGLUT1 mutation causes a muscular dystrophy with reduced Notch signaling and satellite cell loss 
EMBO Molecular Medicine  2016;8(11):1289-1309.
Skeletal muscle regeneration by muscle satellite cells is a physiological mechanism activated upon muscle damage and regulated by Notch signaling. In a family with autosomal recessive limb‐girdle muscular dystrophy, we identified a missense mutation in POGLUT1 (protein O‐glucosyltransferase 1), an enzyme involved in Notch posttranslational modification and function. In vitro and in vivo experiments demonstrated that the mutation reduces O‐glucosyltransferase activity on Notch and impairs muscle development. Muscles from patients revealed decreased Notch signaling, dramatic reduction in satellite cell pool and a muscle‐specific α‐dystroglycan hypoglycosylation not present in patients' fibroblasts. Primary myoblasts from patients showed slow proliferation, facilitated differentiation, and a decreased pool of quiescent PAX7+ cells. A robust rescue of the myogenesis was demonstrated by increasing Notch signaling. None of these alterations were found in muscles from secondary dystroglycanopathy patients. These data suggest that a key pathomechanism for this novel form of muscular dystrophy is Notch‐dependent loss of satellite cells.
PMCID: PMC5090660  PMID: 27807076
muscular dystrophy; Notch; O‐glycosylation; POGLUT1; satellite cell; Development & Differentiation; Musculoskeletal System
2.  Allogeneic haematopoietic stem cell transplantation for mitochondrial neurogastrointestinal encephalomyopathy 
Brain  2015;138(10):2847-2858.
Mitochondrial neurogastrointestinal encephalopathy (MNGIE) is a rare fatal autorecessive disease. Halter et al. report outcomes from all known haematopoietic stem cell transplantations worldwide from sibling or unrelated donors for MNGIE between 2005 and 2011. In some of the recipients, correction of the underlying metabolic defect results in gradual clinical improvement.
Mitochondrial neurogastrointestinal encephalopathy (MNGIE) is a rare fatal autorecessive disease. Halter et al. report outcomes from all known haematopoietic stem cell transplantations worldwide from sibling or unrelated donors for MNGIE between 2005 and 2011. In some of the recipients, correction of the underlying metabolic defect results in gradual clinical improvement.
Haematopoietic stem cell transplantation has been proposed as treatment for mitochondrial neurogastrointestinal encephalomyopathy, a rare fatal autosomal recessive disease due to TYMP mutations that result in thymidine phosphorylase deficiency. We conducted a retrospective analysis of all known patients suffering from mitochondrial neurogastrointestinal encephalomyopathy who underwent allogeneic haematopoietic stem cell transplantation between 2005 and 2011. Twenty-four patients, 11 males and 13 females, median age 25 years (range 10–41 years) treated with haematopoietic stem cell transplantation from related (n = 9) or unrelated donors (n = 15) in 15 institutions worldwide were analysed for outcome and its associated factors. Overall, 9 of 24 patients (37.5%) were alive at last follow-up with a median follow-up of these surviving patients of 1430 days. Deaths were attributed to transplant in nine (including two after a second transplant due to graft failure), and to mitochondrial neurogastrointestinal encephalomyopathy in six patients. Thymidine phosphorylase activity rose from undetectable to normal levels (median 697 nmol/h/mg protein, range 262–1285) in all survivors. Seven patients (29%) who were engrafted and living more than 2 years after transplantation, showed improvement of body mass index, gastrointestinal manifestations, and peripheral neuropathy. Univariate statistical analysis demonstrated that survival was associated with two defined pre-transplant characteristics: human leukocyte antigen match (10/10 versus <10/10) and disease characteristics (liver disease, history of gastrointestinal pseudo-obstruction or both). Allogeneic haematopoietic stem cell transplantation can restore thymidine phosphorylase enzyme function in patients with mitochondrial neurogastrointestinal encephalomyopathy and improve clinical manifestations of mitochondrial neurogastrointestinal encephalomyopathy in the long term. Allogeneic haematopoietic stem cell transplantation should be considered for selected patients with an optimal donor.
PMCID: PMC4836400  PMID: 26264513
mitochondrial neurogastrointestinal encephalomyopathy (MNGIE); allogeneic haematopoietic stem cell transplantation; outcome; risk factors; thymidine phosphorylase
3.  Diagnosis and management of mitochondrial disease: a consensus statement from the Mitochondrial Medicine Society 
The purpose of this statement is to review the literature regarding mitochondrial disease and to provide recommendations for optimal diagnosis and treatment. This statement is intended for physicians who are engaged in diagnosing and treating these patients.
The Writing Group members were appointed by the Mitochondrial Medicine Society. The panel included members with expertise in several different areas. The panel members utilized a comprehensive review of the literature, surveys, and the Delphi method to reach consensus. We anticipate that this statement will need to be updated as the field continues to evolve.
Consensus-based recommendations are provided for the diagnosis and treatment of mitochondrial disease.
The Delphi process enabled the formation of consensus-based recommendations. We hope that these recommendations will help standardize the evaluation, diagnosis, and care of patients with suspected or demonstrated mitochondrial disease.
PMCID: PMC5000852  PMID: 25503498
consensus criteria; Delphi method; mitochondrial disease; mitochondrial medicine; Mitochondrial Medicine Society
4.  Mitochondrial Cardioencephalomyopathy Due to a Novel SCO2 Mutation in a Brazilian Patient 
JAMA neurology  2013;70(2):258-261.
To review all patients with SCO2 mutations and to describe a Brazilian patient with cardioencephalomyopathy carrying compound heterozygous mutations in SCO2, one being the known pathogenic p.E140K mutation and the other a novel 12–base pair (bp) deletion at nucleotides 1519 through 1530 (c.1519_1530del).
Case report and literature review.
University hospital
Infant girl presenting with an encephalomyopathy, inspiratory stridor, ventilator failure, progressive hypotonia, and weakness, leading to death.
Main Outcome Measures
Clinical features, neuro-imaging findings, muscle biopsy with histochemical analysis, and genetic studies.
This infant girl was the first child of healthy, nonconsanguineous parents. She developed progressive muscular hypotonia and ventilatory failure. At the end of the first month of life, she developed cardiomegaly and signs of cardiac failure. Routine blood tests showed lactic acidosis and mild elevation of the creatine kinase level. Brain magnetic resonance imaging showed increased T2 and fluid-attenuated inversion recovery signals in the putamen bilaterally. Nerve conduction studies showed severe axonal sensorimotor neuropathy. Muscle biopsy revealed a neurogenic pattern with mitochondrial proliferation and total absence of cytochrome-c oxidase histochemical stain. Sequencing of SCO2 showed that the patient had compound heterozygote SCO2 mutations: the previously described c.1541G A (p.E140K) mutation and a novel 12-bp deletion at nucleotides 1519 through 1530 (c.1519_1530del). The patient died at age 45 days.
Our findings and the literature review indicate that it is important to consider the diagnosis of mitochondrial disease in newborns with hypotonia and cardiomyopathy. In our case, the accurate diagnosis of SCO2 mutations is particularly important for genetic counseling.
PMCID: PMC4967497  PMID: 23407777
5.  New treatments for mitochondrial disease—no time to drop our standards 
Nature reviews. Neurology  2013;9(8):474-481.
Mitochondrial dysfunction is a common cause of inherited multisystem disease that often involves the nervous system. Despite major advances in our understanding of the pathophysiology of mitochondrial diseases, clinical management of these conditions remains largely supportive. Using a systematic approach, we identified 1,039 publications on treatments for mitochondrial diseases, only 35 of which included observations on more than five patients. Reports of a positive outcome on the basis of a biomarker of unproven clinical significance were more common in nonrandomized and nonblinded studies, suggesting a publication bias toward positive but poorly executed studies. Although trial design is improving, there is a critical need to develop new biomarkers of mitochondrial disease. In this Perspectives article, we make recommendations for the design of future treatment trials in mitochondrial diseases. Patients and physicians should no longer rely on potentially biased data, with the associated costs and risks.
PMCID: PMC4967498  PMID: 23817350
6.  Mutant COQ2 in Multiple-System Atrophy 
PMCID: PMC4961084  PMID: 24988570
7.  A Novel Mutation in PNPLA2 Leading to Neutral Lipid Storage Disease With Myopathy 
Archives of neurology  2012;69(9):1190-1192.
Mutations in PNPLA2, a gene encoding adipose triglyceride lipase, lead to neutral lipid storage disease with myopathy.
To report the clinical and molecular features of a case of neutral lipid storage disease with myopathy resulting from a novel mutation in PNPLA2.
Case report.
University hospital.
A 65-year-old man with progressive muscle weakness and high serum creatine kinase levels.
Direct sequencing of the PNPLA2 gene.
Identification of a novel homozygous mutation in the patient’s PNPLA2 gene confirmed the suspected diagnosis of neutral lipid storage disease with myopathy.
Screening of the PNPLA2 gene should be considered for patients presenting with high levels of creatine kinase, progressive muscle weakness, and systemic lipid accumulation. The presence of Jordans anomaly can be a strong diagnostic clue.
PMCID: PMC4961086  PMID: 22964912
8.  Assessment of Thymidine Phosphorylase Function: Measurement of Plasma Thymidine (and Deoxyuridine) and Thymidine Phosphorylase Activity 
We describe detailed methods to measure thymidine (dThd) and deoxyuridine (dUrd) concentrations and thymidine phosphorylase (TP) activity in biological samples. These protocols allow the detection of TP dysfunction in patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Since the identification of mutations in TϒMP, the gene encoding TP, as the cause of MNGIE (Nishino et al. Science 283:689–692, 1999), the assessment of TP dysfunction has become the best screening method to rule out or confirm MNGIE in patients. TϒMP sequencing, to find the causative mutations, is only needed when TP dysfunction is detected. dThd and dUrd are measured by resolving these compounds with high-performance liquid chromatography (HPLC) followed by the spectrophotometric monitoring of the eluate absorbance at 267 nm (HPLC-UV). TP activity can be measured by an endpoint determination of the thymine formed after 1 h incubation of the buffy coat homogenate in the presence of a large excess of its substrate dThd, either spectrophotometrically or by HPLC-UV.
PMCID: PMC4942128  PMID: 22215544
Thymidine; Deoxyuridine; Thymidine phosphorylase; Mitochondrial neurogastrointestinal encephalomyopathy; MNGIE; HPLC; Biochemical diagnosis
9.  Fhl1 W122S causes loss of protein function and late-onset mild myopathy 
Human Molecular Genetics  2014;24(3):714-726.
A member of the four-and-a-half-LIM (FHL) domain protein family, FHL1, is highly expressed in human adult skeletal and cardiac muscle. Mutations in FHL1 have been associated with diverse X-linked muscle diseases: scapuloperoneal (SP) myopathy, reducing body myopathy, X-linked myopathy with postural muscle atrophy, rigid spine syndrome (RSS) and Emery-Dreifuss muscular dystrophy. In 2008, we identified a missense mutation in the second LIM domain of FHL1 (c.365 G>C, p.W122S) in a family with SP myopathy. We generated a knock-in mouse model harboring the c.365 G>C Fhl1 mutation and investigated the effects of this mutation at three time points (3–5 months, 7–10 months and 18–20 months) in hemizygous male and heterozygous female mice. Survival was comparable in mutant and wild-type animals. We observed decreased forelimb strength and exercise capacity in adult hemizygous male mice starting from 7 to 10 months of age. Western blot analysis showed absence of Fhl1 in muscle at later stages. Thus, adult hemizygous male, but not heterozygous female, mice showed a slowly progressive phenotype similar to human patients with late-onset muscle weakness. In contrast to SP myopathy patients with the FHL1 W122S mutation, mutant mice did not manifest cytoplasmic inclusions (reducing bodies) in muscle. Because muscle weakness was evident prior to loss of Fhl1 protein and without reducing bodies, our findings indicate that loss of function is responsible for the myopathy in the Fhl1 W122S knock-in mice.
PMCID: PMC4342698  PMID: 25274776
10.  MPV17 Loss Causes Deoxynucleotide Insufficiency and Slow DNA Replication in Mitochondria 
PLoS Genetics  2016;12(1):e1005779.
MPV17 is a mitochondrial inner membrane protein whose dysfunction causes mitochondrial DNA abnormalities and disease by an unknown mechanism. Perturbations of deoxynucleoside triphosphate (dNTP) pools are a recognized cause of mitochondrial genomic instability; therefore, we determined DNA copy number and dNTP levels in mitochondria of two models of MPV17 deficiency. In Mpv17 ablated mice, liver mitochondria showed substantial decreases in the levels of dGTP and dTTP and severe mitochondrial DNA depletion, whereas the dNTP pool was not significantly altered in kidney and brain mitochondria that had near normal levels of DNA. The shortage of mitochondrial dNTPs in Mpv17-/- liver slows the DNA replication in the organelle, as evidenced by the elevated level of replication intermediates. Quiescent fibroblasts of MPV17-mutant patients recapitulate key features of the primary affected tissue of the Mpv17-/- mice, displaying virtual absence of the protein, decreased dNTP levels and mitochondrial DNA depletion. Notably, the mitochondrial DNA loss in the patients’ quiescent fibroblasts was prevented and rescued by deoxynucleoside supplementation. Thus, our study establishes dNTP insufficiency in the mitochondria as the cause of mitochondrial DNA depletion in MPV17 deficiency, and identifies deoxynucleoside supplementation as a potential therapeutic strategy for MPV17-related disease. Moreover, changes in the expression of factors involved in mitochondrial deoxynucleotide homeostasis indicate a remodeling of nucleotide metabolism in MPV17 disease models, which suggests mitochondria lacking functional MPV17 have a restricted purine mitochondrial salvage pathway.
Author Summary
Mitochondrial DNA depletion syndrome (MDS) is a genetically heterogeneous condition characterized by a decrease of mitochondrial DNA (mtDNA) copy number and decreased activities of respiratory chain enzymes. Depletion of mtDNA has been associated with mutations in several genes, which encode either proteins directly involved in mtDNA replication or factors regulating the homeostasis of the mitochondrial deoxynucleotide pool. However, for some genes the mechanism linking mutations and mtDNA depletion is not known. One such gene is MPV17, whose loss-of-function causes mtDNA abnormalities in human, mouse and yeast. Here we show that MPV17 dysfunction leads to a shortage of the precursors for DNA synthesis in the mitochondria, slowing DNA replication in the organelle. Not only does mtDNA copy number correlate with dNTP pool size in both mouse tissues and human cells, deoxynucleoside supplementation of the growth medium prevents depletion and restores mtDNA copy number in quiescent MPV17-deficient cells. Hence, our study links MPV17 deficiency, insufficiency of mitochondrial dNTPs, and slow replication in mitochondria to depletion of mtDNA manifesting in the human disease, and places MPV17-related disease firmly in the category of mtDNA disorders caused by deoxynucleotide perturbation. The prevention and reversal of mtDNA loss in MPV17 patient-derived cells identifies potential therapeutic strategy for a currently untreatable disease.
PMCID: PMC4711891  PMID: 26760297
11.  Inhibition of NAPDH Oxidase 2 (NOX2) Prevents Oxidative Stress and Mitochondrial Abnormalities Caused by Saturated Fat in Cardiomyocytes 
PLoS ONE  2016;11(1):e0145750.
Obesity and high saturated fat intake increase the risk of heart failure and arrhythmias. The molecular mechanisms are poorly understood. We hypothesized that physiologic levels of saturated fat could increase mitochondrial reactive oxygen species (ROS) in cardiomyocytes, leading to abnormalities of calcium homeostasis and mitochondrial function. We investigated the effect of saturated fat on mitochondrial function and calcium homeostasis in isolated ventricular myocytes. The saturated fatty acid palmitate causes a decrease in mitochondrial respiration in cardiomyocytes. Palmitate, but not the monounsaturated fatty acid oleate, causes an increase in both total cellular ROS and mitochondrial ROS. Palmitate depolarizes the mitochondrial inner membrane and causes mitochondrial calcium overload by increasing sarcoplasmic reticulum calcium leak. Inhibitors of PKC or NOX2 prevent mitochondrial dysfunction and the increase in ROS, demonstrating that PKC-NOX2 activation is also required for amplification of palmitate induced-ROS. Cardiomyocytes from mice with genetic deletion of NOX2 do not have palmitate-induced ROS or mitochondrial dysfunction. We conclude that palmitate induces mitochondrial ROS that is amplified by NOX2, causing greater mitochondrial ROS generation and partial depolarization of the mitochondrial inner membrane. The abnormal sarcoplasmic reticulum calcium leak caused by palmitate could promote arrhythmia and heart failure. NOX2 inhibition is a potential therapy for heart disease caused by diabetes or obesity.
PMCID: PMC4710525  PMID: 26756466
12.  Thymidine Phosphorylase Participates in Platelet Signaling and Promotes Thrombosis 
Circulation research  2014;115(12):997-1006.
Platelets contain abundant thymidine phosphorylase (TYMP), which is highly expressed in diseases with high risk of thrombosis, such as atherosclerosis and type II diabetes.
Test the hypothesis that TYMP participates in platelet signaling and promotes thrombosis.
Methods and Results
By using a ferric chloride (FeCl3) induced carotid artery injury thrombosis model, we found time to blood flow cessation was significantly prolonged in Tymp−/− and Tymp+/− mice compared to wild type (WT) mice. Bone marrow transplantation and platelet transfusion studies demonstrated that platelet TYMP was responsible for the antithrombotic phenomenon in the TYMP deficient mice. Collagen-, collagen-related peptide (CRP)-, adenosine diphosphate-and/or thrombin-induced platelet aggregation were significantly attenuated in Tymp+/− and Tymp−/− platelets, and in WT or human platelets pretreated with TYMP inhibitor KIN59. Tymp deficiency also significantly decreased agonist-induced P-select in expression. TYMP contains an N-terminal SH3 domain binding proline-rich motif and forms a complex with the tyrosine kinases Lyn, Fyn and Yes in platelets. TYMP-associated Lyn was inactive in resting platelets, and TYMP trapped and diminished active Lyn after collagen stimulation. Tymp/Lyn double haploinsufficiency diminished the antithrombotic phenotype of Tymp+/− mice. TYMP deletion or inhibition of TYMP with KIN59 dramatically increased PECAM-1 tyrosine phosphorylation and diminished CRP or collagen induced AKT phosphorylation. In vivo administration of KIN59 significantly inhibited FeCl3 induced carotid artery thrombosis without affecting hemostasis.
TYMP participates in multiple platelet signaling pathways and regulates platelet activation and thrombosis. Targeting TYMP might be a novel anti-platelet and anti-thrombosis therapy.
PMCID: PMC4258140  PMID: 25287063
Thymidine phosphorylase; platelet; arterial thrombosis; anti-platelet therapy; cell signaling; thrombosis
Muscle & nerve  2014;50(2):292-295.
A 61-year-old woman with a five-year history of progressive muscle weakness and atrophy had a muscle biopsy characterized by a combination of dystrophic features (necrotic fibers and endomysial fibrosis) and mitochondrial alterations [ragged-red cytochrome c oxidase (COX)-negative fibers].
Sequencing of the whole mtDNA, assessment of the mutation load in muscle and in accessible non-muscle tissues, and single fiber polymerase chain reaction (PCR).
Muscle mitochondrial DNA (mtDNA) sequencing revealed a novel heteroplasmic mutation (m.4403G>A) in the gene (MTTM) that encodes tRNAMet. The mutation was not present in accessible non-muscle tissues from the patient or 2 asymptomatic sisters.
The clinical features and muscle morphology in this patient are very similar to those described in a previous patient with a different mutation, also in MTTM, which suggests that mutations in this gene confer a distinctive “dystrophic” quality. This may be a diagnostic clue in patients with isolated mitochondrial myopathy.
PMCID: PMC4107085  PMID: 24711008
mtDNA; tRNAMet; de novo mutation; mitochondrial myopathy; dystrophic features; late-onset weakness
14.  Deoxynucleoside stress exacerbates the phenotype of a mouse model of mitochondrial neurogastrointestinal encephalopathy 
Brain  2014;137(5):1337-1349.
Mitochondrial neurogastrointestinal encephalopathy (MNGIE) is an autosomal recessive disease caused by thymidine phosphorylase mutations. Garcia-Diaz et al. describe a mouse model of MNGIE, in which the clinical phenotype, leukoencephalopathy, and biochemical defects are exacerbated by thymidine and deoxyuridine administration.
Balanced pools of deoxyribonucleoside triphosphate precursors are required for DNA replication, and alterations of this balance are relevant to human mitochondrial diseases including mitochondrial neurogastrointestinal encephalopathy. In this disease, autosomal recessive TYMP mutations cause severe reductions of thymidine phosphorylase activity; marked elevations of the pyrimidine nucleosides thymidine and deoxyuridine in plasma and tissues, and somatic multiple deletions, depletion and site-specific point mutations of mitochondrial DNA. Thymidine phosphorylase and uridine phosphorylase double knockout mice recapitulated several features of these patients including thymidine phosphorylase activity deficiency, elevated thymidine and deoxyuridine in tissues, mitochondrial DNA depletion, respiratory chain defects and white matter changes. However, in contrast to patients with this disease, mutant mice showed mitochondrial alterations only in the brain. To test the hypothesis that elevated levels of nucleotides cause unbalanced deoxyribonucleoside triphosphate pools and, in turn, pathogenic mitochondrial DNA instability, we have stressed double knockout mice with exogenous thymidine and deoxyuridine, and assessed clinical, neuroradiological, histological, molecular, and biochemical consequences. Mutant mice treated with exogenous thymidine and deoxyuridine showed reduced survival, body weight, and muscle strength, relative to untreated animals. Moreover, in treated mutants, leukoencephalopathy, a hallmark of the disease, was enhanced and the small intestine showed a reduction of smooth muscle cells and increased fibrosis. Levels of mitochondrial DNA were depleted not only in the brain but also in the small intestine, and deoxyribonucleoside triphosphate imbalance was observed in the brain. The relative proportion, rather than the absolute amount of deoxyribonucleoside triphosphate, was critical for mitochondrial DNA maintenance. Thus, our results demonstrate that stress of exogenous pyrimidine nucleosides enhances the mitochondrial phenotype of our knockout mice. Our mouse studies provide insights into the pathogenic role of thymidine and deoxyuridine imbalance in mitochondrial neurogastrointestinal encephalopathy and an excellent model to study new therapeutic approaches.
PMCID: PMC3999724  PMID: 24727567
MNGIE; animal model; thymidine; deoxyuridine; deoxynucleotide; mitochondrial DNA
15.  Nuclear localization of SMN and FUS is not altered in fibroblasts from patients with sporadic ALS 
Sporadic amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no established biological marker. Recent observation of a reduced number of gems (survival motor neuron protein (SMN)-positive nuclear bodies) in cells from patients with familial ALS and the mouse models suggests an involvement of SMN in ALS pathology. At a molecular level, fused in sarcoma (FUS), one of the familial ALS-linked proteins, has been demonstrated to directly interact with SMN, while impaired nuclear localization of mutated FUS causes defective gem formation.
To determine whether gems and/or nuclear FUS levels in skin derived-fibroblasts from sporadic ALS patients are consistently reduced and thus could constitute a novel and readily-available biomarker of the disease.
Fibroblasts from 20 patients and 17 age-matched healthy controls were cultured and co-immunostained for SMN and FUS.
No difference was detected between two groups in the number of gems and in expression pattern of FUS. The number of gems negatively correlated with the age at biopsy in both ALS and control subjects.
The expression pattern of SMN and FUS in fibroblasts cannot serve as a biomarker for sporadic ALS. Donor age-dependent gem reduction is a novel observation that links SMN with cellular senescence.
PMCID: PMC4372808  PMID: 24809826
Gem; SMN; FUS; Sporadic ALS; Fibroblast; Donor age
16.  The myopathic form of coenzyme Q10 deficiency is caused by mutations in the electron-transferring-flavoprotein dehydrogenase (ETFDH) gene 
Brain : a journal of neurology  2007;130(0 8):2037-2044.
Coenzyme Q10 (CoQ10) deficiency is an autosomal recessive disorder with heterogenous phenotypic manifestations and genetic background. We describe seven patients from five independent families with an isolated myopathic phenotype of CoQ10 deficiency. The clinical, histological and biochemical presentation of our patients was very homogenous. All patients presented with exercise intolerance, fatigue, proximal myopathy and high serum CK. Muscle histology showed lipid accumulation and subtle signs of mitochondrial myopathy. Biochemical measurement of muscle homogenates showed severely decreased activities of respiratory chain complexes I and II +III, while complex IV (COX) was moderately decreased. CoQ10 was significantly decreased in the skeletal muscle of all patients. Tandem mass spectrometry detected multiple acyl-CoA deficiency, leading to the analysis of the electron-transferring-flavoprotein dehydrogenase (ETFDH) gene, previously shown to result in another metabolic disorder, glutaric aciduria type II (GAII). All of our patients carried autosomal recessive mutations in ETFDH, suggesting that ETFDH deficiency leads to a secondary CoQ10 deficiency. Our results indicate that the late-onset form of GAII and the myopathic form of CoQ10 deficiency are allelic diseases. Since this condition is treatable, correct diagnosis is of the utmost importance and should be considered both in children and in adults. We suggest to give patients both CoQ10 and riboflavin supplementation, especially for long-term treatment.
PMCID: PMC4345103  PMID: 17412732
coenzyme Q10 myopathy; ETFDH mutations; riboflavin and CoQ10 supplementation; late-onset glutaric aciduria type II
17.  Functional characterization of human COQ4, a gene required for Coenzyme Q10 biosynthesis 
Defects in genes involved in coenzyme Q (CoQ) biosynthesis cause primary CoQ deficiency, a severe multisystem disorders presenting as progressive encephalomyopathy and nephropathy.
The COQ4 gene encodes an essential factor for biosynthesis in Saccharomyces cerevisiae. We have identified and cloned its human ortholog, COQ4, which is located on chromosome 9q34.13, and is transcribed into a 795 base-pair open reading frame, encoding a 265 amino acid (aa) protein (Isoform 1) with a predicted N-terminal mitochondrial targeting sequence. It shares 39% identity and 55% similarity with the yeast protein. Coq4 protein has no known enzymatic function, but may be a core component of multisubunit complex required for CoQ biosynthesis.
The human transcript is detected in Northern blots as a ~1.4 kb single band and is expressed ubiquitously, but at high levels in liver, lung, and pancreas. Transcription initiates at multiple sites, located 333–23 nucleotides upstream of the ATG. A second group of transcripts originating inside intron 1 of the gene encodes a 241 aa protein, which lacks the mitochondrial targeting sequence (isoform 2). Expression of GFP-fusion proteins in HeLa cells confirmed that only isoform 1 is targeted to mitochondria. The functional significance of the second isoform is unknown. Human COQ4 isoform 1, expressed from a multicopy plasmid, efficiently restores both growth in glycerol, and CoQ content in COQ4null yeast strains. Human COQ4 is an interesting candidate gene for patients with isolated CoQ10 deficiency.
PMCID: PMC4345104  PMID: 18474229
Coenzyme Q10; Coenzyme Q10 deficiency; Ubiquinone; Biosynthesis
18.  Missense mutation of the COQ2 gene causes defects of bioenergetics and de novo pyrimidine synthesis 
Human molecular genetics  2007;16(9):1091-1097.
Coenzyme Q10 (CoQ10) deficiency has been associated with an increasing number of clinical phenotypes that respond to CoQ10 supplementation. In two siblings with encephalomyopathy, nephropathy and severe CoQ10 deficiency, a homozygous mutation was identified in the CoQ10 biosynthesis gene COQ2, encoding polyprenyl-pHB transferase. To confirm the pathogenicity of this mutation, we have demonstrated that human wild-type, but not mutant COQ2, functionally complements COQ2 defective yeast. In addition, an equivalent mutation introduced in the yeast COQ2 gene also decreases both CoQ6 concentration and growth in respiratory-chain dependent medium. Polyprenyl-pHB transferase activity was 33–45% of controls in COQ2 mutant fibroblasts. CoQ-dependent mitochondrial complexes activities were restored in deficient fibroblasts by CoQ10 supplementation, and growth rate was restored in these cells by either CoQ10 or uridine supplementation. This work is the first direct demonstration of the pathogenicity of a COQ2 mutation involved in human disease, and establishes yeast as a useful model to study human CoQ10 deficiency. Moreover, we demonstrate that CoQ10 deficiency in addition to the bioenergetics defect also impairs de novo pyrimidine synthesis, which may contribute to the pathogenesis of the disease.
PMCID: PMC4345105  PMID: 17374725
19.  Weighing in on Leber hereditary optic neuropathy: effects of mitochondrial mass 
Brain  2014;137(2):308-309.
PMCID: PMC4990816  PMID: 24501072
20.  ANO10 mutations cause ataxia and coenzyme Q10 deficiency 
Journal of Neurology  2014;261(11):2192-2198.
Inherited ataxias are heterogeneous disorders affecting both children and adults, with over 40 different causative genes, making molecular genetic diagnosis challenging. Although recent advances in next-generation sequencing have significantly improved mutation detection, few treatments exist for patients with inherited ataxia. In two patients with adult-onset cerebellar ataxia and coenzyme Q10 (CoQ10) deficiency in muscle, whole exome sequencing revealed mutations in ANO10, which encodes anoctamin 10, a member of a family of putative calcium-activated chloride channels, and the causative gene for autosomal recessive spinocerebellar ataxia-10 (SCAR10). Both patients presented with slowly progressive ataxia and dysarthria leading to severe disability in the sixth decade. Epilepsy and learning difficulties were also present in one patient, while retinal degeneration and cataract were present in the other. The detection of mutations in ANO10 in our patients indicate that ANO10 defects cause secondary low CoQ10 and SCAR10 patients may benefit from CoQ10 supplementation.
Electronic supplementary material
The online version of this article (doi:10.1007/s00415-014-7476-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4221650  PMID: 25182700
Autosomal recessive ataxia; Mitochondrial; Coenzyme Q10 (CoQ10) deficiency; ANO10
22.  Deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency 
EMBO Molecular Medicine  2014;6(8):1016-1027.
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.
Subject Categories Genetics, Gene Therapy & Genetic Disease; Metabolism
PMCID: PMC4154130  PMID: 24968719
deoxycytidine monophosphate; deoxythymidine monophosphate; encephalomyopathy; therapy; thymidine kinase
23.  Limb-girdle muscular dystrophy 1F is caused by a microdeletion in the transportin 3 gene 
Brain  2013;136(5):1508-1517.
In 2001, we reported linkage of an autosomal dominant form of limb-girdle muscular dystrophy, limb-girdle muscular dystrophy 1F, to chromosome 7q32.1-32.2, but the identity of the mutant gene was elusive. Here, using a whole genome sequencing strategy, we identified the causative mutation of limb-girdle muscular dystrophy 1F, a heterozygous single nucleotide deletion (c.2771del) in the termination codon of transportin 3 (TNPO3). This gene is situated within the chromosomal region linked to the disease and encodes a nuclear membrane protein belonging to the importin beta family. TNPO3 transports serine/arginine-rich proteins into the nucleus, and has been identified as a key factor in the HIV-import process into the nucleus. The mutation is predicted to generate a 15-amino acid extension of the C-terminus of the protein, segregates with the clinical phenotype, and is absent in genomic sequence databases and a set of >200 control alleles. In skeletal muscle of affected individuals, expression of the mutant messenger RNA and histological abnormalities of nuclei and TNPO3 indicate altered TNPO3 function. Our results demonstrate that the TNPO3 mutation is the cause of limb-girdle muscular dystrophy 1F, expand our knowledge of the molecular basis of muscular dystrophies and bolster the importance of defects of nuclear envelope proteins as causes of inherited myopathies.
PMCID: PMC3634201  PMID: 23543484
limb-girdle muscular dystrophy 1F; LGMD1F; TNPO3; transportin 3; c.2771del mutation
24.  Haploinsufficiency of COQ4 causes coenzyme Q10 deficiency 
Journal of medical genetics  2012;49(3):187-191.
COQ4 encodes a protein that organises the multienzyme complex for the synthesis of coenzyme Q10 (CoQ10). A 3.9 Mb deletion of chromosome 9q34.13 was identified in a 3-year-old boy with mental retardation, encephalomyopathy and dysmorphic features. Because the deletion encompassed COQ4, the patient was screened for CoQ10 deficiency.
A complete molecular and biochemical characterisation of the patient’s fibroblasts and of a yeast model were performed.
The study found reduced COQ4 expression (48% of controls), CoQ10 content and biosynthetic rate (44% and 43% of controls), and activities of respiratory chain complex II+III. Cells displayed a growth defect that was corrected by the addition of CoQ10 to the culture medium. Knockdown of COQ4 in HeLa cells also resulted in a reduction of CoQ10. Diploid yeast haploinsufficient for COQ4 displayed similar CoQ deficiency. Haploinsufficency of other genes involved in CoQ10 biosynthesis does not cause CoQ deficiency, underscoring the critical role of COQ4. Oral CoQ10 supplementation resulted in a significant improvement of neuromuscular symptoms, which reappeared after supplementation was temporarily discontinued.
Mutations of COQ4 should be searched for in patients with CoQ10 deficiency and encephalomyopathy; patients with genomic rearrangements involving COQ4 should be screened for CoQ10 deficiency, as they could benefit from supplementation.
PMCID: PMC3983946  PMID: 22368301
25.  Longitudinal Clinical Follow-up of a Large Family With the R357P Twinkle Mutation 
JAMA neurology  2013;70(11):1425-1428.
Autosomal dominant progressive external ophthalmoplegia due to PEO1 mutations is considered relatively benign, but no data about long-term progression of this disease have been reported. The aim of this study was to provide a 16-year clinical follow-up of autosomal dominant progressive external ophthalmoplegia due to the p.R357P gene mutation in PEO1.
Twenty-two members of an Irish-American family were examined in 1996, when PEO1 sequencing revealed a c.1071G>C/p.R357P mutation in 9 of them. We reexamined the family in 2012 using a standardized clinical protocol. Autosomal dominant progressive external ophthalmoplegia due to the p.R357P PEO1 mutation is a late-onset ocular myopathy beginning with ptosis and progressing slowly. Ophthalmoparesis, if present, is mild and evident only by neurological examination.
Our results are important for prognosis and genetic counseling.
PMCID: PMC3973017  PMID: 24018892

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