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Background: Mitochondrial diseases are difficult to diagnose and treat. Recent advances in genetic diagnostics and more effective treatment options can improve patient diagnosis and prognosis, but patients with mitochondrial disease typically experience delays in diagnosis and treatment. Here, we describe a unique collaborative practice model among physicians and scientists in Spain focused on identifying TK2 deficiency (TK2d), an ultra-rare mitochondrial DNA depletion and deletions syndrome., Main Body: This collaboration spans research and clinical care, including laboratory scientists, adult and pediatric neuromuscular clinicians, geneticists, and pathologists, and has resulted in diagnosis and consolidation of care for patients with TK2d. The incidence of TK2d is not known; however, the first clinical cases of TK2d were reported in 2001, and only ~ 107 unique cases had been reported as of 2018. This unique collaboration in Spain has led to the diagnosis of more than 30 patients with genetically confirmed TK2d across different regions of the country. Research affiliate centers have led investigative treatment with nucleosides based on understanding of TK2d clinical manifestations and disease mechanisms, which resulted in successful treatment of a TK2d mouse model with nucleotide therapy in 2010. Only 1 year later, this collaboration enabled rapid adoption of treatment with pyrimidine nucleotides (and later, nucleosides) under compassionate use. Success in TK2d diagnosis and treatment in Spain is attributable to two important factors: Spain's fully public national healthcare system, and the designation in 2015 of major National Reference Centers for Neuromuscular Disorders (CSURs). CSUR networking and dissemination facilitated development of a collaborative care network for TK2d disease, wherein participants share information and protocols to request approval from the Ministry of Health to initiate nucleoside therapy. Data have recently been collected in a retrospective study conducted under a Good Clinical Practice-compliant protocol to support development of a new therapeutic approach for TK2d, a progressive disease with no approved therapies., Conclusions: The Spanish experience in diagnosis and treatment of TK2d is a model for the diagnosis and development of new treatments for very rare diseases within an existing healthcare system., (© 2021. The Author(s).)

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare disease caused by recessive mutations in the TYMP gene, which encodes the enzyme thymidine phosphorylase (TP). In this study, the efficient integration of a TYMP transgene into introns of the Tymp and Alb loci of hepatocytes in a murine model of MNGIE was achieved by the coordinated delivery and activity of CRISPR/Cas9 and a TYMP cDNA. CRISPR/Cas9 was delivered either as mRNA using lipid nanoparticle (LNP) or polymeric nanoparticle, respectively, or in an AAV2/8 viral vector; the latter was also used to package the TYMP cDNA. Insertion of the cDNA template downstream of the Tymp and Alb promoters ensured transgene expression. The best in vivo results were obtained using LNP carrying the CRISPR/Cas9 mRNAs. Treated mice showed a consistent long-term (1 year) reduction in plasma nucleoside (thymidine and deoxyuridine) levels that correlated with the presence of TYMP mRNA and functional enzyme in liver cells. In mice with an edited Alb locus, the transgene produced a hybrid Alb-hTP protein that was secreted, with supraphysiological levels of TP activity detected in the plasma. Equivalent results were obtained in mice edited at the Tymp locus. Finally, some degree of gene editing was found in animals treated only with AAV vectors containing the DNA templates, in the absence of nucleases, although there was no impact on plasma nucleoside levels. Overall, these results demonstrate the feasibility of liver-directed genome editing in the long-term correction of MNGIE, with several advantages over other methods.

Pathogenic variants in the mitochondrial tyrosyl-tRNA synthetase gene ( YARS2 ) were associated with myopathy, lactic acidosis, and sideroblastic anemia (MLASA). However, patients can present mitochondrial myopathy, with exercise intolerance and muscle weakness, leading from mild to lethal phenotypes. Genes implicated in mtDNA replication were studied by Next Generation Sequencing (NGS) and whole exome sequence with the TruSeq Rapid Exome kit (Illumina, San Diego, CA, USA). Mitochondrial protein translation was studied following the Sasarman and Shoubridge protocol and oxygen consumption rates with Agilent Seahorse XF24 Analyzer Mitostress Test, (Agilent, Santa Clara, CA, USA). We report two siblings with two novel compound heterozygous pathogenic variants in YARS2 gene: a single nucleotide deletion in exon 1, c.314delG (p.(Gly105Alafs*4)), which creates a premature stop codon in the amino acid 109, and a single nucleotide change in exon 5 c.1391T>C (p.(Ile464Thr)), that cause a missense variant in amino acid 464. We demonstrate the pathogenicity of these new variants associated with reduced YARS2 mRNA transcript, reduced mitochondrial protein translation and dysfunctional organelle function. These pathogenic variants are responsible for late onset MLASA, herein accompanied by pancreatic insufficiency, observed in both brothers, clinically considered as Pearson's syndrome. Molecular study of YARS2 gene should be considered in patients presenting Pearson's syndrome characteristics and MLASA related phenotypes.

Mitochondrial DNA depletion and multiple deletions syndromes (MDDS) constitute a group of mitochondrial diseases defined by dysfunctional mitochondrial DNA (mtDNA) replication and maintenance. As is the case for many other mitochondrial diseases, the options for the treatment of these disorders are rather limited today. Some aggressive treatments such as liver transplantation or allogeneic stem cell transplantation are among the few available options for patients with some forms of MDDS. However, in recent years, significant advances in our knowledge of the biochemical pathomechanisms accounting for dysfunctional mtDNA replication have been achieved, which has opened new prospects for the treatment of these often fatal diseases. Current strategies under investigation to treat MDDS range from small molecule substrate enhancement approaches to more complex treatments, such as lentiviral or adenoassociated vector-mediated gene therapy. Some of these experimental therapies have already reached the clinical phase with very promising results, however, they are hampered by the fact that these are all rare disorders and so the patient recruitment potential for clinical trials is very limited.

Background: Preclinical studies have shown that gene therapy is a feasible approach to treat mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the genetic murine model of the disease (Tymp/Upp1 double knockout, dKO) has a limited functional phenotype beyond the metabolic imbalances, and so the studies showing efficacy of gene therapy have relied almost exclusively on demonstrating correction of the biochemical phenotype. Chronic oral administration of thymidine (dThd) and deoxyuridine (dUrd) to dKO mice deteriorates the phenotype of the animals, providing a better model to test therapy approaches., Methods: dKO mice were treated with both dThd and dUrd in drinking water from weaning until the end of the study. At 8 - 11 weeks of age, mice were treated with several doses of adeno-associated virus (AAV) serotype 8 vector carrying the human TYMP coding sequence under the control of different liver-specific promoters (TBG, AAT, or HLP). The biochemical profile and functional phenotype were studied over the life of the animals., Findings: Nucleoside exposure resulted in 30-fold higher plasma nucleoside levels in dKO mice compared with non-exposed wild type mice. AAV-treatment provided elevated TP activity in liver and lowered systemic nucleoside levels in exposed dKO mice. Exposed dKO mice had enlarged brain ventricles (assessed by magnetic resonance imaging) and motor impairment (rotarod test); both were prevented by AAV treatment. Among all promoters tested, AAT showed the best efficacy., Interpretation: Our results show that AAV-mediated gene therapy restores the biochemical homeostasis in the murine model of MNGIE and, for the first time, demonstrate that this treatment improves the functional phenotype., Funding: This work was funded in part by the Spanish Instituto de Salud Carlos III, and the Generalitat de Catalunya. The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., Competing Interests: Declaration of Competing Interests RM, FV and MM report grants from the Instituto de Salud Carlos III during the conduct of the study; JT reports grants from Departament de Salut, Generalitat de Catalunya (PERIS program), during the conduct of the study; RM, MH, JT and YC report grants and non-financial support from Modis Therapeutics, personal fees and other from Modis Therapeutics, outside the submitted work; in addition, RM and MH have a patent “Deoxynucleoside therapy for diseases caused by unbalanced nucleotide pools including mitochondrial DNA depletion syndromes” (PCT/US16/038110) with royalties paid to Modis Therapeutics; MH reports grants and other support from Entrada Therapeutics, grants from Muscular Dystrophy Association and grants from NIH, outside the submitted work; FM is an employee of Spark Therapeutics; SL reports that the Nuclear Magnetic Resonance facility where she works charged the VHIR a fee for the MRI services; and RM, YC, JT and RC have a patent “Treatment of mitochondrial diseases” (PCT/EP2016/062636) with royalties paid to Modis Therapeutics., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: International patient registries are of particular importance for rare disorders, as they may contribute to overcome the lack of knowledge derived from low number of patients and limited awareness of these diseases, and help to learn more about their geographical or population-based specificities, which is relevant for research purposes and for promoting better standards of care and diagnosis. Our objective was to create and implement a European registry for patients with McArdle disease and other muscle glycogenoses (EUROMAC) and to disseminate the knowledge of these disorders., Results: Teams from nine different countries (United Kingdom, Spain, Italy, France, Germany, Denmark, Greece, Turkey and USA) created a consortium that developed the first European registry dedicated to rare muscle glycogenoses. A work plan was implemented to design the database and platform that constitute the registry, by choosing clinical, genetics and molecular variables of interest, based on experience gained from previous national registries for similar metabolic disorders. Among dissemination activities, several teaching events were organized in different countries, especially those where the consortium considered the awareness of these diseases needs to be promoted among health professionals and patients., Conclusion: EUROMAC represents a step forward in the knowledge of those disorders to which it is dedicated, and will have relevant clinical outcomes at the diagnostic, epidemiological, clinical and research level.

GDF-15 is a biomarker for mitochondrial diseases. We investigated the application of GDF-15 as biomarker of disease severity and response to deoxynucleoside treatment in patients with thymidine kinase 2 (TK2) deficiency and compared it to FGF-21. GDF-15 and FGF-21 were measured in serum from 24 patients with TK2 deficiency treated 1-49 months with oral deoxynucleosides. Patients were grouped according to age at treatment and biomarkers were analyzed at baseline and various time points after treatment initiation. GDF-15 was elevated on average 30-fold in children and 6-fold in adults before the start of treatment. There was a significant correlation between basal GDF-15 and severity based on pretreatment distance walked (6MWT) and weight (BMI). During treatment, GDF-15 significantly declined, and the decrease was accompanied by relevant clinical improvements. The decline was greater in the paediatric group, which included the most severe patients and showed the greatest clinical benefit, than in the adult patients. The decline of FGF-21 was less prominent and consistent. GDF-15 is a potential biomarker of severity and of therapeutic response for patients with TK2 deficiency. In addition, we show evidence of clinical benefit of deoxynucleoside treatment, especially when treatment is initiated at an early age.

Background: Thymidine kinase 2 (TK2) catalyses the phosphorylation of deoxythymidine (dThd) and deoxycytidine (dCtd) within mitochondria. TK2 deficiency leads to mtDNA depletion or accumulation of multiple deletions. In patients, TK2 mutations typically manifest as a rapidly progressive myopathy with infantile onset, leading to respiratory insufficiency and encephalopathy in the most severe clinical presentations. TK2-deficient mice develop the most severe form of the disease and die at average postnatal day 16. dThd+dCtd administration delayed disease progression and expanded lifespan of a knockin murine model of the disease., Methods: We daily administered TK2 knockout mice (Tk2 KO ) from postnatal day 4 with equimolar doses of dThd+dCtd, dTMP+dCMP, dThd alone or dCtd alone. We monitored body weight and survival and studied different variables at 12 or 29 days of age. We determined metabolite levels in plasma and target tissues, mtDNA copy number in tissues, and the expression and activities of enzymes with a relevant role in mitochondrial dNTP anabolism or catabolism., Findings: dThd+dCtd treatment extended average lifespan of Tk2 KO mice from 16 to 34 days, attenuated growth retardation, and rescued mtDNA depletion in skeletal muscle and other target tissues of 12-day-old mice, except in brain. However, the treatment was ineffective in 29-day-old mice that still died prematurely. Bioavailability of dThd and dCtd markedly decreased during mouse development. Activity of enzymes catabolizing dThd and dCtd increased with age in small intestine. Conversely, the activity of the anabolic enzymes decreased in target tissues during mouse development. We also found that administration of dThd alone had the same impact on survival to that of dThd+dCtd, whereas dCtd alone had no influence on lifespan., Interpretation: dThd+dCtd treatment recruits alternative cytosolic salvage pathways for dNTP synthesis, suggesting that this therapy would be of benefit for any Tk2 mutation. dThd accounts for the therapeutic effect of the combined treatment in mice. During the first weeks after birth, mice experience marked tissue-specific metabolic regulations and ontogenetic changes in dNTP metabolism-related enzymes that limit therapeutic efficacy to early developmental stages. FUND: This study was funded by grants from the Spanish Ministry of Industry, Economy and Competitiveness, the Spanish Instituto de Salud Carlos III, the Fundación Inocente, Inocente, AFM Téléthon and the Generalitat de Catalunya. The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., (Copyright © 2019. Published by Elsevier B.V.)

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a devastating disease caused by mutations in TYMP , which encodes thymidine phosphorylase (TP). In MNGIE patients, TP dysfunction results in systemic thymidine and deoxyuridine overload, which interferes with mitochondrial DNA replication. Preclinical studies have shown that gene therapy using a lentiviral vector targeted to hematopoietic stem cells or an adeno-associated virus (AAV) vector transcriptionally targeted to liver are feasible approaches to treat MNGIE. Here, we studied the effect of various promoters (thyroxine-binding globulin [TBG], phosphoglycerate kinase [PGK], hybrid liver-specific promoter [HLP], and alpha-1-antitrypsin [AAT]) and DNA configuration (single stranded or self complementary) on expression of the TYMP transgene in the AAV8 serotype in a murine model of MNGIE. All vectors restored liver TP activity and normalized nucleoside homeostasis in mice. However, the liver-specific promoters TBG, HLP, and AAT were more effective than the constitutive PGK promoter, and the self-complementary DNA configuration did not provide any therapeutic advantage over the single-stranded configuration. Among all constructs, only AAV-AAT was effective in all mice treated at the lowest dose (5 × 10 10 vector genomes/kg). As use of the AAT promoter will likely minimize the dose needed to achieve clinical efficacy as compared to the other promoters tested, we propose using the AAT promoter in the vector eventually designed for clinical use.

Objective: Thymidine kinase 2, encoded by the nuclear gene TK2, is required for mitochondrial DNA maintenance. Autosomal recessive TK2 mutations cause depletion and multiple deletions of mtDNA that manifest predominantly as a myopathy usually beginning in childhood and progressing relentlessly. We investigated the safety and efficacy of deoxynucleoside monophosphate and deoxynucleoside therapies., Methods: We administered deoxynucleoside monophosphates and deoxynucleoside to 16 TK2-deficient patients under a compassionate use program., Results: In 5 patients with early onset and severe disease, survival and motor functions were better than historically untreated patients. In 11 childhood and adult onset patients, clinical measures stabilized or improved. Three of 8 patients who were nonambulatory at baseline gained the ability to walk on therapy; 4 of 5 patients who required enteric nutrition were able to discontinue feeding tube use; and 1 of 9 patients who required mechanical ventilation became able to breathe independently. In motor functional scales, improvements were observed in the 6-minute walk test performance in 7 of 8 subjects, Egen Klassifikation in 2 of 3, and North Star Ambulatory Assessment in all 5 tested. Baseline elevated serum growth differentiation factor 15 levels decreased with treatment in all 7 patients tested. A side effect observed in 8 of the 16 patients was dose-dependent diarrhea, which did not require withdrawal of treatment. Among 12 other TK2 patients treated with deoxynucleoside, 2 adults developed elevated liver enzymes that normalized following discontinuation of therapy., Interpretation: This open-label study indicates favorable side effect profiles and clinical efficacy of deoxynucleoside monophosphate and deoxynucleoside therapies for TK2 deficiency. ANN NEUROL 2019;86:293-303., (© 2019 American Neurological Association.)

Mitochondrial DNA (mtDNA) depletion and deletion syndrome encompasses a group of disorders caused by mutations in genes involved in mtDNA replication and maintenance. The clinical phenotype ranges from fatal infantile hepatocerebral forms to mild adult onset progressive external ophthalmoplegia (PEO). We report the case of a patient with PEO and multiple mtDNA deletions, with two new homozygous mutations in RNASEH1 . The first mutation (c.487T>C) is located in the same catalytic domain as the four previously reported mutations, and the second (c.258_260del) is located in the connection domain, where no mutations have been reported. In silico study of the mutations predicted only the first mutation as pathogenic, but functional studies showed that both mutations cause loss of ribonuclease H1 activity. mtDNA replication dysfunction was demonstrated in patient fibroblasts, which were unable to recover normal mtDNA copy number after ethidium bromide-induced mtDNA depletion. Our results demonstrate the pathogenicity of two new RNASEH1 variants found in a patient with PEO syndrome, multiple deletions, and mild mitochondrial myopathy.

In humans, low brown adipose tissue (BAT) mass and activity have been associated with increased adiposity and fasting glucose levels, suggesting that defective BAT-dependent thermogenesis could contribute to the development of obesity and/or type 2 diabetes. The thermogenic function of BAT relies on a vast network of mitochondria exclusively equipped with UCP1. Mitochondrial biogenesis is exquisitely regulated by a well-defined network of transcription factors that coordinate the expression of nuclear genes required for the formation of functional mitochondria. However, less is known about the mitochondrial factors that control the expression of the genes encoded by the mitochondrial genome. Here, we have studied the role of mitochondrial transcription termination factor-4 (MTERF4) in BAT by using a new mouse model devoid of MTERF4 specifically in adipocytes (MTERF4-FAT-KO mice). Lack of MTERF4 in BAT leads to reduced OxPhos mitochondrial protein levels and impaired assembly of OxPhos complexes I, III and IV due to deficient translation of mtDNA-encoded proteins. As a result, brown adipocytes lacking MTERF4 exhibit impaired respiratory capacity. MTERF4-FAT-KO mice show a blunted thermogenic response and are unable to maintain body temperature when exposed to cold. Despite impaired BAT function, MTERF4-FAT-KO mice do not develop obesity or insulin resistance. Still, MTERF4-FAT-KO mice became resistant to the insulin-sensitizing effects of β 3 -specific adrenergic receptor agonists. Our results demonstrate that MTERF4 regulates mitochondrial protein translation and is essential for proper BAT thermogenic activity. Our study also supports the notion that pharmacological activation of BAT is a plausible therapeutic target for the treatment of insulin resistance., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The endonuclease G gene (Endog), which codes for a mitochondrial nuclease, was identified as a determinant of cardiac hypertrophy. How ENDOG controls cardiomyocyte growth is still unknown. Thus, we aimed at finding the link between ENDOG activity and cardiomyocyte growth. Endog deficiency induced reactive oxygen species (ROS) accumulation and abnormal growth in neonatal rodent cardiomyocytes, altering the AKT-GSK3β and Class-II histone deacethylases (HDAC) signal transduction pathways. These effects were blocked by ROS scavengers. Lack of ENDOG reduced mitochondrial DNA (mtDNA) replication independently of ROS accumulation. Because mtDNA encodes several subunits of the mitochondrial electron transport chain, whose activity is an important source of cellular ROS, we investigated whether Endog deficiency compromised the expression and activity of the respiratory chain complexes but found no changes in these parameters nor in ATP content. MtDNA also codes for humanin, a micropeptide with possible metabolic functions. Nanomolar concentrations of synthetic humanin restored normal ROS levels and cell size in Endog-deficient cardiomyocytes. These results support the involvement of redox signaling in the control of cardiomyocyte growth by ENDOG and suggest a pathway relating mtDNA content to the regulation of cell growth probably involving humanin, which prevents reactive oxygen radicals accumulation and hypertrophy induced by Endog deficiency., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by thymidine phosphorylase (TP) deficiency resulting in systemic accumulation of thymidine (d-Thd) and deoxyuridine (d-Urd) and characterized by early-onset neurological and gastrointestinal symptoms. Long-term effective and safe treatment is not available. Allogeneic bone marrow transplantation may improve clinical manifestations but carries disease and transplant-related risks. In this study, lentiviral vector-based hematopoietic stem cell gene therapy (HSCGT) was performed in Tymp -/- Upp1 -/- mice with the human phosphoglycerate kinase (PGK) promoter driving TYMP . Supranormal blood TP activity reduced intestinal nucleoside levels significantly at low vector copy number (median, 1.3; range, 0.2-3.6). Furthermore, we covered two major issues not addressed before. First, we demonstrate aberrant morphology of brain astrocytes in areas of spongy degeneration, which was reversed by HSCGT. Second, long-term follow-up and vector integration site analysis were performed to assess safety of the therapeutic LV vectors in depth. This report confirms and supplements previous work on the efficacy of HSCGT in reducing the toxic metabolites in Tymp -/- Upp1 -/- mice, using a clinically applicable gene transfer vector and a highly efficient gene transfer method, and importantly demonstrates phenotypic correction with a favorable risk profile, warranting further development toward clinical implementation.

Dechlorane Plus (DP) is a flame retardant used as a substitute of Mirex since 1970s, but it was not detected in the environment until 2006. Since then, this compound and its main relatives, Dechlorane 602, 603 and 604, have been mainly studied in environmental matrices for monitoring purposes, but the dietary exposure to them has been hardly investigated so far. In the present study, we determined this family of compounds in fish and vegetable oil samples from Catalonia (Spain), most of them used as health supplements. Determination was carried out by gas chromatography-high resolution mass spectrometry (GC-HRMS), after a clean up in a multilayer silica column and preparative high performance liquid chromatography (HPLC) equipped with a pyrenyl(ethyl) column. Concentrations of Dechlorane compounds were between below the limit of detection and 384.2 pg g(-1). Although there are only few studies about the presence of these pollutants in food or feed, concentrations obtained indicated that these compounds are in the same order in fish and vegetable oil health supplements as the few other food and feed studies., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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., (© The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Key Points: This is the first study to analyse the effect of muscle glycogen phosphorylase depletion in metabolically different muscle types. In McArdle mice, muscle glycogen phosphorylase is absent in both oxidative and glycolytic muscles. In McArdle mice, the glycogen debranching enzyme (catabolic) is increased in oxidative muscles, whereas the glycogen branching enzyme (anabolic) is increased in glycolytic muscles. In McArdle mice, total glycogen synthase is decreased in both oxidative and glycolytic muscles, whereas the phosphorylated inactive form of the enzyme is increased in both oxidative and glycolytic enzymes. In McArdle mice, glycogen content is higher in glycolytic muscles than in oxidative muscles. Additionally, in all muscles analysed, the glycogen content is higher in males than in females. The maximal endurance capacity of the McArdle mice is significantly lower compared to heterozygous and wild-type mice., Abstract: McArdle disease, caused by inherited deficiency of the enzyme muscle glycogen phosphorylase (GP-MM), is arguably the paradigm of exercise intolerance. The recent knock-in (p.R50X/p.R50X) mouse disease model allows an investigation of the phenotypic consequences of muscle glycogen unavailability and the physiopathology of exercise intolerance. We analysed, in 2-month-old mice [wild-type (wt/wt), heterozygous (p.R50X/wt) and p.R50X/p.R50X)], maximal endurance exercise capacity and the molecular consequences of an absence of GP-MM in the main glycogen metabolism regulatory enzymes: glycogen synthase, glycogen branching enzyme and glycogen debranching enzyme, as well as glycogen content in slow-twitch (soleus), intermediate (gastrocnemius) and glycolytic/fast-twitch (extensor digitorum longus; EDL) muscles. Compared with wt/wt, exercise capacity (measured in a treadmill test) was impaired in p.R50X/p.R50X (∼48%) and p.R50X/wt mice (∼18%). p.R50X/p.R50X mice showed an absence of GP-MM in the three muscles. GP-MM was reduced in p.R50X/wt mice, especially in the soleus, suggesting that the function of 'slow-twitch' muscles is less dependent on glycogen catabolism. p.R50X/p.R50X mice showed increased glycogen debranching enzyme in the soleus, increased glycogen branching enzyme in the gastrocnemius and EDL, as well as reduced levels of mucle glycogen synthase protein in the three muscles (mean ∼70%), reflecting a protective mechanism for preventing deleterious glycogen accumulation. Additionally, glycogen content was highest in the EDL of p.R50X/p.R50X mice. Amongst other findings, the present study shows that the expression of the main muscle glycogen regulatory enzymes differs depending on the muscle phenotype (slow- vs. fast-twitch) and that even partial GP-MM deficiency affects maximal endurance capacity. Our knock-in model might help to provide insights into the importance of glycogen on muscle function., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

McArdle disease is caused by an inherited deficiency of the enzyme myophosphorylase, resulting in exercise intolerance from childhood and acute crises of early fatigue and contractures. In severe cases, these manifestations can be accompanied by rhabdomyolysis, myoglobinuria, and fatal renal failure. Diagnosis of McArdle disease is based on clinical diagnostic tests, together with an absence of myophosphorylase activity in skeletal muscle biopsies and genetic analysis of the myophosphorylase-encoding gene, PYGM. The recently reported association between myophosphorylase and Rac1 GTPase in a T lymphocyte cell line prompted us to study myophosphorylase expression in white blood cells (WBCs) from 20 healthy donors and 30 McArdle patients by flow cytometry using a fluorescent-labeled PYGM antibody. We found that T lymphocytes expressed myophosphorylase in healthy donors, but expression was significantly lower in McArdle patients (p<0.001). PYGM mRNA levels were also lower in white blood cells from McArdle patients. Nevertheless, in 13% of patients (who were either heterozygotes or homozygotes for the most common PYGM pathogenic mutation among Caucasians (p.R50X)), the percentage of myophosphorylase-positive white blood cells was not different compared with the control group. Our findings suggest that analysis of myophosphorylase expression in white blood cells might be a useful, less-invasive, complementary test for diagnosing McArdle disease., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Solute carrier (SLC) transporters mediate the uptake of biologically active compounds in the intestine. Reduced oxygenation (hypoxia) is an important factor influencing intestinal homeostasis. The aim of this study was to investigate the pathophysiological consequences of hypoxia on the expression and function of SLCs in human intestine. Hypoxia was induced in human intestinal epithelial cells (IECs) in vitro (0.2; 1% O2 or CoCl2). For human in vivo studies, duodenal biopsies and serum samples were obtained from individuals (n = 16) acutely exposed to 4,554 meters above sea levels. Expression of relevant targets was analyzed by quantitative PCR, Western blotting, or immunofluorescence. Serum levels of inflammatory mediators and nucleosides were determined by ELISA and LC/MS-MS, respectively. In the duodenum of volunteers exposed to high altitude we observed decreased mRNA levels of apical sodium-dependent bile acid transporter (ASBT), concentrative nucleoside transporters 1/2 (CNT1/2), organic anion transporting polypeptide 2B1 (OATP2B1), organic cation transporter 2 (OCTN2), peptide transporter 1 (PEPT1), serotonin transporter (SERT), and higher levels of IFN-γ, IL-6, and IL-17A. Serum levels of IL-10, IFN-γ, matrix metalloproteinase-2 (MMP-2), and serotonin were elevated, whereas the levels of uridine decreased upon exposure to hypoxia. Hypoxic IECs showed reduced levels of equilibrative nucleoside transporter 2 (ENT2), OCTN2, and SERT mRNAs in vitro, which was confirmed on the protein level and was accompanied by activation of ERK1/2, increase of hypoxia-inducible factor (HIF) proteins, and production of IL-8 mRNA. Costimulation with IFN-γ and IL-6 during hypoxia further decreased the expression of SERT, ENT2, and CNT2 in vitro. Reduced oxygen supply affects the expression pattern of duodenal SLCs that is accompanied by changes in serum levels of proinflammatory cytokines and biologically active compounds demonstrating that intestinal transport is affected during systemic exposure to hypoxia in humans., (Copyright © 2014 the American Physiological Society.)

Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is characterized by a reduction in mtDNA copy number and consequent mitochondrial dysfunction in affected tissues. A subgroup of MDS is caused by mutations in genes that disrupt deoxyribonucleotide metabolism, which ultimately leads to limited availability of one or several deoxyribonucleoside triphosphates (dNTPs), and subsequent mtDNA depletion. Here, using in vitro experimental approaches (primary cell culture of deoxyguanosine kinase-deficient cells and thymidine-induced mtDNA depletion in culture as a model of mitochondrial neurogastrointestinal encephalomyopathy, MNGIE), we show that supplements of those deoxyribonucleosides (dNs) involved in each biochemical defect (deoxyguanosine or deoxycytidine, dCtd) prevents mtDNA copy number reduction. Similar effects can be obtained by specific inhibition of dN catabolism using tetrahydrouridine (THU; inhibitor of cytidine deaminase) or immucillin H (inhibitor of purine nucleoside phosphorylase). In addition, using an MNGIE animal model, we provide evidence that mitochondrial dNTP content can be modulated in vivo by systemic administration of dCtd or THU. In spite of the severity associated with diseases due to defects in mtDNA replication, there are currently no effective therapeutic options available. Only in the case of MNGIE, allogeneic hematopoietic stem cell transplantation has proven efficient as a long-term therapeutic strategy. We propose increasing cellular availability of the deficient dNTP precursor by direct administration of the dN or inhibition of its catabolism, as a potential treatment for mtDNA depletion syndrome caused by defects in dNTP metabolism.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in TYMP, enconding thymidine phosphorylase (TP). TP deficiency results in systemic accumulation of thymidine and deoxyuridine, which interferes with mitochondrial DNA (mtDNA) replication and leads to mitochondrial dysfunction. To date, the only treatment available for MNGIE patients is allogeneic hematopoietic stem cell transplantation, which is associated with high morbidity and mortality. Here, we report that AAV2/8-mediated transfer of the human TYMP coding sequence (hcTYMP) under the control of a liver-specific promoter prevents the biochemical imbalances in a murine model of MNGIE. hcTYMP expression was restricted to liver, and a dose as low as 2 × 10(11) genome copies/kg led to a permanent reduction in systemic nucleoside levels to normal values in about 50% of treated mice. Higher doses resulted in reductions to normal or slightly below normal levels in virtually all mice treated. The nucleoside reduction achieved by this treatment prevented deoxycytidine triphosphate (dCTP) depletion, which is the limiting factor affecting mtDNA replication in this disease. These results demonstrate that the use of AAV to direct TYMP expression in liver is feasible as a potentially safe gene therapy strategy for MNGIE.

Mutations in an increasing number of nuclear genes involved in deoxyribonucleotide homeostasis cause disorders associated with somatic mitochondrial DNA (mtDNA) abnormalities. Dysfunction of the products of these genes leads to limited availability of substrates for mtDNA replication and results in mtDNA depletion, multiple deletions or point mutations; mtDNA depletion is the molecular feature linked to greatest clinical severity. In this review, we discuss recent results demonstrating that enhancement of the salvage pathways by increasing the availability of deoxyribonucleosides needed for each specific genetic defect prevents mtDNA depletion. Hence, we propose administration of selected deoxyribonucleosides and/or inhibitors of their catabolism as a pharmacological strategy to treat these diseases., (Copyright © 2013. Published by Elsevier Ltd.)

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.

McArdle disease (glycogenosis type V), the most common muscle glycogenosis, is a recessive disorder caused by mutations in PYGM, the gene encoding myophosphorylase. Patients with McArdle disease typically experience exercise intolerance manifested as acute crises of early fatigue and contractures, sometimes with rhabdomyolysis and myoblobinuria, triggered by static muscle contractions or dynamic exercises. Currently, there are no therapies to restore myophosphorylase activity in patients. Although two spontaneous animal models for McArdle disease have been identified (cattle and sheep), they have rendered a limited amount of information on the pathophysiology of the disorder; therefore, there have been few opportunities for experimental research in the field. We have developed a knock-in mouse model by replacing the wild-type allele of Pygm with a modified allele carrying the common human mutation, p.R50X, which is the most frequent cause of McArdle disease. Histochemical, biochemical and molecular analyses of the phenotype, as well as exercise tests, were carried out in homozygotes, carriers and wild-type mice. p.R50X/p.R50X mice showed undetectable myophosphorylase protein and activity in skeletal muscle. Histochemical and biochemical analyses revealed massive muscle glycogen accumulation in homozygotes, in contrast to heterozygotes or wild-type mice, which did not show glycogen accumulation in this tissue. Additional characterization confirmed a McArdle disease-like phenotype in p.R50X/p.R50X mice, i.e. they had hyperCKaemia and very poor exercise performance, as assessed in the wire grip and treadmill tests (6% and 5% of the wild-type values, respectively). This model represents a powerful tool for in-depth studies of the pathophysiology of McArdle disease and other neuromuscular disorders, and for exploring new therapeutic approaches for genetic disorders caused by premature stop codon mutations.

Because deoxyribonucleoside triphosphates (dNTPs) are the critical substrates for DNA replication and repair, dNTP pools have been studied in context of multiple basic biochemical processes. Over the last 12 years, interest in dNTPs, and specifically the mitochondrial dNTP pools, has expanded to biomedical science because several mitochondrial diseases have been found to be caused by dysfunctions of several enzymes involved in dNTP catabolism or anabolism. Techniques to reliably measure mitochondrial dNTPs should be sensitive and specific to avoid interference caused by the abundant ribonucleotides. Here, we describe detailed protocols to measure mitochondrial dNTPs from two specific samples, cultured skin fibroblasts and mouse liver. The methods can be easily adapted to other types of samples. The protocol follows a polymerase-based method, which is the most widely used approach to measure dNTP pools. Our description is based on the latest update of the technique, which minimizes the potential interference from ribonucleotides.

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 TYMP, 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. TYMP 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.

Left ventricular mass (LVM) is a highly heritable trait and an independent risk factor for all-cause mortality. So far, genome-wide association studies have not identified the genetic factors that underlie LVM variation, and the regulatory mechanisms for blood-pressure-independent cardiac hypertrophy remain poorly understood. Unbiased systems genetics approaches in the rat now provide a powerful complementary tool to genome-wide association studies, and we applied integrative genomics to dissect a highly replicated, blood-pressure-independent LVM locus on rat chromosome 3p. Here we identified endonuclease G (Endog), which previously was implicated in apoptosis but not hypertrophy, as the gene at the locus, and we found a loss-of-function mutation in Endog that is associated with increased LVM and impaired cardiac function. Inhibition of Endog in cultured cardiomyocytes resulted in an increase in cell size and hypertrophic biomarkers in the absence of pro-hypertrophic stimulation. Genome-wide network analysis unexpectedly implicated ENDOG in fundamental mitochondrial processes that are unrelated to apoptosis. We showed direct regulation of ENDOG by ERR-α and PGC1α (which are master regulators of mitochondrial and cardiac function), interaction of ENDOG with the mitochondrial genome and ENDOG-mediated regulation of mitochondrial mass. At baseline, the Endog-deleted mouse heart had depleted mitochondria, mitochondrial dysfunction and elevated levels of reactive oxygen species, which were associated with enlarged and steatotic cardiomyocytes. Our study has further established the link between mitochondrial dysfunction, reactive oxygen species and heart disease and has uncovered a role for Endog in maladaptive cardiac hypertrophy.

Mitochondrial thymidine kinase (TK2) catalyzes the phosphorylation of thymidine in mitochondria. Its function becomes essential for dTTP synthesis in noncycling cells, where cytosolic dTTP synthesis via R1/R2 ribonucleotide reductase and thymidine kinase 1 is turned down. Mutations in the nuclear gene for TK2 cause a fatal mtDNA depletion syndrome. Only selected cell types are affected, suggesting that the other cells compensate for the TK2 deficiency by adapting the enzyme network that regulates dTTP synthesis outside S-phase. Here we looked for such metabolic adaptation in quiescent cultures of fibroblasts from two TK2-deficient patients with a slow-progressing syndrome. In cell extracts, we measured the activities of TK2, deoxycytidine kinase, thymidine phosphorylase, deoxynucleotidases and the amounts of the three ribonucleotide reductase subunits. Patient cells contained 40% or 5% TK2 activity and unchanged activities of the other enzymes. However, their mitochondrial and cytosolic dTTP pools were unchanged, and also the overall composition of the dNTP pools was normal. TK2-dependent phosphorylation of [(3)H]thymidine in intact cells and the turnover of the dTTP pool showed that even the fibroblasts with 5% residual TK2 activity synthesized dTTP at an almost normal rate. Normal fibroblasts apparently contain more TK2 than needed to maintain dTTP during quiescence, which would explain why TK2-mutated fibroblasts do not manifest mtDNA depletion despite their reduced TK2 activity.

A 29-year-old Spanish man presented with chronic intestinal pseudo-obstruction, progressive external ophthalmoplegia, peripheral neuropathy, and diffuse leukoencephalopathy. This combination of clinical features is characteristic of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Genetic analysis revealed a novel 18-base pair (bp) duplication (5044-5061 dup) in exon 8 of the thymidine phosphorylase (TP) gene. The mutation is predicted to produce a 6 amino acid insertion in the alpha-beta-domain of the protein. This 18-bp insertion in the thymidine phosphorylase gene is the first duplication mutation identified in MNGIE.

Background: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by mutations in the gene encoding thymidine phosphorylase (TP). The clinical manifestations of MNGIE are recognizable and homogeneous, but in the early stages, the disease is often misdiagnosed. This study assesses the reliability of biochemical assays to diagnose MNGIE., Methods: We studied 180 patients with clinical features suggestive of MNGIE, 14 asymptomatic TP mutation carriers, and 20 controls. TP enzyme activity in the buffy coat was determined by a fixed-time method, and the plasma nucleosides thymidine (dThd) and deoxyuridine (dUrd) were assessed by a gradient-elution reversed phase HPLC method. TP was sequenced through standard procedures in patients who met the clinical criteria for MNGIE., Results: Twenty-five of the 180 patients fulfilled the clinical criteria for MNGIE and had homozygous or compound heterozygous TP mutations. All had drastically decreased TP activity [mean (SD), 10 (15) nmol thymine formed. h(-1). (mg protein)(-1) vs 634 (217) nmol thymine formed. h(-1). (mg protein)(-1) for the controls]. Relative to the control mean, TP activities were reduced to 35% in mutation carriers and 65% in MNGIE-like patients. All 25 MNGIE patients had detectable plasma dThd [8.6 (3.4) micromol/L] and dUrd [14.2 (4.4) micromol/L]. Controls, carriers, and MNGIE-like patients showed no detectable plasma dThd and dUrd., Conclusions: We propose a diagnostic algorithm based on the determination of plasma dThd and dUrd, TP activity in buffy coat, or both to make a definitive diagnosis of MNGIE. Increased concentrations of dThd (>3 micromol/L) and dUrd (>5 micromol/L) in plasma or a decrease in buffy coat TP activity to

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by loss-of-function mutations in the gene encoding thymidine phosphorylase (TP). This deficiency of TP leads to increased circulating levels of thymidine (deoxythymidine, dThd) and deoxyuridine (dUrd) and has been associated with multiple deletions and depletion of mitochondrial DNA (mtDNA). Here we describe 36 point mutations in mtDNA of tissues and cultured cells from MNGIE patients. Thirty-one mtDNA point mutations (86%) were T-to-C transitions, and of these, 25 were preceded by 5'-AA sequences. In addition, we identified a single base-pair mtDNA deletion and a TT-to-AA mutation. Next-nucleotide effects and dislocation mutagenesis may contribute to the formation of these mutations. These results provide the first demonstration that alterations of nucleoside metabolism can induce multiple sequence-specific point mutations in humans. We hypothesize that, in patients with TP deficiency, increased levels of dThd and dUrd cause mitochondrial nucleotide pool imbalances, which, in turn, lead to mtDNA abnormalities including site-specific point mutations.

Mutations in the nuclear gene encoding thymidine phosphorylase (TP) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disease with mitochondrial dysfunction and mitochondrial DNA abnormalities. We have demonstrated alterations of thymidine (dThd) metabolism in MNGIE patients. Here, we report the accumulation of another substrate of TP, deoxyuridine (dUrd), whose circulating levels ranged from 5.5 to 24.4 microM (average 14.2) in MNGIE and were undetectable (<0.05 microM) in both TP mutation carriers and controls. The dramatic accumulation of dUrd may contribute to nucleotide pool imbalances and, together with the increased levels of dThd, is likely to contribute to the pathogenesis of MNGIE.