Low aerobic capacity in McArdle disease: A role for mitochondrial network impairment?

BACKGROUND: McArdle disease is caused by myophosphorylase deficiency and results in complete inability for muscle glycogen breakdown. A hallmark of this condition is muscle oxidation impairment (e.g., low peak oxygen uptake (VO2peak)), a phenomenon traditionally attributed to reduced glycolytic flux and Krebs cycle anaplerosis. Here we hypothesized an additional role for muscle mitochondrial network alterations associated with massive intracellular glycogen accumulation. METHODS: We analyzed in depth mitochondrial characteristics-content, biogenesis, ultrastructure-and network integrity in skeletal-muscle from McArdle/control mice and two patients. We also determined VO2peak in patients (both sexes, N = 145) and healthy controls (N = 133). RESULTS: Besides corroborating very poor VO2peak values in patients and impairment in muscle glycolytic flux, we found that, in McArdle muscle: (a) damaged fibers are likely those with a higher mitochondrial and glycogen content, which show major disruption of the three main cytoskeleton components-actin microfilaments, microtubules and intermediate filaments-thereby contributing to mitochondrial network disruption in skeletal muscle fibers; (b) there was an altered subcellular localization of mitochondrial fission/fusion proteins and of the sarcoplasmic reticulum protein calsequestrin-with subsequent alteration in mitochondrial dynamics/function; impairment in mitochondrial content/biogenesis; and (c) several OXPHOS-related complex proteins/activities were also affected. CONCLUSIONS: In McArdle disease, severe muscle oxidative capacity impairment could also be explained by a disruption of the mitochondrial network, at least in those fibers with a higher capacity for glycogen accumulation. Our findings might pave the way for future research addressing the potential involvement of mitochondrial network alterations in the pathophysiology of other glycogenoses.

Influence of Mitochondrial Genetics on the Mitochondrial Toxicity of Linezolid in Blood Cells and Skin Nerve Fibers.

The antibiotic linezolid is a ribosomal inhibitor with excellent efficacy. Although the administration period has been reduced to 28 days, side effects, usually of hematologic or neuropathic origin, are still reported due to secondary inhibition of mitochondrial protein synthesis. Susceptibility to linezolid toxicity remains unknown. Therefore, the objective of this study was to gain an understanding of clinical heterogeneity in response to identical linezolid exposures through exhaustive examination of the molecular basis of tissue-dependent mitotoxicity, consequent cell dysfunction, and the association of mitochondrial genetics with adverse effects of linezolid administered for the recommended period. Peripheral blood mononuclear cells (PBMC) and skin nerve fibers from 19 and 6 patients, respectively, were evaluated before and after a 28-day linezolid treatment in order to assess toxic effects on mitochondria and cells. Mitochondrial DNA haplotypes and single nucleotide polymorphisms (SNPs) in ribosomal sequences where linezolid binds to mitochondrial ribosomes were also analyzed to investigate their genetic contributions. We found that linezolid reduced mitochondrial protein levels, complex IV activity, and mitochondrial mass in PBMC and was associated with a trend toward an increase in the rate of apoptosis. In skin tissue, mitochondrial mass increased within nerve fibers, accompanied by subclinical axonal swelling. Mitochondrial haplogroup U, mutations in 12S rRNA, and the m.2706A→G, m.3197T→C, and m.3010G→A polymorphisms in 16S rRNA showed a trend toward an association with increased mitochondrial and clinical adverse effects. We conclude that even when linezolid is administered for a shorter time than formerly, adverse effects are reported by 63% of patients. Linezolid exerts tissue-dependent mitotoxicity that is responsible for downstream cellular consequences (blood cell death and nerve fiber swelling), leading to adverse hematologic and peripheral nervous side effects. Multicentric studies should confirm genetic susceptibility in larger cohorts.

Identification and biochemical characterization of the novel mutation m.8839G>C in the mitochondrial ATP6 gene associated with NARP syndrome.

Mutations in the ATP6 gene are reported to be associated with Leber hereditary optic neuropathy, bilateral striatal necrosis, coronary atherosclerosis risk and neuropathy, ataxia and retinitis pigmentosa (NARP)/maternally inherited Leigh syndromes. Here, we present a patient with NARP syndrome, in whom a previously undescribed mutation was detected in the ATP6 gene: m.8839G>C. Several observations support the concept that m.8839G>C is pathogenically involved in the clinical phenotype of this patient: (1) the mutation was heteroplasmic in muscle; (2) mutation load was higher in the symptomatic patient than in the asymptomatic carriers; (3) cybrids carrying this mutation presented lower cell proliferation, increased mitochondrial DNA (mtDNA) copy number, increased steady-state OxPhos protein levels and decreased mitochondrial membrane potential with respect to isogenic wild-type cybrids; (4) this change was not observed in 2959 human mtDNAs from different mitochondrial haplogroups; (5) the affected amino acid was conserved in all the ATP6 sequences analyzed; and (6) using in silico prediction, the mutation was classified as 'probably damaging'. However, measurement of ATP synthesis showed no differences between wild-type and mutated cybrids. Thus, we suggest that m.8839G>C may lower the efficiency between proton translocation within F0 and F1 rotation, required for ATP synthesis. Further experiments are needed to fully characterize the molecular mechanisms involved in m.8839G>C pathogenicity.

Effect of an ambulatory diagnostic and treatment programme in patients with sleep apnoea.

The aim of this study to evaluate the efficacy of a home-based programme on clinical response, continuous positive airway pressure (CPAP) compliance and cost in a population of high pre-test probability of suffering obstructive sleep apnoea syndrome (OSAS). Patients were randomised into the following three groups. Group A: home respiratory polygraphy (RP) and home follow-up; group B: hospital polysomnography and hospital follow-up; and group C: home RP and hospital follow-up. Evaluation during 6 months included Epworth Sleepiness Scale (ESS), Functional Outcomes Sleep Questionnaire (FOSQ), and daily activity and symptom questionnaires. Compliance was assessed by memory cards (group A) and using an hourly counter (groups B and C). 66 patients were included (22 per branch), 83% were males, aged mean±sd 52±10 yrs, body mass index 34±7kg·m(-2), apnoea/hypopnoea index 43±20 h(-1), CPAP pressure 8±2 cmH(2)O, with no between-group differences. Clinical response showed an ESS of mean±sd 15±3 to 6±4, a FOSQ of 16±3 to 18±2, symptoms of 43±7 to 25±7, and activity of 37±11 to 25±8. At the end of the study, compliance was: group A 73%, group B 68% and group C 57%. The cost per patient was: group A €590±43, group B €894±11 and group C €644±93 (p<0.001). In conclusion, patients with a high initial probability of having OSAS can be diagnosed and treated in a home setting, with a high level of CPAP compliance and lower cost than using either a hospital-based approach or home RP/hospital follow-up.

Hematopoietic gene therapy restores thymidine phosphorylase activity in a cell culture and a murine model of MNGIE.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in the TYMP gene, which encodes thymidine phosphorylase (TP). TP dysfunction results in systemic thymidine (dThd) and deoxyuridine (dUrd) overload, which selectively impair mitochondrial DNA replication. Allogeneic hematopoietic transplantation has been used to treat MNGIE patients; however, this approach has serious adverse effects, including the toxicity of myeloablative conditioning, graft rejection and graft-versus-host disease. With the aim of testing the feasibility of gene therapy for MNGIE, we transduced TP-deficient B-lymphoblastoid cells from two MNGIE patients, with lentiviral vectors carrying a functional copy of the human TYMP DNA coding sequence. This restored TP activity in the cells, which reduced the excretion of dThd and dUrd and their concentrations when added in excess. Additionally, lentiviral-mediated hematopoietic gene therapy was used in partially myeloablated double Tymp/Upp1 knockout mice. In spite of the relatively low levels of molecular chimerism achieved, high levels of TP activity were observed in the peripheral blood of the transplanted mice, with a concomitant reduction of nucleoside concentrations. Our results suggest that hematopoietic gene therapy could be an alternative treatment for this devastating disorder in the future.

Novel mutations in patients with McArdle disease by analysis of skeletal muscle mRNA.

OBJECTIVE: To identify pathogenic mutant alleles of the PYGM gene in "genetic manifesting heterozygous" patients with McArdle disease-that is, those in whom we could only find a sole mutant allele by genomic DNA analysis. METHODS: We studied four unrelated patients. PCR-RFLP, gene sequencing, and muscle cDNA analysis were performed to search for mutations in the PYGM gene. The effects of the mutations were evaluated by in silico analysis, and gene expression was assessed by real-time polymerase chain reaction (PCR). RESULTS: Patient 1 was a compound heterozygous for the p.G205S missense mutation and for a novel "in frame" mutation, p.Q176_M177insVQ, resulting from a retention of six nucleotides from the 3'-end sequence of intron 4. Patient 2 was heterozygous for the common nonsense mutation p.R50X, and for a 1094 bp, c.1969+214_2177+369del mutation, spanning from intron 16 to intron 17 sequences. Furthermore, mRNA expression level was dramatically reduced consistent with nonsense mediated decay. Patient 3 was heterozygous for the p.R50X substitution, and patient 4 was heterozygous for the relatively common private Spanish mutation p.W798R. These two patients harboured a heterozygous exonic synonymous variant, p.K215K. Quantification of gene transcripts in patient 3 revealed a drastic decrease in the relative expression of the gene, which strongly supports the possibility of nonsense mediated decay. CONCLUSIONS: Our results indicate that skeletal muscle cDNA studies in "genetic manifesting heterozygous" patients with McArdle disease are prone to identify their second mutant allele.

NARP syndrome in a patient harbouring an insertion in the MT-ATP6 gene that results in a truncated protein.

BACKGROUND: Neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP) syndrome have been associated to m.8993T>G/C mutations in the subunit 6 of the ATP synthase (p.MT-ATP6). METHODS: We have performed a mutational screening of the mitochondrial DNA gene encoding for this protein in 62 patients with the disease, that do not carry any of the common mutations described to date. RESULTS: We report clinical and molecular data in one patient who harbours a de novo insertion in the MT-ATP6 gene that results in a truncated subunit. The mutation was heteroplasmic (85%) in muscle DNA and the BN-PAGE analysis showed a clear decrease in the amount of ATP synthase. CONCLUSION: Molecular analysis of NARP patients cannot be limited to the search of the m.8993T>G/C and either the ATP6 or the whole mtDNA should be sequenced.

Mitochondrial DNA oxidation and manganese superoxide dismutase activity in peripheral blood mononuclear cells from type 2 diabetic patients.

AIM: To investigate the balance between parameters of oxidative stress and antioxidant defences in the mitochondria of peripheral blood mononuclear cells (PBMCs) of type 2 diabetic patients with late complications. METHODS: Ten type 2 diabetic patients with late diabetic complications and 10 age-matched healthy volunteers (controls) were prospectively recruited. Mitochondrial DNA (mtDNA) oxidative damage and mtDNA content were measured as indices of oxidative stress. Manganese superoxide dismutase (MnSOD) activity has been used as an index of mitochondrial antioxidant defence. Mitochondrial respiratory-chain function (cytochrome C oxidase activity) was also assessed. RESULTS: Mitochondrial DNA (mtDNA) oxidation was significantly higher in the PBMCs of diabetic patients than in control subjects (P<0.0001) and, although mtDNA content was lower in the diabetic group, this was not statistically significant. MnSOD activity was significantly increased in PBMCs of type 2 diabetic patients compared with healthy controls (1366+/-187 versus 686+/-167 U/g of protein; P=0.01), and was related to mtDNA oxidative damage. No differences in mitochondrial respiratory-chain function were found between diabetic patients and controls. CONCLUSION: PMBCs from type 2 diabetic patients with late diabetic complications exhibit high mtDNA oxidative damage. The degree of mtDNA oxidation was associated with an increase in MnSOD as an adaptive response to oxidative stress. The consequences of mtDNA oxidative damage on PBMC function and the progression of diabetic complications remain to be elucidated.

McArdle disease: another systemic low-inflammation disorder?

McArdle disease is caused by inherited deficit of human muscle glycogen phosphorylase with subsequent blockade in muscle glycogenolysis. Patients usually experience severe exercise intolerance and 'chronic' skeletal muscle damage. We determined circulating levels of 27 cytokines in a group of 31 adult McArdle patients (15 male 16 female; mean (+/-S.E.M.) age: 39+/-3 years) and 29 healthy sedentary controls (14 male, 15 female) before and after an acute exercise bout involving no muscle damage (cycling). Patients had an ongoing state of muscle breakdown even when following a sedentary lifestyle (serum creatine kinase activity at baseline of 2590+/-461 Ul(-1) vs. 97+/-5 Ul(-1) in controls). Under resting conditions, neutrophil count (+20%) and circulating levels of several cytokines were significantly higher (P

AMPD1 genotypes and exercise capacity in McArdle patients.

The purpose of this study was to assess if there exists an association between C34T muscle adenosine monophosphate deaminase ( AMPD1) genotypes (i.e., normal homyzygotes [CC] vs. heterozygotes [ CT]) and directly measured indices of exercise capacity (peak oxygen uptake [VO(2peak)], ventilatory threshold [VT], gross mechanical efficiency [GE], etc.) in 44 Caucasian McArdle patients (23 males, 21 females). All patients performed a graded cycle ergometer test until exhaustion (for VO(2peak) and VT determination) and a 12-min constant-load test at the power output eliciting the VT (for GE determination). We found no significant difference in indices of exercise capacity between CC (n = 18) and CT genotypes (n = 5) in the group of male patients (p > 0.05). In contrast, the VO(2) at the VT was significantly lower (p < 0.05) in CT (n = 4; 7.9 +/- 0.4 ml/kg/min) than in CC female patients (n = 17; 11.0 +/- 0.9 ml/kg/min). In summary, heterozigosity for the C34T allele of the AMPD gene is associated with reduced submaximal aerobic capacity in female patients with McArdle disease and might partly account, in this gender, for the variability that exists in the phenotypic manifestation of the disease.

McArdle disease: molecular genetic update.

McArdle disease or Glycogenosis type V is an autosomal recessive metabolic disorder caused by a deficiency of the muscle isoform of glycogen phosphorylase (myophosphorylase, PYGM), the specific skeletal muscle enzyme that initiates glycogen breakdown. Since the first clinical description by Brian McArdle in 1951, several patients have been identified worldwide and significant advances have been made in the study of molecular genetics of the disease. Molecular heterogeneity has been demonstrated by the identification to date of more than 65 mutations in the PYGM gene.

[Private mutations in the myophosphorylase gene: the first case in a patient of Latin American descent]. / Mutaciones privadas en el gen de la miofosforilasa: primer caso en un paciente de origen latinoamericano.

INTRODUCTION: McArdle's disease (glycogenoses type V) is a common metabolic myopathy caused by deficient myophosphorylase activity. The disease is due to mutations in the myophosphorylase (PYGM) gene and is present in a large number of countries. CASE REPORT: A 13-year-old male who suffered an episode of muscle pain and offered increased levels of creatinkinase in plasma, myoglobinuria and mild weakness of the proximal muscles, after short but vigorous exercise. The patient was born in Ecuador and was adopted by a Spanish family. The myophosphorylase gene was analysed completely and the patient was found to be a carrier of a missense mutation, a homozygous change where a G is replaced by an A in exon 11, changing a valine for a methionine in codon 456 (V456M). The mutation described above affects an amino acid that is conserved in the enzyme and which was not present in the control population that was studied. CONCLUSIONS: These findings show the presence of McArdle's disease in several ethnic groups and confirm that the ethnic origin of the patient is important when it comes to deciding what mutations should be analysed first in molecular diagnosis studies.

Mutaciones privadas en el gen de la miofosforilasa: primer caso en un paciente de origen latinoamericano / Private mutations in the myophosphorylase gene: The first case in a patient of latin american descent

La enfermedad de McArdle (glicogenosis tipo V) es una miopatía metabólica común causada por unadeficiencia de la actividad de la miofosforilasa. La enfermedad se debe a mutaciones en el gen de la miofosforilasa (PYGM) y está presente en un gran número de países. Caso clínico. Varón de 13 años de edad que sufrió un episodio de dolor muscular y presentó unos niveles elevados de creatincinasa en plasma, mioglobinuria y debilidad de la musculatura proximal moderada, después de un corto pero vigoroso ejercicio. El paciente nació en Ecuador y fue adoptado por una familia española.Se analizó completamente el gen de la miofosforilasa y se encontró que el paciente era portador de una mutación consistente en pérdida de sentido, un cambio homocigótico de una G por una A en el exón 11, cambiando una valina por una metionina en el codón 456 (V456M). La mutación previamente descrita afecta a un aminoácido conservado en la enzima y no estaba presenteen la población control estudiada. Conclusiones. Estos hallazgos demuestran la presencia de la enfermedad de McArdle en varios grupos étnicos y sugiere que el origen étnico del paciente es importante para decidir qué mutaciones deberían analizarse primero en los estudios diagnósticos moleculares (AU)

McArdle's disease (glycogenoses type V) is a common metabolic myopathy caused by deficientmyophosphorylase activity. The disease is due to mutations in the myophosphorylase (PYGM) gene and is present in a large number of countries. Case report. A 13-year-old male who suffered an episode of muscle pain and offered increased levels of creatinkinase in plasma, myoglobinuria and mild weakness of the proximal muscles, after short but vigorous exercise. The patient was born in Ecuador and was adopted by a Spanish family. The myophosphorylase gene was analysed completely andthe patient was found to be a carrier of a missense mutation, a homozygous change where a G is replaced by an A in exon 11, changing a valine for a methionine in codon 456 (V456M). The mutation described above affects an amino acid that is conserved in the enzyme and which was not present in the control population that was studied. Conclusions. These findings show thepresence of McArdle’s disease in several ethnic groups and confirm that the ethnic origin of the patient is important when it comes to deciding what mutations should be analysed first in molecular diagnosis studies (AU)

Peginterferon alpha-2b plus ribavirin vs interferon alpha-2b plus ribavirin for chronic hepatitis C in HIV-coinfected patients.

Treatment of chronic hepatitis C in human immunodeficiency virus (HIV)-infected patients is associated with low response rates and high incidence of side effects. One hundred twenty-one hepatitis C virus (HCV)-HIV-coinfected patients were randomized to receive interferon alpha-2b (3 MU thrice weekly; n = 61) or peginterferon alpha-2b (1.5 microg/kg/week; n = 60), plus ribavirin (800 mg daily), for 24 (genotype 2 or 3) or 48 weeks (genotype 1 or 4). We assessed early virological response at 4, 8 and 12 weeks to predict sustained virological response (SVR). Safety assessment included frequent blood lactate measurement and relative quantitation of mitochondrial DNA (mtDNA) content in peripheral blood mononuclear cells. In intention-to-treat analysis, the SVR rate was higher in the peginterferon group (55%vs 26%; P = 0.002). The difference for HCV genotypes 1 and 4 was 45%vs 14% (P = 0.009) and 50%vs 27% (P = 0.387), respectively, and for genotype 2 or 3, 71%vs 43% (P = 0.12) Viral response at 4, 8 and 12 weeks of treatment was highly predictive of SVR. Among genotype 3 patients, 17 of 20 (85%) whose HCV RNA was already undetectable at 4 weeks had an SVR after 24 weeks of treatment. Hyperlactataemia occurred in 22 patients and was clinically significant in six, two of whom died. mtDNA decreased significantly 4-12 weeks after the start of treatment in patients developing clinically significant hyperlactataemia. Peginterferon alpha-2b plus ribavirin was more effective than interferon alpha-2b plus ribavirin in HIV-coinfected patients. Frequent monitoring of virological response may be very helpful to optimize treatment compliance, to tailor treatment duration and to minimize side effects.

Molecular genetics of McArdle's disease.

This review highlights recent advances in our understanding of McArdle's disease, including the mechanisms involved in the regulation of the clinical phenotype. The latest molecular genetic studies have demonstrated the genetic heterogeneity of the disorder, with more than 65 mutations identified to date. There is not a specific treatment for McArdle's disease, but some nutritional treatments in combination with aerobic conditioning could improve the quality of life in most patients.

Can patients with McArdle's disease run?

Patients with McArdle's disease commonly adopt a sedentary lifestyle. This sedentary behaviour, however, usually worsens the limited exercise capacity of these patients. Although eccentric muscle work can be associated with rhabdomyolysis, supervised eccentric training with gradually increasing loads has important advantages compared with conventional concentric work, particularly for patients with a poor cardiorespiratory system. We report the beneficial effects (particularly, increased VO(2peak) (from 14.6 to 30.8 ml/kg/min) and increased gross muscle efficiency (from 13.8% to 17.2%)) induced by a supervised aerobic training programme of 7 months duration including 3-4 running sessions (< or =60 min/session) per week in a 38-year-old patient. These preliminary data suggest the potential therapeutic value of this type of exercise in these patients.

Infusion of platelets transiently reduces nucleoside overload in MNGIE.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by thymidine phosphorylase (TP) deficiency, which leads to toxic accumulations of thymidine (dThd) and deoxyuridine (dUrd). In this work, we report that infusion of platelets from healthy donors to patients with MNGIE restored transiently circulating TP and reduced plasma dThd and dUrd levels, suggesting that treatments to achieve permanent restoration of circulating TP such as allogeneic stem cell transplantation or gene transfer might be therapeutic.

Exercise capacity in a 78 year old patient with McArdle's disease: it is never too late to start exercising.

The case is reported of a 78 year old man with McArdle's disease and a history of treated coronary heart disease. Despite the pre-exercise administration of sucrose allowing the patient to exercise with normal physiological responses, and without typical McArdle's symptoms or biochemical evidence of muscle damage, his exercise capacity was very low (V(O2)peak = 10.7 ml/min/kg), probably attributable to his lifetime of sedentary living. The data suggest that, with pre-exercise sucrose administration, such patients may be candidates for systematic reconditioning, which may improve functional capacity and quality of life.