Gisela Nogales-Gadea

Favorable responses to acute and chronic exercise in McArdle patients.

Objective:This study reports acute exercise responses in a large (N = 46) series of patients with McArdle disease and responses to exercise training in a smaller (n = 9) set of patients. Design:Patients were studied during both incremental and steady-state cycle ergometer exercise, using cardiopulmonary testing, and the patients were compared with age- and gender-matched controls. Setting:The study was performed in a university setting (clinical exercise physiology laboratory). Participants:The 46 patients showed common features of McArdle disease. They were definitively diagnosed by histochemistry, biochemistry, and/or molecular genetic analysis. The 46 controls were healthy, sedentary individuals. Intervention:Nine patients were studied before and after an 8-month supervised aerobic exercise training program (including five weekly sessions of walking and/or cycling exercise with a duration no greater than 60 minutes). Main Outcome Measurements:The main indicators of exercise capacity that we measured were peak power output, peak oxygen uptake (VO2peak), and ventilatory threshold (VT). Results:Exercise capacity (peak power output, 35% control; VO2peak, 44% control; VT, 66% control) was markedly depressed in the patients. The patients who trained improved peak power output (25%), VO2peak (44%), and VT (27%), with no evidence of negative outcomes from training. Although not achieving normal values, the response to training put the patients into the lower limit of normal controls. Conclusions:Under carefully controlled conditions, patients with McArdle disease may perform acute exercise safely, and they may respond favorably to training. This may offer an additional therapeutic option to help normalize the lifestyles of these patients.

Specific Contactin N-Glycans Are Implicated in Neurofascin Binding and Autoimmune Targeting in Peripheral Neuropathies

Background: The glycoproteins contactin and neurofascin-155 are implicated in axo-glial junctions insulating the node of Ranvier. Results: N-Glycosylated contactin is targeted in inflammatory neuropathy. Conclusion: Autoantibodies reveal specific N-glycans that are implicated in adhesive interaction between contactin and neurofascin-155. Significance: Autoantibodies against N-glycosylated contactin may be pathogenic via functional blocking. Cell adhesion molecules (CAMs) play a crucial role in the formation of the nodes of Ranvier and in the rapid propagation of the nerve impulses along myelinated axons. These CAMs are the targets of autoimmunity in inflammatory neuropathies. We recently showed that a subgroup of patients with aggressive chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) shows autoantibodies to contactin (1). The complex of contactin·Caspr·neurofascin-155 (NF155) enables the formation of paranodal junctions, suggesting that antibody attack against paranodes may participate in the severity of CIDP. In the present study, we mapped the molecular determinants of contactin targeted by the autoantibodies. In three patients, immunoreactivity was directed against the Ig domains of contactin and was dependent on N-glycans. The serum of one patient was selectively directed against contactin bearing mannose-rich N-glycans. Strikingly, the oligomannose type sugars of contactin are required for association with its glial partner NF155 (2). To investigate precisely the role of contactin N-glycans, we have mutated each of the nine consensus N-glycosylation sites independently. We found that the mutation of three sites (N467Q/N473Q/N494Q) in Ig domain 5 of contactin prevented soluble NF155-Fc binding. In contrast, these mutations did not abolish cis-association with Caspr. Next, we showed that the cluster of N-glycosylation sites (Asn-467, Asn-473, and Asn-494) was required for immunoreactivity in one patient. Using cell aggregation assays, we showed that the IgGs from the four CIDP patients prevented adhesive interaction between contactin·Caspr and NF155. Importantly, we showed that the anti-contactin autoantibodies induced alteration of paranodal junctions in myelinated neuronal culture. These results strongly suggest that antibodies to CAMs may be pathogenic and induce demyelination via functional blocking activity.

Altered RIG-I/DDX58-mediated innate immunity in dermatomyositis.

We investigated the molecular mechanisms involved in the pathogenesis of three inflammatory myopathies, dermatomyositis (DM), polymyositis (PM) and inclusion body myositis (IBM). We performed microarray experiments† using microdissected pathological muscle fibres from 15 patients with these disorders and five controls. Differentially expressed candidate genes were validated by immunohistochemistry on muscle biopsies, and the altered pathways were analysed in human myotube cultures. Up‐regulation of genes involved in viral and nucleic acid recognition were found in the three myopathies but not in controls. In DM, retinoic acid‐inducible gene 1 (RIG‐I, DDX58) and the novel antiviral factor DDX60, which promotes RIG‐I‐mediated signalling, were significantly up‐regulated, followed by IFIH1 (MDA5) and TLR3. Immunohistochemistry confirmed over‐expression of RIG‐I in pathological muscle fibres in 5/5 DM, 0/5 PM and 0/5 IBM patients, and in 0/5 controls. Stimulation of human myotubes with a ligand of RIG‐I produced a significant secretion of interferon‐β (IFNβ; p < 0.05) and up‐regulation of class I MHC, RIG‐I and TLR3 (p < 0.05) by IFNβ‐dependent and TLR3‐independent mechanisms. RIG‐I‐mediated innate immunity, triggered by a viral or damage signal, plays a significant role in the pathogenesis of DM, but not in that of PM or IBM. Copyright

The I allele of the ACE gene is associated with improved exercise capacity in women with McArdle disease

We assessed the possible association existing between alpha-actinin-3 (ACTN3) R577X genotypes and the capacity for performing aerobic exercise in McArdles patients. Forty adult McArdles disease patients and forty healthy, age and gender-matched sedentary controls (21 men, 19 women in both groups) performed a graded test until exhaustion and a constant-load test on a cycle-ergometer to determine clinically relevant indices of exercise capacity as peak oxygen uptake (VO(2peak)) and the ventilatory threshold (VT). In the group of diseased women, carriers of the X allele had a higher (P<0.01) VO(2peak) (15.0+/-1.2 ml/kg/min) and a higher (P<0.05) oxygen uptake (VO(2)) at the VT (11.2+/-1 ml/kg/min) than R/R homozygotes (VO(2peak): 9.6+/-0.5 ml/kg/min; VO(2) at the VT: 8.2+/-0.7 ml/kg/min). No differences were found in male patients. In women with McArdles disease, ACTN3 genotypes might partly explain the large individual variability that exists in the phenotypic manifestation of this disorder.

Knock-in mice for the R50X mutation in the PYGM gene present with McArdle disease

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.

McArdle Disease: Update of Reported Mutations and Polymorphisms in the PYGM Gene

McArdle disease is an autosomal‐recessive disorder caused by inherited deficiency of the muscle isoform of glycogen phosphorylase (or “myophosphorylase”), which catalyzes the first step of glycogen catabolism, releasing glucose‐1‐phosphate from glycogen deposits. As a result, muscle metabolism is impaired, leading to different degrees of exercise intolerance. Patients range from asymptomatic to severely affected, including in some cases, limitations in activities of daily living. The PYGM gene codifies myophosphoylase and to date 147 pathogenic mutations and 39 polymorphisms have been reported. Exon 1 and 17 are mutational hot‐spots in PYGM and 50% of the described mutations are missense. However, c.148C>T (commonly known as p.R50X) is the most frequent mutation in the majority of the studied populations. No genotype–phenotype correlation has been reported and no mutations have been described in the myophosphorylase domains affecting the phosphorylated Ser‐15, the 280s loop, the pyridoxal 5′‐phosphate, and the nucleoside inhibitor binding sites. A newly generated knock‐in mouse model is now available, which renders the main clinical and molecular features of the disease. Well‐established methods for diagnosing patients in laboratories around the world will shorten the frequent ∼20‐year period stretching from first symptoms appearance to the genetic diagnosis.

Analysis of Serum miRNA Profiles of Myasthenia Gravis Patients

Myasthenia gravis (MG) is an autoimmune disease characterized by the presence of autoantibodies, mainly against the acetylcholine receptor (AChR). The mechanisms triggering and maintaining this chronic disease are unknown. MiRNAs are regulatory molecules that play a key role in the immune system and are altered in many autoimmune diseases. The aim of this study was to evaluate miRNA profiles in serum of 61 AChR MG patients. We studied serum from patients with early onset MG (n = 22), late onset MG (n = 27) and thymoma (n = 12), to identify alterations in the specific subgroups. In a discovery cohort, we analysed 381 miRNA arrays from 5 patients from each subgroup, and 5 healthy controls. The 15 patients had not received any treatment. We found 32 miRNAs in different levels in MG and analysed 8 of these in a validation cohort that included 46 of the MG patients. MiR15b, miR122, miR-140-3p, miR185, miR192, miR20b and miR-885-5p were in lower levels in MG patients than in controls. Our study suggests that different clinical phenotypes in MG share common altered mechanisms in circulating miRNAs, with no additional contribution of the thymoma. MG treatment intervention does not modify the profile of these miRNAs. Novel insights into the pathogenesis of MG can be reached by the analysis of circulating miRNAs since some of these miRNAs have also been found low in MG peripheral mononuclear cells, and have targets with important roles in B cell survival and antibody production.

Proteasome Inhibition with Bortezomib Depletes Plasma Cells and Specific Autoantibody Production in Primary Thymic Cell Cultures from Early-Onset Myasthenia Gravis Patients

Bortezomib is a potent inhibitor of proteasomes currently used to eliminate malignant plasma cells in multiple myeloma patients. It is also effective in depleting both alloreactive plasma cells in acute Ab-mediated transplant rejection and their autoreactive counterparts in animal models of lupus and myasthenia gravis (MG). In this study, we demonstrate that bortezomib at 10 nM or higher concentrations killed long-lived plasma cells in cultured thymus cells from nine early-onset MG patients and consistently halted their spontaneous production not only of autoantibodies against the acetylcholine receptor but also of total IgG. Surprisingly, lenalidomide and dexamethasone had little effect on plasma cells. After bortezomib treatment, they showed ultrastructural changes characteristic of endoplasmic reticulum stress after 8 h and were no longer detectable at 24 h. Bortezomib therefore appears promising for treating MG and possibly other Ab-mediated autoimmune or allergic disorders, especially when given in short courses at modest doses before the standard immunosuppressive drugs have taken effect.

A novel exon 5 mutation (N139H) in the SOD1 gene in a Spanish family associated with incomplete penetrance

BACKGROUND Allelic heterogeneity and phenotype variability-especially in age at onset, penetrance and progression-are reported in ALS1 families. For this reason, SOD1 gene mutation data in ALS1 patients are currently being gathered to better understand the genotype-phenotype relationship in this disorder. Here, we report the clinical and molecular characteristics of a Spanish ALS1 family with incomplete penetrance. PATIENTS AND METHODS Clinical data including age at onset, initial topography, progression and survival were available in three affected members. Erythrocyte SOD1 activity was measured in four individuals. Analysis of the SOD1 gene was performed by PCR and direct sequencing. RESULTS A novel missense mutation in the exon 5 of the SOD1 gene, an A-to-C transversion at nucleotide position 1485 leading to N139H residue change, was identified in three family members. The phenotype was similar in all cases, with initial symptoms in the distal limb muscles and a mean survival time of around 4 years. Incomplete penetrance was observed in our family, as two obligate carriers did not develop any symptoms of amyotrophic lateral sclerosis (ALS). CONCLUSIONS N139H is the fifth SOD1 gene mutation reported in Spain, and the first one presenting with incomplete penetrance. Genetic counseling for at-risk relatives in these low-penetrance families could be difficult as some individuals harbouring the mutation remain asymptomatic throughout their lives. Further genetic characterisation of ALS1 families should provide information regarding the distribution of SOD1 mutants in different ethnic groups.

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.