Identification of genetic risk loci and prioritization of genes and pathways for myasthenia gravis: a genome-wide association study.

Myasthenia gravis is a chronic autoimmune disease characterized by autoantibody-mediated interference of signal transmission across the neuromuscular junction. We performed a genome-wide association study (GWAS) involving 1,873 patients diagnosed with acetylcholine receptor antibody-positive myasthenia gravis and 36,370 healthy individuals to identify disease-associated genetic risk loci. Replication of the discovered loci was attempted in an independent cohort from the UK Biobank. We also performed a transcriptome-wide association study (TWAS) using expression data from skeletal muscle, whole blood, and tibial nerve to test the effects of disease-associated polymorphisms on gene expression. We discovered two signals in the genes encoding acetylcholine receptor subunits that are the most common antigenic target of the autoantibodies: a GWAS signal within the cholinergic receptor nicotinic alpha 1 subunit (CHRNA1) gene and a TWAS association with the cholinergic receptor nicotinic beta 1 subunit (CHRNB1) gene in normal skeletal muscle. Two other loci were discovered on 10p14 and 11q21, and the previous association signals at PTPN22, HLA-DQA1/HLA-B, and TNFRSF11A were confirmed. Subgroup analyses demonstrate that early- and late-onset cases have different genetic risk factors. Genetic correlation analysis confirmed a genetic link between myasthenia gravis and other autoimmune diseases, such as hypothyroidism, rheumatoid arthritis, multiple sclerosis, and type 1 diabetes. Finally, we applied Priority Index analysis to identify potentially druggable genes/proteins and pathways. This study provides insight into the genetic architecture underlying myasthenia gravis and demonstrates that genetic factors within the loci encoding acetylcholine receptor subunits contribute to its pathogenesis.

Intron mutations and early transcription termination in Duchenne and Becker muscular dystrophy.

DMD pathogenic variants for Duchenne and Becker muscular dystrophy are detectable with high sensitivity by standard clinical exome analyses of genomic DNA. However, up to 7% of DMD mutations are deep intronic and analysis of muscle-derived RNA is an important diagnostic step for patients who have negative genomic testing but abnormal dystrophin expression in muscle. In this study, muscle biopsies were evaluated from 19 patients with clinical features of a dystrophinopathy, but negative clinical DMD mutation analysis. Reverse transcription-polymerase chain reaction or high-throughput RNA sequencing methods identified 19 mutations with one of three pathogenic pseudoexon types: deep intronic point mutations, deletions or insertions, and translocations. In association with point mutations creating intronic splice acceptor sites, we observed the first examples of DMD pseudo 3'-terminal exon mutations causing high efficiency transcription termination within introns. This connection between splicing and premature transcription termination is reminiscent of U1 snRNP-mediating telescripting in sustaining RNA polymerase II elongation across large genes, such as DMD. We propose a novel classification of three distinct types of mutations identifiable by muscle RNA analysis, each of which differ in potential treatment approaches. Recognition and appropriate characterization may lead to therapies directed toward full-length dystrophin expression for some patients.

Myasthenia Gravis.

The basic abnormality in myasthenia gravis (MG) is a reduction in acetylcholine receptors (AChRs) at neuromuscular junctions due to the effects of autoantibodies that are directed against the AChRs in most patients, or against neighboring proteins involved in the clustering of AChRs (MuSK, LRP-4, or agrin). Clinically, MG is characterized by muscle weakness and fatigue, often in typical patterns. The diagnosis may be missed early, and depends on the recognition of clinical manifestations, the measurement of autoantibodies, and/or electrophysiological features. The treatment of MG involves the enhancement of neuromuscular transmission by anticholinesterase drugs (pyridostigmine), and by immunotherapy. Therapy should be designed to improve the clinical features quickly, and keep the symptoms in abeyance over the long term. Rapid improvement can be achieved when necessary by the administration of intravenous immunoglobulin or plasma exchange. Intermediate rates of improvement over months involve the use of adrenal corticosteroids, the calcineurin inhibitors cyclosporine or tacrolimus, and in some patients, the B-cell inhibitor rituximab. For long-term treatment, mycophenolate and azathioprine are the most effective agents. A thymectomy may also have long-term beneficial effects. The majority of MG patients can live normal lives, but most patients require lifelong treatment. The physician's skill in managing the immunotherapeutic agents and avoiding adverse side effects is of paramount importance in the treatment of MG.

Prevalence and distribution of regional scar in dysfunctional myocardial segments in Duchenne muscular dystrophy.

BACKGROUND: The segmental relationship between cardiovascular magnetic resonance (CMR) peak circumferential strain (Ecc) and myocardial scar has not been well characterized in Duchenne muscular dystrophy (DMD), and it is unknown whether echocardiography accurately measures Ecc in DMD. We assessed segmental Ecc and scar using CMR with myocardial tissue tagging and late gadolinium enhancement (LGE) in patients with DMD, then compared CMR with echocardiographic velocity vector imaging (VVI) for regional Ecc based on independent observer assessments. RESULTS: Participants enrolled (n = 16; age 8-23) had median left ventricular (LV) ejection fraction of 0.52 (range 0.28-0.69), and 156 basal and mid-cavity myocardial segments from the 13 patients completing the LGE protocol were analyzed for strain and scar. Segmental CMR Ecc in the most negative quartile (quartile 4) ruled out scar in that segment, but scar was present in 46% of segments in the least negative (most dysfunctional) Ecc quartile 1, 33% of Ecc quartile 2 segments, and 15% of Ecc quartile 3 segments. Overall scar prevalence in inferior, inferolateral, and anterolateral segments was eight times higher than in inferoseptal, anteroseptal, and anterior segments (p < 0.001). This increased proportion of scar in lateral versus septal segments was consistent across CMR Ecc quartiles (quartile 1: 76% versus 11%, p = 0.001; quartile 2: 65% versus 9%, p < 0.001; quartile 3: 38% versus 0%, p < 0.001). Echocardiographic analysis could be performed in 12 of 14 patients with CMR exams and had to be limited to mid-cavity slices. Echo segmental Ecc in the most negative quartile made scar by CMR in that segment highly unlikely, but the correlation in segmental Ecc between CMR and echo was limited (r = 0.27; p = 0.02). CONCLUSIONS: The relationship between scar and Ecc in DMD is complex. Among myocardial segments with depressed Ecc, scar prevalence was much higher in inferior, inferolateral, and anterolateral segments, indicating a regionally dependent association between abnormal Ecc and scar, with free wall segments commonly developing dysfunction with scar and septal segments developing dysfunction without scar. Although normal echocardiographic Ecc predicted absence of scar, regional echocardiographic Ecc by VVI has only a limited association with CMR Ecc in DMD.

Epidemiological evidence for a hereditary contribution to myasthenia gravis: a retrospective cohort study of patients from North America.

OBJECTIVES: To approximate the rate of familial myasthenia gravis and the coexistence of other autoimmune disorders in the patients and their families. DESIGN: Retrospective cohort study. SETTING: Clinics across North America. PARTICIPANTS: The study included 1032 patients diagnosed with acetylcholine receptor antibody (AChR)-positive myasthenia gravis. METHODS: Phenotype information of 1032 patients diagnosed with AChR-positive myasthenia gravis was obtained from clinics at 14 centres across North America between January 2010 and January 2011. A critical review of the epidemiological literature on the familial rate of myasthenia gravis was also performed. RESULTS: Among 1032 patients, 58 (5.6%) reported a family history of myasthenia gravis. A history of autoimmune diseases was present in 26.6% of patients and in 28.4% of their family members. DISCUSSION: The familial rate of myasthenia gravis was higher than would be expected for a sporadic disease. Furthermore, a high proportion of patients had a personal or family history of autoimmune disease. Taken together, these findings suggest a genetic contribution to the pathogenesis of myasthenia gravis.

Anti-HMGCR myopathy may resemble limb-girdle muscular dystrophy.

Objective: To determine the prevalence and clinical features of anti-HMGCR myopathy among patients with presumed limb-girdle muscular dystrophy (LGMD) in whom genetic testing has failed to elucidate causative mutations. Methods: Patients with presumed LGMD and unrevealing genetic testing were selected based on a few clinico-pathologic features and tested for anti-HMGCR autoantibodies (n = 11). These clinico-pathologic features are peak creatine kinase (CK) greater than 1,000 IU/L and at least 3 of the following features: (1) limb-girdle pattern of weakness, (2) selective involvement of posterior thigh on clinical examination or muscle imaging, (3) dystrophic changes on muscle biopsy, and (4) no family history of muscular dystrophy. Results: Six patients tested positive for anti-HMGCR autoantibodies. In 4, there was a presymptomatic phase, lasting as long as 10 years, characterized by elevated CK levels without weakness. Muscle biopsies revealed variable degrees of a dystrophic pathology without prominent inflammation. In an independent cohort of patients with anti-HMGCR myopathy, 17 of 51 (∼33%) patients were initially presumed to have a form of LGMD based on clinico-pathologic features but were ultimately found to have anti-HMGCR myopathy. Most of these patients responded favorably to immunomodulatory therapies, evidenced by reduction of CK levels and improved strength. Conclusions: Anti-HMGCR myopathy can resemble LGMD. Diagnosis of patients with a LGMD-like presentation of anti-HMGCR myopathy is critical because these patients may respond favorably to immunotherapy, especially those with shorter disease duration.

Rebooting the immune system with high-dose cyclophosphamide for treatment of refractory myasthenia gravis.

A small but important proportion of patients with myasthenia gravis (MG) are refractory to conventional immunotherapy. We have treated 12 such patients by "rebooting" the immune system with high-dose cyclophosphamide (Hi Cy, 200 mg/kg), which largely eliminates the mature immune system, while leaving hematopoietic precursors intact. The objective of this report is to describe the clinical and immunologic results of Hi Cy treatment of refractory MG. We have followed 12 patients clinically for 1-9 years, and have analyzed their humoral and cellular immunologic parameters. Hi Cy is safe and effective. All but one of the patients experienced dramatic clinical improvement for variable periods from 5 months to 7.5 years, lasting for more than 1 year in seven of the patients. Two patients are still in treatment-free remission at 5.5 and 7.5 years, and five have achieved responsiveness to immunosuppressive agents that were previously ineffective. Hi Cy typically reduced, but did not completely eliminate, antibodies to the autoantigen AChR or to tetanus or diphtheria toxin; re-immunization with tetanus or diphtheria toxoid increased the antibody levels. Despite prior thymectomy, T cell receptor excision circles, generally considered to reflect thymic emigrant T cells, were produced by all patients. Hi Cy treatment results in effective, but often not permanent, remission in most refractory myasthenic patients, suggesting that the immune system is in fact "rebooted," but not "reformatted." We therefore recommend that treatment of refractory MG with Hi Cy be followed with maintenance immunotherapy.

Gene transfer of baculoviral p35 by adenoviral vector protects human cerebral neurons from apoptosis.

Apoptosis plays an important role in neuronal cell death in both chronic and acute human neurological diseases, including ALS, Huntington's disease, cerebral ischemia, and HIV encephalopathy. We evaluated the ability of an extremely powerful antiapoptotic agent, baculoviral p35, to prevent apoptosis and cell death of human cerebral neurons that undergo severe neurotoxic changes in a culture system when treated with agents that are implicated in human neurological disorders, that is, tumor necrosis factor (TNFalpha) and the HIV proteins Tat and gp120. P35 is a potent broad-spectrum antiapoptotic protein derived from baculovirus, that inhibits nearly all caspases, and has other antiapoptotic actions as well. Adenoviral vectors expressing p35 (Ad. p35) or a control gene (lacZ) efficiently transduced human neurons. Treatment of control cultures with the toxic agents TNFalpha, TNFalpha plus Actinomycin D, or Tat and gp120, induced neurotoxicity and death of neurons. Transduction of neurons with Ad. p35 blocked apoptosis, and eliminated cell death due to TNFalpha, or Tat and gp120. Viral vector transfer of the p35 gene efficiently protects human neurons from TNFalpha, or Tat and gp120-induced apoptosis and cell death. These results suggest that p35 transduction of neurons by viral vectors could be therapeutically useful in the treatment of human neurodegenerative diseases.

Repeated treatment with high dose cyclophosphamide for severe autoimmune diseases.

High dose cyclophosphamide (HiCY) without stem cell rescue has been shown to induce remissions in patients with severe autoimmune disorders (SADS). However, up to 80% of these patients ultimately relapse. Here we review the outcomes of seven patients treated with two cycles and one patient treated with three cycles of HiCY. The diseases re-treated were scleroderma, multiple sclerosis, three patients with severe aplastic anemia (SAA), and three patients with myasthenia gravis (MG). All but two patients with SAA had received standard immunomodulatory therapy for their disease up front and had been refractory. All patients had complete hematologic recovery. Overall survival in this cohort was 100%. All patients relapsed after the initial cycle but event free survival thereafter was 93.3%. All are still in remission except two MG patients, one of whom relapsed after a severe GI infection requiring hospitalization, and the other relapsed 3 years after the second treatment and she did not respond to the third treatment. This shows that HiCY can be safely re-administered in patients with SAA and refractory SADS. The quality and duration of second remissions appears to be equal or superior to the first remission.

High-dose cyclophosphamide without stem cell rescue in 207 patients with aplastic anemia and other autoimmune diseases.

High-dose cyclophosphamide has long been used as an anticancer agent, a conditioning regimen for hematopoietic stem cell transplantation, and a potent immunosuppressive agent in autoimmune diseases including aplastic anemia. High-dose cyclophosphamide is highly toxic to lymphocytes but spares hematopoietic stem cells because of their abundant levels of aldehyde dehydrogenase, the major mechanism of cyclophosphamide inactivation. High-dose cyclophosphamide therapy induces durable remissions in most patients with acquired aplastic anemia. Moreover, high-dose cyclophosphamide without hematopoietic stem cell rescue has shown activity in a variety of other severe autoimmune diseases. Here we review the history of cyclophosphamide as it applies to aplastic anemia and other autoimmune diseases. We include historical data from early patients treated for aplastic anemia as well as data from 140 patients from an observational retrospective study in a single tertiary care hospital. This latter component was designed to assess the safety and efficacy of high-dose cyclophosphamide therapy without stem cell rescue in patients with refractory autoimmune diseases. We analyzed the 140 patients with severe, progressive autoimmune diseases treated. All patients discussed here received cyclophosphamide, 50 mg/kg per day for 4 consecutive days. Response, relapse, and overall survival were measured. Response was defined as a decrease in disease activity in conjunction with a decrease or elimination of immune-modulating drugs. Relapse was defined as worsening disease activity and/or a requirement for an increase in dose of, or administration of new, immunosuppressive medications. Hematologic recovery occurred in all patients. The overall response rate was 94%, and 44% of those patients remained progression free with a median follow-up of 36 months (range, 1-120 mo) for the 140 patients analyzed together. The overall actuarial and event-free survival across all diseases at 60 months was 90.7% and 20.6%, respectively. High-dose cyclophosphamide without stem cell rescue is well tolerated and induces a high rate of remission in severe autoimmune diseases.

Strategy for treating motor neuron diseases using a fusion protein of botulinum toxin binding domain and streptavidin for viral vector access: work in progress.

Although advances in understanding of the pathogenesis of amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) have suggested attractive treatment strategies, delivery of agents to motor neurons embedded within the spinal cord is problematic. We have designed a strategy based on the specificity of botulinum toxin, to direct entry of viral vectors carrying candidate therapeutic genes into motor neurons. We have engineered and expressed fusion proteins consisting of the binding domain of botulinum toxin type A fused to streptavidin (SAv). This fusion protein will direct biotinylated viral vectors carrying therapeutic genes into motor nerve terminals where they can enter the acidified endosomal compartments, be released and undergo retrograde transport, to deliver the genes to motor neurons. Both ends of the fusion proteins are shown to be functionally intact. The binding domain end binds to mammalian nerve terminals at neuromuscular junctions, ganglioside GT1b (a target of botulinum toxin), and a variety of neuronal cells including primary chick embryo motor neurons, N2A neuroblastoma cells, NG108-15 cells, but not to NG CR72 cells, which lack complex gangliosides. The streptavidin end binds to biotin, and to a biotinylated Alexa 488 fluorescent tag. Further studies are in progress to evaluate the delivery of genes to motor neurons in vivo, by the use of biotinylated viral vectors.

Reduction of disease activity and disability with high-dose cyclophosphamide in patients with aggressive multiple sclerosis.

OBJECTIVE: To explore the safety and effectiveness of high-dose cyclophosphamide (HiCy) without bone marrow transplantation in patients with aggressive multiple sclerosis (MS). DESIGN: A 2-year open-label trial of patients with aggressive relapsing-remitting multiple sclerosis (RRMS) given an immunoablative regimen of HiCy (50 mg/kg/d for 4 consecutive days) with no subsequent immunomodulatory therapy unless disease activity reappeared that required rescue therapy. SETTING: The Johns Hopkins University Multiple Sclerosis Center, Baltimore, Maryland. Patients A total of 21 patients with RRMS were screened for eligibility and 9 patients were enrolled in the trial. Patients were required to have 2 or more gadolinium-enhancing lesions on each of 2 pretreatment magnetic resonance imaging scans, at least 1 clinical exacerbation in the 12 months prior to HiCy treatment, or a sustained increase of 1.0 point or higher on the Expanded Disability Status Scale (EDSS) in the preceding year. Intervention Patients received 50 mg/kg/d of cyclophosphamide intravenously for 4 consecutive days, followed by 5 mug/kg/d of granulocyte colony-stimulating factor 6 days after completion of HiCy treatment, until the absolute neutrophil count exceeded 1.0 x 10(9) cells/L for 2 consecutive days. MAIN OUTCOME MEASURES: The primary outcome of the study was the safety and tolerability of HiCy in patients with RRMS. Secondary outcome measures included a change in gadolinium-enhancing lesions on magnetic resonance images and a change in disability measures (EDSS and Multiple Sclerosis Functional Composite). RESULTS: Nine patients were treated and followed up for a mean period of 23 months. Eight patients had failed conventional therapy and 1 was treatment naive. The median age at time of entry was 29 years (range, 20-47 years). All patients developed transient total or near-total pancytopenia as expected, followed by hematopoietic recovery in 10 to 17 days, stimulated by granulocyte colony-stimulating factor. There were no deaths or unexpected serious adverse events. There was a statistically significant reduction in disability (EDSS) at follow-up (mean [SD] decrease, 2.11 [1.97]; 39.4%; P = .02). The mean (SD) number of gadolinium-enhancing lesions on the 2 pretreatment scans were 6.5 (2.1) and 1.2 (2.3) at follow-up (81.4% reduction; P = .01). Two patients required rescue treatment with other immunomodulatory therapies during the study owing to MS exacerbations. CONCLUSION: Treatment with HiCy was safe and well tolerated in our patients with MS. Patients experienced a pronounced reduction in disease activity and disability after HiCy treatment. This immunoablative regimen of cyclophosphamide for patients with aggressive MS is worthy of further study and may be an alternative to bone marrow transplantation. Published online June 9, 2008 (doi:10.1001/archneurol.65.8.noc80042).