Myasthenia Gravis and Thymectomy

Myasthenia gravis (MG) is a rare autoimmune neuromuscular disorder. Though MG was diagnosed four centuries ago, its rational management started in 1930s. In the present era, MG is managed by multimodality care including pharmacological agents, plasmapheresis, intravenous immunoglobulins, and surgical thymectomy. Thymectomy has evolved from open trans-sternal to video-assisted thoracoscopic and robotic thymectomy. In this article, the concise history of MG, its clinical features, diagnosis, and management are described.

Adeno-Associated Virus (AAV)-Mediated Gene Therapy for Duchenne Muscular Dystrophy: The Issue of Transgene Persistence

Duchenne muscular dystrophy (DMD) is an X-linked recessive, infancy-onset neuromuscular disorder characterized by progressive muscle weakness and atrophy, leading to delay of motor milestones, loss of autonomous ambulation, respiratory failure, cardiomyopathy, and premature death. DMD originates from mutations in the DMD gene that result in a complete absence of dystrophin. Dystrophin is a cytoskeletal protein which belongs to the dystrophin-associated protein complex, involved in cellular signaling and myofiber membrane stabilization. To date, the few available therapeutic options are aimed at lessening disease progression, but persistent loss of muscle tissue and function and premature death are unavoidable. In this scenario, one of the most promising therapeutic strategies for DMD is represented by adeno-associated virus (AAV)-mediated gene therapy. DMD gene therapy relies on the administration of exogenous micro-dystrophin, a miniature version of the dystrophin gene lacking unnecessary domains and encoding a truncated, but functional, dystrophin protein. Limited transgene persistence represents one of the most significant issues that jeopardize the translatability of DMD gene replacement strategies from the bench to the bedside. Here, we critically review preclinical and clinical studies of AAV-mediated gene therapy in DMD, focusing on long-term transgene persistence in transduced tissues, which can deeply affect effectiveness and sustainability of gene replacement in DMD. We also discuss the role played by the overactivation of the immune host system in limiting long-term expression of genetic material. In this perspective, further studies aimed at better elucidating the need for immune suppression in AAV-treated subjects are warranted in order to allow for life-long therapy in DMD patients.

Glucocorticoid-Induced Hypokalemic Periodic Paralysis after Short-Term Use of Tenofovir with Hypophosphatemia: A Case Report

Hypokalemic periodic paralysis (HPP) is a neuromuscular disorder associated with muscular dysfunction caused by hypokalemia. There are various causes of HPPs and rarely, HPP appears to be relevant to tenofovir or glucocorticoid treatment. There have been several case reports of tenofovir-related nephrotoxicity or tenofovir-induced HPP. However, a case report of glucocorticoid-induced HPP in a patient using tenofovir temporarily has not been reported. Herein, we report a case of glucocorticoid-induced HPP with short-term use of tenofovir. A 28-year-old man visited the emergency room with decreased muscle power in all extremities (2/5 grade). In their past medical history, the patient was treated with tenofovir for two months for a hepatitis B virus infection. At the time of the visit, the drug had been discontinued for four months. The day before visiting the emergency room, betamethasone was administered at a local clinic for herpes on the lips. Laboratory tests showed hypokalemia, hypophosphatemia, and mild metabolic acidosis. However, urinalysis revealed no abnormal findings. Consequently, it can be postulated that this patient developed HPP by glucocorticoids after taking tenofovir temporarily. This is the first case report of glucocorticoid-induced HPP in a patient using tenofovir. Clinicians who prescribe tenofovir should be aware of HPP occurring when glucocorticoids are used.

Screening of Chimeric GAA Variants in a Preclinical Study of Pompe Disease Results in Candidate Vector for Hematopoietic Stem Cell Gene Therapy

Pompe disease is a rare genetic neuromuscular disorder caused by acid alpha-glucosidase (GAA) deficiency resulting in lysosomal glycogen accumulation and progressive myopathy. Enzyme replacement therapy (ERT) is the current standard of care, which prolongs the quality of life for Pompe patients. However, ERT has limitations due to lack of enzyme penetration into the central nervous system (CNS) and skeletal muscles, immunogenicity against the recombinant enzyme, and requires life-long biweekly infusions. In a preclinical mouse model, a clinically relevant promoter to drive lentiviral vector-mediated expression of engineered GAA in autologous hematopoietic stem and progenitor cells (HSPC) was tested with nine unique human chimeric GAA coding sequences incorporating distinct peptide tags and codon-optimization iterations. Vectors including glycosylation independent lysosomal targeting (GILT) tags resulted in effective GAA enzyme delivery into key disease tissues with enhanced reduction of glycogen, myofiber and CNS vacuolation, compared to non-tagged GAA in Gaa knockout mice, a model of Pompe disease. Genetically modified microglial cells in brains were detected at low levels, but provided robust correction. Furthermore, an aminoacid substitution in the tag added to reduced capacity to induce insulin signaling and there was no evidence of off-target effects. This study demonstrated the therapeutic potential of lentiviral HSPC gene therapy exploiting optimized GAA tagged coding sequences to reverse Pompe disease pathology in a preclinical mouse model providing a promising vector candidate for further investigation.

A novel zebrafish model of SPEG-related centronuclear myopathy (CNM): characterization and comparison with other CNM model zebrafish

Centronuclear myopathy (CNM) is a congenital neuromuscular disorder caused by pathogenic variation in genes associated with membrane trafficking and excitation-contraction coupling (ECC). Bi-allelic autosomal recessive mutations in striated muscle enriched protein kinase (SPEG) account for a subset of CNM patients. Previous research has been limited by the perinatal lethality of Speg knockout mice. Thus, the precise biological role of SPEG in skeletal muscle remains unknown. To address this issue, we generated zebrafish spega, spegb, and spega/spegb (speg-DKO) mutant lines. We demonstrate that speg-DKO zebrafish faithfully recapitulate multiple phenotypes associated with human CNM, including disruption of the ECC protein machinery, dysregulation of calcium homeostasis during ECC, and impairment of muscle performance. Taking advantage of the availability of zebrafish models of multiple CNM genetic subtypes, we compared novel and known disease markers in speg-DKO with mtm1-KO and DNM2-S619L transgenic zebrafish. We observed desmin (DES) accumulation common to all CNM subtypes, and Dnm2 upregulation in muscle of both speg-DKO and mtm1-KO zebrafish. In all, we establish a new model of SPEG-related CNM, and identify abnormalities in this model suitable for defining disease pathomechanisms and evaluating potential therapies.

Generation of Dystrophin Short Product-Specific Tag-Insertion Mouse: Distinct Dp71 Glycoprotein Complexes at Inhibitory Postsynapse and Glia Limitans

Duchenne muscular dystrophy (DMD), the most severe form of dystrophinopathies, is a fatal X-linked recessive neuromuscular disorder characterized by progressive muscle degeneration and various extents of intellectual disabilities. Physiological and pathological roles of the responsible gene, dystrophin, in the brain remain elusive due to the presence of multiple dystrophin products, mainly full-length dystrophin, Dp427, and the short product, Dp71. In this study, we generated a Dp71-specific hemagglutinin (HA) peptide tag-insertion mice to enable specific detection of intrinsic Dp71 expression by anti-HA tag antibodies. Immunohistochemical detections in the transgenic mice demonstrated Dp71 expression not only at the blood-brain barrier, where astrocytic endfeet surround the microvessels, but also at the inhibitory postsynapse of hippocampal dentate granule neurons. Interestingly, hippocampal cornu ammonis (CA)1 pyramidal neurons were negative for Dp71 although Dp427 detected by anti-dystrophin antibody was clearly present at the inhibitory postsynapse, suggesting cell-type dependent dystrophin expressions. Precise examination using the primary hippocampal culture validated exclusive localization of Dp71 at the inhibitory postsynaptic compartment but not at the excitatory synapse in neurons. We further performed interactome analysis and found that Dp71 formed distinct molecular complexes, i.e. synapse-associated Dp71 interacted with dystroglycan (Dg) and dystrobrevinb (Dtnb) whereas glia-associated Dp71 did with Dg and dystrobrevina (Dtna). Thus, our data indicates that Dp71 and its binding partners are relevant to the inhibitory postsynaptic function of hippocampal granule neurons and the novel Dp71-transgenic mouse provides a valuable tool to understand precise physiological expressions and functions of Dp71 and its interaction proteins in vivo and in vitro.

In Vitro Fertilization Using Preimplantation Genetic Testing in a Romanian Couple Carrier of Mutations in the TTN Gene: A Case Report and Literature Review

Severe congenital myopathy with fatal cardiomyopathy (EOMFC) is a rare genetic neuromuscular disorder inherited in an autosomal recessive manner. Here we presented a successful pregnancy obtained by in vitro fertilization (IVF) using preimplantation genetic testing (PGT) in one young Romanian carrier couple that already lost mutation(s) within the TNN gene and whose first baby passed away due to multiple complications. It was delivered via emergency C-section at 36 weeks and fully dependent on artificial ventilation for a couple of months, weighing 2200 g and an APGAR score of 3. The aCGH + SNP analysis revealed an abnormal profile of the first newborn; three areas associated with loss of heterozygosity on chromosome 1 (q25.1–q25.3) of 6115 kb, 5 (p15.2–p15.1) of 2589 kb and 8 (q11.21–q11.23) of 4830 kb, a duplication of 1104 kb on chromosome 10 in the position q11.22, and duplication of 1193 kb on chromosome 16 in the position p11.2p11.1. Subsequently, we proceeded to test the parents and showed that both parents are carriers; confirmed by Sanger and NGS sequencing—father—on Chr2(GRCh37):g.179396832_179396833del—TTN variant c.104509_104510del p.(Leu34837Glufs*12)—exon 358 and mother—on Chr2(GRCh37):g.179479653G>C—TTN variant c.48681C>G p.(Tyr16227*)—exon 260. Their first child died shortly after birth due to multiple organ failures, possessing both parent’s mutations; weighing 2200 g at birth and received an APGAR score of 3 following premature delivery via emergency C-section at 36 weeks. Two embryos were obtained following the IVF protocol; one possessed the mother’s mutation, and the other had no mutations and was normal (WT). In contrast with the first birth, the second one was uneventful. A healthy female baby weighing 2990 g was delivered by C-section at 38 weeks, receiving an APGAR score of 9.

Clinical and Genetic Characteristics in Young, Glucocorticoid-Naive Boys With Duchenne Muscular Dystrophy

Background and ObjectivesDuchenne muscular dystrophy (DMD) is a paediatric neuromuscular disorder caused by mutations in the dystrophin gene. Geneotype-phenotype associations have been examined in glucocorticoid treated boys, but there are few data on the young glucocorticoid-naïve DMD population. A sample of young glucocorticoid-naïve DMD boys is described and genotype-phenotype associations are investigated.MethodsScreening and baseline data were collected for all the participants in the Finding the Optimum Corticosteroid Regime for Duchenne Muscular Dystrophy (FOR-DMD)study, an international, multi-centre, randomized, double-blind, clinical trial comparing three glucocorticoid regimens in glucocorticoid-naïve, genetically confirmed boys with DMD between 4 and <8 years of age.ResultsOne hundred and ninety-six boys were recruited. The meanage at randomization (+ standard deviation) was 5.8+ 1.0 years. The predominant mutation type was out of frame deletions 67.4%, (130/193) of which 68.5% (89/130) were amenable to exon skipping. The most frequent mutations were deletions amenable to exon 51 skipping 13.0% (25/193). Stop codon mutations accounted for 10.4% (20/193).The mean age at first parental concerns was 29.8 + 18.7 months, the mean age at genetic diagnosis was 53.9 + 21.9 months and the mean diagnostic delay was 25.9 + 18.2 months. The mean diagnostic delay for boys diagnosed following an incidental finding of isolated hyperCKemia (n=19) was 6.4 + 7.4 months. The mean ages at independent walking and talking in sentences were 17.1 + 4.2 and 29.0 + 10.7 months, respectively. Median height percentiles were below the 25th percentile regardless of age group. No genotype-phenotype associations were identified expect for boys with an exon 8 skippable deletions who had better performance on time to walk/run 10 meters (p=0.02)compared to boys with deletions not amenable to skipping.DiscussionThis study describes clinical and genetic characteristics of a sample of young glucocorticoid-naïve boys with DMD. A low threshold for CK testing can lead to an earlier diagnosis. Motor and speech delay were common presenting symptoms.The effects of low, pre-treatment height on growth and adults height requires further study. These findings may promote earlier recognition of DMD and inform study design for future clinical trials.

Incidence and clinical feature of Myasthenia gravis: A five-year data analysis in Ulaanbaatar, Mongolia

Myasthenia gravis (MG) is a rare neuromuscular disorder. Till now, there are no studies on the prevalence and incidence of MG in Mongolia. The current study aimed to elucidate the incidence of MG in Ulaanbaatar, the age of onset, and the gender distribution of Mongolian patients with MG. We conducted a cross-sectional, hospital-based study involving MG patients (n=48) all around Ulaanbaatar from 1 January 2015 to 1 January 2020. The clinical diagnosis was assessed with the Myasthenia Gravis Foundation of America (MGFA) classification system. The disease severity was evaluated by using Osserman’s classification. The diagnosis was confirmed with serological and electrophysiological tests. Statistical analysis was performed using SPSS software. A total of 30 patients with MG were registered for the last five years in Ulaanbaatar. The average annual incidence of MG in Ulaanbaatar was 0.65 per 100,000 populations (95%CI 0.26-1.34), 0.60 in males (95%CI 0.25-1.28), and 0.69 in females (95%CI 0.33-1.46). The cumulative incidence in the study period was 3.2 per 100,000 populations. The ratio of males to females was 1:1,3. The median age for onset of MG was 33 years (ranging from 27 to 46 years); 43.3% of patients had ocular and 56.7% generalized symptoms at the disease onset. Only 23.3% of patients remained with purely ocular symptoms (Osserman I stage). The average incidence of MG between 2015 and 2020 was 6,5 per 1.000.000 population, and the annual incidence was relatively stable. Although ocular and generalized symptoms were observed each in about half of the cases, only one-fourth remained with pure ocular signs at the end of the review period.