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Congenital myasthenic syndromes (CMS) are a group of heterogeneous disorders caused by mutations in genes encoding for proteins that are essential for neuromuscular transmission. All CMS share the clinical feature of fatigable muscle weakness. The differential diagnosis of CMS is wide, with a range of diseases going from autoimmune myasthenia gravis to muscle disorders. In this thesis, it was shown that measuring antibodies to clustered acetylcholine receptors (AChRs) by cell-based assay is helpful in the differential diagnosis of CMS. The findings of the current investigations showed that mutations in COL13A1, encoding the Collagen Type XIII α1 chain, were responsible for the symptoms of several patients with previously undefined myasthenias. In addition, this work described the clinical and complementary features of a novel CMS subtype due to mutations in the glycosylation pathway gene GMPPB. Investigations on a novel MUSK missense mutation (p.Ala617Val) uncovered previously unrecognised mechanisms of how levels of MuSK phosphorylation are critical to maintain synaptic structure, and guided suitable treatment for the patient. The study on the clinical and molecular basis of stridor, a novel clinical feature recently identified in patients with DOK7-CMS, prompted the identification of a novel DOK7 isoform, which warrants further investigation to elucidate its role in AChR clustering. Finally, the therapy of patients with severe AChR-deficiency was optimised thanks to a case series study that showed a robust improvement following the addition of β2-adrenergic agonists to their long-term treatment regime that included pyridostigmine.

<p>Myasthenia gravis (MG) is an autoimmune neuromuscular disorder that causes fluctuating muscle weakness. MG may be divided into an ocular form and a generalized form based on the involved muscles. Treatment differs between these different MG forms. The majority (80%) of patients with generalized MG are seropositive for antibodies against the acetylcholine receptor (AChR-Ab). Recently a new antibody was detected against muscle specific tyrosine kinase (MuSK) in about 40% of patients who are AChR-Ab seronegative. A few patients with MuSK-Abs have muscular atrophies, as well as electrophysiological myopathy.</p><p>In this thesis we have characterized MuSK-Ab seropositive [MuSK(+)] patients using clinical parameters, including health-related quality of life (hrQoL), neurophysiology and muscle morphology, and compared them to patients with and without AChR-Abs. The question concerned which factors contribute to their muscle weakness. Additionally, we wanted to determine if single-fiber electromyography (SFEMG) in a limb muscle has any predictive value for generalization of ocular MG. </p><p>Our results suggest that MuSK(+) patients more often have a myopathic electromyography pattern, although this pattern is found also in other immunological subtypes of MG. The myopathic pattern may be associated with the frequently found mitochondrial abnormalities. However, disturbed neuromuscular transmission is the primary cause of muscle weakness in the majority of MuSK(+) patients, as well as in AChR-Ab seropositive patients. The disease-specific hrQoL MG questionnaire was successfully validated into Swedish and these scores correlated with disturbed neuromuscular transmission in a proximal arm muscle. Abnormal SFEMG findings occur also in muscles outside of the facial area in ocular MG, although this is not predictive of subsequent generalization. </p><p>MuSK (+) patients have little or no beneficial effect of acetylcholine esterase inhibitors (AChEI). On the contrary AChEI may produce profound adverse effects. We present the hypothesis that this effect of AChEI is due to abnormal receptor morphology in MuSK(+) patients.</p>

Myasthenia gravis (MG) is an autoimmune neuromuscular disorder that causes fluctuating muscle weakness. MG may be divided into an ocular form and a generalized form based on the involved muscles. Treatment differs between these different MG forms. The majority (80%) of patients with generalized MG are seropositive for antibodies against the acetylcholine receptor (AChR-Ab). Recently a new antibody was detected against muscle specific tyrosine kinase (MuSK) in about 40% of patients who are AChR-Ab seronegative. A few patients with MuSK-Abs have muscular atrophies, as well as electrophysiological myopathy. In this thesis we have characterized MuSK-Ab seropositive [MuSK(+)] patients using clinical parameters, including health-related quality of life (hrQoL), neurophysiology and muscle morphology, and compared them to patients with and without AChR-Abs. The question concerned which factors contribute to their muscle weakness. Additionally, we wanted to determine if single-fiber electromyography (SFEMG) in a limb muscle has any predictive value for generalization of ocular MG. Our results suggest that MuSK(+) patients more often have a myopathic electromyography pattern, although this pattern is found also in other immunological subtypes of MG. The myopathic pattern may be associated with the frequently found mitochondrial abnormalities. However, disturbed neuromuscular transmission is the primary cause of muscle weakness in the majority of MuSK(+) patients, as well as in AChR-Ab seropositive patients. The disease-specific hrQoL MG questionnaire was successfully validated into Swedish and these scores correlated with disturbed neuromuscular transmission in a proximal arm muscle. Abnormal SFEMG findings occur also in muscles outside of the facial area in ocular MG, although this is not predictive of subsequent generalization. MuSK (+) patients have little or no beneficial effect of acetylcholine esterase inhibitors (AChEI). On the contrary AChEI may produce profound adverse effects. We present the hypothesis that this effect of AChEI is due to abnormal receptor morphology in MuSK(+) patients.

Die Fetale Akinesia Deformation Sequence (FADS) umfasst ein breites klinisches Spektrum. Dieses reicht von Tot- und Fehlgeburten, fetalen Ödemen bis hin zu Kontrakturen, Pterygien und Atemschwäche. Die Ätiologie der FADS ist sehr heterogen. Der Fokus dieser Forschungsarbeit lag in der Mutationsanalyse in Genen des Acetylcholin-Rezeptor-Pathways. Bekannt war das homozygote missense und nonsense Mutationen in den Genen der fetalen Untereinheit CHRNG des Rezeptors mit dem klinischen Bild des multiple Pterygien-Syndrom (MPS) und Letalen multiplen Pterygien-Syndrom (LMPS) einhergehen. Missense Mutationen in weiteren Genen des AChR-Komplexes präsentieren sich klinisch als Congenitales Myasthenes Syndrom (CMS). Vermutet, aber nicht bewiesen war das homozygote nonsense Mutationen in diesen weiteren Genen letal verlaufen und ursächlich für das letale Pterygiensyndrom sein können. Wir konnten diese Hypothese bestätigen.:1. Einführung in das Thema 1.1. Prävalenz und Relevanz fetaler Bewegungsstörungen und angeborener Kontrakturen 1.2. Das klinische Bild 1.3. Ursachen verminderter fetaler Bewegung 1.4. Der nicotinerge Acetylcholinrezeptor 1.5. Mutationssuche in den Genen der α1-, β1- und δ-Untereinheit (CHRNA1, CHRNB1 und CHRND) sowie in dem Rezeptor assoziierten RAPSN-Gen 2. Publikation 3. Zusammenfassung der Arbeit Literaturverzeichnis Anlagen Darstellung des eigenen wissenschaftlichen Beitrags Erklärung über die eigenständige Abfassung der Arbeit

Congenital myasthenic syndrome (CMS) and myasthenia gravis (MG) are, respectively, inherited or autoimmunological disorders caused by aberrant neuromuscular transmission, which manifests as fatiguable muscle weakness. A novel subtype of CMS, resulting from mutations in GFPT1 and characterised by a limb girdle pattern of muscle weakness, has been described. The gene encodes L glutamine:D fructose-6-phosphate amidotransferase 1 (GFAT1) – a key rate limiting enzyme in the hexosamine biosynthetic pathway, providing building blocks for glycosylation of proteins and lipids. The research focused on the molecular bases of the CMS resulting from mutations in the ubiquitously expressed gene, but with symptoms largely restricted to the neuromuscular junction (NMJ). The work has established a link between the NMJ and GFPT1 CMS by demonstrating that the AChR cell surface is decreased in GFPT1 patient muscle cells and in GFPT1-silenced cell lines. The decrease is likely to be caused by reduced steady-state levels of individual AChR α, δ and ε, but not β, subunits. To optimise treatment for myasthenic disorders, a comparative in vivo trial of therapy with pyridostigmine bromide and salbutamol sulphate, and pyridostigmine bromide alone, was conducted. Supplementation of the AChE inhibitor-based therapy with the β2-adrenergic receptor agonist had a beneficial effect. This offers promise for more effective treatments for CMS and MG affected individuals. Molecular causes of MG were also investigated. The search for novel antibody targets was conducted with the use of a designed cell-based assay for the detection of anti COLQ autoimmunoglobulins in MG patient sera. The antibodies were detected in 24 out of 418 analysed samples, but their pathogenicity has not been determined.

RNA interference (RNAi) is a post transcriptional gene silencing mechanism that allows potent and specific silencing of cognate mRNA transcripts. Selective silencing can be used to dissect complex polygenic diseases, elucidate the function of known genes and provide a tool for genetic therapy. Its use in the case of dominant inherited disorders including disorders of the central nervous system, depends on its ability to confer single nucleotide discrimination between normal and mutant gene alleles. In this thesis the ability of RNAi effector molecules to provide single nucleotide specificity was examined by targeting two dominant inherited mutations of the acetylcholine receptor that cause slow-channel syndrome. Allele-specific silencing was achieved for one mutation. The other mutation was also silenced but not in an allele specific way despite employing known techniques for increasing single-nucleotide specificity. The model used in this thesis is the congenital myasthenia slow-channel syndrome. This is a dominant inherited disorder of the neuromuscular junction which is both well-characterised and more readily accessible compared to the central nervous system, thus provides a prototype for development of allele-specific RNAi therapeutics. Here we describe a new transgenic animal model of the slow-channel syndrome and show good representation of the human disorder. The need for defining the characteristics that determine the effectiveness and the specificity of RNAi effectors at single-nucleotide level, along with the future uses of the newly described animal model are discussed.