Myoclonus-dystonia (M-D) is a rare hyperkinetic movement disorder. Patients generally present with sudden, brief, shock-like jerks called myoclonus and with dystonic symptoms, which are repetitive twisting movements or abnormal postures. Symptoms in M-D mainly affect the upper part of the body and manifest in the first or second decade of life. Alcohol responsiveness of motor symptoms is a common feature in M-D as well as psychiatric abnormalities such as depression and anxiety syndromes. Mutations in the epsilon-sarcoglycan (SGCE) gene have been identified in several M-D patients and families. M-D inheritance follows an autosomal dominant pattern with a reduced penetrance owing to imprinting. The SGCE gene is maternally imprinted meaning that symptoms only manifest upon paternal transmission. The SGCE protein is a ubiquitously expressed transmembrane glycoprotein located at the plasma membrane. The function of SGCE is unknown. Also, M-D pathomechanisms remain largely elusive; there is evidence for involvement of the basal ganglia as well as the cerebellum. This thesis comprises different studies aiming at understanding the role of SGCE in M-D by studying the phenotypic spectrum of SGCE mutations, the SGCE mRNA expression and splicing pattern throughout the human brain and the function of the SGCE protein.

Chapter 1 reviews current knowledge on M-D, its genetic basis and pathophysiology and elaborates the main open questions.
In 43% of reported M-D families dystonia manifested as writer´s cramp. In order to elucidate the phenotypic spectrum of M-D, we investigated whether writer´s cramp as presenting symptom is associated with SGCE mutations in chapter 2. In a large cohort of writer´s cramp patients it became apparent that testing for SGCE mutations should only be considered if writer´s cramp is accompanied by myoclonic jerks. We did not find evidence for involvement of other dystonia genes, DYT1 (TOR1A) and DYT16 (PRKRA), in this patient group. However, intronic and regulatory regions have not been investigated. With the current knowledge, we cannot conclude which dystonia genes need to be tested in patients presenting with isolated writer´s cramp. Course of disease and disease spread in the patient and family members may provide an indication for which genes need to be examined. 
In chapter 3 the need for strict clinical criteria for genetic testing in M-D is discussed and corroborated by studying the phenotypic spectrum of SGCE mutations in a large Dutch M-D cohort. In literature, SGCE mutations were identified in only about 23% of M-D patients. This low frequency can partly be attributed to the lack of screening for large SGCE deletions or insertions and to the lack of comprehensive clinical criteria; many patients that do not present with the definite M-D symptoms are tested for SGCE mutations. Therefore, we applied recently introduced clinical criteria to our large M-D cohort and grouped them into definite, probable, and possible M-D. We showed that only definite patients (early-onset myoclonus and dystonia or isolated myoclonus predominantly in the upper body half and a positive family history for myoclonus and/or dystonia) should be considered for genetic testing. Exceptions should be made for patients with the typical phenotype and a young onset with a negative family history as well as for patients with the typical phenotype, late onset and a positive family history. Several different types of mutations were identified in our definite M-D cohort such as nonsense, missense, and splice site mutations and one multi-exonic deletion. There were no genotype-phenotype correlations; all reported mutations are thought to lead to loss of function explaining this lack of association. In our cohort, large exonic deletions played a minor role, but several exonic rearrangements have been reported in literature, and testing for large exonic deletions should be performed in standard diagnostic settings for definite M-D patients. Alcohol responsiveness, psychiatric abnormalities, and a paternal transmission were common features in our SGCE-associated patient group. Patients showing dystonic features only did not belong to the SGCE mutation spectrum. Despite implementation of the strict inclusion criteria, we did identify a SGCE mutation in only 50% of definite patients, suggesting genetic heterogeneity or unknown factors or mutations affecting the SGCE gene or its expression. 
Chapter 4 addresses an important question in M-D pathomechanisms: why do loss-of-function mutations in the widely expressed SGCE gene lead to exclusively neurological symptoms? We hypothesised that a brain-specific SGCE isoform plays a role in disease pathomechanisms, whereas the function of ubiquitous SGCE is redundant. Therefore, we systematically investigated the tissue-specific SGCE mRNA exon structure, and conducted an ultra-deep amplicon sequencing study. Using this approach we showed that exon 11b is the major brain-specific alternatively spliced exon. Inclusion of this alternatively spliced exon results in an alternative intracellular C-terminus containing a potential binding site for proteins. We showed that transcripts containing exon 11b are differentially expressed throughout the human brain with moderate to low expression in the basal ganglia and notably high levels in the cerebellum. By isoform-specific in situ hybridizations, we confirmed cerebellar expression of the brain-specific SGCE isoform in Purkinje cells and neurons of the dentate nucleus. We hypothesised that loss of function of brain-specific SGCE leads to the specific M-D symptoms and provided evidence for a role of the cerebellum in M-D pathogenesis.
Based on these findings, we proceeded with the investigation of SGCE protein interactions in the cerebellum and focused on interactions with the brain-specific intracellular SGCE C-terminus in chapter 5. We performed immunoprecipitation and isoform-specific pull-down experiments using biotinylated SGCE peptides encompassing the ubiquitous or the brain-specific C-terminus. As SGCE was enriched in synaptosomal fractions, we used this fraction from mouse and human cerebellum for our assays and performed mass spectrometric analysis to identify precipitated proteins. Two binding candidates emerged from immunoprecipitations as well as isoform-specific SGCE peptide pull-down experiments, synapsin I and synapsin II. Both proteins were detected in independent experiments by both approaches, for both species, and only with the brain-specific and not ubiquitous SGCE C-terminal peptide. To validate these two candidates, we performed (1) co-immunoprecipitations in HEK293 cells and in the motor neuron-like cell line NSC-34 and (2) immunofluorescence and confocal microscopy to study the (co-) localisation of both candidates in differentiated NSC-34 cells. We confirmed binding of synapsin I and II with brain-specific and not ubiquitous Sgce by immunoprecipitating the complex using a synapsin antibody in differentiated NSC-34 cells. Synapsin immunoprecipitation in HEK293 cells gave variable results, which implies that both proteins are not direct binding partners, but may be part of the same complex and/or that certain neuronal factors are required for the interaction. Confocal microscopy revealed co-localisation of the synapsins with Sgce at the plasma membrane and tips of dendrites of differentiated NSC-34 cells. Synapsins are involved in the modulation of neurotransmitter release and play a role in synaptic function and plasticity. Synapsins anchor synaptic vesicles to the extracellular matrix and regulate their release upon an action potential. SGCE may play a modulatory role in this pathway and loss of this modulatory function may explain the abnormal, hyperkinetic movements in M-D by “lack of tuning” of neurotransmitter release in the cerebellum.

Studies on the transcriptome have been highly facilitated by next generation sequence technologies. The potential to detect unknown and rare transcripts is one of the advantages of this new technology. In chapter 4 we presented a unique ultra-deep sequencing approach to study tissue-specific alternative splicing events of the SGCE gene. We detected several novel low frequency variants that we further characterised in chapter 7. Most of these variants can be attributed to conventional alternative splicing except for two. These were variations affecting multiple exons and intra-exonic deletions (Figure 1). Both type of variants lack canonical splice sites, but do show short identical sequences of 1 to 8 nucleotides at the junction. We identified these low frequency variants in different genes, tissues, and species. We excluded that they represent an artifact of PCR, reverse transcription, or sequencing. The expression profile of the two novel events showed tissue-specific trends: they were predominantly expressed in the brain and muscle tissue and to a lower extent or even absent for one gene in blood. Intramolecular transcriptional slippage is the most plausible mechanism that can explain these events. In this model, the pre-mRNA molecule dissociates from the DNA template, followed by reannealing, which can occur at the same (intramolecular slippage) or at a different template (intermolecular slippage).1 The position at which reannealing occurs is probably dependent on the presence of short identical sequences. Intramolecular slippage is a process similar to intermolecular slippage. The latter is thought to generate chimeric RNAs. This was the first study reporting intramolecular transcriptional slippage. Here we discuss and speculate about implications of transcriptional slippage.