[Principles of translational gene therapy for neuromuscular diseases]. / Prinzipien der translationalen Gentherapie für neuromuskuläre Erkrankungen.

BACKGROUND: In recent years the theoretical hope has become reality and the first hereditary neuromuscular diseases have become causally treatable. Neuromuscular diseases have thus become the pacemaker of this form of therapy for the whole of neurology. AIMS: This article describes the principles of precision gene therapy for neurogenetic diseases using examples of neuromuscular diseases. DISCUSSION: Various strategies of gene therapy have become established and are being tested in preclinical and clinical trials and evaluated as approved forms for long-term efficacy. The aim of every gene therapy is the modification or introduction of the target gene with initiation of a degradation of dysfunctional proteins. Various techniques, such as gene transfer, gene substitution or gene editing in vivo and ex vivo are now usable. For example, a modification of the pre-mRNA using antisense oligonucleotides or RNA interference (siRNA) can be used for exon skipping. An initiation of gene expression to produce the target protein can be based on a modification of the DNA by means of gene replacement, cell-based therapy (iPS cells), regulation by compensatory proteins or pharmacological treatment with so-called small molecules. Each method has advantages and complex disadvantages that must be individually evaluated. Phenotypic peculiarities of a rare disease often only become apparent through specific translational therapy. It is already becoming obvious that a very early point in timing of gene therapy is probably the most effective. Newborn screening is therefore gaining additional importance as early diagnosis can achieve the best possible success of therapies, possibly even preventively.

Dyslexia and cognitive impairment in adult patients with myotonic dystrophy type 1: a clinical prospective analysis.

BACKGROUND: Cognitive impairments in patients with myotonic dystrophy type 1 (DM1) have often been described, however, there are only few studies differentiating between partial performance disorders and mental retardation in common. This study focused on the evaluation of reading performance and the frequency of dyslexia in adult DM1 patients. METHODS: We performed a prospective cohort study including genetically confirmed adult DM1 patients registered in the DM registry of Germany or the internal database of the Friedrich-Baur-Institute, Munich, Germany. For the assessment of the patients' reading and spelling performance, we used the standardized and validated test 'Salzburger Lese- und Rechtschreibtest' (SLRT II). The 'CFT-20 R Grundintelligenztest Skala 2' in revised ("R") version (CFT 20-R), determining the intelligence level, was appropriate to differentiate between dyslexia and general mental retardation. The diagnosis of dyslexia, the combined reading and spelling disorder, was based on the guidelines for diagnosis and therapy of children and adolescents with dyslexia 2015 (S3-guideline) providing (1) the criterion of the divergence from age level and (2) the criterion of IQ-divergence. RESULTS: Fifty-seven DM1 patients participated in our study. Evaluating the reading performance, 16 patients fulfilled the divergence criteria of the age level and 2 patients the IQ-divergence criteria. In total, the diagnosis of a reading disorder was given in 18 DM1 patients (31.6 %). In 11 out of these 18 patients with a reading disorder, a relevant impairment of spelling performance was observed with at least three spelling errors. As there are no normative values for adults in spelling performance, we assume a combined reading disorder and dyslexia, in those 11 DM1 patients (19.3 %). Regarding the separate analyses of the test procedures, in the SLRT II the performance was below average in 40.4 % of all patients for 'word reading' and in 61.4 % of all patients for 'pseudoword reading'. There was a significant positive correlation between the CTG expansion size and a reading disorder (p=0.027). The average IQ of 17 examined DM1 patients was in the lower normal range (86.1 ± 19.1). 54.5 % of patients with reading disorder had a normal IQ. CONCLUSION: The calculated prevalence of dyslexia in the DM1 study cohort was 19.3 % and thus considerably increased compared to the normal German population. As dyslexia is not equivalent to a general cognitive impairment, it is important not to miss dyslexic features in cognitive inconspicuous DM1 patients. Case-by-case one should consider a differential diagnostic approach, as individualized therapies can be offered to support dyslexic patients in their performance.

An integrative correlation of myopathology, phenotype and genotype in late onset Pompe disease.

AIMS: Pompe disease is caused by pathogenic mutations in the alpha 1,4-glucosidase (GAA) gene and in patients with late onset Pome disease (LOPD), genotype-phenotype correlations are unpredictable. Skeletal muscle pathology includes glycogen accumulation and altered autophagy of various degrees. A correlation of the muscle morphology with clinical features and the genetic background in GAA may contribute to the understanding of the phenotypic variability. METHODS: Muscle biopsies taken before enzyme replacement therapy were analysed from 53 patients with LOPD. On resin sections, glycogen accumulation, fibrosis, autophagic vacuoles and the degree of muscle damage (morphology-score) were analysed and the results were compared with clinical findings. Additional autophagy markers microtubule-associated protein 1A/1B-light chain 3, p62 and Bcl2-associated athanogene 3 were analysed on cryosections from 22 LOPD biopsies. RESULTS: The myopathology showed a high variability with, in most patients, a moderate glycogen accumulation and a low morphology-score. High morphology-scores were associated with increased fibrosis and autophagy highlighting the role of autophagy in severe stages of skeletal muscle damage. The morphology-score did not correlate with the patient's age at biopsy, disease duration, nor with the residual GAA enzyme activity or creatine-kinase levels. In 37 patients with LOPD, genetic analysis identified the most frequent mutation, c.-32-13T>G, in 95%, most commonly in combination with c.525delT (19%). No significant correlation was found between the different GAA genotypes and muscle morphology type. CONCLUSIONS: Muscle morphology in LOPD patients shows a high variability with, in most cases, moderate pathology. Increased pathology is associated with more fibrosis and autophagy.

[Innovative therapeutic approaches for hereditary neuromuscular diseases]. / Innovative Therapieansätze bei hereditären neuromuskulären Erkrankungen.

Advances in the understanding of the genetic mechanisms and pathophysiology of neuromuscular diseases have recently led to the development of new, innovative and often mutation-specific therapeutic approaches. Methods used include splicing modification by antisense oligonucleotides, read-through of premature stopcodons, use of viral vectors to introduce genetic information, or optimizing the effectiveness of enzyme replacement therapies. The first drugs have already been approved for the treatment of Duchenne muscular dystrophy and spinal muscular atrophy. For other diseases, such as myotubular myopathy, myotonic dystrophy, facioscapulohumeral muscular dystrophy and Pompe disease, new promising approaches are in preclinical or clinical development. As these are rare diseases with a broad spectrum of clinical severity, drug approval is often based on a limited amount of evidence. Therefore, systematic follow-up in the postmarketing period is particularly important to assess the safety and efficacy of these new and often high-priced orphan drugs.

A zebrafish model for FHL1-opathy reveals loss-of-function effects of human FHL1 mutations.

Missense mutations in the four and a half LIM domain 1 (FHL1) gene were found to cause X-linked inherited myopathies of both skeletal and heart muscles. However, the mechanisms by which FHL1 mutations impact on FHL1 function and lead to alteration of muscle structure and function have not been deciphered yet. We generated here by Morpholino-modified antisense oligonucleotide-mediated gene knockdown fHL1-deficient zebrafish embryos. Similar to the human situation, fhl1a-morphants zebrafish displayed severe skeletal and heart muscle myopathy. Whereas ectopic expression of wild-type FHL1 (FHL1 wt) suppressed both skeletal and heart muscle myopathy in fhl1a-morphants zebrafish, overexpression of the FHL1-opathy associated human mutations FHL1-H123Y, FHL1-C132F or FHL1-C224W did not rescue skeletal and heart muscle myopathy in fhl1a-morphants. Overexpression of FHL1-H123Y, FHL1-C132F or FHL1-C224W in wild-type zebrafish did not induce myopathy in a dominant-negative mode. Altogether these results indicate that FHL1 mutations found to cause X-linked FHL1-opathies in humans consistently lead to severely impaired FHL1 function.

Falls and resulting fractures in Myotonic Dystrophy: Results from a multinational retrospective survey.

Myotonic Dystrophy type 1 multisystem involvement leads to functional impairment with an increased risk of falling. This multinational study estimates the prevalence of falls and fall-associated fractures. A web-based survey among disease-specific registries (Germany, UK and The Netherlands) was carried out among DM1 ambulant adults with a total of 573 responses retrieved. Results provided a risk ratio estimation of 30%-72% for falls and of 11%-17% for associated fractures. There was no significant difference for falls between male and female, but there was for fall-related fractures with a higher prevalence in women. Balance and leg weakness were the most commonly reported causes for falling. This study is based on a voluntary retrospective survey with naturally inherent limitations; however, the sample size allows for robust comparisons. The estimated risk of falls in this cohort with a mean age of 46 years compares to a previous estimation for a healthy population of over 65 years of age. These results suggest a premature-ageing DM1 phenotype with an increased risk of falling depending on age and disease severity that, so far, might have been underestimated. This may have clinical implications for the development of care guidelines and when testing new interventions in this population.

Early-Onset Myopathies: Clinical Findings, Prevalence of Subgroups and Diagnostic Approach in a Single Neuromuscular Referral Center in Germany.

BACKGROUND: Early-onset myopathies are a heterogeneous group of neuromuscular diseases with broad clinical, genetic and histopathological overlap. The diagnostic approach has considerably changed since high throughput genetic methods (next generation sequencing, NGS) became available. OBJECTIVE: We present diagnostic subgroups in a single neuromuscular referral center and describe an algorithm for the diagnostic work-up. METHODS: The diagnostic approach of 98 index patients was retrospectively analysed. In 56 cases targeted sequencing of a known gene was performed, in 44 patients NGS was performed using large muscle specific panels, and in 12 individuals whole exome sequencing (WES) was undertaken. One patient was diagnosed via array CGH. Clinical features of all patients are provided. RESULTS: The final diagnosis could be found in 63 out of 98 patients (64%) with molecular genetic analysis. In 55% targeted gene sequencing could establish the genetic diagnosis. However, this rate largely depended on the presence of distinct histological or clinical features. NGS (large myopathy-related panels and WES) revealed genetic diagnosis in 58.5% (52% and 67%, respectively). The genes detected by WES in our cohort of patients were all covered by the panels. Based on our findings we propose an algorithm for a practical diagnostic approach.Prevalences:MTM1- and LAMA2-patients are the two biggest subgroups, followed by SEPN1-, RYR1- and Collagen VI-related diseases. 31% of genetically confirmed cases represents a group with overlap between "congenital myopathies (CM)" and "congenital muscular dystrophies (CMD)". In 36% of the patients a specific genetic diagnosis could not be assigned. CONCLUSIONS: A final diagnosis can be confirmed by high throughput genetic analysis in 58.5% of the cases, which is a higher rate than reported in the literature for muscle biopsy and should in many cases be considered as a first diagnostic tool. NGS cannot replace neuromuscular expertise and a close discussion with the geneticists on NGS is mandatory. Targeted candidate gene sequencing still plays a role in selected cases with highly suspicious clinical or histological features. There is a relevant clinical and genetic overlap between the entities CM and CMD.

European consensus for starting and stopping enzyme replacement therapy in adult patients with Pompe disease: a 10-year experience.

BACKGROUND AND PURPOSE: Pompe disease is a rare inheritable muscle disorder for which enzyme replacement therapy (ERT) has been available since 2006. Uniform criteria for starting and stopping ERT in adult patients were developed and reported here. METHODS: Three consensus meetings were organized through the European Pompe Consortium, a network of experts from 11 European countries in the field of Pompe disease. A systematic review of the literature was undertaken to determine the effectiveness of ERT in adult patients on a range of clinical outcome measures and quality of life. A narrative synthesis is presented. RESULTS: Consensus was reached on how the diagnosis of Pompe disease should be confirmed, when treatment should be started, reasons for stopping treatment and the use of ERT during pregnancy. This was based on expert opinion and supported by the literature. One clinical trial and 43 observational studies, covering a total of 586 individual adult patients, provided evidence of a beneficial effect of ERT at group level. At individual patient level, the response to treatment varied, but factors associated with a patient's response to ERT were not described in many studies. Eleven observational studies focused on more severely affected patients, suggesting that ERT can also be beneficial in these patients. There are no studies on the effects of ERT in pre-symptomatic patients. CONCLUSIONS: This is the first European consensus recommendation for starting and stopping ERT in adult patients with Pompe disease, based on the extensive experience of experts from different countries.

[Educational and Professional Qualifications of Adults With Myotonic Dystrophies - A Misleading Perception by the Myopathic Face?]. / Ausbildung und berufliche Qualifikation von Erwachsenen mit Myotonen Dystrophien - eine fehlgeleitete Wahrnehmung durch die Facies myopathica?

BACKGROUND: Myotonic dystrophies types 1 and 2 (DM1 / DM2) are the most frequent inherited progressive, segmental progeroid, multisystemic neuromuscular diseases in adulthood. The executive impairment is one of the key disease features. The myopathic face triggers the general perception of DM1 patients being associated with a low educational level. METHODS: We used a standardized questionnaire to evaluate educational levels in adults with genetically confirmed DM1 and DM2 in comparison to data of the general population. Investigated topics included the level of education, e. g. the highest university degree aquired. RESULTS: Out of a total cohort of 546 DM patients, 125 DM1 and 156 DM2 patients (51 %) participated in this study. There was no statistically significant difference between the two collectives as far as high school levels are concerned. 50.4 % of DM1 and 48.3 % of DM2 patients obtained the higher education entrance qualification compared to 29.6 % of the normal German population. However, there were significant differences between the two collectives in "spelling problems" (DM1 cohort: p = 0.039), "difficulty in mental arithmetic" (p = 0.043), and classification of patients "with learning difficulties" (p = 0.012). DISCUSSION: Misled by a myopathic face, many physicians associate myotonic dystrophy with cognitive deficiency. Based on our study, the minimal deviation between DM1 and DM2 and the normal German population indicates that the multisystemic disease does not significantly influence the maximum attainable level of education in adults with DM1. CONCLUSION: In summary, physicians should be aware that the general educational levels are rather normal in patients with myotonic dystrophy type 1 and rethink their perception of DM1 patients.

New insights into the protein aggregation pathology in myotilinopathy by combined proteomic and immunolocalization analyses.

INTRODUCTION: Myofibrillar myopathies are characterized by progressive muscle weakness and impressive abnormal protein aggregation in muscle fibers. In about 10 % of patients, the disease is caused by mutations in the MYOT gene encoding myotilin. The aim of our study was to decipher the composition of protein deposits in myotilinopathy to get new information about aggregate pathology. RESULTS: Skeletal muscle samples from 15 myotilinopathy patients were included in the study. Aggregate and control samples were collected from muscle sections by laser microdissection and subsequently analyzed by a highly sensitive proteomic approach that enables a relative protein quantification. In total 1002 different proteins were detected. Seventy-six proteins showed a significant over-representation in aggregate samples including 66 newly identified aggregate proteins. Z-disc-associated proteins were the most abundant aggregate components, followed by sarcolemmal and extracellular matrix proteins, proteins involved in protein quality control and degradation, and proteins with a function in actin dynamics or cytoskeletal transport. Forty over-represented proteins were evaluated by immunolocalization studies. These analyses validated our mass spectrometric data and revealed different regions of protein accumulation in abnormal muscle fibers. Comparison of data from our proteomic analysis in myotilinopathy with findings in other myofibrillar myopathy subtypes indicates a characteristic basic pattern of aggregate composition and resulted in identification of a highly sensitive and specific diagnostic marker for myotilinopathy. CONCLUSIONS: Our findings i) indicate that main protein components of aggregates belong to a network of interacting proteins, ii) provide new insights into the complex regulation of protein degradation in myotilinopathy that may be relevant for new treatment strategies, iii) imply a combination of a toxic gain-of-function leading to myotilin-positive protein aggregates and a loss-of-function caused by a shift in subcellular distribution with a deficiency of myotilin at Z-discs that impairs the integrity of myofibrils, and iv) demonstrate that proteomic analysis can be helpful in differential diagnosis of protein aggregate myopathies.

"Orbiting around" the orbital myositis: clinical features, differential diagnosis and therapy.

Orbital myositis (OM) is a rare disease whose clinical heterogeneity and different treatment options represent a diagnostic and therapeutic challenge. We aim to review the state of knowledge on OM, also describing a cohort of patients diagnosed in our centre, to highlight some remarkable clinical features. A literature review was conducted in PubMed and Medline databases. The herein described cohort is composed of seven OM patients, diagnosed according to clinical, laboratory and neuroradiological features, whose clinical data were retrospectively analysed. OM is a non-infectious, inflammatory process primarily involving extraocular eye-muscles. It typically presents as an acute to sub-acute, painful ophthalmoplegia with signs of ocular inflammation, but atypical cases without pain or with a chronic progression have been described. The wide range of OM mimicking diseases make a prompt diagnosis challenging but orbit MRI provides valuable clues for differential diagnosis. Timely treatment is greatly important as OM promptly responds to steroids; nevertheless, partial recovery or relapses often occur. In refractory, recurrent or steroid-intolerant cases other therapeutic options (radiotherapy, immunosuppressants, immunoglobulins) can be adopted, but the most effective therapeutic management is yet to be established. In this review, we provide a detailed clinical description of OM, considering the main differential diagnoses and suggesting the most useful investigations. In light of the currently available data on therapy efficacy, we propose a therapeutic algorithm that may guide neurologists in OM patients' management.

[The Spectrum of Neuromyotonia: Clinics, Therapy and Outcome]. / Das Spektrum der Neuromyotonie: Klinik, Therapie und Langzeitverlauf.

BACKGROUND: Neuromyotonia (NM), Isaacs-Zschoke-Mertens syndrome or continuous muscle fiber activity (CMFA), is a rare condition associated with VGKC-antibodies. Clinically, fasciculations, myokymias, muscle stiffness and a myotonic appearance of movements after contraction are typical findings. In addition, CNS-symptoms vary from moderate fatigue, poor concentration and autonomic symptoms to severe encephalopathy in Morvan's syndrome. In electromyography, spontaneous irregular discharges can be found frequently with typical di-, tri- or multiplet single motor unit discharges. In up to 60 %, serum antibodies against VGKC-complexes can be detected. METHODS: Patients with neuromyotonia were evaluated for clinical symptoms, response to treatment and outcome over a five-year period of follow-up. For evaluation, we used video recording of clinical symptoms, electroneurography, electromyography and myosonography as well as immunological tests (VGKC-complex antibody including CASPR2 and IGL1). Furthermore, cerebral fluid and screening for neoplasias were done. Patients with evidence for neuropathy, myopathy or motor neuron disease, even if diagnosed in the follow-up, were excluded. RESULTS: In 3 of 5 patients, neuromyotonia was diagnosed by electromyography and positive VGKC antibodies. In two patients, diagnosis was based on typical clinical symptoms and electromyographical changes. Anticonvulsants (carbamazepine) for symptomatic treatment were moderately effective in four patients; treatment with i. v. immunoglobulins was highly successful in one patient with high positive VGKC-complex antibody titers. In one patient with low-titer VGKC antibodies, neither anticonvulsants nor i. v. immunoglobulins nor prednisone was a successful treatment. CONCLUSIONS: Neuromyotonia is a rare, treatable condition. However, due to the high variability of symptoms, response to therapy and outcome, neuromyotonia treatment needs to be highly individualized.

Sleep-related symptoms and sleep-disordered breathing in adult Pompe disease.

BACKGROUND AND PURPOSE: Respiratory muscle weakness is the major cause of early death in patients with adult Pompe disease. It first manifests as nocturnal hypercapnia, eventually leading to sleep disruption. Sleep-related symptoms along with motor performance, forced vital capacity (FVC) and respiratory symptoms were investigated in 65 adult patients with Pompe disease. METHODS: Patients answered the Pittsburgh Sleep Quality Index (PSQI), the Epworth Sleepiness Scale, the Fatigue Severity Scale, the Rotterdam Nine-item Handicap Scale, the SF-36 health-related quality of life questionnaire, and a respiratory symptom questionnaire. In all patients, the 6-min walk test was performed and FVC was obtained. Polysomnography and oxycapnometry results were available in 31 patients. RESULTS: Sixty patients received enzyme replacement therapy, and 32 individuals were on home ventilatory support. Reduced sleep quality was highly prevalent (PSQI > 5; 43.1%) and correlated with both excessive daytime sleepiness (Epworth Sleepiness Scale > 10; 24.6%) and fatigue (Fatigue Severity Scale > 4; 72.3%). The SF-36 health-related quality of life questionnaire was reduced in the physical domains, and was inversely correlated with sleep quality, FVC and motor performance. In 11 out of 17 non-ventilated patients with polysomnography records, sleep-disordered breathing was present, and duration of nocturnal oxygen desaturation (SaO2 < 90%) was significantly correlated to the PSQI global score. CONCLUSIONS: In adult Pompe disease, sleep disturbances are a common cause of excessive daytime sleepiness and fatigue. Sleep-related symptoms may be indicative of respiratory muscle weakness and should give rise to further work-up of sleep-disordered breathing.

Muscle ultrasound in classic infantile and adult Pompe disease: a useful screening tool in adults but not in infants.

A cohort of 4 infantile and 15 adult Pompe patients has been investigated regarding correlation between strength and ultrasound of skeletal musculature. In adults, muscle ultrasound is useful to assess clinical and subclinical involvement of muscles. In this study, visible sonographic changes were found in every clinically affected muscle, using a modified Heckmatt scale. In some muscles morphologic changes preceded weakness. Regarding the anatomical pattern of involvement, our findings do not support the hypothesis of a specific pattern with a higher vulnerability of vastus intermedius than rectus femoris, which has been postulated before. A frequent sparing of triceps brachii could be confirmed. Intramuscular abnormalities occurred in a focal, a diffuse, or an intermediate pattern, with characteristics of both. In contrast to muscular dystrophies, bone echogencity was not markedly decreased in Pompe disease even in an advanced stage. In infants, muscle ultrasound showed no distinct pathology even in clinically severely affected children and should not be used as a screening method for infantile Pompe disease.

[Diagnosis and therapy of late onset Pompe disease]. / Diagnose und Therapie des Late-onset-Morbus-Pompe.

As Pompe disease glycogen storage disease type 2 with a severely reduced life expectancy is now a treatable disorder, accurate diagnostic procedures and evidence-based indications for therapy are mandatory. We screened the literature for consensus reports and published trial data of late-onset Pompe disease. These data were summarized in a Delphi consensus method approach. The clinical suspicion of late-onset Pompe disease should be substantiated by the validated dry blood spot test measurement for acid α-glucosidase activity. Alternatively, enzyme activity analysis in lymphocytes is also feasible. Glucosidase α gene sequencing for verifying the diagnosis is recommended. A muscle biopsy including measurements of acid α-glucosidase activity and glycogen concentration is warranted for differential diagnosis in selected cases. The confirmed diagnosis should lead to a multidisciplinary treatment approach, possibly including enzyme replacement therapy.

Pain in adult patients with Pompe disease: a cross-sectional survey.

BACKGROUND: Pompe disease is a rare hereditary metabolic myopathy caused by a deficiency of acid-α-glucosidase. We investigated the presence and severity of pain and its interference with daily activities in a large group of adults with Pompe disease, who we compared with an age-matched control group. METHODS: Data were collected in a cross-sectional survey in Germany and The Netherlands. Pain was assessed using the short-form brief pain inventory (BPI). Patients also completed the Short Form-36 item (SF-36v2), the Hospital Anxiety and Depression Scale (HADS) and the Rotterdam Handicap Scale (RHS). RESULTS: Forty-five percent of the 124 adult Pompe patients reported having had pain in the previous 24h, against 27% of the 111 controls (p=0.004). The median pain severity score in Pompe patients reporting pain was 3.1 (on a scale from 0 to 10), indicating mild pain; against 2.6 amongst controls (p=0.06). The median score of pain interference with daily activities in patients who reported pain was 3.3, against 1.3 in controls (p=0.001). Relative to patients without pain, those with pain had lower RHS scores (p=0.02), lower SF-36 Physical and Mental component summary scores (p<0.001 and p=0.049), and higher levels of depression and anxiety (p=0.005 and p=0.003). CONCLUSIONS: To date, this is one of the largest studies on pain in a specific neuromuscular disorder. Nearly one in two Pompe patients had experienced pain in the previous 24h. Although pain severity and its interference with daily life were mild, pain was related to a reduced quality of life, less participation in daily life, and greater depression and anxiety. Its management should therefore be seen as part of clinical practice involving Pompe patients.