Pregnancy outcome in Charcot-Marie-Tooth disease: results of the CMT-NET cohort study in Germany.

BACKGROUND AND PURPOSE: Systematic research on the effect of Charcot-Marie-Tooth (CMT) disease on the outcome of pregnancy and conversely the effect of pregnancy on neuropathy is still sparse. METHODS: A clinical cohort study and cross-sectional study within the German CMT-NET was conducted between 2016 and 2019. Inclusion criteria were a confirmed diagnosis of CMT and at least one completed pregnancy after 1990. All participants agreed to fill in questionnaires and have their medical files reviewed. RESULTS: The study group comprised 54 women with a total of 98 pregnancies. The mean age at onset of CMT disease was 12.6 years (range 0-37 years). Fifty (92%) patients had autosomal dominant CMT; two patients each (4%) had X-linked and autosomal recessive CMT. Forty patients (74%) had a PMP22 gene duplication (CMT1A). Obstetric complications did not differ significantly from a German reference population, neither in the whole group nor in the CMT1A group. Overall there was no increased newborn morbidity and mortality. About one-third of patients reported exacerbation of CMT disease in or after pregnancy. No adverse effects of anaesthesia were reported. Most participants stressed a positive attitude and awareness of challenges associated with pregnancy. Important issues were assistance and support in caring for the family. DISCUSSION: In line with findings from our previous study undertaken in the 1990s, there were no increased complication rates for pregnancy and delivery. These results are reassuring for the vast majority of CMT patients and are important for family planning and clinical care.

[Risk genes in myopathies and mitochondrial diseases]. / Risikogene bei Myopathien und mitochondrialen Erkrankungen.

Myopathies and mitochondrial diseases pose a major challenge in diagnosis due to the multitude of different entities and - in the case of mitochondriopathies - the possible involvement of multiple organs. Furthermore, there is broad clinical variability within particular diseases; patients with hereditary myopathy, for example, can show great phenotypic variability despite identical genetic defects. In addition to environmental factors, gender-specific influences, and the degree of heteroplasmy in mitochondrial diseases, the existence of disease-modifying genes has long been assumed as an explanation. In recent years, risk genes, which can influence the course of disease, have been identified for some myopathies and mitochondrial diseases. The precise role of these disease-modifying genes in the pathogenesis of the diseases is largely unexplained and requires further research.

Cyclosporine A treatment for Ullrich congenital muscular dystrophy: a cellular study of mitochondrial dysfunction and its rescue.

Mutations in COL6A1, COL6A2 and COL6A3, the genes which encode the extra-cellular matrix component collagen VI, lead to Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD). Although the Col6a1(-/-) null mouse has an extremely mild neuromuscular phenotype, a mitochondrial defect has been demonstrated, linked to dysregulation of the mitochondrial permeability transition pore (PTP) opening. This finding has been replicated in UCMD muscle cells in culture, providing justification for a clinical trial using cyclosporine A, an inhibitor of PTP opening. We investigated whether PTP dysregulation could be detected in UCMD fibroblasts (the predominant source of muscle collagen VI), in myoblast cells from patients with other diseases and its response to rescue agents other than collagen VI. Although we confirm the presence of PTP dysregulation in muscle-derived cultures from two UCMD patients, fibroblasts from the same patients and the majority of fibroblasts from other well-characterized UCMD patients behave normally. PTP dysregulation is found in limb girdle muscular dystrophy (LGMD) type 2B myoblasts but not in myoblasts from patients with Bethlem myopathy, merosin-deficient congenital muscular dystrophy, LGMD2A, Duchenne muscular dystrophy and Leigh syndrome. In addition to rescue by cyclosporine A and collagen VI, this cellular phenotype was also rescued by other extra-cellular matrix constituents (laminin and collagen I). As the muscle derived cultures demonstrating PTP dysregulation shared poor growth in culture and lack of desmin labelling, we believe that PTP dysregulation may be a particular characteristic of the state of these cells in culture and is not specific to the collagen VI defect, and can in any case be rescued by a range of extra-cellular matrix components. Further work is needed on the relationship of PTP dysregulation with UCMD pathology.

Measuring Outcomes in Adults with Spinal Muscular Atrophy - Challenges and Future Directions - Meeting Report.

Spinal muscular atrophy (SMA) is a progressive autosomal recessive motor neuron disease which affects 1 in 6,000-10,000 live births, caused by loss of the survival motor neuron 1 gene (SMN1). A major focus of therapeutic developments has been on increasing the full-length SMN protein by increasing the inclusion of exon 7 in SMN2 transcripts, enhancing SMN2 gene expression, stabilizing the SMN protein or replacing the SMN1 gene.In June 2017, FDA and EMA have approved the antisense oligonucleotide Nusinersen as the first treatment for all SMA subtypes without age restriction. While prominent treatment effects have been observed in the earlier stages of the disease and in patients up to 15 years of age, there is only limited data from clinical trials in adult SMA patients. First real-world data from neuromuscular clinical centers suggest a therapeutic benefit of nusinersen with a favourable safety profile also in adult SMA patients: in several cases, relevant improvements of motor function is achieved, which might lead to enhanced autonomy in daily life activities and improved quality of life. Systematic follow-up of the motor status with validated instruments is crucial for an adequate monitoring of the therapeutic effects but most of the widely used scales and scores have been developed and evaluated for the pediatric population only. International neuromuscular experts have met in Frankfurt/Main, Germany in May 2019 to discuss relevant aspects of the diagnostic pathway and patient management in adult SMA. The recommendations and challenges in this patient population are discussed.

Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.

Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific nucleases offers new options for restoring the DMD reading frame, resulting in expression of a shortened but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of DMD (DMDΔ52), as well as in a corresponding patient-derived induced pluripotent stem cell model. In DMDΔ52 pigs1, intramuscular injection of adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 (ref. 2) and a pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) induced expression of a shortened dystrophin (DMDΔ51-52) and improved skeletal muscle function. Moreover, systemic application of AAV9-Cas9-gE51 led to widespread dystrophin expression in muscle, including diaphragm and heart, prolonging survival and reducing arrhythmogenic vulnerability. Similarly, in induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a patient lacking DMDΔ52, AAV6-Cas9-g51-mediated excision of exon 51 restored dystrophin expression and amelioreate skeletal myotube formation as well as abnormal cardiomyocyte Ca2+ handling and arrhythmogenic susceptibility. The ability of Cas9-mediated exon excision to improve DMD pathology in these translational models paves the way for new treatment approaches in patients with this devastating disease.

No muscle involvement in myoclonus-dystonia caused by epsilon-sarcoglycan gene mutations.

Mutations in the epsilon-sarcoglycan gene (SGCE) can cause autosomal dominant inherited myoclonus-dystonia (M-D). Defects in other sarcoglycans; alpha-, beta-, gamma-, and delta can cause autosomal recessive inherited limb girdle muscular dystrophies. epsilon- and alpha-sarcoglycans are very homologous and may substitute for one-another in different tissues. We therefore investigated whether mutations in SGCE also cause abnormalities of skeletal and myocardial muscle. Six patients with clinically and genetically verified M-D and no signs of limb-girdle muscular dystrophy were included. Skeletal muscle biopsies were obtained from all patients, and endomyocardial muscle biopsy from one of the patients. Morphological and immunohistological investigations were performed and compared with controls. Histological and immunohistological investigations of muscle and clinical assessment of muscle strength and mass showed no difference between M-D patients and controls. Our findings indicate that patients with M-D have no signs or symptoms of muscle disease. This suggests a different role of the sarcoglycan complex epsilonbetagammadelta versus alphabetagammadelta complex in humans, as earlier suggested in rodents.

Scapuloperoneal syndrome type Kaeser and a wide phenotypic spectrum of adult-onset, dominant myopathies are associated with the desmin mutation R350P.

In 1965, an adult-onset, autosomal dominant disorder with a peculiar scapuloperoneal distribution of weakness and atrophy was described in a large, multi-generation kindred and named 'scapuloperoneal syndrome type Kaeser' (OMIM #181400). By genetic analysis of the original kindred, we discovered a heterozygous missense mutation of the desmin gene (R350P) cosegregating with the disorder. Moreover, we detected DES R350P in four unrelated German families allowing for genotype-phenotype correlations in a total of 15 patients carrying the same mutation. Large clinical variability was recognized, even within the same family, ranging from scapuloperoneal (n = 2, 12%), limb girdle (n = 10, 60%) and distal phenotypes (n = 3, 18%) with variable cardiac (n = 7, 41%) or respiratory involvement (n = 7, 41%). Facial weakness, dysphagia and gynaecomastia were frequent additional symptoms. Overall and within each family, affected men seemingly bear a higher risk of sudden, cardiac death as compared to affected women. Moreover, histological and immunohistochemical examination of muscle biopsy specimens revealed a wide spectrum of findings ranging from near normal or unspecific pathology to typical, myofibrillar changes with accumulation of desmin. This study reveals that the clinical and pathological variability generally observed in desminopathies may not be attributed to the nature of the DES mutation alone, but may be influenced by additional genetic and epigenetic factors such as gender. In addition, mutations of the desmin gene should be considered early in the diagnostic work-up of any adult-onset, dominant myopathy, even if specific myofibrillar pathology is absent.

Phenotypic variability in siblings with calpainopathy (LGMD2A).

Calpainopathy is an autosomal-recessive limb girdle muscular dystrophy (LGMD2A) characterized by selective atrophy and weakness of proximal limb girdle muscles. The clinical phenotype of the disease is highly variable inter-familial, but little is known about intra-familial variability. This study reports the phenotypic variability in eight sibling pairs with genetically proven LGMD2A. Although siblings with identical mutations were often similarly affected, in some families the age of onset and the clinical course varied considerably.

Brain imaging and neuropsychology in late-onset dementia due to a novel mutation (R93C) of valosin-containing protein.

Inclusion body myopathy with Paget disease of bone and frontotemporal dementia (IBMPFD, MIM 167320) is a recently identified autosomal dominant disorder due to mutations in the valosin-containing protein (VCP) that affects muscle, bone and brain. Brain involvement and neuropsychological findings of IBMPFD have not been described in detail. A patient carried a novel heterozygous base pair change, 47832C>T, in the VCP gene that resulted in substitution of an arginine residue by cysteine at position 93 (R93C). He presented first with myopathy while bone involvement remained subclinical. The patient developed behavioral abnormalities in his 60s and showed frank personality change with fluent empty speech at the age of 74 years. This syndrome was best classified as semantic dementia. Magnetic resonance imaging disclosed slight but progressive cerebral atrophy with prominent callosal and frontal white matter loss. Positron emission tomography demonstrated glucose hypometabolism of the frontal and temporal lobes disproportionate to their structural involvement. This first comprehensive clinical and neuroimaging study in IBMPFD may raise the awareness among clinicians as well as basic scientists for this exemplary genetic model of dementia.

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.

Facing the genetic heterogeneity in neuromuscular disorders: linkage analysis as an economic diagnostic approach towards the molecular diagnosis.

The identification of an ever increasing number of gene defects in patients with neuromuscular disorders has disclosed both marked phenotype and genotype variability and considerable disease overlap. In order to offer an economic strategy to characterise the molecular defect in patients with unclassified neuromuscular disorders, we designed DNA marker sets for linkage analysis of 62 distinct neuromuscular disorders gene loci, including all known muscular dystrophies, congenital myopathies, congenital myasthenic syndromes and myotonias. Genotyping of marker loci of 140 clinically well-characterised families with unclassified neuromuscular disorders reduced the number of candidates to one or two genes in 49 % of the families. Subsequent mutation analysis and genome-wide scans enabled the determination of the genetic defect in 31 % of the families including the identification of a new gene and a new mutation in an unexpected candidate gene. This highlights the effective application of this approach both for diagnostic strategies as well as for the identification of new loci and genes.

LGMD 2I due to the common mutation 826C>A in the FKRP gene presenting as myopathy with vacuoles and paired-helical filaments.

We report on two unrelated patients clinically presenting with late-onset progressive limb girdle weakness; cardiomyopathy was seen in one patient. Muscle biopsy revealed a necrotic myopathy with numerous rimmed vacuoles, ultrastructurally typical paired-helical filaments, and reduced immunohistochemical staining for alpha-dystroglycan. Quadriceps sparing hereditary inclusion body myopathy due to mutations in GNE gene, and OPMD due to PABPN1 mutations were excluded, genetically. We detected a homozygous mutation of the FKRP gene (826C>A) in both patients. Mutations of FKRP have been reported in congenital muscular dystrophies, LGMD2I, cardiomyopathy and hyperCKemia, but not in myopathies with vacuoles and paired-helical filaments. Therefore, our findings further extend the morphological variability of muscular dystrophies due to FKRP mutations.

The Clinical Outcome Study for dysferlinopathy: An international multicenter study.

OBJECTIVE: To describe the baseline clinical and functional characteristics of an international cohort of 193 patients with dysferlinopathy. METHODS: The Clinical Outcome Study for dysferlinopathy (COS) is an international multicenter study of this disease, evaluating patients with genetically confirmed dysferlinopathy over 3 years. We present a cross-sectional analysis of 193 patients derived from their baseline clinical and functional assessments. RESULTS: There is a high degree of variability in disease onset, pattern of weakness, and rate of progression. No factor, such as mutation class, protein expression, or age at onset, accounted for this variability. Among patients with clinical diagnoses of Miyoshi myopathy or limb-girdle muscular dystrophy, clinical presentation and examination was not strikingly different. Respiratory impairment and cardiac dysfunction were observed in a minority of patients. A substantial delay in diagnosis was previously common but has been steadily reducing, suggesting increasing awareness of dysferlinopathies. CONCLUSIONS: These findings highlight crucial issues to be addressed for both optimizing clinical care and planning therapeutic trials in dysferlinopathy. This ongoing longitudinal study will provide an opportunity to further understand patterns and variability in disease progression and form the basis for trial design.

Long-term MRI and clinical follow-up of symptomatic and presymptomatic carriers of dysferlin gene mutations.

We report the results of a longitudinal study involving MRI and clinical follow-up in nine siblings from four families with Miyoshi myopathy (MM). All individuals carried pathogenic dysferlin gene (DYSF) mutations with six of them suffering from symptomatic disease and three being presymptomatic. In presymptomatic subjects, MRI was sensitive to detect alterations in muscle tissue years before disease onset. The first MRI alteration to disclose was evidence for myoedema in dorsal compartment muscles of the legs followed by fatty degeneration. Moreover, MRI changes anticipated the topography of subsequent clinical muscle involvement and progressed from distal to proximal dorsal leg muscles. In symptomatic subjects, MRI changes reflected the pattern and severity of clinical muscle involvement. MRI evidence, however, suggests that muscle involvement is much more prominent in early disease stages than clinically seen. Clinical follow-up up to 8 years made evident that MM onset occurs at a mean age of 18.4 years. The most prominent initial deficit was impaired tiptoe gait due to muscle plantarflexor dysfunction followed by impaired dorsiflexor function. Dorsal compartments were predominantly affected not only in distal but also in proximal leg muscles, and a more rapid progression was noticed during the early phase of the disease. Our data suggest that MRI is a helpful diagnostic tool for an early diagnosis of MM and other distal myopathies since it provides sensitive and topographic information about initial and even preclinical muscle involvement. This is of particular relevance in Miyoshi myopathy because distinct CK elevation is present long before its clinical onset and often misdiagnosed as "idiopathic".

Vitamin B-6 metabolism as affected by exercise in trained and untrained women fed diets differing in carbohydrate and vitamin B-6 content.

To assess the effect of exercise on vitamin B-6 metabolism in women, five young-trained, five young-untrained, and five postmenopausal-untrained women were alternately fed four diets and then exercised (80% VO2 max; 20 min) after each diet. The following diets were fed: 2 wk moderate carbohydrate (CHO) (49%) (2.3 mg B-6); 1 wk high CHO (64%) (2.4 mg B-6); 2 wk moderate CHO + B-6 (10.3 mg B-6); 1 wk high CHO + B-6); 1 wk high CHO + B-6 (10.4 mg B-6). Blood was drawn pre- and postexercise and post-30 min and post-60 min of exercise and analyzed for plasma pyridoxal 5'-phosphate (PLP) and total B-6. Twenty-four-hour urines were analyzed for 4-pyridoxic acid and urinary B-6. PLP and total B-6 increased significantly with exercise and decreased significantly from post- to post-60 min for all diets. Four-pyridoxic acid increased significantly from the preexercise day to the day of exercise for all diets. Although there was a significant change in B-6 metabolism with exercise, neither training, increased CHO, nor age had an effect on this change.

Creatine monohydrate in muscular dystrophies: A double-blind, placebo-controlled clinical study.

The authors assessed the safety and efficacy of creatine monohydrate (Cr) in various types of muscular dystrophies in a double-blind, crossover trial. Thirty-six patients (12 patients with facioscapulohumeral dystrophy, 10 patients with Becker dystrophy, 8 patients with Duchenne dystrophy, and 6 patients with sarcoglycan-deficient limb girdle muscular dystrophy) were randomized to receive Cr or placebo for 8 weeks. There was mild but significant improvement in muscle strength and daily-life activities by Medical Research Council scales and the Neuromuscular Symptom Score. Cr was well tolerated throughout the study period.

Genetic identity of Marinesco-Sjögren/myoglobinuria and CCFDN syndromes.

OBJECTIVE AND BACKGROUND: To describe three Gypsy families with Marinesco-Sjögren syndrome (MSS), demyelinating neuropathy, and recurrent episodes of myoglobinuria in five of the six affected subjects. Because these families originated from the same genetically isolated founder population as did patients with congenital cataracts facial dysmorphism neuropathy (CCFDN) syndrome, and because the two syndromes have clinical manifestations in common, we hypothesized that the two related, albeit distinct, syndromes may represent clinical variants of a single genetic disorder. METHODS: Clinical studies were conducted and linkage and haplotype analyses were performed for the three families. A total of 16 individuals, including the 6 with MSS and 10 unaffected relatives, were genotyped for six polymorphic microsatellite markers from the CCFDN region on 18qter. RESULTS: Linkage analysis of markers in the 18qter region, where we previously had located the CCFDN gene, produced a lod score of 3.55, demonstrating colocalization of the gene responsible for MSS with demyelinating neuropathy and myoglobinuria with the CCFDN gene. Moreover, the patients with MSS shared the conserved marker haplotype found in CCFDN chromosomes. CONCLUSIONS: These data suggest that Marinesco-Sjögren syndrome with peripheral neuropathy and myoglobinuria, and congenital cataracts facial dysmorphism neuropathy syndrome are genetically identical and are caused by a single founder mutation.

Novel approaches to treat muscular dystrophies.

Muscular dystrophies (MD) are a clinically and genetically heterogeneous group of skeletal muscle-wasting diseases. Mutations in the dystrophin gene result in dystrophin deficiency, which constitutes the pathogenic basis of Duchenne and Becker MD (DMD and BMD). Several MD are caused by mutations in other recently identified genes coding for proteins linked to the sarcolemma, the nuclear envelope or the contractile apparatus. In addition, several MD have been mapped to different chromosomal loci and for most of them, the identification of the molecular defect is underway. The immediate result is an ongoing reclassification of the MD into disorders defined not by clinical characteristics but specific genetic mutations. At present, therapy of MD is based on symptomatic treatment and supportive care. Convincing evidence for clinical efficacy is only available for corticosteroids that also suffer from frequent and severe side effects. Up to now, curative therapy is not available, although promising new molecular therapies are under investigation in animal models of MD. Current treatment strategies are discussed and a perspective for effective molecular therapy is given.

High-dose immunoglobulin therapy in sporadic inclusion body myositis: a double-blind, placebo-controlled study.

Sporadic inclusion body myositis (s-IBM) is an acquired inflammatory muscle disease of unknown cause. In general, s-IBM presents with slowly progressive, asymmetric weakness, and atrophy of skeletal muscle. There is a mild transitory or nil responsiveness to standard immunosuppressive treatment. A controlled cross-over study of 22 s-IBM patients over 3 months showed a partial improvement in those treated with high-dose intravenous immunoglobulin therapy (IVIG) versus placebo. The present study included 22 patients aged 32-75 years and with a mean duration of disease of 5.2+/-3.6 years. They were randomized by a double-blind, placebo-controlled, cross-over design to monthly infusions of 2 g/kg bodyweight IVIG or to placebo for 6 months each, followed by the alternative treatment. After 6 and 12 months the response to treatment was evaluated, using a modified Medical Research Council scale, Neuromuscular Symptom Score (NSS), the patient's own assessment of improvement, arm outstretched time, and electromyography. No serious side effects were seen, in particular no viral infection and no major cardiac or neurological complications. Overall there was no progression of the disease in 90% of patients, unlike that which might have been expected in untreated patients. A mild and significant improvement (11%) in clinical symptoms was found using NSS, but not with other test procedures. There was a trend to mild improvement in treated patients when using other tests. Individual responses to treatment was heterogeneous. The validity of this study may be reduced by mismatch of groups with regard to age at onset and variability in disease expression. The findings of this study largely confirm those of a previous IVIG trial. Treatment with IVIG may be mildly effective in s-IBM by preventing disease progression or inducing mild improvement. Long-term studies are needed to evaluate further the benefit of IVIG therapy in s-IBM.