What does DMD 1

Muscular dystrophies

Clinical symptoms

Muscular dystrophies (MD) form a clinically and genetically heterogeneous group of genetically determined, progressive diseases of the muscle. The common symptom of all MD is progressive muscle weakness and atrophy, which differs significantly in distribution pattern and severity between the various MD forms. Typical histological findings show increased fiber caliber variations, a coexistence of degenerating and regenerating muscle fibers and significant endo- and perimysial fibrosis even in the early stages. Usually the morphological examination allows an unambiguous assignment to the diagnosis MD, but without allowing a classification into the various diagnoses within this group. MD can occur at any age; For example, children who suffer from a form of congenital MD (MDC) are noticeable at birth or shortly afterwards with generalized hypotension ("floppy infant"), while in patients with oculopharyngeal MD (OPMD) the first symptoms usually only appear between the Show 50th and 60th year of life.

According to conservative estimates, the prevalence of MD in both sexes is around 286 x 10-6, i.e. one in 3500 of the German population can be assumed to have an inherited neuromuscular disease that manifests itself at birth or later in life. The most common form is the X-linked recessive inherited DMD, first described by G. Duchenne in 1861. In 1986 the underlying gene, and subsequently also the gene product, was discovered, which enables the molecular diagnosis of DMD and the milder allelic variant BMD has been. About 50 years later, Batten published the first cases of congenital muscular dystrophy (MDC). In contrast to the Duchenne / Becker phenotype, weakness and dystrophic changes in the muscle are already present at birth.

The term limb girdle muscular dystrophy (LGMD) was coined in the mid-20th century when it became clear that an additional large group of non-congenital muscular dystrophies existed, which differ from both the X-linked dystrophinopathies (DMD / BMD) as well as from the autosomal dominant facial scapulo-humeral muscular dystrophy (FSHD). In the meantime, the term LGMD has changed from a diagnosis of embarrassment to an ever-expanding, detailed list of LGMD subtypes (currently 22, and the number is rising!) For which accurate molecular diagnostics are available.

The onset of the disease extends from early childhood to late adulthood; the same genetic defect can cause allelic forms of congenital and limb girdle forms, as has been shown for the fukutin-related protein gene (FKRP) and the POMT1 gene.

The discovery of Emerin, the gene responsible for the X-linked muscular dystrophy Emery-Dreifuss (EDMD1, X-EDMD), and the description of an autosomal dominant variant (EDMD2) based on mutations in the lamin A / C gene (LMNA) showed the importance of the “nuclear envelope” for neuromuscular diseases. Mutations in LMNA also lead to dilated cardiomyopathy with conduction disorders (CMD1A), girdle dystrophy 1B (LGMD1B), Charcot – Marie – Tooth disease type 2B1 (CMT2B1), and a variety of other non-neuromuscular diseases such as familial partial lipodystrophy, the Dunnigan type mandibuloacral dysplasia, or premature aging syndromes such as Hutchinson-Gilford progeria and atypical Werner syndrome. Furthermore, a new laminopathy phenotype with combined myopathy and progeria was described.

The identification of the genetic cause and the defective protein in a number of MD has revolutionized the classification of these diseases in recent years and allows new insights into the pathophysiological relationships.

Diagnosis

The growing number of gene locations, specific genes and gene products that play a role in the pathogenesis of muscular dystrophies makes it impossible for a single clinical center or laboratory to cover the entire spectrum of molecular genetic diagnostics. A precise diagnosis enables predictions about the course and prognosis of the disease, influences the patient's choice of profession, serves to prevent complications (respiratory insufficiency, cardiac arrhythmias, cardiomyopathy), is a prerequisite for any genetic family counseling, a possible prenatal diagnosis and for the inclusion of the patient in clinical studies and for future molecular therapies. The diagnosis should be carried out gradually, based on a detailed medical history including family history (beginning distal / proximal, contractures, cardiac / pulmonary involvement, recessive / dominant / X-linked inheritance). During the physical examination, particular attention should be paid to the severity and distribution pattern of the paresis. Determination of the creatine kinase (CK) in the serum and an electromyographic examination (pathological spontaneous activity, myopathic pattern) are also useful. Myosonography and magnetic resonance tomography help to find a suitable place for a diagnostic muscle biopsy in advanced paresis and atrophy. In the case of an informative family structure, a linkage analysis (haplotyping) helps to exclude or narrow down gene locations so that one or a few genes can be specifically examined for mutations (Rocha et al., 2010).

In some cases, a primary molecular diagnosis bypassing the muscle biopsy is possible. In around 60% of dystrophinopathies with a typical phenotype (DMD / BMD), a larger deletion / duplication can be detected. New molecular methods have increased the accuracy even further: Even with FSHD, myotonic dystrophies type 1 and 2 (DM1, DM2 / PROMM), OPMD, Emery tripod dystrophies (EMD1, EMD2 / LGMD1B) and LGMD2I a primary genetic diagnosis is already routine. In the case of all other forms of MD, a primary genetic diagnosis of further family members can be carried out as soon as the diagnosis of an affected family member has been confirmed by molecular genetics.

Histologically, the muscle biopsy leads to the confirmation of the diagnosis “muscular dystrophy” or to the exclusion of other, possibly causally treatable diagnoses. Defects in MD-associated proteins can be detected by means of detailed protein diagnostics (immunohistochemistry, Western blot). The result often allows a specific classification of the muscular dystrophy present, but the combination of protein diagnostics and molecular genetic analysis is often of decisive importance. If all of these examinations are negative, the classification of the MD must remain open for the time being, especially in sporadic cases and in small families. The definitive diagnosis is only possible when the defective genes and protein products have been characterized.

The muscular dystrophy network MD-NET offers a Germany-wide overview with laboratory locations and addresses for genetic diagnostics in muscular dystrophies.

Therapy options

Symptomatic therapy

Although no causal therapy is currently available, the life expectancy and quality of MD patients have improved in recent years. Primarily symptomatic therapies and aids contribute to this; the primary goal should be to improve the quality of life. These include:

  • physical therapy
  • Aid supplies, especially orthotics and wheelchairs
  • If necessary, remedial education, speech therapy support or occupational therapy depending on the level of development
  • Orthopedic support, contracture-relieving interventions and, in the event of inability to walk, correction of spinal deformities
  • Cardiological care and symptomatic therapy, e.g. for cardiomyopathies
  • Regular pulmonological diagnostics including body plethysmography (vital capacity) and / or, in younger children, polysomnography. Depending on the extent of the restrictive ventilation disorder, non-invasive ventilation or ventilation via a tracheostoma, if necessary. New data show that the early use of non-invasive ventilation can decisively improve symptoms and quality of life.
  • If there are problems with food intake, insert a PEG tube
  • Appropriate anticonvulsant therapy for cerebral seizures / epilepsy
  • Ophthalmological care
  • Human genetic counseling and, if necessary, prenatal diagnostics

Pharmacotherapy

Corticosteroids

For DMD, oral corticosteroids such as prednisone and deflazacort are currently the standard pharmacotherapy. Randomized clinical studies with oral prednisone have shown improvements in muscle strength and function in patients with DMD, which is reflected in the increased ability to walk and a lower prevalence of scoliosis. A daily dose of 0.75 mg / kg body weight prednisone is usually started at the age of 4-6 years and usually continued until the patient loses the ability to walk. In the event of severe side effects during treatment with corticosteroids (weight gain, growth retardation, osteoporosis, cataract, behavioral problems), alternate administration or a reduction in the dose should be considered. Compared to prednisone, side effects seem to occur less frequently with Deflazacort, and Deflazacort probably has a cardioprotective effect. A large, multicenter, randomized study comparing different therapy regimens with prednisone and deflazacort is currently being planned.

The exact mechanism of action of the corticosteroids is unclear; membrane-stabilizing and anti-inflammatory effects are discussed. In other forms of MD, corticosteroids have not yet been systematically tested, although case reports describe a clinical response to corticosteroids in some sarcoglycanopathies. The immunosuppressive effect is apparently not a determining factor, since other immunosuppressants such as azathioprine hardly had any noteworthy effects in controlled studies.

Cyclosporine A

The Muscular Dystrophy Network (MD-NET) is a research network for neuromuscular diseases funded by the BMBF. As part of this network, a multicenter, placebo-controlled, double-blind study on the effect of cyclosporine A in patients with Duchenne muscular dystrophy has recently been carried out. A total of 153 ambulatory patients at the 11 participating centers in Germany, Austria and Switzerland took part in the study. Initially, the patients received cyclosporine A (3.5-4 mg / kg body weight) or placebo over a period of three months. Thereafter, all patients were additionally treated for twelve months with intermittent cortisone (0.75 mg / kg body weight prednisone, each 10 days with a subsequent 10-day break). Cyclosporine A was well tolerated, but could not improve muscle strength or functional abilities in ambulatory DMD patients either as monotherapy or in combination with intermittent steroid administration.

Creatine monohydrate and beta-2 sympathomimetics

Therapy with food supplements (e.g. creatine monohydrate) and beta-2 sympathomimetics can be useful in individual cases; studies have confirmed a small but significant improvement in strength in patients with various muscular dystrophies (Duchenne, Becker, LGMD, FSHD).

ACE inhibitors and beta blockers

During an international workshop of the European Neuromuscular Center (ENMC) guidelines for the treatment of cardiac involvement in various muscular dystrophies were recently established. Dilated cardiomyopathy is common among dystrophinopathies (DMD / BMD). All patients should therefore receive an ECG and a UKG every 24 months at the time of the initial diagnosis and before any surgical intervention, patients with DMD also up to the age of 10 years, and every 12 months from the age of 10 years. Screening for cardiomyopathy every 2 years is sufficient in patients with BMD. As soon as abnormalities appear, patients should be treated with ACE inhibitors and, if necessary, beta blockers. In DMD patients, prophylactic treatment with the ACE inhibitor perindopril can delay the development of left ventricular dysfunction.

Molecular Therapy

Stop Codon Readthrough (Aminoglycoside Antibiotics)

A 1999 study showed that nonsense mutations that lead to premature stop codons and thus to the termination of translation are skipped after administration of gentamycin both in myotubes in vitro and in mdx mice in vivo. This leads to the expression of functional dystrophin molecules and the partial restoration of muscle function. In a study with four dystrophinopathy patients, however, no positive effect could be achieved, although the aminoglycoside serum levels achieved corresponded to those of the mdx mice. It appears that certain nonsense mutations are refractory to suppression with aminoglycosides. A nonsense mutation comparable to the mdx mouse is the cause of the disease in a maximum of 10-20% of DMD patients. The therapeutic effect, which has not yet been proven in humans, would also have to be weighed against the side effects (e.g. hearing loss and nephrotoxicity).

Stop codon readthrough (Translarna ™, active ingredient: ataluren)

The drug Ataluren (trade name TranslarnaTM) also works via a similar mechanism. During translation, the binding of the active substance to the RNA means that the ribosome can skip the premature stop codon. The ribosome no longer receives the information about the stop, but rather continues reading normally during the translation up to the correct end point. In this way a functional protein can be formed again. This drug has already been investigated in a 48-week multicenter, international, double-blind, placebo-controlled study with 174 DMD patients with the 6-minute walk test as the primary outcome parameter. The results of this study suggest a delay in disease progression in the ataluren-treated group; after one year of treatment, a longer walking distance within 6 minutes was measurable in these patients than in the placebo-treated patients (Bushby K et al, Muscle Nerve 2014). Based on these results, the drug received conditional approval from the European authorities for the treatment of Duchenne muscular dystrophy. Further studies will follow. Since July 23, 2018, this medication can be prescribed by your doctor or muscle center to patients with DMD with nonsense mutations (approx. 13% of DMD patients carry nonsense mutations), who are still able to walk and who are older than 2 years.

Exon skipping by antisense oligonucleotides

One strategy to intervene in the disease process in DMD at the molecular level is the use of so-called antisense oligonucleotides (AON). These are constructed in such a way that they effect the correction of the translational reading frame by binding to the exons flanking a mutation. If an affected patient has a deletion of one or more exons of the dystrophin gene so that the formation of functional dystrophin molecules is prevented by shifting the reading frame, a truncated but functional protein can be formed in this way, which then corresponds to the image of a BMD. Deletions and duplications of whole or even several exons are the most common genetic cause of DMD, so that this therapeutic strategy can be used in a large proportion of these patients. Direct intramuscular injection of AON into mdx mouse muscle resulted in a significant increase in the number of dystrophin-positive fibers; the expression lasted for about 2 months and resulted in an increase in strength without any adverse immune reactions being registered. Systemic application of AON in adeno-associated vectors was even able to demonstrate dystrophin expression in 50-80% of the muscle fibers of adult mdx mice. In a recent clinical study with AON injected locally into the anterior tibial muscle of 4 DMD patients, dystrophin expression was successfully detected 28 days later. Due to the limited half-life of the AON, the mRNA and the dystrophin, the treatment must be repeated at regular intervals. Clinical studies with systemic application of AON in DMD are currently being planned. However, its use in other hereditary myopathies is limited, since in most cases there are point mutations rather than deletions.

Morpholinos

Morpholino oligos or morpholinos for short are nucleic acid analogues that are used as tools in molecular biology to knock down genes. Morpholinos can interfere in the mRNA splicing process by preventing the formation of the nuclear ribonucleoprotein complex (snRNP) or by interacting with the binding sites for other regulatory proteins. They mediate the exclusion of exons from the mature mRNA in a similar way to AONs, but because of their unnatural structure they have an advantage over AONs, since they are not recognized by cellular proteins and not broken down by nucleases, do not generate an immune response and do not modify DNA methylation. Morpholinos have already been used to induce exon skipping in young mdx mice; a decrease in the proportion of fibers with central nuclei and the infiltration of inflammatory cells was observed, comparable to the results obtained with AON. However, relevant studies on the safety of morpholinos for use in patients are still required.

Viral vectors

The possibility of treating DMD through various viral vectors by means of gene transfer to the dystrophic muscle has raised high hopes in recent years.Adenoviral vectors are free of viral genes and have a large clonogenic capacity, but are difficult to manufacture and do not persist permanently in the muscle. Adeno-associated viruses (AAV) are small, non-enveloped, single-stranded DNA viruses that require co-infection with a helper virus for replication and have a smaller clonogenic capacity (5kb). Most of the vectors in use today are encased RNA retroviruses. Lentiviral vectors can take up a maximum of 9 kb cargo DNA, sufficient for e.g. minidystrophin, markers and promoters that are required for expression. They can cross the nuclear membrane and integrate into host cell chromosomes. Their biosafety has been improved through the development of self-inactivating vectors. Furthermore, these vectors transduce and express transgenes stably in various cell types and are considered the system of choice for cell-based gene transfer. Lentiviral vectors were already used in the first gene replacement experiments with DMD, in which attempts were made to transduce proliferating myoblasts in vitro or in vivo by means of direct injection of a mini-dystrophin-carrying murine retrovirus. However, this approach was limited by the occurrence of immunological problems. Improved technologies have allowed lentiviruses to be used as the most suitable vectors for transduction of a range of tissue and cell types. However, there were also serious problems with regard to the biosafety of these vectors and the risk of harmful activating or inactivating insertions. Five patients with severe X-linked combined immunodeficiency (X-SCID) developed leukemia following a study of retroviral gene therapy as a result of insertion of the gene-carrying retrovirus near an oncogene. Consequently, follow-up experiments have been directed towards a more detailed understanding of the integration patterns of retroviruses and the molecular mechanisms that regulate the choice of integration site, indispensable for the design of safe and effective gene transfer vectors. Nevertheless, lentiviral vectors could be well suited for ex vivo gene therapy strategies, e.g. transfer of the dystrophin gene into stem cells isolated from DMD patients.

Myogenic stem cells

In recent years, attention has turned to stem cells as a source of donor myoblasts with high replicability, which is believed to improve the survival of the donor cells and their ability to expand and fuse with dystrophic muscle cells. Various forms of stem cells have already been investigated at the cell culture level for the treatment of muscular dystrophy, but there are still no studies on the question of a functional proof of effectiveness in humans. Stem cells have the advantage that only a small number of cells and a corresponding stimulation signal are required for expansion in order to achieve a therapeutic effect, and that systemic administration is possible.

Research, clinical studies and patient registries

The institute's research projects on muscular dystrophies include genotype / phenotype correlations in various MD subtypes, especially in limb girdle dystrophies (LGMD), clinical studies in various forms of muscular dystrophy, and molecular therapies (see the research section).

As part of the TREAT-NMD project funded in the 6th EU framework program, patient registries are being set up worldwide to facilitate the planning and implementation of multicenter clinical studies. The registry for German and Austrian patients with muscular dystrophy Duchenne, Becker or spinal muscular atrophy as well as the international registry for patients with FKRPopathies (MDC1C / LGMD2I) is located at the Friedrich-Baur-Institut. Registration takes place online at www.dmd-register.de, www.sma-register.de or www.fkrp-register.de.

Contact person in the institute

Prof. Dr. Maggie C. Walter