The importance of genetic diagnosis for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy are caused by mutations in the dystrophin-encoding DMD gene. Large deletions and duplications are most common, but small mutations have been found as well. Having a correct diagnosis is important for family planning and providing proper care to patients according to published guidelines. With mutation-specific therapies under development for DMD, a correct diagnosis is now also important for assessing whether patients are eligible for treatments. This review discusses different mutations causing DMD, diagnostic techniques available for making a genetic diagnosis for children suspected of DMD and the importance of having a specific genetic diagnosis in the context of emerging genetic therapies for DMD.

Reduced cerebral gray matter and altered white matter in boys with Duchenne muscular dystrophy.

OBJECTIVE: Duchenne muscular dystrophy (DMD) is characterized by progressive muscle weakness caused by DMD gene mutations leading to absence of the full-length dystrophin protein in muscle. Multiple dystrophin isoforms are expressed in brain, but little is known about their function. DMD is associated with specific learning and behavioral disabilities that are more prominent in patients with mutations in the distal part of the DMD gene, predicted to affect expression of shorter protein isoforms. We used quantitative magnetic resonance (MR) imaging to study brain microstructure in DMD. METHODS: T1-weighted and diffusion tensor images were obtained on a 3T MR scanner from 30 patients and 22 age-matched controls (age = 8-18 years). All subjects underwent neuropsychological examination. Group comparisons on tissue volume and diffusion tensor imaging parameters were made between DMD patients and controls, and between 2 DMD subgroups that were classified according to predicted Dp140 isoform expression (DMD_Dp140(+) and DMD_Dp140(-) ). RESULTS: DMD patients had smaller total brain volume, smaller gray matter volume, lower white matter fractional anisotropy, and higher white matter mean and radial diffusivity than healthy controls. DMD patients also performed worse on neuropsychological examination. Subgroup analyses showed that DMD_Dp140(-) subjects contributed most to the gray matter volume differences and performed worse on information processing. INTERPRETATION: Both gray and white matter is affected in boys with DMD at a whole brain level. Differences between the DMD_Dp140(-) subgroup and controls indicate an important role for the Dp140 dystrophin isoform in cerebral development.

Cardiac involvement in Dutch patients with sarcoglycanopathy: a cross-sectional cohort and follow-up study.

INTRODUCTION: The aim of this study is to describe the frequency, nature, severity, and progression of cardiac abnormalities in a cohort of Dutch sarcoglycanopathy patients. METHODS: In this cross-sectional cohort study, patients were interviewed using a standardized questionnaire and assigned a functional score. Electrocardiography (ECG), echocardiography, and 24-h ECG were performed. RESULTS: Twenty-four patients with sarcoglycanopathy had a median age of 25 years (range, 8-59 years). Beta blockers were used by 13%, and 17% used angiotensin-converting enzyme inhibitors. ECG abnormalities were present in 5 (21%), and 4 (17%) fulfilled the criteria for dilated cardiomyopathy (DCM). There were no significant differences in median age or severity of disease between patients with or without DCM. Eleven patients were examined earlier. Median follow-up time was 10 years. Two of the 11 patients (18%) developed DCM during follow-up. CONCLUSIONS: Seventeen percent of the patients with sarcoglycanopathy were found to have dilated cardiomyopathy. We recommend biannual cardiac monitoring, including ECG and echocardiography.

Elucidation of the Genetic Cause in Dutch Limb Girdle Muscular Dystrophy Families: A 27-Year's Journey.

BACKGROUND: A Dutch cohort of 105 carefully selected limb girdle muscular dystrophy (LGMD) patients from 68 families has been subject to genetic testing over the last 20 years. After subsequent targeted gene analysis around two thirds (45/68) of the families had received a genetic diagnosis in 2013. OBJECTIVE: To describe the results of further genetic testing in the remaining undiagnosed limb girdle muscular dystrophy families in this cohort. METHODS: In the families of the cohort for whom no genetic diagnosis was established (n = 23) further testing using Sanger sequencing, next generation sequencing with gene panel analysis or whole-exome sequencing was performed. In one case DNA analysis for facioscapulohumeral dystrophy type 1 was carried out. RESULTS: In eight families no additional genetic tests could be performed. In 12 of the remaining 15 families in which additional testing could be performed a genetic diagnosis was established: two LGMDR1 calpain3-related families with CAPN3 mutations, one LGMDR2 dysferlin-related family with DYSF mutations, three sarcoglycanopathy families (LGMDR3-5 α-, ß- and γ-sarcoglycan-related) with SGCA/SGCB/SGCG mutations, one LGMDR8 TRIM 32-related family with TRIM32 mutations, two LGMDR19 GMPPB-related families with GMPPB mutations, one family with MICU1-related myopathy, one family with FLNC-related myopathy and one family with facioscapulohumeral dystrophy type 1. At this moment a genetic diagnosis has been made in 57 of the 60 families of which DNA was available (95%). CONCLUSION: A genetic diagnosis is obtained in 95% of the families of the original Dutch LGMD cohort of which DNA was available.

Local dystrophin restoration with antisense oligonucleotide PRO051.

BACKGROUND: Duchenne's muscular dystrophy is associated with severe, progressive muscle weakness and typically leads to death between the ages of 20 and 35 years. By inducing specific exon skipping during messenger RNA (mRNA) splicing, antisense compounds were recently shown to correct the open reading frame of the DMD gene and thus to restore dystrophin expression in vitro and in animal models in vivo. We explored the safety, adverse-event profile, and local dystrophin-restoring effect of a single, intramuscular dose of an antisense oligonucleotide, PRO051, in patients with this disease. METHODS: Four patients, who were selected on the basis of their mutational status, muscle condition, and positive exon-skipping response to PRO051 in vitro, received a dose of 0.8 mg of PRO051 injected into the tibialis anterior muscle. A biopsy was performed 28 days later. Safety measures, composition of mRNA, and dystrophin expression were assessed. RESULTS: PRO051 injection was not associated with clinically apparent adverse events. Each patient showed specific skipping of exon 51 and sarcolemmal dystrophin in 64 to 97% of myofibers. The amount of dystrophin in total protein extracts ranged from 3 to 12% of that found in the control specimen and from 17 to 35% of that of the control specimen in the quantitative ratio of dystrophin to laminin alpha2. CONCLUSIONS: Intramuscular injection of antisense oligonucleotide PRO051 induced dystrophin synthesis in four patients with Duchenne's muscular dystrophy who had suitable mutations, suggesting that further studies might be feasible.

In tandem analysis of CLCN1 and SCN4A greatly enhances mutation detection in families with non-dystrophic myotonia.

Non-dystrophic myotonias (NDMs) are caused by mutations in CLCN1 or SCN4A. The purpose of the present study was to optimize the genetic characterization of NDM in The Netherlands by analysing CLCN1 and SCN4A in tandem. All Dutch consultant neurologists and the Dutch Patient Association for Neuromuscular Diseases (Vereniging Spierziekten Nederland) were requested to refer patients with an initial diagnosis of NDM for clinical assessment and subsequent genetic analysis over a full year. Based on clinical criteria, sequencing of either CLCN1 or SCN4A was performed. When previously described mutations or novel mutations were identified in the first gene under study, the second gene was not sequenced. If no mutations were detected in the first gene, the second gene was subsequently also analysed. Underlying NDM mutations were explored in 54 families. In total, 20% (8 of 40) of our probands with suspected chloride channel myotonia showed no CLCN1 mutations but subsequent SCN4A screening revealed mutations in all of them. All 14 probands in whom SCN4A was primarily sequenced showed a mutation. In total, CLCN1 mutations were identified in 32 families (59%) and SCN4A in 22 (41%), resulting in a diagnostic yield of 100%. The yield of mutation detection was 93% with three recessive and three sporadic cases not yielding a second mutation. Among these mutations, 13 in CLCN1 and 3 in SCN4A were novel. In conclusion, the current results show that in tandem analysis of CLCN1 and SCN4A affords high-level mutation ascertainment in families with NDM.

What can we learn from assisted bicycle training in a girl with dystrophinopathy? A case study.

In this case study, a 9-year-old ambulatory girl with dystrophinopathy due to a mosaic translocation mutation participated in dynamic training. Because the role of exercise is unclear in both boys and girls with dystrophinopathy, a recently developed assisted bicycle training regimen was evaluated for its feasibility and effectiveness in this girl. The girl trained at home, first 15 minutes with her legs and then 15 minutes with her arms, 5 times a week, for 24 weeks. This case study showed that the training was feasible and safe. In addition, we found that no physical deterioration occurred during the training period: the Motor Function Measure and the Assisted 6-Minute Cycling Test results remained stable. Slight improvements in quantitative muscle ultrasound intensity were found, indicating less fatty infiltration in the muscles. These results suggest that physical training could be beneficial in females with dystrophinopathy who express low levels of dystrophin.

DGGE-based whole-gene mutation scanning of the dystrophin gene in Duchenne and Becker muscular dystrophy patients.

Duchenne and Becker muscular dystrophy (DMD and BMD) are caused by mutations in the dystrophin gene. Large rearrangements in the gene are found in about two-thirds of DMD patients, with approximately 60% carrying deletions and 5-10% carrying duplications. Most of the remaining 30-35% of patients are expected to have small nucleotide substitutions, insertions, or deletions. To detect these subtle changes within the coding and splice site determining sequences of the dystrophin gene, we established a semiautomated denaturing gradient gel electrophoresis (DGGE) mutation scanning system. The DGGE scan covers the dystrophin gene with 95 amplicons, PCRed either individually or in a multiplex setup. PCR and pooling were performed semiautomatically, using a pipetting robot and 384-well plates, enabling concurrent amplification of DNA of four patients in one run. Amplification of individual fragments was performed using one PCR program. The products were pooled just before gel loading; DGGE requires only a single gel condition. Validation was performed using DNA samples harboring 39 known DMD variants, all of which could be readily detected. DGGE mutation scanning was applied to analyze 135 DMD/BMD patients and potential DMD carriers without large deletions or duplications. In DNA from 25 out of 44 DMD patients (57%) and from 5 out of 39 BMD patients (13%), we identified clear pathogenic changes. All mutations were different, with the exception of one DMD mutation, which occurred twice. In DNA from 10 out of 44 potential DMD carriers, including four obligate carriers, we detected causative changes, including one pathogenic change in every obligate carrier. In addition to these pathogenic changes, we detected 15 unique unclassified variants, i.e., changes for which a pathogenic nature is uncertain.

Novel mutations in three patients with LGMD2C with phenotypic differences.

Limb-girdle muscular dystrophy type 2C is an autosomal-recessive disorder caused by mutations in gamma-sarcoglycan encoding gene. This disease is characterized by childhood onset of progressive muscular dystrophy. Because of the clinical presentation, this disorder may be misdiagnosed as a dystrophinopathy. Two males (Patients A and B) from one Turkish family and one male (Patient C) from a Moroccan family had progressive walking disturbances for several years, exercise intolerance, and leg pains. Clinical examination revealed limb-girdle weakness and calf hypertrophy. Serum creatine kinase levels ranged from 1100 to 19000 U/L. The initial findings and course of the disease were less severe in Patient B compared with his brother (Patient A) at the same age. By means of immunohistochemistry on muscle biopsy all patients manifested reduced expression of alpha-, beta-, gamma-, and delta-sarcoglycans. DNA sequence analysis revealed a homozygous splice site mutation in exon 5 (IVS5+2T>C) in the Turkish family. In the patient from the Moroccan family a homozygous nonsense mutation in exon 2 (93G>A;Trp31X) was present. In conclusion, this report describes the clinical, histologic, and immunohistochemical characteristics of three children with limb-girdle muscular dystrophy type 2C. Two novel mutations in the gamma-sarcoglycan gene were present. We found phenotypic differences in two brothers.

Clinical experience with long-term acetazolamide treatment in children with nondystrophic myotonias: a three-case report.

BACKGROUND: Today, treatment of the nondystrophic myotonias consists of mexiletine, although care has to be taken because of the proarrhythmogenic potential of this drug. In this article, we report years of experience with the carbonic anhydrase inhibitor acetazolamide. PATIENTS: We present three children with nondystrophic myotonias. RESULTS: During acetazolamide treatment, symptoms and signs of myotonia decreased in our children. CONCLUSIONS: Based on this clinical experience and the favorable pharmacologic profile of acetazolamide, it may be a good treatment option for children with nondystrophic myotonias.

ANO5 mutations in the Dutch limb girdle muscular dystrophy population.

A Dutch cohort of 105 limb girdle muscular dystrophy (LGMD) patients were subject to subsequent genetic investigations. In half the families a causative mutation was found. Recently mutations were identified in ANO5 causing LGMD2L and Miyoshi-like myopathy (MMD3), but could also be found in patients with hyperCKemia only. Therefore, we analysed the index cases of the remaining 31 as yet undiagnosed families from our previously described cohort of LGMD patients for the presence of ANO5 mutations. Detailed history and neurological examination were available for all patients. Serum creatine kinase (CK) activity, skeletal muscle computed tomography (CT) and cardiological investigations were performed. Mutations in ANO5 were found in 16% of the families: 11 index patients and two sibs, eight males and five females. The founder mutation c.191dupA was present in 8 out of 13 patients. Ten different pathogenic mutations were identified of which seven were novel: five missense and two splice site mutations. The age of these patients ranged from 26 to 69 years and the age of onset varied from 21 to 57 years. Symptoms at onset were related to proximal leg weakness. The weakness was slowly progressive. Calf hypertrophy was present in three patients. Males were more severely affected than females. Serum CK activity was highly elevated in the early stage of disease and moderately increased in later stages. Muscle biopsy showed predominantly dystrophic changes. One patient had hypertrophic cardiomyopathy, two others had intraventricular septum thickening.

Therapeutic exon skipping for dysferlinopathies?

Antisense-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy (DMD) currently tested in clinical trials. The aim is to reframe dystrophin transcripts using antisense oligonucleotides (AONs). These hide an exon from the splicing machinery to induce exon skipping, restoration of the reading frame and generation of internally deleted, but partially functional proteins. It thus relies on the characteristic of the dystrophin protein, which has essential N- and C-terminal domains, whereas the central rod domain is largely redundant. This approach may also be applicable to limb-girdle muscular dystrophy type 2B (LGMD2B), Myoshi myopathy (MM) and distal myopathy with anterior tibial onset (DMAT), which are caused by mutations in the dysferlin-encoding DYSF gene. Dysferlin has a function in repairing muscle membrane damage. Dysferlin contains calcium-dependent C2 lipid binding (C2) domains and an essential transmembrane domain. However, mildly affected patients in whom one or a large number of DYSF exons were missing have been described, suggesting that internally deleted dysferlin proteins can be functional. Thus, exon skipping might also be applicable as a LGMD2B, MM and DMAT therapy. In this study we have analyzed the dysferlin protein domains and DYSF mutations and have described what exons are promising targets with regard to applicability and feasibility. We also show that DYSF exon skipping seems to be as straightforward as DMD exon skipping, as AONs to induce efficient skipping of four DYSF exons were readily identified.

Rapid and cost effective detection of small mutations in the DMD gene by high resolution melting curve analysis.

Duchenne/Becker muscular dystrophy (DMD/BMD) is caused by large deletions or duplications in two-thirds of the cases. The remaining one-third DMD patients have small mutations in the DMD gene. Screening for such small mutations is a daunting and costly task. High resolution melting curve analysis (HR-MCA) followed by sequencing for amplicons with altered melting profiles can be used to scan DNA for small alterations. We first validated the technique as screening procedure for the DMD gene and then screened a group of unrelated 22 DMD/BMD patients and 11 females. We managed to identify all previously found mutations by means of HR-MCA, which provided its validation. Furthermore, 17 different pathogenic mutations were found in the screening group, of which 10 were novel. Our results provide validation of HR-MCA as a powerful and inexpensive pre-sequencing scanning method. This technology is now ready for routine diagnostic use on DMD/BMD patients and female carriers.

CAV3 gene mutation analysis in patients with idiopathic hyper-CK-emia.

As caveolin-3 deficiencies may explain persistent hyper-CK-emia, we performed CAV3 gene mutation analysis and immunohistochemistry for caveolin-3 in 31 patients with idiopathic hyper-CK-emia. In 2 of 29 patients who donated blood, variants in the CAV3 gene were detected. Although immunohistochemical analysis strongly suggested that caveolin-3 was properly localized in the muscle tissue of the two affected patients, it may not function normally and could thus explain their persistent hyper-CK-emia. Our findings contribute to the clarification of unexplained persistent hyper-CK-emia, but further research is needed before CAV3 gene mutation analysis becomes part of the routine evaluation of these patients.

Dystrophin analysis in carriers of Duchenne and Becker muscular dystrophy.

Associations between clinical phenotype (muscle weakness, dilated cardiomyopathy) and dystrophin abnormalities in muscle tissue among definite carriers of Duchenne (DMD) and Becker muscular dystrophy (BMD) were investigated. No associations between dystrophin abnormalities and clinical variables in DMD/BMD carriers were found. Because 26% of nonmanifesting carriers have dystrophin-negative fibers, this might be used in suspected DMD/BMD carriers in whom DNA analysis fails to give an answer about their carrier risk.