Luisa Politano

North Star Ambulatory Assessment, 6-minute walk test and timed items in ambulant boys with Duchenne muscular dystrophy

The North Star Ambulatory Assessment is a functional scale specifically designed for ambulant boys affected by Duchenne muscular dystrophy (DMD). Recently the 6-minute walk test has also been used as an outcome measure in trials in DMD. The aim of our study was to assess a large cohort of ambulant boys affected by DMD using both North Star Assessment and 6-minute walk test. More specifically, we wished to establish the spectrum of findings for each measure and their correlation. This is a prospective multicentric study involving 10 centers. The cohort included 112 ambulant DMD boys of age ranging between 4.10 and 17 years (mean 8.18±2.3 DS). Ninety-one of the 112 were on steroids: 37/91 on intermittent and 54/91 on daily regimen. The scores on the North Star assessment ranged from 6/34 to 34/34. The distance on the 6-minute walk test ranged from 127 to 560.6 m. The time to walk 10 m was between 3 and 15 s. The time to rise from the floor ranged from 1 to 27.5 s. Some patients were unable to rise from the floor. As expected the results changed with age and were overall better in children treated with daily steroids. The North Star assessment had a moderate to good correlation with 6-minute walk test and with timed rising from floor but less with 10 m timed walk/run test. The 6-minute walk test in contrast had better correlation with 10 m timed walk/run test than with timed rising from floor. These findings suggest that a combination of these outcome measures can be effectively used in ambulant DMD boys and will provide information on different aspects of motor function, that may not be captured using a single measure.

Mutation analysis of the lamin A/C gene (LMNA) among patients with different cardiomuscular phenotypes

Laminopathies represent a heterogeneous group of genetic disorders characterised by mutations in the LMNA gene, which encodes two lamins, A and C, by alternative splicing of the primary transcript.1 Lamins belong to the intermediate filament multigene family and form the nuclear lamina, a mesh-like structure adjacent to the nucleoplasmic side of the inner nuclear membrane.2 They interact with emerin, the proteins encoded by the gene for the X-linked (X EDMD) form of EDMD, with several nuclear envelope proteins and with chromatin. Despite their widespread distribution and their role in nuclear architecture, alterations of lamin A/C are responsible for a number of very specific but quite heterogeneous disorders. The first laminopathy was the autosomal dominant form of Emery-Dreifuss muscular dystrophy (EDMD), a genetic disorder characterised by the clinical triad of early onset contractures, progressive muscular wasting and weakness with humeroperoneal distribution and cardiac conduction defects.3 The finding that emerin, an inner nuclear envelope protein, and LMNA were both involved in EDMD suggested that the lamins may represent specific and relevant factors in cardiac and skeletal muscle and that integrity of the nuclear membrane and associated structures is specifically required for muscle function. However, later on it was found that besides autosomal dominant Emery–Dreifuss muscular dystrophy (AD-EDMD), mutations in LMNA are responsible for six other disorders: limb girdle muscular dystrophy 1B, (LGMD1B),4,5 dilated cardiomyopathy with conduction system disease, (DCM-CD),6 Dunningan-type familial partial lipodystrophy,7–9 one recessive axonal form of Charcot-Marie-Tooth neuropathy,10 mandibuloacral dysplasia,11 and Hutchinson Gilford progeria.12,13 Despite the very different phenotypic consequences of mutations in LMNA , and the quite large number of mutations identified, no genotype/phenotype correlation has been demonstrated, pointing to the role of factors other than lamins A and C in determining the different tissue specific phenotypes. …

Long Term Natural History Data in Ambulant Boys with Duchenne Muscular Dystrophy: 36-Month Changes

The 6 minute walk test has been recently chosen as the primary outcome measure in international multicenter clinical trials in Duchenne muscular dystrophy ambulant patients. The aim of the study was to assess the spectrum of changes at 3 years in the individual measures, their correlation with steroid treatment, age and 6 minute walk test values at baseline. Ninety-six patients from 11 centers were assessed at baseline and 12, 24 and 36 months after baseline using the 6 minute walk test and the North Star Ambulatory Assessment. Three boys (3%) lost the ability to perform the 6 minute walk test within 12 months, another 13 between 12 and 24 months (14%) and 11 between 24 and 36 months (12%). The 6 minute walk test showed an average overall decline of −15.8 (SD 77.3) m at 12 months, of −58.9 (SD 125.7) m at 24 months and −104.22 (SD 146.2) m at 36 months. The changes were significantly different in the two baseline age groups and according to the baseline 6 minute walk test values (below and above 350 m) (p<0.001). The changes were also significantly different according to steroid treatment (p = 0.01). Similar findings were found for the North Star Ambulatory Assessment. These are the first 36 month longitudinal data using the 6 minute walk test and North Star Ambulatory Assessment in Duchenne muscular dystrophy. Our findings will help not only to have a better idea of the progression of the disorder but also provide reference data that can be used to compare with the results of the long term extension studies that are becoming available.

24 Month Longitudinal Data in Ambulant Boys with Duchenne Muscular Dystrophy

Objectives The aim of the study was i) to assess the spectrum of changes over 24 months in ambulant boys affected by Duchenne muscular dystrophy, ii) to establish the difference between the first and the second year results and iii) to identify possible early markers of loss of ambulation. Methods One hundred and thirteen patients (age range 4.1–17, mean 8.2) fulfilled the inclusion criteria, 67 of the 113 were on daily and 40 on intermittent steroids, while 6 were not on steroids. All were assessed using the 6 Minute Walk Test (6MWT), the North Star Ambulatory Assessment (NSAA) and timed test. Results On the 6MWT there was an average overall decline of −22.7 (SD 81.0) in the first year and of −64.7 (SD 123.1) in the second year. On the NSAA the average overall decline was of −1.86 (SD 4.21) in the first year and of −2.98 (SD 5.19) in the second year. Fourteen children lost ambulation, one in the first year and the other 13 in the second year of the study. A distance of at least 330 meters on the 6MWT, or a NSAA score of 18 at baseline reduced significantly the risk of losing ambulation within 2 years. Conclusions These results can be of help at the time of using inclusion criteria for a study in ambulant patients in order to minimize the risk of patients who may lose ambulation within the time of the trial.

Affinity proteomics within rare diseases: a BIO-NMD study for blood biomarkers of muscular dystrophies

Despite the recent progress in the broad‐scaled analysis of proteins in body fluids, there is still a lack in protein profiling approaches for biomarkers of rare diseases. Scarcity of samples is the main obstacle hindering attempts to apply discovery driven protein profiling in rare diseases. We addressed this challenge by combining samples collected within the BIO‐NMD consortium from four geographically dispersed clinical sites to identify protein markers associated with muscular dystrophy using an antibody bead array platform with 384 antibodies. Based on concordance in statistical significance and confirmatory results obtained from analysis of both serum and plasma, we identified eleven proteins associated with muscular dystrophy, among which four proteins were elevated in blood from muscular dystrophy patients: carbonic anhydrase III (CA3) and myosin light chain 3 (MYL3), both specifically expressed in slow‐twitch muscle fibers and mitochondrial malate dehydrogenase 2 (MDH2) and electron transfer flavoprotein A (ETFA). Using age‐matched sub‐cohorts, 9 protein profiles correlating with disease progression and severity were identified, which hold promise for the development of new clinical tools for management of dystrophinopathies.

A preliminary randomized study of growth hormone administration in Becker and Duchenne muscular dystrophies

AIM Since growth hormone (GH) has proven beneficial in experimental heart failure, and the natural history of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) is frequently complicated by the development of dilated cardiomyopathy, we administered GH to six patients with DMD and 10 with BMD, with the evidence of cardiac involvement. METHODS AND RESULTS Patients were randomized to receive for 3 months either placebo or recombinant human GH, in a double-blind fashion. In GH-treated patients, left ventricular (LV) mass increased by 16% in BMD and by 29% in DMD (both p<0.01), with a significant increase of relative wall thickness (+19%). Systemic blood pressure remained unchanged, while LV end-systolic stress fell significantly by 13% in BMD and by 33% in DMD, with a slight increase of systolic function indexes. No changes were observed related to cardiac arrhythmias and skeletal muscle function in the patient groups during the treatment period, nor any side effects were observed. Brain natriuretic peptide, interleukin-6, and tumor necrosis factor-alpha circulating levels were elevated at baseline. While brain natriuretic peptide decreased by 40%, cytokine levels did not exhibit significant variations during the treatment period. CONCLUSIONS The 3-month GH therapy in patients with DMD and BMD induces a hypertrophic response associated with a significant reduction of brain natriuretic peptide plasma levels and a slight improvement of systolic function, no changes in skeletal muscle function, and no side effects.

6 Minute walk test in Duchenne MD patients with different mutations: 12 month changes.

Objective In the last few years some of the therapeutical approaches for Duchenne muscular dystrophy (DMD) are specifically targeting distinct groups of mutations, such as deletions eligible for skipping of individual exons. The aim of this observational study was to establish whether patients with distinct groups of mutations have different profiles of changes on the 6 minute walk test (6MWT) over a 12 month period. Methods The 6MWT was performed in 191 ambulant DMD boys at baseline and 12 months later. The results were analysed using a test for heterogeneity in order to establish possible differences among different types of mutations (deletions, duplications, point mutations) and among subgroups of deletions eligible to skip individual exons. Results At baseline the 6MWD ranged between 180 and 560,80 metres (mean 378,06, SD 74,13). The 12 month changes ranged between −325 and 175 (mean −10.8 meters, SD 69.2). Although boys with duplications had better results than those with the other types of mutations, the difference was not significant. Similarly, boys eligible for skipping of the exon 44 had better baseline results and less drastic changes than those eligible for skipping exon 45 or 53, but the difference was not significant. Conclusions even if there are some differences among subgroups, the mean 12 month changes in each subgroup were all within a narrow Range: from the mean of the whole DMD cohort. This information will be of help at the time of designing clinical trials with small numbers of eligible patients.

Muscular dystrophy with marked Dysferlin deficiency is consistently caused by primary dysferlin gene mutations.

Dysferlin is a 237-kDa transmembrane protein involved in calcium-mediated sarcolemma resealing. Dysferlin gene mutations cause limb-girdle muscular dystrophy (LGMD) 2B, Miyoshi myopathy (MM) and distal myopathy of the anterior tibialis. Considering that a secondary Dysferlin reduction has also been described in other myopathies, our original goal was to identify cases with a Dysferlin deficiency without dysferlin gene mutations. The dysferlin gene is huge, composed of 55 exons that span 233 140 bp of genomic DNA. We performed a thorough mutation analysis in 65 LGMD/MM patients with ≤20% Dysferlin. The screening was exhaustive, as we sequenced both genomic DNA and cDNA. When required, we used other methods, including real-time PCR, long PCR and array CGH. In all patients, we were able to recognize the primary involvement of the dysferlin gene. We identified 38 novel mutation types. Some of these, such as a dysferlin gene duplication, could have been missed by conventional screening strategies. Nonsense-mediated mRNA decay was evident in six cases, in three of which both alleles were only detectable in the genomic DNA but not in the mRNA. Among a wide spectrum of novel gene defects, we found the first example of a ‘nonstop’ mutation causing a dysferlinopathy. This study presents the first direct and conclusive evidence that an amount of Dysferlin ≤20% is pathogenic and always caused by primary dysferlin gene mutations. This demonstrates the high specificity of a marked reduction of Dysferlin on western blot and the value of a comprehensive molecular approach for LGMD2B/MM diagnosis.

Risk of arrhythmia in type I Myotonic Dystrophy: the role of clinical and genetic variables

Objective: To examine the association between the presence of arrhythmia in type 1 myotonic dystrophy (DM1) and clinical–genetic variables, evaluating their role as predictors of the risk of arrhythmia. Methods: 245 patients with genetically proven DM1 underwent clinical and non-invasive cardiological evaluation. Severity of muscular involvement was assessed according to the 5 point Muscular Disability Rating Score (MDRS). Data were analysed by univariate and multivariate models. Results: 245 patients were examined and cardiac arrhythmias were found in 63 subjects, 40 of whom required a device implant. Statistical analyses revealed that men had more than double the risk of developing arrhythmias compared with women (p = 0.018). Addition of each year of age caused an increased risk of arrhythmia equal to 3% (p = 0.030). Subjects with MDRS 5 had a risk of arrhythmia 12 times higher than patients with MDRS 1–2 (p<0.001). Although all of these variables were significantly associated with cardiac rhythm dysfunction, they had a low sensitivity for the prediction of arrhythmic risk Conclusion: Male sex, age and muscular disability were strongly associated with the development of arrhythmia in DM1. However, all of these variables were weak predictors of arrhythmic risk. These results suggest that other factors may be involved in the development of cardiac conduction abnormalities in DM1.

Risk of arrhythmias in myotonic dystrophy: trial design of the RAMYD study.

Objective Myotonic dystrophy type 1 (DM1) is the most frequent muscular dystrophy in adults. DM1 is a multisystem disorder also affecting the heart with an increased incidence of sudden death, which has been explained with the common impairment of the conduction system often requiring pacemaker implantation; however, the occurrence of sudden death despite pacemaker implantation and the observation of major ventricular arrhythmias generated the hypothesis that ventricular arrhythmias may play a causal role as well. The aim of the study was to assess the 2-year cumulative incidence and the value of noninvasive and invasive findings as predictive factors for sudden death, resuscitated cardiac arrest, ventricular fibrillation, sustained ventricular tachycardia and severe sinus dysfunction or high-degree atrioventricular block. Methods/design More than 500 DM1 patients will be evaluated at baseline with a clinical interview, 12-lead ECG, 24-h ECG and echocardiogram. Conventional and nonconventional indications to electrophysiological study, pacemaker, implantable cardioverter defibrillator or loop recorder implantation have been developed. In the case of an indication to electrophysiological study, pacemaker, implantable cardioverter defibrillator or loop recorder implant at baseline or at follow-up, the patient will be referred for the procedure. At the end of 2-year follow-up, all candidate prognostic factors will be tested for their association with the endpoints. Trial registration: ClinicalTrials.gov ID NCT00127582. Conclusion The available evidence supports the hypothesis that both bradyarrhythmias and tachyarrhythmias may cause sudden death in DM1, but the course of cardiac disease in DM1 is still unclear. We expect that this large, prospective, multicenter study will provide evidence to improve diagnostic and therapeutic strategies in DM1.