Adele D'Amico

De novo LMNA mutations cause a new form of congenital muscular dystrophy

To describe a new entity of congenital muscular dystrophies caused by de novo LMNA mutations.

miRNAs as serum biomarkers for Duchenne muscular dystrophy

Dystrophin absence in Duchenne muscular dystrophy (DMD) causes severe muscle degeneration. We describe that, as consequence of fibre damage, specific muscle‐miRNAs are released in to the bloodstream of DMD patients and their levels correlate with the severity of the disease. The same miRNAs are abundant also in the blood of mdx mice and recover to wild‐type levels in animals ‘cured’ through exon skipping. Even though creatine kinase (CK) blood levels have been utilized as diagnostic markers of several neuromuscular diseases, including DMD, we demonstrate that they correlate less well with the disease severity. Although the analysis of a larger number of patients should allow to obtain more refined correlations with the different stages of disease progression, we propose that miR‐1, miR‐133, and miR‐206 are new and valuable biomarkers for the diagnosis of DMD and possibly also for monitoring the outcomes of therapeutic interventions in humans. Despite many different DMD therapeutic approaches are now entering clinical trials, a unifying method for assessing the benefit of different treatments is still lacking.

Randomized, double-blind, placebo-controlled trial of phenylbutyrate in spinal muscular atrophy

Objective: To assess the efficacy of phenylbutyrate (PB) in patients with spinal muscular atrophy in a randomized, double-blind, placebo-controlled trial involving 10 Italian centers. Methods: One hundred seven children were assigned to receive PB (500 mg/kg/day) or matching placebo on an intermittent regimen (7 days on/7 days off) for 13 weeks. The Hammersmith functional motor scale (primary outcome measure), myometry, and forced vital capacity were assessed at baseline and at weeks 5 and 13. Results: Between January and September 2004, 107 patients aged 30 to 154 months were enrolled. PB was well tolerated, with only one child withdrawing because of adverse events. Mean improvement in functional score was 0.60 in the PB arm and 0.73 in placebo arm (p = 0.70). Changes in the secondary endpoints were also similar in the two study arms. Conclusions: Phenylbutyrate was not effective at the regimen, schedule, and duration used in this study.

Pilot trial of phenylbutyrate in spinal muscular atrophy

The aim of this study was to evaluate tolerability and efficacy of phenylbutyrate (PB) in patients with spinal muscular atrophy (SMA). Ten patients with SMA type II confirmed by DNA studies (age range 2.6-12.7 years, mean age 6.01) were started on oral PB (triButyrate) in powder or tablets. The dosage was 500 mg/kg per day (maximum dose 19 g/d), divided in five doses (every 4 h, skipping one night-dose) using an intermittent schedule (7 days on and 7 days off). Measures of efficacy were the change in motor function from baseline to 3 and 9 weeks, by means of the Hammersmith functional motor scale. In children older than 5 years, muscle strength, assessed by myometry, and forced vital capacity were also measured. We found a significant increase in the scores of the Hammersmith functional scale between the baseline and both 3-weeks (P < 0.012) and 9-weeks assessments (P < 0.004). Our results indicate that PB might be beneficial to SMA patients without producing any major side effect. Larger prospective randomised, double-blind, placebo controlled trials are needed to confirm these preliminary findings.

Functional changes in Duchenne muscular dystrophy A 12-month longitudinal cohort study

Objective: The aim of the study was to assess different outcome measures in a cohort of ambulant boys with Duchenne muscular dystrophy (DMD) over 12 months in order to establish the spectrum of possible changes in relation to age and steroid treatment. Methods: The study is a longitudinal multicentric cohort study. A total of 106 ambulant patients with DMD were assessed using the 6-minute walk test (6MWT) and North Star Ambulatory Assessment (NSAA) at baseline and 12 months. Clinical data including age and steroid treatment were collected. Results: During the 12 months of the study, we observed a mean decline of 25.8 meters in the 6MWT with a SD of 74.3 meters. On NSAA, the mean decline was 2.2 points with a SD of 3.7. Not all the boys with DMD in our cohort showed a decline over the 12 months, with young boys showing some improvement in their 6MWT and NSAA scores up to the age of 7. NSAA and the 6MWT had the highest correlation (r = 0.52, p < 0.001). Conclusions: This study provides longitudinal data of NSAA and 6MWT over a 12-month period. These data can be useful when designing a clinical trial.

Spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease characterized by degeneration of alpha motor neurons in the spinal cord, resulting in progressive proximal muscle weakness and paralysis. Estimated incidence is 1 in 6,000 to 1 in 10,000 live births and carrier frequency of 1/40-1/60. This disease is characterized by generalized muscle weakness and atrophy predominating in proximal limb muscles, and phenotype is classified into four grades of severity (SMA I, SMAII, SMAIII, SMA IV) based on age of onset and motor function achieved. This disease is caused by homozygous mutations of the survival motor neuron 1 (SMN1) gene, and the diagnostic test demonstrates in most patients the homozygous deletion of the SMN1 gene, generally showing the absence of SMN1 exon 7. The test achieves up to 95% sensitivity and nearly 100% specificity. Differential diagnosis should be considered with other neuromuscular disorders which are not associated with increased CK manifesting as infantile hypotonia or as limb girdle weakness starting later in life.Considering the high carrier frequency, carrier testing is requested by siblings of patients or of parents of SMA children and are aimed at gaining information that may help with reproductive planning. Individuals at risk should be tested first and, in case of testing positive, the partner should be then analyzed. It is recommended that in case of a request on carrier testing on siblings of an affected SMA infant, a detailed neurological examination should be done and consideration given doing the direct test to exclude SMA. Prenatal diagnosis should be offered to couples who have previously had a child affected with SMA (recurrence risk 25%). The role of follow-up coordination has to be managed by an expert in neuromuscular disorders and in SMA who is able to plan a multidisciplinary intervention that includes pulmonary, gastroenterology/nutrition, and orthopedic care. Prognosis depends on the phenotypic severity going from high mortality within the first year for SMA type 1 to no mortality for the chronic and later onset forms.

Phenotypic clustering of lamin A/C mutations in neuromuscular patients

Background: Mutations in the LMNA gene, encoding human lamin A/C, have been associated with an increasing number of disorders often involving skeletal and cardiac muscle, but no clear genotype/phenotype correlation could be established to date. Methods: We analyzed the LMNA gene in a large cohort of patients mainly affected by neuromuscular or cardiac disease and clustered mutated patients in two groups to unravel possible correlations. Results: We identified 28 variants, 9 of which reported for the first time. The two groups of patients were characterized by clinical and genetic differences: 1) patients with childhood onset displayed skeletal muscle involvement with predominant scapuloperoneal and facial weakness associated with missense mutations; 2) patients with adult onset mainly showed cardiac disorders or myopathy with limb girdle distribution, often associated with frameshift mutations presumably leading to a truncated protein. Conclusions: Our findings, supported by meta-analysis of previous literature, suggest the presence of two different pathogenetic mechanisms: late onset phenotypes may arise through loss of function secondary to haploinsufficiency, while dominant negative or toxic gain of function mechanisms may explain the severity of early phenotypes. This model of patient stratification may help patient management and facilitate future studies aimed at deciphering lamin A/C pathogenesis.

GM1 gangliosidosis and Morquio B disease: an update on genetic alterations and clinical findings

GM1 gangliosidosis and Morquio B syndrome, both arising from beta-galactosidase (GLB1) deficiency, are very rare lysosomal storage diseases with an incidence of about 1:100,000-1:200,000 live births worldwide. Here we report the beta-galactosidase gene (GLB1) mutation analysis of 21 unrelated GM1 gangliosidosis patients, and of 4 Morquio B patients, of whom two are brothers. Clinical features of the patients were collected and compared with those in literature. In silico analyses were performed by standard alignments tools and by an improved version of GLB1 three-dimensional models. The analysed cohort includes remarkable cases. One patient with GM1 gangliosidosis had a triple X syndrome. One patient with juvenile GM1 gangliosidosis was homozygous for a mutation previously identified in Morquio type B. A patient with infantile GM1 gangliosidosis carried a complex GLB1 allele harbouring two genetic variants leading to p.R68W and p.R109W amino acid changes, in trans with the known p.R148C mutation. Molecular analysis showed 27 mutations, 9 of which are new: 5 missense, 3 microdeletions and a nonsense mutation. We also identified four new genetic variants with a predicted polymorphic nature that was further investigated by in silico analyses. Three-dimensional structural analysis of GLB1 homology models including the new missense mutations and the p.R68W and p.R109W amino acid changes showed that all the amino acid replacements affected the resulting protein structures in different ways, from changes in polarity to folding alterations. Genetic and clinical associations led us to undertake a critical review of the classifications of late-onset GM1 gangliosidosis and Morquio B disease.

Attention Deficit Hyperactivity Disorder and Cognitive Function in Duchenne Muscular Dystrophy: Phenotype-Genotype Correlation

OBJECTIVES To assess attention deficit hyperactivity disorder (ADHD) in boys affected by Duchenne muscular dystrophy (DMD) and to explore the relationship with cognitive abilities and genetic findings. STUDY DESIGN Boys with DMD (n = 103; 4-17 years of age, mean: 12.6) were assessed using a cognitive test (Wechsler scales). Assessment of ADHD was based on the Diagnostic Statistical Manual, Fourth Edition, Text Revision criteria and on the long version of the Conners Parents and Teachers Rating Scales. RESULTS ADHD was found in 33 of the 103 boys with DMD. Attention problems together with hyperactivity (17/33) or in isolation (15/33) were more frequent than hyperactivity alone, which was found in 1 patient. Intellectual disability (ID) was found in 27/103 (24.6%). Sixty-two of the 103 boys had no ID and no ADHD, 9 had ID but no ADHD, 14 had ADHD but no ID, and 18 had both. ADHD occurred more frequently in association with mutations predicted to affect Dp140 expression (exon 45-55) and in those with mutations predicted to affect all dystrophin product, including Dp71 (ie, those that have promoter region and specific first exon between exons 62 and 63 but were also relatively frequent). CONCLUSIONS Our results suggest that ADHD is a frequent feature in DMD. The risk of ADHD appears to be higher in patients carrying mutations predicted to affect dystrophin isoforms expressed in the brain and are known to be associated with higher risk of cognitive impairment.

Salbutamol increases survival motor neuron (SMN) transcript levels in leucocytes of spinal muscular atrophy (SMA) patients: relevance for clinical trial design

Background Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by mutations of the SMN1 gene. Based on severity, three forms of SMA are recognised (types I–III). All patients usually have 2–4 copies of a highly homologous gene (SMN2) which produces insufficient levels of functional survival motor neuron (SMN) protein. Recently, evidence has been provided that SMN2 expression can be enhanced in vitro by salbutamol, a β2-adrenergic agonist. This compound has also been shown to improve motor function of SMA patients in two different pilot trials. Aim To evaluate the in vivo molecular efficacy of salbutamol in SMA patients. Methods Twelve type II–III patients took salbutamol orally for 6 months. SMN2 full length transcript levels were determined in peripheral blood leucocytes by absolute real-time PCR, at baseline and after 3 and 6 months of treatment. Results A significant and constant increase in SMN2 full length transcript levels was detected; the response was directly proportional to SMN2 gene copy number. Conclusions The data strongly support salbutamol as a candidate for treating SMA, and suggest that SMN2 copy number may predict the molecular response to treatment and may be a useful randomisation parameter in a double blind placebo controlled clinical trial design.