A. Armaroli

Dystrophin quantification and clinical correlations in Becker muscular dystrophy: implications for clinical trials.

Duchenne muscular dystrophy is caused by mutations in the DMD gene that disrupt the open reading frame and prevent the full translation of its protein product, dystrophin. Restoration of the open reading frame and dystrophin production can be achieved by exon skipping using antisense oligonucleotides targeted to splicing elements. This approach aims to transform the Duchenne muscular dystrophy phenotype to that of the milder disorder, Becker muscular dystrophy, typically caused by in-frame dystrophin deletions that allow the production of an internally deleted but partially functional dystrophin. There is ongoing debate regarding the functional properties of the different internally deleted dystrophins produced by exon skipping for different mutations; more insight would be valuable to improve and better predict the outcome of exon skipping clinical trials. To this end, we have characterized the clinical phenotype of 17 patients with Becker muscular dystrophy harbouring in-frame deletions relevant to on-going or planned exon skipping clinical trials for Duchenne muscular dystrophy and correlated it to the levels of dystrophin, and dystrophin-associated protein expression. The cohort of 17 patients, selected exclusively on the basis of their genotype, included 4 asymptomatic, 12 mild and 1 severe patient. All patients had dystrophin levels of >40% of control and significantly higher dystrophin (P = 0.013), β-dystroglycan (P = 0.025) and neuronal nitric oxide synthase (P = 0.034) expression was observed in asymptomatic individuals versus symptomatic patients with Becker muscular dystrophy. Furthermore, grouping the patients by deletion, patients with Becker muscular dystrophy with deletions with an end-point of exon 51 (the skipping of which could rescue the largest group of Duchenne muscular dystrophy deletions) showed significantly higher dystrophin levels (P = 0.034) than those with deletions ending with exon 53. This is the first quantitative study on both dystrophin and dystrophin-associated protein expression in patients with Becker muscular dystrophy with deletions relevant for on-going exon skipping trials in Duchenne muscular dystrophy. Taken together, our results indicate that all varieties of internally deleted dystrophin assessed in this study have the functional capability to provide a substantial clinical benefit to patients with Duchenne muscular dystrophy.

Early corticosteroid treatment in 4 Duchenne muscular dystrophy patients: 14-year follow-up.

Corticosteroid treatment is the standard of care in Duchenne muscular dystrophy (DMD), but the optimal age to initiate treatment and dosage pattern remain a matter of discussion.

Genetic characterization in symptomatic female DMD carriers: lack of relationship between X-inactivation, transcriptional DMD allele balancing and phenotype

BackgroundAlthough Duchenne and Becker muscular dystrophies, X-linked recessive myopathies, predominantly affect males, a clinically significant proportion of females manifesting symptoms have also been reported. They represent an heterogeneous group characterized by variable degrees of muscle weakness and/or cardiac involvement. Though preferential inactivation of the normal X chromosome has long been considered the principal mechanism behind disease manifestation in these females, supporting evidence is controversial.MethodsEighteen females showing a mosaic pattern of dystrophin expression on muscle biopsy were recruited and classified as symptomatic (7) or asymptomatic (11), based on the presence or absence of muscle weakness. The causative DMD gene mutations were identified in all cases, and the X-inactivation pattern was assessed in muscle DNA. Transcriptional analysis in muscles was performed in all females, and relative quantification of wild-type and mutated transcripts was also performed in 9 carriers. Dystrophin protein was quantified by immunoblotting in 2 females.ResultsThe study highlighted a lack of relationship between dystrophic phenotype and X-inactivation pattern in females; skewed X-inactivation was found in 2 out of 6 symptomatic carriers and in 5 out of 11 asymptomatic carriers. All females were characterized by biallelic transcription, but no association was found between X-inactivation pattern and allele transcriptional balancing. Either a prevalence of wild-type transcript or equal proportions of wild-type and mutated RNAs was observed in both symptomatic and asymptomatic females. Moreover, very similar levels of total and wild-type transcripts were identified in the two groups of carriers.ConclusionsThis is the first study deeply exploring the DMD transcriptional behaviour in a cohort of female carriers. Notably, no relationship between X-inactivation pattern and transcriptional behaviour of DMD gene was observed, suggesting that the two mechanisms are regulated independently. Moreover, neither the total DMD transcript level, nor the relative proportion of the wild-type transcript do correlate with the symptomatic phenotype.

Cyclosporine A in Ullrich congenital muscular dystrophy: long-term results.

Six individuals with Ullrich congenital muscular dystrophy (UCMD) and mutations in the genes-encoding collagen VI, aging 5–9, received 3–5 mg/kg of cyclosporine A (CsA) daily for 1 to 3.2 years. The primary outcome measure was the muscle strength evaluated with a myometer and expressed as megalimbs. The megalimbs score showed significant improvement (P = 0.01) in 5 of the 6 patients. Motor function did not change. Respiratory function deteriorated in all. CsA treatment corrected mitochondrial dysfunction, increased muscle regeneration, and decreased the number of apoptotic nuclei. Results from this study demonstrate that long-term treatment with CsA ameliorates performance in the limbs, but not in the respiratory muscles of UCMD patients, and that it is well tolerated. These results suggest considering a trial of CsA or nonimmunosuppressive cyclosporins, that retains the PTP-desensitizing properties of CsA, as early as possible in UCMD patients when diaphragm is less compromised.

Biochemical characterization of patients with in-frame or out-of-frame DMD deletions pertinent to exon 44 or 45 skipping

IMPORTANCE In Duchenne muscular dystrophy (DMD), the reading frame of an out-of-frame DMD deletion can be repaired by antisense oligonucleotide (AO)-mediated exon skipping. This creates a shorter dystrophin protein, similar to those expressed in the milder Becker muscular dystrophy (BMD). The skipping of some exons may be more efficacious than others. Patients with exon 44 or 45 skippable deletions (AOs in clinical development) have a less predictable phenotype than those skippable for exon 51, a group in advanced clinical trials. A way to predict the potential of AOs is the study of patients with BMD who have deletions that naturally mimic those that would be achieved by exon skipping. OBJECTIVE To quantify dystrophin messenger RNA (mRNA) and protein expression in patients with DMD deletions treatable by, or mimicking, exon 44 or 45 skipping. DESIGN, SETTING, AND PARTICIPANTS Retrospective study of nondystrophic controls (n = 2), patients with DMD (n = 5), patients with intermediate muscular dystrophy (n = 3), and patients with BMD (n = 13) at 4 university-based academic centers and pediatric hospitals. Biochemical analysis of existing muscle biopsies was correlated with the severity of the skeletal muscle phenotype. MAIN OUTCOMES AND MEASURES Dystrophin mRNA and protein expression. RESULTS Patients with DMD who have out-of-frame deletions skippable for exon 44 or 45 had an elevated number of revertant and trace dystrophin expression (approximately 19% of control, using quantitative immunohistochemistry) with 4 of 9 patients presenting with an intermediate muscular dystrophy phenotype (3 patients) or a BMD-like phenotype (1 patient). Corresponding in-frame deletions presented with predominantly mild BMD phenotypes and lower dystrophin levels (approximately 42% of control) than patients with BMD modeling exon 51 skipping (approximately 80% of control). All 12 patients with in-frame deletions had a stable transcript compared with 2 of 9 patients with out-of-frame deletions (who had intermediate muscular dystrophy and BMD phenotypes). CONCLUSIONS AND RELEVANCE Exon 44 or 45 skipping will likely yield lower levels of dystrophin than exon 51 skipping, although the resulting protein is functional enough to often maintain a mild BMD phenotype. Dystrophin transcript stability is an important indicator of dystrophin expression, and transcript instability in DMD compared with BMD should be explored as a potential biomarker of response to AOs. This study is beneficial for the planning, execution, and analysis of clinical trials for exon 44 and 45 skipping.

A patient with limb girdle muscular dystrophy carries a TRIM32 deletion, detected by a novel CGH array, in compound heterozygosis with a nonsense mutation

Limb girdle muscular dystrophy 2H is a rare autosomal recessive muscular dystrophy, clinically highly variable, caused by mutations in the TRIM32 gene. Here we describe a 35-years-old who experienced progressive muscle weakness. The muscle biopsy revealed an unspecific pattern of atrophic and hypertrophic fibers; the immunohistochemistry for several proteins was normal. Comparative genomic hybridization (CGH) analysis showed a heterozygous deletion of the entire TRIM32 gene. On the other allele we identified the R316X nonsense mutation. The genetic diagnosis of LGMD2H in this case was reached by using a novel high throughput diagnostic tool.

Recessive mutations in MSTO1 cause mitochondrial dynamics impairment, leading to myopathy and ataxia

We report here the first families carrying recessive variants in the MSTO1 gene: compound heterozygous mutations were identified in two sisters and in an unrelated singleton case, who presented a multisystem complex phenotype mainly characterized by myopathy and cerebellar ataxia. Human MSTO1 is a poorly studied protein, suggested to have mitochondrial localization and to regulate morphology and distribution of mitochondria. As for other mutations affecting genes involved in mitochondrial dynamics, no biochemical defects typical of mitochondrial disorders were reported. Studies in patients’ fibroblasts revealed that MSTO1 protein levels were strongly reduced, the mitochondrial network was fragmented, and the fusion events among mitochondria were decreased, confirming the deleterious effect of the identified variants and the role of MSTO1 in modulating mitochondrial dynamics. We also found that MSTO1 is mainly a cytosolic protein. These findings indicate recessive mutations in MSTO1 as a new cause for inherited neuromuscular disorders with multisystem features.

Deep RNA profiling identified CLOCK and molecular clock genes as pathophysiological signatures in collagen VI myopathy

ABSTRACT Collagen VI myopathies are genetic disorders caused by mutations in collagen 6 A1, A2 and A3 genes, ranging from the severe Ullrich congenital muscular dystrophy to the milder Bethlem myopathy, which is recapitulated by collagen-VI-null (Col6a1−/−) mice. Abnormalities in mitochondria and autophagic pathway have been proposed as pathogenic causes of collagen VI myopathies, but the link between collagen VI defects and these metabolic circuits remains unknown. To unravel the expression profiling perturbation in muscles with collagen VI myopathies, we performed a deep RNA profiling in both Col6a1−/− mice and patients with collagen VI pathology. The interactome map identified common pathways suggesting a previously undetected connection between circadian genes and collagen VI pathology. Intriguingly, Bmal1−/− (also known as Arntl) mice, a well-characterized model displaying arrhythmic circadian rhythms, showed profound deregulation of the collagen VI pathway and of autophagy-related genes. The involvement of circadian rhythms in collagen VI myopathies is new and links autophagy and mitochondrial abnormalities. It also opens new avenues for therapies of hereditary myopathies to modulate the molecular clock or potential gene–environment interactions that might modify muscle damage pathogenesis. Summary: A new association between hereditary myopathy and CLOCK circuit genes opens novel avenues of research on circadian genes and their role in skeletal muscle hereditary pathologies.

Becker muscular dystrophy due to an intronic splicing mutation inducing a dual dystrophin transcript.

We describe a 29-year-old patient who complained of left thigh muscle weakness since he was 23 and of moderate proximal weakness of both lower limbs with difficulty in climbing stairs and running since he was 27. Mild weakness of iliopsoas and quadriceps muscles and muscle atrophy of both the distal forearm and thigh were observed upon clinical examination. He harboured a novel c.1150-3C>G substitution in the DMD gene, affecting the intron 10 acceptor splice site and causing exon 11 skipping and an out-of-frame transcript. However, protein of normal molecular weight but in reduced amounts was observed on Western Blot analysis. Reverse transcription analysis on muscle RNA showed production, via alternative splicing, of a transcript missing exon 11 as well as a low abundant full-length transcript which is enough to avoid the severe Duchenne phenotype. Our study showed that a reduced amount of full length dystrophin leads to a mild form of Becker muscular dystrophy. These results confirm earlier findings that low amounts of dystrophin can be associated with a milder phenotype, which is promising for therapies aiming at dystrophin restoration.

Huntington's disease-like presentation in Spinocerebellar ataxia type 12.

Spinocerebellar ataxia type 12 (SCA12), caused by a CAG repeat expansion in PPP2R2B gene, is characterized by mild, slowly progressive cerebellar syndrome with postural and action tremor as the presenting and preeminent sign. Cognitive decline is rare and occurs late in the disease. The reported psychiatric disturbances consist of mild depression and anxiety. Additional features are parkinsonism and hyper-reflexia. Neuroimaging shows a prevalent atrophy of the cerebral cortex with relative sparing of the cerebellum; this aspect is considered a hallmark of SCA12. In the European population, SCA12 has been originally described in German patients. In Italy, Brussino and colleagues identified 2 SCA12 patients, both coming from the city of Ferrara in the northeast of the country (Supporting Table 1). A 53-year-old man developed postural instability and occasional choreodystonic movements. From the age of 30, he presented “bizarre” behavior with selfand other-directed aggressiveness. At the age of 54, he became impulsive and violent, presented motor and vocal tics and stereotypies involving face, trunk, and limbs. Choreoathetoid movements in the lower limbs became more pronounced. Cerebellar dysartria, dysmetria, gait ataxia, and mild bradikynesia were also evident. Cognitive function assessment revealed moderate encoding difficulties, severe recall memory disturbance, fatuity, and verbal disinhibition. At the age of 56, he was completely dependent, frontal in nature, with frequent falls. He refused any symptomatic medication. Brain MRI revealed diffuse cortical and subcortical atrophy in absence of significant cerebellar atrophy (Fig. 1A,B). The proband’s mother, at the age of 50, showed mild choreic mixed with stereotypic and dystonic movements at upper limbs, became severely depressed, rapidly demented, and wheelchair bound. A similar condition was referred in several deceased maternal relatives (Fig. 1C). Based on the early clinical presentation, Huntington’s disease (HD) was molecularly ruled out as first. Extensive investigations, which included ceruloplasmin, acanthocytes, lipoproteins, hereditary metabolic screen, and genetic testing for FRAX-A, DRPLA, SCA1, 2, 3, 6, 7, and 17, FRDA, PRNP, and C9Orf72 genes, were also normal. Despite the atypical presentation, SCA12 was considered in light of the geographical origin (Ferrara) of the family. Heterozygosis for an expanded allele with 63 CAG repeats within the 50 untranslated region region of the PPP2R2B gene was found in the proband and in his mother (genotype CAG (63)/CAG(10)[3]; see Video). Differently from the American-German descendent index case, the majority of Indian, and the Italian SCA12 probands, none of our cases manifested tremor. In our proband, the onset was in the third decade with rapidly progressive psychiatric and cognitive impairment and choreodystonic movements. In literature, slow progression with normal life span is described, whereas our patients became fully dependent in less than 5 years. The reported case represents the third SCA12 family of Italian origin and is from the same municipality of the 2 previously reported patients, suggesting a possible founder effect and prompting SCA12 inclusion within “SCAs screening genetic panel” for this population. Furthermore, the HDlike presentation expands the phenotypic spectrum of SCA12, enlarging the clinical indication for SCA12 genetic testing in ataxic patients.