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Clinical trial preparedness in facioscapulohumeral muscular dystrophy: Clinical, tissue, and imaging outcome measures 29–30 May 2015, Rochester, New York Rabi Tawil *, George W. Padberg , Dennis W. Shaw , Silvere M. van der Maarel , Stephen J. Tapscott , The FSHD Workshop Participants 1 a Department of Neurology, University of Rochester Medical Center, Rochester, NY, USA b Department of Neurology, Radboud University Medical Center, Nijmegen, The Netherlands c Department of Radiology, Seattle Children’s Hospital, Seattle, WA, USA d Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands e Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

Laing distal myopathy is an autosomal dominant disease due to mutations in the gene encoding for the human slow-β myosin heavy chain, MYH7. Most reports describe it as a mild, early onset myopathy with involvement usually restricted to foot extensors, hand finger extensors and neck flexors, and unspecific findings on muscle biopsy. We identified the first two Italian families with Laing distal myopathy, harboring two novel mutations in the MYH7 gene and performed clinical, neurophysiological, pathological, muscle MRI and cardiological investigations on affected members from the two families. Subjects from one family presented a moderate-severe phenotype, with proximal together with distal involvement and even loss of ambulation at advanced age. One patient displayed atypical muscle biopsy findings including cytoplasmic bodies and myofibrillar myopathy-like features. Affected members from the second family shared a very mild phenotype, with weakness largely limited to long toe and foot extensors and/or late onset. No patient showed any sign of heart involvement. Our study significantly broadens the clinical and pathological spectrum of Laing distal myopathy. We suggest that MYH7 screening should be considered in undiagnosed late-onset distal myopathy or cytoplasmic body myopathy patients.

Charcot-Marie-Tooth disease (CMT) is a group of clinically and genetically heterogeneous neuropathies classically divided into demyelinating (CMT1) and axonal forms (CMT2). The most common demyelinating form is CMT1A, due to a duplication in the gene encoding the peripheral myelin protein 22 (PMP22). Less frequently, mutations in the myelin protein zero gene (MPZ/P0) account for demyelinating CMT1B. Herein, we report a patient presenting with an isolated hyperCKemia in whom electrophysiological and pathological findings revealed a demyelinating and axonal neuropathy. Sequencing of the MPZ gene revealed a 306delA at codon 102 in the proband and in two relatives. This mutation has been already described in association with paucisymptomatic CMT without hyperCKemia.

Ageing is thought to participate to the pathogenesis of sporadic inclusion-body myositis (s-IBM). Although the regenerative potential of s-IBM muscle is reduced in vivo, age-related abnormalities of satellite cells possibly accounting for the decline of muscle repair have not been demonstrated. Here we show that proliferation rate and clonogenicity of s-IBM myoblasts are significantly lower and doubling time is longer than normal age-matched controls, indicating that proliferative capacity of s-IBM muscles becomes exhausted earlier. Telomere shortening is detected in s-IBM cells suggesting premature senescence. Differently from controls, s-IBM myoblasts show increased active β-catenin mainly localized within myonuclei, indicating active Wnt stimulation. After many rounds of muscle growth, only s-IBM myoblasts accumulate congophilic inclusions and immunoreactive Aβ 1–40 deposits. Therefore, s-IBM myoblasts seem to have a constitutively impaired regenerative capacity and the intrinsic property, upon sufficient aging in vitro, to accumulate Aβ. Our results might be valuable in understanding molecular mechanisms associated with muscle aging underlying the defective regeneration of s-IBM muscle and provide new clues for future therapeutic strategies.

Facioscapulohumeral dystrophy (FSHD) is a muscular disease usually spreading from upper to lower body and characterised by asymmetric muscle weakness. Walking ability is compromised in these patients, with a consequent high risk of falls. A quantitative analysis of the upper body oscillations may unveil useful information about the capacity of these patients to stabilise the head, maintain balance, and compensate for lower limb muscle weakness during walking. This study involved 13 patients with FSHD and 13 healthy volunteers. The trajectories of three points located on the cranio-caudal axis, at head, shoulder, and pelvis levels, during level walking, were analysed. The range of motion of these three points and the attenuation of the relevant accelerations going from pelvis to head level were used to describe the upper body movements during walking. The patients had wider and less symmetrical oscillations than the healthy controls both in antero-posterior and medio-lateral directions. Furthermore, the capacity of the patients to attenuate the accelerations going from pelvis to head level was reduced. These features may be related not only to upper body muscle weakness, but also to a strategy functional to the compensation of proximal leg muscle weakness. In conclusion, this study highlighted that the control of upper body oscillations and of head stability is reduced in patients with FSHD, suggesting that the assessment of the upper body movements should be included in the treatment decision process.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most frequent forms of muscular dystrophy. The aims of this study were: 1) to evaluate the prevalence of sleep disordered breathing (SDB) in patients with FSHD; 2) to define the sleep-related respiratory patterns in FSHD patients with SDB; and 3) to find the clinical predictors of SDB. Fifty-one consecutive FSHD patients were enrolled, 23 women, mean age 45.7+/-12.3 years (range: 26-72). The diagnosis of FSHD was confirmed by genetic tests. All patients underwent medical and neurological evaluations, subjective evaluation of sleep and full-night laboratory-based polysomnography. Twenty patients presented SDB: 13 presented obstructive apneas, four presented REM related oxygen desaturations and three showed a mixed pattern. Three patients needed positive airways pressure. SDB was not related to the severity of the disease. Body mass index, neck circumference and daytime sleepiness did not allow prediction of SDB. In conclusion, the results suggest a high prevalence of SDB in patients with FSHD. The presence of SDB does not depend on the clinical severity of the disease. SDB is often asymptomatic, and no clinical or physical measure can reliably predict its occurrence. A screening of SDB should be included in the clinical assessment of FSHD.

Mutations in POMT1 and POMT2 genes were originally identified in Walker–Warburg syndrome (WWS) and subsequently reported in patients with milder phenotypes characterised by mental retardation with or without brain abnormalities and without ocular malformations. As part of a multicentric Italian study we screened the POMT1 and POMT2 genes in 61 congenital muscular dystrophy (CMD) patients with α-dystroglycan reduction on muscle biopsy and/or clinical and radiological findings suggestive of the known forms of CMD with α-dystroglycan deficiency. The aim of the study was to establish how frequently mutations in POMT1 and POMT2 occur in CMD patients in the Italian population and to evaluate the spectrum of associated phenotypes. Thirteen patients showed mutations in POMT1 and five harboured mutations in POMT2 , accounting for a total of 20 different mutations, eight of which were novel (two in POMT1 and six in POMT2 ). Normal brain MRI associated with mental retardation and microcephaly was the most frequent finding in patients with mutations in POMT1 (six out of 13), but was also found in a patient with POMT2 mutations. Predominant cerebellar hypoplasia was also frequent both in patients with POMT1 (three out of 13) and POMT2 (three out of 5) mutations. A MEB phenotype with frontal cortical dysplasia and pons abnormalities was found in two patients with POMT1 and in one with POMT2 mutations, while a WWS phenotype was only found in a case with mutations in POMT1 . Mutations causing frameshifts and stop codons were responsible for the more severe phenotypes. Our results provide further evidence that, as previously reported for FKRP , the array of mutations in POMT1 and POMT2 is ample and the spectrum of associated phenotypes is wider than initially thought.

Alpha-latrotoxin (alpha-ltx), a component of the venom of black widow spiders (BWSV), binds to higher vertebrates presynaptic nerve terminals, stimulating massive neurotransmitter release. This neurotoxic protein is responsible for most of the symptoms elicited in men by the bite of black widow spider (BWS), i.e. a neurological syndrome named latrodectism. By reasoning that targeting this single component would abrogate most of the effect of BWS envenomation, we took advantage of the antibody phage display technology to generate monoclonal Fab fragments able to bind and neutralize the alpha-ltx. To this aim, we immunized Balb/c mice with purified toxin and cloned their antibody repertoire in the pCombIII phage display vector. By combining a high-stringency affinity selection with a sensitive 45Ca(2+) uptake assay, we isolated a Fab fragment (FM1) able to bind the alpha-ltx in the low nM range and neutralize its ionophore activity, in vitro and in vivo. After the onset of overt symptomatology, administration of FM1 to experimentally envenomed mice induced remission of symptoms and prevented lethality. Since alpha-ltx is the only molecule responsible for the great toxicity of BWS bites in mammals, the FM1 Fab, highly effective in neutralizing the toxin in vivo, represents a promising immunotherapy reagent for treating latrodectic patients.

Objective To evaluate the subjective sleep quality, the prevalence of daytime sleepiness and the risk of sleep-related upper airways obstruction in patients with genetically proven Facioscapulohumeral muscular dystrophy (FSHD). FSHD is an autosomal dominant myopathy, characterized by an early involvement of facial and scapular muscles with eventual spreading to pelvic and lower limb muscles. Patients and methods Forty-six patients were enrolled, 27 women and 19 men, mean age 43.6 ± 14.1 years. Study protocol included: a Clinical Severity Scale (CSS) for FSHD, Pittsburgh Sleep Quality Index (PSQI), Italian version of the Epworth Sleepiness Scale (ESS) and the search for clinical predictors of sleep-related airways obstruction. Results Twenty-seven patients presented snoring, 12 reported respiratory pauses during sleep. One half (23/46) had PSQI scores above the normal threshold (= 5). Correlations were found between the CSS and: the total PSQI score, the components C1 sleep quality, C5 sleep disturbances, C7 daytime dysfunction. Conclusion Our data support the hypothesis that patients with FSHD have an impaired sleep quality, and that this impairment is directly related to the severity of the disease. A systematic polysomnographic evaluation of these patients will be necessary to confirm the presence of sleep disruption and to clarify its pathogenesis.

ObjectivesOur goal was to identify early changes in myocardial physical properties in children with Duchenne muscular dystrophy (DMDch).BackgroundDuchenne muscular dystrophy (DMD) is caused by the absence of dystrophin, which triggers complex molecular and biological events in skeletal and cardiac muscle tissues. Although about 30% of patients display overt signs of cardiomyopathy in the late stage of the disease, it is unknown whether changes in myocardial physical properties can be detected in the early (preclinical) stages of the disease.MethodsWe performed an ultrasonic tissue characterization (UTC) analysis of myocardium in DMDch with normal systolic myocardial function and no signs of cardiomyopathy. Both the cyclic variation of integrated backscatter (cvIBS) and the calibrated integrated backscatter (cIBS) were assessed in 8 myocardial regions of 20 DMDch, age 7 ± 2 years (range 4 to 10 years), and in 20 age-matched healthy controls.ResultsWe found large differences in the UTC data between DMDch and controls; the mean value of cvIBS was 4.4 ± 1.5 dB versus 8.8 ± 0.8 dB, whereas the mean value of cIBS was 36.4 ± 7.1 dB versus 26.9 ± 2.0 dB (p < 10−6for both). In DMDch, all eight sampled segments showed cIBS mean values to be significantly higher and cvIBS mean values to be significantly lower than those in the controls. Finally, interindividual differences were greater in DMDch than in controls for both parameters.ConclusionsThe myocardium in DMDch displays UTC features different from those in healthy controls. These results show that lack of dystrophin is commonly associated with changes in myocardial features well before the onset of changes of systolic function and overt cardiomyopathy.

Emery Dreifuss muscular dystrophy is a genetically heterogeneous disorder characterized by the clinical triad of early onset contractures, progressive muscular wasting and weakness with humeroperoneal distribution and cardiac conduction defects. Mutations in the Lamin A/C (LMNA) gene are responsible for the autosomal dominant and the autosomal recessive forms. Familiar and sporadic patients carrying mutations in the LMNA gene show high variability in the clinical symptomatology and age of onset. In this report, we describe four families harboring missense mutations in the LMNA gene and we show that the effect of mutations ranges from silent to fully penetrant. We suggest that incomplete penetrance of dominant mutations in the LMNA gene is a common feature and we emphasize the significance of mutational analysis in relatives of sporadic cases of laminopathies, as asymptomatic carriers face high risk of sudden cardiac death.

Background Previous studies reported an impairment of nervous autonomic activity in patients with Duchenne muscular dystrophy (DMD). However, the relationship of the autonomic dysfunction to the impairment of cardiac mechanical function and of respiratory failure is not completely understood. Methods We evaluated cardiac autonomic function by time- and frequency-domain heart rate variability (HRV) analysis on 24-hour Holter recordings in 60 patients with DMD (16.8 ± 4.8 years) and 28 healthy control patients (15.2 ± 4.6 years, P = not significant). The circadian rhythm of R-R interval, low frequency, high frequency, and low-frequency/highfrequency ratio was also assessed. In all patients, left ventricular ejection fraction was measured by 2D echocardiography; respiratory function was assessed by spirometry. Results All HRV parameters were lower in patients with DMD than in control subjects, with the percentage of differences between adjacent R-R intervals >50 ms (11.6% ± 8.5% vs 27.3% ± 14.1%, P =.00001) and high frequency (23.9 ± 10.3 ms vs 36.1 ± 12.2 ms, P =.0001) showing the strongest differences. A significant circadian rhythm of HRV variables was present in both groups, but it was considerably flattened in patients with DMD. There was no correlation between left ventricular ejection fraction and HRV indexes except for a weak correlation with high frequency (r = 0.30, P =.02) and with low-frequency to high-frequency ratio (r = –0.29, P

In the majority of facioscapulohumeral muscular dystrophy (FSHD) families (about 95%) the genetic defect has been identified as a deletion of a variable number of KpnI repeats in the 4q35 region, although no specific transcripts from this locus have been isolated so far. Molecular diagnosis is based on the detection by probe p13E–11 of EcoRI small fragments, in the range 10–28 kb, that are resistant to BlnI digestion. In family studies this probe is used with other 4q35 polymorphic markers to assign the haplotype associated with the disease. So far, we performed DNA analysis in 145 FSHD families and identified the 4q35 DNA rearrangement not only in affected individuals, but also in healthy subjects at risk of transmitting the disease, such as non-penetrant gene carriers and somatic mosaics. In addition we applied prenatal tests to 19 fetuses, using DNA extracted from chorionic villi samples (CVS) at 10–11 weeks of gestation. The FSHD status, as determined by the presence of BlnI-resistant small fragments associated with the at risk haplotype, was assessed in nine fetuses; in the remaining 10 cases the disease was excluded. Our results show that molecular analysis of 4q35 rearrangements is a reliable indirect method to perform diagnostic, predictive and prenatal tests in FSHD.

Thirty-nine patients with mitochondrial diseases were studied with somatosensory and motor evoked potentials. Sixteen patients (41%) had clinical and 12 (31%) had neuroradiological evidence of central nervous system involvement. The overall incidence of electrophysiological abnormalities was 64%. Abnormal evoked potentials were also found in a significant percentage (33%) of patients with pure myopathic forms of mitochondrial diseases and in an asymptomatic carrier of MERRF mutation. Of the individual tests, somatosensory evoked potentials were abnormal in 49% of the patients and motor evoked potentials were abnormal in 46% of the patients. The outcome is that electrophysiological evidence of central nervous system involvement is present in a high percentage of patients with mitochondrial disorders, and that the threshold for central nervous system electrophysiological abnormalities is well below that for clinical and/or radiological manifestations.

We report a new mutation, a T-->C transition at nt.4285 in the mitochondrial tRNA(Ile) gene, in a sporadic case of progressive external ophtalmoplegia (PEO) and ragged-red fibers (RRF). The mutation, involving a highly conserved base-pair in the anticodon stem, was detected in high percentages (91%) in muscle, but not in blood. It has never been reported in literature in normal subjects and it was not found in any of 80 controls studied in our laboratory. The absence of the mutation in leukocytes in this case with pure muscle involvement confirms the importance of performing mtDNA studies in PEO patients preferentially on muscle rather than blood, which could give false negative results. Other mutations in the tRNA(Ile) gene associated with different phenotypes have been previously reported. Thus, tRNA(Ile) gene is confirmed to be another "hot spot" region for mtDNA mutations.

The interactions of a gaseous phase with a liquid metallic surface are analysed in terms of surface tension, adsorption and chemical reactivity. It is shown that thermodynamic equilibrium considerations are not sufficient to interpret surface tension measurements especially at high temperatures, and that diffusion and interactions in the vapour phase must also be considered. Surface tension isotherms are discussed and models allowing calculations of maximum adsorption and surface tension values are presented. These are of particular interest when dealing with systems forming very stable oxides. The influence of surface thermodynamic quantities on chemical reactivity is also shown by means of classical thermodynamic reasoning. One of the main results presented here is that reactions favoured in the bulk phase may not take place at the surface, where the presence of the excess free energy term may favour another reaction, thermodynamically impossible in the bulk phase.

Limb-girdle muscular dystrophies (LGMDs) are a highly heterogeneous group of muscle disorders affecting the pelvic and the shoulder girdle musculature. Until now, over thirty disease genes are known. Even if the age of onset and other clinical features could address the diagnosis towards the specific LGMD form, the phenotypic heterogeneity within each form hampers an easy and rapid molecular identification of causative mutations. We developed a unique and exhaustive platform, based on Next Generation Sequencing on Illumina HiSeq, to analyze 89 muscular disease genes at very high coverage. This regains 20–30% of missing sequences when compared with whole exome sequencing of the same DNA samples. Using this protocol, we have sequenced 312 patients with a LGMD phenotype. Most patients have been previously studied using a gene-by-gene approach without success. Thus, these cases are somewhat enriched for unknown and elusive mutations. Nevertheless, in about 20% of patients we found typical causative mutations in predicted genes, missed by Sanger sequencing or generally poorly studied. An additional 30% of patients carry other novel pathogenic variations. As easily predictable, the diagnostic rate sensibly increases in naive samples not previously screened. Our extensive procedure allowed us to obtain a full comprehensive view of all sequence variants in these samples and to refine the genotype-phenotype correlation. More interestingly, we found in each patient at least five rare mutations (

Eleven FSHD patients (PG: seven males, 40 11 years, 84 24 kg, 1.75 0.11 m) and 11 control subjects (CG: seven males, 32 12 years, 71 24 kg, 1.73 0.36 m) participated in the study. The number of years since first symptoms occurred (SY) was recorded. Manual muscle testing was performed on upper and lower limbs to determine a clinical severity factor (CF [2]), ranging from 0.5 (facial weakness) to 5 (wheelchair bound). A stereophotogrammetric system was used to reconstruct the 3D position of 39 skin markers placed by a single operator. Two force plates were also used. The shoulders’ range of motion (ROM) was computed during repeated arm flexion (FROM)

To assess the pattern and degree of involvement of scapular girdle muscles in Facioscapulohumeral muscular dystrophy (FSHD). We standardized an MRI protocol that allowed the identification and evaluation of 14 muscle groups of the neck and upper girdle. 108 FSHD patients were included in the study. The involvement in FSHD was compared with that of limb girdle muscular dystrophies and other myopathies with upper girdle weakness. We were able to identify the most affected and spared muscles in FSHD. Asymmetry was a consistent feature, similarly to what is found in lower limb muscles, and T2-STIR hyperintensities could be detected as well. The pattern of involvement significantly differed from that encountered in other myopathies. Upper girdle imaging is a helpful tool in guiding the clinical diagnosis of FSHD. Moreover, the detailed knowledge of single muscle involvement provides information useful for understanding and following the natural history of the disease and developing rehabilitation protocols.

Exon skipping using antisense oligonucleotides (AONs) is a promising therapy for Duchenne muscular dystrophy (DMD) which aims to genocopy the milder Becker muscular dystrophy (BMD) by restoring the reading frame of an out-of-frame deletion. The less severe phenotype of BMD patients suggests that internally truncated dystrophin retains some functionality. We previously demonstrated that dystrophin and dystrophin-associated protein expression is correlated to clinical severity in BMD patients with in-frame deletions relevant to current exon skipping strategies (exons 51 and 53), and that BMD patients with deletions ending in exon 51 have significantly higher dystrophin levels than those ending in exon 53. We have now studied deletions relevant for skipping exons 44 and 45 which are now being considered for clinical trials. We studied a total of 25 patients; 11 patients had out-of-frame mutations correctable by skipping exons 44 or 45, and 14 patients had in-frame deletions corresponding to the skipping of exon 45. We quantified dystrophin and dystrophin-associated protein expression using immunohistochemistry and total dystrophin mRNA levels using qRT-PCR. DMD patients skippable for exons 44 and 45 had high levels of revertant and trace dystrophin expression (mean: 14% of control) with 6/11 out-of-frame patients presenting with an intermediate phenotype. Corresponding in frame deletions presented with phenotypes ranging from mild to severe BMD, and showed remarkably lower dystrophin levels (∼42% of control) than patients with deletions modelling the skipping of exons 51 or 53 (80% and 56% of control, respectively). Our study provides valuable insights into the stability and/or function of internally truncated dystrophin suggesting that higher levels of the dystrophin produced by skipping exons 44 and 45 are required to achieve the same level of functionality observed with exon 51 and 53 skipping studies.

Molecular diagnosis of childhood proximal spinal muscular atrophy has been enhanced by the discovery of the survival motor neuron (SMN) gene, which is absent or truncated in 98.6% of patients. To determine whether deletion analysis of the SMN gene may also be diagnostic for adult-onset disease, we studied six patients and found deletions in all. This finding will facilitate the diagnosis of adult-onset spinal muscular atrophy, and provides evidence for genetic homogeneity between the clinically diverse adult and childhood forms of the disease.