Targeted Re-Sequencing Emulsion PCR Panel for Myopathies: Results in 94 Cases.

BACKGROUND: Molecular diagnostics in the genetic myopathies often requires testing of the largest and most complex transcript units in the human genome (DMD, TTN, NEB). Iteratively targeting single genes for sequencing has traditionally entailed high costs and long turnaround times. Exome sequencing has begun to supplant single targeted genes, but there are concerns regarding coverage and needed depth of the very large and complex genes that frequently cause myopathies. OBJECTIVE: To evaluate efficiency of next-generation sequencing technologies to provide molecular diagnostics for patients with previously undiagnosed myopathies. METHODS: We tested a targeted re-sequencing approach, using a 45 gene emulsion PCR myopathy panel, with subsequent sequencing on the Illumina platform in 94 undiagnosed patients. We compared the targeted re-sequencing approach to exome sequencing for 10 of these patients studied. RESULTS: We detected likely pathogenic mutations in 33 out of 94 patients with a molecular diagnostic rate of approximately 35%. The remaining patients showed variants of unknown significance (35/94 patients) or no mutations detected in the 45 genes tested (26/94 patients). Mutation detection rates for targeted re-sequencing vs. whole exome were similar in both methods; however exome sequencing showed better distribution of reads and fewer exon dropouts. CONCLUSIONS: Given that costs of highly parallel re-sequencing and whole exome sequencing are similar, and that exome sequencing now takes considerably less laboratory processing time than targeted re-sequencing, we recommend exome sequencing as the standard approach for molecular diagnostics of myopathies.

Treatment of Duchenne muscular dystrophy with ciclosporin A: a randomised, double-blind, placebo-controlled multicentre trial.

BACKGROUND: Duchenne muscular dystrophy is a rare X-linked progressive disease characterised by loss of ambulation at about age 10 years, with death in early adulthood due to respiratory and cardiac insufficiency. Steroids are effective at slowing the progression of muscle weakness; however, their use is limited by side-effects, prompting the search for alternatives. We assessed the effect of ciclosporin A as monotherapy and in combination with intermittent prednisone for the treatment of ambulant patients with this disorder. METHODS: Our study was a parallel-group, placebo-controlled, double-blind, multicentre trial at trial sites of the German muscular dystrophy network, MD-NET, over 36 months. Ambulant patients with Duchenne muscular dystrophy who were aged 5 years or older were randomly assigned to receive either ciclosporin A (3·5-4·0 mg/kg per day) or matching placebo. Allocation was done centrally with computer-generated random numbers. Patients and investigators were masked to the allocated treatment. After 3 months of treatment, both groups were also given intermittent prednisone for a further 12 months (0·75 mg/kg, alternating 10 days on with 10 days off). All patients who received at least one dose of study drug or placebo were included in the primary analysis. The primary outcome measure was manual muscle strength measured on the Medical Research Council (MRC) scale. This trial is registered with the German clinical trial register DRKS, number DRKS00000445. FINDINGS: 77 patients were randomly assigned to the ciclosporin A group and 76 to the placebo group; 73 patients on ciclosporin A and 73 on placebo received at least one dose and were available for efficacy analyses. 3 months of treatment with ciclosporin A alone did not show any significant improvement in primary outcome measures (mean change in the proportion of a possible total MRC score [%MRC] was -2·6 [SD 6·0] for patients on ciclosporin A and -0·8 [4·9] for patients on placebo; adjusted group difference estimate -0·88, 97·5% CI -2·6 to 0·9; p=0·26). The combination of ciclosporin A with intermittent steroids was not better than intermittent steroids alone over 12 months (mean change in %MRC was 0·7 [7·1] for patients on ciclosporin A and -0·3 [7·9] for patients on placebo; adjusted group difference estimate -0·85, -3·6 to 1·9; p=0·48). Numbers of adverse events (75 in patients on ciclosporin A and 74 on placebo) and serious adverse events (four with ciclosporin A and four with placebo) did not differ significantly between groups. INTERPRETATION: Ciclosporin A alone or in combination with intermittent prednisone does not improve muscle strength or functional abilities in ambulant boys with Duchenne muscular dystrophy, but is safe and well tolerated. FUNDING: German Federal Ministry of Education and Research, Action Benni and co eV, Novartis Pharma AG, and Deutsche Gesellschaft für Muskelkranke eV.

Subcortical somatosensory evoked potentials after posterior tibial nerve stimulation in children.

We report our normative data of somatosensory evoked potentials (SEP) after posterior tibial nerve (PTN) stimulation from a group of 89 children and 18 adults, 0.4-29.2 years of age. We recorded near-field potentials from the peripheral nerve, the cauda equina, the lumbar spinal cord and the somatosensory cortex. Far-field potentials were recorded from the scalp electrodes with a reference at the ipsilateral ear. N8 (peripheral nerve) and P40 (cortex) were present in all children but one. N20 (cauda equina) and N22 (lumbar spinal cord) were recorded in 94 and 106 subjects, respectively. P30 and N33 (both waveforms probably generated in the brainstem) were recorded in 103 and 101 subjects, respectively. Latencies increased with age, while central conduction times including the cortical component, decreased with age (up to about age 10 years). The amplitudes of all components were very variable in each age group. We report our normative data of the interpeak latencies N8-N22 (peripheral conduction time), N22-P30 (spinal conduction time), N22-P40 (central conduction time) and P30-P40 (intracranial conduction time). These interpeak latencies should be useful to assess particular parts of the pathway. The subcortical PTN-SEPs might be of particular interest in young or retarded children and during intraoperative monitoring, when the cortical peaks are influenced by sedation and sleep, or by anesthesia.

Infantile postural asymmetry and osteopathic treatment: a randomized therapeutic trial.

The aim of this study was to assess the therapeutic efficacy of osteopathic treatment in infants with postural asymmetry. A randomized clinical trial of efficacy with blinded videoscoring was performed. Sixty-one infants with postural asymmetry aged 6 to 12 weeks (mean 9wks) were recruited. Thirty-two infants (18 males, 14 females) with a gestational age of at least 36 weeks were found to be eligible and randomly assigned to the intervention groups, 16 receiving osteopathic treatment and 16 sham therapy. After a treatment period of 4 weeks the outcome was measured using a standardized scale (4-24 points). With sham therapy, five infants improved (at least 3 points), eight infants were unchanged (within 3 points), and three infants deteriorated (not more than -3 points); the mean improvement was 1.2 points (SD 3.5). In the osteopathic group, 13 infants improved and three remained unchanged; the mean improvement was 5.9 points (SD 3.8). The difference was significant (p=0.001). We conclude that osteopathic treatment in the first months of life improves the degree of asymmetry in infants with postural asymmetry.

Considerations to the policy of future clinical therapeutic trials in DMD.

In spite of rapidly increasing insight into the molecular basis of neuromuscular diseases, treatment still relies on convention and clinical studies. Experience with a multicentre double blind treatment study in Duchenne muscular dystrophy and with consecutive steroid treatment documentation for up to 8 years enables us to identify a series of crucial points on which to focus while planning such clinical trials. The most important seem to be: a carefully structured, detailed study, clear-cut aims and objectives, expertise of investigators, sufficient training of examiners, and careful monitoring. If patients with neuromuscular diseases are treated outside structured studies, their course should be monitored comparably. Examples of the impact of such documentation are available from the ongoing German multicentre trial on the treatment of Duchenne muscular dystrophy.

Topiramate and metabolic acidosis in infants and toddlers.

PURPOSE: Topiramate (TPM) inhibits carbonic anhydrase, with metabolic acidosis as a possible side effect, although this has been reported in only two adult cases. We investigated the acid-base metabolism in infants and toddlers treated with TPM. METHODS: Nine infants and toddlers aged 5 months to 2.3 years (median, 6 months) were treated with TPM at maximal doses of 8.2-26 mg/kg/day (median, 11 mg/kg/day). The maximal TPM dose was achieved after 8-35 days (median, 17 days). TPM was given in addition to other antiepileptic drugs (AEDs) in five cases and as a sole AED in four patients with refractory epilepsy resistant to multiple AEDs. The diagnoses were infantile spasms (n = 5), epilepsia partialis continua (n = 1), infantile epileptic encephalopathy (n = 1), and Lennox-Gastaut syndrome (n = 2). RESULTS: The blood gases were normal before treatment with TPM in all nine children. Metabolic acidosis developed in eight children after 8-26 days (median, 14 days) of TPM treatment with a minimum of serum bicarbonate between 15 to 18 mM (median, 17 mM), a minimal base excess between -6.2 and -11.2 mM (median, -7.9 mM), and pH between 7.22 and 7.40 (median, 7.35). Four of nine children showed clinical signs of hyperventilation and received oral sodium bicarbonate (1-2 mmol/kg), while TPM was still effective. CONCLUSIONS: Because metabolic acidosis developed in eight of the nine infants and toddlers taking TPM, we would suggest that the acid-base metabolism be monitored in young children who receive TPM.