Janbernd Kirschner

Hexosamine biosynthetic pathway mutations cause neuromuscular transmission defect.

Neuromuscular junctions (NMJs) are synapses that transmit impulses from motor neurons to skeletal muscle fibers leading to muscle contraction. Study of hereditary disorders of neuromuscular transmission, termed congenital myasthenic syndromes (CMS), has helped elucidate fundamental processes influencing development and function of the nerve-muscle synapse. Using genetic linkage, we find 18 different biallelic mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) in 13 unrelated families with an autosomal recessive CMS. Consistent with these data, downregulation of the GFPT1 ortholog gfpt1 in zebrafish embryos altered muscle fiber morphology and impaired neuromuscular junction development. GFPT1 is the key enzyme of the hexosamine pathway yielding the amino sugar UDP-N-acetylglucosamine, an essential substrate for protein glycosylation. Our findings provide further impetus to study the glycobiology of NMJ and synapses in general.

Peripheral Nerve Demyelination Caused by a Mutant Rho GTPase Guanine Nucleotide Exchange Factor, Frabin/FGD4

GTPases of the Rho subfamily are widely involved in the myelination of the vertebrate nervous system. Rho GTPase activity is temporally and spatially regulated by a set of specific guanine nucleotide exchange factors (GEFs). Here, we report that disruption of frabin/FGD4, a GEF for the Rho GTPase cell-division cycle 42 (Cdc42), causes peripheral nerve demyelination in patients with autosomal recessive Charcot-Marie-Tooth (CMT) neuropathy. These data, together with the ability of frabin to induce Cdc42-mediated cell-shape changes in transfected Schwann cells, suggest that Rho GTPase signaling is essential for proper myelination of the peripheral nervous system.

Intravenously Administered Immunoglobulin in the Treatment of Childhood Guillain-Barré Syndrome: A Randomized Trial

Objective. To determine the optimal treatment for childhood Guillain-Barré syndrome (GBS). Methods. We performed a randomized, multicenter study of GBS according to international diagnostic criteria. In study 1 (early treatment), children able to walk unaided for 5 meters were randomized for 1 g/kg intravenously administered immunoglobulin (IVIG) over 2 days or no treatment. The primary outcome measure was the degree of disability at nadir. In study 2 (treatment for severe GBS), children unable to walk 5 meters unaided were randomized for 1 g/kg IVIG over 2 days or 0.4 g/kg IVIG over 5 days. The primary outcome measure was the number of days needed to regain the ability to walk unaided. Children randomized for no treatment in study 1 could enter study 2 if loss of unaided walking occurred. Results. Ninety-five children with GBS were registered in 40 months. Twenty-one children were randomized in study 1 and 51 in study 2 (5 after deterioration in study 1). Twenty-eight children were not randomized for various reasons. Eleven of 21 patients in study 1 lost the ability to walk unassisted and 6 were bedridden, with no statistically significant difference between the children initially randomized for treatment versus no treatment. Recovery occurred faster in the group randomized for early treatment. In study 2, recovery did not differ significantly between the children treated for 2 days versus 5 days (median time to unaided walking: 19 days vs 13 days). Secondary transient deterioration in the disability score occurred more frequently in the group with the 2-day regimen than in the group treated for 5 days (5 of 23 patients vs 0 of 23 patients). Multivariate analysis with Cox regression showed that disease severity at the nadir was the only prognostic factor for recovery. Conclusions. Treatment with IVIG before loss of unaided walking did not give rise to a less severe course, but recovery occurred somewhat faster. However, given the small number of patients, the power of this conclusion is low. For treatment after loss of unaided walking, there was no significant difference in the effectiveness of 2 g/kg IVIG administered over 2 days versus 5 days. Early “relapses” occurred more frequently after the shorter treatment regimen.

Clinical and genetic findings in a large cohort of patients with ryanodine receptor 1 gene-associated myopathies.

Ryanodine receptor 1 (RYR1) mutations are a common cause of congenital myopathies associated with both dominant and recessive inheritance. Histopathological findings frequently feature central cores or multi‐minicores, more rarely, type 1 predominance/uniformity, fiber‐type disproportion, increased internal nucleation, and fatty and connective tissue. We describe 71 families, 35 associated with dominant RYR1 mutations and 36 with recessive inheritance. Five of the dominant mutations and 35 of the 55 recessive mutations have not been previously reported. Dominant mutations, typically missense, were frequently located in recognized mutational hotspot regions, while recessive mutations were distributed throughout the entire coding sequence. Recessive mutations included nonsense and splice mutations expected to result in reduced RyR1 protein. There was wide clinical variability. As a group, dominant mutations were associated with milder phenotypes; patients with recessive inheritance had earlier onset, more weakness, and functional limitations. Extraocular and bulbar muscle involvement was almost exclusively observed in the recessive group. In conclusion, our study reports a large number of novel RYR1 mutations and indicates that recessive variants are at least as frequent as the dominant ones. Assigning pathogenicity to novel mutations is often difficult, and interpretation of genetic results in the context of clinical, histological, and muscle magnetic resonance imaging findings is essential. Hum Mutat 33:981–988, 2012.

Mitochondrial Tubulopathy in Tenofovir Disoproxil Fumarate-Treated Rats

Objectives:Tenofovir disoproxil fumarate (tenofovir DF) use has been associated with renal dysfunction and Fanconi syndrome. Tenofovir is taken up into renal tubules by anion transporters where high intracellular drug concentration may induce a functionally relevant depletion of mitochondrial DNA (mtDNA). We investigated if tenofovir may induce renal mtDNA depletion and respiratory chain dysfunction. Methods:Rats (n = 8) were gavaged daily with 100 mg·kg−1·d−1 of tenofovir DF or didanosine. Kidneys and livers were examined after 8 weeks of treatment. Results:The tenofovir group had significantly lower body and kidney weights than rats exposed to water or didanosine. Proximal but not distal tubules were of increased diameter and contained small lipid droplets. Tubular mitochondria were enlarged, and their crystal architecture was disrupted. Tenofovir-exposed kidneys contained low mtDNA copy numbers and impaired expression of mtDNA-encoded cytochrome c oxidase (COX) I but not nucleus-encoded COX IV subunits. Histochemistry demonstrated low tubular COX and nicotinamide adenine dinucleotide dehydrogenase (NADH-DH) activities, whereas succinate dehydrogenase activity was preserved. COX activity was preserved in the glomeruli of tenofovir-exposed rats. Didanosine did not elicit renal effects but, unlike tenofovir, depleted mtDNA in liver (by 52%). Conclusions:Tenofovir DF induces an organ-specific nephrotoxicity with mtDNA depletion and dysfunction of mtDNA-encoded respiratory chain subunits. The data do not support nephrotoxicity of didanosine.

Adult-onset cerebellar ataxia due to mutations in CABC1/ADCK3

Objective Inherited ataxias are heterogeneous disorders affecting both children and adults. The primary cause can be identified in about half of the patients and only very few can receive causative therapy. Methods The authors performed sequencing of known Coenzyme Q10 (CoQ10) deficiency genes in 22 patients with unexplained recessive or sporadic ataxia. Results CABC1/ADCK3 mutations were detected in four patients and two siblings presenting with cerebellar ataxia, epilepsy and muscle symptoms. Spasticity, dystonia, tremor and migraine were variably present; cognitive impairment was severe in early childhood cases, but was absent in adults. In contrast to previous reports, two of the patients had a later-onset, very mild phenotype and remained ambulatory in their late forties. Muscle biopsy revealed lipid accumulation, mitochondrial proliferation and cytochrome c oxidase-deficient fibres, but no typical ragged red fibres. Respiratory-chain enzyme activities and CoQ10 were decreased in severely affected patients but remained normal in a mildly affected patient at 46u2005years of age. Conclusions These observations highlight the importance of screening for a potentially treatable cause, CABC1/ADCK3 mutations, not only in severe childhood-onset ataxia, but also in patients with mild cerebellar ataxia in adult life.

Elevated satellite cell number in Duchenne muscular dystrophy

The regenerative potential of muscle tissue relies mostly on satellite cells situated between the muscular basal membrane and the sarcolemma. The regeneration of muscle tissue comprises proliferation, the propagation of satellite cells, and their subsequent differentiation with the expression of multiple muscle-specific proteins. However, in Duchenne muscular dystrophy (DMD), regeneration cannot compensate for the loss of muscle tissue. To examine the regenerative potential in DMD, satellite cell nuclei number and markers of differentiation in DMD muscle from various disease states were compared with control muscle. Differentiation of satellite cells is characterized by the helix-loop-helix factor myogenin, which is never co-expressed with Pax7, whereas MyoD1 and Myf5 are co-expressed with Pax7, with Myf5 being present even in muscle of controls. The results indicate that satellite cell number is elevated in DMD in comparison with control muscle, even in advanced stages of dystrophy, suggesting that exhaustion of satellite cells is not the primary cause for failed regeneration. The expression of myogenin is correlated neither with fibrosis nor with age. We suggest variable factors influencing the differentiation of satellite cells in DMD.

A Combined Laser Microdissection and Mass Spectrometry Approach Reveals New Disease Relevant Proteins Accumulating in Aggregates of Filaminopathy Patients

Filaminopathy is a subtype of myofibrillar myopathy caused by mutations in FLNC, the gene encoding filamin C, and histologically characterized by pathologic accumulation of several proteins within skeletal muscle fibers. With the aim to get new insights in aggregate composition, we collected aggregates and control tissue from skeletal muscle biopsies of six myofibrillar myopathy patients harboring three different FLNC mutations by laser microdissection and analyzed the samples by a label-free mass spectrometry approach. A total of 390 proteins were identified, and 31 of those showed significantly higher spectral indices in aggregates compared with patient controls with a ratio >1.8. These proteins included filamin C, other known myofibrillar myopathy associated proteins, and a striking number of filamin C binding partners. Across the patients the patterns were extremely homogeneous. Xin actin-binding repeat containing protein 2, heat shock protein 27, nebulin-related-anchoring protein, and Rab35 could be verified as new filaminopathy biomarker candidates. In addition, further experiments identified heat shock protein 27 and Xin actin-binding repeat containing protein 2 as novel filamin C interaction partners and we could show that Xin actin-binding repeat containing protein 2 and the known interaction partner Xin actin-binding repeat containing protein 1 simultaneously associate with filamin C. Ten proteins showed significant lower spectral indices in aggregate samples compared with patient controls (ratio <0.56) including M-band proteins myomesin-1 and myomesin-2. Proteomic findings were consistent with previous and novel immunolocalization data. Our findings suggest that aggregates in filaminopathy have a largely organized structure of proteins also interacting under physiological conditions. Different filamin C mutations seem to lead to almost identical aggregate compositions. The finding that filamin C was detected as highly abundant protein in aggregates in filaminopathy indicates that our proteomic approach may be suitable to identify new candidate genes among the many MFM patients with so far unknown mutation.

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

BACKGROUNDnDuchenne 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.nnnMETHODSnOur 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.nnnFINDINGSn77 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.nnnINTERPRETATIONnCiclosporin 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.nnnFUNDINGnGerman Federal Ministry of Education and Research, Action Benni and co eV, Novartis Pharma AG, and Deutsche Gesellschaft für Muskelkranke eV.

Medium-term functional benefits in children with cerebral palsy treated with botulinum toxin type A: 1-year follow-up using gross motor function measure

One of the main goals when treating spasticity is to relieve pain and improve function. Intramuscular injection of botulinum toxin type A (BTX‐A) has gained widespread acceptance in the treatment of spastic cerebral palsy. Several studies have clearly shown the short‐term functional benefit of BTX‐A treatment. Information is limited, however, on the efficacy of medium and long‐term regimens, using repeated injection of BTX‐A. The aim of the present open‐label, prospective study was to evaluate functional outcome in children with spastic cerebral palsy after 1u2003year of treatment with BTX‐A, using the Gross Motor Function Measure (GMFM) as a validated outcome measure. Patients (n=25, age 1.5–15.5u2003years) were treated with BTX‐A for adductor spasm (n=12) or pes equinus (n=13). The local effect was evaluated using passive range of motion and modified Ashworth Scale. Apart from a significant improvement in joint mobility and reduction of spasticity compared to pretreatment values (Pu2003<u20030.01), we demonstrated a significant improvement of gross motor function after 12u2003months of treatment, with a median gain of 6% in total and goal scores (Pu2003<u20030.001). An increase in GMFM scores was particularly evident in younger and moderately impaired children (Gross Motor Function Classification System level III). Whether the observed improvement in gross motor function in children with cerebral palsy is specifically related to therapy with BTX‐A or represents at least in part the natural course of motor development still needs clarification.