A zebrafish model for FHL1-opathy reveals loss-of-function effects of human FHL1 mutations.

Missense mutations in the four and a half LIM domain 1 (FHL1) gene were found to cause X-linked inherited myopathies of both skeletal and heart muscles. However, the mechanisms by which FHL1 mutations impact on FHL1 function and lead to alteration of muscle structure and function have not been deciphered yet. We generated here by Morpholino-modified antisense oligonucleotide-mediated gene knockdown fHL1-deficient zebrafish embryos. Similar to the human situation, fhl1a-morphants zebrafish displayed severe skeletal and heart muscle myopathy. Whereas ectopic expression of wild-type FHL1 (FHL1 wt) suppressed both skeletal and heart muscle myopathy in fhl1a-morphants zebrafish, overexpression of the FHL1-opathy associated human mutations FHL1-H123Y, FHL1-C132F or FHL1-C224W did not rescue skeletal and heart muscle myopathy in fhl1a-morphants. Overexpression of FHL1-H123Y, FHL1-C132F or FHL1-C224W in wild-type zebrafish did not induce myopathy in a dominant-negative mode. Altogether these results indicate that FHL1 mutations found to cause X-linked FHL1-opathies in humans consistently lead to severely impaired FHL1 function.

New insights into the protein aggregation pathology in myotilinopathy by combined proteomic and immunolocalization analyses.

INTRODUCTION: Myofibrillar myopathies are characterized by progressive muscle weakness and impressive abnormal protein aggregation in muscle fibers. In about 10 % of patients, the disease is caused by mutations in the MYOT gene encoding myotilin. The aim of our study was to decipher the composition of protein deposits in myotilinopathy to get new information about aggregate pathology. RESULTS: Skeletal muscle samples from 15 myotilinopathy patients were included in the study. Aggregate and control samples were collected from muscle sections by laser microdissection and subsequently analyzed by a highly sensitive proteomic approach that enables a relative protein quantification. In total 1002 different proteins were detected. Seventy-six proteins showed a significant over-representation in aggregate samples including 66 newly identified aggregate proteins. Z-disc-associated proteins were the most abundant aggregate components, followed by sarcolemmal and extracellular matrix proteins, proteins involved in protein quality control and degradation, and proteins with a function in actin dynamics or cytoskeletal transport. Forty over-represented proteins were evaluated by immunolocalization studies. These analyses validated our mass spectrometric data and revealed different regions of protein accumulation in abnormal muscle fibers. Comparison of data from our proteomic analysis in myotilinopathy with findings in other myofibrillar myopathy subtypes indicates a characteristic basic pattern of aggregate composition and resulted in identification of a highly sensitive and specific diagnostic marker for myotilinopathy. CONCLUSIONS: Our findings i) indicate that main protein components of aggregates belong to a network of interacting proteins, ii) provide new insights into the complex regulation of protein degradation in myotilinopathy that may be relevant for new treatment strategies, iii) imply a combination of a toxic gain-of-function leading to myotilin-positive protein aggregates and a loss-of-function caused by a shift in subcellular distribution with a deficiency of myotilin at Z-discs that impairs the integrity of myofibrils, and iv) demonstrate that proteomic analysis can be helpful in differential diagnosis of protein aggregate myopathies.

Muscle ultrasound in classic infantile and adult Pompe disease: a useful screening tool in adults but not in infants.

A cohort of 4 infantile and 15 adult Pompe patients has been investigated regarding correlation between strength and ultrasound of skeletal musculature. In adults, muscle ultrasound is useful to assess clinical and subclinical involvement of muscles. In this study, visible sonographic changes were found in every clinically affected muscle, using a modified Heckmatt scale. In some muscles morphologic changes preceded weakness. Regarding the anatomical pattern of involvement, our findings do not support the hypothesis of a specific pattern with a higher vulnerability of vastus intermedius than rectus femoris, which has been postulated before. A frequent sparing of triceps brachii could be confirmed. Intramuscular abnormalities occurred in a focal, a diffuse, or an intermediate pattern, with characteristics of both. In contrast to muscular dystrophies, bone echogencity was not markedly decreased in Pompe disease even in an advanced stage. In infants, muscle ultrasound showed no distinct pathology even in clinically severely affected children and should not be used as a screening method for infantile Pompe disease.

Familial reducing body myopathy with cytoplasmic bodies and rigid spine revisited: identification of a second LIM domain mutation in FHL1.

OBJECTIVE: Reducing body myopathy (RBM) is a rare progressive disorder of muscle characterized by intracytoplasmic inclusions, which stain strongly with menadione-NBT (nitroblue tetrazolium). We recently identified the four and a half LIM domain gene FHL1 located on chromosome Xq26 as the causative gene for RBM. So far eight familial cases and 21 sporadic patients with RBM have been reported in the literature. METHODS: We ascertained a total of 8 members of a German family initially reported by Goebel et al. as a mixed myopathy with rigid spine myopathy and reducing as well as cytoplasmic bodies. Clinical findings in the original and additional family members have been reviewed. Mutation detection was performed by direct sequencing of FHL1 exons. RESULTS: We identified a novel mutation (p.C150R) in the second LIM domain of FHL1 in six family members (1 male, 5 females). The male index patient was the most affected member presenting with rigid spine, followed by rapidly progressive muscle weakness. He died from the consequences of respiratory insufficiency at the age of 29.5 years. His sister, mother, grandmother, aunt and female cousin all carried the mutation in the heterozygous state. The sister is clinically unaffected; their mother had myopathic changes in her muscle biopsy, while the grandmother showed first signs of weakness at 50 years of age. The 54-year-old aunt and her daughter are clinically asymptomatic. CONCLUSION: We report a novel LIM2 domain mutation in FHL1 in a previously reported family with RBM with cytoplasmic bodies and spinal rigidity. While the male index patient was significantly affected, female carriers show varying manifestations and may be asymptomatic, likely reflecting varying degrees of X-inactivation. RBM continues to be associated with mutations in the LIM2 domain of FHL1. We also confirm our earlier observation that mutations at the N-terminal end of the LIM2 domain seem to be milder compared to mutations seen at the C-terminal part of the domain which cause severe disease even in female carriers.

Muscle MRI in FHL1-linked reducing body myopathy.

Reducing body myopathy is a rare progressive myopathy identified by characteristic pathological findings and secondary to dominantly acting mutations in the X-linked FHL1 gene. We report muscle MRI findings in two patients affected by reducing body myopathy and in their carrier mothers. All four showed a distinctive pattern of muscle alteration, with a predominant involvement of postero-medial muscle at thigh level and of soleus at calf level, with a striking sparing of glutei muscles that also appeared to be hypertrophic. These findings may help in the differential diagnosis of these disorders.

Phenotypic variability in siblings with calpainopathy (LGMD2A).

Calpainopathy is an autosomal-recessive limb girdle muscular dystrophy (LGMD2A) characterized by selective atrophy and weakness of proximal limb girdle muscles. The clinical phenotype of the disease is highly variable inter-familial, but little is known about intra-familial variability. This study reports the phenotypic variability in eight sibling pairs with genetically proven LGMD2A. Although siblings with identical mutations were often similarly affected, in some families the age of onset and the clinical course varied considerably.

Clinical presentation and course of childhood Guillain-Barré syndrome: a prospective multicentre study.

Presenting symptoms, clinical course and paraclinical findings in childhood Guillain-Barré syndrome (GBS) have rarely been investigated prospectively. We performed a multicentre study in GBS diagnosed according to international criteria. Clinical findings were recorded using an ordinal GBS score and additional scores for arm, cranial nerve and vegetative function, and pain. Electrophysiological and CSF investigations followed individual procedures in the local hospitals. Ninety-five children with a median age of 6.2 years were registered over 40 months (53 boys, 42 girls). 70 had suffered an infection and 8 had been vaccinated during the previous 6 weeks. The first symptom was usually a disturbance of gait or neuropathic pain. The symptoms progressed for a median of 7 days. At the height of the disease, 60% of patients were unable to walk and 24% could not use their arms. 46% showed cranial nerve involvement, and 51% autonomous dysfunction. 13% required artificial ventilation. 79% complained of neuropathic pain, half of them to a severe degree. Electrophysiological examination showed demyelination in 74%, and 26% of these presented with very low amplitude compound action potentials. Purely axonal changes were found in 11%. All but eight were treated with I.V. immunoglobulin. Improvement began on day 13 after the first symptom (median). Ability to walk unaided returned after 27 days. In the children observed over the long-term, it took 118 days for them to be free of symptoms. Transient deterioration after immunoglobulin treatment occurred in seven patients, two suffered relapsing GBS, and three developed CIDP. At the end of the observation period (288 days), 75% of patients were free of symptoms. 21% suffered residual symptoms having no effect on daily functioning. The more severely disabled 4% either suffered from CIDP or concurrent myelitis. With this prospective study, the results of earlier retrospective investigations are confirmed. Besides pareses and respiratory compromise, severe neuropathic pain frequently is a therapeutic challenge during the acute phase of the disease. The long-term prognosis is good for most children. However, a change to CIDP and concurrent myelitis can give rise to a worse prognosis.

Prospective study on anti-ganglioside antibodies in childhood Guillain-Barré syndrome.

BACKGROUND: Antiganglioside antibodies have been reported to play a part in the pathophysiology of Guillain-Barré syndrome (GBS). AIMS: To investigate the prevalence and correlation of anti-ganglioside antibodies with clinical data in children with GBS in a multicentre clinical trial. METHODS: Immunoglobin (Ig)G and IgM to GM1, GM1b, GD1a, GalNAc-GD1a, GD1b, GT1a, and GQ1b were measured by ELISA in sera obtained before treatment. In addition, serological testing for Campylobacter jejuni was carried out. In parallel, a group of adults with GBS and a control group of children without GBS or other inflammatory diseases were evaluated. RESULTS: Sera from 63 children with GBS, 36 adults with GBS and 41 children without GBS were evaluated. Four of the children with GBS showed positive IgG to GM1, in one case combined with anti-GalNAc-GD1a and in one with anti-GD1b. Two others showed isolated positive IgG to GD1b and GT1a. One showed increased anti-GalNAc-GD1a IgM. In 5 of the 63 children, serological evidence of a recent infection with C jejuni was found, and this correlated significantly with the raised antibodies (p = 0.001). In the control group without GBS, no child showed positive IgG, but one showed anti-GalNAc-GD1a IgM. Compared with the adults with GBS, the frequency of antibodies in children was insignificantly lower. In our study, patients with positive antibodies did not show a more severe GBS course or worse outcome than those who were seronegative, and we could not show an increased incidence of axonal dysfunction. CONCLUSIONS: In some children with GBS, one can detect raised IgG against various gangliosides, similar to that in adults. A recent infection with C jejuni is markedly associated with the presence of these antibodies. However, in contrast with what has been reported in adults, in this study we were unable to show a negative effect of these findings on the clinical course.

Low level of intracortical inhibition in children shown by transcranial magnetic stimulation.

Transcranial magnetic stimulation (TMS) is an established neurophysiological tool to evaluate the integrity and maturation of the corticospinal tract. TMS was used in this study to compare intracortical inhibition (ICI) in children, adolescents, and adults. The paired-pulse technique of TMS with interstimulus intervals of 2 ms was used to determine the ratio of conditioned (cMEP) and unconditioned amplitudes (ucMEP) that measures ICI. In experiment 1 (Exp 1) stimulus intensity was adapted to motor threshold (50 healthy subjects; 24 male, 26 female, median age 13.5 years, range 6.3 - 34 years) and in experiment 2 (Exp 2) stimulus intensity was adapted to the ucMEP (200 - 400 microV). Children (quotient of cMEP and ucMEP: Exp. 1: 0.71 +/- 0.41, Exp. 2: 0.82 +/- 0.25) had significantly less ICI compared to adults (Exp. 1: 0.21 +/- 0.19, mean +/- STD, Exp. 2: 0.35 +/- 0.22, in both experiments p < 0.001). Recently, ICI has been linked to the regulating function of GABAergic cortical interneurons on practice-dependent neuronal plasticity. Therefore, the lower ICI in children points to maturation processes that may have implications for the greater capacity of practice-dependent neuronal plasticity in children.