[Transition from neuropediatrics to neurology in neuromuscular diseases]. / Transition von der Neuropädiatrie zur Neurologie bei neuromuskulären Erkrankungen.

BACKGROUND: Neuromuscular diseases in childhood, adolescence and adulthood are rare or very rare diseases and for many of them the prevalence and incidence are unknown. Causal therapies are currently used for individual disease entities only. Nevertheless, new genetic methods, a better understanding of the pathophysiology and multidisciplinary treatment concepts help to improve patient life expectancy and quality of life. As a result, more and more patients with an early disease onset reach adulthood and further care in adult medicine is necessary. This imposes new challenges particularly on neurology and the requirements for interdisciplinary cooperation in adult medicine are increased. OBJECTIVE: How can transition be made meaningful? Where do structural and content problems stand out? MATERIAL AND METHOD: Using the example of Duchenne muscular dystrophy, the content and structural requirements for transition are presented and important aspects and possible problems are pointed out. CONCLUSION: The transition process is complex and requires time and personnel resources. If carried out sensibly, it can lead to a better and more efficient care of patients in the long term and thus can also become economically more effective.

Detection of single-nucleotide polymorphisms in Plasmodium falciparum by PCR primer extension and lateral flow immunoassay.

The resistance of Plasmodium falciparum to some antimalarial drugs is linked to single-nucleotide polymorphisms (SNPs). Currently, there are no methods for the identification of resistant parasites that are sufficiently simple, cheap, and fast enough to be performed at point-of-care, i.e., in local hospitals where drugs are prescribed. Primer extension methods (PEXT) were developed to identify 4 SNPs in P. falciparum positioned at amino acids 86, 184, and 1246 of the P. falciparum multidrug resistance 1 gene (pfmdr1) and amino acid 76 of the chloroquine resistance transporter gene (pfcrt). The PEXT products were visualized by a nucleic acid lateral flow immunoassay (NALFIA) with carbon nanoparticles as the detection labels. PCR-PEXT-NALFIAs showed good correlation to the reference methods, quantitative PCR (qPCR) or direct amplicon sequence analysis, in an initial open-label evaluation with 17 field samples. The tests were further evaluated in a blind study design in a set of 150 patient isolates. High specificities of 98 to 100% were found for all 4 PCR-PEXT genotyping assays. The sensitivities ranged from 75% to 100% when all PEXT-positive tests were considered. A number of samples with a low parasite density were successfully characterized by the reference methods but failed to generate a result in the PCR-PEXT-NALFIA, particularly those samples with microscopy-negative subpatent infections. This proof-of principle study validates the use of PCR-PEXT-NALFIA for the detection of resistance-associated mutations in P. falciparum, particularly for microscopy-positive infections. Although it requires a standard thermal cycler, the procedure is cheap and rapid and thus a potentially valuable tool for point-of-care detection in developing countries.

Integrated high pressure microhydraulic actuation and control for surgical instruments.

To reduce the surgical trauma to the patient, minimally invasive surgery is gaining considerable importance since the eighties. More recently, robot assisted minimally invasive surgery was introduced to enhance the surgeon's performance in these procedures. This resulted in an intensive research on the design, fabrication and control of surgical robots over the last decades. A new development in the field of surgical tool manipulators is presented in this article: a flexible manipulator with distributed degrees of freedom powered by microhydraulic actuators. The tool consists of successive flexible segments, each with two bending degrees of freedom. To actuate these compliant segments, dedicated fluidic actuators are incorporated, together with compact hydraulic valves which control the actuator motion. Especially the development of microvalves for this application was challenging, and are the main focus of this paper. The valves distribute the hydraulic power from one common high pressure supply to a series of artificial muscle actuators. Tests show that the angular stroke of the each segment of this medical instrument is 90°.

Carbon nanoparticles in lateral flow methods to detect genes encoding virulence factors of Shiga toxin-producing Escherichia coli.

The use of carbon nanoparticles is shown for the detection and identification of different Shiga toxin-producing Escherichia coli virulence factors (vt1, vt2, eae and ehxA) and a 16S control (specific for E. coli) based on the use of lateral flow strips (nucleic acid lateral flow immunoassay, NALFIA). Prior to the detection with NALFIA, a rapid amplification method with tagged primers was applied. In the evaluation of the optimised NALFIA strips, no cross-reactivity was found for any of the antibodies used. The limit of detection was higher than for quantitative PCR (q-PCR), in most cases between 10(4) and 10(5) colony forming units/mL or 0.1-0.9 ng/µL DNA. NALFIA strips were applied to 48 isolates from cattle faeces, and results were compared to those achieved by q-PCR. E. coli virulence factors identified by NALFIA were in very good agreement with those observed in q-PCR, showing in most cases sensitivity and specificity values of 1.0 and an almost perfect agreement between both methods (kappa coefficient larger than 0.9). The results demonstrate that the screening method developed is reliable, cost-effective and user-friendly, and that the procedure is fast as the total time required is <1 h, which includes amplification.

Quality of care of VLBW neonates: relationship between unit volume and outcome is different between metropolitan and rural regions.

BACKGROUND: Recent studies from predominantly rural areas in Germany show that neonatal outcome of very low birth weight (VLBW) neonates is (on average) inferior with lower NICU (neonatal intensive care unit) volume. However, there are no data available which show that study results of one specific region can be transferred to other areas with possibly different medical infrastructure and needs. AIM: It was investigated whether a systematic difference of treatment quality between smaller (1000-2000 births/year; < or =20 neonatal beds) vs. larger neonatal centres in Berlin (>3000 births/year; >20 neonatal beds) exists. Furthermore, the results are compared to data from a rural region in order to discuss transferability between regions. METHODS: Retrospectively, completely, and for the first time, the data of all centres which treat VLBW neonates (< or =1500 g birth weight) in the city-state of Berlin, Germany, from the years 2003/2004 were reviewed. RESULTS: Our study showed no difference in the treatment quality of smaller vs. larger neonatal units in Berlin. This result differs from those of a study in Baden-Württemberg, a predominantly rural state, with different medical infrastructure than Berlin. CONCLUSION: The present study suggests that regional investigations on the infrastructure vs. treatment outcome are not transferable between areas. Patient volume/unit appears inadequate for predicting the future treatment quality of neonatal departments. Direct quality indicators are stable for the assessed departments and should be preferably used to organize medical infrastructure.

Facing the genetic heterogeneity in neuromuscular disorders: linkage analysis as an economic diagnostic approach towards the molecular diagnosis.

The identification of an ever increasing number of gene defects in patients with neuromuscular disorders has disclosed both marked phenotype and genotype variability and considerable disease overlap. In order to offer an economic strategy to characterise the molecular defect in patients with unclassified neuromuscular disorders, we designed DNA marker sets for linkage analysis of 62 distinct neuromuscular disorders gene loci, including all known muscular dystrophies, congenital myopathies, congenital myasthenic syndromes and myotonias. Genotyping of marker loci of 140 clinically well-characterised families with unclassified neuromuscular disorders reduced the number of candidates to one or two genes in 49 % of the families. Subsequent mutation analysis and genome-wide scans enabled the determination of the genetic defect in 31 % of the families including the identification of a new gene and a new mutation in an unexpected candidate gene. This highlights the effective application of this approach both for diagnostic strategies as well as for the identification of new loci and genes.

Platelets in atherothrombosis: lessons from mouse models.

Platelets play a central role in hemostasis and thrombosis but also in the initiation of atherosclerosis, making platelet receptors and their intracellular signaling pathways important molecular targets for antithrombotic and anti-inflammatory therapy. Historically, much of the knowledge about hemostasis and thrombosis has been derived from patients suffering from bleeding and thrombotic disorders and the identification of the underlying molecular defects. In recent years, the availability of genetically modified mouse strains with defined defects in platelet function and the development of in vivo models to assess platelet-related physiologic and pathophysiologic processes have opened new ways to identify the individual roles and the interplay of platelet proteins in adhesion, activation, aggregation, secretion, and procoagulant activity in vitro and in vivo. This review will summarize key findings made by these approaches and discuss them in the context of human disease.

Loss of Gq/11 family G proteins in the nervous system causes pituitary somatotroph hypoplasia and dwarfism in mice.

Heterotrimeric G proteins of the Gq/11 family transduce signals from a variety of neurotransmitter and hormone receptors and have therefore been implicated in various functions of the nervous system. Using the Cre/loxP system, we generated mice which lack the genes coding for the alpha subunits of the two main members of the Gq/11 family, gnaq and gna11, selectively in neuronal and glial precursor cells. Mice with defective gnaq and gna11 genes were morphologically normal, but they died shortly after birth. Mice carrying a single gna11 allele survived the early postnatal period but died within 3 to 6 weeks as anorectic dwarfs. In these mice, postnatal proliferation of pituitary somatotroph cells was strongly impaired, and plasma growth hormone (GH) levels were reduced to 15%. Hypothalamic levels of GH-releasing hormone (GHRH), an important stimulator of somatotroph proliferation, were strongly decreased, and exogenous administration of GHRH restored normal proliferation. The hypothalamic effects of ghrelin, a regulator of GHRH production and food intake, were reduced in these mice, suggesting that an impairment of ghrelin receptor signaling might contribute to GHRH deficiency and abnormal eating behavior. Taken together, our findings show that Gq/11 signaling is required for normal hypothalamic function and that impairment of this signaling pathway causes somatotroph hypoplasia, dwarfism, and anorexia.

CNS disease as the main manifestation of hemophagocytic lymphohistiocytosis in two children.

Hemophagocytic lymphohistiocytosis is a rare and fatal disorder of early infancy, which affects predominantly the mononuclear phagocyte system and is characterized by the presence of fever, hepatosplenomegaly and cytopenia. Neurological symptoms can be extremely variable, ranging from irritability, and convulsions to focal neurological signs. They often develop during disease progression, but can also be the leading initial symptoms. Early diagnosis is mandatory, because new treatments, including bone marrow transplantation, appear to be promising. Here we present the clinical, neuroradiological and histopathological findings from two children with progressive CNS disease as the main clinical manifestation of hemophagocytic lymphohistiocytosis. Both children died and diagnosis was only obtained in retrospect after careful review of the histopathological material.

Clinical and genetic heterogeneity in megalencephalic leukoencephalopathy with subcortical cysts (MLC).

BACKGROUND: Megalencephalic leukoencephalopathy with subcortical cysts (MLC) was recently localized on chromosome 22q (tel) and 26 different mutations of the gene MLC1 have been found. We report three siblings of non-consanguineous parents who presented with characteristic features of MLC, but did not have MLC1 mutations. MEYHODS: Clinical, laboratory and neuro-imaging findings of the siblings are described and similar patients with MLC are reviewed. RESULTS: All three siblings suffered from ataxia, progressive severe tetraparesis, dysarthria, dysphagia and epilepsy. Generalized dystonia occurred in one patient. Mental deterioration progressed more slowly than motor deterioration. The youngest male was the most severely affected and died at the age of 23 years. The two older females are now 34 and 35 years old. Our patients are among the oldest described with this clinical entity. No mutation of the MLC1 gene was found in our siblings and linkage with the MLC1 locus was excluded. CONCLUSIONS: The genetic findings in our patients suggest at least a second gene locus for MLC.

Large cerebral vessel occlusive disease in Lyme neuroborreliosis.

We report on a 12-year-old, previously healthy girl with an acute hemiparesis as the predominant clinical manifestation of Lyme neuroborreliosis (LNB). The diagnosis of LNB was based on cerebrospinal fluid (CSF) studies, laboratory findings and the clinical course whereas the patient's history and the lack of characteristic skin lesions obscured the diagnosis in the beginning. After four weeks of antibiotic and physiotherapeutic treatment, the hemiparetic symptoms had completely resolved. Although evidence of vasculitic and perivascular inflammation in LNB has been described in the literature, large cerebral vessel occlusive disease represents a rare finding. Appropriate treatment strategies can lead to good clinical rehabilitation, as shown in this case, making the timely diagnosis a crucial issue. We conclude that LNB should be considered in every stroke-like episode of unknown origin in children, even in the absence of a history of a tick bite or typical skin lesions.

Autosomal dominant glut-1 deficiency syndrome and familial epilepsy.

Glut-1 deficiency syndrome was first described in 1991 as a sporadic clinical condition, later shown to be the result of haploinsufficiency. We now report a family with Glut-1 deficiency syndrome affecting 5 members over 3 generations. The syndrome behaves as an autosomal dominant condition. Affected family members manifested mild to severe seizures, developmental delay, ataxia, hypoglycorrhachia, and decreased erythrocyte 3-O-methyl-D-glucose uptake. Seizure frequency and severity were aggravated by fasting, and responded to a carbohydrate load. Glut-1 immunoreactivity in erythrocyte membranes was normal. A heterozygous R126H missense mutation was identified in the 3 patients available for testing, 2 brothers (Generation 3) and their mother (Generation 2). The sister and her father were clinically and genotypically normal. In vitro mutagenesis studies in Xenopus laevis oocytes demonstrated significant decreases in the transport of 3-O-methyl-D-glucose and dehydroascorbic acid. Xenopus oocyte membranes expressed high amounts of the R126H mutant Glut-1. Kinetic analysis indicated that replacement of arginine-126 by histidine in the mutant Glut-1 resulted in a lower Vmax. These studies demonstrate the pathogenicity of the R126H missense mutation and transmission of Glut-1 deficiency syndrome as an autosomal dominant trait.

Functional hemizygosity of PAFAH1B3 due to a PAFAH1B3-CLK2 fusion gene in a female with mental retardation, ataxia and atrophy of the brain.

We report on the molecular characterization of a translocation t(1;19)(q21.3;q13.2) in a female with mental retardation, ataxia and atrophy of the brain. Sequence analysis of the breakpoints revealed an ALU:-repeat-mediated mechanism of recombination that led to truncation of two genes: the kinase CLK2 and PAFAH1B3, the gene product of which interacts with LIS1 as part of a heterotrimeric G protein complex PAF-AH1B. In addition, two reciprocal fusion genes are present. One expressed fusion gene encodes the first 136 amino acids of PAFAH1B3 followed by the complete CLK2 protein. Truncated PAFAH1B3 protein lost its potential to interact with LIS1 whereas CLK2 activity was conserved within the fusion protein. These data emphasize the importance of PAF-AH1B in brain development and functioning and demonstrate the first fusion gene apparently not associated with cancer.

Ataxia with vitamin E deficiency: biochemical effects of malcompliance with vitamin E therapy.

To prevent neuronal damage, patients with ataxia with isolated vitamin E deficiency need lifelong supplementation with high doses of vitamin E. Short interruptions of therapy, such as occur in malcompliance, do not lead to clinical symptoms. However, the authors show that even short withdrawals may cause a prolonged decrease of the total radical trapping capacity of plasma; its major contributors, such as urate and sulfhydryl groups, fail to compensate for the missing vitamin E.

Heterozygous myogenic factor 6 mutation associated with myopathy and severe course of Becker muscular dystrophy.

Myogenic factors (MYF) belong to the basic helix-loop-helix (bHLH) transcription factor family and regulate myogenesis and muscle regeneration. The physiological importance of both functions was demonstrated in homozygous Myf knockout mice and mdx mice. Myf5 and Myod are predominantly expressed in proliferating myoblasts while Myf4 and Myf6 are involved in differentiation of myotubes. In a boy with myopathy and an increase of muscle fibres with central nuclei we detected a heterozygous 387G-->T nucleotide transversion in the MYF6 gene (MIM*159991). Protein-protein interaction of mutant MYF6 was reduced, and DNA-binding potential and transactivation capacity were abolished, thus demonstrating MYF6 haploinsufficiency. The boy's father carried the identical mutation and, in addition, an in-frame deletion of exons 45-47 in his dystrophin gene. This mutation is normally associated with a mild to moderate course of Becker muscular dystrophy but the father suffered from a severe course of Becker muscular dystrophy suggesting MYF6 as a modifier.

Mutations in LHX3 result in a new syndrome revealed by combined pituitary hormone deficiency.

Combined pituitary hormone deficiency (CPHD) has been linked with rare abnormalities in genes encoding transcription factors necessary for pituitary development. We have isolated LHX3, a gene involved in a new syndrome, using a candidate-gene approach developed on the basis of documented pituitary abnormalities of a recessive lethal mutation in mice generated by targeted disruption of Lhx3 (ref. 2). LHX3, encoding a member of the LIM class of homeodomain proteins, consists of at least six exons located at 9q34. We identified a homozygous LHX3 defect in patients of two unrelated consanguineous families displaying a complete deficit in all but one (adrenocorticotropin) anterior pituitary hormone and a rigid cervical spine leading to limited head rotation. Two of these patients also displayed a severe pituitary hypoplasia, whereas one patient presented secondarily with an enlarged anterior pituitary. These LHX3 mutations consist of a missense mutation (Y116C) in the LIM2 domain at a phylogenetically conserved residue and an intragenic deletion predicting a severely truncated protein lacking the entire homeodomain. These data are consistent with function of LHX3 in the proper development of all anterior pituitary cell types, except corticotropes, and extrapituitary structures.

Elevated p21 mRNA level in skeletal muscle of DMD patients and mdx mice indicates either an exhausted satellite cell pool or a higher p21 expression in dystrophin-deficient cells per se.

Abnormalities in proliferation and differentiation of the dystrophin-deficient muscle are a controversial aspect of the pathogenesis of Duchenne muscular dystrophy (DMD). Analyses of molecules involved in cell cycle modulation do not exist in this context. Cells withdrawn from the cell cycle permanently express p21. The fact that p2 1, in contrast to other cell cycle proteins, is not diminished when myotubes are reexposed to growth media, allocates this cyclin-dependent kinase inhibitor a special function. Here we report for the first time statistically increased p21 mRNA levels in dystrophin-deficient muscle tissue. Only 42% of conventional RT-PCRs from six muscle samples of human controls yielded positive results but almost all skeletal muscle biopsy samples (87%) from DMD patients (n=5). For p21 mRNA quantification in murine muscle samples we were able to use the exact real-time TaqMan PCR method due to generally higher p21 mRNA levels than in human muscles. In addition, contamination with fibroblasts can be excluded for the murine samples because they do not demonstrate fibrosis at the age of 350 days but start to lose their regenerative capacity. In accord with the results in humans, we observed p21 mRNA levels in mdx mice that were approx. four times as high as those in control mice. Elevated p21 mRNA level may indicate a shift in cell composition towards differentiated p21 expressing cells as a result of an exhausted pool of undifferentiated, non-p21-expressing satellite cells due to previous cycles of de- and regeneration. Alternatively, dystrophin-deficient cells per se may express higher p21 levels for unknown reasons. Although we cannot distinguish between these possibilities, the eventual transfec tion of a patient's own satellite cells with p21 antisense oligonucleotides may enable the dystrophic process to be influenced.

Dystrophinopathy in a boy with Chediak-Higashi syndrome.

Chediak-Higashi syndrome (CHS) is a hereditary, biphasic immunodeficiency syndrome which usually leads to early death, during the first decade. The second phase is characterized by a lymphoproliferative syndrome with histiocytic infiltrations in various tissues. Recently the gene has been identified on chromosome 1q43. In the patient presented here, a mutation within codon 3197 was found, resulting in a frame-shift. Additionally, Duchenne muscular dystrophy (DMD) was diagnosed by immunostaining of the muscle. Unusual for both CHS and DMD muscle weakness and hypotonia became evident during the first months of life. Compared to typical DMD cases we found an increased histiocytic infiltration in the muscle. The underlying muscular dystrophy probably predisposes to the affection of muscle in the second phase of CHS. This patient is presented as an example of modification of the phenotype by a second genetic disease.