Introduction: (Sub)acute myelitis is often caused by CNS antibody-triggered autoimmune processes, so that the detection of an elevated specific antibody strongly suggests a causative autoimmune disease. However, re-evaluation of this hypothesis is needed, especially when the disease course is unexpected despite correct treatment. The newly described very rare Mitchell syndrome mimics autoimmune disease and may only be recognized in the course of time.

Glycine constitutes a major inhibitory neurotransmitter predominantly in caudal regions of the CNS. The extracellular glycine concentration is regulated synergistically by two high affinity, large capacity transporters GlyT1 and GlyT2. Both proteins are encoded by single genes SLC6A9 and SLC6A5, respectively. Mutations within the SLC6A5 gene encoding for GlyT2 have been demonstrated to be causative for hyperekplexia (OMIM #614618), a complex neuromuscular disease, in humans. In contrast, mutations within the SLC6A9 gene encoding for GlyT1 have been associated with GlyT1 encephalopathy (OMIM #601019), a disease causing severe postnatal respiratory deficiency, muscular hypotonia and arthrogryposis. The consequences of the respective GlyT1 mutations on the function of the transporter protein, however, have not yet been analysed. In this study we present the functional characterisation of three previously published GlyT1 mutations, two mutations predicted to cause truncation of GlyT1 (GlyT1(Q573*) and Gly(T1K310F+fs*31)) and one predicted to cause an amino acid exchange within transmembrane domain 7 of the transporter (GlyT1(S407G)), that are associated with GlyT1 encephalopathy. Additionally, the characterization of a novel mutation predicted to cause an amino acid exchange within transmembrane domain 1 (GlyT1(V118M)) identified in two fetuses showing increased nuchal translucency and arthrogryposis in routine ultrasound scans is demonstrated. We show that in recombinant systems the two presumably truncating mutations resulted in an intracellular retained GlyT1 protein lacking the intracellular C terminal domain. In both cases this truncated protein did not show any residual transport activity. The point mutations, hGlyT1(S407G) and hGlyT1(V118M), were processed correctly, but showed severely diminished activity, thus constituting a functional knock-out in-vivo. Taken together our data demonstrate that all analysed mutations of GlyT1 that have been identified in GlyT1 encephalopathy patients cause severe impairment of transporter function. This is consistent with the idea that loss of GlyT1 function is indeed causal for the disease phenotype.

Im Kontrast zur bisherigen Beschreibung einer Balkenagenesie mit peripherer Neuropathie bei Veränderungen im SLC12A6- Gen wurden nun Fälle einer isolierten sensomotorischen Polyneuropathie bei heterozygoten de novo Veränderungen im SLC12A6- Gen berichtet [1]. Wir zeigen hier einen 11-jährigen Patienten mit heterozygoter, bislang noch nicht beschriebener Sequenzveränderung im SLC12A6- Gen.

Among the clusters of imprinted genes in humans, one of the most relevant regions involved in human growth is localised in 11p15. Opposite epigenetic and genomic disturbances in this chromosomal region contribute to two distinct imprinting disorders associated with disturbed growth, Silver–Russell and Beckwith–Wiedemann syndromes. Due to the complexity of the 11p15 imprinting regions and their interactions, the interpretation of the copy number variations in that region is complicated. The clinical outcome in case of microduplications or microdeletions is therefore influenced by the size, the breakpoint positions and the parental inheritance of the imbalance as well as by the imprinting status of the affected genes. Based on their own new cases and those from the literature, the authors give an overview on the genotype–phenotype correlation in chromosomal rearrangements in 11p15 as the basis for a directed genetic counselling. The detailed characterisation of patients and families helps to further delineate risk figures for syndromes associated with 11p15 disturbances. Furthermore, these cases provide us with profound insights in the complex regulation of the (imprinted) factors localised in 11p15.

Mutations in the LARGE gene are a rare condition causing Walker-Warburg-syndrome and other forms of a-dystroglycanopathies. We report on a girl with clinical features of Walker-Warburg Syndrome – the most severe form of a-dystroglycanopathy. We found an interstitial amplification (4 copies) of a 132 kb segment within the LARGE-Gen by 6.0 SNP-Array analysis. The 19 month old girl suffers from very severe global developmental delay and severe hypotonia. She is not able to turn her head or move any part of her body. She has got brain malformations including large dilatation of the supratentoral ventricle system with monoventriculare configuration of lateral ventrikels and missing septum, pachymicrogyria, agenesis of dorsal corpus callosum, aquaduct stenosis and hydrocephalus occlusus, cerebellar vermis agenesy of the caudal part. She has got microphthalmy and cataract at the left eye and proximale ureter stenosis (Ureterabgangsstenose) with hydronephrosis. She started myoclonic seizures at the age of 6 weeks. CK was highly elevated (>2000U/l). The parents are first cousins. This is first case of Walker-Warburg Syndrome caused by a large interstitial duplication in the LARGE-gene.

Seit Juli 2009 wird in Tübingen eine genetische Testung der Risikogene BRCA1 und BRCA2 durchgeführt. BRCA1/2 negative Patienten, in deren Familien sowohl Brustkrebs als auch Eierstockkrebs auftreten, werden zudem seit Juni 2010 auf Veränderungen im RAD51C-Gen getestet. Eine Zwischenbilanz dieser genetischen Analysen verfolgte das Ziel, Detektionsraten für pathogene Mutationen und Unklassifizierte Varianten (UV) in den drei genannten Genen im untersuchten Patientenkollektiv angeben zu können. Weiterhin werden alle begleitenden Varianten dokumentiert, um auch zur Einschätzung von UVs weitere Evidenz beizutragen.

Spinocerebellar Ataxia Type 3 (SCA3) or Machado-Joseph-Disease (MJD) is an autosomal dominantly inherited neurodegenerative disorder caused by the expansion of a CAG stretch in the MJD1 gene encoding a polyglutamine repeat in the respective ataxin-3-protein. In order to study the course of the disease, we generated an inducible transgenic mouse model using the “Tet-Off-System“. This system is based on two constructs: The promoter construct controls the expression of the so called tTA (Tetracycline transactivator) gene product. The binding of this protein to a Tetracycline responsive element (TRE) in the responder construct induces the transcription of the gene of interest. The expression can be blocked by the addition of Tetracycline or Doxycycline which allosterically inhibits the tTA protein. For the ataxin-3-responder mouse lines, a full length constructs containing an expanded repeat with the pathological length of 77 glutamine repeats was used. The use of two different promoter mouse lines with known expression in the brain (Prion protein (Prp) promoter, Ca2+/Calmoduline-dependent protein kinase II (CamKII) promoter) allows us to target the transgene expression to different brain regions. In preceding promoter analyses we identified and characterized these brain regions in detail. Using the CamKII promoter, the trangene is targeted mainly to the frontal brain and mice display first symptoms at the age of six months. However, when we use the Prp promoter, the transgene is widely expressed in the brain with pronounced expression in the cerebellum and the brain stem. In these mice, first motor symptoms can be detected at the age of two months. We then stopped the expression of the transgene by treatment with doxycyclin and compared treated and untreated mice at different levels: We first analysed the phenotype in behavioural studies. Afterwards we performed immunohistochemical analyses of brain sections to study the expression of the transgene and neurodegenerative processes in the brain. These analyses revealed that turning off the pathogenic transgene at least slows down the progression of symptoms but did not reverse the phenotype. Comparable approaches in human patients -if available- would therefore require early initiation.

Spinocerebellar Ataxia Type 3 (SCA3) or Machado-Joseph-Disease (MJD) is an autosomal dominantly inherited neurodegenerative disorder caused by the expansion of a CAG stretch in the MJD1 gene encoding a polyglutamine repeat in the respective ataxin-3-protein. In order to study the course of the disease we generated an inducible transgenic mouse model using the „Tet-Off-System“ developed by Dr. Bujard (Heidelberg). This system is based on two constructs: The promoter construct controls the expression of the so called tTA (Tetracycline transactivator) gene product. The binding of this protein to a Tetracycline responsive element (TRE) in the responder construct induces the transcription of the gene of interest. The expression can be blocked by the addition of Tetracycline which allosterically inhibits the tTA protein.

Alpha-synuclein, a small soluble protein is expressed primarily at presynaptic terminals in the central nervous system. It has been implicated in the pathophysiology of many neurodegenerative disorders, including Parkinson's disease and dementia with Lewy-bodies, collectively referred to as synucleinopathies. As neurodegenerative disorders like Parkinson's disease are charecterized by progressive neuropathological and clinical symptoms, conditional models of these neurodegenerative diseases are powerful means to analyze the relationship between transgene expression and progression of the disease. We have created a conditional mouse model of synucleinopathies by using the tet-off gene system combined with expression of human wildtype or mutated (A30P) alpha-synuclein. We investigated the behavioral and neuropathological effects of overexpression of human alpha-synuclein under conditional control of both the PrP promotor and the CaMKIIalpha promotor. Western blot analyses revealed that double-transgenic mice express alpha-synuclein at different levels in specific brain regions. The expression-pattern and level of human alpha-synuclein in the brain of double-transgenic mice depend on both the neuron-specific promotor and the integration site of the human alpha-synuclein construct. Histological analysis of the brainsof transgenic mice showed aberrant expression of the protein in cell soma, axons and synapses, but no Lewy body-like alpha-synuclein inclusions could be identified, yet. We currently investigate, whether focal neurodegeneration, which we observed in one 9 month-old CaMKIIalpha-h[wt]alpha-synuclein double-transgenic mouse could be reproduced. Double-transgenic males of this line performerd significantly weaker on accelerated rotarod than age-matched control animals. HPLC-analyses of double-transgenic mice that showed expression of the transgene limited to the olfactory bulb, demonstrate significantly lower levels of Dopamin, DOPAC and HVA in this brain region. In all lines administration of doxycycline downregulates alpha-synuclein expression to basal level. Our conditional mouse-model may help to define the role of human alpha-synuclein in synucleinopathies and might be used to demonstrate whether neuropathological symptoms of these diseases are reversible.

Das alpha-Synuklein ist ein niedermolekulares, lösliches Protein, welches primär in präsynaptischen Nervenendigungen exprimiert wird. Es ist an der Pathophysiologie einer ganzen Gruppe neurodegenerativer Erkrankungen wie bspw. dem Morbus Parkinson und der Demenz mit Lewy-Körperchen beteiligt, die man heute unter dem Begriff "Synukleinopathien" zusammenfasst. Neurodegenerative Erkrankungen, wie der Morbus Parkinson, sind durch progrediente neuropathologische und klinische Symptome charakterisiert. Deshalb stellen konditionelle Modelle solcher Erkrankungen ein wichtiges Hilfsmittel zur Analyse der Beziehung zwischen der Expression des Transgens und dem progressiven Verlauf der Neurodegeneration, dar. Aus diesem Grund erzeugten wir ein konditionelles Mausmodell für Synukleinopathien mithilfe des sog. Tet-off-Systems. Hierfür wurden Konstrukte für das humane Wildtyp-alpha-Synuklein und das mutierte (A30P) alpha-Synuklein eingesetzt. Wir analysierten Verhaltensauffälligkeiten und neuropathologische Effekte der sowohl durch den PrP-Promotor als auch den CaMKIIalpha-Promotor induzierten Überexpression des humanen alpha-Synukleins im transgenen Mausgehirn. Die Western-Blot-Analyse transgener Mäusen zeigte, dass doppelt-transgene Tiere humanes alpha-Synuklein in unterschiedlicher Stärke in spezifischen Hirnregionen exprimierten. Dabei ist sowohl der neuronenspezifische Promotor als auch die Integrationsstelle des humanen alpha-Synukleins im Mausgenom für das jeweilige Expressionsmuster entscheidend. Die histologische Untersuchung des transgenen Mausgehirns ergab eine starke Proteinexpression im neuronalen Zellsoma, Axonen und Synapsen. Eine Lewy-Körperchen ähnliche Pathologie konnte jedoch bisher noch nicht nachgewiesen werden. Momentan untersuchen wir, ob eine fokale Neurodegeneration, die in einer für CaMKIIalpha-Promotor und das humane Wildtyp-alpha-Synuklein doppelt-transgenen Maus beobachtet wurde, in einer zweiten Maus derselben Linie ebenfalls auftritt. Die Gabe von Doxyzyklin regulierte die alpha-Synuklein Expression im Gehirn doppelt-transgener Mäuse auf einen basalen Level herab. Ebenso führten wir Microarray-Expressionsanalysen durch, um Einblicke in die Pathomechanismen zu bekommen, die einer Überexpression des humanen alpha-Synukleins im Gehirn der transgenen Mäuse zu Grunde liegen. Unser konditionelles Mausmodell könnte für die Aufklärung der Rolle des alpha-Synukleins bei Entstehung von Synukleinopathien entscheidend sein und aufzeigen, ob neuropathologische Symptome dieser Erkrankungen reversibel sind.