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Chung S.-K.,University of Swansea | Bode A.,University of Queensland | Cushion T.D.,University of Swansea | Thomas R.H.,University of Swansea | And 23 more authors.
Human Molecular Genetics | Year: 2013

Glycinergic neurotransmission is a major inhibitory influence in the CNS and its disruption triggers a paediatric and adult startle disorder, hyperekplexia. The postsynaptic α1-subunit (GLRA1) of the inhibitory glycine receptor (GlyR) and the cognate presynaptic glycine transporter (SLC6A5/GlyT2) are well-established genes of effect in hyperekplexia. Nevertheless, 52% of cases (117 from 232) remain gene negative and unexplained. Ligand-gated heteropentameric GlyRs form chloride ion channels that contain the a1 and b-subunits (GLRB) in a 2α1:3β configuration and they form the predominant population of GlyRs in the postnatal and adult human brain, brainstem and spinal cord. We screened GLRB through 117 GLRA1- and SLC6A5-negative hyperekplexia patients using a multiplex-polymerase chain reaction and Sanger sequencing approach. The screening identified recessive and dominant GLRB variants in 12 unrelated hyperekplexia probands. This primarily yielded homozygous null mutations, with nonsense (n=3), small indel (n=1), a large 95 kb deletion (n=1), frameshifts (n 5 1) and one recurrent splicing variant found in four cases. A further three cases were found with two homozygous and one dominant GLRB missense mutations. We provide strong evidence for the pathogenicity of GLRB mutations using splicing assays, deletion mapping, cell-surface biotinylation, expression studies and molecular modelling. This study describes the definitive assignment of GLRB as the third major gene for hyperekplexia and impacts on the genetic stratification and biological causation of this neonatal/paediatric disorder. Driven principally by consanguineous homozygosity of GLRB mutations, the study reveals long-term additive phenotypic outcomes for affected cases such as severe apnoea attacks, learning difficulties and developmental delay. © The Author 2012. Published by Oxford University Press. All rights reserved.


Iesalnieks I.,University of Regensburg | Machens A.,Martin Luther University of Halle Wittenberg | Bures C.,Krankenanstalt Rudolfstiftung | Krenz D.,Klinikum Dritter Orden | And 14 more authors.
Annals of Surgical Oncology | Year: 2015

Conclusions: Although significant improvement of local disease control in patients with thyroid metastases of RCC has been achieved during the last decade, overall outcome continues to be poor for patients with locally invasive thyroid metastases.Background: Most investigations of thyroidectomy for metastatic renal cell carcinoma (RCC) are case studies or small series. This study was conducted to determine the contribution of clinical and histopathologic variables to local recurrence in the neck and overall survival after thyroidectomy for RCC metastases.Methods: The medical records of 140 patients with thyroidectomy for metastatic RCC performed between 1979 and 2012 at 25 institutions in Germany and Austria were analyzed.Results: The median interval between nephrectomy and thyroidectomy was 120 months. Concurrence of thyroid and pancreatic metastases was present in 23 % of the patients and concurrence of thyroid and adrenal metastases in 13 % of the patients. Clinical outcome data were available for 130 patients with a median follow-up period of 34 months. The 5-year overall survival rate was 46 %, and 28 % of patients developed a local neck recurrence at a median of 12 months after thyroidectomy. Multivariate analysis showed that invasion of adjacent cervical structures (hazard ratio [HR] 3.2; p = 0.001), patient age exceeding 70 years (HR 2.5; p = 0.004), and current or past evidence of metastases to nonendocrine organs (HR 2.4; p = 0.003) were independent determinants of inferior overall survival. Conversely, invasion of adjacent cervical structures (HR 12.1; p < 0.0001) and year of thyroidectomy (HR 5.7 before 2000; p < 0.0001) were shown to be independently associated with local recurrence in the neck by multivariate analysis. © 2014, Society of Surgical Oncology.


Tro-Baumann B.,Epilepsy Center | Von Spiczak S.,University of Kiel | Von Spiczak S.,Northern German Epilepsy Center for Children and Adolescents | Lotte J.,Epilepsy Center | And 11 more authors.
Epilepsia | Year: 2011

Dravet syndrome is a severe epileptic encephalopathy starting in the first year of life. Mutations in SCN1A can be identified in the majority of patients, and epileptic seizures in the setting of fever are a clinical hallmark. Fever is also commonly seen after vaccinations and provocation of epileptic seizures by vaccinations in patients with Dravet syndrome has been reported, but not systematically assessed. In a retrospective evaluation of 70 patients with Dravet syndrome and SCN1A mutations, seizures following vaccinations were reported in 27%. In 58% of these patients vaccination-related seizures represented the first clinical manifestation. The majority of seizures occurred after DPT vaccinations and within 72 h after vaccination. Two-thirds of events occurred in the context of fever. Our findings highlight seizures after vaccinations as a common feature in Dravet syndrome and emphasize the need for preventive measures for seizures triggered by vaccination or fever in these children. © 2010 International League Against Epilepsy.


Howard M.F.,University of Oxford | Murakami Y.,Osaka University | Pagnamenta A.T.,University of Oxford | Daumer-Haas C.,Pranatal Medizin Munich | And 22 more authors.
American Journal of Human Genetics | Year: 2014

Glycosylphophatidylinositol (GPI)-anchored proteins play important roles in many biological processes, and mutations affecting proteins involved in the synthesis of the GPI anchor are reported to cause a wide spectrum of intellectual disabilities (IDs) with characteristic additional phenotypic features. Here, we describe a total of five individuals (from three unrelated families) in whom we identified mutations in PGAP3, encoding a protein that is involved in GPI-anchor maturation. Three siblings in a consanguineous Pakistani family presented with profound developmental delay, severe ID, no speech, psychomotor delay, and postnatal microcephaly. A combination of autozygosity mapping and exome sequencing identified a 13.8 Mb region harboring a homozygous c.275G>A (p.Gly92Asp) variant in PGAP3 region 17q11.2-q21.32. Subsequent testing showed elevated serum alkaline phosphatase (ALP), a GPI-anchored enzyme, in all three affected children. In two unrelated individuals in a cohort with developmental delay, ID, and elevated ALP, we identified compound-heterozygous variants c.439dupC (p.Leu147Profs-16) and c.914A>G (p.Asp305Gly) and homozygous variant c.314C>G (p.Pro105Arg). The 1 bp duplication causes a frameshift and nonsense-mediated decay. Further evidence supporting pathogenicity of the missense mutations c.275G>A, c.314C>G, and c.914A>G was provided by the absence of the variants from ethnically matched controls, phylogenetic conservation, and functional studies on Chinese hamster ovary cell lines. Taken together with recent data on PGAP2, these results confirm the importance of the later GPI-anchor remodelling steps for normal neuronal development. Impairment of PGAP3 causes a subtype of hyperphosphatasia with ID, a congenital disorder of glycosylation that is also referred to as Mabry syndrome. © 2014 The American Society of Human Genetics.


Schumann H.,Albert Ludwigs University of Freiburg | Schumann H.,Catholic University of Applied Sciences at Freiburg | Kiritsi D.,Albert Ludwigs University of Freiburg | Pigors M.,Albert Ludwigs University of Freiburg | And 10 more authors.
British Journal of Dermatology | Year: 2013

Background Integrin α6β4 is a transmembrane receptor and a key component of the hemidesmosome anchoring complex. It is involved in cell-matrix adhesion and signalling in various tissues. Mutations in the ITGA6 and ITGB4 genes coding for α6β4 integrin compromise dermal-epidermal adhesion and are associated with skin blistering and pyloric atresia (PA), a disorder known as epidermolysis bullosa with PA (EB-PA). Objectives To elucidate the molecular pathology of skin fragility in eight cases, disclose the underlying ITGA6 and ITGB4 mutations and study genotype-phenotype correlations. Methods DNA was isolated from ethylenediaminetetraacetic acid-blood samples, and the coding exons and exon-intron boundaries of ITGA6 and ITGB4 were amplified by polymerase chain reaction (PCR), and directly sequenced. Skin samples were submitted to immunofluorescence mapping with antibodies to adhesion proteins of the dermal-epidermal junction. Primary keratinocytes were isolated, and used for RNA and protein extraction, reverse transcription PCR and immunoblotting. Ultrastructural analysis of the skin was performed in one patient. Results We disclose 10 novel mutations, one in ITGA6 and nine in ITGB4. Skin cleavage was either intraepidermal or junctional. Lethal outcome and PA correlated with loss-of-function mutations in two cases. Solely mild skin involvement was associated with deletion of the C-terminus of β4 integrin. Combinations of missense, nonsense or frameshift mutations caused severe urinary tract involvement in addition to skin fragility in five cases. Conclusions The present study reveals novel ITGA6 and ITGB4 gene mutations and supports previous reports showing that the phenotype may lack PA and be limited to skin and nail involvement. In four out of six cases of EB-PA, life expectancy was not impaired. A high frequency of urinary tract involvement was found in this study, and represented the main cause of morbidity. Low levels of β4 integrin expression were compatible with hemidesmosomal integrity and a mild skin phenotype. What's already known about this topic? Epidermolysis bullosa (EB) with pyloric atresia (PA) is a rare EB type caused by mutations in the genes coding for α6β4 integrin or plectin, disrupting the hemidesmosome adhesion complex. A reduced life expectancy is predicted in most cases of EB with PA. α6β4 integrin gene mutations are not always associated with PA. What does this study add? This study identifies 10 novel ITGB4 and ITGA6 mutations causing a spectrum of phenotypes, ranging from mild skin blistering to a lethal multisystem disorder with skin, urinary and gastrointestinal involvement. Urinary tract involvement is relatively common (five out of eight cases in this study). Low levels of α6β4 integrin are sufficient for hemidesmosomal integrity and are associated with a mild skin phenotype. © 2013 The Authors BJD © 2013 British Association of Dermatologists.


Gotzberger M.,Ludwig Maximilians University of Munich | Rosch T.,UKE | Geisenhof S.,Klinikum Dritter Orden | Gulberg V.,Ludwig Maximilians University of Munich | And 6 more authors.
Endoscopy | Year: 2011

Background and study aims: Training standards in gastrointestinal endoscopy are poorly defined even though different simulators are increasingly used for skills training. In 2001 a new training concept called GATE gastroenterological education training endoscopy was established, which provides a combination of background theory, video demonstrations, and simulator training. We aimed to evaluate the acceptance and training effect of this training model. Methods: In total, 98 physicians participating in four training courses were included. Data were collected on baseline characteristics, acceptance (5-point Likert scale), and pre- and post-course knowledge through a structured questionnaire (A-type and Pick-N multiple choice questions). A total of 13 trainees were randomly selected for additional simulator assessment of training effects on manual skills (5-point Likert scale). Results: A total of 78 trainees (80%) provided complete data sets. The evaluation showed a positive acceptance of the training program (value 1 and 2, Likert scale); for example, 88% of participants suggested the inclusion of the GATE course as an obligatory part of endoscopic education. There was a significant improvement in theoretical knowledge in the post-test set compared with the pre-test set (mean 3.27 ±1.30 vs. 1.69 ±1.01 points; P<0.001). The training effect on practical skill showed a significant reduction in time needed for a procedure (445 ±189s vs. 274 ±129s; P<0.01). The mean assessment rating for practical skills improved from 3.05 ±0.65 at baseline to 2.52 ±0.59 on Likert scale (P=0.085). Conclusions: The integrated GATE training improved theoretical knowledge and manual skill. The GATE courses have been accredited by the German Society of Gastroenterology, underlining the demand for implementing preclinical training courses in endoscopic training. © Georg Thieme Verlag KGStuttgart, New York.


Olzowy B.,University of Rostock | Abendroth S.,Klinikum Dritter Orden | Von Gleichenstein G.,Klinik fur Neurologie | Mees K.,Ludwig Maximilians University of Munich | Stelter K.,Ludwig Maximilians University of Munich
High Altitude Medicine and Biology | Year: 2014

No evidence of intracranial hypertension in trekkers with acute mountain sickness when assessed noninvasively with distortion product otoacoustic emissions. High Alt Med Biol 15:364-370, 2014. - Aims: The role of intracranial hypertension in acute mountain sickness (AMS) is a matter of debate. Distortion product otoacoustic emissions (DPOAEs) can be used to monitor the intracranial pressure (ICP) noninvasively with a level decrease at the frequencies f2=1 and 1.5 kHz indicating elevated ICP. Methods: DPOAEs (f2=1, 1.5, 2, 3, and 4 kHz), oxygen saturation (Sao2) and the Lake Louise score (LLS) to assess AMS were measured in trekking tourists on the Mount Everest trek in Nepal at 2610 m and 5170 m. Results: Paired data of both altitudes could be obtained of 187 subjects. All results are given in mean±SD. Sao2was 94.8±2.7% at 2610 m and 79.0±6.9% at 5170 m. While at 2610 m, none of the study subjects had AMS (LLS 0.04±0.02), at 5170 m 82 (43.9%) had AMS when defined as LLS>2, and 31 (16.6%) when defined as LLS>4 (LLS 2.8±2.2). DPOAE levels decreased at altitude in all frequencies without a difference between trekkers with AMS and without AMS. Low Sao2correlated with high LLS. Low Sao2correlated with larger DPOAE level decrease only at f2=1 kHz, while the other frequencies showed no correlation. DPOAE level decrease and LLS showed no correlation. Conclusions: Our data suggest that subjects with AMS symptoms did not have higher ICP compared to healthy subjects. Consequently, it seems unlikely that intracranial hypertension accounts for the symptoms of AMS. © 2014 Mary Ann Liebert, Inc.


Jacobs C.,Ludwig Maximilians University of Munich | Duewell P.,Ludwig Maximilians University of Munich | Heckelsmiller K.,Ludwig Maximilians University of Munich | Wei J.,Ludwig Maximilians University of Munich | And 10 more authors.
International Journal of Cancer | Year: 2011

Vaccines based on immune stimulatory complexes (ISCOM) induce T-cell responses against tumor antigen (Ag). However, immune responses are impaired in pancreatic cancer patients. We investigated the efficacy of an ISCOM vaccine in a murine pancreatic carcinoma model. Panc02 cells expressing OVA as a model Ag were induced subcutaneously or orthotopically in the pancreas of C57BL/6 mice. Treatment consisted of an OVA containing ISCOM vaccine, either used alone or in combination with the TLR9 agonist CpG. The ISCOM vaccine effectively induced Ag-specific CTL capable of killing tumor cells. However, in mice with established tumors CTL induction by the vaccine was inefficient and did not affect tumor growth. Lack of efficacy correlated with increased numbers of Treg. Depletion of Treg with anti-CD25 mAb restored CTL induction and prolonged survival. Adding low-dose CpG to the ISCOM vaccine reduced Treg numbers, enhanced CTL responses and induced regression of pancreatic tumors in a CD8 + T cell-dependent manner. Mice cured from the primary tumor mounted a memory T-cell response against wild-type Panc02 tumors, indicative of epitope spreading. Combining ISCOM vaccines with TLR agonists is a promising strategy for breaking tumor immune evasion and deserves further evaluation for the treatment of pancreatic carcinoma. Copyright © 2010 UICC.


Bauer C.,Ludwig Maximilians University of Munich | Sterzik A.,Ludwig Maximilians University of Munich | Bauernfeind F.,Ludwig Maximilians University of Munich | Duewell P.,Ludwig Maximilians University of Munich | And 6 more authors.
Cancer Immunology, Immunotherapy | Year: 2014

Background: Multiple studies have shown that dendritic cell (DC)-based vaccines can induce antitumor immunity. Previously, we reported that gemcitabine enhances the efficacy of DC vaccination in a mouse model of pancreatic carcinoma. The present study aimed at investigating the influence of gemcitabine on vaccine-induced anti-tumoral immune responses in a syngeneic pancreatic cancer model. Materials and methods: Subcutaneous or orthotopic pancreatic tumors were induced in C57BL/6 mice using Panc02 cells expressing the model antigen OVA. Bone marrow-derived DC were loaded with soluble OVA protein (OVA-DC). Animals received gemcitabine twice weekly. OVA-specific CD8 + T-cells and antibody titers were monitored by FACS analysis and ELISA, respectively. Results: Gemcitabine enhanced clinical efficacy of the OVA-DC vaccine. Interestingly, gemcitabine significantly suppressed the vaccine-induced frequency of antigen-specific CD8+ T-cells and antibody titers. DC migration to draining lymph nodes and antigen cross-presentation were unaffected. Despite reduced numbers of tumor-reactive T-cells in peripheral blood, in vivo cytotoxicity assays revealed that cytotoxic T-cell (CTL)-mediated killing was preserved. In vitro assays revealed sensitization of tumor cells to CTL-mediated lysis by gemcitabine. In addition, gemcitabine facilitated recruitment of CD8+ T-cells into tumors in DC-vaccinated mice. T- and B-cell suppression by gemcitabine could be avoided by starting chemotherapy after two cycles of DC vaccination. Conclusions: Gemcitabine enhances therapeutic efficacy of DC vaccination despite its negative influence on vaccine-induced T-cell proliferation. Quantitative analysis of tumor-reactive T-cells in peripheral blood may thus not predict vaccination success in the setting of concomitant chemotherapy. © 2013 Springer-Verlag.

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