Dresden, Germany
Dresden, Germany

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Hanniger S.,Max Planck Institute for Chemical Ecology | Dumas P.,University of Amsterdam | Schofl G.,DKMS Life Science Laboratory | Gebauer-Jung S.,Max Planck Institute for Chemical Ecology | And 5 more authors.
BMC Evolutionary Biology | Year: 2017

Background: Very little is known on how changes in circadian rhythms evolve. The noctuid moth Spodoptera frugiperda (Lepidoptera: Noctuidae) consists of two strains that exhibit allochronic differentiation in their mating time, which acts as a premating isolation barrier between the strains. We investigated the genetic basis of the strain-specific timing differences to identify the molecular mechanisms of differentiation in circadian rhythms. Results: Through QTL analyses we identified one major Quantitative trait chromosome (QTC) underlying differentiation in circadian timing of mating activity. Using RADtags, we identified this QTC to be homologous to Bombyx mori C27, on which the clock gene vrille is located, which thus became the major candidate gene. In S. frugiperda, vrille showed strain-specific polymorphisms. Also, vrille expression differed significantly between the strains, with the rice-strain showing higher expression levels than the corn-strain. In addition, RT-qPCR experiments with the other main clock genes showed that pdp1, antagonist of vrille in the modulatory feedback loop of the circadian clock, showed higher expression levels in the rice-strain than in the corn-strain. Conclusions: Together, our results indicate that the allochronic differentiation in the two strains of S. frugiperda is associated with differential transcription of vrille or a cis-acting gene close to vrille, which contributes to the evolution of prezygotic isolation in S. frugiperda. © 2017 The Author(s).


Jovanovic M.,University of Zürich | Jovanovic M.,DKMS Life Science Laboratory | Reiter L.,University of Zürich | Reiter L.,Biognosys | And 21 more authors.
Nature Methods | Year: 2010

Efficient experimental strategies are needed to validate computationally predicted microRNA (miRNA) target genes. Here we present a large-scale targeted proteomics approach to validate predicted miRNA targets in Caenorhabditis elegans. Using selected reaction monitoring (SRM), we quantified 161 proteins of interest in extracts from wild-type and let-7 mutant worms. We demonstrate by independent experimental downstream analyses such as genetic interaction, as well as polysomal profiling and luciferase assays, that validation by targeted proteomics substantially enriched for biologically relevant let-7 interactors. For example, we found that the zinc finger protein ZTF-7 was a bona fide let-7 miRNA target. We also validated predicted miR-58 targets, demonstrating that this approach is adaptable to other miRNAs. We propose that targeted mass spectrometry can be applied generally to validate candidate lists generated by computational methods or in large-scale experiments, and that the described strategy should be readily adaptable to other organisms. © 2010 Nature America, Inc. All rights reserved.


Lange V.,DKMS Life Science Laboratory | Bohme I.,DKMS Life Science Laboratory | Hofmann J.,German Bone Marrow Center | Lang K.,DKMS Life Science Laboratory | And 14 more authors.
BMC Genomics | Year: 2014

Background: A close match of the HLA alleles between donor and recipient is an important prerequisite for successful unrelated hematopoietic stem cell transplantation. To increase the chances of finding an unrelated donor, registries recruit many hundred thousands of volunteers each year. Many registries with limited resources have had to find a trade-off between cost and resolution and extent of typing for newly recruited donors in the past. Therefore, we have taken advantage of recent improvements in NGS to develop a workflow for low-cost, high-resolution HLA typing.Results: We have established a straightforward three-step workflow for high-throughput HLA typing: Exons 2 and 3 of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 are amplified by PCR on Fluidigm Access Array microfluidic chips. Illumina sequencing adapters and sample specific tags are directly incorporated during PCR. Upon pooling and cleanup, 384 samples are sequenced in a single Illumina MiSeq run. We developed " neXtype" for streamlined data analysis and HLA allele assignment. The workflow was validated with 1140 samples typed at 6 loci. All neXtype results were concordant with the Sanger sequences, demonstrating error-free typing of more than 6000 HLA loci. Current capacity in routine operation is 12,000 samples per week.Conclusions: The workflow presented proved to be a cost-efficient alternative to Sanger sequencing for high-throughput HLA typing. Despite the focus on cost efficiency, resolution exceeds the current standards of Sanger typing for donor registration. © 2014 Lange et al.; licensee BioMed Central Ltd.


Schmidt A.H.,German Bone Marrow Donor Center | Solloch U.V.,German Bone Marrow Donor Center | Pingel J.,German Bone Marrow Donor Center | Baier D.,German Bone Marrow Donor Center | And 7 more authors.
Human Immunology | Year: 2011

We present high-resolution allele and haplotype frequency (HF) estimations of the Polish population based on more than 20,000 registered stem cell donors. Sequencing-based donor human leukocyte antigen (HLA) typing led to unambiguous typing results in most cases (between 94.3% for HLA-DRB1 and 96.9% for HLA-B). HF estimations were carried out with a new, validated implementation of the expectation-maximization algorithm that allowed processing of data with ambiguities. Our results confirm several earlier results, for example, the relative commonness of the haplotype A*25:01g, B*18:01g, C*12:03, DRB1*04:01 in the Polish population. Because of the large sample size, we were able to obtain results of unprecedented accuracy. The estimated population-specific HFs were then used to analyze questions of strategic donor registry planning. Simulated matching probabilities by donor file size suggest that there is a need for intense donor recruitment efforts in Poland despite the large German donor registry and the genetic relatedness of both populations. Based on the current German registry size of approximately 4 million donors, the recruitment of 100,000 Polish donors would produce a stronger increase in matching probabilities for Polish patients than the recruitment of 3.3 million additional German donors. © 2011 American Society for Histocompatibility and Immunogenetics.


Schofl G.,DKMS Life Science Laboratory | Schmidt A.H.,German Bone Marrow Donor Center | Lange V.,DKMS Life Science Laboratory
Human Immunology | Year: 2016

While modern high-throughput sequence-based HLA genotyping methods generally provide highly accurate typing results, artefacts may nonetheless arise for numerous reasons, such as sample contamination, sequencing errors, read misalignments, or PCR amplification biases. To help detecting spurious typing results, we tested the performance of two probabilistic classifiers (binary logistic regression and random forest models) based on population-specific genotype frequencies. We trained the model using high-resolution typing results for HLA-DRB1, DQB1, and DPB1 from large samples of German, Polish and UK-based donors. The high predictive capacity of the best models replicated both in 10-fold cross-validation for each gene and in using independent evaluation data (AUC 0.820-0.893). While genotype frequencies alone provide enough predictive power to render the model generally useful for highlighting potentially spurious typing results, the inclusion of workflow-specific predictors substantially increases prediction specificity. Low initial DNA concentrations in combination with low-volume PCR reactions form a major source of stochastic error specific to the Fluidigm chip-based workflow at DKMS Life Science Lab. The addition of DNA concentrations as a predictor variable thus substantially increased AUC (0.947-0.959) over purely frequency-based models. © 2016 American Society for Histocompatibility and Immunogenetics.


Pingel J.,German Bone Marrow Donor Center | Solloch U.V.,German Bone Marrow Donor Center | Hofmann J.A.,German Bone Marrow Donor Center | Lange V.,DKMS Life Science Laboratory | And 3 more authors.
Human Immunology | Year: 2013

In hematopoietic stem cell transplantation, human leukocyte antigens (HLA), usually HLA loci A, B, C, DRB1 and DQB1, are required to check histocompatibility between a potential donor and the recipient suffering from a malignant or non-malignant blood disease. As databases of potential unrelated donors are very heterogeneous with respect to typing resolution and number of typed loci, donor registries make use of haplotype frequency-based algorithms to provide matching probabilities for each potentially matching recipient/donor pair. However, it is well known that HLA allele and haplotype frequencies differ significantly between populations. We estimated high-resolution HLA-A, -B, -C, -DRB1 haplotype and allele frequencies of donors within DKMS German Bone Marrow Donor Center with parentage from 17 different countries: Turkey, Poland, Italy, Russian Federation, Croatia, Greece, Austria, Kazakhstan, France, The Netherlands, Republic of China, Romania, Portugal, USA, Spain, United Kingdom and Bosnia and Herzegovina. 5-locus haplotypes including HLA-DQB1 are presented for Turkey, Poland, Italy and Russian Federation. We calculated linkage disequilibria for each sample. Genetic distances between included countries could be shown to reflect geography. We further demonstrate how genetic differences between populations are reflected in matching probabilities of recipient/donor pairs and how they influence the search for unrelated donors as well as strategic donor center typings. © 2012 American Society for Histocompatibility and Immunogenetics.


Zhao S.,TU Dresden | Wehner R.,TU Dresden | Bornhauser M.,TU Dresden | Bornhauser M.,Center for Regenerative Therapies Dresden | And 6 more authors.
Stem Cells and Development | Year: 2010

Bone marrow-derived mesenchymal stromal cells (MSCs) represent a population of nonhematopoietic cells, which play a crucial role in supporting hematopoiesis and can differentiate into various cell types such as osteocytes, chondrocytes, adipocytes, and myocytes. Due to their differentiation capability, MSCs emerge as promising candidates for therapeutic applications in tissue engineering. In addition, they display immunomodulatory properties that have prompted consideration of their potential use for treatment modalities aimed at the inhibition of immune responses. In this context, MSCs efficiently inhibit maturation, cytokine production, and T-cell stimulatory capacity of dendritic cells (DCs). They also markedly impair proliferation, cytokine secretion, and cytotoxic potential of natural killer cells and T lymphocytes. Furthermore, MSCs are able to inhibit the proliferation of B cells and their capacity to produce antibodies. Various animal models confirm the immunomodulatory properties of MSCs. Thus, administered MSCs prolong the survival of skin and cardiac allografts and ameliorate acute graft-versus-host disease (GVHD) as well as experimental autoimmune encephalomyelitis. Clinical studies enrolling patients with severe acute GVHD reveal that the administration of MSCs results in significant clinical responses. Due to their immunomodulatory capability and their low immunogenicity, MSCs represent promising candidates for the prevention and treatment of immune-mediated diseases. © 2010, Mary Ann Liebert, Inc.


Schmidt A.H.,German Bone Marrow Donor Center | Solloch U.V.,German Bone Marrow Donor Center | Baier D.,German Bone Marrow Donor Center | Stahr A.,German Bone Marrow Donor Center | And 4 more authors.
Tissue Antigens | Year: 2010

We analyzed regional differences in human leukocyte antigen (HLA)-A, -B, and -DR antigen and haplotype frequencies based on a sample of approximately 320,000 German donors in order to identify regions that are especially suited for ongoing stem cell donor recruitment. Geographic partitioning was carried out by postal code regions. Analysis of genetic distances suggests the existence of three regional clusters in South (regions 6-9), East (0-1), and Northwest (2-5) Germany. The southern cluster shows most favorable characteristics with respect to haplotypic and phenotypic diversity and the occurrence of rare HLA antigens. The opposite behavior is shown by regions 2-4 of the northwestern cluster. As a result of lower HLA diversity, completeness of a regional donor file in region 4 with 100,000 donors would be higher than that of a file in region 7 with 170,000 donors. This fact shows the relevance of regional HLA differences for practical donor registry planning. Results such as those presented in this work can be used to diminish the problem of decreasing marginal benefit of donor recruitment, as more than 13 million donors are registered worldwide today. © 2010 John Wiley & Sons A/S.


PubMed | German Bone Marrow Donor Center and DKMS Life Science Laboratory
Type: Journal Article | Journal: Human immunology | Year: 2016

While modern high-throughput sequence-based HLA genotyping methods generally provide highly accurate typing results, artefacts may nonetheless arise for numerous reasons, such as sample contamination, sequencing errors, read misalignments, or PCR amplification biases. To help detecting spurious typing results, we tested the performance of two probabilistic classifiers (binary logistic regression and random forest models) based on population-specific genotype frequencies. We trained the model using high-resolution typing results for HLA-DRB1, DQB1, and DPB1 from large samples of German, Polish and UK-based donors. The high predictive capacity of the best models replicated both in 10-fold cross-validation for each gene and in using independent evaluation data (AUC 0.820-0.893). While genotype frequencies alone provide enough predictive power to render the model generally useful for highlighting potentially spurious typing results, the inclusion of workflow-specific predictors substantially increases prediction specificity. Low initial DNA concentrations in combination with low-volume PCR reactions form a major source of stochastic error specific to the Fluidigm chip-based workflow at DKMS Life Science Lab. The addition of DNA concentrations as a predictor variable thus substantially increased AUC (0.947-0.959) over purely frequency-based models.


PubMed | National Hospital for Neurology, TU Munich, DKMS Life Science Laboratory, Max Planck Institute of Psychiatry and German Bone Marrow Center
Type: Journal Article | Journal: Journal of neurology | Year: 2016

Few regional and seasonal Guillain-Barr syndrome (GBS) clusters have been reported so far. It is unknown whether patients suffering from sporadic GBS differ from GBS clusters with respect to clinical and paraclinical parameters, HLA association and antibody response to glycosphingolipids and Campylobacter jejuni (Cj). We examined 40 consecutive patients with GBS from the greater Munich area in Germany with 14 of those admitted within a period of 3months in fall 2010 defining a cluster of GBS. Sequencing-based HLA typing of the HLA genes DRB1, DQB1, and DPB1 was performed, and ELISA for anti-glycosphingolipid antibodies was carried out. Clinical and paraclinical findings (Cj seroreactivity, cerebrospinal fluid parameters, and electrophysiology) were obtained and analyzed. GBS cluster patients were characterized by a more severe clinical phenotype with more patients requiring mechanical ventilation and higher frequencies of autoantibodies against sulfatide, GalC and certain ganglioside epitopes (54%) as compared to sporadic GBS cases (13%, p=0.017). Cj seropositivity tended to be higher within GBS cluster patients (69%) as compared to sporadic cases (46%, p=0.155). We noted higher frequencies of HLA class II allele DQB1*05:01 in the cluster cohort (23%) as compared to sporadic GBS patients (3%, p=0.019). Cluster of severe GBS was defined by higher frequencies of autoantibodies against glycosphingolipids. HLA class II allele DQB1*05:01 might contribute to clinical worsening in the cluster patients.

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