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Stuttgart, Germany

Diethelm M.,Bavarian State Research Center for Agriculture | Rhiel M.,Justus Liebig University | Wagner C.,IMGM Laboratories GmbH | Mikolajewski S.,Bavarian State Research Center for Agriculture | And 4 more authors.
Euphytica | Year: 2012

Fusarium head blight (FHB) is a highly destructive disease of wheat and other cereals which causes serious mycotoxin contaminations of grain. A number of molecular mapping studies led to the detection of QTL with small to moderate effects on FHB resistance in European winter wheat. Genes involved in the defence reaction of these genotypes remain largely unknown. WIR1 (wheat induced resistance 1) genes have been shown to be upregulated in cereals during attack of various fungal pathogens; however, their role in resistance is ambiguous. In this study, the expression of three WIR1 genes and a gene with high sequence similarity to WIR1 was investigated in European winter wheat genotypes after inoculation with Giberella zeae. Floret tissues of four winter wheat genotypes (Dream, Lynx, G16-92, Hussar) were challenged with G. zeae conidia or water (control) and sampled six times during 0-96 h after inoculation. Quantitative real-time PCR showed that all four genes were highly upregulated in the resistant genotypes compared to the susceptible ones. WIR1b and a gene with sequence similarity to WIR1 genes mapped to chromosome 5DS in the G16-92/Hussar mapping population. Two genes annotated as WIR1a mapped in the interval of a FHB resistance QTL on chromosome 7BS in the Dream/Lynx mapping population. These could be considered possible candidate genes for quantitative FHB resistance. © 2011 Springer Science+Business Media B.V. Source

Meyer S.U.,TU Munich | Meyer S.U.,Ludwig Maximilians University of Munich | Kaiser S.,Ludwig Maximilians University of Munich | Wagner C.,IMGM Laboratories GmbH | And 2 more authors.
PLoS ONE | Year: 2012

Background: Adequate normalization minimizes the effects of systematic technical variations and is a prerequisite for getting meaningful biological changes. However, there is inconsistency about miRNA normalization performances and recommendations. Thus, we investigated the impact of seven different normalization methods (reference gene index, global geometric mean, quantile, invariant selection, loess, loessM, and generalized procrustes analysis) on intra- and inter-platform performance of two distinct and commonly used miRNA profiling platforms. Methodology/Principal Findings: We included data from miRNA profiling analyses derived from a hybridization-based platform (Agilent Technologies) and an RT-qPCR platform (Applied Biosystems). Furthermore, we validated a subset of miRNAs by individual RT-qPCR assays. Our analyses incorporated data from the effect of differentiation and tumor necrosis factor alpha treatment on primary human skeletal muscle cells and a murine skeletal muscle cell line. Distinct normalization methods differed in their impact on (i) standard deviations, (ii) the area under the receiver operating characteristic (ROC) curve, (iii) the similarity of differential expression. Loess, loessM, and quantile analysis were most effective in minimizing standard deviations on the Agilent and TLDA platform. Moreover, loess, loessM, invariant selection and generalized procrustes analysis increased the area under the ROC curve, a measure for the statistical performance of a test. The Jaccard index revealed that inter-platform concordance of differential expression tended to be increased by loess, loessM, quantile, and GPA normalization of AGL and TLDA data as well as RGI normalization of TLDA data. Conclusions/Significance: We recommend the application of loess, or loessM, and GPA normalization for miRNA Agilent arrays and qPCR cards as these normalization approaches showed to (i) effectively reduce standard deviations, (ii) increase sensitivity and accuracy of differential miRNA expression detection as well as (iii) increase inter-platform concordance. Results showed the successful adoption of loessM and generalized procrustes analysis to one-color miRNA profiling experiments. © 2012 Meyer et al. Source

Waldmuller S.,University of Tubingen | Schroeder C.,University of Tubingen | Sturm M.,University of Tubingen | Scheffold T.,MediClin MVZ Lahr | And 10 more authors.
Molecular and Cellular Probes | Year: 2015

With the implementation of high-throughput sequencing protocols, the exhaustive scanning of known and candidate disease genes has become a feasible approach to genetic testing of patients with cardiomyopathy. A primary objective of the present study was to assess the performance characteristics of a 46-gene next-generation sequencing (NGS) assay that targets well-established cardiomyopathy genes. A total of 25 samples were analyzed. Twelve of those had previously been sequenced using resequencing arrays and served as reference samples for the assessment of the assay's performance characteristics. The remaining 13 samples were derived from consecutive patients. Both the analytical sensitivity and the specificity of the assay were 100% and the percentage of low-coverage bases was 0.4%, at an average read depth of 210×. In order to assess the diagnostic yield of the test, 13 consecutive samples representing cases of Dilated (n = 7), Hypertrophic (n = 4) and Left Ventricular Non-Compaction Cardiomyopathy (n = 2), were subjected to the 46-gene NGS assay. Including predicted pathogenic variants in the gene TTN, a total of 22 variants (11 novel) were detected in 10 patients, with a clear preponderance of variants of unknown pathogenicity (class 3 variants, 21/22, 95%). Of the seven DCM cases, two were digenic, involving variants in the genes MYH7 and RBM20 in one case and in DSP and TTN in the other case. Three other patients carried single TTN variants predicted to be pathogenic. Of the four HCM patients, one was trigenic (. LAMA4, PKP2 and TTN) and three were digenic (. DSP and TTN, MYH7 and NEXN, NEXN and TTN, respectively). As to LVNC, one of the two patients had one variant in the gene ABCC9 and two predicted pathogenic variants in the gene TTN. Strikingly, out of the thirteen investigated cases, only a single case exhibited a likely pathogenic or pathogenic variant justifying a positive test report. The percentage of inconclusive cases thus amounted to 69%. Three cases were devoid of any relevant variant. Two of these "negative" cases were subsequently taken to initially evaluate the use of an alternative NGS assay addressing 4813 genes previously implicated in genetic diseases (the so-called clinical exome). Although showing similar sensitivity and specificity values, the coverage of the 46 established cardiomyopathy genes was less efficient (low-coverage bases: 5%). In a case of DCM, the assay revealed a disruptive variant in the gene encoding the adrenoreceptor beta 2 ( ADRB2), a protein implicated in signal transduction and energy metabolism in the heart. In conclusion, the 46 gene assay is applicable to routine genetic diagnostics of cardiomyopathy. The test detects many variants of unknown pathogenicity which need to be followed-up in order to gain benefit for the patients and their families. Samples devoid of any relevant variant may be subjected to a clinical exome assay, in order to identify interesting novel candidate genes. © 2015 Elsevier Ltd. Source

Riede O.,Mologen AG | Seifert K.,London School of Hygiene and Tropical Medicine | Oswald D.,Mologen AG | Endmann A.,Mologen AG | And 7 more authors.
Gene Therapy | Year: 2015

The leishmaniases are a complex of vector-borne diseases caused by protozoan parasites of the genus Leishmania. LEISHDNAVAX is a multi-antigen, T-cell epitope-enriched DNA vaccine candidate against human leishmaniasis. The vaccine candidate has been proven immunogenic and showed prophylactic efficacy in preclinical studies. Here, we describe the safety testing of LEISHDNAVAX in naive mice and rats, complemented by the demonstration of tolerability in Leishmania-infected mice. Biodistribution and persistence were examined following single and repeated intradermal (i.d.) administration to rats. DNA vectors were distributed systemically but did not accumulate upon repeated injections. Although vector DNA was cleared from most other tissues within 60 days after the last injection, it persisted in skin at the site of injection and in draining lymph nodes. Evaluation of single-dose and repeated-dose toxicity of the vaccine candidate after i.d. administration to naive, non-infected mice did not reveal any safety concerns. LEISHDNAVAX was also well tolerated in Leishmania-infected mice. Taken together, our results substantiate a favorable safety profile of LEISHDNAVAX in both naive and infected animals and thus, support the initiation of clinical trials for both preventive and therapeutic applications of the vaccine. © 2015 Macmillan Publishers Limited All rights reserved. Source

Kehrer M.,Applied Genomics | Schaferhoff K.,Applied Genomics | Bonin M.,Applied Genomics | Bonin M.,IMGM Laboratories GmbH | And 4 more authors.
American Journal of Medical Genetics, Part A | Year: 2015

Interstitial deletions encompassing chromosome bands 1p32.1p32.3 are rare. Only nine unrelated patients with partially overlapping 1p32.1p32.3 deletions of variable size and position have been reported to date. We report on a 17-month-old boy with choanal atresia, hearing loss, urogenital anomalies, and microcephaly in whom an interstitial de novo deletion of 6.4Mb was detected in 1p32.1p32.3 (genomic position chr1:54,668,618-61,113,264 according to GRCh37/hg19). The deleted region harbors 31 RefSeq genes. Notable genes in the region are PCSK9, haploinsufficiency of which caused low LDL cholesterol plasma levels in the patient, and DAB1, which is a candidate gene for cognitive deficits, microcephaly, and cerebral abnormalities such as ventriculomegaly and agenesis of the corpus callosum. Choanal atresia, microcephaly, and severe hearing loss were previously not known to be associated with 1p32 deletions. Our reported patient thus broadens the spectrum of clinical findings in this chromosome region and further facilitates genotype-phenotype correlations. Additional patients with overlapping deletions and/or point mutations in genes of this region need to be identified to elucidate the role of individual genes for the complex clinical manifestations. © 2015 Wiley Periodicals, Inc. Source

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