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.
Ehret J.K.,University of Bonn |
Engels H.,University of Bonn |
Cremer K.,University of Bonn |
Becker J.,University of Bonn |
And 23 more authors.
Molecular Cytogenetics | Year: 2015
Background: Most microdeletions involving chromosome sub-bands 9q33.3-9q34.11 to this point have been detected by analyses focused on STXBP1, a gene known to cause early infantile epileptic encephalopathy 4 and other seizure phenotypes. Loss-of-function mutations of STXBP1 have also been identified in some patients with intellectual disability without epilepsy. Consequently, STXBP1 is widely assumed to be the gene causing both seizures and intellectual disability in patients with 9q33.3-q34.11 microdeletions. Results: We report five patients with overlapping microdeletions of chromosome 9q33.3-q34.11, four of them previously unreported. Their common clinical features include intellectual disability, psychomotor developmental delay with delayed or absent speech, muscular hypotonia, and strabismus. Microcephaly and short stature are each present in four of the patients. Two of the patients had seizures. De novo deletions range from 1.23 to 4.13 Mb, whereas the smallest deletion of 432 kb in patient 3 was inherited from her mother who is reported to have mild intellectual disability. The smallest region of overlap (SRO) of these deletions in 9q33.3 does not encompass STXBP1, but includes two genes that have not been previously associated with disease, RALGPS1 and GARNL3. Sequencing of the two SRO genes RALGPS1 and GARNL3 in at least 156 unrelated patients with mild to severe idiopathic intellectual disability detected no causative mutations. Gene expression analyses in our patients demonstrated significantly reduced expression levels of GARNL3, RALGPS1 and STXBP1 only in patients with deletions of the corresponding genes. Thus, reduced expression of STXBP1 was ruled out as a cause for seizures in our patient whose deletion did not encompass STXBP1. Conclusions: We suggest that microdeletions of this region on chromosome 9q cause a clinical spectrum including intellectual disability, developmental delay especially concerning speech, microcephaly, short stature, mild dysmorphisms, strabismus, and seizures of incomplete penetrance, and may constitute a new contiguous gene deletion syndrome which cannot completely be explained by deletion of STXBP1. © 2015 Ehret et al.
Waldmuller S.,University of Tübingen |
Schroeder C.,University of Tübingen |
Sturm M.,University of Tübingen |
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.
Kotschote S.,IMGM Laboratories GmbH |
Wagner C.,IMGM Laboratories GmbH |
Marschall C.,Center for Human Genetics |
Mayer K.,Center for Human Genetics |
And 5 more authors.
LaboratoriumsMedizin | Year: 2010
In the past 5 years, next-generation sequencing (NGS) has been established as a valuable tool for several research applications. Commercially available platforms from Helicos, Illumina, Life Technologies, Pacific Biosciencies, and Roche allow for massively parallel sequencing and analysis in the fields of genomics, transcriptomics, and epigenomics. As in most projects, data throughput of the sequencers is not the limiting factor; genomic DNA samples are directly prepared for sequencing without prior conditioning. However, there are some applications such as targeted resequencing that do not require sequencing of whole genomes. Therefore, a technology called target enrichment was established more than 2 years ago. Different PCR- or hybridization-based approaches were further commercially developed and refined. The combination of this method with NGS can improve analysis of disease-related gene sets in molecular diagnostics by reducing time and costs. By taking advantage of the enormous data output, several genes and patients can be analyzed in parallel in one single instrument run. © 2010 by Walter de Gruyter Berlin New York.
Harasim T.,Center for Human Genetics and Laboratory Diagnostics |
Rost I.,Zentrum For Humangenetik Und Laboratoriumsdiagnostik Dr Klein |
Klein H.-G.,IMGM Laboratories GmbH
LaboratoriumsMedizin | Year: 2016
The introduction of non-invasive prenatal testing (NIPT) into prenatal care represents a paradigm shift. With the absence of any intervention risk in contrast to invasive diagnostic procedures, NIPT has been widely adopted for the detection of fetal trisomy 13, 18 and 21. Additionally, fetal sex chromosome aneuploidy testing and sex determination are available, but can be compromised by both, medical and legal factors. Available validation studies were predominantly based on patients with a high a priori aneuploidy risk, determined by trimester screening or invasive diagnostics. In this review, we discuss the interpretation of NIPT results in context of patient specific risk constellations, the available performance data and dominant methodical approaches of NIPT including necessary content of genetic counseling. © 2016 Walter de Gruyter GmbH, Berlin/Boston.
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.
Rucker O.,IMGM Laboratories GmbH |
Dangel A.,IMGM Laboratories GmbH |
Klein H.-G.,IMGM Laboratories GmbH |
Klein H.-G.,Center For Human Genetics And Laboratory Medicine Dr Klein And Dr Rost
LaboratoriumsMedizin | Year: 2013
The intense research focused on the human microbiome during the last years has shed some light on this mostly uncharacterized part of the human body. The constantly improving sequencing technologies have additionally eased the process of collecting a large amount of genome data from metagenomics samples. Using these methods, large studies with sufficient number of subjects have started to reveal the implications of our microbiome in health and disease. Here, we present a review on the last developments of sequencing technology together with an overview on the findings in this fast-evolving field of science.
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.
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.
PubMed | MOLOGEN AG, Animal Health and Veterinary Laboratories Agency, London School of Hygiene and Tropical Medicine, LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG and IMGM Laboratories GmbH
Type: Journal Article | Journal: 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.