This study evaluates the performance of the antigen-based anterior nasal screening programme impl... more This study evaluates the performance of the antigen-based anterior nasal screening programme implemented in all Austrian schools to detect SARS-CoV-2 infections. We combined nationwide antigen-based screening data obtained in March 2021 from 5,370 schools (Grade 1–8) with an RT-qPCR-based prospective cohort study comprising a representative sample of 244 schools. Considering a range of assumptions, only a subset of infected individuals are detected with the programme (low to moderate sensitivity) and non-infected individuals mainly tested negative (very high specificity).
De novo mutations (DNMs) are important players in heritable diseases and evolution. Of particular... more De novo mutations (DNMs) are important players in heritable diseases and evolution. Of particular interest are highly recurrent DNMs associated with congenital disorders that have been described as selfish mutations expanding in the male germline, thus becoming more frequent with age. Here, we have adapted duplex sequencing (DS), an ultradeep sequencing method that renders sequence information on both DNA strands; thus, one mutation can be reliably called in millions of sequenced bases. With DS, we examined ∼4.5 kb of the FGFR3 coding region in sperm DNA from older and younger donors. We identified sites with variant allele frequencies (VAFs) of 10−4 to 10−5, with an overall mutation frequency of the region of ∼6 × 10−7. Some of the substitutions are recurrent and are found at a higher VAF in older donors than in younger ones or are found exclusively in older donors. Also, older donors harbor more mutations associated with congenital disorders. Other mutations are present in both ag...
The nonclassical HLA-G class I gene is expressed by extravill- ous cytotrophoblast that invades d... more The nonclassical HLA-G class I gene is expressed by extravill- ous cytotrophoblast that invades decidua in uterine pregnancy, suggesting that it may contribute to the immunological mechanisms that protect the fetus against maternal alloimmune response and/or pathogen infections. We first addressed the question of whether HLA-G expression was dependent on maternal tissue environment by comparing uterine and ectopic tubal preg- nancies. Using HLA-G-specific mAb on placental cryosections, we found by immunohistochemistry that all subtypes of extravillous cytotrophoblast similarly expressed HLA-G in pregnant tubes, demonstrating that its ex- pression was independent of the site of implantation. We next compared by immunohistochemistry the phenotype of maternal leukocytes recruited in both pregnant tissues. In contrast to decidua, pregnant tubes were character- ized firstly, by the lack of natural killer (NK) cells and of cells expressing CD94 receptor specific for HLA-E, secondly, by a ...
The Human Major Histocompatibility Complex (MHC) is a highly polymorphic region full of immunoreg... more The Human Major Histocompatibility Complex (MHC) is a highly polymorphic region full of immunoregulatory genes. The MHC codes for the human leukocyte antigens (HLA), proteins that present on the cellular surface and that are involved in self-non-self recognition. For matching donors and recipients for organ and stem-cell transplants it is important to know an individual's HLA haplotype determinable in this region. Now, as next-generation sequencing (NGS) platforms mature and become more and more accepted as a standard method, NGS applications have spread from research laboratories to the clinic, where they provide valid genetic insights. Here, we describe a cost-effective microarray-based sequence capture, enrichment, and NGS sequencing approach to characterize MHC haplotypes. Using this approach, ~4 MB of MHC sequence for four DNA samples (donor, recipient and the parents of the recipient) were sequenced in parallel in one NGS instrument run. We complemented this approach using...
Colorectal cancer (CRC) is the third most frequent cancer worldwide and the second cause of cance... more Colorectal cancer (CRC) is the third most frequent cancer worldwide and the second cause of cancer deaths. Increasing evidences supports the idea that the poor prognosis of patients is related to the presence of cancer stem cells (CSCs), a cell population able to drive cancer recurrence and metastasis. The deregulation of microRNAs (miRNAs) plays a role in the formation of CSC. We investigated the role of hsa-miR-486-5p (miR-486-5p) in CRC, CSCs, and metastasis, in order to reach a better understanding of the biomolecular and epigenetic mechanisms mir-486-5p-related. The expression of miR-486-5p was investigated in three different matrices from CRC patients and controls and in CSCs obtained from the CRC cell lines HCT-116, HT-29, and T-84. In the human study, miR-486-5p was up-regulated in serum and stool of CRC patients in comparison with healthy controls but down-regulated in tumor tissue when compared with normal mucosa. miR-486-5p was also down-regulated in the sera of metastati...
BackgroundThe role of schools in the SARS-CoV-2 pandemic is much debated. We aimed to quantify re... more BackgroundThe role of schools in the SARS-CoV-2 pandemic is much debated. We aimed to quantify reliably the prevalence of SARS-CoV-2 infections at schools detected with reverse-transcription quantitative polymerase-chain-reaction (RT-qPCR).MethodsThis nationwide prospective cohort study monitors a representative sample of pupils (grade 1-8) and teachers at Austrian schools throughout the school year 2020/2021. We repeatedly test participants for SARS-CoV-2 infection using a gargling solution and RT-qPCR. We herein report on the first two rounds of examinations. We used mixed-effect logistic regression to estimate odds ratios and robust 95% confidence intervals (95% CI).FindingsWe analysed data on 10734 participants from 245 schools (9465 pupils, 1269 teachers). Prevalence of SARS-CoV-2 infection increased from 0.39% at round 1 (95% CI 0.28-0·55%, 29 September-22 October 2020) to 1·39% at round 2 (95% CI 1·04-1·85%, 10-16 November). Odds ratios for SARS-CoV-2 infection were 2·26 (95%...
Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by peripheral cytopenia, an a... more Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by peripheral cytopenia, an accumulation of dysplastic cells in the bone marrow (BM) and a high risk of transformation to acute myeloid leukemia. Transfusion-dependent anemia develops in most patients suffering from MDS. In patients with refractory anemias the accumulation of dysplastic erythroid BM progenitor cells (EryPC) which paradoxically accompanies peripheral anemia, is a typical finding. Although various hypotheses have been discussed, the biochemical basis of the maturation defect of erythroid cells and their abnormal accumulation in the BM in MDS remains unknown. In order to learn more about abnormally regulated and functionally relevant genes expressed in EryPC, we have recently established a screening program involving mRNA expression profiling studies of EryPC in patients with low-risk MDS (IPSS-R score<7) and control BM samples obtained from patients with other BM neoplasms as well as patients with ...
Background: Next-generation sequencing allows for determining the genetic composition of a mixed ... more Background: Next-generation sequencing allows for determining the genetic composition of a mixed sample. For instance, when performing resistance testing for BCR-ABL1 it is necessary to identify clones and define compound mutations; together with an exact quantification this may complement diagnosis and therapy decisions with additional information. Moreover, that applies not only to oncological issues but also determination of viral, bacterial or fungal infection. The efforts to retrieve multiple haplotypes (more than two) and proportion information from data with conventional software are difficult, cumbersome and demand multiple manual steps. Results: Therefore, we developed a tool called cFinder that is capable of automatic detection of haplotypes and their accurate quantification within one sample. BCR-ABL1 samples containing multiple clones were used for testing and our cFinder could identify all previously found clones together with their abundance and even refine some results. Additionally, reads were simulated using GemSIM with multiple haplotypes, the detection was very close to linear (R 2 = 0.96). Our aim is not to deduce haploblocks over statistics, but to characterize one sample's composition precisely. As a result the cFinder reports the connections of variants (haplotypes) with their readcount and relative occurrence (percentage). Download is available at http://sourceforge.net/projects/cfinder/. Conclusions: Our cFinder is implemented in an efficient algorithm that can be run on a low-performance desktop computer. Furthermore, it considers paired-end information (if available) and is generally open for any current nextgeneration sequencing technology and alignment strategy. To our knowledge, this is the first software that enables researchers without extensive bioinformatic support to designate multiple haplotypes and how they constitute to a sample.
We have recently identified lymphatic endothelial cells (LECs) to form two morphologically differ... more We have recently identified lymphatic endothelial cells (LECs) to form two morphologically different populations, exhibiting significantly different surface protein expression levels of podoplanin, a major surface marker for this cell type. In vitro shockwave treatment (IVSWT) of LECs resulted in enrichment of the podoplanin high cell population and was accompanied by markedly increased cell proliferation, as well as 2D and 3D migration. Gene expression profiles of these distinct populations were established using Affymetrix microarray analyses. Here we provide additional details about our dataset (NCBI GEO accession number GSE62510) and describe how we analyzed the data to identify differently expressed genes in these two LEC populations.
Special attention has recently been drawn to the molecular network of different genes that are re... more Special attention has recently been drawn to the molecular network of different genes that are responsible for the development of erythroid cells. The aim of the present study was to establish in detail the immunophenotype of early erythroid cells and to compare the gene expression profile of freshly isolated early erythroid precursors with that of the CD34-positive (CD34(+)) compartment. Multiparameter flow cytometric analyses of human bone marrow mononuclear cell fractions (n=20) defined three distinct early erythroid stages. The gene expression profile of sorted early erythroid cells was analyzed by Affymetrix array technology. For 4524 genes, a differential regulation was found in CD105-positive erythroid cells as compared with the CD34(+) progenitor compartment (2362 upregulated genes). A highly significant difference was observed in the expression level of genes involved in transcription, heme synthesis, iron and mitochondrial metabolism and transforming growth factor-β signal...
Extracorporeal shockwave treatment was shown to improve orthopaedic diseases and wound healing an... more Extracorporeal shockwave treatment was shown to improve orthopaedic diseases and wound healing and to stimulate lymphangiogenesis in vivo. The aim of this study was to investigate in vitro shockwave treatment (IVSWT) effects on lymphatic endothelial cell (LEC) behavior and lymphangiogenesis. We analyzed migration, proliferation, vascular tube forming capability and marker expression changes of LECs after IVSWT compared with HUVECs. Finally, transcriptome- and miRNA analyses were conducted to gain deeper insight into the IVSWT-induced molecular mechanisms in LECs. The results indicate that IVSWT-mediated proliferation changes of LECs are highly energy flux density-dependent and LEC 2D as well as 3D migration was enhanced through IVSWT. IVSWT suppressed HUVEC 3D migration but enhanced vasculogenesis. Furthermore, we identified podoplaninhigh and podoplaninlow cell subpopulations, whose ratios changed upon IVSWT treatment. Transcriptome- and miRNA analyses on these populations showed d...
The detection of KRAS mutations in codons 12 and 13 is critical for anti-EGFR therapy strategies;... more The detection of KRAS mutations in codons 12 and 13 is critical for anti-EGFR therapy strategies; however, only those methodologies with high sensitivity, specificity, and accuracy as well as the best cost and turnaround balance are suitable for routine daily testing. Here we compared the performance of compact sequencing using the novel hybcell technology with 454 next-generation sequencing (454-NGS), Sanger sequencing, and pyrosequencing, using an evaluation panel of 35 specimens. A total of 32 mutations and 10 wild-type cases were reported using 454-NGS as the reference method. Specificity ranged from 100% for Sanger sequencing to 80% for pyrosequencing. Sanger sequencing and hybcell-based compact sequencing achieved a sensitivity of 96%, whereas pyrosequencing had a sensitivity of 88%. Accuracy was 97% for Sanger sequencing, 85% for pyrosequencing, and 94% for hybcell-based compact sequencing. Quantitative results were obtained for 454-NGS and hybcell-based compact sequencing da...
A novel procedure for DNA methylation analysis is described that characterizes the extent of DNA ... more A novel procedure for DNA methylation analysis is described that characterizes the extent of DNA methylation in CpG islands. The basic concept relies on direct immunodetection of 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; methylcytosines (5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; mCs) without the need for bisulfite treatment utilizing a microarray format. This system is designed for the application of immunofluorescence using a monoclonal antibody that specifically recognizes 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; mC in single-stranded DNA hybridized to oligonucleotide microarrays. An ultrasensitive fluorescence scanner and 170-mum thin aldehyde-functionalized glass slides are used to optimize the signal-to-noise ratio and to minimize autofluorescence. These methodological improvements allow for the direct detection of 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; mC in genomic DNA hybridized to microarrays without prior PCR amplification with high analytical sensitivity.
Background and Objective Genotyping may be applied for rare blood group polymorphisms in a high-t... more Background and Objective Genotyping may be applied for rare blood group polymorphisms in a high-throughput mode as well for the molecular determination of blood groups due to unclear serological results. Material and Methods We developed and validated a DNA typing method for the determination of KEL1/2, JK1/2, FY1/2, FY0, MNS1/2, MNS3/4, DO1/2, CO1/2 and LU1/2 alleles using a melting curve analysis downstream from a fully automated DNA extraction. All assays were validated in terms of specificity, sensitivity, assay variability and robustness. The usability was proven by a batch of 200 blood samples with partially known phenotype. Results Assays for all blood groups were within the range of specificity (100%), assay variability and robustness (coefficient of variance < 3%). Genotypes of 200 random platelet donors were fully consistent with existing phenotype data. The obtained genotype distribution is in complete concordance with existing data for the European population underlined by a complete absence of CO2 homozygous donors and the FY0 allele among the cohort. Conclusion We introduce an approach for blood group genotyping of particular samples or gene loci in glass capillary format and for medium-throughput analysis in 96/384-well format. The advantages of this real-time polymerase chain reaction method are its automation potential, the flexibility regarding hardware and the rapid cycling time.
Background and objectives Molecular variations of the RHD gene may result in the reduced expressi... more Background and objectives Molecular variations of the RHD gene may result in the reduced expression of the D antigen and altered Rh phenotypes. In many occasions, they cannot be typed reliably by standard serological methods. Sequencebased typing is the gold standard to determine rare and unknown RHD genotypes. For this pilot study, sequence-based typing by standard Sanger sequencing was compared to a newly established next-generation sequencing approach based on pyrosequencing. Materials and methods Twenty-six DNA samples were selected after primary serological testing exhibiting a weak reaction in Rh phenotype. Parallel sequence analysis of the complete coding sequence including adjacent intronic sequences allowed a comparison of the methodical potency in mutation detection of Sanger with next-generation sequencing. Results Sanger sequencing revealed 39 RHD polymorphisms in 21 of 26 samples in the RHD coding region, while pyrosequencing detected all but two alterations resulting in a concordance rate of 94AE9% and clearly revealed a heterozygous compound mutation in one sample with RHDw and Weak D type 4 alleles. The resolution of cis ⁄ trans linkage of polymorphisms and exact characterization of a 37 bp duplication was achieved by next-generation sequencing. Conclusion Our data suggest that next-generation sequencing offers a new development for high-throughput and clonal sequencing for molecular RHD genotyping. However, further attempts in the methodical setup have to be undertaken prior to validation and introduction as a routine service.
Background. Hematopoietic stem-cell transplantation is a well-established treatment in various he... more Background. Hematopoietic stem-cell transplantation is a well-established treatment in various hematologic malignancies, but the outcome depends on disease relapse, infections, and the development and severity of acute and chronic graft-versus-host disease. Some evidence has revealed an important role for the nonclassical major histocompatibility complex class I molecules in transplantation, most notably human leukocyte antigen (HLA)-E. This study evaluates the impact of HLA-E alleles on transplantation outcome after HLA-matched allogeneic HSCT. Methods. We genotyped DNA for HLA-E polymorphism from 83 recipients and their respective donors by real-time polymerase chain reaction after melting curve analysis and compared the results with clinical outcome. Results. HLA-E*0103 homozygous patients showed a higher probability of overall survival (Pϭ0.003) and disease-free survival (Pϭ0.001) in a univariate model. Cox regression analysis confirmed HLA-E*0103, 0103 (Pϭ0.006; relative risk 1.12; 95% confidence interval 0.31-1.94) and early stage of disease (Pϭ0.005; relative risk 1.16; 95% confidence interval 0.45-1.86) as independent factors improving overall survival. Moreover, homozygosity for HLA-E*0103 was associated with a significant decreased incidence of transplant-related mortality (Pϭ0.01). Conclusions. We found an association between HLA-E*0103 homozygosity and the significant reduction of transplant-related mortality in related and unrelated HSCT. The risk of posttransplant complications was significantly reduced when the donor possesses the HLA-E*0103, 0103 genotype, and this was translated in a better overall survival.
The human genome project triggered the introduction of next generation sequencing (NGS) systems. ... more The human genome project triggered the introduction of next generation sequencing (NGS) systems. Although originally developed for total genome sequencing, metagenomics and plant genetics, the ultra-deep sequencing feature of NGS was utilized for diagnostic purposes in HIV resistance and tropism as well in detecting new mutations and tumor clones in oncology. Recent publications exploited the feature of clonal sequencing for immunogenetics to dissolve the growing number of ambiguities. This concept is quite reliable if all exons of interest are tested and the amplification region includes flanking introns. Challenging questions on quality control, cost effectiveness, workflow, and management of enormous loads of data remain if NGS is considered as routine method in the immunogenetics laboratory. If solved, NGS has big potential to have a major impact on immunogenetics by way of providing ambiguity-free HLA-typing results faster, but will also have a great influence on how immunogenetics testing and workflows are organized. Schlüsselwörter Histokompatibilität • Massively-Parallel-Sequenzierung • HLA-Typisierung Zusammenfassung Das humane Genom-Projekt hat Entwicklungen ausgelöst, die zur nächsten Generation von Sequenziermethoden (next generation sequencing, NGS) geführt hat. Ursprünglich als Methode für das Sequenzieren gesamter Genome, die Metagenomik und die Pflanzengenetik eingerichtet, wurde die Möglichkeit zum «Ultra-Deep»-Sequenzieren auch für die Diagnose von HIV-Resistenzen und-Tropismen, aber auch für die Erkennung neuer Mutationen und Tumorklone eingesetzt. Neuere Literatur hat die Möglichkeit zur klonalen Sequenzierung aufgezeigt, mit der die wachsende Anzahl von Ambiguitäten in der Immungenetik aufgelöst werden kann. Dieses Konzept ist dann zuverlässig, wenn möglichst viele Exons in die Analyse mit einbezogen werden (insbesondere jene, die für die Polymorphismen in Frage kommen) und wenn die Amplifikationsregion auch die flankierenden Introns umfasst. Herausragende Fragen verbleiben noch für die Qualitätskontrolle, die Kosteneffizienz, den Arbeitsablauf, und die Bewältigung der enormen Datenflut bevor Überlegungen zur Einführung dieser Methode in der Routine angestellt werden können. Wenn diese Fragen gelöst sind, hat NGS ein hohes Potenzial, die Patienten schneller und sicherer mit eindeutigen HLA-Typisierungsergebnissen zu versorgen, aber auch einen großen Einfluss auf die zukünftige Organisation von Abläufen und Tests in der Immungenetik.
This study evaluates the performance of the antigen-based anterior nasal screening programme impl... more This study evaluates the performance of the antigen-based anterior nasal screening programme implemented in all Austrian schools to detect SARS-CoV-2 infections. We combined nationwide antigen-based screening data obtained in March 2021 from 5,370 schools (Grade 1–8) with an RT-qPCR-based prospective cohort study comprising a representative sample of 244 schools. Considering a range of assumptions, only a subset of infected individuals are detected with the programme (low to moderate sensitivity) and non-infected individuals mainly tested negative (very high specificity).
De novo mutations (DNMs) are important players in heritable diseases and evolution. Of particular... more De novo mutations (DNMs) are important players in heritable diseases and evolution. Of particular interest are highly recurrent DNMs associated with congenital disorders that have been described as selfish mutations expanding in the male germline, thus becoming more frequent with age. Here, we have adapted duplex sequencing (DS), an ultradeep sequencing method that renders sequence information on both DNA strands; thus, one mutation can be reliably called in millions of sequenced bases. With DS, we examined ∼4.5 kb of the FGFR3 coding region in sperm DNA from older and younger donors. We identified sites with variant allele frequencies (VAFs) of 10−4 to 10−5, with an overall mutation frequency of the region of ∼6 × 10−7. Some of the substitutions are recurrent and are found at a higher VAF in older donors than in younger ones or are found exclusively in older donors. Also, older donors harbor more mutations associated with congenital disorders. Other mutations are present in both ag...
The nonclassical HLA-G class I gene is expressed by extravill- ous cytotrophoblast that invades d... more The nonclassical HLA-G class I gene is expressed by extravill- ous cytotrophoblast that invades decidua in uterine pregnancy, suggesting that it may contribute to the immunological mechanisms that protect the fetus against maternal alloimmune response and/or pathogen infections. We first addressed the question of whether HLA-G expression was dependent on maternal tissue environment by comparing uterine and ectopic tubal preg- nancies. Using HLA-G-specific mAb on placental cryosections, we found by immunohistochemistry that all subtypes of extravillous cytotrophoblast similarly expressed HLA-G in pregnant tubes, demonstrating that its ex- pression was independent of the site of implantation. We next compared by immunohistochemistry the phenotype of maternal leukocytes recruited in both pregnant tissues. In contrast to decidua, pregnant tubes were character- ized firstly, by the lack of natural killer (NK) cells and of cells expressing CD94 receptor specific for HLA-E, secondly, by a ...
The Human Major Histocompatibility Complex (MHC) is a highly polymorphic region full of immunoreg... more The Human Major Histocompatibility Complex (MHC) is a highly polymorphic region full of immunoregulatory genes. The MHC codes for the human leukocyte antigens (HLA), proteins that present on the cellular surface and that are involved in self-non-self recognition. For matching donors and recipients for organ and stem-cell transplants it is important to know an individual's HLA haplotype determinable in this region. Now, as next-generation sequencing (NGS) platforms mature and become more and more accepted as a standard method, NGS applications have spread from research laboratories to the clinic, where they provide valid genetic insights. Here, we describe a cost-effective microarray-based sequence capture, enrichment, and NGS sequencing approach to characterize MHC haplotypes. Using this approach, ~4 MB of MHC sequence for four DNA samples (donor, recipient and the parents of the recipient) were sequenced in parallel in one NGS instrument run. We complemented this approach using...
Colorectal cancer (CRC) is the third most frequent cancer worldwide and the second cause of cance... more Colorectal cancer (CRC) is the third most frequent cancer worldwide and the second cause of cancer deaths. Increasing evidences supports the idea that the poor prognosis of patients is related to the presence of cancer stem cells (CSCs), a cell population able to drive cancer recurrence and metastasis. The deregulation of microRNAs (miRNAs) plays a role in the formation of CSC. We investigated the role of hsa-miR-486-5p (miR-486-5p) in CRC, CSCs, and metastasis, in order to reach a better understanding of the biomolecular and epigenetic mechanisms mir-486-5p-related. The expression of miR-486-5p was investigated in three different matrices from CRC patients and controls and in CSCs obtained from the CRC cell lines HCT-116, HT-29, and T-84. In the human study, miR-486-5p was up-regulated in serum and stool of CRC patients in comparison with healthy controls but down-regulated in tumor tissue when compared with normal mucosa. miR-486-5p was also down-regulated in the sera of metastati...
BackgroundThe role of schools in the SARS-CoV-2 pandemic is much debated. We aimed to quantify re... more BackgroundThe role of schools in the SARS-CoV-2 pandemic is much debated. We aimed to quantify reliably the prevalence of SARS-CoV-2 infections at schools detected with reverse-transcription quantitative polymerase-chain-reaction (RT-qPCR).MethodsThis nationwide prospective cohort study monitors a representative sample of pupils (grade 1-8) and teachers at Austrian schools throughout the school year 2020/2021. We repeatedly test participants for SARS-CoV-2 infection using a gargling solution and RT-qPCR. We herein report on the first two rounds of examinations. We used mixed-effect logistic regression to estimate odds ratios and robust 95% confidence intervals (95% CI).FindingsWe analysed data on 10734 participants from 245 schools (9465 pupils, 1269 teachers). Prevalence of SARS-CoV-2 infection increased from 0.39% at round 1 (95% CI 0.28-0·55%, 29 September-22 October 2020) to 1·39% at round 2 (95% CI 1·04-1·85%, 10-16 November). Odds ratios for SARS-CoV-2 infection were 2·26 (95%...
Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by peripheral cytopenia, an a... more Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by peripheral cytopenia, an accumulation of dysplastic cells in the bone marrow (BM) and a high risk of transformation to acute myeloid leukemia. Transfusion-dependent anemia develops in most patients suffering from MDS. In patients with refractory anemias the accumulation of dysplastic erythroid BM progenitor cells (EryPC) which paradoxically accompanies peripheral anemia, is a typical finding. Although various hypotheses have been discussed, the biochemical basis of the maturation defect of erythroid cells and their abnormal accumulation in the BM in MDS remains unknown. In order to learn more about abnormally regulated and functionally relevant genes expressed in EryPC, we have recently established a screening program involving mRNA expression profiling studies of EryPC in patients with low-risk MDS (IPSS-R score<7) and control BM samples obtained from patients with other BM neoplasms as well as patients with ...
Background: Next-generation sequencing allows for determining the genetic composition of a mixed ... more Background: Next-generation sequencing allows for determining the genetic composition of a mixed sample. For instance, when performing resistance testing for BCR-ABL1 it is necessary to identify clones and define compound mutations; together with an exact quantification this may complement diagnosis and therapy decisions with additional information. Moreover, that applies not only to oncological issues but also determination of viral, bacterial or fungal infection. The efforts to retrieve multiple haplotypes (more than two) and proportion information from data with conventional software are difficult, cumbersome and demand multiple manual steps. Results: Therefore, we developed a tool called cFinder that is capable of automatic detection of haplotypes and their accurate quantification within one sample. BCR-ABL1 samples containing multiple clones were used for testing and our cFinder could identify all previously found clones together with their abundance and even refine some results. Additionally, reads were simulated using GemSIM with multiple haplotypes, the detection was very close to linear (R 2 = 0.96). Our aim is not to deduce haploblocks over statistics, but to characterize one sample's composition precisely. As a result the cFinder reports the connections of variants (haplotypes) with their readcount and relative occurrence (percentage). Download is available at http://sourceforge.net/projects/cfinder/. Conclusions: Our cFinder is implemented in an efficient algorithm that can be run on a low-performance desktop computer. Furthermore, it considers paired-end information (if available) and is generally open for any current nextgeneration sequencing technology and alignment strategy. To our knowledge, this is the first software that enables researchers without extensive bioinformatic support to designate multiple haplotypes and how they constitute to a sample.
We have recently identified lymphatic endothelial cells (LECs) to form two morphologically differ... more We have recently identified lymphatic endothelial cells (LECs) to form two morphologically different populations, exhibiting significantly different surface protein expression levels of podoplanin, a major surface marker for this cell type. In vitro shockwave treatment (IVSWT) of LECs resulted in enrichment of the podoplanin high cell population and was accompanied by markedly increased cell proliferation, as well as 2D and 3D migration. Gene expression profiles of these distinct populations were established using Affymetrix microarray analyses. Here we provide additional details about our dataset (NCBI GEO accession number GSE62510) and describe how we analyzed the data to identify differently expressed genes in these two LEC populations.
Special attention has recently been drawn to the molecular network of different genes that are re... more Special attention has recently been drawn to the molecular network of different genes that are responsible for the development of erythroid cells. The aim of the present study was to establish in detail the immunophenotype of early erythroid cells and to compare the gene expression profile of freshly isolated early erythroid precursors with that of the CD34-positive (CD34(+)) compartment. Multiparameter flow cytometric analyses of human bone marrow mononuclear cell fractions (n=20) defined three distinct early erythroid stages. The gene expression profile of sorted early erythroid cells was analyzed by Affymetrix array technology. For 4524 genes, a differential regulation was found in CD105-positive erythroid cells as compared with the CD34(+) progenitor compartment (2362 upregulated genes). A highly significant difference was observed in the expression level of genes involved in transcription, heme synthesis, iron and mitochondrial metabolism and transforming growth factor-β signal...
Extracorporeal shockwave treatment was shown to improve orthopaedic diseases and wound healing an... more Extracorporeal shockwave treatment was shown to improve orthopaedic diseases and wound healing and to stimulate lymphangiogenesis in vivo. The aim of this study was to investigate in vitro shockwave treatment (IVSWT) effects on lymphatic endothelial cell (LEC) behavior and lymphangiogenesis. We analyzed migration, proliferation, vascular tube forming capability and marker expression changes of LECs after IVSWT compared with HUVECs. Finally, transcriptome- and miRNA analyses were conducted to gain deeper insight into the IVSWT-induced molecular mechanisms in LECs. The results indicate that IVSWT-mediated proliferation changes of LECs are highly energy flux density-dependent and LEC 2D as well as 3D migration was enhanced through IVSWT. IVSWT suppressed HUVEC 3D migration but enhanced vasculogenesis. Furthermore, we identified podoplaninhigh and podoplaninlow cell subpopulations, whose ratios changed upon IVSWT treatment. Transcriptome- and miRNA analyses on these populations showed d...
The detection of KRAS mutations in codons 12 and 13 is critical for anti-EGFR therapy strategies;... more The detection of KRAS mutations in codons 12 and 13 is critical for anti-EGFR therapy strategies; however, only those methodologies with high sensitivity, specificity, and accuracy as well as the best cost and turnaround balance are suitable for routine daily testing. Here we compared the performance of compact sequencing using the novel hybcell technology with 454 next-generation sequencing (454-NGS), Sanger sequencing, and pyrosequencing, using an evaluation panel of 35 specimens. A total of 32 mutations and 10 wild-type cases were reported using 454-NGS as the reference method. Specificity ranged from 100% for Sanger sequencing to 80% for pyrosequencing. Sanger sequencing and hybcell-based compact sequencing achieved a sensitivity of 96%, whereas pyrosequencing had a sensitivity of 88%. Accuracy was 97% for Sanger sequencing, 85% for pyrosequencing, and 94% for hybcell-based compact sequencing. Quantitative results were obtained for 454-NGS and hybcell-based compact sequencing da...
A novel procedure for DNA methylation analysis is described that characterizes the extent of DNA ... more A novel procedure for DNA methylation analysis is described that characterizes the extent of DNA methylation in CpG islands. The basic concept relies on direct immunodetection of 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; methylcytosines (5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; mCs) without the need for bisulfite treatment utilizing a microarray format. This system is designed for the application of immunofluorescence using a monoclonal antibody that specifically recognizes 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; mC in single-stranded DNA hybridized to oligonucleotide microarrays. An ultrasensitive fluorescence scanner and 170-mum thin aldehyde-functionalized glass slides are used to optimize the signal-to-noise ratio and to minimize autofluorescence. These methodological improvements allow for the direct detection of 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; mC in genomic DNA hybridized to microarrays without prior PCR amplification with high analytical sensitivity.
Background and Objective Genotyping may be applied for rare blood group polymorphisms in a high-t... more Background and Objective Genotyping may be applied for rare blood group polymorphisms in a high-throughput mode as well for the molecular determination of blood groups due to unclear serological results. Material and Methods We developed and validated a DNA typing method for the determination of KEL1/2, JK1/2, FY1/2, FY0, MNS1/2, MNS3/4, DO1/2, CO1/2 and LU1/2 alleles using a melting curve analysis downstream from a fully automated DNA extraction. All assays were validated in terms of specificity, sensitivity, assay variability and robustness. The usability was proven by a batch of 200 blood samples with partially known phenotype. Results Assays for all blood groups were within the range of specificity (100%), assay variability and robustness (coefficient of variance < 3%). Genotypes of 200 random platelet donors were fully consistent with existing phenotype data. The obtained genotype distribution is in complete concordance with existing data for the European population underlined by a complete absence of CO2 homozygous donors and the FY0 allele among the cohort. Conclusion We introduce an approach for blood group genotyping of particular samples or gene loci in glass capillary format and for medium-throughput analysis in 96/384-well format. The advantages of this real-time polymerase chain reaction method are its automation potential, the flexibility regarding hardware and the rapid cycling time.
Background and objectives Molecular variations of the RHD gene may result in the reduced expressi... more Background and objectives Molecular variations of the RHD gene may result in the reduced expression of the D antigen and altered Rh phenotypes. In many occasions, they cannot be typed reliably by standard serological methods. Sequencebased typing is the gold standard to determine rare and unknown RHD genotypes. For this pilot study, sequence-based typing by standard Sanger sequencing was compared to a newly established next-generation sequencing approach based on pyrosequencing. Materials and methods Twenty-six DNA samples were selected after primary serological testing exhibiting a weak reaction in Rh phenotype. Parallel sequence analysis of the complete coding sequence including adjacent intronic sequences allowed a comparison of the methodical potency in mutation detection of Sanger with next-generation sequencing. Results Sanger sequencing revealed 39 RHD polymorphisms in 21 of 26 samples in the RHD coding region, while pyrosequencing detected all but two alterations resulting in a concordance rate of 94AE9% and clearly revealed a heterozygous compound mutation in one sample with RHDw and Weak D type 4 alleles. The resolution of cis ⁄ trans linkage of polymorphisms and exact characterization of a 37 bp duplication was achieved by next-generation sequencing. Conclusion Our data suggest that next-generation sequencing offers a new development for high-throughput and clonal sequencing for molecular RHD genotyping. However, further attempts in the methodical setup have to be undertaken prior to validation and introduction as a routine service.
Background. Hematopoietic stem-cell transplantation is a well-established treatment in various he... more Background. Hematopoietic stem-cell transplantation is a well-established treatment in various hematologic malignancies, but the outcome depends on disease relapse, infections, and the development and severity of acute and chronic graft-versus-host disease. Some evidence has revealed an important role for the nonclassical major histocompatibility complex class I molecules in transplantation, most notably human leukocyte antigen (HLA)-E. This study evaluates the impact of HLA-E alleles on transplantation outcome after HLA-matched allogeneic HSCT. Methods. We genotyped DNA for HLA-E polymorphism from 83 recipients and their respective donors by real-time polymerase chain reaction after melting curve analysis and compared the results with clinical outcome. Results. HLA-E*0103 homozygous patients showed a higher probability of overall survival (Pϭ0.003) and disease-free survival (Pϭ0.001) in a univariate model. Cox regression analysis confirmed HLA-E*0103, 0103 (Pϭ0.006; relative risk 1.12; 95% confidence interval 0.31-1.94) and early stage of disease (Pϭ0.005; relative risk 1.16; 95% confidence interval 0.45-1.86) as independent factors improving overall survival. Moreover, homozygosity for HLA-E*0103 was associated with a significant decreased incidence of transplant-related mortality (Pϭ0.01). Conclusions. We found an association between HLA-E*0103 homozygosity and the significant reduction of transplant-related mortality in related and unrelated HSCT. The risk of posttransplant complications was significantly reduced when the donor possesses the HLA-E*0103, 0103 genotype, and this was translated in a better overall survival.
The human genome project triggered the introduction of next generation sequencing (NGS) systems. ... more The human genome project triggered the introduction of next generation sequencing (NGS) systems. Although originally developed for total genome sequencing, metagenomics and plant genetics, the ultra-deep sequencing feature of NGS was utilized for diagnostic purposes in HIV resistance and tropism as well in detecting new mutations and tumor clones in oncology. Recent publications exploited the feature of clonal sequencing for immunogenetics to dissolve the growing number of ambiguities. This concept is quite reliable if all exons of interest are tested and the amplification region includes flanking introns. Challenging questions on quality control, cost effectiveness, workflow, and management of enormous loads of data remain if NGS is considered as routine method in the immunogenetics laboratory. If solved, NGS has big potential to have a major impact on immunogenetics by way of providing ambiguity-free HLA-typing results faster, but will also have a great influence on how immunogenetics testing and workflows are organized. Schlüsselwörter Histokompatibilität • Massively-Parallel-Sequenzierung • HLA-Typisierung Zusammenfassung Das humane Genom-Projekt hat Entwicklungen ausgelöst, die zur nächsten Generation von Sequenziermethoden (next generation sequencing, NGS) geführt hat. Ursprünglich als Methode für das Sequenzieren gesamter Genome, die Metagenomik und die Pflanzengenetik eingerichtet, wurde die Möglichkeit zum «Ultra-Deep»-Sequenzieren auch für die Diagnose von HIV-Resistenzen und-Tropismen, aber auch für die Erkennung neuer Mutationen und Tumorklone eingesetzt. Neuere Literatur hat die Möglichkeit zur klonalen Sequenzierung aufgezeigt, mit der die wachsende Anzahl von Ambiguitäten in der Immungenetik aufgelöst werden kann. Dieses Konzept ist dann zuverlässig, wenn möglichst viele Exons in die Analyse mit einbezogen werden (insbesondere jene, die für die Polymorphismen in Frage kommen) und wenn die Amplifikationsregion auch die flankierenden Introns umfasst. Herausragende Fragen verbleiben noch für die Qualitätskontrolle, die Kosteneffizienz, den Arbeitsablauf, und die Bewältigung der enormen Datenflut bevor Überlegungen zur Einführung dieser Methode in der Routine angestellt werden können. Wenn diese Fragen gelöst sind, hat NGS ein hohes Potenzial, die Patienten schneller und sicherer mit eindeutigen HLA-Typisierungsergebnissen zu versorgen, aber auch einen großen Einfluss auf die zukünftige Organisation von Abläufen und Tests in der Immungenetik.
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Papers by Johannes Pröll