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An in-frame heterozygous large deletion of exons 4 through 34 of the von Willebrand factor (VWF) gene was identified in a type 3 von Willebrand disease (VWD) index patient (IP), as the only VWF variant. The IP exhibited severe bleeding episodes despite prophylaxis treatment, with a short VWF half-life after infusion of VWF/factor VIII concentrates. Transcript analysis confirmed transcription of normal VWF messenger RNA besides an aberrant deleted transcript. The IP endothelial colony-forming cells (ECFCs) exhibited a defect in the VWF multimers and Weibel-Palade bodies (WPBs) biogenesis, although demonstrating normal VWF secretion compared with healthy cells. Immunostaining of IP-ECFCs revealed subcellular mislocalization of WPBs pro-inflammatory cargos angiopoietin-2 (Ang2, nuclear accumulation) and P-selectin. Besides, the RNA-sequencing (RNA-seq) analysis showed upregulation of pro-inflammatory and proangiogenic genes, P-selectin, interleukin 8 (IL-8), IL-6, and GROα, copackaged with VWF into WPBs. Further, whole-transcriptome RNA-seq and subsequent gene ontology (GO) enrichment analysis indicated the most enriched GO-biological process terms among the differentially expressed genes in IP-ECFCs were regulation of cell differentiation, cell adhesion, leukocyte adhesion to vascular endothelial, blood vessel morphogenesis, and angiogenesis, which resemble downstream signaling pathways associated with inflammatory stimuli and Ang2 priming. Accordingly, our functional experiments exhibited an increased endothelial cell adhesiveness and interruption in endothelial cell–cell junctions of the IP-ECFCs. In conclusion, the deleted VWF has a dominant-negative impact on multimer assembly and the biogenesis of WPBs, leading to altered trafficking of their pro-inflammatory cargos uniquely, which, in turn, causes changes in cellular signaling pathways, phenotype, and function of the endothelial cells.

An in-frame heterozygous large deletion of exons 4 through 34 of the von Willebrand factor (VWF) gene was identified in a type 3 von Willebrand disease (VWD) index patient (IP), as the only VWF variant. The IP exhibited severe bleeding episodes despite prophylaxis treatment, with a short VWF half-life after infusion of VWF/factor VIII concentrates. Transcript analysis confirmed transcription of normal VWF messenger RNA besides an aberrant deleted transcript. The IP endothelial colony-forming cells (ECFCs) exhibited a defect in the VWF multimers and Weibel-Palade bodies (WPBs) biogenesis, although demonstrating normal VWF secretion compared with healthy cells. Immunostaining of IP-ECFCs revealed subcellular mislocalization of WPBs pro-inflammatory cargos angiopoietin-2 (Ang2, nuclear accumulation) and P-selectin. Besides, the RNA-sequencing (RNA-seq) analysis showed upregulation of pro-inflammatory and proangiogenic genes, P-selectin, interleukin 8 (IL-8), IL-6, and GROα, copackaged with VWF into WPBs. Further, whole-transcriptome RNA-seq and subsequent gene ontology (GO) enrichment analysis indicated the most enriched GO-biological process terms among the differentially expressed genes in IP-ECFCs were regulation of cell differentiation, cell adhesion, leukocyte adhesion to vascular endothelial, blood vessel morphogenesis, and angiogenesis, which resemble downstream signaling pathways associated with inflammatory stimuli and Ang2 priming. Accordingly, our functional experiments exhibited an increased endothelial cell adhesiveness and interruption in endothelial cell–cell junctions of the IP-ECFCs. In conclusion, the deleted VWF has a dominant-negative impact on multimer assembly and the biogenesis of WPBs, leading to altered trafficking of their pro-inflammatory cargos uniquely, which, in turn, causes changes in cellular signaling pathways, phenotype, and function of the endothelial cells.

Pilocytic astrocytoma is the most frequently occurring brain tumor during childhood. It is classified as grade I by the World Health Organization and may rarely evolve into higher-grade tumors. Frequent genetic abnormalities documented in astrocytomas in children are gains on chromosomal arm 7q. Duplications at 7q34 lead to a fusion between genes KIAA1549and BRAFresulting in constitutive activation of the BRAF kinase. The BRAFgene is located on chromosome 7q34 and a pseudogene has been identified on chromosome Xq13. We have developed a simple and sensitive pyrosequencing method for the determination of the BRAFcopy number in clinical samples. The approach is based on the simultaneous amplification of a DNA fragment contained in exon 11 of BRAFand the respective pseudogene that is used as an internal control. Three different bases in the PCR product allow precise sequence assessment of products originating from the BRAFgene and the respective pseudogene and a calculation of gene copy numbers. After the calibration of the assay on 78 control DNA samples, 42 clinical PA samples were analyzed for variation in copy numbers by pyrosequencing and for fusion gene expression by reverse transcription-polymerase chain reaction. The results obtained from tumor DNA by the developed assay and the established reverse transcription-polymerase chain reaction assays show a high concordance. In summary, we have established a pyrosequencing-based assay allowing precise detection of BRAF copy numbers in DNA extracted from clinical samples.

Resistance to chemotherapy is a major complication during treatment of cancer patients. Hypermethylation of the MGMTgene alters DNA repair and is associated with longer survival of glioblastoma patients treated with alkylating agents. Therefore, MGMTpromoter methylation plays an important role as a predictive biomarker for chemotherapy resistance. To adopt this established correlation into a molecular diagnosis procedure, we compared and optimized three experimental techniques [combined bisulfite restriction analysis, a primer extension- and denaturing high-performance liquid chromatography-based method named SIRPH (SNuPE ion pair-reverse phase high-performance liquid chromatography), and pyrosequencing] with regard to their accuracy of detecting MGMTpromoter methylation. Initially, bisulfite sequencing was used to obtain a comprehensive methylation profile of the MGMTpromoter region in 22 glioblastoma samples and in three normal brain controls. Next, we statistically identified CpG sites that best discriminate between methylated and unmethylated MGMTpromoters. These results were then used to design optimal combined bisulfite restriction analysis, SIRPH, and pyrosequencing assays for accurate and cost-efficient assessment of MGMTpromoter methylation. We compared all three techniques with regard to their reliability and reproducibility on well-characterized tumor samples. The optimized pyrosequencing assay performed best and provides a sensitive, robust, and easy-to-use method for quantitative assessment of MGMTmethylation, for both snap-frozen and paraffin-embedded specimens.

Hemophilia A (HA) is caused by partial or total deficiency of F8 protein activity. In a small group, about 1.8% of patients with HA, no mutation is found in the F8 gene. Among this group, we report here on one patient with severe HA in whom no mRNA of the F8 gene was detected. Using 2 common polymorphisms in F8 exon 14, we were able to show that the same allele shared by the patient, his mother, and his sister was not detected by reverse transcription–polymerase chain reaction (RT-PCR) from total blood mRNA. Skewed X-chromosome inactivation in both the mother and the sister was excluded by studying the methylation profile of the androgen receptor gene (HUMARA locus). These findings strongly suggest that the cause of HA in this patient is either absence or rapid degradation of the F8 mRNA, which points to a novel mechanism leading to HA.

Hemophilia A (HA) is caused by partial or total deficiency of F8 protein activity. In a small group, about 1.8% of patients with HA, no mutation is found in the F8gene. Among this group, we report here on one patient with severe HA in whom no mRNA of the F8gene was detected. Using 2 common polymorphisms in F8exon 14, we were able to show that the same allele shared by the patient, his mother, and his sister was not detected by reverse transcription–polymerase chain reaction (RT-PCR) from total blood mRNA. Skewed X-chromosome inactivation in both the mother and the sister was excluded by studying the methylation profile of the androgen receptor gene (HUMARAlocus). These findings strongly suggest that the cause of HA in this patient is either absence or rapid degradation of the F8mRNA, which points to a novel mechanism leading to HA.

Classical hemophilia A (HA) is characterized by absence or decrease in factor VIII activity. F8 is an essential cofactor of F9 to activate F10. Most known genetic mutations that lead to HA phenotype can be traced to the F8 gene itself. However, in some cases, mutations in chaperon proteins, such as LMNA1 and MCFD2, cause a decrease in secretion of both F5 and F8 resulting in a combined F5/F8 deficiency. Moreover, mutations in the domain of von Willebrand factor (VWF) that interact with F8 cause a HA-like syndrome known as type 2N (Normandy) von Willebrand disease (VWD-2N). Still, in a minority of HA cases, no mutations could be attributed to any of the genes known to be involved in the F8 pathway. In this article we will give an overview of these cases and outline future efforts needed to identify the molecular defects in such patients.

Oldenburg: SOBI: Consultancy.

Different endothelial cells share common features but also acquire organ specificity: for example the liver sinusoidal endothelial cells (LSECs) are major site of F8 secretion as compared to other endothelial cells. To decipher this specificity in LSECs and to understand the molecular mechanism of F8 synthesis and secretion, we have compared genome wide expression and epigenetic data of fetal LSECs (N=3) and non-liver endothelial cells (HCMEC, HPAEC, HPMEC and HUVEC; N=3), hepatocytes (N=3) and adult total blood (N=3). At a false discovery rate of <5% we found 4134 differentially methylated CpG sites and 663 differentially expressed probes (corresponding to 623 genes/loci) between LSECs and other endothelial cells. The loci were found by ingenuity pathway analysis (IPA) to be enriched in EIF2, eIF4, p7056K and mTOR signaling pathways together with LXR/RXR activation pathway. On the other hand, only 21 different loci and 152 CpGs were found to be statistically different (at <5% FDR) between hepatocytes and LSECs. According to IPA these were found enriched in interferon signaling, activation of IRF by cytosolic pattern recognition receptors and role of pattern recognition receptor in recognition of bacteria and viruses. Additionally, gene ontology analysis showed enrichment in GO-terms such as regulation of cellular metabolic process for LSECs vs. endothelial cells and blood vessel development, cell adhesion and regulation of cell migration for LSECs vs. hepatocytes. Moreover, in each of the GO sub categories of general coagulation (GO:0007596) and transcription factors (GO:003700) a small number of loci successfully differentially identify the LSECs from other endothelial cells. The above described results show specific molecular signature that characterize the F8 secreting cells and highlight specific cellular pathways associated with this process.

The intron 22 inversion represents the most prevalentfactor VIII gene defect in severe hemophilia A, accounting for about 40% of all mutations. It is hypothesized that the inversion mutations occur almost exclusively in germ cells during meiotic cell division by intrachromosomal recombination between 1 of 2 telomeric copies of the Int22h region and its intragenic homologue. The majority of inversion mutations originate in male germ cells, where the lack of bivalent formation may facilitate flipping of the telomeric end of the single X chromosome. This is the first intron 22 inversion that presents as a somatic mosaicism in a female, affecting only about 50% of lymphocyte and fibroblast cells of the proposita. Supposing a post-zygotic de novo mutation as the usual cause of somatic mosaicism, the finding would imply that the intron 22 inversion mutation is not restricted to meiotic cell divisions but can also occur during mitotic cell divisions, either in germ cell precursors or in somatic cells.

The intron 22 inversion represents the most prevalentfactor VIIIgene defect in severe hemophilia A, accounting for about 40% of all mutations. It is hypothesized that the inversion mutations occur almost exclusively in germ cells during meiotic cell division by intrachromosomal recombination between 1 of 2 telomeric copies of the Int22h region and its intragenic homologue. The majority of inversion mutations originate in male germ cells, where the lack of bivalent formation may facilitate flipping of the telomeric end of the single X chromosome. This is the first intron 22 inversion that presents as a somatic mosaicism in a female, affecting only about 50% of lymphocyte and fibroblast cells of the proposita. Supposing a post-zygotic de novo mutation as the usual cause of somatic mosaicism, the finding would imply that the intron 22 inversion mutation is not restricted to meiotic cell divisions but can also occur during mitotic cell divisions, either in germ cell precursors or in somatic cells.

Haemophilia A (HA) is an X-linked bleeding disorder caused by heterogeneous mutations in the coagulation factor VIII (F8) gene. Despite applying sensitive methods for mutation detection, and after excluding the inversions mutations a causative mutation is not identified in F8 gene in about 2,5% of severe HA patients (53 patients out of 2350 German patients). Analysis of mRNA from a small group of such (German) patients has excluded mutations deep in the introns that may affect normal splicing or mechanisms causing some unknown rearrangements of the F8 gene as the cause of HA. Among this group, in one patient no F8 mRNA was detected. Using two common polymorphisms in F8 exon 14, we were able to show that the same allele shared by the patient, his mother and his sister was not detected by reverse transcription PCR (RT-PCR) from total blood mRNA. These findings strongly suggest that the cause of HA in this patient is either absence or rapid degradation of the F8 mRNA which points to a novel mechanism leading to HA. To further investigate the cause of Hemophilia A in these patients an international multi-center study was established. The aim of this study to assemble a large collection of such families that could provide clues for novel factors/mechanisims that are important for the F8 molecule biogenesis and protein regulation as well as F8 expression. This multi-center study is therefore suposed to identify novel mechanisms causing HA.

Haemophilia A (HA) is an X-linked bleeding disorder caused by heterogeneous mutations in the coagulation factor VIII (F8) gene. Despite applying sensitive methods for mutation detection, and after excluding the inversions mutations a causative mutation is not identified in F8 gene in about 2,5% of severe HA patients (53 patients out of 2350 German patients). Analysis of mRNA from a small group of such (German) patients has excluded mutations deep in the introns that may affect normal splicing or mechanisms causing some unknown rearrangements of the F8 gene as the cause of HA. Among this group, in one patient no F8 mRNA was detected. Using two common polymorphisms in F8 exon 14, we were able to show that the same allele shared by the patient, his mother and his sister was not detected by reverse transcription PCR (RT-PCR) from total blood mRNA. These findings strongly suggest that the cause of HA in this patient is either absence or rapid degradation of the F8 mRNA which points to a novel mechanism leading to HA. To further investigate the cause of Hemophilia A in these patients an international multi-center study was established. The aim of this study to assemble a large collection of such families that could provide clues for novel factors/mechanisims that are important for the F8 molecule biogenesis and protein regulation as well as F8 expression. This multi-center study is therefore suposed to identify novel mechanisms causing HA.