Search

Neurofibromas and schwannomas are benign Schwann cell-derived peripheral nerve sheath tumors arising sporadically and within neurofibromatoses. Multiple tumors are a hallmark of neurofibromatosis type 1 (NF1) and type 2 (NF2) and schwannomatosis. Neurofibromas in NF1 and schwannomas in NF2 or schwannomatosis are defined by distinctive molecular hits. Among these, multiple hybrid neurofibromas/schwannomas may also appear, not yet being defined by a molecular background. We therefore performed molecular analysis of 22 hybrid neurofibromas/schwannomas using array comparative genomic hybridization, immunohistochemistry, quantitative RT-PCR, and functional analyses of cultured Schwann cells. Furthermore, we analyzed SMARCB1 by fluorescence in situ hybridization and multiplex ligation-dependent probe. Monosomy 22 was identified in 44% of tumors of tested patients with hybrid neurofibromas/schwannomas. In addition, in a single case, we detected focal deletion of the α-T-catenin/CTNNA3 gene (10q21.3). To further characterize this candidate, transient knockdown of α-T-catenin in Schwann cells was performed. CTNNA3 depleted cells showed cytoskeletal abnormalities and reduced E-cadherin expression, indicating epithelial-mesenchymal transition-like abnormalities. To conclude, we uncovered loss of chromosome 22 in almost half of all cases with hybrid neurofibromas/schwannomas of patients with multiple peripheral nerve sheath tumors. We tagged α-T-catenin/CTNNA3 as a novel candidate gene. Our functional investigations might indicate involvement of α-T-catenin/CTNNA3 in the biology of peripheral nerve sheath tumors., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Unlabelled: Essentials Von Willebrand disease IIC Miami features high von Willebrand factor (VWF) with reduced function. We aimed to identify and characterize the elusive underlying mutation in the original family. An inframe duplication of VWF exons 9-10 was identified and characterized. The mutation causes a defect in VWF multimerization and decreased VWF clearance from the circulation., Summary: Background A variant of von Willebrand disease (VWD) type 2A, phenotype IIC (VWD2AIIC), is characterized by recessive inheritance, low von Willebrand factor antigen (VWF:Ag), lack of VWF high-molecular-weight multimers, absence of VWF proteolytic fragments and mutations in the VWF propeptide. A family with dominantly inherited VWD2AIIC but markedly elevated VWF:Ag of > 2 U L(-1) was described as VWD type IIC Miami (VWD2AIIC-Miami) in 1993; however, the molecular defect remained elusive. Objectives To identify the molecular mechanism underlying the phenotype of the original VWD2AIIC-Miami. Patients and Methods We studied the original family with VWD2AIIC-Miami phenotypically and by genotyping. The identified mutation was recombinantly expressed and characterized by standard techniques, confocal imaging and in a mouse model, respectively. Results By Multiplex ligation-dependent probe amplification we identified an in-frame duplication of VWF exons 9-10 (c.998_1156dup; p.Glu333_385dup) in all patients. Recombinant mutant (rm)VWF only presented as a dimer. Co-expressed with wild-type VWF, the multimer pattern was indistinguishable from patients' plasma VWF. Immunofluorescence studies indicated retention of rmVWF in unusually large intracellular granules in the endoplasmic reticulum. ADAMTS-13 proteolysis of rmVWF under denaturing conditions was normal; however, an aberrant proteolytic fragment was apparent. A decreased ratio of VWF propeptide to VWF:Ag and a 1-desamino-8-d-arginine vasopressin (DDAVP) test in one patient indicated delayed VWF clearance, which was supported by clearance data after infusion of rmVWF into VWF(-/-) mice. Conclusion The unique phenotype of VWD2 type IIC-Miami results from dominant impairment of multimer assembly, an aberrant structure of mutant mature VWF and reduced clearance in vivo., (© 2016 International Society on Thrombosis and Haemostasis.)

Rhabdoid tumors are rare but highly aggressive malignancies of infancy and early childhood with a generally unfavorable prognosis. Despite a wide variety of anatomic locations rhabdoid tumors share mutational inactivation of the SWI/SNF (SWItch/Sucrose NonFermentable) core component gene SMARCB1 (also known as INI1, hSNF5 or BAF47) in chromosome 22. As this inactivation usually results in loss of SMARCB1 expression, detectable by an antibody against the SMARCB1 protein, the accurate diagnosis of a rhabdoid tumor may be more distinctly and frequently made. Several reports on rhabdoid tumors presenting in various anatomic sites outside the kidneys and CNS are on record. We report two cases of rhabdoid tumors originating in the heart (cardiac tissue), which were entered into the European Rhabdoid Registry (EU-RHAB). The first case presented with intracardial and -cranial lesions as well as malignant ascites, while the second patient demonstrated an isolated cardiac tumor. This induced a different therapeutic approach and subsequently different clinical course (death 7 weeks after diagnosis in patient 1). Patient 2 presented with a bifocal intracardial tumor without metastases and remains in complete remission for 46 months since diagnosis following multimodal therapy. The second case demonstrates that even in a potentially futile clinical situation early and accurate diagnosis followed by prompt and intensive multimodal therapy may offer prolonged survival, potential cure and improved quality of life., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Atypical teratoid rhabdoid tumor (AT/RT), a rare and highly malignant tumor entity of the central nervous system that presents in early childhood, has a poor prognosis. AT/RTs are characterized by biallelic inactivating mutations of the gene SMARCB1 in 98% of patients; these mutations may serve as molecular markers for residual tumor cell detection in liquid biopsies. We developed a marker-specific method to detect residual AT/RT cells. Seven of 150 patient samples were selected, each with a histological and genetically ascertained diagnosis of AT/RT. Tumor tissue was either formalin fixed or fresh frozen. DNA was extracted from the patients' peripheral blood leukocytes (PBL) and cerebrospinal fluid (CSF). Multiplex ligation-dependent probe amplification, DNA sequencing, and fluorescence in situ hybridization were used to characterize the tumors' mutations. Residual tumor cell detection used mutation-specific primers and real-time PCR. The detection limit for the residual tumor cell search was 1-18%, depending on the quality of the template provided. The residual tumor cell search in PBL and CSF was negative for all seven patients. The SMARCB1 region of chromosome 22 is prone to DNA double-strand breaks. The individual breakpoints and breakpoint-specific PCR offer the option to detect minimal residual tumor cells in CSF or blood. Even if we did not detect minimal residual tumor cells in the investigated material, proof of principle for this method was confirmed., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Type 3 von Willebrand disease (VWD) is an autosomal recessive bleeding disorder, characterized by virtually undetectable plasma von Willebrand factor (VWF) and consequently reduced plasma factor VIII levels. Genetic mutations responsible for type 3 VWD are very heterogeneous, scattered throughout the VWF gene and show high variability among different populations., Methods: Twenty-five severe VWD patients were studied by direct sequencing of the 51 coding exons of the VWF gene. The total number of VWD type 3 families in Hungary is 24, of which 23 were investigated., Results: Fifteen novel mutations were identified in 31 alleles, five being nonsense mutations (p.Q1238X, p.Q1898X, p.Q1931X, p.S2505X and p.S2568X), four small deletions and insertions resulting in frame shifts (c.1992insC, c.3622delT, c.5315insGA and c.7333delG), one a large partial deletion (delExon1-3) of the 5'-region, four candidate missense mutations (p.C35R, p.R81G, p.C295S, p.C623T) and one a candidate splice site mutation (c.1730-10C>A). Six previously described mutations were detected in 17 alleles, including the repeatedly found c.2435delC, p.R1659X and p.R1853X. Only one patient developed alloantibodies to VWF, carrying a homozygous c.3622delT., Conclusion: We report the genetic background of the entire Hungarian type 3 VWD population. A large novel deletion, most probably due to a founder effect, seems to be unique to Hungarian type 3 VWD patients with high allele frequency. In contrast to previous reports, none of the five patients homozygous for the large partial deletion developed inhibitors to VWF. This discrepancy raises the possibility of selection bias in some of the reports., (© 2011 International Society on Thrombosis and Haemostasis.)

Among the different phenotypes of von Willebrand disease (VWD) type 2A, we identified a particular subgroup with a high frequency of 29%, characterized by a relative decrease of large von Willebrand factor (VWF) multimers and decreased A Disintegrin And Metalloproteinase with ThromboSpondin type 1 motifs, member 13 (ADAMTS13)-mediated proteolysis previously described in a single family as VWD type IIE (VWD2A/IIE). Phenotype and genotype of 57 patients from 38 unrelated families displaying a particular multimer pattern resembling the original VWD2A/IIE were studied. Pathogenicity of candidate mutations was confirmed by expression studies and phenotypic characterization of recombinant mutants. Specific mutations were identified in all patients. Twenty-two different mutations, most of them affecting cysteine residues, 17 of them being novel, are clustering mainly in the VWF D3 domain and correlate with the VWD2A/IIE phenotype. An intracellular retention of most mutants and/or a defect of multimerization seem to be the main pathogenic molecular mechanisms. ADAMTS13 proteolysis of mutant VWF was not different from wild-type VWF in a static assay, suggesting that reduced in vivo proteolysis is not an intrinsic property of mutant VWF. Our study identified a distinct VWD subtype with a common molecular background which contributes significantly to the heterogeneous spectrum of VWD.

Background: We studied 24 Hungarian patients from 23 unrelated families to identify the genetic background of the entire type 3 von Willebrand disease (VWD) population in this country. The current report focuses on the molecular characterization of a novel large deletion., Results: A large partial deletion (delExon1-3) of the 5'-region of the von Willebrand factor gene (VWF) was detected in 12/48 alleles (25% of all type 3 alleles). The 5'-deletion breakpoint is located in the untranslated region between VWF and CD9, whereas the 3' breakpoint is in intron 3 of VWF. Analysis of the breakpoints showed Alu Y and Alu SP repetitive sequences at the ends of the deletion, suggesting that a recombination event caused the subsequent loss of the 35-kb fragment. DelExon1-3 was not found in any of the other screened populations., Conclusion: We report a large novel deletion including exons 1, 2 and 3 of VWF commonly causing type 3 VWD in the Hungarian population. This mutation, probably caused by an Alu-mediated recombination event, and subsequently distributed in Hungary by a founder effect, seems to be unique to Hungarian patients with a high allele frequency. Together, delExon1-3 and 2435delC make up 37.5% of the genetic defects in Hungarian patients with VWD type 3. This offers a rational approach to molecular testing of relevant families in Hungary.

Background: Severe von Willebrand disease (VWD) type 3 is caused by large deletions, insertions, small truncating mutations, splice site mutations and missense mutations of the VWF gene, respectively. Large deletions have been regarded as being a rare cause of VWD type 3. Complete gene deletions have only been identified in Italian and German patients to date. However, their extent and breakpoints have not been determined yet., Objectives: To identify the breakpoints of complete VWF deletions in patients with VWD type 3., Patients/methods: Five index patients with large deletions from two unrelated German and three Italian families were investigated by polymerase chain reaction (PCR) and primer walking. Haplotypes were composed of eight deletion flanking markers., Results: After initial characterization of a homozygous 253,246 bp deletion (Delta253 k) in a German patient, with the centromeric breakpoint located between CD9 and VWF and the telomeric breakpoint in intron 3 of TMEM16B, respectively, we identified the same Delta253 k in an additional two homozygous and two compound-heterozygous patients, and in their heterozygous parents. All patients share the same deletion-associated marker haplotype. The genomic structure of the breakpoint regions favors DNA double-strand breaks followed by non-homologous end-joining repair as an underlying molecular mechanism rather than a homologous recombination event., Conclusions: Our results suggest a single genetic origin of Delta253 k. Homozygosity for Delta253 k in non-consanguineous families from two different countries may indicate a higher incidence of large deletions in VWD type 3 than previously thought. The availability of a Delta253k-specific assay allows simple and rapid detection of even heterozygous patients and carriers.

Thrombotic thrombocytopenic purpura (TTP) is caused by the persistence of the highly reactive high-molecular-weight multimers of von Willebrand factor (VWF) due to deficiency of the specific VWF-cleaving protease (VWF-CP) ADAMTS13, resulting in microangiopathic disease. The acquired form is caused by autoantibodies against VWF-CP, whereas homozygous or compound heterozygous mutations of ADAMTS13 are responsible for recessively inherited TTP. We investigated 83 children with hemolytic or thrombocytopenic episodes with or without additional neurologic symptoms or renal failure. The presumed diagnosis was chronic idiopathic thrombocytopenic purpura (ITP; n = 50), TTP (n = 8), hemolytic uremic syndrome (HUS; n = 24), and Evans syndrome (n = 1). A severe deficiency of VWF-CP (< or = 5%) was found in all investigated patients with TTP and in none of those with HUS. Additionally, 2 of 50 patients with a prior diagnosis of ITP were deficient for VWF-CP. Antibodies against VWF-CP were found in 4 children. Mutation analysis of the ADAMTS13 gene in the patients deficient in VWF-CP by direct sequencing of all 29 exons identified 8 different mutations, suggesting the hereditary form of TTP in 1 patient with ITP, in the patient with Evans syndrome, and in 5 of the 8 patients with TTP. The phenotype of TTP in childhood can be rather variable. Besides the classical clinical picture, oligosymptomatic forms may occur that can delay the identification of patients at risk.