5 results on '"Triantafyllia Brozou"'
Search Results
2. The Role of Adult Cancer Predisposition Genes in Hematological Malignancies of Childhood
- Author
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Triantafyllia Brozou, Rabea Wagener, Danielle Brandes, Ute Fischer, and Arndt Borkhardt
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
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3. Novel Germline POT1 Variant Predisposes to Childhood Acute Myeloid Leukemia
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Rabea Wagener, Carolin Walter, Julia Hauer, Martin Dugas, Franziska Auer, Triantafyllia Brozou, Ulrike Anne Friedrich, Friedrich Stölzel, Pia Michler, and Arndt Borkhardt
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business.industry ,Immunology ,Childhood Acute Myeloid Leukemia ,Cancer research ,Medicine ,Cell Biology ,Hematology ,business ,Biochemistry ,Germline - Abstract
Introduction: Current studies indicate a contribution of germline predisposition in the development of approximately 8.5% of childhood cancers (Zhang J. et al., N Engl J Med, 2015), although their apparent rate is estimated to be much higher. Understanding tumor evolution based on a predisposed cell can open unknown doors for prevention and therapy of childhood cancer e.g., leukemia. Here we present a novel rare (MAF Methods: Whole exome sequencing (WES) was implemented to identify germline variants. To assess the effect of POT1 p.Q199*, patient's fibroblast and stably transfected HEK293T cells were used as cell models. The variant's functional impact was experimentally tested performing yH2AX and 53BP1 immunofluorescence assays for DNA damage detection, qRT-PCR for telomere length measurement and telomere FISH to assess chromosomal instability. Results: Utilizing WES to detect variants within shelterin complex genes we analyzed genomic data of an unselected German parent-child cohort of children with cancer (n=60, TRIO-DD), as well as a recently published parent-child pediatric cancer cohort (n=158, TRIO-D) (Wagener R. et al., Eur. J. Hum. Genet, 2021). Here, we identified a novel germline POT1 variant in a boy affected with Myelodysplastic syndrome (MDS) and secondary AML (7q-). This novel germline variant constitutes a stop-gain mutation causing a substitution of the amino acid Glutamine by a stop codon (p.Q199*). QRT-PCR analysis within the patient's fibroblasts showed a significant (student's t-test p=0.0037) reduction of POT1 mRNA expression to ≈0.5 compared to POT1 wildtype. Western Blot analysis revealed reduced POT1 levels, confirming the loss of one POT1 allele mediated by p.Q199*. Thereupon, POT1 p.Q199* cloning and stable transfection into Hek293T cells was performed to test the variant's cooperative functionality in a controlled environment. Subsequently, POT1 p.Q199* lead to a drastically significant (student's t-test p= In addition, we detected dysregulation of telomere length maintenance. Here, relative telomere length measurement by means of qRT-PCR indicated significant (student's t-test p=0.019) telomere elongation in POT1 p.Q199* fibroblast cells. Furthermore telomere FISH on metaphase chromosomes was performed to analyse chromosomal stability. In POT1 p.Q199* Hek293T cells we identified a significant (student's t-test p=0.002) increase in telomere fragility compared to POT1 WT cells. Conclusion: Taken together, we present the functional effects of POT1 p.Q199* leading to a significant increase of DNA damage, telomere length and chromosomal instability. Our results on functional dysregulation strengthen a potential genetic predisposition to childhood AML mediated by germline POT1 variants. Disclosures No relevant conflicts of interest to declare. more...
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- 2021
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4. Recurrent Germline Variant in the Cohesin Complex Gene RAD21 Predisposes Children to Lymphoblastic Leukemia and Lymphoma
- Author
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Anne Schedel, Ulrike Anne Friedrich, Rabea Wagener, Juha Mehtonen, Claudia Saitta, Triantafyllia Brozou, Pia Michler, Carolin Walter, Peter Horak, Nagarajan Paramasivam, Grazia Fazio, Stefan Fröhling, Martin Dugas, Daniela Richter, Hanno Glimm, Merja Heinäniemi, Rolf Jessberger, Giovanni Cazzaniga, Arndt Borkhardt, Julia Hauer, and Franziska Auer more...
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Abstract
Introduction: Cohesin complex genes are commonly mutated in cancer particularly in myeloid malignancies. Yet patients with germline mutations in cohesin genes, leading to cohesinopathies like Cornelia-de-Lange syndrome (CdLS) are generally not known to be tumor-prone. The complex plays a major role in chromosome alignment and segregation (Uhlmann, Nature Reviews Molecular Cell Biology, 2016), homologous recombination-driven DNA repair (Ström et al., Molecular Cell, 2004) and regulation of gene expression (Busslinger et al., Nature, 2017). To deepen the understanding of cohesin variants in cancer predisposition, we performed TRIO Sequencing in two independent pediatric cancer cohorts. Thereby, we identified a novel recurrent heterozygous germline variant in the cohesin gene RAD21 not described in CdLS patients , located in the binding domain of the cofactors WAPL and PDS5B . Methods: Whole exome sequencing (WES) in a TRIO (child-parent datasets) setting was carried out in two independent, unselected cancer cohorts (TRIO-D, n=158 (Wagener et al., European Journal of Human Genetics, 2021) and TRIO-DD, n=60). To investigate the oncogenic potential of the novel RAD21 variant molecular and functional assessment was performed focusing on potential implications on the complex. Results: The newly identified RAD21 variant at amino acid position 298 resulting in a Proline to Serine (p.P298S) and a Proline to Alanine exchange, respectively, (p.P298A) is only rarely mutated in the general population (gnomAD database n=118,479; RAD21 p.P298S MAF To assess the influence of RAD21 p.P298S/A on the binding capacity of the complex, RAD21 variants and the wildtype (WT) were cloned and transfected into HEK293T cells, respectively. Immunoprecipitation analysis of RAD21 with the cofactors WAPL and PDS5B showed no differential binding between the WT and the variants, suggesting that RAD21 p.P298S/A does not impact the formation of the complex. Nevertheless, on a transcriptional level 83 genes were significantly differentially expressed in RAD21 p.P298S and p.P298A compared to the wildtype (fc>1.5, adj. p-value For cross-validation of the germline variant RAD21 p.P298S/A and its potential role in pediatric lymphoblastic malignancies, we analysed a third cohort of 150 children with relapsed ALL (IntReALL) for RAD21 p.P298S/A. We again identified RAD21 p.P298A in a boy (12y) with B-cell precursor acute lymphoblastic leukemia. To compare our data to a non-pediatric cancer setting, a cohort of 2300 young adults ( Conclusion: Taken together, we present for the first time the potential role of RAD21 germline variants in pediatric lymphoblastic malignancies. This may shed new light on the many roles of the cohesin complex and its implication outside the typical syndromal presentation. Disclosures No relevant conflicts of interest to declare. more...
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- 2021
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5. Family Trio-Based Whole Genome Optical Mapping Identifies Candidate Structural Variations Predisposing Children to Acute Lymphoblastic Leukemia
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Arndt Borkhardt, Ute Fischer, Julia Täubner, Triantafyllia Brozou, and Layal Yasin
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Genetics ,Cancer-Predisposing Gene ,Immunology ,Genomics ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Genome ,Gene duplication ,Human genome ,Copy-number variation ,Exome sequencing ,Reference genome - Abstract
Germline mutations account for a substantial proportion of childhood cancer and may critically affect disease characteristics, therapy efficacy, severity of treatment side effects and patient outcome. To date, only 8-10% of childhood cancer cases can be explained by germline mutations identified in known cancer predisposing genes. This is in part due to the technical limitation of next generation short read sequencing, which detects single nucleotide variants, small deletions/insertions or simple copy number variations, but is not a reliable tool to identify larger structural variations (SVs, >500 bp) which are frequent in the human genome and may impact on disease predisposition. Using whole genome optical mapping (WGOM) we aimed at identification of de novo and inherited germline SVs in a cohort of patients with clinically suspected cancer predisposition but without informative findings in short read sequencing analyses. After informed consent we performed family trio based short read (2x 100 bp) whole exome sequencing (WES) on a HiSeq2500 (Illumina) and collected clinical and demographic data for a cohort of >100 families with children affected by cancer who were treated in our hospital. About 25% of the patients either (1) had a family history indicative of cancer susceptibility, or (2) had accompanying clinical findings (e.g. developmental delay, congenital anomalies) or (3) experienced excessive toxicity during chemotherapy. From this subgroup we selected four patients with acute lymphoblastic leukemia whose sequencing data and routine genetic workup were not informative of a known cancer predisposing syndrome and employed family trio-based next generation WGOM on a Saphyr instrument equipped with Access software (Bionano Genomics) to identify genomic SVs. To this end, we extracted and labeled high molecular weight DNA molecules at specific hexamer sequence motifs (average distance: 5 kb) using a DNA methyltransferase-based direct labeling reaction. Imaging was carried out on single-molecule level and each sample genome was de novo assembled from molecule data. Consensus genome maps were clustered into two alleles and diploid assemblies created. Genomes of patients were compared to parental genomes and the GRCh38 reference genome. SVs were inferred from de novo assemblies and genome comparisons with respect to quality scores, overall molecule coverage, fraction of molecules displaying the SV event, and chimeric DNA fragment mapping. Specific SV calls were compared to a set of > 160 human control samples (provided by Bionano Genomics) to filter against common SVs and potential artifacts. Filtered SVs were annotated using structural variant and gene databases. Employing WGOM we analyzed DNA molecules 300.000 bp long on average and achieved genomic coverage ranging from 90-132x corresponding to 330-480 Gbp. For instance, for one patient, we obtained 1751 insertions, 624 deletions, 77 inversions, 21 duplications, 1 intra- and 2 inter-chromosomal translocations before filtering. The majority of these events (78%) were inherited from both parents. 20% were inherited from either father or mother and 2% were generated de novo. As the family history of this patient was inconspicuous for tumor diseases, we removed all inherited events and filtered against common variants. This resulted in only two candidate de novo lesions: a heterozygous 129,495 bp deletion framed by inversions (chr9: 66,156,733-66,622,623) in a gene-less region and a heterozygous inverted 352,667 bp duplication (chr22: 15,522,454-15.875,120) that spanned the genes OR11H, POTEH, POTEH-AS1, LINC01297, DUXAP8, and BMS1P22. Of these genes DUXAP8 is an oncogenic non-coding RNA of the homeobox gene family that has been associated with increased tumor growth and poorer prognosis in a wide variety of somatic cancers. It functions as a regulator of transcription by binding to key components of the developmental regulator epigenetic polycomb repressive complex 2 and may thus account for additional presentations of the child (dwarfism, accelerated skeletal age, linguistic developmental delay, morphological traits). Our results indicate that WGOM is a useful technology to identify candidate SVs in children predisposed to cancer and developmental syndromes. Several candidates are currently being tested and the results will be presented. Disclosures No relevant conflicts of interest to declare. more...
- Published
- 2019
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