| 1. |
- Elander, Johanna, et al.
(författare)
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Extended genetic diagnostics for children with profound sensorineural hearing loss by implementing massive parallel sequencing. Diagnostic outcome, family experience and clinical implementation
- 2022
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Ingår i: International Journal of Pediatric Otorhinolaryngology. - : Elsevier BV. - 0165-5876. ; 159
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: The aim of this study was to investigate genetic outcomes, analyze the family experience, and describe the process of implementing genetic sequencing for children with profound sensorineural hearing loss (SNHL) at a tertial audiological center in southern Sweden. Design: This is a prospective pilot study including eleven children with profound bilateral SNHL who underwent cochlear implant surgery. Genetic diagnostic investigation was performed with whole exome sequencing (WES) complemented with XON-array to identify copy number variants, using a manually curated gene panel incorporating 179 genes associated with non-syndromic and syndromic SNHL. Mitochondrial DNA (mtDNA) from blood was examined separately. A patient reported experience measures (PREM) questionnaire was used to evaluate parental experience. We also describe here the process of implementing WES in an audiology department. Results: Six female and five male children (mean 3.4 years, SD 3.5 years), with profound bilateral SNHL were included. Genetic variants of interest were found in six subjects (55%), where three (27%) could be classified as pathogenic or likely pathogenic. Among the six cases, one child was found to have a homozygous pathogenic variant in MYO7A and two children had homozygous likely pathogenic variants in SLC26A4 and PCDH15, respectively. One was carrying a compound heterozygote frameshift variant of uncertain significance (VUS) on one allele and in trans, a likely pathogenic deletion on the other allele in PCDH15. Two subjects had homozygous VUS in PCDH15 and ADGRV1, respectively. In five of the cases the variants were in genes associated with Usher syndrome. For one of the likely pathogenic variants, the finding was related to Pendred syndrome. No mtDNA variants related to SNHL were found. The PREM questionnaire revealed that the families had difficulty in fully understanding the results of the genetic analysis. However, the parents of all eleven (100%) subjects still recommended that other families with children with SNHL should undergo genetic testing. Specifically addressed referrals for prompt complementary clinical examination and more individualized care were possible, based on the genetic results. Close clinical collaboration between different specialists, including physicians of audiology, audiologists, clinical geneticists, ophthalmologists, pediatricians, otoneurologists, physiotherapists and hearing habilitation teams was initiated during the implementation of the new regime. For all professionals involved, a better knowledge of the diversity of the genetic background of hearing loss was achieved. Conclusions: Whole exome sequencing and XON-array using a panel of genes associated with SNHL had a high diagnostic yield, added value to the families, and provided guidance for further examinations and habilitation for the child. Great care should be taken to thoroughly inform parents about the genetic test result. Collaborations between departments were intensified and knowledge of hearing genomics was increased among the staff.
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| 4. |
- Montano, Giorgia, et al.
(författare)
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The hematopoietic tumor suppressor interferon regulatory factor 8 (IRF8) is upregulated by the antimetabolite cytarabine in leukemic cells involving the zinc finger protein ZNF224, acting as a cofactor of the Wilms' tumor gene 1 (WT1) protein.
- 2016
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Ingår i: Leukemia Research: A Forum for Studies on Leukemia and Normal Hemopoiesis. - : Elsevier BV. - 1873-5835. ; 40:1, s. 60-67
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Tidskriftsartikel (refereegranskat)abstract
- The transcription factor interferon regulatory factor-8 (IRF8) is highly expressed in myeloid progenitors, while most myeloid leukemias show low or absent expression. Loss of IRF8 in mice leads to a myeloproliferative disorder, indicating a tumor-suppressive role of IRF8. The Wilms tumor gene 1 (WT1) protein represses the IRF8-promoter. The zinc finger protein ZNF224 can act as a transcriptional co-factor of WT1 and potentiate the cytotoxic response to the cytostatic drug cytarabine. We hypothesized that cytarabine upregulates IRF8 and that transcriptional control of IRF8 involves WT1 and ZNF224. Treatment of leukemic K562 cells with cytarabine upregulated IRF8 protein and mRNA, which was correlated to increased expression of ZNF224. Knock down of ZNF224 with shRNA suppressed both basal and cytarabine-induced IRF8 expression. While ZNF224 alone did not affect IRF8 promoter activity, ZNF224 partially reversed the suppressive effect of WT1 on the IRF8 promoter, as judged by luciferase reporter experiments. Coprecipitation revealed nuclear binding of WT1 and ZNF224, and by chromatin immunoprecipitation (ChIP) experiments it was demonstrated that WT1 recruits ZNF224 to the IRF8 promoter. We conclude that cytarabine-induced upregulation of the IRF8 in leukemic cells involves increased levels of ZNF224, which can counteract the repressive activity of WT1 on the IRF8-promoter.
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| 5. |
- Nilsson, Helena Jernmark, et al.
(författare)
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The transcriptional coregulator NAB2 is a target gene for the Wilms' tumor gene 1 protein (WT1) in leukemic cells
- 2017
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Ingår i: Oncotarget. - : Impact Journals, LLC. - 1949-2553. ; 8:50, s. 87136-87150
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Tidskriftsartikel (refereegranskat)abstract
- The Wilms' tumor gene 1 (WT1) is recurrently mutated in acute myeloid leukemia. Mutations and high expression of WT1 associate with a poor prognosis. In mice, WT1 cooperates with the RUNX1/RUNX1T1 (AML1/ETO) fusion gene in the induction of acute leukemia, further emphasizing a role for WT1 in leukemia development. Molecular mechanisms for WT1 are, however, incompletely understood. Here, we identify the transcriptional coregulator NAB2 as a target gene of WT1. Analysis of gene expression profiles of leukemic samples revealed a positive correlation between the expression of WT1 and NAB2, as well as a non-zero partial correlation. Overexpression of WT1 in hematopoietic cells resulted in increased NAB2 levels, while suppression of WT1 decreased NAB2 expression. WT1 bound and transactivated the proximal NAB2 promoter, as shown by ChIP and reporter experiments, respectively. ChIP experiments also revealed that WT1 can recruit NAB2 to the IRF8 promoter, thus modulating the transcriptional activity of WT1, as shown by reporter experiments. Our results implicate NAB2 as a previously unreported target gene of WT1 and that NAB2 acts as a transcriptional cofactor of WT1.
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| 6. |
- Ullmark, Tove, et al.
(författare)
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Anti-apoptotic quinolinate phosphoribosyltransferase (QPRT) is a target gene of Wilms' tumor gene 1 (WT1) protein in leukemic cells
- 2017
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 0006-291X. ; 482:4, s. 802-807
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Tidskriftsartikel (refereegranskat)abstract
- Wilms' tumor gene 1 (WT1) is a zinc finger transcription factor that has been implicated as an oncogene in leukemia and several other malignancies. When investigating possible gene expression network partners of . WT1 in a large acute myeloid leukemia (AML) patient cohort, one of the genes with the highest correlation to . WT1 was quinolinate phosphoribosyltransferase (QPRT), a key enzyme in the . de novo nicotinamide adenine dinucleotide (NAD+) synthesis pathway. To investigate the possible relationship between . WT1 and . QPRT, we overexpressed . WT1 in hematopoietic progenitor cells and cell lines, resulting in an increase of . QPRT expression. WT1 knock-down gave a corresponding decrease in . QPRT gene and protein expression. Chromatin-immunoprecipitation revealed WT1 binding to a conserved site in the first intron of the . QPRT gene. Upon overexpression in leukemic K562 cells, QPRT conferred partial resistance to the anti-leukemic drug imatinib, indicating possible anti-apoptotic functions, consistent with previous reports on glioma cells. Interestingly, the rescue effect of QPRT overexpression was not correlated to increased NAD + levels, suggesting NAD + independent mechanisms. We conclude that . QPRT, encoding a protein with anti-apoptotic properties, is a novel and direct target gene of WT1 in leukemic cells.
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| 7. |
- Ullmark, Tove, et al.
(författare)
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Distinct global binding patterns of the Wilms' tumor gene 1 (WT1) -KTS and +KTS isoforms in leukemic cells
- 2017
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Ingår i: Haematologica. - : Ferrata Storti Foundation (Haematologica). - 1592-8721 .- 0390-6078. ; 102:2, s. 336-345
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Tidskriftsartikel (refereegranskat)abstract
- The zinc finger transcription factor Wilms' tumor gene 1 (WT1) acts as an oncogene in acute myeloid leukemia. A naturally occurring alternative splice event between zinc fingers three and four, removing or retaining three amino acids (+/-KTS), is believed to change the DNA binding affinity of WT1, although there are conflicting data regarding the binding affinity and motifs of the different isoforms. Increased expression of WT1 -KTS at the expense of WT1 +KTS isoform associates with poor prognosis in acute myeloid leukemia. We determined the genome-wide binding pattern of WT1 -KTS and WT1 +KTS in leukemic K562 cells by chromatin immunoprecipitation and deep sequencing (ChIP-seq). Motif discovery revealed distinct binding motifs for the isoforms, some of which have been previously reported as WT1 binding sites. We discovered that the WT1 -KTS isoform predominantly binds close to transcription start sites and to enhancers, in a similar fashion to other transcription factors, whereas WT1 +KTS binding is rather enriched within gene bodies. We observed a significant overlap between WT1 -KTS and WT1 +KTS target genes, despite the binding sites being distinct. Motif discovery revealed distinct binding motifs for the isoforms, some of which have been previously reported as WT1 binding sites. Additional analyses showed that both WT1 -KTS and WT1 +KTS target genes are more likely to be transcribed than non-targets, and are involved in cell proliferation, cell death, and development. Our study provides evidence that WT1 -KTS and WT1 +KTS share target genes yet still bind distinct locations, indicating isoform-specific regulation in transcription of genes related to cell proliferation and differentiation, consistent with involvement of WT1 in acute myeloid leukemia.
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| 8. |
- Ullmark, Tove, et al.
(författare)
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DNA and RNA binding by the Wilms' tumour gene 1 (WT1) protein +KTS and −KTS isoforms—From initial observations to recent global genomic analyses
- 2018
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Ingår i: European Journal of Haematology. - : Wiley. - 0902-4441. ; 100:3, s. 229-240
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Forskningsöversikt (refereegranskat)abstract
- The Wilms' tumour gene 1 protein (WT1) is a zinc finger transcription factor found indispensable for foetal development. WT1 has also been implicated in the development of tumours in several organ systems, including acute myeloid leukaemia (AML). Four main WT1 isoforms come from 2 alternative splice events. One alternative splice results in the inclusion or exclusion of 3 amino acids, KTS, between zinc fingers 3 and 4 in the WT1 protein. The KTS insert has been extensively investigated due to the functional implications for DNA and RNA binding. In this review, we provide an overview of the research into the isoforms containing or lacking the KTS insert in leukaemic cells, as well as the research into the binding patterns of the WT1 −KTS and +KTS isoforms to DNA and RNA. Finally, we connect the results of the DNA binding research to the ChIP-CHIP and ChIP-Seq investigations into the global genomic binding of the WT1 protein that have recently been performed.
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| 9. |
- Ullmark, Tove, et al.
(författare)
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Global binding pattern of the Wilms' tumor gene 1 (WT1) +17AA -KTS isoform in leukemic cells
- 2016
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Ingår i: Cancer Research. - 1538-7445. ; 76:14 Suppl.
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Konferensbidrag (refereegranskat)abstract
- The aim of this study was to investigate the global DNA-binding pattern of Wilms' tumor gene 1 (WT1) in leukemic cells. Clinical and preclinical data indicate the zinc finger transcription factor WT1 as an oncogene, but the full target gene repertoire of WT1 in leukemic cells has not been previously characterized. The -KTS isoforms (excluding the three amino acid (KTS) insert between zinc finger three and four) are considered as the most efficient DNA-binders. Among these, the 17AA isoform (including 17 amino acids encoded by exon 5) is the most abundant one. To specifically analyze the DNA-binding of WT1(+17AA/-KTS) in leukemic cells, we generated a K562 clone that stably expressed BIO-tagged WT1(+17AA/-KTS), as well as the biotinylating enzyme Bir A. From the cells chromatin immunoprecipitation (ChIP) by streptavidin capture was performed followed by sequencing with a minimum of 50 million reads per sample. After alignment to the genome and peak calling, peaks were characterized and compared to available K562 tracks in the ENCODE database. We found that 45% of identified WT1(+17AA/-KTS) peaks are in the proximity of transcription start sites (promoter area, first exon or first intron) of target genes, whereas only 11% of randomized peaks were found here. Within the peaks we show strong enrichment for three different previously published WT1-binding motifs. Comparison to ENCODE tracks showed that WT1(+17AA/-KTS) peaks are in close proximity to binding sites of other transcription factors, to histone marks for actively transcribed genes, and to binding sites of chromatin modifiers. Considering peaks within promoters and gene bodies only (for safe assignment to a target gene), Gene Ontology (GO) analysis revealed enrichment of GO groups important for proliferation, cell death, embryonic development, and cell motility. In conclusion, WT1(+17AA/-KTS) binds close to transcription start sites in areas of active transcription. The target genes implicated in proliferation, cell death, cell signaling and motility adds to the growing evidence of WT1 as an effector gene in leukemia.
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| 10. |
- Vidovic, Karina, et al.
(författare)
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Leukemia associated mutant Wilms' tumor gene 1 protein promotes expansion of human hematopoietic progenitor cells.
- 2013
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Ingår i: Leukemia Research: A Forum for Studies on Leukemia and Normal Hemopoiesis. - : Elsevier BV. - 1873-5835 .- 0145-2126. ; 37:10, s. 1341-1349
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Tidskriftsartikel (refereegranskat)abstract
- The transcription factor Wilms' tumor gene 1 (WT1) is highly expressed in the majority of leukemias, suggesting a role in leukemogenesis. Acquired WT1 mutations are reported as an independent predictor of poor clinical outcome, and mutations resulting in deletion of the entire DNA-binding zinc-finger domain (WT1delZ), is the most common type. The aim of this study was to study cellular effects of WT1(delZ) that may contribute to an oncogenic phenotype. We found that expression of WT1(delZ) supported proliferation of human hematopoietic CD34(+) progenitor cells. Moreover, WT1(delZ) transduced cells expressed erythroid markers, including raised levels of STAT5, independently of addition of erythropoietin. At the global gene expression level, WT1(delZ) caused upregulation of genes related to cell division and genes associated with erythroid maturation, in the absence of added erythropoietin. Our results indicate that WT1(delZ) promotes cell proliferation and expansion of progenitor cells, consistent with a possible role in leukemogenesis.
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