Heritable GATA2 Mutations Associated with Familial Myelodysplastic Syndrome and Acute Myeloid Leukemia
Identifieur interne : 004707 ( Pmc/Corpus ); précédent : 004706; suivant : 004708Heritable GATA2 Mutations Associated with Familial Myelodysplastic Syndrome and Acute Myeloid Leukemia
Auteurs : Christopher N. Hahn ; Chan-Eng Chong ; Catherine L. Carmichael ; Ella J. Wilkins ; Peter J. Brautigan ; Xiao-Chun Li ; Milena Babic ; Ming Lin ; Amandine Carmagnac ; Young K. Lee ; Chung H. Kok ; Lucia Gagliardi ; Kathryn L. Friend ; Paul G. Ekert ; Carolyn M. Butcher ; Anna L. Brown ; Ian D. Lewis ; L. Bik To ; Andrew E. Timms ; Jan Storek ; Sarah Moore ; Meryl Altree ; Robert Escher ; Peter G. Bardy ; Graeme K. Suthers ; Richard J. D Ndrea ; Marshall S. Horwitz ; Hamish S. ScottSource :
- Nature genetics [ 1061-4036 ] ; 2011.
Abstract
We report the discovery of the
Url:
DOI: 10.1038/ng.913
PubMed: 21892162
PubMed Central: 3184204
Links to Exploration step
PMC:3184204Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Heritable <italic>GATA2</italic> Mutations Associated with Familial Myelodysplastic Syndrome and Acute Myeloid Leukemia</title><author><name sortKey="Hahn, Christopher N" sort="Hahn, Christopher N" uniqKey="Hahn C" first="Christopher N." last="Hahn">Christopher N. Hahn</name><affiliation><nlm:aff id="A1">Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A2">School of Medicine, University of Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Chong, Chan Eng" sort="Chong, Chan Eng" uniqKey="Chong C" first="Chan-Eng" last="Chong">Chan-Eng Chong</name><affiliation><nlm:aff id="A1">Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A2">School of Medicine, University of Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Carmichael, Catherine L" sort="Carmichael, Catherine L" uniqKey="Carmichael C" first="Catherine L." last="Carmichael">Catherine L. Carmichael</name><affiliation><nlm:aff id="A3">Molecular Medicine Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Vic, Australia</nlm:aff></affiliation></author><author><name sortKey="Wilkins, Ella J" sort="Wilkins, Ella J" uniqKey="Wilkins E" first="Ella J." last="Wilkins">Ella J. Wilkins</name><affiliation><nlm:aff id="A3">Molecular Medicine Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Vic, Australia</nlm:aff></affiliation></author><author><name sortKey="Brautigan, Peter J" sort="Brautigan, Peter J" uniqKey="Brautigan P" first="Peter J." last="Brautigan">Peter J. Brautigan</name><affiliation><nlm:aff id="A1">Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Li, Xiao Chun" sort="Li, Xiao Chun" uniqKey="Li X" first="Xiao-Chun" last="Li">Xiao-Chun Li</name><affiliation><nlm:aff id="A1">Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Babic, Milena" sort="Babic, Milena" uniqKey="Babic M" first="Milena" last="Babic">Milena Babic</name><affiliation><nlm:aff id="A1">Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Lin, Ming" sort="Lin, Ming" uniqKey="Lin M" first="Ming" last="Lin">Ming Lin</name><affiliation><nlm:aff id="A1">Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Carmagnac, Amandine" sort="Carmagnac, Amandine" uniqKey="Carmagnac A" first="Amandine" last="Carmagnac">Amandine Carmagnac</name><affiliation><nlm:aff id="A3">Molecular Medicine Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Vic, Australia</nlm:aff></affiliation></author><author><name sortKey="Lee, Young K" sort="Lee, Young K" uniqKey="Lee Y" first="Young K." last="Lee">Young K. Lee</name><affiliation><nlm:aff id="A1">Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Kok, Chung H" sort="Kok, Chung H" uniqKey="Kok C" first="Chung H." last="Kok">Chung H. Kok</name><affiliation><nlm:aff id="A4">Department of Haematology and Oncology, The Queen Elizabeth Hospital, Woodville, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A5">Department of Haematology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Gagliardi, Lucia" sort="Gagliardi, Lucia" uniqKey="Gagliardi L" first="Lucia" last="Gagliardi">Lucia Gagliardi</name><affiliation><nlm:aff id="A1">Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Friend, Kathryn L" sort="Friend, Kathryn L" uniqKey="Friend K" first="Kathryn L." last="Friend">Kathryn L. Friend</name><affiliation><nlm:aff id="A6">Department of Paediatric and Reproductive Genetics, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Ekert, Paul G" sort="Ekert, Paul G" uniqKey="Ekert P" first="Paul G." last="Ekert">Paul G. Ekert</name><affiliation><nlm:aff id="A7">Cell Signalling and Cell Death Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Vic, Australia</nlm:aff></affiliation></author><author><name sortKey="Butcher, Carolyn M" sort="Butcher, Carolyn M" uniqKey="Butcher C" first="Carolyn M." last="Butcher">Carolyn M. Butcher</name><affiliation><nlm:aff id="A4">Department of Haematology and Oncology, The Queen Elizabeth Hospital, Woodville, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A5">Department of Haematology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Brown, Anna L" sort="Brown, Anna L" uniqKey="Brown A" first="Anna L." last="Brown">Anna L. Brown</name><affiliation><nlm:aff id="A5">Department of Haematology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Lewis, Ian D" sort="Lewis, Ian D" uniqKey="Lewis I" first="Ian D." last="Lewis">Ian D. Lewis</name><affiliation><nlm:aff id="A2">School of Medicine, University of Adelaide, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A5">Department of Haematology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="To, L Bik" sort="To, L Bik" uniqKey="To L" first="L. Bik" last="To">L. Bik To</name><affiliation><nlm:aff id="A2">School of Medicine, University of Adelaide, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A5">Department of Haematology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Timms, Andrew E" sort="Timms, Andrew E" uniqKey="Timms A" first="Andrew E." last="Timms">Andrew E. Timms</name><affiliation><nlm:aff id="A8">Department of Pathology, University of Washington School of Medicine, Seattle, WA, USA</nlm:aff></affiliation></author><author><name sortKey="Storek, Jan" sort="Storek, Jan" uniqKey="Storek J" first="Jan" last="Storek">Jan Storek</name><affiliation><nlm:aff id="A9">Department of Medicine, University of Calgary, Calgary, Alberta, Canada</nlm:aff></affiliation></author><author><name sortKey="Moore, Sarah" sort="Moore, Sarah" uniqKey="Moore S" first="Sarah" last="Moore">Sarah Moore</name><affiliation><nlm:aff id="A1">Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Altree, Meryl" sort="Altree, Meryl" uniqKey="Altree M" first="Meryl" last="Altree">Meryl Altree</name><affiliation><nlm:aff id="A10">SA Clinical Genetics Service, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Escher, Robert" sort="Escher, Robert" uniqKey="Escher R" first="Robert" last="Escher">Robert Escher</name><affiliation><nlm:aff id="A3">Molecular Medicine Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Vic, Australia</nlm:aff></affiliation></author><author><name sortKey="Bardy, Peter G" sort="Bardy, Peter G" uniqKey="Bardy P" first="Peter G." last="Bardy">Peter G. Bardy</name><affiliation><nlm:aff id="A5">Department of Haematology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Suthers, Graeme K" sort="Suthers, Graeme K" uniqKey="Suthers G" first="Graeme K." last="Suthers">Graeme K. Suthers</name><affiliation><nlm:aff id="A10">SA Clinical Genetics Service, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A11">Department of Paediatrics, University of Adelaide, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="D Ndrea, Richard J" sort="D Ndrea, Richard J" uniqKey="D Ndrea R" first="Richard J." last="D Ndrea">Richard J. D Ndrea</name><affiliation><nlm:aff id="A2">School of Medicine, University of Adelaide, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A4">Department of Haematology and Oncology, The Queen Elizabeth Hospital, Woodville, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A5">Department of Haematology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Horwitz, Marshall S" sort="Horwitz, Marshall S" uniqKey="Horwitz M" first="Marshall S." last="Horwitz">Marshall S. Horwitz</name><affiliation><nlm:aff id="A8">Department of Pathology, University of Washington School of Medicine, Seattle, WA, USA</nlm:aff></affiliation></author><author><name sortKey="Scott, Hamish S" sort="Scott, Hamish S" uniqKey="Scott H" first="Hamish S." last="Scott">Hamish S. Scott</name><affiliation><nlm:aff id="A1">Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A2">School of Medicine, University of Adelaide, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A3">Molecular Medicine Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Vic, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A12">School of Molecular and Biomedical Science, University of Adelaide, SA, Australia</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">21892162</idno><idno type="pmc">3184204</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3184204</idno><idno type="RBID">PMC:3184204</idno><idno type="doi">10.1038/ng.913</idno><date when="2011">2011</date><idno type="wicri:Area/Pmc/Corpus">004707</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">004707</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Heritable <italic>GATA2</italic> Mutations Associated with Familial Myelodysplastic Syndrome and Acute Myeloid Leukemia</title><author><name sortKey="Hahn, Christopher N" sort="Hahn, Christopher N" uniqKey="Hahn C" first="Christopher N." last="Hahn">Christopher N. Hahn</name><affiliation><nlm:aff id="A1">Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A2">School of Medicine, University of Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Chong, Chan Eng" sort="Chong, Chan Eng" uniqKey="Chong C" first="Chan-Eng" last="Chong">Chan-Eng Chong</name><affiliation><nlm:aff id="A1">Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A2">School of Medicine, University of Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Carmichael, Catherine L" sort="Carmichael, Catherine L" uniqKey="Carmichael C" first="Catherine L." last="Carmichael">Catherine L. Carmichael</name><affiliation><nlm:aff id="A3">Molecular Medicine Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Vic, Australia</nlm:aff></affiliation></author><author><name sortKey="Wilkins, Ella J" sort="Wilkins, Ella J" uniqKey="Wilkins E" first="Ella J." last="Wilkins">Ella J. Wilkins</name><affiliation><nlm:aff id="A3">Molecular Medicine Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Vic, Australia</nlm:aff></affiliation></author><author><name sortKey="Brautigan, Peter J" sort="Brautigan, Peter J" uniqKey="Brautigan P" first="Peter J." last="Brautigan">Peter J. Brautigan</name><affiliation><nlm:aff id="A1">Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Li, Xiao Chun" sort="Li, Xiao Chun" uniqKey="Li X" first="Xiao-Chun" last="Li">Xiao-Chun Li</name><affiliation><nlm:aff id="A1">Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Babic, Milena" sort="Babic, Milena" uniqKey="Babic M" first="Milena" last="Babic">Milena Babic</name><affiliation><nlm:aff id="A1">Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Lin, Ming" sort="Lin, Ming" uniqKey="Lin M" first="Ming" last="Lin">Ming Lin</name><affiliation><nlm:aff id="A1">Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Carmagnac, Amandine" sort="Carmagnac, Amandine" uniqKey="Carmagnac A" first="Amandine" last="Carmagnac">Amandine Carmagnac</name><affiliation><nlm:aff id="A3">Molecular Medicine Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Vic, Australia</nlm:aff></affiliation></author><author><name sortKey="Lee, Young K" sort="Lee, Young K" uniqKey="Lee Y" first="Young K." last="Lee">Young K. Lee</name><affiliation><nlm:aff id="A1">Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Kok, Chung H" sort="Kok, Chung H" uniqKey="Kok C" first="Chung H." last="Kok">Chung H. Kok</name><affiliation><nlm:aff id="A4">Department of Haematology and Oncology, The Queen Elizabeth Hospital, Woodville, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A5">Department of Haematology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Gagliardi, Lucia" sort="Gagliardi, Lucia" uniqKey="Gagliardi L" first="Lucia" last="Gagliardi">Lucia Gagliardi</name><affiliation><nlm:aff id="A1">Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Friend, Kathryn L" sort="Friend, Kathryn L" uniqKey="Friend K" first="Kathryn L." last="Friend">Kathryn L. Friend</name><affiliation><nlm:aff id="A6">Department of Paediatric and Reproductive Genetics, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Ekert, Paul G" sort="Ekert, Paul G" uniqKey="Ekert P" first="Paul G." last="Ekert">Paul G. Ekert</name><affiliation><nlm:aff id="A7">Cell Signalling and Cell Death Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Vic, Australia</nlm:aff></affiliation></author><author><name sortKey="Butcher, Carolyn M" sort="Butcher, Carolyn M" uniqKey="Butcher C" first="Carolyn M." last="Butcher">Carolyn M. Butcher</name><affiliation><nlm:aff id="A4">Department of Haematology and Oncology, The Queen Elizabeth Hospital, Woodville, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A5">Department of Haematology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Brown, Anna L" sort="Brown, Anna L" uniqKey="Brown A" first="Anna L." last="Brown">Anna L. Brown</name><affiliation><nlm:aff id="A5">Department of Haematology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Lewis, Ian D" sort="Lewis, Ian D" uniqKey="Lewis I" first="Ian D." last="Lewis">Ian D. Lewis</name><affiliation><nlm:aff id="A2">School of Medicine, University of Adelaide, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A5">Department of Haematology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="To, L Bik" sort="To, L Bik" uniqKey="To L" first="L. Bik" last="To">L. Bik To</name><affiliation><nlm:aff id="A2">School of Medicine, University of Adelaide, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A5">Department of Haematology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Timms, Andrew E" sort="Timms, Andrew E" uniqKey="Timms A" first="Andrew E." last="Timms">Andrew E. Timms</name><affiliation><nlm:aff id="A8">Department of Pathology, University of Washington School of Medicine, Seattle, WA, USA</nlm:aff></affiliation></author><author><name sortKey="Storek, Jan" sort="Storek, Jan" uniqKey="Storek J" first="Jan" last="Storek">Jan Storek</name><affiliation><nlm:aff id="A9">Department of Medicine, University of Calgary, Calgary, Alberta, Canada</nlm:aff></affiliation></author><author><name sortKey="Moore, Sarah" sort="Moore, Sarah" uniqKey="Moore S" first="Sarah" last="Moore">Sarah Moore</name><affiliation><nlm:aff id="A1">Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Altree, Meryl" sort="Altree, Meryl" uniqKey="Altree M" first="Meryl" last="Altree">Meryl Altree</name><affiliation><nlm:aff id="A10">SA Clinical Genetics Service, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Escher, Robert" sort="Escher, Robert" uniqKey="Escher R" first="Robert" last="Escher">Robert Escher</name><affiliation><nlm:aff id="A3">Molecular Medicine Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Vic, Australia</nlm:aff></affiliation></author><author><name sortKey="Bardy, Peter G" sort="Bardy, Peter G" uniqKey="Bardy P" first="Peter G." last="Bardy">Peter G. Bardy</name><affiliation><nlm:aff id="A5">Department of Haematology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Suthers, Graeme K" sort="Suthers, Graeme K" uniqKey="Suthers G" first="Graeme K." last="Suthers">Graeme K. Suthers</name><affiliation><nlm:aff id="A10">SA Clinical Genetics Service, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A11">Department of Paediatrics, University of Adelaide, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="D Ndrea, Richard J" sort="D Ndrea, Richard J" uniqKey="D Ndrea R" first="Richard J." last="D Ndrea">Richard J. D Ndrea</name><affiliation><nlm:aff id="A2">School of Medicine, University of Adelaide, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A4">Department of Haematology and Oncology, The Queen Elizabeth Hospital, Woodville, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A5">Department of Haematology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation></author><author><name sortKey="Horwitz, Marshall S" sort="Horwitz, Marshall S" uniqKey="Horwitz M" first="Marshall S." last="Horwitz">Marshall S. Horwitz</name><affiliation><nlm:aff id="A8">Department of Pathology, University of Washington School of Medicine, Seattle, WA, USA</nlm:aff></affiliation></author><author><name sortKey="Scott, Hamish S" sort="Scott, Hamish S" uniqKey="Scott H" first="Hamish S." last="Scott">Hamish S. Scott</name><affiliation><nlm:aff id="A1">Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A2">School of Medicine, University of Adelaide, SA, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A3">Molecular Medicine Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Vic, Australia</nlm:aff></affiliation><affiliation><nlm:aff id="A12">School of Molecular and Biomedical Science, University of Adelaide, SA, Australia</nlm:aff></affiliation></author></analytic><series><title level="j">Nature genetics</title><idno type="ISSN">1061-4036</idno><idno type="eISSN">1546-1718</idno><imprint><date when="2011">2011</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p id="P1">We report the discovery of the <italic>GATA2</italic> gene as a new myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) predisposition gene. We found the same, novel heterozygous c.1061C>T (p.Thr354Met) missense mutation in the <italic>GATA2</italic> transcription factor gene segregating with the multigenerational transmission of MDS/AML in three families, and a <italic>GATA2</italic> c.1063_1065delACA (p.Thr355del) mutation at an adjacent codon in a fourth MDS/AML family. The mutations reside within the second zinc finger of GATA2 which mediates DNA-binding and protein-protein interactions. We show differential effects of the mutants on transactivation of target genes, cellular differentiation, apoptosis and global gene expression. Identification of such predisposing genes to familial forms of MDS and AML is critical for more effective diagnosis and prognosis, counselling, selection of related bone marrow transplant donors, and development of therapies.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Vardiman, Jw" uniqKey="Vardiman J">JW Vardiman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Barzi, A" uniqKey="Barzi A">A Barzi</name></author><author><name sortKey="Sekeres, Ma" uniqKey="Sekeres M">MA Sekeres</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Owen, C" uniqKey="Owen C">C Owen</name></author><author><name sortKey="Barnett, M" uniqKey="Barnett M">M Barnett</name></author><author><name sortKey="Fitzgibbon, J" uniqKey="Fitzgibbon J">J Fitzgibbon</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhang, P" uniqKey="Zhang P">P 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<front><journal-meta><journal-id journal-id-type="nlm-journal-id">9216904</journal-id><journal-id journal-id-type="pubmed-jr-id">2419</journal-id><journal-id journal-id-type="nlm-ta">Nat Genet</journal-id><journal-id journal-id-type="iso-abbrev">Nat. Genet.</journal-id><journal-title-group><journal-title>Nature genetics</journal-title></journal-title-group><issn pub-type="ppub">1061-4036</issn><issn pub-type="epub">1546-1718</issn></journal-meta><article-meta><article-id pub-id-type="pmid">21892162</article-id><article-id pub-id-type="pmc">3184204</article-id><article-id pub-id-type="doi">10.1038/ng.913</article-id><article-id pub-id-type="manuscript">NIHMS315105</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Heritable <italic>GATA2</italic> Mutations Associated with Familial Myelodysplastic Syndrome and Acute Myeloid Leukemia</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Hahn</surname><given-names>Christopher N.</given-names></name><xref ref-type="aff" rid="A1">1</xref><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Chong</surname><given-names>Chan-Eng</given-names></name><xref ref-type="aff" rid="A1">1</xref><xref ref-type="aff" rid="A2">2</xref><xref rid="FN4" ref-type="author-notes">15</xref></contrib><contrib contrib-type="author"><name><surname>Carmichael</surname><given-names>Catherine L.</given-names></name><xref ref-type="aff" rid="A3">3</xref><xref rid="FN4" ref-type="author-notes">15</xref></contrib><contrib contrib-type="author"><name><surname>Wilkins</surname><given-names>Ella J.</given-names></name><xref ref-type="aff" rid="A3">3</xref><xref rid="FN2" ref-type="author-notes">13</xref></contrib><contrib contrib-type="author"><name><surname>Brautigan</surname><given-names>Peter J.</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Xiao-Chun</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Babic</surname><given-names>Milena</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Lin</surname><given-names>Ming</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Carmagnac</surname><given-names>Amandine</given-names></name><xref ref-type="aff" rid="A3">3</xref></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Young K.</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kok</surname><given-names>Chung H.</given-names></name><xref ref-type="aff" rid="A4">4</xref><xref ref-type="aff" rid="A5">5</xref></contrib><contrib contrib-type="author"><name><surname>Gagliardi</surname><given-names>Lucia</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Friend</surname><given-names>Kathryn L.</given-names></name><xref ref-type="aff" rid="A6">6</xref></contrib><contrib contrib-type="author"><name><surname>Ekert</surname><given-names>Paul G.</given-names></name><xref ref-type="aff" rid="A7">7</xref></contrib><contrib contrib-type="author"><name><surname>Butcher</surname><given-names>Carolyn M.</given-names></name><xref ref-type="aff" rid="A4">4</xref><xref ref-type="aff" rid="A5">5</xref></contrib><contrib contrib-type="author"><name><surname>Brown</surname><given-names>Anna L.</given-names></name><xref ref-type="aff" rid="A5">5</xref></contrib><contrib contrib-type="author"><name><surname>Lewis</surname><given-names>Ian D.</given-names></name><xref ref-type="aff" rid="A2">2</xref><xref ref-type="aff" rid="A5">5</xref></contrib><contrib contrib-type="author"><name><surname>To</surname><given-names>L. Bik</given-names></name><xref ref-type="aff" rid="A2">2</xref><xref ref-type="aff" rid="A5">5</xref></contrib><contrib contrib-type="author"><name><surname>Timms</surname><given-names>Andrew E.</given-names></name><xref ref-type="aff" rid="A8">8</xref></contrib><contrib contrib-type="author"><name><surname>Storek</surname><given-names>Jan</given-names></name><xref ref-type="aff" rid="A9">9</xref></contrib><contrib contrib-type="author"><name><surname>Moore</surname><given-names>Sarah</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Altree</surname><given-names>Meryl</given-names></name><xref ref-type="aff" rid="A10">10</xref></contrib><contrib contrib-type="author"><name><surname>Escher</surname><given-names>Robert</given-names></name><xref ref-type="aff" rid="A3">3</xref><xref rid="FN3" ref-type="author-notes">14</xref></contrib><contrib contrib-type="author"><name><surname>Bardy</surname><given-names>Peter G.</given-names></name><xref ref-type="aff" rid="A5">5</xref></contrib><contrib contrib-type="author"><name><surname>Suthers</surname><given-names>Graeme K.</given-names></name><xref ref-type="aff" rid="A10">10</xref><xref ref-type="aff" rid="A11">11</xref></contrib><contrib contrib-type="author"><name><surname>D’Andrea</surname><given-names>Richard J.</given-names></name><xref ref-type="aff" rid="A2">2</xref><xref ref-type="aff" rid="A4">4</xref><xref ref-type="aff" rid="A5">5</xref><xref rid="FN5" ref-type="author-notes">16</xref></contrib><contrib contrib-type="author"><name><surname>Horwitz</surname><given-names>Marshall S.</given-names></name><xref ref-type="aff" rid="A8">8</xref></contrib><contrib contrib-type="author"><name><surname>Scott</surname><given-names>Hamish S.</given-names></name><xref ref-type="aff" rid="A1">1</xref><xref ref-type="aff" rid="A2">2</xref><xref ref-type="aff" rid="A3">3</xref><xref ref-type="aff" rid="A12">12</xref><xref rid="FN5" ref-type="author-notes">16</xref></contrib></contrib-group><aff id="A1"><label>1</label>Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</aff><aff id="A2"><label>2</label>School of Medicine, University of Adelaide, SA, Australia</aff><aff id="A3"><label>3</label>Molecular Medicine Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Vic, Australia</aff><aff id="A4"><label>4</label>Department of Haematology and Oncology, The Queen Elizabeth Hospital, Woodville, SA, Australia</aff><aff id="A5"><label>5</label>Department of Haematology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia</aff><aff id="A6"><label>6</label>Department of Paediatric and Reproductive Genetics, SA Pathology, Adelaide, SA, Australia</aff><aff id="A7"><label>7</label>Cell Signalling and Cell Death Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Vic, Australia</aff><aff id="A8"><label>8</label>Department of Pathology, University of Washington School of Medicine, Seattle, WA, USA</aff><aff id="A9"><label>9</label>Department of Medicine, University of Calgary, Calgary, Alberta, Canada</aff><aff id="A10"><label>10</label>SA Clinical Genetics Service, SA Pathology, Adelaide, SA, Australia</aff><aff id="A11"><label>11</label>Department of Paediatrics, University of Adelaide, Adelaide, SA, Australia</aff><aff id="A12"><label>12</label>School of Molecular and Biomedical Science, University of Adelaide, SA, Australia</aff><author-notes><corresp id="FN1">Correspondence should be addressed to H.S.S. (<email>hamish.scott@health.sa.gov.au</email>)</corresp><fn id="FN2" fn-type="present-address"><label>13</label><p>Present Address: Neurogenetics Laboratory, Howard Florey Institute, Parkville, VIC, Australia.</p></fn><fn id="FN3" fn-type="present-address"><label>14</label><p>Present Address: Medical Clinic, Regional Hospital Emmental, Burgdorf, Switzerland.</p></fn><fn id="FN4" fn-type="equal"><label>15</label><p>These authors contributed equally to this work.</p></fn><fn id="FN5"><label>16</label><p>These authors jointly supervised this work.</p></fn></author-notes><pub-date pub-type="nihms-submitted"><day>1</day><month>8</month><year>2011</year></pub-date><pub-date pub-type="epub"><day>04</day><month>9</month><year>2011</year></pub-date><pub-date pub-type="collection"><month>10</month><year>2011</year></pub-date><pub-date pub-type="pmc-release"><day>01</day><month>4</month><year>2012</year></pub-date><volume>43</volume><issue>10</issue><fpage>1012</fpage><lpage>1017</lpage><pmc-comment>elocation-id from pubmed: 10.1038/ng.913</pmc-comment>
<permissions><license><license-p>Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
<uri xlink:type="simple" xlink:href="http://www.nature.com/authors/editorial_policies/license.html#terms">http://www.nature.com/authors/editorial_policies/license.html#terms</uri></license-p></license></permissions><abstract><p id="P1">We report the discovery of the <italic>GATA2</italic> gene as a new myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) predisposition gene. We found the same, novel heterozygous c.1061C>T (p.Thr354Met) missense mutation in the <italic>GATA2</italic> transcription factor gene segregating with the multigenerational transmission of MDS/AML in three families, and a <italic>GATA2</italic> c.1063_1065delACA (p.Thr355del) mutation at an adjacent codon in a fourth MDS/AML family. The mutations reside within the second zinc finger of GATA2 which mediates DNA-binding and protein-protein interactions. We show differential effects of the mutants on transactivation of target genes, cellular differentiation, apoptosis and global gene expression. Identification of such predisposing genes to familial forms of MDS and AML is critical for more effective diagnosis and prognosis, counselling, selection of related bone marrow transplant donors, and development of therapies.</p></abstract></article-meta></front><body><p id="P2">AML is the most common form of sporadic leukemia in adults <sup><xref rid="R1" ref-type="bibr">1</xref></sup> while MDS is a clonal disorder of hematopoietic stem cells characterized by ineffective hematopoiesis, with a tendency to progress to AML<sup><xref rid="R2" ref-type="bibr">2</xref></sup>. The study of families predisposed to particular malignancies is a successful strategy for discovering causative oncogenes and tumour suppressor genes (TSG). While rare, dozens of families developing non-syndromic forms of MDS and AML (<italic>i.e.</italic> lacking other systemic manifestations) have been described. To date, only two MDS/AML predisposition genes have been recognized: Runt-related transcription factor 1 (<italic>RUNX1</italic>/<italic>AML1</italic>) and CCAAT-enhancer binding protein α (<italic>CEBPA</italic>) (reviewed in<sup><xref rid="R3" ref-type="bibr">3</xref></sup>).</p><p id="P3">Here we report a highly specific p.Thr354Met heritable mutation in GATA2 co-segregating with with early onset MDS/AML in three families. We also report a family with MDS with a 3 bp heritable deletion in the <italic>GATA2</italic> gene (p.Thr355del) deleting the second threonine in this sequence.</p><p id="P4">We determined the genomic DNA sequence of all RefSeq exons in 50 candidate genes (<xref rid="SD1" ref-type="supplementary-material">Supplementary Table 1</xref>) from patients representing five pedigrees with predisposition to MDS/AML, prescreened for absence of <italic>RUNX1</italic> or <italic>CEBPA</italic> germline mutations. In three families, there was an identical heritable heterozygous variation in the transcription factor <italic>GATA2</italic>, c.1061C>T (p.Thr354Met) (<xref rid="F1" ref-type="fig">Fig. 1</xref>, <xref rid="SD1" ref-type="supplementary-material">Supplementary Fig. 1</xref>). In all three families p.Thr354Met segregated with the disease for the samples tested, and no family members had AML or MDS who did not also carry p.Thr354Met (<xref rid="F1" ref-type="fig">Fig. 1a</xref>, <xref rid="SD1" ref-type="supplementary-material">Supplementary table 2</xref>). There were also members in each family who carried this variant but were unaffected (Pedigree 1: III-5 and III-8; Pedigree 2: II-6; Pedigree 3: III-9).</p><p id="P5">We recently identified a fourth family in which a father and son, both affected by MDS, shared a heterozygous heritable deletion of 3 bp in <italic>GATA2</italic> (c.1063-1065delACA) resulting in p.Thr355del (<xref rid="F1" ref-type="fig">Fig. 1</xref>, <xref rid="SD1" ref-type="supplementary-material">Supplementary Fig. 1</xref>). This codon is adjacent to the codon mutated in the first three families, and encodes the second of the five consecutive threonines.</p><p id="P6">p.Thr354 and p.Thr355del are the first two of five consecutive threonine residues in a highly conserved region of the GATA2 protein (<xref rid="SD1" ref-type="supplementary-material">Supplementary Fig. 1b</xref>) encoding zinc finger 2 (ZF2), which is involved in DNA binding, homodimerization and interaction with transcription factor PU.1<sup><xref rid="R4" ref-type="bibr">4</xref>,<xref rid="R5" ref-type="bibr">5</xref></sup>. PolyPhen-2 predicts that p.Thr354Met and p.Thr355del are likely to affect GATA2 function. Somatic mutations in ZF2 of GATA2 have also been reported during chronic myeloid leukaemia (CML) blast crisis (BC) (p.Leu359Val, p.Ala341_Gly346del)<sup><xref rid="R6" ref-type="bibr">6</xref></sup> and recently in ZF1 and ZF2 in AML-M5<sup><xref rid="R7" ref-type="bibr">7</xref></sup> (<xref rid="F1" ref-type="fig">Fig. 1b,c</xref>). Somatic mutations in the corresponding ZF2 of the related protein family member GATA3 are found in breast cancer<sup><xref rid="R8" ref-type="bibr">8</xref></sup> (<xref rid="F1" ref-type="fig">Fig. 1c</xref>).</p><p id="P7">High resolution melt (HRM) analysis did not detect p.Thr354Met, p.Thr355del or other variants in exon 5 of <italic>GATA2</italic> in 695 non-leukemic, ethnically-matched normal controls (<italic>i.e.</italic> 1390 chromosomes) (<xref rid="SD1" ref-type="supplementary-material">Supplementary Fig. 2a</xref>, <xref rid="SD1" ref-type="supplementary-material">Supplementary Note</xref>). Thus it is improbable that these variants represent rare polymorphisms. These variants were also not present in dbSNP132 or 1000 Genomes Project (January, 2011; URLs). Together with the disease segregation data, these results indicate that the GATA2 p.Thr354Met and p.Thr355del variants are the predisposing mutation in these families with familial MDS/AML.</p><p id="P8">A distinguishing feature of our families with <italic>GATA2</italic> mutation was a lack of apparent “accessory” phenotype inside or outside the hematopoietic system, akin to the thrombocytopenia and eosinophilia seen in AML-predisposed families due to <italic>RUNX1</italic> and <italic>CEBPA</italic> mutations, respectively<sup><xref rid="R9" ref-type="bibr">9</xref>,<xref rid="R10" ref-type="bibr">10</xref></sup>. In all 4 families, the <italic>GATA2</italic> mutations were associated with early-onset MDS and/or AML displaying highly penetrant autosomal dominant inheritance and resulted in a poor outcome unless successfully transplanted (<italic>e.g.</italic> Pedigree 1: age of death from AML – 10 – 50 years; 2 individuals of age 58 and 62 years have mutation but no disease; all other siblings without the mutation are alive or have lived beyond 53 years) (<xref rid="SD1" ref-type="supplementary-material">Supplementary Table 2 and 3</xref>, <xref rid="SD1" ref-type="supplementary-material">Supplementary Note</xref>). For Pedigrees 1 and 3<sup><xref rid="R11" ref-type="bibr">11</xref>,<xref rid="R12" ref-type="bibr">12</xref></sup>, the presentation varied with some displaying protracted MDS and others acute onset; the FAB subtype and karyotypic features of AML varied. In Pedigree 4, MDS was first diagnosed at age 13 in the son, who was treated with allogeneic bone marrow transplant at age 15; MDS was later diagnosed at age 53 in the father, who underwent allogeneic bone marrow transplantation.</p><p id="P9">Heritable <italic>GATA2</italic> coding variations were not found in samples from another 8 families with multiple cases of AML, or in another 27 families with multiple occurrences of various lymphoid malignancies (11 NHL, 5 HL, 3 ALL, 7 CLL, 1 Multiple myeloma families). Also, no mutations were detected in <italic>GATA2</italic> in 15 hematopoietic cell lines (<xref rid="SD1" ref-type="supplementary-material">Supplementary Table 4</xref>). No sequence variations were detected in the entire <italic>GATA2</italic> coding region of 268 sporadic AML patient sample DNAs except a single c.182C>T (p.Ala61Val) variant in exon 2 (<xref rid="SD1" ref-type="supplementary-material">Supplementary Fig. 2c</xref>), which was assessed to be benign using PolyPhen-2 (URLs). Together, this suggests that point mutations and small indels in the <italic>GATA2</italic> coding sequence are not frequent in sporadic AML.</p><p id="P10">Haplotype mapping using 8 informative single nucleotide polymorphisms (SNPs) within and surrounding the <italic>GATA2</italic> gene demonstrated that the c.1061C>T (p.Thr354Met) mutation segregated within two distinct haplotypes (<xref rid="SD1" ref-type="supplementary-material">Supplementary Table 5</xref>) indicating that this mutation has arisen at least twice among the three families in which it is found.</p><p id="P11">GATA2 is a DNA-binding transcription factor which localizes predominantly to the nucleus. We generated cDNAs for the p.Thr354Met and p.Thr355del mutant GATA2 proteins and the acquired CML BC p.Leu359Val mutant<sup><xref rid="R6" ref-type="bibr">6</xref></sup>. Wildtype (WT) and mutant proteins expressed at comparable levels when transiently expressed in HEK293 fibroblasts (<xref rid="F2" ref-type="fig">Fig. 2a</xref>) and when induced to express from a 4HT-responsive dual vector lentivirus system in stably transduced HL-60 promyelocytes (<xref rid="SD1" ref-type="supplementary-material">Supplementary Fig. 3a</xref>). Mutant proteins were expressed at comparable levels to wildtype (WT) GATA2 when transiently introduced into WT and mutant proteins appropriately localized to the nucleus (<xref rid="F2" ref-type="fig">Fig. 2b</xref>, <xref rid="SD1" ref-type="supplementary-material">Supplementary Fig. 3b</xref>). However, the p.Thr354Met mutation dramatically reduced the ability of GATA2 to bind its consensus WGATAR DNA motif while p.Thr355del almost completely ablated DNA binding (<xref rid="F2" ref-type="fig">Fig. 2c</xref>, <xref rid="SD1" ref-type="supplementary-material">Supplementary Fig. 3c</xref>).</p><p id="P12">Molecular modeling of GATA2 ZF2 (<xref rid="SD1" ref-type="supplementary-material">Supplementary Fig. 4</xref>, <xref rid="SD1" ref-type="supplementary-material">Supplementary Note</xref>) demonstrated that the p.Thr354 residue does not contact DNA, but rather makes polar contact with adjacent threonines, and via its amino group with p.Cys349 which coordinates the zinc atom. Replacement of p.Thr354 with the bulky methionine moiety is predicted to alter the overall structure of this zinc finger by affecting zinc contacts. This may explain reduced binding of p.Thr354Met to DNA (<xref rid="F2" ref-type="fig">Fig. 2c</xref>, <xref rid="SD1" ref-type="supplementary-material">Supplementary Fig. 3c</xref>). In contrast, p.Thr355del shortens the conserved threonine string, likely impacting the orientation and position of p.Leu359, which directly contacts DNA. These observations likely explain the almost-complete ablation of DNA binding.</p><p id="P13">Luciferase reporter assay experiments show that GATA2 p.Thr354Met and p.Thr355del had significantly reduced transactivation ability compared to WT on known <italic>GATA2</italic> responsive enhancers (<italic>RUNX1</italic> and <italic>CD34</italic>) and the <italic>LYL1</italic> promoter (<xref rid="F3" ref-type="fig">Fig. 3a,b,c</xref>). Experiments mixing WT with p.Thr354Met or p.Thr355del at a 1:1 ratio, mimicking heterozygosity, demonstrated a dominant negative effect of the mutants over WT transcription activation in multiple systems (<xref rid="F3" ref-type="fig">Fig. 3d,e</xref>, <xref rid="SD1" ref-type="supplementary-material">Supplementary Fig. 5</xref>). Interestingly, WT and PU.1 transactivated the <italic>CSF1R</italic> (<italic>M-CSF-R</italic>) promoter 2.4 and 2.5-fold, respectively, but together synergized to induce 18-fold (<xref rid="F3" ref-type="fig">Fig. 3e</xref>). While p.Leu359Val was similar to WT GATA2, p.Thr354Met and p.Thr355del gave dramatically reduced induction alone (1.5- and 0.9-fold) or with PU.1 (7- and 9-fold, respectively, compared to 18-fold with WT). p.Thr354Met and p.Thr355del also displayed dominant negative activity with transactivation by WT GATA2 reduced to 9- and 10-fold, respectively in the presence of these mutants (only marginally above the 7- and 9-fold with mutants alone). Hence, p.Thr354Met and p.Thr355del perturb the transactivation ability of GATA2, presumably by disrupting association with PU.1 or other interacting transcription factors, and are likely impact expression of downstream targets. Interestingly, while WT GATA2 displayed different responses in HEK293 versus Cos-7 cells on the <italic>RUNX1</italic> enhancer (activating versus repressing, respectively), p.Thr354Met displayed loss-of-function activity in both cell types (<xref rid="SD1" ref-type="supplementary-material">Supplementary Fig. 6</xref>). Thus, on multiple GATA responsive elements, p.Thr354Met and p.Thr355del show loss-of-function and also dominant negative effects.</p><p id="P14">HL-60 promyelocytes differentiate into granulocytes upon exposure to all-<italic>trans</italic> retinoic acid (ATRA), resulting in upregulation of CD11b, cessation of proliferation and subsequent promotion of apoptosis (<xref rid="F4" ref-type="fig">Fig. 4</xref>). When expressed at equivalent levels under non-differentiating conditions, unlike WT and p.Leu359Val which inhibited proliferation and promoted apoptosis, p.Thr354Met and p.Thr355del acted as loss-of-function mutants (<xref rid="F4" ref-type="fig">Fig. 4b,f,j</xref>). However, in the presence of ATRA, p.Thr354Met alone enabled cell proliferation/survival (<xref rid="F4" ref-type="fig">Fig. 4h</xref>), while simultaneously inhibiting differentiation and apoptosis (<xref rid="F4" ref-type="fig">Fig. 4d,m</xref>, <xref rid="SD1" ref-type="supplementary-material">Supplementary Fig. 7</xref>). p.Thr355del appeared to be a null mutant under these conditions.</p><p id="P15">In order to better understand the effects of the GATA2 mutants on gene expression, microarray analysis was performed to compare global gene expression in HL-60 cells expressing WT GATA2 and the three GATA2 mutants (<xref rid="SD1" ref-type="supplementary-material">Supplementary Table 6</xref> and <xref rid="SD1" ref-type="supplementary-material">Supplementary Fig. 8</xref>). The data clearly showed that p.Thr355del and p.Thr354Met are almost total loss-of-function mutants (<xref rid="SD1" ref-type="supplementary-material">Supplementary Fig. 8</xref>). Note that, p.Leu359Val exhibits gain-of-function (1,253 newly regulated genes compared to WT GATA2) and partial loss-of-function (457 genes no longer regulated) while retaining 786 genes commonly regulated. These results are consistent with EMSA-western blot and transactivation assays. Further bioinformatics analysis indicated that <italic>MYC</italic> may be among key target which is repressed by GATA2 WT but not p.Thr354Met and p.Thr355del (<xref rid="SD1" ref-type="supplementary-material">Supplementary Table 6, Supplementary Note</xref>)</p><p id="P16">Interestingly, recurrent p.Leu359Val mutation in ZF2 of GATA2 was reported in 8/85 cases of CML BC<sup><xref rid="R6" ref-type="bibr">6</xref></sup>, a disease often phenotypically indistinguishable from AML. As shown in <xref rid="F1" ref-type="fig">Fig. 1b,c, p</xref>.Thr354Met is situated between the deleted residues (p.Ala341_Gly346del), also observed in CML BC, and the p.Leu359 residue. p.Leu359 contacts DNA at the guanine residue of the WGATAR consensus motif and based on <italic>in vitro</italic> DNA binding and transactivation assays, p.Leu359Val has previously been reported to be a gain-of-function mutation while p.Ala341_Gly346del appears to be a partial loss-of-function mutation. p.Met354 or deletion of p.Thr355 may affect overall ZF structure (<xref rid="SD1" ref-type="supplementary-material">Supplementary Fig. 4</xref>) although we cannot exclude disrupted heterodimerization with GATA2’s interacting partners of GATA2 (<xref rid="SD1" ref-type="supplementary-material">Supplementary Table 7</xref>). We speculate that aberrant protein partnerships may explain dominant negative activity and adversely influences expression of genes critical to myelopoiesis.</p><p id="P17">The MDS/AML observed within these families is clinically heterogeneous, and demonstrate a variety of somatic chromosomal abnormalities, including monosomy 7, trisomy 8, and trisomy 21 (<xref rid="SD1" ref-type="supplementary-material">Supplementary Table 2</xref>). As such it is similar to familial MDS/AML with monosomy 7<sup><xref rid="R3" ref-type="bibr">3</xref>,<xref rid="R13" ref-type="bibr">13</xref></sup>. Moreover, <italic>GATA2</italic> mutations were not detected in 8 MDS/AML families in which <italic>RUNX1</italic> and <italic>CEPBA</italic> mutations were excluded and 27 families presenting with lymphoid malignancy. Mutations at this position within ZF2 are likely to initiate an exclusively myeloid pathway of oncogenesis in which subsequent gene-specific, somatically acquired mutations probably define the particular type of disease that ultimately arises.</p><p id="P18">The mechanism by which GATA2 p.Thr354Met and p.Thr355del mutations function is distinct to that generally described for <italic>RUNX1</italic> and <italic>CEBPA</italic>, which commonly act as classical TSG with a wide-range of mutations and requiring functional disruption of both alleles. Transcription factors are well characterized as targets of dominant negative or constitutively active mutations in cancer<sup><xref rid="R14" ref-type="bibr">14</xref>,<xref rid="R15" ref-type="bibr">15</xref></sup>, with <italic>RUNX1</italic> mutations leading to a spectrum of outcomes including AML and ALL consistent with both TSG and dominant oncogene models<sup><xref rid="R16" ref-type="bibr">16</xref>,<xref rid="R17" ref-type="bibr">17</xref></sup>. While we have only been able to detect single allele <italic>GATA2</italic> germline mutations in affected samples, we cannot rule out the possibility of acquired mutations in the “normal” allele.</p><p id="P19"><italic>GATA2</italic> is indispensable for hematopoiesis<sup><xref rid="R17" ref-type="bibr">17</xref>, <xref rid="R18" ref-type="bibr">18</xref>,<xref rid="R19" ref-type="bibr">19</xref></sup>. It associates with, regulates or is regulated by transcription factors implicated in myeloid malignancy (<xref rid="SD1" ref-type="supplementary-material">Supplementary Table 7</xref>). Many of these interactions involve ZF2 in which the p.Thr354Met and p.Thr355del mutations reside, and it is likely that changes in the nature of these interactions play an important role in predisposition to MDS/AML. Indeed, our co-transfection studies are consistent with altered transactivation by p.Thr354Met and p.Thr355del with PU.1 (<xref rid="F3" ref-type="fig">Fig. 3e</xref>).</p><p id="P20">p.Thr354Met, p.Thr355del or any other mutations in <italic>GATA2</italic> were absent in our heterogeneous cohort of sporadic AML patients, although we cannot rule out possible mutations in samples with low percentage blasts. This is consistent with other recent studies, however, suggesting that somatic <italic>GATA2</italic> mutations in both ZF1 and ZF2 could be acquired only in specific AML subtypes such as AML-M5<sup><xref rid="R7" ref-type="bibr">7</xref>,<xref rid="R20" ref-type="bibr">20</xref>,<xref rid="R21" ref-type="bibr">21</xref></sup>. <italic>GATA2</italic> is, however, overexpressed in many cases of sporadic MDS<sup><xref rid="R22" ref-type="bibr">22</xref></sup> and AML, particularly in <italic>FLT3-ITD</italic><sup>+</sup> AML<sup><xref rid="R23" ref-type="bibr">23</xref></sup>, suggesting that alterations to <italic>GATA2</italic> expression, rather than direct mutation, may occur more commonly.. Further, chromosomal aberrations at the 3q21 breakpoint cluster encompassing a presumptive <italic>GATA2</italic> regulatory region resulted in upregulated <italic>GATA2</italic> expression in MDS and AML<sup><xref rid="R22" ref-type="bibr">22</xref>,<xref rid="R24" ref-type="bibr">24</xref>–<xref rid="R26" ref-type="bibr">26</xref></sup>. In addition, retroviral insertional mutagenesis in <italic>NUP98-HOXD13</italic> mice, a model for MDS/AML, identified <italic>Gata2</italic> as a common insertion site in induced AML, all of which overexpressed <italic>Gata2</italic><sup><xref rid="R27" ref-type="bibr">27</xref></sup>. Hence, accumulating evidence suggests that aberrant activation or overexpression of <italic>GATA2</italic> contributes to AML.</p><p id="P21">In this study, we show that <italic>GATA2</italic> is a new predisposition gene for familial MDS/AML and demonstrate functional changes due to mutations within a highly conserved threonine repeat located in the second zinc finger. Our findings highlight the power of approaches investigating familial predispositions to cancer, and have implications for diagnostic genetic testing. The poor outcome associated with these mutations may suggest that an aggressive treatment strategy is appropriate for individuals carrying <italic>GATA2</italic> mutations.</p><sec id="S1"><title>ONLINE METHODS</title><sec id="S2" sec-type="subjects"><title>Patients</title><p id="P22">Families (<xref rid="SD1" ref-type="supplementary-material">Supplementary Table 2, Supplementary Note</xref>) were recruited and sample use approved through institutional human ethics review board approved protocols from the Australian Familial Haematological Cancer Study (Royal Adelaide Hospital (RAH) #091203 and #100702, and Children, Youth and Women’s Health Service #REC1542/12/12, Adelaide, SA Australia), The Queen Elizabeth Hospital and the University of Washington (Seattle, WA USA).</p></sec><sec id="S3"><title>Sequence analysis of candidate genes</title><p id="P23">To identify germline and somatic mutations in patients with familial AML, a panel of 50 hematopoietic candidate genes, incorporating a total of 638 exons, was assembled (<xref rid="SD1" ref-type="supplementary-material">Supplementary Table 1</xref>). Primer design, PCR amplification, and dideoxy sequencing of genomic DNA purified from lymphoblastoid cells of probands from 7 MDS/AML pedigrees were performed by the Australian Genome Research Facility (AGRF). Sequences were aligned with NCBI RefSeq sequences using Mutation Surveyor (SoftGenetics) and variants compared to the UCSC and NCBI SNP databases for novelty. Sequence changes were confirmed by re-sequencing in both directions. Primer sequences are available upon request. Screening of control and sporadic AML populations was performed using high resolution melt (HRM) analysis (<xref rid="SD1" ref-type="supplementary-material">Supplementary Table 8</xref> and <xref rid="SD1" ref-type="supplementary-material">Supplementary Note</xref>).</p></sec><sec id="S4"><title>Cell culture</title><p id="P24">HEK293, 293T and Cos-7 cells were cultured in DMEM with 10% fetal bovine serum (FBS) (JRH Biosciences) and transient transfections were performed using Lipofectamine 2000 (Invitrogen). HL-60 promyelocytic cells were cultured in RPMI containing 10% FBS. All cultures contained 50 units/ml penicillin and 50 μg/ml streptomycin (Sigma).</p></sec><sec id="S5"><title>Generation of mutant <italic>GATA2</italic> plasmid and lentiviral expression constructs</title><p id="P25">An expression clone (pCMV6-XL6-GATA2) containing a 3.7 kb <italic>GATA2</italic> cDNA insert was obtained from OriGene, and p.Thr354Met, p.Thr355del and p.Leu359Val mutants were generated by site directed mutagenesis. The coding regions of wildtype (WT), p.Thr354Met and p.Leu359Val were cloned into a dual lentiviral vector system which was used to generate HL-60 cells expressing <italic>GATA2</italic> WT or mutants upon addition of 4-hydroxytamoxifen (4HT) (see <xref rid="SD1" ref-type="supplementary-material">Supplementary Note</xref>).</p></sec><sec id="S6"><title>GATA2-responsive promoter and enhancer studies</title><p id="P26">The <italic>GATA2</italic>-responsive promoter (<italic>LYL1</italic>) and enhancer (<italic>RUNX</italic>) were PCR amplified and cloned into pGL4.12[<italic>luc2CP</italic>] (<italic>Sfi</italic>I) and pGL3-Promoter (<italic>Kpn</italic>I/<italic>Bgl</italic>II) (Promega), respectively. The CSF1R (M-CSF-R) promoter was PCR amplified and cloned into pGL4.12[<italic>luc2CP</italic>] (<italic>Sfi</italic>I). See <xref rid="SD1" ref-type="supplementary-material">Supplementary Table 9</xref> for PCR primers used. The GATA2-responsive <italic>CD34</italic> enhancer-luciferase construct (CD34x2/Luc) and one with the GATA binding sites mutated (mutant CD34x2/Luc)<sup><xref rid="R31" ref-type="bibr">31</xref></sup> were kindly provided by Tariq Enver, Weatherall Institute of Molecular Medicine, Oxford, U.K. HEK293 or Cos-7 cells were transfected at 90% confluence with Lipofectamine 2000. In all experiments, the molar equivalents of EV constructs were used to balance gene expressing constructs to avoid squelching artifacts. After 20 h, cells were harvested and luciferase activity determined with the Dual-Luciferase Reporter Assay System (Promega) using a GloMax®-Multi Detection System (Promega). All assays were performed a minimum of three times in triplicate. All results were analysed using Student’s t-test, and reported as mean ± s.e.m. with significance, p<0.05 (asterisk).</p></sec><sec id="S7"><title>Cell differentiation and proliferation assays</title><p id="P27">HL-60 cells were plated at 1.25 × 10<sup>4</sup> cells/ml and treated with or without 30 nm 4-hyrdoxy tamoxifen (4-HT) for 24 h and then with or without 2 μM all-<italic>trans</italic> retinoic acid (ATRA). Cell numbers were determined by manual counting and FACS analysis (Phycoerythrin anti-mouse CD11b and Phycoerythrin rat IgG2b isotype control) (eBioscience) was performed 6 days after addition of ATRA (Sigma). The cells were also stained with hematoxylin and eosin for assessing morphological changes.</p></sec><sec id="S8"><title>Haplotyping</title><p id="P28">Haplotype mapping was performed by PCR amplification and sequencing of amplicons containing 50 single nucleotide polymorphisms (SNP) within and surrounding the position of the p.Thr354Met variant of the <italic>GATA2</italic> gene (<xref rid="SD1" ref-type="supplementary-material">Supplementary Table 10</xref>). All amplicons were generated using AmpliTaq Gold (Applied Biosystems) according to the manufacturer’s protocol using 2 mM MgCl<sub>2</sub> and the following cycle strategy; 95°C, 10 min; 95°C, 30 s, 66°C – 58°C, 20 s (touchdown, 0.8°C/cycle for 10 cycles), 72°C, 45 s (total of 40 cycles); 72°C, 3 min.</p></sec><sec id="S9"><title>Generation of mutant GATA2 plasmids and lentiviral expression constructs</title><p id="P29">An expression clone (pCMV6-XL6-GATA2) containing a 3.7 kb <italic>GATA2</italic> cDNA insert was obtained from OriGene (Cat. No. SC125368). p.Thr354Met, p.Thr355del and p.Leu359Val mutants were generated by QuikChange mutagenesis (Stratagene) using the primers T354M-F and T354M-R, 355delT-F and 355delT-R, and L359V-F and L359V-R (<xref rid="SD1" ref-type="supplementary-material">Supplementary Table 11</xref>), respectively. For the generation of lentiviral expression constructs, the regulatable pF 5xUAS W SV40 Puro (5xUAS)<sup><xref rid="R32" ref-type="bibr">32</xref></sup> was used. GATA2 WT or mutants were PCR amplified from the above pCMV6 plasmid vectors using the primers (KOZAK-GATA2-F and either GATA2-FLAG-R or GATA2-R) (<xref rid="SD1" ref-type="supplementary-material">Supplementary Table 12</xref>) and Pfu Turbo (Stratagene), excised with <italic>Xba</italic>I and cloned into the unique <italic>Xba</italic>I site of 5xUAS.</p></sec><sec id="S10"><title>Generation of regulatable GATA2 expressing HL-60 cell lines</title><p id="P30">A dual lentiviral vector system was used to generate HL-60 cells expressing GATA2 WT or mutants upon addition of 4-hydroxytamoxifen (4HT) (Sigma). Infectious third generation lentivirus was made by cotransfecting 293T cells with either 5xUAS-GATA2 (WT or mutants) or pF GEV16 Super PGKHygro (GEV16)<sup><xref rid="R33" ref-type="bibr">33</xref></sup> plasmid and the three packaging plasmids pHCMVwhvgagpolml, pHCMV-G and pHCMVwhvrevml<sup><xref rid="R34" ref-type="bibr">34</xref></sup> (mass ratio 50:5:2.5:1). Supernatants were harvested 24 h later and filtered (Nalgene 45 μm syringe filter) (Nalge Nunc Int.). HL-60 cells were firstly transduced with GEV16 lentiviral supernatant including 4 μg/ml polybrene and 2.5 μg/ml fungizone. After 48 h, HL-60GEV cells were selected in 1 mg/ml hygromycin (Roche). These cells were subsequently transduced with the GATA2 (WT, p.Thr354Met, p.Thr355del and p.Leu359Val) or EV lentiviral supernatant and selected in 3 μM Puromycin (Sigma).</p></sec><sec id="S11"><title>Immunofluorescence staining</title><p id="P31">HL-60 cells carrying stably transduced 4HT-regulatable GATA2 (WT, p.Thr354Met, p.Thr355del and p.Leu359Val) were treated with and without 100nM 4HT. After 24 h, the cells were fixed with 4% of paraformaldehyde for 10 min. The cells were permeabilized with 0.1% Triton/PBS, for 10 min and blocked with 2% BSA for 30 min. The cells were then stained with rabbit α-GATA2 antibody (Santa Cruz Biotechnology, Inc) (1:1000) for 1 h followed by Alexa 594-conjugated goat anti-rabbit secondary antibody (Molecular Probes) (2 μg/ml) for 20 min. The slides were mounted in Vectashield<sup>®</sup> mounting medium with DAPI (Vector Laboratories, Inc). Cells without primary antibody served as negative controls. All incubations were performed at room temperature.</p></sec><sec id="S12"><title>Western blot analysis</title><p id="P32">HL-60 cells carrying stably transduced 4HT-regulatable GATA2 (WT, p.Thr354Met, p.Thr355del and p.Leu359Val) were treated with and without 100 nM 4HT. After 24 h, the cells were harvested in RIPA buffer (50 mM Tris-Cl pH 7.6, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate with protease inhibitor (cOmplete Mini EDTA free protease inhibitor tablets, Roche Diagnostics)). Samples were loaded onto the 10% acrylamide gels, electrophoresed and transferred onto Hybond-P PVDF membranes (Amersham). Membranes were probed with antibodies using standard techniques and visualised using ECL plus detection reagents (Amersham) on x-ray film (Amersham Hyperfilm<sup>™</sup> MP).</p></sec><sec id="S13"><title>Apoptosis Assays</title><p id="P33">HL-60 cells were stained for surface Annexin V and propidium iodide according to the manufacturer’s protocol (#556547, Becton Dickinson).</p></sec><sec id="S14"><title>Electromobility shift assay (EMSA) and EMSA-Western Blot</title><p id="P34">HEK293 cells were transfected with GATA2 WT or mutants using Lipofectamine<sup>™</sup> 2000. After 24 h, nuclear extracts were prepared using a NE-PER<sup>®</sup> Nuclear and Cytoplasmic Extraction kit (Pierce) according to the manufacturer’s protocol. Double stranded DNA oligonucleotides containing two GATA binding sites (Human TCRδ enhancer) or a single GATA binding site (GATA Consensus and Human GM-CSF-153 promoter) were synthesized (<xref rid="SD1" ref-type="supplementary-material">Supplementary Table 13</xref>). Each single stranded oligomer was labeled using a Biotin 3′ End DNA Labeling kit (Pierce) and annealed according to manufacturer’s protocol. Electrophoretic mobility shift assays were performed using a modified protocol from Kumar <italic>et al</italic> 2008<sup><xref rid="R35" ref-type="bibr">35</xref></sup> and visualized using a Chemiluminescent Nucleic Acid Detection Module (Pierce) according to the manufacturer’s protocol. Double stranded labeled probes (100 fmol) were incubated with 3 μg of nuclear extract for 20 min in 1x binding buffer containing 20 mM HEPES-KOH, pH 7.9, 100 mM KCl, 2 mM MgCl<sub>2</sub>, 10 μM ZnSO<sub>4</sub>, 10 mM 2 mercaptoethanol, 0.1% NP-40, 10% glycerol, 0.2 mM EDTA and 5 μg/ml sheared salmon sperm DNA. Polyclonal rabbit α-GATA2 (H-116) antibody (Cat. No. sc-9008; Santa Cruz Biotechnology, Inc) (1:100) was added to nuclear lysates for 20 min prior to addition of probe to demonstrate GATA2 as the binding protein. To assess the specificity of the binding, 200-fold excess of each unlabeled probe was used as competitor. The mixtures were resolved in 6% non-denaturing polyacrylamide gels made in 0.5x TGE buffer (12.5 mM Tris-HCl, pH 8.5, 85 mM glycine and 0.5 mM EDTA) and the electrophoresis was performed at 4°C. For EMSA-Western blots, the experiment was carried as described above, except that the shifted DNA oligonucleotides-protein complexes were transferred onto nitrocellulose membrane, instead of PVDF. The membrane was probed with monoclonal mouse α-GATA2 (CG2-96) antibody (Cat. No. sc-267; Santa Cruz Biotechnology, Inc) and detection was performed as mentioned above.</p></sec><sec id="S15"><title>Determination of genes differentially expressed in the presence of GATA2 mutants</title><p id="P35">HL-60 cell lines were treated with 100 nM 4HT to turn on GATA2 WT and mutant protein expression. After 24 h, gene expression levels were determined by microarray(<xref rid="SD1" ref-type="supplementary-material">Supplementary Note</xref>).</p></sec></sec><sec sec-type="supplementary-material" id="S16"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="SD1"><label>1</label><media xlink:href="NIHMS315105-supplement-1.pdf" mimetype="application" mime-subtype="pdf" orientation="portrait" xlink:type="simple" id="d37e1104" position="anchor"></media></supplementary-material></sec></body><back><ack id="S17"><p>The authors would like to thank the families for their participation in our studies. Thanks also to Junia Melo for provision of CML cell lines and Raman Sharma for help with EMSA. Large scale Sanger sequencing was performed by the Australian Genome Research Facility, which was established through the Commonwealth-funded Major National Research Facilities program. This work was supported by grants from the National Health and Medical Research Council of Australia (program grants 257501 (HSS) and 219176 (HSS), fellowships 171601 (HSS) and 461204 (HSS), and a Dora Lush Postgraduate Award (CLC), Leukaemia Foundation of Australia (Grant in Aid to HSS, Postdoctoral fellowship to CLC), the Cancer Council of South Australia (HSS), and MedVet Pty Ltd (HSS and RDA) and NIH grant R01DK058161 (MSH).</p></ack><fn-group><fn id="FN6"><p><bold>URLs.</bold> dbSNP132, <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/SNP/">http://www.ncbi.nlm.nih.gov/SNP/</ext-link>; 1000 Genomes Project, <ext-link ext-link-type="uri" xlink:href="http://www.1000genomes.org/">http://www.1000genomes.org/</ext-link>; PolyPhen-2, <ext-link ext-link-type="uri" xlink:href="http://genetics.bwh.harvard.edu/pph2/">http://genetics.bwh.harvard.edu/pph2/</ext-link>.</p></fn><fn id="FN7"><p><bold>Accession numbers.</bold> Affymetrix expression data are available from Gene Expression Omnibus under accession GSE29276. Human <italic>GATA2</italic> cDNA sequence is available from Genbank under accession number NM_032638.4.</p></fn><fn id="FN8" fn-type="con"><p><bold>AUTHOR CONTRIBUTIONS</bold></p><p>C.N.H., R.J.D., M.S.H. and H.S.S. managed the project. C.N.H., C.L.C., E.J.W., C-E.C., P.J.B., X-C.L., M.S., M.L., A.C., Y.K.L., C.M.B., K.L.F. and A.E.T. performed the experiments. C.H.K. and R.J.D. performed structural modeling and C.N.H., C.H.K. and L.G. performed data analysis. L.B.T., M.A., J.S., P.G.B., G.K.S., R.J.D., M.S.H. and H.S.S. collected families with MDS/AML, and provided clinical data and samples. R.E. and P.G.E. participated in experimental design and provided critical reagents. A.L.B., I.D.L., S.M., L.B.T. provided sporadic AML samples and correlative clinical data. C.N.H., R.J.D., M.S.H. and H.S.S. wrote the manuscript.</p></fn><fn id="FN9" fn-type="conflict"><p><bold>COMPETING FINANCIAL INTERESTS</bold></p><p>The authors declare no competing financial interests.</p></fn><fn id="FN10"><p>While this manuscript was under consideration, Hsu et al have identified germline <italic>GATA2</italic> mutations, including p.Thr354Met and other ZF2 mutations in “MonoMAC syndrome”, an autosomal dominant syndrome associated with myelodysplasia and myeloid leukemias as well as monocytopenia, B and NK cell lymphopenia and mycobacterial, fungal and viral infections<sup><xref rid="R28" ref-type="bibr">28</xref></sup>. 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One family from Australia (Pedigree 1) and two from the USA (Pedigrees 2 and 3) display the p.Thr354Met variant segregating with MDS-AML, and one USA family (Pedigree 4) contains a p.Thr355del variant that segregates with MDS. The genotype of tested individuals is shown; T354, (Thr354/Thr354); T354M, (Thr354/Met354). <bold>b.</bold> Domain structure of GATA2 showing positions of mutations. The positions of the p.Thr354Met, p.Thr355del, AML-M5<sup><xref rid="R7" ref-type="bibr">7</xref></sup> (green) and CML BC<sup><xref rid="R6" ref-type="bibr">6</xref></sup> (black) mutations are shown with respect to zinc finger (ZF) 1 and 2, transactivation domain (TA) and nuclear localization signal (NLS). <bold>c.</bold> Zinc finger 2 (ZF2) domain of GATA2 and GATA3 contains mutations associated with leukemia and breast cancer. The primary sequence is that of human GATA2 with the two alternative residues in GATA3 ZF2 shown (light grey with black letters). The position of p.Thr354Met and p.Thr355del is highlighted along with mutations found in GATA2 in AML-M5<sup><xref rid="R7" ref-type="bibr">7</xref></sup> (green) and CML BC<sup><xref rid="R6" ref-type="bibr">6</xref></sup> (black), and in GATA3 in breast cancer (summarized in <sup><xref rid="R8" ref-type="bibr">8</xref></sup>) (mutated residues in the corresponding GATA3 ZF2; grey with white letters).</p></caption><graphic xlink:href="nihms315105f1"></graphic></fig><fig id="F2" orientation="portrait" position="float"><label>Figure 2</label><caption><title>Subcellular localisation and DNA binding properties of GATA2 WT and mutants</title><p>HEK293 cells were transiently transfected with EV (pCMV-XL6 empty vector), WT, p.Thr354Met, p.Thr355del or p.Leu359Val and harvested after 24 h. <bold>a.</bold> Western blot analysis of GATA2 expression in nuclear lysates. Nuclear lysates were prepared and western blots performed, probing for GATA2. <bold>b.</bold> Cells were stained for GATA2 (red) and DAPI (blue). Scale bars, 10 μm. <bold>c.</bold> Electromobility shift assay (EMSA) of GATA2 WT and mutants. Nuclear lysates were prepared and bound to the TCRδ enhancer (contains GATA binding site) oligonucleotide in the absence or presence of 200-fold unlabeled competitor oligonucleotide (<bold>D</bold>, human TCRδ enhancer; <bold>C</bold>, GATA consensus; <bold>G</bold>, GM-CSF promoter). The probes were visualised using chemiluminescence (top panel). Note, GATA2 & NS relates to a band that contains both GATA2 and a non-specific (NS) protein. To visualise GATA2 alone, an EMSA-western blot was performed probing with polyclonal α-GATA2 antibody (bottom panel), showing the level of binding of GATA2 WT and mutants. A neutralizing α-GATA2 antibody in the far right lane removes GATA2, but not the non-specific binding protein (NS) (top panel), and the specificity of GATA2 is confirmed in the bottom panel.</p></caption><graphic xlink:href="nihms315105f2"></graphic></fig><fig id="F3" orientation="portrait" position="float"><label>Figure 3</label><caption><title>p.Thr354Met and p.Thr355del cause altered transactivation via target GATA2 response elements</title><p>p.Thr354Met and p.Thr355del act as a loss-of-function mutations on GATA2 target promoter and enhancer elements. HEK293 cells were cotransfected with 1) GATA2-responsive <italic>CD34</italic> (mut – <italic>CD34</italic> enhancer with GATA binding sites mutated<sup><xref rid="R31" ref-type="bibr">31</xref></sup>) (<bold>a</bold>) and <italic>RUNX1</italic> (<bold>b</bold>) enhancer elements linked to a LUC reporter, and 2) GATA2 (WT, p.Thr354Met, p.Thr355del or p.Leu359Val) expression constructs or pCMV6-XL6 empty vector (EV). Similarly, Cos-7 cells were cotransfected using <italic>LYL1</italic> promoter LUC as reporter (<bold>c</bold>). After 20 h, cells were harvested and luciferase assays performed and plotted as fold (mean ± s.e.m.) compared to EV control. Pairwise comparisons are shown (*p<0.05, n = 3). <bold>d.</bold> p.Thr354Met and p.Thr355del act as dominant negative mutations over WT GATA2. HEK293 cells were cotransfected with: 1) <italic>CD34</italic> enhancer-LUC reporter, and equivalent mole ratios of 2) WT to 3) p.Thr354Met or p.Thr355del. After 20 h, cells were harvested and luciferase assays performed. Pairwise comparisons are shown (*p<0.05; NS -not significant, n=3). <bold>e.</bold> p.Thr354Met has reduced ability to co-activate the <italic>CSF1R</italic> (<italic>M-CSF-R</italic>) promoter with PU.1. Cos-7 cells were cotransfected with 1) <italic>CSF1R</italic> promoter-LUC reporter, 2) PU.1 expression construct, and 3) WT, p.Thr354Met, p.Thr355del or p.Leu359Val expression constructs or EV. After 20 h, luciferase assays were performed and plotted as fold compared to EV. Pairwise comparisons are shown (*p<0.05, compared to WT plus PU.1; **p<0.05 compared to WT plus PU.1, but not significant when compared to p.Thr354Met or p.Thr355del plus PU.1, respectively). In all comparisons, a Student’s t-test was used.</p></caption><graphic xlink:href="nihms315105f3"></graphic></fig><fig id="F4" orientation="portrait" position="float"><label>Figure 4</label><caption><title>p.Thr354Met inhibits differentiation and apoptosis while allowing accumulation of cells in the presence of ATRA-induced differentiation</title><p>HL-60 cells carrying stably transduced 4HT-regulatable GATA2 (WT, p.Thr354Met, p.Thr355del and p.Leu359Val) or EV were treated with or without 30 nM 4HT for 24 h and then with or without 2 μM ATRA for 6 days. <bold>a–d.</bold> Differentiation of HL-60 cells into granulocytes. Differentiation was measured by FACS analysis for percentage of CD11b positive cells (mean ± s.e.m.) (see also <xref rid="SD1" ref-type="supplementary-material">Supplementary Fig. 7b</xref>). <bold>e–h.</bold> Cell numbers following differentiation. Cells were counted after 6 days (mean ± s.e.m.). <bold>i–m.</bold> Apoptosis following differentiation with ATRA. Cells were FACS analysed following staining with FITC anti-Annexin V and propidium iodide (PI). Annexin V<sup>+</sup>, PI<sup>−</sup> (black) or Annexin V<sup>+</sup>, PI<sup>+</sup> (white). Indicative FACS plots (<xref rid="SD1" ref-type="supplementary-material">Supplementary Fig. 7c</xref>). <bold>a,e,i.</bold> −4HT, −ATRA; <bold>b,f,j.</bold> +4HT, −ATRA; <bold>c,g,k.</bold> −4HT, +ATRA; <bold>d,h,m.</bold> +4HT, +ATRA.(*p<0.05; **p<0.01, compared to WT). In all comparisons, a Student’s t-test was used.</p></caption><graphic xlink:href="nihms315105f4"></graphic></fig></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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