Scholarly Works - Haematology
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Item Open Access Accepted version Peer-reviewed Real-space refinement in PHENIX for cryo-EM and crystallography.(International Union of Crystallography (IUCr), 2018-06-01) Afonine, Pavel V; Poon, Billy K; Read, Randy J; Sobolev, Oleg V; Terwilliger, Thomas C; Urzhumtsev, Alexandre; Adams, Paul D; Afonine, Pavel V [0000-0002-5052-991X]; Poon, Billy K [0000-0001-9633-6067]; Read, Randy J [0000-0001-8273-0047]This article describes the implementation of real-space refinement in the phenix.real_space_refine program from the PHENIX suite. The use of a simplified refinement target function enables very fast calculation, which in turn makes it possible to identify optimal data-restraint weights as part of routine refinements with little runtime cost. Refinement of atomic models against low-resolution data benefits from the inclusion of as much additional information as is available. In addition to standard restraints on covalent geometry, phenix.real_space_refine makes use of extra information such as secondary-structure and rotamer-specific restraints, as well as restraints or constraints on internal molecular symmetry. The re-refinement of 385 cryo-EM-derived models available in the Protein Data Bank at resolutions of 6 Å or better shows significant improvement of the models and of the fit of these models to the target maps.Item Open Access Published version Peer-reviewed Branched-chain amino acid depletion conditions bone marrow for hematopoietic stem cell transplantation avoiding amino acid imbalance-associated toxicity.(Elsevier BV, 2018-07) Wilkinson, Adam C; Morita, Maiko; Nakauchi, Hiromitsu; Yamazaki, SatoshiHematopoietic stem cells (HSCs) are used clinically in bone marrow (BM) transplantation due to their unique ability to reform the entire hematopoietic system. Recently, we reported that HSCs are highly sensitive to valine, one of the three branched-chain amino acids (BCAAs) in addition to isoleucine and leucine. Dietary depletion of valine could even be used as a conditioning regimen for HSC transplantation. Here, we report that HSCs are highly sensitive to the balance of BCAAs, with both proliferation and survival reduced by BCAA imbalance. However, low but balanced BCAA levels failed to rescue HSC maintenance. Importantly, in vivo depletion of all three BCAAs was significantly less toxic than depletion of valine only. We demonstrate that BCAA depletion can replace valine depletion as a safer alternative to BM conditioning. In summary, by determining HSC metabolic requirements, we can improve metabolic approaches to BM conditioning.Item Open Access Exploiting distant homologues for phasing through the generation of compact fragments, local fold refinement and partial solution combination.(International Union of Crystallography (IUCr), 2018-04-01) Millán, Claudia; Sammito, Massimo Domenico; McCoy, Airlie J; Nascimento, Andrey F Ziem; Petrillo, Giovanna; Oeffner, Robert D; Domínguez-Gil, Teresa; Hermoso, Juan A; Read, Randy J; Usón, Isabel; Millán, Claudia [0000-0002-9283-2220]; Oeffner, Robert D [0000-0003-3107-2202]; Read, Randy J [0000-0001-8273-0047]Macromolecular structures can be solved by molecular replacement provided that suitable search models are available. Models from distant homologues may deviate too much from the target structure to succeed, notwithstanding an overall similar fold or even their featuring areas of very close geometry. Successful methods to make the most of such templates usually rely on the degree of conservation to select and improve search models. ARCIMBOLDO_SHREDDER uses fragments derived from distant homologues in a brute-force approach driven by the experimental data, instead of by sequence similarity. The new algorithms implemented in ARCIMBOLDO_SHREDDER are described in detail, illustrating its characteristic aspects in the solution of new and test structures. In an advance from the previously published algorithm, which was based on omitting or extracting contiguous polypeptide spans, model generation now uses three-dimensional volumes respecting structural units. The optimal fragment size is estimated from the expected log-likelihood gain (LLG) values computed assuming that a substructure can be found with a level of accuracy near that required for successful extension of the structure, typically below 0.6 Å root-mean-square deviation (r.m.s.d.) from the target. Better sampling is attempted through model trimming or decomposition into rigid groups and optimization through Phaser's gyre refinement. Also, after model translation, packing filtering and refinement, models are either disassembled into predetermined rigid groups and refined (gimble refinement) or Phaser's LLG-guided pruning is used to trim the model of residues that are not contributing signal to the LLG at the target r.m.s.d. value. Phase combination among consistent partial solutions is performed in reciprocal space with ALIXE. Finally, density modification and main-chain autotracing in SHELXE serve to expand to the full structure and identify successful solutions. The performance on test data and the solution of new structures are described.Item Open Access Published version Peer-reviewed Critical Modulation of Hematopoietic Lineage Fate by Hepatic Leukemia Factor.(Elsevier BV, 2017-11-21) Wahlestedt, Martin; Ladopoulos, Vasileios; Hidalgo, Isabel; Sanchez Castillo, Manuel; Hannah, Rebecca; Säwén, Petter; Wan, Haixia; Dudenhöffer-Pfeifer, Monika; Magnusson, Mattias; Norddahl, Gudmundur L; Göttgens, Berthold; Bryder, David; Hannah, Rebecca [0000-0003-0477-7792]; Gottgens, Berthold [0000-0001-6302-5705]A gradual restriction in lineage potential of multipotent stem/progenitor cells is a hallmark of adult hematopoiesis, but the underlying molecular events governing these processes remain incompletely understood. Here, we identified robust expression of the leukemia-associated transcription factor hepatic leukemia factor (Hlf) in normal multipotent hematopoietic progenitors, which was rapidly downregulated upon differentiation. Interference with its normal downregulation revealed Hlf as a strong negative regulator of lymphoid development, while remaining compatible with myeloid fates. Reciprocally, we observed rapid lymphoid commitment upon reduced Hlf activity. The arising phenotypes resulted from Hlf binding to active enhancers of myeloid-competent cells, transcriptional induction of myeloid, and ablation of lymphoid gene programs, with Hlf induction of nuclear factor I C (Nfic) as a functionally relevant target gene. Thereby, our studies establish Hlf as a key regulator of the earliest lineage-commitment events at the transition from multipotency to lineage-restricted progeny, with implications for both normal and malignant hematopoiesis.Item Open Access Published version Peer-reviewed Single-cell RNA-sequencing uncovers transcriptional states and fate decisions in haematopoiesis.(Springer Science and Business Media LLC, 2017-12-11) Athanasiadis, Emmanouil I; Botthof, Jan G; Andres, Helena; Ferreira, Lauren; Lio, Pietro; Cvejic, Ana; Athanasiadis, Emmanouil I [0000-0002-2771-5562]The success of marker-based approaches for dissecting haematopoiesis in mouse and human is reliant on the presence of well-defined cell surface markers specific for diverse progenitor populations. An inherent problem with this approach is that the presence of specific cell surface markers does not directly reflect the transcriptional state of a cell. Here, we used a marker-free approach to computationally reconstruct the blood lineage tree in zebrafish and order cells along their differentiation trajectory, based on their global transcriptional differences. Within the population of transcriptionally similar stem and progenitor cells, our analysis reveals considerable cell-to-cell differences in their probability to transition to another committed state. Once fate decision is executed, the suppression of transcription of ribosomal genes and upregulation of lineage-specific factors coordinately controls lineage differentiation. Evolutionary analysis further demonstrates that this haematopoietic programme is highly conserved between zebrafish and higher vertebrates.Item Open Access Published version Peer-reviewed A genome-wide association search for type 2 diabetes genes in African Americans.(Public Library of Science (PLoS), 2012) Palmer, Nicholette D; McDonough, Caitrin W; Hicks, Pamela J; Roh, Bong H; Wing, Maria R; An, S Sandy; Hester, Jessica M; Cooke, Jessica N; Bostrom, Meredith A; Rudock, Megan E; Talbert, Matthew E; Lewis, Joshua P; DIAGRAM Consortium; MAGIC Investigators; Ferrara, Assiamira; Lu, Lingyi; Ziegler, Julie T; Sale, Michele M; Divers, Jasmin; Shriner, Daniel; Adeyemo, Adebowale; Rotimi, Charles N; Ng, Maggie CY; Langefeld, Carl D; Freedman, Barry I; Bowden, Donald W; Voight, Benjamin F; Scott, Laura J; Steinthorsdottir, Valgerdur; Morris, Andrew P; Dina, Christian; Welch, Ryan P; Zeggini, Eleftheria; Huth, Cornelia; Aulchenko, Yurii S; Thorleifsson, Gudmar; McCulloch, Laura J; Ferreira, Teresa; Grallert, Harald; Amin, Najaf; Wu, Guanming; Willer, Cristen J; Raychaudhuri, Soumya; McCarroll, Steve A; Langenberg, Claudia; Hofmann, Oliver M; Dupuis, Josée; Qi, Lu; Segrè, Ayellet V; van Hoek, Mandy; Navarro, Pau; Ardlie, Kristin; Balkau, Beverley; Benediktsson, Rafn; Bennett, Amanda J; Blagieva, Roza; Boerwinkle, Eric; Bonnycastle, Lori L; Boström, Kristina Bengtsson; Bravenboer, Bert; Bumpstead, Suzannah; Burtt, Noël P; Charpentier, Guillaume; Chines, Peter S; Cornelis, Marilyn; Couper, David J; Crawford, Gabe; Doney, Alex SF; Elliott, Katherine S; Elliott, Amanda L; Erdos, Michael R; Fox, Caroline S; Franklin, Christopher S; Ganser, Martha; Gieger, Christian; Grarup, Niels; Green, Todd; Griffin, Simon; Groves, Christopher J; Guiducci, Candace; Hadjadj, Samy; Hassanali, Neelam; Herder, Christian; Isomaa, Bo; Jackson, Anne U; Johnson, Paul RV; Jørgensen, Torben; Kao, Wen HL; Klopp, Norman; Kong, Augustine; Kraft, Peter; Kuusisto, Johanna; Lauritzen, Torsten; Li, Man; Lieverse, Aloysius; Lindgren, Cecilia M; Lyssenko, Valeriya; Marre, Michel; Meitinger, Thomas; Midthjell, Kristian; Morken, Mario A; Narisu, Narisu; Nilsson, Peter; Owen, Katharine R; Payne, Felicity; Perry, John RB; Petersen, Ann-Kristin; Platou, Carl; Proença, Christine; Prokopenko, Inga; Rathmann, Wolfgang; Rayner, N William; Robertson, Neil R; Rocheleau, Ghislain; Roden, Michael; Sampson, Michael J; Saxena, Richa; Shields, Beverley M; Shrader, Peter; Sigurdsson, Gunnar; Sparsø, Thomas; Strassburger, Klaus; Stringham, Heather M; Sun, Qi; Swift, Amy J; Thorand, Barbara; Tichet, Jean; Tuomi, Tiinamaija; van Dam, Rob M; van Haeften, Timon W; van Herpt, Thijs; van Vliet-Ostaptchouk, Jana V; Walters, G Bragi; Weedon, Michael N; Wijmenga, Cisca; Witteman, Jacqueline; Bergman, Richard N; Cauchi, Stephane; Collins, Francis S; Gloyn, Anna L; Gyllensten, Ulf; Hansen, Torben; Hide, Winston A; Hitman, Graham A; Hofman, Albert; Hunter, David J; Hveem, Kristian; Laakso, Markku; Mohlke, Karen L; Morris, Andrew D; Palmer, Colin NA; Pramstaller, Peter P; Rudan, Igor; Sijbrands, Eric; Stein, Lincoln D; Tuomilehto, Jaakko; Uitterlinden, Andre; Walker, Mark; Wareham, Nicholas J; Watanabe, Richard M; Abecasis, Goncalo R; Boehm, Bernhard O; Campbell, Harry; Daly, Mark J; Hattersley, Andrew T; Hu, Frank B; Meigs, James B; Pankow, James S; Pedersen, Oluf; Wichmann, H-Erich; Barroso, Inês; Florez, Jose C; Frayling, Timothy M; Groop, Leif; Sladek, Rob; Thorsteinsdottir, Unnur; Wilson, James F; Illig, Thomas; Froguel, Philippe; van Duijn, Cornelia M; Stefansson, Kari; Altshuler, David; Boehnke, Michael; McCarthy, Mark I; Soranzo, Nicole; Wheeler, Eleanor; Glazer, Nicole L; Bouatia-Naji, Nabila; Mägi, Reedik; Randall, Joshua; Johnson, Toby; Elliott, Paul; Rybin, Denis; Henneman, Peter; Dehghan, Abbas; Hottenga, Jouke Jan; Song, Kijoung; Goel, Anuj; Egan, Josephine M; Lajunen, Taina; Doney, Alex; Kanoni, Stavroula; Cavalcanti-Proença, Christine; Kumari, Meena; Timpson, Nicholas J; Zabena, Carina; Ingelsson, Erik; An, Ping; O'Connell, Jeffrey; Luan, Jian'an; Elliott, Amanda; McCarroll, Steven A; Roccasecca, Rosa Maria; Pattou, François; Sethupathy, Praveen; Ariyurek, Yavuz; Barter, Philip; Beilby, John P; Ben-Shlomo, Yoav; Bergmann, Sven; Bochud, Murielle; Bonnefond, Amélie; Borch-Johnsen, Knut; Böttcher, Yvonne; Brunner, Eric; Bumpstead, Suzannah J; Chen, Yii-Der Ida; Chines, Peter; Clarke, Robert; Coin, Lachlan JM; Cooper, Matthew N; Crisponi, Laura; Day, Ian NM; de Geus, Eco JC; Delplanque, Jerome; Fedson, Annette C; Fischer-Rosinsky, Antje; Forouhi, Nita G; Frants, Rune; Franzosi, Maria Grazia; Galan, Pilar; Goodarzi, Mark O; Graessler, Jürgen; Grundy, Scott; Gwilliam, Rhian; Hallmans, Göran; Hammond, Naomi; Han, Xijing; Hartikainen, Anna-Liisa; Hayward, Caroline; Heath, Simon C; Hercberg, Serge; Hicks, Andrew A; Hillman, David R; Hingorani, Aroon D; Hui, Jennie; Hung, Joe; Jula, Antti; Kaakinen, Marika; Kaprio, Jaakko; Kesaniemi, Y Antero; Kivimaki, Mika; Knight, Beatrice; Koskinen, Seppo; Kovacs, Peter; Kyvik, Kirsten Ohm; Lathrop, G Mark; Lawlor, Debbie A; Le Bacquer, Olivier; Lecoeur, Cécile; Li, Yun; Mahley, Robert; Mangino, Massimo; Manning, Alisa K; Martínez-Larrad, María Teresa; McAteer, Jarred B; McPherson, Ruth; Meisinger, Christa; Melzer, David; Meyre, David; Mitchell, Braxton D; Mukherjee, Sutapa; Naitza, Silvia; Neville, Matthew J; Oostra, Ben A; Orrù, Marco; Pakyz, Ruth; Paolisso, Giuseppe; Pattaro, Cristian; Pearson, Daniel; Peden, John F; Pedersen, Nancy L; Perola, Markus; Pfeiffer, Andreas FH; Pichler, Irene; Polasek, Ozren; Posthuma, Danielle; Potter, Simon C; Pouta, Anneli; Province, Michael A; Psaty, Bruce M; Rayner, Nigel W; Rice, Kenneth; Ripatti, Samuli; Rivadeneira, Fernando; Rolandsson, Olov; Sandbaek, Annelli; Sandhu, Manjinder; Sanna, Serena; Sayer, Avan Aihie; Scheet, Paul; Seedorf, Udo; Sharp, Stephen J; Shields, Beverley; Sijbrands, Eric JG; Silveira, Angela; Simpson, Laila; Singleton, Andrew; Smith, Nicholas L; Sovio, Ulla; Swift, Amy; Syddall, Holly; Syvänen, Ann-Christine; Tanaka, Toshiko; Tönjes, Anke; Uitterlinden, André G; van Dijk, Ko Willems; Varma, Dhiraj; Visvikis-Siest, Sophie; Vitart, Veronique; Vogelzangs, Nicole; Waeber, Gérard; Wagner, Peter J; Walley, Andrew; Ward, Kim L; Watkins, Hugh; Wild, Sarah H; Willemsen, Gonneke; Witteman, Jaqueline CM; Yarnell, John WG; Zelenika, Diana; Zethelius, Björn; Zhai, Guangju; Zhao, Jing Hua; Zillikens, M Carola; Borecki, Ingrid B; Loos, Ruth JF; Meneton, Pierre; Magnusson, Patrik KE; Nathan, David M; Williams, Gordon H; Silander, Kaisa; Salomaa, Veikko; Smith, George Davey; Bornstein, Stefan R; Schwarz, Peter; Spranger, Joachim; Karpe, Fredrik; Shuldiner, Alan R; Cooper, Cyrus; Dedoussis, George V; Serrano-Ríos, Manuel; Lind, Lars; Palmer, Lyle J; Franks, Paul W; Ebrahim, Shah; Marmot, Michael; Kao, WH Linda; Pramstaller, Peter Paul; Wright, Alan F; Stumvoll, Michael; Hamsten, Anders; Buchanan, Thomas A; Valle, Timo T; Rotter, Jerome I; Siscovick, David S; Penninx, Brenda WJH; Boomsma, Dorret I; Deloukas, Panos; Spector, Timothy D; Ferrucci, Luigi; Cao, Antonio; Scuteri, Angelo; Schlessinger, David; Uda, Manuela; Ruokonen, Aimo; Jarvelin, Marjo-Riitta; Waterworth, Dawn M; Vollenweider, Peter; Peltonen, Leena; Mooser, Vincent; Sladek, Robert; Langenberg, Claudia [0000-0002-5017-7344]; Griffin, Simon [0000-0002-2157-4797]; Wareham, Nicholas [0000-0003-1422-2993]; Soranzo, Nicole [0000-0003-1095-3852]; Wheeler, Eleanor [0000-0002-8616-6444]; Luan, Jian'an [0000-0003-3137-6337]; Forouhi, Nita [0000-0002-5041-248X]; Sharp, Stephen [0000-0003-2375-1440]; Sovio, Ulla [0000-0002-0799-1105]African Americans are disproportionately affected by type 2 diabetes (T2DM) yet few studies have examined T2DM using genome-wide association approaches in this ethnicity. The aim of this study was to identify genes associated with T2DM in the African American population. We performed a Genome Wide Association Study (GWAS) using the Affymetrix 6.0 array in 965 African-American cases with T2DM and end-stage renal disease (T2DM-ESRD) and 1029 population-based controls. The most significant SNPs (n = 550 independent loci) were genotyped in a replication cohort and 122 SNPs (n = 98 independent loci) were further tested through genotyping three additional validation cohorts followed by meta-analysis in all five cohorts totaling 3,132 cases and 3,317 controls. Twelve SNPs had evidence of association in the GWAS (P<0.0071), were directionally consistent in the Replication cohort and were associated with T2DM in subjects without nephropathy (P<0.05). Meta-analysis in all cases and controls revealed a single SNP reaching genome-wide significance (P<2.5×10(-8)). SNP rs7560163 (P = 7.0×10(-9), OR (95% CI) = 0.75 (0.67-0.84)) is located intergenically between RND3 and RBM43. Four additional loci (rs7542900, rs4659485, rs2722769 and rs7107217) were associated with T2DM (P<0.05) and reached more nominal levels of significance (P<2.5×10(-5)) in the overall analysis and may represent novel loci that contribute to T2DM. We have identified novel T2DM-susceptibility variants in the African-American population. Notably, T2DM risk was associated with the major allele and implies an interesting genetic architecture in this population. These results suggest that multiple loci underlie T2DM susceptibility in the African-American population and that these loci are distinct from those identified in other ethnic populations.Item Open Access Accepted version Peer-reviewed Identification of an imprinted master trans regulator at the KLF14 locus related to multiple metabolic phenotypes.(Springer Science and Business Media LLC, 2011-06) Small, Kerrin S; Hedman, Asa K; Grundberg, Elin; Nica, Alexandra C; Thorleifsson, Gudmar; Kong, Augustine; Thorsteindottir, Unnur; Shin, So-Youn; Richards, Hannah B; GIANT Consortium; MAGIC Investigators; DIAGRAM Consortium; Soranzo, Nicole; Ahmadi, Kourosh R; Lindgren, Cecilia M; Stefansson, Kari; Dermitzakis, Emmanouil T; Deloukas, Panos; Spector, Timothy D; McCarthy, Mark I; MuTHER Consortium; Soranzo, Nicole [0000-0003-1095-3852]Genome-wide association studies have identified many genetic variants associated with complex traits. However, at only a minority of loci have the molecular mechanisms mediating these associations been characterized. In parallel, whereas cis regulatory patterns of gene expression have been extensively explored, the identification of trans regulatory effects in humans has attracted less attention. Here we show that the type 2 diabetes and high-density lipoprotein cholesterol-associated cis-acting expression quantitative trait locus (eQTL) of the maternally expressed transcription factor KLF14 acts as a master trans regulator of adipose gene expression. Expression levels of genes regulated by this trans-eQTL are highly correlated with concurrently measured metabolic traits, and a subset of the trans-regulated genes harbor variants directly associated with metabolic phenotypes. This trans-eQTL network provides a mechanistic understanding of the effect of the KLF14 locus on metabolic disease risk and offers a potential model for other complex traits.Item Open Access Published version Peer-reviewed On the application of the expected LLG to decision making in molecular replacementMcCoy, Airlie; Read, Randy; Read, Randy [0000-0001-8273-0047]olecular replacement phasing of macromolecular crystal structures is often fast, but if a molecular replacement solution is not immediately obtained, the crystallographer must judge whether to pursue molecular replacement or attempt experimental phasing as the quickest path to structure solution. The introduction of eLLG (expected log-likelihood gain (McCoy et al., 2017)) has given the crystallographer a powerful new tool to aid in making this decision. The eLLG is LLGI (log-likelihood gain on intensity (Read & McCoy, 2016)) expected from a correctly placed model. It is calculated as a sum over the reflections of a function dependent on the fraction of the scattering for which the model accounts, the estimated model coordinate error, and the measurement errors in the data. We show how eLLG is used to answer the question "Can I solve my structure by molecular replacement?" However, this is only the most obvious of the applications of eLLG. We also discuss how eLLG is used for determining search order, minimal data requirements for obtaining a molecular replacement solution using a given model, and for decision making in fragment-based molecular replacement, single-atom molecular replacement, and likelihood-guided model pruning.Item Open Access Accepted version Peer-reviewed Abnormal differentiation of B cells and megakaryocytes in patients with Roifman syndrome.(Elsevier BV, 2018-08) Heremans, Jessica; Garcia-Perez, Josselyn E; Turro, Ernest; Schlenner, Susan M; Casteels, Ingele; Collin, Roxanne; de Zegher, Francis; Greene, Daniel; Humblet-Baron, Stephanie; Lesage, Sylvie; Matthys, Patrick; Penkett, Christopher J; Put, Karen; Stirrups, Kathleen; National Institute for Health Research BioResource; Thys, Chantal; Van Geet, Chris; Van Nieuwenhove, Erika; Wouters, Carine; Meyts, Isabelle; Freson, Kathleen; Liston, Adrian; Turro Bassols, Ernest [0000-0002-1820-6563]; Stirrups, Kathleen [0000-0002-6823-3252]; Liston, Adrian [0000-0002-6272-4085]BACKGROUND: Roifman syndrome is a rare inherited disorder characterized by spondyloepiphyseal dysplasia, growth retardation, cognitive delay, hypogammaglobulinemia, and, in some patients, thrombocytopenia. Compound heterozygous variants in the small nuclear RNA gene RNU4ATAC, which is necessary for U12-type intron splicing, were identified recently as driving Roifman syndrome. OBJECTIVE: We studied 3 patients from 2 unrelated kindreds harboring compound heterozygous or homozygous stem II variants in RNU4ATAC to gain insight into the mechanisms behind this disorder. METHODS: We systematically profiled the immunologic and hematologic compartments of the 3 patients with Roifman syndrome and performed RNA sequencing to unravel important splicing defects in both cell lineages. RESULTS: The patients exhibited a dramatic reduction in B-cell numbers, with differentiation halted at the transitional B-cell stage. Despite abundant B-cell activating factor availability, development past this B-cell activating factor-dependent stage was crippled, with disturbed minor splicing of the critical mitogen-activated protein kinase 1 signaling component. In the hematologic compartment patients with Roifman syndrome demonstrated defects in megakaryocyte differentiation, with inadequate generation of proplatelets. Platelets from patients with Roifman syndrome were rounder, with increased tubulin and actin levels, and contained increased α-granule and dense granule markers. Significant minor intron retention in 354 megakaryocyte genes was observed, including DIAPH1 and HPS1, genes known to regulate platelet and dense granule formation, respectively. CONCLUSION: Together, our results provide novel molecular and cellular data toward understanding the immunologic and hematologic features of Roifman syndrome.Item Open Access Accepted version Peer-reviewed Ruxolitinib-induced defects in DNA repair cause sensitivity to PARP inhibitors in myeloproliferative neoplasms.(American Society of Hematology, 2017-12-28) Nieborowska-Skorska, Margaret; Maifrede, Silvia; Dasgupta, Yashodhara; Sullivan, Katherine; Flis, Sylwia; Le, Bac Viet; Solecka, Martyna; Belyaeva, Elizaveta A; Kubovcakova, Lucia; Nawrocki, Morgan; Kirschner, Martin; Zhao, Huaqing; Prchal, Josef T; Piwocka, Katarzyna; Moliterno, Alison R; Wasik, Mariusz; Koschmieder, Steffen; Green, Tony R; Skoda, Radek C; Skorski, Tomasz; Green, Tony [0000-0002-9795-0218]Myeloproliferative neoplasms (MPNs) often carry JAK2(V617F), MPL(W515L), or CALR(del52) mutations. Current treatment options for MPNs include cytoreduction by hydroxyurea and JAK1/2 inhibition by ruxolitinib, both of which are not curative. We show here that cell lines expressing JAK2(V617F), MPL(W515L), or CALR(del52) accumulated reactive oxygen species-induced DNA double-strand breaks (DSBs) and were modestly sensitive to poly-ADP-ribose polymerase (PARP) inhibitors olaparib and BMN673. At the same time, primary MPN cell samples from individual patients displayed a high degree of variability in sensitivity to these drugs. Ruxolitinib inhibited 2 major DSB repair mechanisms, BRCA-mediated homologous recombination and DNA-dependent protein kinase-mediated nonhomologous end-joining, and, when combined with olaparib, caused abundant accumulation of toxic DSBs resulting in enhanced elimination of MPN primary cells, including the disease-initiating cells from the majority of patients. Moreover, the combination of BMN673, ruxolitinib, and hydroxyurea was highly effective in vivo against JAK2(V617F)+ murine MPN-like disease and also against JAK2(V617F)+, CALR(del52)+, and MPL(W515L)+ primary MPN xenografts. In conclusion, we postulate that ruxolitinib-induced deficiencies in DSB repair pathways sensitized MPN cells to synthetic lethality triggered by PARP inhibitors.Item Open Access Published version Peer-reviewed Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34+ progenitors.(Ferrata Storti Foundation (Haematologica), 2017-02) Poggi, Marjorie; Canault, Matthias; Favier, Marie; Turro, Ernest; Saultier, Paul; Ghalloussi, Dorsaf; Baccini, Veronique; Vidal, Lea; Mezzapesa, Anna; Chelghoum, Nadjim; Mohand-Oumoussa, Badreddine; Falaise, Céline; Favier, Rémi; Ouwehand, Willem H; Fiore, Mathieu; Peiretti, Franck; Morange, Pierre Emmanuel; Saut, Noémie; Bernot, Denis; Greinacher, Andreas; BioResource, Nihr; Nurden, Alan T; Nurden, Paquita; Freson, Kathleen; Trégouët, David-Alexandre; Raslova, Hana; Alessi, Marie-Christine; Turro Bassols, Ernest [0000-0002-1820-6563]; Ouwehand, Willem [0000-0002-7744-1790]Variants in ETV6, which encodes a transcription repressor of the E26 transformation-specific family, have recently been reported to be responsible for inherited thrombocytopenia and hematologic malignancy. We sequenced the DNA from cases with unexplained dominant thrombocytopenia and identified six likely pathogenic variants in ETV6, of which five are novel. We observed low repressive activity of all tested ETV6 variants, and variants located in the E26 transformation-specific binding domain (encoding p.A377T, p.Y401N) led to reduced binding to corepressors. We also observed a large expansion of megakaryocyte colony-forming units derived from variant carriers and reduced proplatelet formation with abnormal cytoskeletal organization. The defect in proplatelet formation was also observed in control CD34+ cell-derived megakaryocytes transduced with lentiviral particles encoding mutant ETV6. Reduced expression levels of key regulators of the actin cytoskeleton CDC42 and RHOA were measured. Moreover, changes in the actin structures are typically accompanied by a rounder platelet shape with a highly heterogeneous size, decreased platelet arachidonic response, and spreading and retarded clot retraction in ETV6 deficient platelets. Elevated numbers of circulating CD34+ cells were found in p.P214L and p.Y401N carriers, and two patients from different families suffered from refractory anemia with excess blasts, while one patient from a third family was successfully treated for acute myeloid leukemia. Overall, our study provides novel insights into the role of ETV6 as a driver of cytoskeletal regulatory gene expression during platelet production, and the impact of variants resulting in platelets with altered size, shape and function and potentially also in changes in circulating progenitor levels.Item Open Access Accepted version Peer-reviewed High-throughput sequencing approaches for diagnosing hereditary bleeding and platelet disorders.(Elsevier BV, 2017-07) Freson, K; Turro, E; Turro, E [0000-0002-1820-6563]Hereditary bleeding and platelet disorders (BPDs) are characterized by marked genetic heterogeneity, far greater than previously appreciated. The list of genes involved in the regulation of megakaryopoiesis, platelet formation, platelet function and bleeding has been growing rapidly since the introduction of high-throughput sequencing (HTS) approaches in research. Thanks to the gradual adoption of HTS in diagnostic practice, these discoveries are improving the diagnostic yield for BPD patients, who may or may not present with bleeding problems and often have other clinical symptoms unrelated to the blood system. However, it was previously found that screening for all known etiologies gives a diagnostic yield of over 90% when the phenotype closely matches a known BPD but drops to 10% when the phenotype is indicative of a novel disorder. Thus, further research is needed to identify currently unknown etiologies for BPDs. Novel genes are likely to be found to be implicated in BPDs. New modes of inheritance, including digenic inheritance, are likely to play a role in some cases. Additionally, identifying and interpreting pathogenic variants outside exons is a looming challenge that can only be tackled with an improved understanding of the regulatory landscape of relevant cell types and with the transition from targeted sequencing to whole-genome sequencing in the clinic.Item Open Access Accepted version Peer-reviewed Expanded repertoire of RASGRP2 variants responsible for platelet dysfunction and severe bleeding.(American Society of Hematology, 2017-08-24) Westbury, Sarah K; Canault, Matthias; Greene, Daniel; Bermejo, Emilse; Hanlon, Katharine; Lambert, Michele P; Millar, Carolyn M; Nurden, Paquita; Obaji, Samya G; Revel-Vilk, Shoshana; Van Geet, Chris; Downes, Kate; Papadia, Sofia; Tuna, Salih; Watt, Christopher; NIHR BioResource–Rare Diseases Consortium; Freson, Kathleen; Laffan, Michael A; Ouwehand, Willem H; Alessi, Marie-Christine; Turro, Ernest; Mumford, Andrew D; Downes, Kate [0000-0003-0366-1579]; Papadia, Sofia [0000-0002-9222-3812]; Tuna, Salih [0000-0003-3606-4367]; Ouwehand, Willem [0000-0002-7744-1790]; Turro Bassols, Ernest [0000-0002-1820-6563]Heritable platelet function disorders (PFDs) are genetically heterogeneous and poorly characterized. Pathogenic variants in RASGRP2, which encodes calcium and diacylglycerol-regulated guanine exchange factor I (CalDAG-GEFI), have been reported previously in 3 pedigrees with bleeding and reduced platelet aggregation responses. To better define the phenotype associated with pathogenic RASGRP2 variants, we compared high-throughput sequencing and phenotype data from 2042 cases in pedigrees with unexplained bleeding or platelet disorders to data from 5422 controls. Eleven cases harbored 11 different, previously unreported RASGRP2 variants that were biallelic and likely pathogenic. The variants included 5 high-impact variants predicted to prevent CalDAG-GEFI expression and 6 missense variants affecting the CalDAG-GEFI CDC25 domain, which mediates Rap1 activation during platelet inside-out αIIbβ3 signaling. Cases with biallelic RASGRP2 variants had abnormal mucocutaneous, surgical, and dental bleeding from childhood, requiring ≥1 blood or platelet transfusion in 78% of cases. Platelets displayed reduced aggregation in response to adenosine 5'-diphosphate and epinephrine, but variable aggregation defects with other agonists. There were no other consistent clinical or laboratory features. These data enable definition of human CalDAG-GEFI deficiency as a nonsyndromic, recessive PFD associated with a moderate or severe bleeding phenotype and complex defects in platelet aggregation.Item Open Access Accepted version Peer-reviewed Altered fibrinolysis in autosomal dominant thrombomodulin-associated coagulopathy.(American Society of Hematology, 2016-10-06) Burley, Kate; Whyte, Claire S; Westbury, Sarah K; Walker, Mary; Stirrups, Kathleen E; Turro, Ernest; NIHR BioResource; Chapman, Oliver G; Reilly-Stitt, Christopher; Mutch, Nicola J; Mumford, Andrew D; Stirrups, Kathleen [0000-0002-6823-3252]; Turro Bassols, Ernest [0000-0002-1820-6563]Thrombomodulin-associated coagulopathy (TM-AC) is a newly recognized dominant bleeding disorder in which a p.Cys537Stop variant in the thrombomodulin (TM) gene THBD, results in high plasma TM levels and protein C-mediated suppression of thrombin generation. Thrombin in complex with TM also activates thrombin-activatable fibrinolysis inhibitor (TAFI). However, the effect of the high plasma TM on fibrinolysis in TM-AC is unknown. Plasma from TM-AC cases and high-TM model control samples spiked with recombinant soluble TM showed reduced tissue factor-induced thrombin generation. Lysis of plasma clots from TM-AC cases was significantly delayed compared with controls but was completely restored when TM/thrombin-mediated TAFI activation was inhibited. Clots formed in blood from TM-AC cases had the same viscoelastic strength as controls but also showed a TAFI-dependent delay in fibrinolysis. Delayed fibrinolysis was reproduced in high-TM model plasma and blood samples. Partial restoration of thrombin generation with recombinant activated factor VII or activated prothrombin complex concentrate did not alter the delayed fibrinolysis in high-TM model blood. Our finding of a previously unrecognized fibrinolytic phenotype indicates that bleeding in TM-AC has a complex pathogenesis and highlights the pivotal role of TM as a regulator of hemostasis.Item Open Access Published version Peer-reviewed PIGO deficiency: palmoplantar keratoderma and novel mutations.(2017-05-25) Morren, Marie-Anne; Jaeken, Jaak; Visser, Gepke; Salles, Isabelle; Van Geet, Chris; NIHR BioResource,; Simeoni, Ilenia; Turro Bassols, Ernest; Freson, Kathleen; Jaeken, Jaak [0000-0003-1571-9707]; Salles, Isabelle [0000-0001-7394-0587]; Simeoni, Ilenia [0000-0001-5039-2194]; Turro, Ernest [0000-0002-1820-6563]; Freson, Kathleen [0000-0002-4381-2442]Item Open Access Published version Peer-reviewed The integrated stress response regulates BMP signalling through effects on translation.(BioMed Central, 2018-04-03) Malzer, Elke; Dominicus, Caia S; Chambers, Joseph; Dickens, Jennifer; Mookerjee, Souradip; Marciniak, Stefan; Chambers, Joseph [0000-0003-4675-0053]; Mookerjee, Souradip [0000-0003-4904-1324]; Marciniak, Stefan [0000-0001-8472-7183]Background: Developmental pathways must be responsive to the environment. Phosphorylation of eIF2α enables a family of stress sensing kinases to trigger the integrated stress response (ISR), which has pro-survival and developmental consequences. Bone Morphogenetic Proteins (BMPs) regulate multiple developmental processes in organisms from insects to mammals. Results: Here we show in Drosophila that GCN2 antagonises BMP signaling through direct effects on translation and indirectly via the transcription factor crc (dATF4). Expression of a constitutively active GCN2 or loss of the eIF2α phosphatase dPPP1R15 impair developmental BMP signaling in flies. In cells, inhibition of translation by GCN2 blocks downstream BMP signaling. Moreover, loss of d4E-BP, a target of crc, augments BMP signaling in vitro and rescues tissue development in vivo. Conclusion: These results identify a novel mechanism by which the ISR modulates BMP signaling during development.Item Open Access Published version Peer-reviewed Single-Cell Sequencing in Normal and Malignant Hematopoiesis.(Wiley, 2018) Wilson, Nicola K; Göttgens, Berthold; Wilson, Nicola [0000-0003-0865-7333]; Gottgens, Berthold [0000-0001-6302-5705]Hematopoiesis is one of the best studied adult stem-cell systems, with a differentiation hierarchy progressing from immature hematopoietic stem cells to over 10 distinct mature cell types. Recent technological breakthroughs now make it possible to define transcriptional profiles in thousands of individual cells. Facilitated by the wealth of prior data on cell purification and analysis strategies, hematopoiesis has been one of the earliest experimental systems to which many of these new single-cell sequencing technologies have been applied. In this review, the authors focus on recent studies, which have shed light on heterogeneity within individual populations as well as the relationships between populations, and also attempt to characterize the differences between normal and disease/perturbed states.Item Open Access Published version Peer-reviewed Determination of complex subclonal structures of hematological malignancies by multiplexed genotyping of blood progenitor colonies.(Elsevier BV, 2018-01) Nice, Francesca L; Massie, Charlie E; Klampfl, Thorsten; Green, Anthony R; Massie, Charles [0000-0003-2314-4843]; Green, Tony [0000-0002-9795-0218]Current next-generation sequencing (NGS) technologies allow unprecedented insights into the mutational profiles of tumors. Recent studies in myeloproliferative neoplasms have further demonstrated that, not only the mutational profile, but also the order in which these mutations are acquired is relevant for our understanding of the disease. Our ability to assign mutation order from NGS data alone is, however, limited. Here, we present a strategy of highly multiplexed genotyping of burst forming unit-erythroid colonies based on NGS results to assess subclonal tumor structure. This allowed for the generation of complex clonal hierarchies and determination of order of mutation acquisition far more accurately than was possible from NGS data alone.Item Open Access Accepted version Peer-reviewed The unfolded protein response governs integrity of the haematopoietic stem-cell pool during stress.(Springer Science and Business Media LLC, 2014-06-12) van Galen, Peter; Kreso, Antonija; Mbong, Nathan; Kent, David G; Fitzmaurice, Timothy; Chambers, Joseph E; Xie, Stephanie; Laurenti, Elisa; Hermans, Karin; Eppert, Kolja; Marciniak, Stefan J; Goodall, Jane C; Green, Anthony R; Wouters, Bradly G; Wienholds, Erno; Dick, John E; Kent, David [0000-0001-7871-8811]; Fitzmaurice, Tim [0000-0003-1403-2495]; Chambers, Joseph [0000-0003-4675-0053]; Laurenti, Elisa [0000-0002-9917-9092]; Marciniak, Stefan [0000-0001-8472-7183]; Goodall, Jane [0000-0002-3761-161X]; Green, Tony [0000-0002-9795-0218]The blood system is sustained by a pool of haematopoietic stem cells (HSCs) that are long-lived due to their capacity for self-renewal. A consequence of longevity is exposure to stress stimuli including reactive oxygen species (ROS), nutrient fluctuation and DNA damage. Damage that occurs within stressed HSCs must be tightly controlled to prevent either loss of function or the clonal persistence of oncogenic mutations that increase the risk of leukaemogenesis. Despite the importance of maintaining cell integrity throughout life, how the HSC pool achieves this and how individual HSCs respond to stress remain poorly understood. Many sources of stress cause misfolded protein accumulation in the endoplasmic reticulum (ER), and subsequent activation of the unfolded protein response (UPR) enables the cell to either resolve stress or initiate apoptosis. Here we show that human HSCs are predisposed to apoptosis through strong activation of the PERK branch of the UPR after ER stress, whereas closely related progenitors exhibit an adaptive response leading to their survival. Enhanced ER protein folding by overexpression of the co-chaperone ERDJ4 (also called DNAJB9) increases HSC repopulation capacity in xenograft assays, linking the UPR to HSC function. Because the UPR is a focal point where different sources of stress converge, our study provides a framework for understanding how stress signalling is coordinated within tissue hierarchies and integrated with stemness. Broadly, these findings reveal that the HSC pool maintains clonal integrity by clearance of individual HSCs after stress to prevent propagation of damaged stem cells.Item Open Access Accepted version Peer-reviewed Glutaminolysis is a metabolic dependency in FLT3ITD acute myeloid leukemia unmasked by FLT3 tyrosine kinase inhibition.(American Society of Hematology, 2018-04-12) Gallipoli, Paolo; Giotopoulos, George; Tzelepis, Konstantinos; Costa, Ana SH; Vohra, Shabana; Medina-Perez, Paula; Basheer, Faisal; Marando, Ludovica; Di Lisio, Lorena; Dias, Joao ML; Yun, Haiyang; Sasca, Daniel; Horton, Sarah J; Vassiliou, George; Frezza, Christian; Huntly, Brian JP; Gallipoli, Paolo [0000-0001-7254-2253]; Huntly, Brian JP [0000-0003-0312-161X]FLT3 internal tandem duplication (FLT3ITD) mutations are common in acute myeloid leukemia (AML) associated with poor patient prognosis. Although new-generation FLT3 tyrosine kinase inhibitors (TKI) have shown promising results, the outcome of FLT3ITD AML patients remains poor and demands the identification of novel, specific, and validated therapeutic targets for this highly aggressive AML subtype. Utilizing an unbiased genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screen, we identify GLS, the first enzyme in glutamine metabolism, as synthetically lethal with FLT3-TKI treatment. Using complementary metabolomic and gene-expression analysis, we demonstrate that glutamine metabolism, through its ability to support both mitochondrial function and cellular redox metabolism, becomes a metabolic dependency of FLT3ITD AML, specifically unmasked by FLT3-TKI treatment. We extend these findings to AML subtypes driven by other tyrosine kinase (TK) activating mutations and validate the role of GLS as a clinically actionable therapeutic target in both primary AML and in vivo models. Our work highlights the role of metabolic adaptations as a resistance mechanism to several TKI and suggests glutaminolysis as a therapeutically targetable vulnerability when combined with specific TKI in FLT3ITD and other TK activating mutation-driven leukemias.