ARTICLES Functional analysis of a chromosomal deletion associated with myelodysplastic syndromes using isogenic human induced pluripotent stem cells Andriana G Kotini1–3, Chan-Jung Chang1–3,19, Ibrahim Boussaad4,5,19, Jeffrey J Delrow6, Emily K Dolezal7, Abhinav B Nagulapally5,8, Fabiana Perna9, Gregory A Fishbein5,10, Virginia M Klimek11, R David Hawkins5,8, Danwei Huangfu12, Charles E Murry5,13–16, Timothy Graubert17, Stephen D Nimer7 & Eirini P Papapetrou1–5,13,18 Chromosomal deletions associated with human diseases, such as cancer, are common, but synteny issues complicate modeling of these deletions in mice. We use cellular reprogramming and genome engineering to functionally dissect the loss of chromosome 7q (del(7q)), a somatic cytogenetic abnormality present in myelodysplastic syndromes (MDS). We derive del(7q)- and isogenic karyotypically normal induced pluripotent stem cells (iPSCs) from hematopoietic cells of MDS patients and show that the del(7q) iPSCs recapitulate disease-associated phenotypes, including impaired hematopoietic differentiation. These disease phenotypes are rescued by spontaneous dosage correction and can be reproduced in karyotypically normal cells by engineering hemizygosity of defined chr7q segments in a 20-Mb region. We use a phenotype-rescue screen to identify candidate haploinsufficient genes that might mediate the del(7q)- hematopoietic defect. Our approach highlights the utility of human iPSCs both for functional mapping of disease-associated large-scale chromosomal deletions and for discovery of haploinsufficient genes. Large hemizygous deletions are found in most tumors and might if the mechanism fits a “classic” recessive (satisfying Knudson’s “two- be both hallmarks and drivers of cancer1. Hemizygous segmental hit” hypothesis) or a haploinsufficiency model and, finally, identifying chromosomal deletions are also frequent in normal genomes2. Apart the specific genetic elements that are lost. Classic tumor suppressor Nature America, Inc. All rights reserved. America, Inc. Nature from rare prototypic deletion syndromes (e.g., Smith-Magenis, genes were discovered through physical mapping of homozygous 5 Williams-Beuren, 22q11 deletion syndromes), genome-wide asso- deletions5. More recent data suggest that sporadic tumor suppressor ciation studies (GWAS) have implicated genomic deletions in neuro- genes are more likely to be mono-allelically lost and to function © 201 developmental diseases like schizophrenia and autism3, prompting through haploinsufficiency (wherein a single functional copy of a the hypothesis that deletions might account for an important source gene is insufficient to maintain normal function)6,7. of the ‘missing heritability’ of complex diseases3,4. MDS are clonal hematologic disorders characterized by ineffec- npg Unlike translocations or point mutations, chromosomal deletions are tive hematopoiesis and a propensity for progression to acute myeloid difficult to study with existing tools because primary patient material leukemia (AML)8. Somatic loss of one copy of the long arm of chro- is often scarce, and incomplete conservation of synteny (homologous mosome 7 (del(7q)) is a characteristic cytogenetic abnormality in genetic loci can be present on different chromosomes or in different MDS, well-recognized for decades as a marker of unfavorable progno- physical locations relative to each other within a chromosome across sis. However, the role of del(7q) in the pathogenesis of MDS remains species) complicate modeling in mice. Dissecting the role of specific elusive. The deletions are typically large and dispersed along the entire chromosomal deletions in specific cancers entails, first, determin- long arm of chr7 (ref. 9). Homology for human chr7q maps to four ing if a deletion has phenotypic consequences; second, determining different mouse chromosomes. 1Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA. 2The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA. 3The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA. 4Division of Hematology, Department of Medicine, University of Washington, Seattle, Washington, USA. 5Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, USA. 6Genomics Resource, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. 7Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, Florida, USA. 8Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, Washington, USA. 9Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. 10Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA. 11Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. 12Developmental Biology Program, Sloan-Kettering Institute, New York, New York, USA. 13Department of Pathology, University of Washington, Seattle, Washington, USA. 14Center for Cardiovascular Biology, University of Washington, Seattle, Washington, USA. 15Department of Bioengineering University of Washington, Seattle, Washington, USA. 16Division of Cardiology, Department of Medicine, University of Washington, Seattle, Washington, USA. 17MGH Cancer Center, Massachusetts General Hospital, Boston, Massachusetts, USA. 18Division of Hematology and Medical Oncology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA. 19These authors contributed equally to this work. Correspondence should be addressed to E.P.P. ([email protected]). Received 6 November 2014; accepted 13 February 2015; published online 23 March 2015; doi:10.1038/nbt.3178 646 VOLUME 33 NUMBER 6 JUNE 2015 NATURE BIOTECHNOLOGY ARTICLES Genetic engineering of human pluripotent stem cells (hPSCs) has in BMMCs and fibroblasts, and variants with variant allele frequency been used to model point mutations causing monogenic diseases in >1% in fibroblasts or <10% in BMMCs (Fig. 1e and Supplementary an isogenic setting10,11, but not disease-associated genomic deletions. Table 4). Similarly, 73 and 48 somatic SNVs were identified in the We used reprogramming and chromosome engineering to model del(7q)- and isogenic normal iPSC line, respectively. The del(7q)- del(7q) in an isogenic setting in hPSCs. Using different isogenic pairs iPSC line contained all 34 variants of the MDS clone, whereas the iso- of hPSCs harboring one or two copies of chr7q, we characterized genic normal line harbored none. These variants included two genes hematopoietic defects mediated by del(7q). We used spontaneous found recurrently mutated in MDS, SRSF2 and PHF6. Resequencing rescue and genome editing experiments to show that these pheno- of the rest of the iPSC lines derived from this patient (Supplementary types are mediated by a haploid dose of chr7q material, consistent Table 2) showed that all of the del(7q)- (6 out of 6) and none of with haploinsufficiency of one or more genes. We functionally map a the karyotypically normal iPSC lines (0 out of 15) harbored these 20-Mb fragment spanning cytobands 7q32.3–7q36.1 as the crucial mutations. These results unequivocally demonstrate that the isogenic region and identify candidate disease-specific haploinsufficient genes iPSCs represent normal cells that contain no premalignant lesions and using a phenotype-rescue screen. Finally, we show that the hemato- that the del(7q)- iPSCs accurately capture the genetic composition of poietic defect is mediated by the combined haploinsufficiency of the MDS clone. EZH2, in cooperation with one or more out of three additional candi- date genes residing in this region. The strategy reported here could be Decreased hematopoietic differentiation of del(7q)- MDS iPSCs applied to study the phenotypic consequences of segmental chromo- To assess the hematopoietic differentiation potential of the MDS somal deletions and for haploinsufficient gene discovery in a variety iPSCs we optimized an embryoid body–based differentiation pro- of human cancers, and neurological and developmental diseases. tocol and monitored the course of differentiation using CD34, a hemato-endothelial marker, and CD45, the key marker of definitive RESULTS hematopoietic cells15. In this differentiation protocol, normal human Generation of del(7q)- and normal isogenic iPSCs embryonic stem cell (hESC) and iPSC lines typically express CD34 We derived iPSC lines from hematopoietic cells of two patients (patients by day 6. Co-expression of CD45 appears by day 10, peaks at no. 2 and no. 3) with del(7q)- MDS (Fig. 1 and Supplementary Tables 1 day 14, and is followed by loss of CD34, as the cells become more and 2). We reasoned that residual normal hematopoietic cells are differentiated along hematopoietic lineages. By day 18, typically likely to co-exist with the MDS clone in the bone marrow of MDS more than 90% of cells are CD45+ (Supplementary Fig. 2a). We patients and that we could exploit this to reprogram both del(7q)- assessed the intra- (Supplementary Fig. 2a,b, panels I, II and c) and and isogenic karyotypically normal cells in parallel. We were able to inter- (Supplementary Fig. 2a,b, panels III, IV) line variation in this derive del(7q)- and karyotypically normal (N-) iPSC lines at the same differentiation process using normal iPSC lines derived from bone reprogramming round from both