Publications by authors named "Jacob C Ulirsch"

41 Publications

Genome-wide enhancer maps link risk variants to disease genes.

Nature 2021 05 7;593(7858):238-243. Epub 2021 Apr 7.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Genome-wide association studies (GWAS) have identified thousands of noncoding loci that are associated with human diseases and complex traits, each of which could reveal insights into the mechanisms of disease. Many of the underlying causal variants may affect enhancers, but we lack accurate maps of enhancers and their target genes to interpret such variants. We recently developed the activity-by-contact (ABC) model to predict which enhancers regulate which genes and validated the model using CRISPR perturbations in several cell types. Here we apply this ABC model to create enhancer-gene maps in 131 human cell types and tissues, and use these maps to interpret the functions of GWAS variants. Across 72 diseases and complex traits, ABC links 5,036 GWAS signals to 2,249 unique genes, including a class of 577 genes that appear to influence multiple phenotypes through variants in enhancers that act in different cell types. In inflammatory bowel disease (IBD), causal variants are enriched in predicted enhancers by more than 20-fold in particular cell types such as dendritic cells, and ABC achieves higher precision than other regulatory methods at connecting noncoding variants to target genes. These variant-to-function maps reveal an enhancer that contains an IBD risk variant and that regulates the expression of PPIF to alter the membrane potential of mitochondria in macrophages. Our study reveals principles of genome regulation, identifies genes that affect IBD and provides a resource and generalizable strategy to connect risk variants of common diseases to their molecular and cellular functions.
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http://dx.doi.org/10.1038/s41586-021-03446-xDOI Listing
May 2021

Tractor uses local ancestry to enable the inclusion of admixed individuals in GWAS and to boost power.

Nat Genet 2021 02 18;53(2):195-204. Epub 2021 Jan 18.

Analytic and Translational Genetics Unit, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA.

Admixed populations are routinely excluded from genomic studies due to concerns over population structure. Here, we present a statistical framework and software package, Tractor, to facilitate the inclusion of admixed individuals in association studies by leveraging local ancestry. We test Tractor with simulated and empirical two-way admixed African-European cohorts. Tractor generates accurate ancestry-specific effect-size estimates and P values, can boost genome-wide association study (GWAS) power and improves the resolution of association signals. Using a local ancestry-aware regression model, we replicate known hits for blood lipids, discover novel hits missed by standard GWAS and localize signals closer to putative causal variants.
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http://dx.doi.org/10.1038/s41588-020-00766-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7867648PMC
February 2021

Prioritizing disease and trait causal variants at the TNFAIP3 locus using functional and genomic features.

Nat Commun 2020 03 6;11(1):1237. Epub 2020 Mar 6.

Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA.

Genome-wide association studies have associated thousands of genetic variants with complex traits and diseases, but pinpointing the causal variant(s) among those in tight linkage disequilibrium with each associated variant remains a major challenge. Here, we use seven experimental assays to characterize all common variants at the multiple disease-associated TNFAIP3 locus in five disease-relevant immune cell lines, based on a set of features related to regulatory potential. Trait/disease-associated variants are enriched among SNPs prioritized based on either: (1) residing within CRISPRi-sensitive regulatory regions, or (2) localizing in a chromatin accessible region while displaying allele-specific reporter activity. Of the 15 trait/disease-associated haplotypes at TNFAIP3, 9 have at least one variant meeting one or both of these criteria, 5 of which are further supported by genetic fine-mapping. Our work provides a comprehensive strategy to characterize genetic variation at important disease-associated loci, and aids in the effort to identify trait causal genetic variants.
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http://dx.doi.org/10.1038/s41467-020-15022-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060350PMC
March 2020

Control of human hemoglobin switching by LIN28B-mediated regulation of BCL11A translation.

Nat Genet 2020 02 20;52(2):138-145. Epub 2020 Jan 20.

Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.

Increased production of fetal hemoglobin (HbF) can ameliorate the severity of sickle cell disease and β-thalassemia. BCL11A represses the genes encoding HbF and regulates human hemoglobin switching through variation in its expression during development. However, the mechanisms underlying the developmental expression of BCL11A remain mysterious. Here we show that BCL11A is regulated at the level of messenger RNA (mRNA) translation during human hematopoietic development. Despite decreased BCL11A protein synthesis earlier in development, BCL11A mRNA continues to be associated with ribosomes. Through unbiased genomic and proteomic analyses, we demonstrate that the RNA-binding protein LIN28B, which is developmentally expressed in a pattern reciprocal to that of BCL11A, directly interacts with ribosomes and BCL11A mRNA. Furthermore, we show that BCL11A mRNA translation is suppressed by LIN28B through direct interactions, independently of its role in regulating let-7 microRNAs, and that BCL11A is the major target of LIN28B-mediated HbF induction. Our results reveal a previously unappreciated mechanism underlying human hemoglobin switching that illuminates new therapeutic opportunities.
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http://dx.doi.org/10.1038/s41588-019-0568-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031047PMC
February 2020

Genetic predisposition to mosaic Y chromosome loss in blood.

Nature 2019 11 20;575(7784):652-657. Epub 2019 Nov 20.

Genetics of Complex Traits, University of Exeter Medical School, University of Exeter, Exeter, UK.

Mosaic loss of chromosome Y (LOY) in circulating white blood cells is the most common form of clonal mosaicism, yet our knowledge of the causes and consequences of this is limited. Here, using a computational approach, we estimate that 20% of the male population represented in the UK Biobank study (n = 205,011) has detectable LOY. We identify 156 autosomal genetic determinants of LOY, which we replicate in 757,114 men of European and Japanese ancestry. These loci highlight genes that are involved in cell-cycle regulation and cancer susceptibility, as well as somatic drivers of tumour growth and targets of cancer therapy. We demonstrate that genetic susceptibility to LOY is associated with non-haematological effects on health in both men and women, which supports the hypothesis that clonal haematopoiesis is a biomarker of genomic instability in other tissues. Single-cell RNA sequencing identifies dysregulated expression of autosomal genes in leukocytes with LOY and provides insights into why clonal expansion of these cells may occur. Collectively, these data highlight the value of studying clonal mosaicism to uncover fundamental mechanisms that underlie cancer and other ageing-related diseases.
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http://dx.doi.org/10.1038/s41586-019-1765-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6887549PMC
November 2019

Transcriptional States and Chromatin Accessibility Underlying Human Erythropoiesis.

Cell Rep 2019 06;27(11):3228-3240.e7

Division of Hematology/Oncology, Boston Children's Hospital, and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address:

Human erythropoiesis serves as a paradigm of physiologic cellular differentiation. This process is also of considerable interest for better understanding anemias and identifying new therapies. Here, we apply deep transcriptomic and accessible chromatin profiling to characterize a faithful ex vivo human erythroid differentiation system from hematopoietic stem and progenitor cells. We reveal stage-specific transcriptional states and chromatin accessibility during various stages of erythropoiesis, including 14,260 differentially expressed genes and 63,659 variably accessible chromatin peaks. Our analysis suggests differentiation stage-predominant roles for specific master regulators, including GATA1 and KLF1. We integrate chromatin profiles with common and rare genetic variants associated with erythroid cell traits and diseases, finding that variants regulating different erythroid phenotypes likely act at variable points during differentiation. In addition, we identify a regulator of terminal erythropoiesis, TMCC2, more broadly illustrating the value of this comprehensive analysis to improve our understanding of erythropoiesis in health and disease.
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http://dx.doi.org/10.1016/j.celrep.2019.05.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6579117PMC
June 2019

Gene-centric functional dissection of human genetic variation uncovers regulators of hematopoiesis.

Elife 2019 05 9;8. Epub 2019 May 9.

Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, United States.

Genome-wide association studies (GWAS) have identified thousands of variants associated with human diseases and traits. However, the majority of GWAS-implicated variants are in non-coding regions of the genome and require in depth follow-up to identify target genes and decipher biological mechanisms. Here, rather than focusing on causal variants, we have undertaken a pooled loss-of-function screen in primary hematopoietic cells to interrogate 389 candidate genes contained in 75 loci associated with red blood cell traits. Using this approach, we identify 77 genes at 38 GWAS loci, with most loci harboring 1-2 candidate genes. Importantly, the hit set was strongly enriched for genes validated through orthogonal genetic approaches. Genes identified by this approach are enriched in specific and relevant biological pathways, allowing regulators of human erythropoiesis and modifiers of blood diseases to be defined. More generally, this functional screen provides a paradigm for gene-centric follow up of GWAS for a variety of human diseases and traits.
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http://dx.doi.org/10.7554/eLife.44080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534380PMC
May 2019

HRI coordinates translation necessary for protein homeostasis and mitochondrial function in erythropoiesis.

Elife 2019 04 29;8. Epub 2019 Apr 29.

Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, United States.

Iron and heme play central roles in the production of red blood cells, but the underlying mechanisms remain incompletely understood. Heme-regulated eIF2α kinase (HRI) controls translation by phosphorylating eIF2α. Here, we investigate the global impact of iron, heme, and HRI on protein translation in vivo in murine primary erythroblasts using ribosome profiling. We validate the known role of HRI-mediated translational stimulation of integratedstressresponse mRNAs during iron deficiency in vivo. Moreover, we find that the translation of mRNAs encoding cytosolic and mitochondrial ribosomal proteins is substantially repressed by HRI during iron deficiency, causing a decrease in cytosolic and mitochondrial protein synthesis. The absence of HRI during iron deficiency elicits a prominent cytoplasmic unfolded protein response and impairs mitochondrial respiration. Importantly, ATF4 target genes are activated during iron deficiency to maintain mitochondrial function and to enable erythroid differentiation. We further identify GRB10 as a previously unappreciated regulator of terminal erythropoiesis.
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http://dx.doi.org/10.7554/eLife.46976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533081PMC
April 2019

Impaired human hematopoiesis due to a cryptic intronic splicing mutation.

J Exp Med 2019 05 26;216(5):1050-1060. Epub 2019 Mar 26.

Division of Hematology/Oncology, The Manton Center for Orphan Disease Research, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA

Studies of allelic variation underlying genetic blood disorders have provided important insights into human hematopoiesis. Most often, the identified pathogenic mutations result in loss-of-function or missense changes. However, assessing the pathogenicity of noncoding variants can be challenging. Here, we characterize two unrelated patients with a distinct presentation of dyserythropoietic anemia and other impairments in hematopoiesis associated with an intronic mutation in that is 24 nucleotides upstream of the canonical splice acceptor site. Functional studies demonstrate that this single-nucleotide alteration leads to reduced canonical splicing and increased use of an alternative splice acceptor site that causes a partial intron retention event. The resultant altered GATA1 contains a five-amino acid insertion at the C-terminus of the C-terminal zinc finger and has no observable activity. Collectively, our results demonstrate how altered splicing of GATA1, which reduces levels of the normal form of this master transcription factor, can result in distinct changes in human hematopoiesis.
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http://dx.doi.org/10.1084/jem.20181625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504223PMC
May 2019

Interrogation of human hematopoiesis at single-cell and single-variant resolution.

Nat Genet 2019 04 11;51(4):683-693. Epub 2019 Mar 11.

Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.

Widespread linkage disequilibrium and incomplete annotation of cell-to-cell state variation represent substantial challenges to elucidating mechanisms of trait-associated genetic variation. Here we perform genetic fine-mapping for blood cell traits in the UK Biobank to identify putative causal variants. These variants are enriched in genes encoding proteins in trait-relevant biological pathways and in accessible chromatin of hematopoietic progenitors. For regulatory variants, we explore patterns of developmental enhancer activity, predict molecular mechanisms, and identify likely target genes. In several instances, we localize multiple independent variants to the same regulatory element or gene. We further observe that variants with pleiotropic effects preferentially act in common progenitor populations to direct the production of distinct lineages. Finally, we leverage fine-mapped variants in conjunction with continuous epigenomic annotations to identify trait-cell type enrichments within closely related populations and in single cells. Our study provides a comprehensive framework for single-variant and single-cell analyses of genetic associations.
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http://dx.doi.org/10.1038/s41588-019-0362-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441389PMC
April 2019

Lineage Tracing in Humans Enabled by Mitochondrial Mutations and Single-Cell Genomics.

Cell 2019 03 28;176(6):1325-1339.e22. Epub 2019 Feb 28.

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA. Electronic address:

Lineage tracing provides key insights into the fate of individual cells in complex organisms. Although effective genetic labeling approaches are available in model systems, in humans, most approaches require detection of nuclear somatic mutations, which have high error rates, limited scale, and do not capture cell state information. Here, we show that somatic mutations in mtDNA can be tracked by single-cell RNA or assay for transposase accessible chromatin (ATAC) sequencing. We leverage somatic mtDNA mutations as natural genetic barcodes and demonstrate their utility as highly accurate clonal markers to infer cellular relationships. We track native human cells both in vitro and in vivo and relate clonal dynamics to gene expression and chromatin accessibility. Our approach should allow clonal tracking at a 1,000-fold greater scale than with nuclear genome sequencing, with simultaneous information on cell state, opening the way to chart cellular dynamics in human health and disease.
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http://dx.doi.org/10.1016/j.cell.2019.01.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6408267PMC
March 2019

The Genetic Landscape of Diamond-Blackfan Anemia.

Am J Hum Genet 2018 12 29;103(6):930-947. Epub 2018 Nov 29.

Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 7 out of 1,000,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole-exome sequencing (WES). We identified relevant rare and predicted damaging mutations for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and located in 1 of 19 previously reported ribosomal protein (RP)-encoding genes. Using exon coverage estimates, we identified and validated 31 deletions in RP genes. We also observed an enrichment for extended splice site mutations and validated their diverse effects using RNA sequencing in cell lines obtained from individuals with DBA. Leveraging the size of our cohort, we observed robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. We further identified rare mutations in seven previously unreported RP genes that may cause DBA, as well as several distinct disorders that appear to phenocopy DBA, including nine individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain >5% of DBA-affected case subjects. Overall, this report should inform not only clinical practice for DBA-affected individuals, but also the design and analysis of rare variant studies for heterogeneous Mendelian disorders.
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http://dx.doi.org/10.1016/j.ajhg.2018.10.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6288280PMC
December 2018

Detecting genome-wide directional effects of transcription factor binding on polygenic disease risk.

Nat Genet 2018 10 3;50(10):1483-1493. Epub 2018 Sep 3.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Biological interpretation of genome-wide association study data frequently involves assessing whether SNPs linked to a biological process, for example, binding of a transcription factor, show unsigned enrichment for disease signal. However, signed annotations quantifying whether each SNP allele promotes or hinders the biological process can enable stronger statements about disease mechanism. We introduce a method, signed linkage disequilibrium profile regression, for detecting genome-wide directional effects of signed functional annotations on disease risk. We validate the method via simulations and application to molecular quantitative trait loci in blood, recovering known transcriptional regulators. We apply the method to expression quantitative trait loci in 48 Genotype-Tissue Expression tissues, identifying 651 transcription factor-tissue associations including 30 with robust evidence of tissue specificity. We apply the method to 46 diseases and complex traits (average n = 290 K), identifying 77 annotation-trait associations representing 12 independent transcription factor-trait associations, and characterize the underlying transcriptional programs using gene-set enrichment analyses. Our results implicate new causal disease genes and new disease mechanisms.
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http://dx.doi.org/10.1038/s41588-018-0196-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202062PMC
October 2018

The NORAD lncRNA assembles a topoisomerase complex critical for genome stability.

Nature 2018 09 27;561(7721):132-136. Epub 2018 Aug 27.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

The human genome contains thousands of long non-coding RNAs, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen. A specific long non-coding RNA-non-coding RNA activated by DNA damage (NORAD)-has recently been shown to be required for maintaining genomic stability, but its molecular mechanism is unknown. Here we combine RNA antisense purification and quantitative mass spectrometry to identify proteins that directly interact with NORAD in living cells. We show that NORAD interacts with proteins involved in DNA replication and repair in steady-state cells and localizes to the nucleus upon stimulation with replication stress or DNA damage. In particular, NORAD interacts with RBMX, a component of the DNA-damage response, and contains the strongest RBMX-binding site in the transcriptome. We demonstrate that NORAD controls the ability of RBMX to assemble a ribonucleoprotein complex-which we term NORAD-activated ribonucleoprotein complex 1 (NARC1)-that contains the known suppressors of genomic instability topoisomerase I (TOP1), ALYREF and the PRPF19-CDC5L complex. Cells depleted for NORAD or RBMX display an increased frequency of chromosome segregation defects, reduced replication-fork velocity and altered cell-cycle progression-which represent phenotypes that are mechanistically linked to TOP1 and PRPF19-CDC5L function. Expression of NORAD in trans can rescue defects caused by NORAD depletion, but rescue is significantly impaired when the RBMX-binding site in NORAD is deleted. Our results demonstrate that the interaction between NORAD and RBMX is important for NORAD function, and that NORAD is required for the assembly of the previously unknown topoisomerase complex NARC1, which contributes to maintaining genomic stability. In addition, we uncover a previously unknown function for long non-coding RNAs in modulating the ability of an RNA-binding protein to assemble a higher-order ribonucleoprotein complex.
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http://dx.doi.org/10.1038/s41586-018-0453-zDOI Listing
September 2018

Common α-globin variants modify hematologic and other clinical phenotypes in sickle cell trait and disease.

PLoS Genet 2018 03 28;14(3):e1007293. Epub 2018 Mar 28.

Department of Epidemiology, University of Washington, Seattle, Washington, United States of America.

Co-inheritance of α-thalassemia has a significant protective effect on the severity of complications of sickle cell disease (SCD), including stroke. However, little information exists on the association and interactions for the common African ancestral α-thalassemia mutation (-α3.7 deletion) and β-globin traits (HbS trait [SCT] and HbC trait) on important clinical phenotypes such as red blood cell parameters, anemia, and chronic kidney disease (CKD). In a community-based cohort of 2,916 African Americans from the Jackson Heart Study, we confirmed the expected associations between SCT, HbC trait, and the -α3.7 deletion with lower mean corpuscular volume/mean corpuscular hemoglobin and higher red blood cell count and red cell distribution width. In addition to the recently recognized association of SCT with lower estimated glomerular filtration rate and glycated hemoglobin (HbA1c), we observed a novel association of the -α3.7 deletion with higher HbA1c levels. Co-inheritance of each additional copy of the -α3.7 deletion significantly lowered the risk of anemia and chronic kidney disease among individuals with SCT (P-interaction = 0.031 and 0.019, respectively). Furthermore, co-inheritance of a novel α-globin regulatory variant was associated with normalization of red cell parameters in individuals with the -α3.7 deletion and significantly negated the protective effect of α-thalassemia on stroke in 1,139 patients with sickle cell anemia from the Cooperative Study of Sickle Cell Disease (CSSCD) (P-interaction = 0.0049). Functional assays determined that rs11865131, located in the major alpha-globin enhancer MCS-R2, was the most likely causal variant. These findings suggest that common α- and β-globin variants interact to influence hematologic and clinical phenotypes in African Americans, with potential implications for risk-stratification and counseling of individuals with SCD and SCT.
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http://dx.doi.org/10.1371/journal.pgen.1007293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5891078PMC
March 2018

Ribosome Levels Selectively Regulate Translation and Lineage Commitment in Human Hematopoiesis.

Cell 2018 03 15;173(1):90-103.e19. Epub 2018 Mar 15.

Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address:

Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation.
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http://dx.doi.org/10.1016/j.cell.2018.02.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866246PMC
March 2018

Whole-exome sequencing identifies an α-globin cluster triplication resulting in increased clinical severity of β-thalassemia.

Cold Spring Harb Mol Case Stud 2017 Nov 21;3(6). Epub 2017 Nov 21.

Departments of Hematology-Oncology, Schneider Children's Medical Center of Israel, Petach Tivka 49202, Israel.

Whole-exome sequencing (WES) has been increasingly useful for the diagnosis of patients with rare causes of anemia, particularly when there is an atypical clinical presentation or targeted genotyping approaches are inconclusive. Here, we describe a 20-yr-old man with a lifelong moderate-to-severe anemia with accompanying splenomegaly who lacked a definitive diagnosis. After a thorough clinical workup and targeted genetic sequencing, we identified a paternally inherited β-globin mutation (:c.93-21G>A, IVS-I-110:G>A), a known cause of β-thalassemia minor. As this mutation alone was inconsistent with the severity of the anemia, we performed WES. Although we could not identify any relevant pathogenic single-nucleotide variants (SNVs) or small indels, copy-number variant (CNV) analyses revealed a likely triplication of the entire α-globin cluster, which was subsequently confirmed by multiplex ligation-dependent probe amplification. Treatment and follow-up was redefined according to the diagnosis of β-thalassemia intermedia resulting from a single β-thalassemia mutation in combination with an α-globin cluster triplication. Thus, we describe a case where the typical WES-based analysis of SNVs and small indels was unrevealing, but WES-based CNV analysis resulted in a definitive diagnosis that informed clinical decision-making. More generally, this case illustrates the value of performing CNV analysis when WES is otherwise unable to elucidate a clear genetic diagnosis.
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http://dx.doi.org/10.1101/mcs.a001941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701307PMC
November 2017

Confounding in ex vivo models of Diamond-Blackfan anemia.

Blood 2017 08 14;130(9):1165-1168. Epub 2017 Jun 14.

Division of Hematology/Oncology, The Manton Center for Orphan Disease Research, Boston Children's Hospital, and.

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http://dx.doi.org/10.1182/blood-2017-05-783191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5580274PMC
August 2017

Developmentally-faithful and effective human erythropoiesis in immunodeficient and Kit mutant mice.

Am J Hematol 2017 Sep 19;92(9):E513-E519. Epub 2017 Jul 19.

Division of Hematology/Oncology, The Manton Center for Orphan Disease Research, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts.

Immunodeficient mouse models have been valuable for studies of human hematopoiesis, but high-fidelity recapitulation of erythropoiesis in most xenograft recipients remains elusive. Recently developed immunodeficient and Kit mutant mice, however, have provided a suitable background to achieve higher-level human erythropoiesis after long-term hematopoietic engraftment. While there has been some characterization of human erythropoiesis in these models, a comprehensive analysis from various human developmental stages has not yet been reported. Here, we have utilized cell surface phenotypes, morphologic analyses, and molecular studies to fully characterize human erythropoiesis from multiple developmental stages in immunodeficient and Kit mutant mouse models following long-term hematopoietic stem and progenitor cell engraftment. We show that human erythropoiesis in such models demonstrates complete maturation and enucleation, as well as developmentally appropriate globin gene expression. These results provide a framework for future studies to utilize this model system for interrogating disorders affecting human erythropoiesis and for developing improved therapeutic approaches.
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http://dx.doi.org/10.1002/ajh.24805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5546987PMC
September 2017

Functional Selectivity in Cytokine Signaling Revealed Through a Pathogenic EPO Mutation.

Cell 2017 03;168(6):1053-1064.e15

Division of Hematology/Oncology, The Manton Center for Orphan Disease Research, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address:

Cytokines are classically thought to stimulate downstream signaling pathways through monotonic activation of receptors. We describe a severe anemia resulting from a homozygous mutation (R150Q) in the cytokine erythropoietin (EPO). Surprisingly, the EPO R150Q mutant shows only a mild reduction in affinity for its receptor but has altered binding kinetics. The EPO mutant is less effective at stimulating erythroid cell proliferation and differentiation, even at maximally potent concentrations. While the EPO mutant can stimulate effectors such as STAT5 to a similar extent as the wild-type ligand, there is reduced JAK2-mediated phosphorylation of select downstream targets. This impairment in downstream signaling mechanistically arises from altered receptor dimerization dynamics due to extracellular binding changes. These results demonstrate how variation in a single cytokine can lead to biased downstream signaling and can thereby cause human disease. Moreover, we have defined a distinct treatable form of anemia through mutation identification and functional studies.
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http://dx.doi.org/10.1016/j.cell.2017.02.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5376096PMC
March 2017

Comprehensive population-based genome sequencing provides insight into hematopoietic regulatory mechanisms.

Proc Natl Acad Sci U S A 2017 01 28;114(3):E327-E336. Epub 2016 Dec 28.

Broad Institute of MIT and Harvard, Cambridge, MA 02142;

Genetic variants affecting hematopoiesis can influence commonly measured blood cell traits. To identify factors that affect hematopoiesis, we performed association studies for blood cell traits in the population-based Estonian Biobank using high-coverage whole-genome sequencing (WGS) in 2,284 samples and SNP genotyping in an additional 14,904 samples. Using up to 7,134 samples with available phenotype data, our analyses identified 17 associations across 14 blood cell traits. Integration of WGS-based fine-mapping and complementary epigenomic datasets provided evidence for causal mechanisms at several loci, including at a previously undiscovered basophil count-associated locus near the master hematopoietic transcription factor CEBPA The fine-mapped variant at this basophil count association near CEBPA overlapped an enhancer active in common myeloid progenitors and influenced its activity. In situ perturbation of this enhancer by CRISPR/Cas9 mutagenesis in hematopoietic stem and progenitor cells demonstrated that it is necessary for and specifically regulates CEBPA expression during basophil differentiation. We additionally identified basophil count-associated variation at another more pleiotropic myeloid enhancer near GATA2, highlighting regulatory mechanisms for ordered expression of master hematopoietic regulators during lineage specification. Our study illustrates how population-based genetic studies can provide key insights into poorly understood cell differentiation processes of considerable physiologic relevance.
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http://dx.doi.org/10.1073/pnas.1619052114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5255587PMC
January 2017

A novel pathogenic mutation in RPL11 identified in a patient diagnosed with diamond Blackfan anemia as a young adult.

Blood Cells Mol Dis 2016 10 17;61:46-7. Epub 2016 Aug 17.

Division of Genetics and Genomics, The Manton Center for Orphan Disease Research, Boston Children's Hospital, Boston, MA, USA.

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http://dx.doi.org/10.1016/j.bcmd.2016.08.001DOI Listing
October 2016

Exome sequencing results in successful diagnosis and treatment of a severe congenital anemia.

Cold Spring Harb Mol Case Stud 2016 Jul;2(4):a000885

Division of Hematology/Oncology, The Manton Center for Orphan Disease Research, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA;; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA;; Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA;

Whole-exome sequencing is increasingly used for diagnosis and identification of appropriate therapies in patients. Here, we present the case of a 3-yr-old male with a lifelong and severe transfusion-dependent anemia of unclear etiology, despite an extensive clinical workup. Given the difficulty of making the diagnosis and the potential side effects from performing interventions in patients with a congenital anemia of unknown etiology, we opted to perform whole-exome sequencing on the patient and his parents. This resulted in the identification of homozygous loss-of-function mutations in the EPB41 gene, encoding erythrocyte protein band 4.1, which therefore causes a rare and severe form of hereditary elliptocytosis in the patient. Based on prior clinical experience in similar patients, a surgical splenectomy was performed that resulted in subsequent transfusion independence in the patient. This case illustrates how whole-exome sequencing can lead to accurate diagnoses (and exclusion of diagnoses where interventions, such as splenectomy, would be contraindicated), thereby resulting in appropriate and successful therapeutic intervention-a major goal of precision medicine.
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http://dx.doi.org/10.1101/mcs.a000885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4990811PMC
July 2016

Systematic Functional Dissection of Common Genetic Variation Affecting Red Blood Cell Traits.

Cell 2016 Jun;165(6):1530-1545

Division of Hematology/Oncology, The Manton Center for Orphan Disease Research, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA. Electronic address:

Genome-wide association studies (GWAS) have successfully identified thousands of associations between common genetic variants and human disease phenotypes, but the majority of these variants are non-coding, often requiring genetic fine-mapping, epigenomic profiling, and individual reporter assays to delineate potential causal variants. We employ a massively parallel reporter assay (MPRA) to simultaneously screen 2,756 variants in strong linkage disequilibrium with 75 sentinel variants associated with red blood cell traits. We show that this assay identifies elements with endogenous erythroid regulatory activity. Across 23 sentinel variants, we conservatively identified 32 MPRA functional variants (MFVs). We used targeted genome editing to demonstrate endogenous enhancer activity across 3 MFVs that predominantly affect the transcription of SMIM1, RBM38, and CD164. Functional follow-up of RBM38 delineates a key role for this gene in the alternative splicing program occurring during terminal erythropoiesis. Finally, we provide evidence for how common GWAS-nominated variants can disrupt cell-type-specific transcriptional regulatory pathways.
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http://dx.doi.org/10.1016/j.cell.2016.04.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4893171PMC
June 2016

Insight into GATA1 transcriptional activity through interrogation of cis elements disrupted in human erythroid disorders.

Proc Natl Acad Sci U S A 2016 Apr 4;113(16):4434-9. Epub 2016 Apr 4.

Division of Hematology/Oncology, Manton Center for Orphan Disease Research, Boston Children's Hospital, Boston, MA 02115; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA; 02115; The Broad Institute, Cambridge, MA 02142;

Whole-exome sequencing has been incredibly successful in identifying causal genetic variants and has revealed a number of novel genes associated with blood and other diseases. One limitation of this approach is that it overlooks mutations in noncoding regulatory elements. Furthermore, the mechanisms by which mutations in transcriptionalcis-regulatory elements result in disease remain poorly understood. Here we used CRISPR/Cas9 genome editing to interrogate three such elements harboring mutations in human erythroid disorders, which in all cases are predicted to disrupt a canonical binding motif for the hematopoietic transcription factor GATA1. Deletions of as few as two to four nucleotides resulted in a substantial decrease (>80%) in target gene expression. Isolated deletions of the canonical GATA1 binding motif completely abrogated binding of the cofactor TAL1, which binds to a separate motif. Having verified the functionality of these three GATA1 motifs, we demonstrate strong evolutionary conservation of GATA1 motifs in regulatory elements proximal to other genes implicated in erythroid disorders, and show that targeted disruption of such elements results in altered gene expression. By modeling transcription factor binding patterns, we show that multiple transcription factors are associated with erythroid gene expression, and have created predictive maps modeling putative disruptions of their binding sites at key regulatory elements. Our study provides insight into GATA1 transcriptional activity and may prove a useful resource for investigating the pathogenicity of noncoding variants in human erythroid disorders.
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http://dx.doi.org/10.1073/pnas.1521754113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4843446PMC
April 2016

Advances in understanding erythropoiesis: evolving perspectives.

Br J Haematol 2016 Apr 5;173(2):206-18. Epub 2016 Feb 5.

Division of Hematology/Oncology, The Manton Center for Orphan Disease Research, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.

Red blood cells (RBCs) are generated from haematopoietic stem and progenitor cells (HSPCs) through the step-wise process of differentiation known as erythropoiesis. In this review, we discuss our current understanding of erythropoiesis and highlight recent advances in this field. During embryonic development, erythropoiesis occurs in three distinct waves comprising first, the yolk sac-derived primitive RBCs, followed sequentially by the erythro-myeloid progenitor (EMP) and HSPC-derived definitive RBCs. Recent work has highlighted the complexity and variability that may exist in the hierarchical arrangement of progenitors responsible for erythropoiesis. Using recently defined cell surface markers, it is now possible to enrich for erythroid progenitors and precursors to a much greater extent than has been possible before. While a great deal of knowledge has been gained on erythropoiesis from model organisms, our understanding of this process is currently being refined through human genetic studies. Genes mutated in erythroid disorders can now be identified more rapidly by the use of next-generation sequencing techniques. Genome-wide association studies on erythroid traits in healthy populations have also revealed new modulators of erythropoiesis. All of these recent developments have significant promise not only for increasing our understanding of erythropoiesis, but also for improving our ability to intervene when RBC production is perturbed in disease.
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http://dx.doi.org/10.1111/bjh.13938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833665PMC
April 2016

Targeted Application of Human Genetic Variation Can Improve Red Blood Cell Production from Stem Cells.

Cell Stem Cell 2016 Jan 22;18(1):73-78. Epub 2015 Oct 22.

Division of Hematology/Oncology, Boston Children's Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.

Multipotent and pluripotent stem cells are potential sources for cell and tissue replacement therapies. For example, stem cell-derived red blood cells (RBCs) are a potential alternative to donated blood, but yield and quality remain a challenge. Here, we show that application of insight from human population genetic studies can enhance RBC production from stem cells. The SH2B3 gene encodes a negative regulator of cytokine signaling and naturally occurring loss-of-function variants in this gene increase RBC counts in vivo. Targeted suppression of SH2B3 in primary human hematopoietic stem and progenitor cells enhanced the maturation and overall yield of in-vitro-derived RBCs. Moreover, inactivation of SH2B3 by CRISPR/Cas9 genome editing in human pluripotent stem cells allowed enhanced erythroid cell expansion with preserved differentiation. Our findings therefore highlight the potential for combining human genome variation studies with genome editing approaches to improve cell and tissue production for regenerative medicine.
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http://dx.doi.org/10.1016/j.stem.2015.09.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4707983PMC
January 2016
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