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An effector index to predict target genes at GWAS loci

Forgetta, Vincenzo; Jiang, Lai; Vulpescu, Nicholas A; Hogan, Megan S; Chen, Siyuan; Morris, John A; Grinek, Stepan; Benner, Christian; Jang, Dong-Keun; Hoang, Quy; Burtt, Noel; Flannick, Jason A; McCarthy, Mark I; Fauman, Eric; Greenwood, Celia M T; Maurano, Matthew T; Richards, J Brent
Drug development and biological discovery require effective strategies to map existing genetic associations to causal genes. To approach this problem, we selected 12 common diseases and quantitative traits for which highly powered genome-wide association studies (GWAS) were available. For each disease or trait, we systematically curated positive control gene sets from Mendelian forms of the disease and from targets of medicines used for disease treatment. We found that these positive control genes were highly enriched in proximity of GWAS-associated single-nucleotide variants (SNVs). We then performed quantitative assessment of the contribution of commonly used genomic features, including open chromatin maps, expression quantitative trait loci (eQTL), and chromatin conformation data. Using these features, we trained and validated an Effector Index (Ei), to map target genes for these 12 common diseases and traits. Ei demonstrated high predictive performance, both with cross-validation on the training set, and an independently derived set for type 2 diabetes. Key predictive features included coding or transcript-altering SNVs, distance to gene, and open chromatin-based metrics. This work outlines a simple, understandable approach to prioritize genes at GWAS loci for functional follow-up and drug development, and provides a systematic strategy for prioritization of GWAS target genes.
PMID: 35147782
ISSN: 1432-1203
CID: 5156932

Synthetic regulatory reconstitution reveals principles of mammalian Hox cluster regulation

Pinglay, Sudarshan; Bulajić, Milica; Rahe, Dylan P; Huang, Emily; Brosh, Ran; Mamrak, Nicholas E; King, Benjamin R; German, Sergei; Cadley, John A; Rieber, Lila; Easo, Nicole; Lionnet, Timothée; Mahony, Shaun; Maurano, Matthew T; Holt, Liam J; Mazzoni, Esteban O; Boeke, Jef D
Precise Hox gene expression is crucial for embryonic patterning. Intra-Hox transcription factor binding and distal enhancer elements have emerged as the major regulatory modules controlling Hox gene expression. However, quantifying their relative contributions has remained elusive. Here, we introduce "synthetic regulatory reconstitution," a conceptual framework for studying gene regulation, and apply it to the HoxA cluster. We synthesized and delivered variant rat HoxA clusters (130 to 170 kilobases) to an ectopic location in the mouse genome. We found that a minimal HoxA cluster recapitulated correct patterns of chromatin remodeling and transcription in response to patterning signals, whereas the addition of distal enhancers was needed for full transcriptional output. Synthetic regulatory reconstitution could provide a generalizable strategy for deciphering the regulatory logic of gene expression in complex genomes.
PMID: 35771912
ISSN: 1095-9203
CID: 5268842

A conditional counterselectable Piga knockout in mouse embryonic stem cells for advanced genome writing applications

Zhang, Weimin; Brosh, Ran; McCulloch, Laura H; Zhu, Yinan; Ashe, Hannah; Ellis, Gwen; Camellato, Brendan R; Kim, Sang Yong; Maurano, Matthew T; Boeke, Jef D
Overwriting counterselectable markers is an efficient strategy for removing wild-type DNA or replacing it with payload DNA of interest. Currently, one bottleneck of efficient genome engineering in mammals is the shortage of counterselectable (negative selection) markers that work robustly without affecting organismal developmental potential. Here, we report a conditional Piga knockout strategy that enables efficient proaerolysin-based counterselection in mouse embryonic stem cells. The conditional Piga knockout cells show similar proaerolysin resistance as full (non-conditional) Piga deletion cells, which enables the use of a PIGA transgene as a counterselectable marker for genome engineering purposes. Native Piga function is readily restored in conditional Piga knockout cells to facilitate subsequent mouse development. We also demonstrate the generality of our strategy by engineering a conditional knockout of endogenous Hprt. Taken together, our work provides a new tool for advanced mouse genome writing and mouse model establishment.
PMID: 35692632
ISSN: 2589-0042
CID: 5282452

Genomic context sensitivity of insulator function

Ribeiro-Dos-Santos, André M; Hogan, Megan S; Luther, Raven D; Brosh, Ran; Maurano, Matthew T
The specificity of interactions between genomic regulatory elements and potential target genes is influenced by the binding of insulator proteins such as CTCF, which can act as potent enhancer blockers when interposed between an enhancer and a promoter in a reporter assay. But not all CTCF sites genome-wide function as insulator elements, depending on cellular and genomic context. To dissect the influence of genomic context on enhancer blocker activity, we integrated reporter constructs with promoter-only, promoter and enhancer, and enhancer blocker configurations at hundreds of thousands of genomic sites using the Sleeping Beauty transposase. Deconvolution of reporter activity by genomic position reveals distinct expression patterns subject to genomic context, including a compartment of enhancer blocker reporter integrations with robust expression. The high density of integration sites permits quantitative delineation of characteristic genomic context sensitivity profiles, and their decomposition into sensitivity to both local and distant DNase I hypersensitive sites. Furthermore, using a single-cell expression approach to test the effect of integrated reporters for differential expression of nearby endogenous genes reveals that CTCF insulator elements do not completely abrogate reporter effects on endogenous gene expression. Collectively, our results lend new insight to genomic regulatory compartmentalization and its influence on the determinants of promoter-enhancer specificity.
PMID: 35082140
ISSN: 1549-5469
CID: 5154592

De novo mutations in childhood cases of sudden unexplained death that disrupt intracellular Ca2+ regulation

Halvorsen, Matthew; Gould, Laura; Wang, Xiaohan; Grant, Gariel; Moya, Raquel; Rabin, Rachel; Ackerman, Michael J; Tester, David J; Lin, Peter T; Pappas, John G; Maurano, Matthew T; Goldstein, David B; Tsien, Richard W; Devinsky, Orrin
Sudden unexplained death in childhood (SUDC) is an understudied problem. Whole-exome sequence data from 124 "trios" (decedent child, living parents) was used to test for excessive de novo mutations (DNMs) in genes involved in cardiac arrhythmias, epilepsy, and other disorders. Among decedents, nonsynonymous DNMs were enriched in genes associated with cardiac and seizure disorders relative to controls (odds ratio = 9.76, P = 2.15 × 10-4). We also found evidence for overtransmission of loss-of-function (LoF) or previously reported pathogenic variants in these same genes from heterozygous carrier parents (11 of 14 transmitted, P = 0.03). We identified a total of 11 SUDC proband genotypes (7 de novo, 1 transmitted parental mosaic, 2 transmitted parental heterozygous, and 1 compound heterozygous) as pathogenic and likely contributory to death, a genetic finding in 8.9% of our cohort. Two genes had recurrent missense DNMs, RYR2 and CACNA1C Both RYR2 mutations are pathogenic (P = 1.7 × 10-7) and were previously studied in mouse models. Both CACNA1C mutations lie within a 104-nt exon (P = 1.0 × 10-7) and result in slowed L-type calcium channel inactivation and lower current density. In total, six pathogenic DNMs can alter calcium-related regulation of cardiomyocyte and neuronal excitability at a submembrane junction, suggesting a pathway conferring susceptibility to sudden death. There was a trend for excess LoF mutations in LoF intolerant genes, where ≥1 nonhealthy sample in denovo-db has a similar variant (odds ratio = 6.73, P = 0.02); additional uncharacterized genetic causes of sudden death in children might be discovered with larger cohorts.
PMID: 34930847
ISSN: 1091-6490
CID: 5108732

De novo assembly and delivery to mouse cells of a 101 kb functional human gene

Mitchell, Leslie A; McCulloch, Laura H; Pinglay, Sudarshan; Berger, Henri; Bosco, Nazario; Brosh, Ran; Bulajić, Milica; Huang, Emily; Hogan, Megan S; Martin, James A; Mazzoni, Esteban O; Davoli, Teresa; Maurano, Matthew T; Boeke, Jef D
Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here we describe a workflow for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kilobase pairs (kb). The DNA assembly step is supported by an integrated robotic workcell. We demonstrate assembly of the 101 kb human HPRT1 gene in yeast from 3 kb building blocks, precision delivery of the resulting construct to mouse embryonic stem cells, and subsequent expression of the human protein from its full-length human gene in mouse cells. This workflow provides a framework for mammalian genome writing. We envision utility in producing designer variants of human genes linked to disease and their delivery and functional analysis in cell culture or animal models.
PMID: 33742653
ISSN: 1943-2631
CID: 4821992

Tissue context determines the penetrance of regulatory DNA variation

Halow, Jessica M; Byron, Rachel; Hogan, Megan S; Ordoñez, Raquel; Groudine, Mark; Bender, M A; Stamatoyannopoulos, John A; Maurano, Matthew T
Functional assessment of disease-associated sequence variation at non-coding regulatory elements is complicated by their high degree of context sensitivity to both the local chromatin and nuclear environments. Allelic profiling of DNA accessibility across individuals has shown that only a select minority of sequence variation affects transcription factor (TF) occupancy, yet low sequence diversity in human populations means that no experimental assessment is available for the majority of disease-associated variants. Here we describe high-resolution in vivo maps of allelic DNA accessibility in liver, kidney, lung and B cells from 5 increasingly diverged strains of F1 hybrid mice. The high density of heterozygous sites in these hybrids enables precise quantification of effect size and cell-type specificity for hundreds of thousands of variants throughout the mouse genome. We show that chromatin-altering variants delineate characteristic sensitivity profiles for hundreds of TF motifs. We develop a compendium of TF-specific sensitivity profiles accounting for genomic context effects. Finally, we link maps of allelic accessibility to allelic transcript levels in the same samples. This work provides a foundation for quantitative prediction of cell-type specific effects of non-coding variation on TF activity, which will facilitate both fine-mapping and systems-level analyses of common disease-associated variation in human genomes.
PMID: 33990600
ISSN: 2041-1723
CID: 4868242

Dispersal dynamics of SARS-CoV-2 lineages during the first epidemic wave in New York City

Dellicour, Simon; Hong, Samuel L; Vrancken, Bram; Chaillon, Antoine; Gill, Mandev S; Maurano, Matthew T; Ramaswami, Sitharam; Zappile, Paul; Marier, Christian; Harkins, Gordon W; Baele, Guy; Duerr, Ralf; Heguy, Adriana
During the first phase of the COVID-19 epidemic, New York City rapidly became the epicenter of the pandemic in the United States. While molecular phylogenetic analyses have previously highlighted multiple introductions and a period of cryptic community transmission within New York City, little is known about the circulation of SARS-CoV-2 within and among its boroughs. We here perform phylogeographic investigations to gain insights into the circulation of viral lineages during the first months of the New York City outbreak. Our analyses describe the dispersal dynamics of viral lineages at the state and city levels, illustrating that peripheral samples likely correspond to distinct dispersal events originating from the main metropolitan city areas. In line with the high prevalence recorded in this area, our results highlight the relatively important role of the borough of Queens as a transmission hub associated with higher local circulation and dispersal of viral lineages toward the surrounding boroughs.
PMID: 34015049
ISSN: 1553-7374
CID: 4877512

A versatile platform for locus-scale genome rewriting and verification

Brosh, Ran; Laurent, Jon M; Ordoñez, Raquel; Huang, Emily; Hogan, Megan S; Hitchcock, Angela M; Mitchell, Leslie A; Pinglay, Sudarshan; Cadley, John A; Luther, Raven D; Truong, David M; Boeke, Jef D; Maurano, Matthew T
Routine rewriting of loci associated with human traits and diseases would facilitate their functional analysis. However, existing DNA integration approaches are limited in terms of scalability and portability across genomic loci and cellular contexts. We describe Big-IN, a versatile platform for targeted integration of large DNAs into mammalian cells. CRISPR/Cas9-mediated targeting of a landing pad enables subsequent recombinase-mediated delivery of variant payloads and efficient positive/negative selection for correct clones in mammalian stem cells. We demonstrate integration of constructs up to 143 kb, and an approach for one-step scarless delivery. We developed a staged pipeline combining PCR genotyping and targeted capture sequencing for economical and comprehensive verification of engineered stem cells. Our approach should enable combinatorial interrogation of genomic functional elements and systematic locus-scale analysis of genome function.
PMID: 33649239
ISSN: 1091-6490
CID: 4801272

SARS-CoV-2 genomic characterization and clinical manifestation of the COVID-19 outbreak in Uruguay

Elizondo, Victoria; Harkins, Gordon W; Mabvakure, Batsirai; Smidt, Sabine; Zappile, Paul; Marier, Christian; Maurano, Matthew; Perez, Victoria; Mazza, Natalia; Beloso, Carolina; Ifran, Silvana; Fernandez, Mariana; Santini, Andrea; Perez, Veronica; Estevez, Veronica; Nin, Matilde; Manrique, Gonzalo; Perez, Leticia; Ross, Fabiana; Boschi, Susana; Zubillaga, Maria Noel; Balleste, Raquel; Dellicour, Simon; Heguy, Adriana; Duerr, Ralf
COVID-19 is a respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and declared by the World Health Organization a global public health emergency. Among the severe outbreaks across South America, Uruguay has become known for curtailing SARS-CoV-2 exceptionally well. To understand the SARS-CoV-2 introductions, local transmissions, and associations with genomic and clinical parameters in Uruguay, we sequenced the viral genomes of 44 outpatients and inpatients in a private healthcare system in its capital, Montevideo, from March to May 2020. We performed a phylogeographic analysis using sequences from our cohort and other studies that indicate a minimum of 23 independent introductions into Uruguay, resulting in five major transmission clusters. Our data suggest that most introductions resulting in chains of transmission originate from other South American countries, with the earliest seeding of the virus in late February 2020, weeks before the borders were closed to all non-citizens and a partial lockdown implemented. Genetic analyses suggest a dominance of S and G clades (G, GH, GR) that make up >90% of the viral strains in our study. In our cohort, lethal outcome of SARS-CoV-2 infection significantly correlated with arterial hypertension, kidney failure, and ICU admission (FDR < 0.01), but not with any mutation in a structural or non-structural protein, such as the spike D614G mutation. Our study contributes genetic, phylodynamic, and clinical correlation data about the exceptionally well-curbed SARS-CoV-2 outbreak in Uruguay, which furthers the understanding of disease patterns and regional aspects of the pandemic in Latin America.
PMID: 33306459
ISSN: 2222-1751
CID: 4709432