Cancer genomes are frequently characterized by numerical and structural chromosomal abnormalities. Here we integrated a centromere-specific inactivation approach with selection for a conditionally essential gene, a strategy termed CEN-SELECT, to systematically interrogate the structural landscape of mis-segregated chromosomes. We show that single-chromosome mis-segregation into a micronucleus can directly trigger a broad spectrum of genomic rearrangement types. Cytogenetic profiling revealed that mis-segregated chromosomes exhibit 120-fold-higher susceptibility to developing seven major categories of structural aberrations, including translocations, insertions, deletions, and complex reassembly through chromothripsis coupled to classical non-homologous end joining. Whole-genome sequencing of clonally propagated rearrangements identified random patterns of clustered breakpoints with copy-number alterations resulting in interspersed gene deletions and extrachromosomal DNA amplification events. We conclude that individual chromosome segregation errors during mitotic cell division are sufficient to drive extensive structural variations that recapitulate genomic features commonly associated with human disease.
Subscribe to Journal
Get full journal access for 1 year
only $18.75 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
The data that support the findings of this study are available from the corresponding authors upon request. Whole-genome-sequencing data have been deposited in the European Genome-phenome Archive under accession number EGAD00001004163.
Campbell, P. J. et al. Identification of somatically acquired rearrangements in cancer using genome-wide massively parallel paired-end sequencing. Nat. Genet. 40, 722–729 (2008).
Stephens, P. J. et al. Complex landscapes of somatic rearrangement in human breast cancer genomes. Nature 462, 1005–1010 (2009).
Carvalho, C. M. & Lupski, J. R. Mechanisms underlying structural variant formation in genomic disorders. Nat. Rev. Genet. 17, 224–238 (2016).
Liu, P. et al. An organismal CNV mutator phenotype restricted to early human development. Cell 168, 830–842.e7 (2017).
Redin, C. et al. The genomic landscape of balanced cytogenetic abnormalities associated with human congenital anomalies. Nat. Genet. 49, 36–45 (2017).
Bass, A. J. et al. Genomic sequencing of colorectal adenocarcinomas identifies a recurrent VTI1A-TCF7L2 fusion. Nat. Genet. 43, 964–968 (2011).
Yang, L. et al. Diverse mechanisms of somatic structural variations in human cancer genomes. Cell 153, 919–929 (2013).
Baca, S. C. et al. Punctuated evolution of prostate cancer genomes. Cell 153, 666–677 (2013).
Notta, F. et al. A renewed model of pancreatic cancer evolution based on genomic rearrangement patterns. Nature 538, 378–382 (2016).
Anderson, N. D. et al. Rearrangement bursts generate canonical gene fusions in bone and soft tissue tumors. Science 361, eaam8419 (2018).
Cogen, P. H., Daneshvar, L., Metzger, A. K. & Edwards, M. S. Deletion mapping of the medulloblastoma locus on chromosome 17p. Genomics 8, 279–285 (1990).
Nowell, P. C. The minute chromosome (Phl) in chronic granulocytic leukemia. Blut 8, 65–66 (1962).
Rowley, J. D. Letter: A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining. Nature 243, 290–293 (1973).
de Klein, A. et al. A cellular oncogene is translocated to the Philadelphia chromosome in chronic myelocytic leukaemia. Nature 300, 765–767 (1982).
Gao, Q. et al. Driver fusions and their implications in the development and treatment of human cancers. Cell Rep. 23, 227–238 e3 (2018).
Northcott, P. A. et al. Enhancer hijacking activates GFI1 family oncogenes in medulloblastoma. Nature 511, 428–434 (2014).
Zhang, X. et al. Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers. Nat. Genet. 48, 176–182 (2016).
Hnisz, D. et al. Activation of proto-oncogenes by disruption of chromosome neighborhoods. Science 351, 1454–1458 (2016).
Weischenfeldt, J. et al. Pan-cancer analysis of somatic copy-number alterations implicates IRS4 and IGF2 in enhancer hijacking. Nat. Genet. 49, 65–74 (2017).
Dixon, J. R. et al. Integrative detection and analysis of structural variation in cancer genomes. Nat. Genet. 50, 1388–1398 (2018).
Stephens, P. J. et al. Massive genomic rearrangement acquired in a single catastrophic event during cancer development. Cell 144, 27–40 (2011).
Lombard, D. B. et al. DNA repair, genome stability, and aging. Cell 120, 497–512 (2005).
Lin, C. et al. Nuclear receptor-induced chromosomal proximity and DNA breaks underlie specific translocations in cancer. Cell 139, 1069–1083 (2009).
Henssen, A. G. et al. PGBD5 promotes site-specific oncogenic mutations in human tumors. Nat. Genet. 49, 1005–1014 (2017).
Garaycoechea, J. I. et al. Alcohol and endogenous aldehydes damage chromosomes and mutate stem cells. Nature 553, 171–177 (2018).
Ly, P. & Cleveland, D. W. Rebuilding chromosomes after catastrophe: emerging mechanisms of chromothripsis. Trends Cell Biol. 27, 917–930 (2017).
Janssen, A., van der Burg, M., Szuhai, K., Kops, G. J. & Medema, R. H. Chromosome segregation errors as a cause of DNA damage and structural chromosome aberrations. Science 333, 1895–1898 (2011).
Soto, M. et al. p53 prohibits propagation of chromosome segregation errors that produce structural aneuploidies. Cell Rep. 19, 2423–2431 (2017).
Santaguida, S. et al. Chromosome mis-segregation generates cell-cycle-arrested cells with complex karyotypes that are eliminated by the immune system. Dev. Cell 41, 638–651.e5 (2017).
Crasta, K. et al. DNA breaks and chromosome pulverization from errors in mitosis. Nature 482, 53–58 (2012).
Hatch, E. M., Fischer, A. H., Deerinck, T. J. & Hetzer, M. W. Catastrophic nuclear envelope collapse in cancer cell micronuclei. Cell 154, 47–60 (2013).
Kato, H. & Sandberg, A. A. Chromosome pulverization in human cells with micronuclei. J. Natl Cancer Inst. 40, 165–179 (1968).
Ly, P. et al. Selective Y centromere inactivation triggers chromosome shattering in micronuclei and repair by non-homologous end joining. Nat. Cell Biol. 19, 68–75 (2017).
Zhang, C. Z. et al. Chromothripsis from DNA damage in micronuclei. Nature 522, 179–184 (2015).
Garsed, D. W. et al. The architecture and evolution of cancer neochromosomes. Cancer Cell 26, 653–667 (2014).
Mitchell, T. J. et al. Timing the landmark events in the evolution of clear cell renal cell cancer: TRACERx Renal. Cell 173, 611–623.e17 (2018).
Cortés-Ciriano, I. et al. Comprehensive analysis of chromothripsis in 2,658 human cancers using whole-genome sequencing. Preprint at https://www.biorxiv.org/content/early/2018/05/30/333617 (2018).
Kloosterman, W. P. et al. Chromothripsis as a mechanism driving complex de novo structural rearrangements in the germline. Hum. Mol. Genet. 20, 1916–1924 (2011).
Kloosterman, W. P. et al. Constitutional chromothripsis rearrangements involve clustered double-stranded DNA breaks and nonhomologous repair mechanisms. Cell Rep. 1, 648–655 (2012).
Chiang, C. et al. Complex reorganization and predominant non-homologous repair following chromosomal breakage in karyotypically balanced germline rearrangements and transgenic integration. Nat. Genet. 44, 390–397 (2012).
Weckselblatt, B., Hermetz, K. E. & Rudd, M. K. Unbalanced translocations arise from diverse mutational mechanisms including chromothripsis. Genome Res. 25, 937–947 (2015).
Cretu Stancu, M. et al. Mapping and phasing of structural variation in patient genomes using nanopore sequencing. Nat. Commun. 8, 1326 (2017).
Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T. & Kanemaki, M. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat. Methods 6, 917–922 (2009).
Holland, A. J., Fachinetti, D., Han, J. S. & Cleveland, D. W. Inducible, reversible system for the rapid and complete degradation of proteins in mammalian cells. Proc. Natl Acad. Sci. USA 109, E3350–E3357 (2012).
Sun, C. et al. An azoospermic man with a de novo point mutation in the Y-chromosomal gene USP9Y. Nat. Genet. 23, 429–432 (1999).
Masumoto, H., Masukata, H., Muro, Y., Nozaki, N. & Okazaki, T. A human centromere antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromeric satellite. J. Cell. Biol. 109, 1963–1973 (1989).
Burma, S., Chen, B. P., Murphy, M., Kurimasa, A. & Chen, D. J. ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J. Biol. Chem. 276, 42462–42467 (2001).
Zhao, S. et al. Functional link between ataxia-telangiectasia and Nijmegen breakage syndrome gene products. Nature 405, 473–477 (2000).
Wu, X. et al. ATM phosphorylation of Nijmegen breakage syndrome protein is required in a DNA damage response. Nature 405, 477–482 (2000).
Schultz, L. B., Chehab, N. H., Malikzay, A. & Halazonetis, T. D. p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks. J. Cell Biol. 151, 1381–1390 (2000).
Anderson, L., Henderson, C. & Adachi, Y. Phosphorylation and rapid relocalization of 53BP1 to nuclear foci upon DNA damage. Mol. Cell. Biol. 21, 1719–1729 (2001).
Terradas, M., Martin, M., Tusell, L. & Genesca, A. DNA lesions sequestered in micronuclei induce a local defective-damage response. DNA Repair 8, 1225–1234 (2009).
Miake-Lye, R. & Kirschner, M. W. Induction of early mitotic events in a cell-free system. Cell 41, 165–175 (1985).
Muhlhausser, P. & Kutay, U. An in vitro nuclear disassembly system reveals a role for the RanGTPase system and microtubule-dependent steps in nuclear envelope breakdown. J. Cell Biol. 178, 595–610 (2007).
Soto, M., Garcia-Santisteban, I., Krenning, L., Medema, R. H. & Raaijmakers, J. A. Chromosomes trapped in micronuclei are liable to segregation errors. J. Cell Sci. 131, jcs214742 (2018).
Skaletsky, H. et al. The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature 423, 825–837 (2003).
Santaguida, S., Tighe, A., D’Alise, A. M., Taylor, S. S. & Musacchio, A. Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine. J. Cell Biol. 190, 73–87 (2010).
Li, Y. et al. Constitutional and somatic rearrangement of chromosome 21 in acute lymphoblastic leukaemia. Nature 508, 98–102 (2014).
Willis, N. A. et al. Mechanism of tandem duplication formation in BRCA1-mutant cells. Nature 551, 590–595 (2017).
Nik-Zainal, S. et al. Mutational processes molding the genomes of 21 breast cancers. Cell 149, 979–993 (2012).
Maciejowski, J., Li, Y., Bosco, N., Campbell, P. J. & de Lange, T. Chromothripsis and kataegis induced by telomere crisis. Cell 163, 1641–1654 (2015).
Behjati, S. et al. Recurrent mutation of IGF signalling genes and distinct patterns of genomic rearrangement in osteosarcoma. Nat. Commun. 8, 15936 (2017).
Turner, K. M. et al. Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity. Nature 543, 122–125 (2017).
deCarvalho, A. C. et al. Discordant inheritance of chromosomal and extrachromosomal DNA elements contributes to dynamic disease evolution in glioblastoma. Nat. Genet. 50, 708–717 (2018).
Rausch, T. et al. Genome sequencing of pediatric medulloblastoma links catastrophic DNA rearrangements with TP53 mutations. Cell 148, 59–71 (2012).
Liu, S. et al. Nuclear envelope assembly defects link mitotic errors to chromothripsis. Nature 561, 551–555 (2018).
Truong, L. N. et al. Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells. Proc. Natl Acad. Sci. USA 110, 7720–7725 (2013).
Taylor, B. J. et al. DNA deaminases induce break-associated mutation showers with implication of APOBEC3B and 3A in breast cancer kataegis. eLife 2, e00534 (2013).
Ly, P. & Cleveland, D. W. Interrogating cell division errors using random and chromosome-specific missegregation approaches. Cell Cycle 16, 1252–1258 (2017).
Mardin, B. R. et al. A cell-based model system links chromothripsis with hyperploidy. Mol. Syst. Biol. 11, 828 (2015).
Leibowitz, M. L., Zhang, C. Z. & Pellman, D. Chromothripsis: a new mechanism for rapid karyotype evolution. Annu. Rev. Genet. 49, 183–211 (2015).
Murray, A. W. Cell cycle extracts. Methods Cell Biol. 36, 581–605 (1991).
Shankaran, S. S., Mackay, D. R. & Ullman, K. S. A time-lapse imaging assay to study nuclear envelope breakdown. Methods Mol. Biol. 931, 111–122 (2013).
Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics 26, 589–595 (2010).
Raine, K. M. et al. ascatNgs: identifying somatically acquired copy-number alterations from whole-genome sequencing data. Curr. Protoc. Bioinformatics 56, 15 9 1–15 9 17 (2016).
Nik-Zainal, S. et al. Landscape of somatic mutations in 560 breast cancer whole-genome sequences. Nature 534, 47–54 (2016).
We thank H. Skaletsky for advice on targeting the Y chromosome, P. Rao and T. Senaratne for assistance with initial cytogenetic experiments, P. Mischel for helpful discussions, J. Santini and M. Erb for assistance with super-resolution imaging, the UCSD School of Medicine Microscopy Core (grant no. P30 NS047101) and A. Shiau for shared use of equipment, and the UCSD Animal Care Program for irradiator use. This work was supported by the US National Institutes of Health (grant no. K99 CA218871 to P.L. and no. R35 GM122476 to D.W.C.); the Wellcome Trust (grant no. WT088340MA to P.J.C. and no. 110104/Z/15/Z to S.B.); Hope Funds for Cancer Research (grant no. HFCR-14-06-06 to P.L.); the Swiss National Science Foundation (grant no. P2SKP3-171753 and no. P400PB-180790 to S.F.B.); St. Baldricks Foundation (Robert J. Arceci Innovation Award to S.B.); the NIHR UCLH Biomedical Research Centre, the UCL Experimental Cancer Centre, and the RNOH NHS Trust (to A.M.F., who is an NIHR Senior Investigator); and the Howard Hughes Medical Institute (to D.C.P.). D.W.C. receives salary support from the Ludwig Institute for Cancer Research.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Supplementary Figure 1 CRISPR–Cas9-mediated engineering of a neomycin-resistance gene (NeoR) into the Y-chromosome AZFa locus.
a) Step-by-step outline of genome editing strategies used to generate DLD-1 cells and their corresponding clonal derivatives used in this study. NeoR, neomycin-resistance gene. b) Schematic of CRISPR–Cas9-mediated integration of NeoR into the Y-chromosome AZFa locus. The locations of PCR primers used to identify clones with correct NeoR integration are shown. Forward primers outside the left homology arm (HA) produced a PCR product specific for correct integration. As a control, another set of forward primers within the left HA amplified both correct and non-specific integration events. Coordinates represent reference assembly GRCh38. c) PCR using the primers shown in b or the indicated sequence-tagged site (STS). Four correctly targeted clones were identified, of which the indicated clone was chosen for further modifications, as shown in a. d) Representative colony formation plate scans (n = 3 biological replicates) from 3 independent DLD-1 clones carrying a CENP-AC–H3 rescue gene, which were generated as described in a, following the indicated treatment conditions.
Supplementary Figure 2 Endogenous CENP-A reaccumulates and localizes to the Y-chromosome centromere after doxycycline/auxin washout.
a–c) DLD-1 cells carrying a CENP-AC–H3 rescue gene were treated with or without DOX/IAA followed by washout for the indicated duration. a) Whole-cell lysates were immunoblotted with antibodies against CENP-A or GAPDH as a loading control. b,c) Representative immunofluorescence images using antibodies against CENP-A and GFP on b) interphase cells or c) mitotic cells. Anti-GFP antibody specifically recognizes endogenous, wild-type CENP-A fused to an EYFP-AID tag. Scale bars, 5 µm. d) DLD-1 cells carrying a CENP-AC–H3 rescue gene were treated with DOX/IAA for 3 d followed by 4-d washout and selection in G418 for 10 d. Cells were processed for immunofluorescence with a GFP antibody, followed by DNA FISH using a Y centromere-specific probe. Magnified insets show positive GFP signals detected at the Y centromere. Scale bar, 10 µm.
Supplementary Figure 3 Recognition of micronuclear DNA damage without active DNA double-strand-break repair.
a) Representative immunofluorescence images of DLD-1 cells treated with 1 µM doxorubicin for 1 h or DOX/IAA for 3 d stained with antibodies against the indicated DNA damage components. Scale bar, 5 µm. b) Representative immuno-FISH images of DLD-1 cells treated with 1 µM doxorubicin for 1 h or DOX/IAA for 3 d. Cells were processed for immunofluorescence with γH2AX and 53BP1 antibodies followed by DNA FISH using a Y chromosome paint probe. Scale bar, 5 µm.
Supplementary Figure 4 Most micronuclei disassemble their nuclear envelopes with normal kinetics during mitotic entry.
a) Experimental schematic for nuclear envelope breakdown assay using Xenopus egg extracts, which contain abundant kinases whose activity can disassemble typical nuclear membranes and promote chromosome condensation, the latter visualized by H2B–mRFP. b) Time-lapse imaging of semi-permeabilized RPE-1 cells expressing lamin A–GFP and H2B–mRFP following the addition of interphase or CSF-arrested egg extracts. Scale bar, 5 µm. c) Lamin A–GFP disassembly kinetics following the addition of interphase or CSF-arrested egg extract (mean ± SEM). d-e) Lamin A–GFP disassembly kinetics following CSF-arrested egg extract addition for d) primary nuclei and e) micronuclei. Inset depicts the overlay between primary nuclei and micronuclei with normal disassembly. Green lines represent mean ± SEM, and red lines indicate individual examples of micronuclei with partial or delayed lamin A–GFP disassembly. f) Example image series of a micronucleus that fails to efficiently disassemble its nuclear envelope upon addition of CSF-arrested egg extracts. Scale bar, 2 µm. g) Quantification of the frequency of micronuclei undergoing normal or delayed disassembly in the presence of CSF-arrested egg extracts. Data in c–e and g represent n = number of nuclei or micronuclei as indicated.
Supplementary Figure 5 Schematic for the generation of alternating FISH patterns by chromothripsis or a series of inversions.
(associated with Fig. 2d)
a) Experimental schematic for chronic centromere inactivation. p, passage; p0, starting population; p0’, final passage of cells selected with G418 without Y centromere inactivation. b-c) Measurements of b) cumulative cell doublings and c) population doubling time at each passage. d) Quantification of Y chromosome location from interphase cells hybridized to whole Y chromosome paint probes (n = number of cells examined). e) Quantification of Y chromosome structural status from metaphase spreads hybridized to MSY/YqH FISH probes (n = number of metaphases examined). f) Examples of a rare ring chromosome and uncharacterized derivative chromosome following prolonged CEN-SELECT.
Supplementary Figure 7 Chromosomal rearrangements generated from ionizing-radiation exposure or mitotic spindle assembly checkpoint inactivation.
(associated with Fig. 4a). a-b) Increasing doses of ionizing radiation were used to induce widespread DNA damage, which produced co-localized nuclear foci of γH2AX and 53BP1. DLD-1 cells were treated with the indicated doses of radiation and fixed after recovery for 30 min. Cells were processed for immunofluorescence using antibodies against γH2AX and 53BP1. Quantification of co-localized foci (n = number of cells examined per dose) is shown in a, and representative images are shown in b. Scale bar, 5 µm. c-d) An inhibitor of the Mps1 mitotic kinase (reversine) was used to inactivate the spindle assembly checkpoint during mitosis to drive premature anaphase onset. DLD-1 cells stained with SiR-DNA were treated with the indicated doses of reversine and imaged by time-lapse microscopy. Quantification of mitotic duration is shown in c (n = number of cells examined per dose; NEBD, nuclear envelope breakdown), and the frequency of mitotic events developing chromosome segregation errors is shown in d. At the highest concentration used (800 nM), nearly half of mitoses resulted in errors with micronuclei detected in ~10% of cells. e) Experimental schematic for Fig. 4a. f) Examples of irradiated cells showing structural rearrangements for the indicated chromosomes by metaphase FISH. Scale bar, 5 µm. g) Sample sizes for each condition and set of probes for Fig. 4a. h) The total number of inter- and intra-chromosomal rearrangements observed for the indicated conditions.
Supplementary Figure 8 Molecular sequence features of chromosomal-rearrangement breakpoint junctions.
a) Bar graph depicting the total number of structural variants detected per chromosome normalized to chromosome size and copy-number. Data were pooled from all 20 sequenced clones. Inner pie chart shows the distribution of each Y-Y junction orientation. b) Scatter plot depicting the absolute number of structural variants per chromosome compared to its length. c) Distribution of 71 breakpoints reconstructed from 817 split-end reads with the indicated lengths of microhomology for Y-chromosome specific or non-Y chromosome junctions compared to microhomology lengths from 7,100 randomly simulated junctions (see Methods). Data were compiled from all sequenced clones. d) Distribution of microhomology or insertion lengths with each dot representing an individually reconstructed Y-Y breakpoint junction. e) Schematic of intra-DSB distance measurements (left) and distribution of Y chromosome fragment sizes pooled across all sequenced clones (right). f) Rainfall plot of substitution patterns across the MSY region from four clones. Red dots below the line represent sites of consecutive C>T basepair substitutions with a 1 kb intermutation distance.
Supplementary Figure 9 Cytogenetic verification of rearrangement partners identified by whole-genome sequencing.
Representative metaphase FISH images of the indicated clones hybridized to the corresponding chromosome paint probes. Scale bar, 5 µm.
Supplementary Figure 10 Y chromosome rearrangement and DNA copy-number profiles of additional clones.
Red vertical line indicates position of the integrated NeoR gene. All three inter-chromosomal translocations shown were confirmed by metaphase FISH. X-axes are clipped at 30 Mb to exclude the Yq heterochromatic region.
Supplementary Figure 11 Patients with osteosarcoma presenting complex Y chromosome rearrangements coupled to interchromosomal translocations.
a–c) X-axes are clipped at 30 Mb to exclude the Yq heterochromatic region. Vertical grey lines represent inter-chromosomal rearrangements with a) chromosome 11, b) chromosomes 1, 19, and 22, and c) chromosome 15. d) Distribution of each Y-Y junction orientation (n = number of breakpoint junctions). e) Distribution of microhomology or insertion lengths with each dot representing an individually reconstructed Y-Y breakpoint junction. f) Distribution of intra-DSB distances pooled across all three examples.
a) DNA copy-number plots for KDM5D and EIF1AY across all clones obtained by sequencing. b) Magnified DNA copy-number profiles at the KDM5D or EIF1AY gene regions, represented by red shading, in the indicated clones. c) Reverse transcription polymerase chain reaction (RT–PCR) analysis of KDM5D, EIF1AY, or GAPDH transcripts from the indicated clones or female HEK-293 cells as a negative control. Bar graphs show the copy-number status of the respective gene.
Supplementary Figures 1–12 and Supplementary Note
Summary of DLD-1 clones analysed by cytogenetics and whole-genome sequencing.
Breakpoint junctions with microhomology or non-templated DNA insertion sequences.
List of primer sequences.
Normal micronuclear envelope disassembly during mitosis.
Aberrant micronuclear envelope disassembly during mitosis.
About this article
Cite this article
Ly, P., Brunner, S.F., Shoshani, O. et al. Chromosome segregation errors generate a diverse spectrum of simple and complex genomic rearrangements. Nat Genet 51, 705–715 (2019) doi:10.1038/s41588-019-0360-8