Abstract

Heterogeneity in early language development in autism spectrum disorder (ASD) is clinically important and may reflect neurobiologically distinct subtypes. Here, we identified a large-scale association between multiple coordinated blood leukocyte gene coexpression modules and the multivariate functional neuroimaging (fMRI) response to speech. Gene coexpression modules associated with the multivariate fMRI response to speech were different for all pairwise comparisons between typically developing toddlers and toddlers with ASD and poor versus good early language outcome. Associated coexpression modules were enriched in genes that are broadly expressed in the brain and many other tissues. These coexpression modules were also enriched in ASD-associated, prenatal, human-specific, and language-relevant genes. This work highlights distinctive neurobiology in ASD subtypes with different early language outcomes that is present well before such outcomes are known. Associations between neuroimaging measures and gene expression levels in blood leukocytes may offer a unique in vivo window into identifying brain-relevant molecular mechanisms in ASD.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Data availability

The raw data that support the findings from this study are publicly available from the NIH National Database for Autism Research (NDAR). Raw blood leukocyte gene expression data are publicly available via Gene Expression Omnibus (GEO) (GSE42133; GSE111175). Songbird Area X gene expression data are publicly available via GEO (GSE34819). GTEx data are publicly available at https://gtexportal.org/. ASD postmortem cortical gene expression can be found at https://github.com/dhglab/Genome-wide-changes-in-lncRNA-alternative-splicing-and-cortical-patterning-in-autism/.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

    Geschwind, D. H. & Levitt, P. Autism spectrum disorders: developmental disconnection syndromes. Curr. Opin. Neurobiol. 17, 103–111 (2007).

  2. 2.

    Happé, F., Ronald, A. & Plomin, R. Time to give up on a single explanation for autism. Nat. Neurosci. 9, 1218–1220 (2006).

  3. 3.

    Lai, M. C., Lombardo, M. V., Chakrabarti, B. & Baron-Cohen, S. Subgrouping the autism “spectrum”: reflections on DSM-5. PLoS Biol. 11, e1001544 (2013).

  4. 4.

    Kapur, S., Phillips, A. G. & Insel, T. R. Why has it taken so long for biological psychiatry to develop clinical tests and what to do about it? Mol. Psychiatry 17, 1174–1179 (2012).

  5. 5.

    Lombardo, M. V., Lai, M. C. & Baron-Cohen, S. Big data approaches to decomposing heterogeneity across the autism spectrum. Preprint at https://www.biorxiv.org/content/early/2018/03/17/278788 (2018).

  6. 6.

    Kjelgaard, M. M. & Tager-Flusberg, H. An investigation of language impairment in autism: implications for genetic subgroups. Lang. Cogn. Process. 16, 287–308 (2001).

  7. 7.

    Tager-Flusberg, H. & Kasari, C. Minimally verbal school-aged children with autism spectrum disorder: the neglected end of the spectrum. Autism Res. 6, 468–478 (2013).

  8. 8.

    Howlin, P. Outcome in high-functioning adults with autism with and without early language delays: implications for the differentiation between autism and Asperger syndrome. J. Autism. Dev. Disord. 33, 3–13 (2003).

  9. 9.

    Perry, A. et al. Predictors of outcome for children receiving intensive behavioral intervention in a large, community-based program. Res. Autism Spectr. Disord. 5, 592–603 (2011).

  10. 10.

    Gotham, K., Pickles, A. & Lord, C. Trajectories of autism severity in children using standardized ADOS scores. Pediatrics 130, e1278–e1284 (2012).

  11. 11.

    Venter, A., Lord, C. & Schopler, E. A follow-up study of high-functioning autistic children. J. Child Psychol. Psychiatry 33, 489–507 (1992).

  12. 12.

    Szatmari, P. et al. Similar developmental trajectories in autism and Asperger syndrome: from early childhood to adolescence. J. Child Psychol. Psychiatry 50, 1459–1467 (2009).

  13. 13.

    Courchesne, E. et al. TheASD Living Biology: from cell proliferation to clinical phenotype. Mol. Psychiatry https://doi.org/10.1038/s41380-018-0056-y (2018).

  14. 14.

    Pramparo, T. et al. Cell cycle networks link gene expression dysregulation, mutation, and brain maldevelopment in autistic toddlers. Mol. Syst. Biol. 11, 841 (2015).

  15. 15.

    Pramparo, T. et al. Prediction of autism by translation and immune/inflammation coexpressed genes in toddlers from pediatric community practices. JAMA Psychiatry 72, 386–394 (2015).

  16. 16.

    Geschwind, D. H. & State, M. W. Gene hunting in autism spectrum disorder: on the path to precision medicine. Lancet. Neurol. 14, 1109–1120 (2015).

  17. 17.

    Sandin, S. et al. The heritability of autism spectrum disorder. J. Am. Med. Assoc. 318, 1182–1184 (2017).

  18. 18.

    Battle, A., Brown, C. D., Engelhardt, B. E. & Montgomery, S. B. Genetic effects on gene expression across human tissues. Nature 550, 204–213 (2017).

  19. 19.

    Lombardo, M. V. et al. Different functional neural substrates for good and poor language outcome in autism. Neuron 86, 567–577 (2015).

  20. 20.

    Boyle, E. A., Li, Y. I. & Pritchard, J. K. An expanded view of complex traits: from polygenic to omnigenic. Cell 169, 1177–1186 (2017).

  21. 21.

    Wray, N. R., Wijmenga, C., Sullivan, P. F., Yang, J. & Visscher, P. M. Common disease is more complex than implied by the core gene omnigenic model. Cell 173, 1573–1580 (2018).

  22. 22.

    Cremers, H. R., Wager, T. D. & Yarkoni, T. The relation between statistical power and inference in fMRI. PLoS ONE. 12, e0184923 (2017).

  23. 23.

    Langfelder, P. & Horvath, S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 9, 559 (2008).

  24. 24.

    Hickok, G. & Poeppel, D. The cortical organization of speech processing. Nat. Rev. Neurosci. 8, 393–402 (2007).

  25. 25.

    Konopka, G. & Roberts, T. F. Insights into the neural and genetic basis of vocal communication. Cell 164, 1269–1276 (2016).

  26. 26.

    Pfenning, A. R. et al. Convergent transcriptional specializations in the brains of humans and song-learning birds. Science 346, 1256846 (2014).

  27. 27.

    Kelleher, R. J. III & Bear, M. F. The autistic neuron: troubled translation? Cell 135, 401–406 (2008).

  28. 28.

    De Rubeis, S. et al. Synaptic, transcriptional and chromatin genes disrupted in autism. Nature 515, 209–215 (2014).

  29. 29.

    Bernier, R. et al. Disruptive CHD8 mutations define a subtype of autism early in development. Cell 158, 263–276 (2014).

  30. 30.

    Morgan, J. T. et al. Microglial activation and increased microglial density observed in the dorsolateral prefrontal cortex in autism. Biol. Psychiatry 68, 368–376 (2010).

  31. 31.

    Vargas, D. L., Nascimbene, C., Krishnan, C., Zimmerman, A. W. & Pardo, C. A. Neuroglial activation and neuroinflammation in the brain of patients with autism. Ann. Neurol. 57, 67–81 (2005).

  32. 32.

    Voineagu, I. et al. Transcriptomic analysis of autistic brain reveals convergent molecular pathology. Nature 474, 380–384 (2011).

  33. 33.

    Hilliard, A. T., Miller, J. E., Fraley, E. R., Horvath, S. & White, S. A. Molecular microcircuitry underlies functional specification in a basal ganglia circuit dedicated to vocal learning. Neuron 73, 537–552 (2012).

  34. 34.

    Konopka, G. et al. Human-specific transcriptional networks in the brain. Neuron 75, 601–617 (2012).

  35. 35.

    Liu, X. et al. Disruption of an evolutionarily novel synaptic expression pattern in autism. PLoS Biol. 14, e1002558 (2016).

  36. 36.

    Eising, E. et al. A set of regulatory genes co-expressed in embryonic human brain is implicated in disrupted speech development. Mol. Psychiatry https://doi.org/10.1038/s41380-018-0020-x (2018).

  37. 37.

    Parikshak, N. N. et al. Integrative functional genomic analyses implicate specific molecular pathways and circuits in autism. Cell 155, 1008–1021 (2013).

  38. 38.

    Willsey, A. J. et al. Coexpression networks implicate human midfetal deep cortical projection neurons in the pathogenesis of autism. Cell 155, 997–1007 (2013).

  39. 39.

    Miller, J. A. et al. Transcriptional landscape of the prenatal human brain. Nature 508, 199–206 (2014).

  40. 40.

    Parikshak, N. N. et al. Genome-wide changes in lncRNA, splicing, and regional gene expression patterns in autism. Nature 540, 423–427 (2016).

  41. 41.

    Rossignol, D. A. & Frye, R. E. Mitochondrial dysfunction in autism spectrum disorders: a systematic review and meta-analysis. Mol. Psychiatry 17, 290–314 (2012).

  42. 42.

    Sanders, S. J. et al. Insights into autism spectrum disorder genomic architecture and biology from 71 risk loci. Neuron 87, 1215–1233 (2015).

  43. 43.

    Marchetto, M. C. N. et al. Differential L1 regulation in pluripotent stem cells of humans and apes. Nature 503, 525–529 (2013).

  44. 44.

    Nuttle, X. et al. Emergence of a Homo sapiens–specific gene family and chromosome 16p11.2 CNV susceptibility. Nature 536, 205–209 (2016).

  45. 45.

    Hippolyte, L. et al. The number of genomic copies at the 16p11.2 locus modulates language, verbal memory, and inhibition. Biol. Psychiatry 80, 129–139 (2016).

  46. 46.

    Demopoulos, C. et al. Abnormal speech motor control in individuals with 16p11.2 deletions. Sci. Rep. 8, 1274 (2018).

  47. 47.

    Berman, J. I. et al. Abnormal auditory and language pathways in children with 16p11.2 deletion. Neuroimage Clin. 9, 50–57 (2015).

  48. 48.

    Kosmicki, J. A. et al. Refining the role of de novo protein-truncating variants in neurodevelopmental disorders by using population reference samples. Nat. Genet. 49, 504–510 (2017).

  49. 49.

    Lek, M. et al. Analysis of protein-coding genetic variation in 60,706 humans. Nature 536, 285–291 (2016).

  50. 50.

    Abrahams, B. S. et al. SFARI Gene 2.0: a community-driven knowledgebase for the autism spectrum disorders (ASDs). Mol. Autism 4, 36 (2013).

  51. 51.

    Pierce, K., Conant, D., Hazin, R., Stoner, R. & Desmond, J. Preference for geometric patterns early in life as a risk factor for autism. Arch. Gen. Psychiatry 68, 101–109 (2011).

  52. 52.

    Pierce, K. et al. Eye tracking reveals abnormal visual preference for geometric images as an early biomarker of an autism spectrum disorder subtype associated with increased symptom severity. Biol. Psychiatry 79, 657–666 (2016).

  53. 53.

    Pierce, K. et al. Detecting, studying, and treating autism early: the One-Year Well-Baby Check-Up Approach. J. Pediatr. 159, 458–465.e6 (2011).

  54. 54.

    Wetherby, A. & Prizant, B. Communication and Symbolic Behavior Scales Developmental Profile, First Normed Edition (Paul H. Brookes, Baltimore, 2002).

  55. 55.

    Wetherby, A. M., Brosnan-Maddox, S., Peace, V. & Newton, L. Validation of the Infant-Toddler Checklist as a broadband screener for autism spectrum disorders from 9 to 24 months of age. Autism 12, 487–511 (2008).

  56. 56.

    Lord, C. et al. The autism diagnostic observation schedule-generic: a standard measure of social and communication deficits associated with the spectrum of autism. J. Autism. Dev. Disord. 30, 205–223 (2000).

  57. 57.

    Mullen, E. M. Mullen Scales of Early Learning (American Guidance Service, Circle Pine, MN, USA, 1995).

  58. 58.

    Sparrow, S., Cicchetti, D. & Balla, D. Vineland-II Scales Of Adaptive Behavior: Survey Form Manual (American Guidance Service, Circle Pines, MN, USA, 2005).

  59. 59.

    Schroeder, A. et al. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC. Mol. Biol. 7, 3 (2006).

  60. 60.

    Du, P., Kibbe, W. A. & Lin, S. M. lumi: a pipeline for processing Illumina microarray. Bioinformatics 24, 1547–1548 (2008).

  61. 61.

    Gentleman, R. C. et al. Bioconductor: open software development for computational biology and bioinformatics. Genome. Biol. 5, R80 (2004).

  62. 62.

    Chikina, M., Zaslavsky, E. & Sealfon, S. C. CellCODE: a robust latent variable approach to differential expression analysis for heterogeneous cell populations. Bioinformatics 31, 1584–1591 (2015).

  63. 63.

    Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).

  64. 64.

    Storey, J. D. A direct approach to false discovery rates. J. R. Statist. Soc. B 64, 479–498 (2002).

  65. 65.

    Zhang, B. & Horvath, S. A general framework for weighted gene co-expression network analysis. Stat. Appl. Genet. Mol. Biol. 4, Article17 (2005).

  66. 66.

    Oldham, M. C., Horvath, S. & Geschwind, D. H. Conservation and evolution of gene coexpression networks in human and chimpanzee brains. Proc. Natl Acad. Sci. USA 103, 17973–17978 (2006).

  67. 67.

    Langfelder, P., Luo, R., Oldham, M. C. & Horvath, S. Is my network module preserved and reproducible? PLoS. Comput. Biol. 7, e1001057 (2011).

  68. 68.

    Eyler, L. T., Pierce, K. & Courchesne, E. A failure of left temporal cortex to specialize for language is an early emerging and fundamental property of autism. Brain 135, 949–960 (2012).

  69. 69.

    Redcay, E. & Courchesne, E. Deviant functional magnetic resonance imaging patterns of brain activity to speech in 2-3-year-old children with autism spectrum disorder. Biol. Psychiatry 64, 589–598 (2008).

  70. 70.

    Redcay, E., Haist, F. & Courchesne, E. Functional neuroimaging of speech perception during a pivotal period in language acquisition. Dev. Sci. 11, 237–252 (2008).

  71. 71.

    Dehaene-Lambertz, G., Dehaene, S. & Hertz-Pannier, L. Functional neuroimaging of speech perception in infants. Science 298, 2013–2015 (2002).

  72. 72.

    Genovese, C. R., Lazar, N. A. & Nichols, T. Thresholding of statistical maps in functional neuroimaging using the false discovery rate. Neuroimage 15, 870–878 (2002).

  73. 73.

    Yarkoni, T., Poldrack, R. A., Nichols, T. E., Van Essen, D. C. & Wager, T. D. Large-scale automated synthesis of human functional neuroimaging data. Nat. Methods 8, 665–670 (2011).

  74. 74.

    Krishnan, A., Williams, L. J., McIntosh, A. R. & Abdi, H. Partial Least Squares (PLS) methods for neuroimaging: a tutorial and review. Neuroimage 56, 455–475 (2011).

  75. 75.

    McIntosh, A. R. & Lobaugh, N. J. Partial least squares analysis of neuroimaging data: applications and advances. Neuroimage 23, S250–S263 (2004).

  76. 76.

    Cotney, J. et al. The autism-associated chromatin modifier CHD8 regulates other autism risk genes during human neurodevelopment. Nat. Commun. 6, 6404 (2015).

  77. 77.

    Darnell, J. C. et al. FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism. Cell 146, 247–261 (2011).

  78. 78.

    Sugathan, A. et al. CHD8 regulates neurodevelopmental pathways associated with autism spectrum disorder in neural progenitors. Proc. Natl Acad. Sci. USA 111, E4468–E4477 (2014).

  79. 79.

    Greenblatt, E. J. & Spradling, A. C. Fragile X mental retardation 1 gene enhances the translation of large autism-related proteins. Science 361, 709–712 (2018).

Download references

Acknowledgements

This research was supported by grants to E.C. and K.P. (NIMH R01-MH080134 (K.P.), NIMH R01-MH104446 (K.P.), NFAR grant (K.P.), NIMH Autism Center of Excellence grant P50-MH081755 (E.C. and K.P.), NIMH R01-MH036840 (E.C.), NIMH R01-MH110558 (E.C. and N.E.L.), NIMH U01-MH108898 (E.C.), NIDCD R01-DC016385 (E.C., K.P., and M.V.L.), CDMRP AR130409 (E.C.), and Simons Foundation 176540 (E.C.)). The work was additionally supported by an ERC Starting Grant (ERC-2017-STG; 755816) to M.V.L. and a grant from the Brain & Behavior Research Foundation (NARSAD) to T.P. We thank R. Znamirowski, C. Ahrens-Barbeau, S. Solso, K. Campbell, M. Mayo, and J. Young for help with data collection, and S. Spendlove and M. Weinfeld for assistance with clinical characterization of subjects.

Author information

Affiliations

  1. Department of Psychology, University of Cyprus, Nicosia, Cyprus

    • Michael V. Lombardo
  2. Autism Research Centre, Department of Psychiatry, University of Cambridge, Cambridge, UK

    • Michael V. Lombardo
    • , Varun Warrier
    •  & Richard A. I. Bethlehem
  3. Department of Neuroscience, University of California, San Diego, La Jolla, CA, USA

    • Tiziano Pramparo
    • , Cynthia Carter Barnes
    • , Linda Lopez
    • , Karen Pierce
    •  & Eric Courchesne
  4. Department of Pediatrics, University of California, San Diego, La Jolla, CA, USA

    • Vahid Gazestani
    •  & Nathan E. Lewis
  5. Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA

    • Nathan E. Lewis
  6. Novo Nordisk Foundation Center for Biosustainability at the University of California, San Diego, La Jolla, CA, USA

    • Nathan E. Lewis
  7. Department of Psychiatry, University of California, San Diego, La Jolla, CA, USA

    • Lisa Eyler
  8. Veterans Affairs San Diego Healthcare System, San Diego, CA, USA

    • Lisa Eyler

Authors

  1. Search for Michael V. Lombardo in:

  2. Search for Tiziano Pramparo in:

  3. Search for Vahid Gazestani in:

  4. Search for Varun Warrier in:

  5. Search for Richard A. I. Bethlehem in:

  6. Search for Cynthia Carter Barnes in:

  7. Search for Linda Lopez in:

  8. Search for Nathan E. Lewis in:

  9. Search for Lisa Eyler in:

  10. Search for Karen Pierce in:

  11. Search for Eric Courchesne in:

Contributions

E.C., K.P., L.E., M.V.L., and T.P. conceived the idea and designed the study. M.V.L. conceived and performed all analyses. T.P., V.G., V.W., R.A.I.B., and N.E.L. aided in data analyses. E.C., K.P., L.E., L.L., and C.C.B. collected data. E.C., K.P., L.E., N.E.L., T.P., and M.V.L. obtained grant funding. M.V.L. and E.C. wrote the manuscript. All authors contributed to editing the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Michael V. Lombardo or Eric Courchesne.

Integrated supplementary information

  1. Supplementary Figure 1 Preservation of ASD blood leukocyte modules in ASD frontal and temporal cortical tissue.

    Zsummary preservation statistics for all ASD blood leukocyte modules. Zsummary > 10 indicates strong preservation, while 2 < Zsummary < 10 indicates moderate preservation. Non-zero modules M2, M8, and M11 are annotated on this plot, since they show Zsummary > 2. M2 has Zsummary = 8.1, which indicates high-moderate preservation.

  2. Supplementary Figure 2 Enrichments between non-zero and zero modules with vocal learning, human-specific, and ASD-associated gene lists after broadly expressed genes are removed.

    Numbers within the cells indicate the enrichment odds ratio, while the color indicates the -log10(p-value). Cells highlighted in green pass multiple comparison correction at FDR q < 0.05.

  3. Supplementary Figure 3 Soft power, TOM dendrogram, and module preservation plots for comparisons of each group.

    Panel A shows the soft power plot for the main WGCNA analysis including data from all groups. A horizontal red line depicts soft power topology model fit R2 of 0.9, where the chosen soft power of 16 is located. Panel B shows the TOM dendrogram with modules labeled at the bottom. Panel C shows the module preservation Zsummary statistic for WGCNA analyses run separately on each group in order to show that networks are highly preserved (Zsummary > 10) across groups.

  4. Supplementary Figure 4 Plots of module eigengenes for all modules and all groups.

    For all comparisons shown below, the sample sizes are TD n = 37 (blue), ASD Good n = 40 (pink), ASD Poor n = 41 (green). The box in the boxplots indicates the interquartile range (IQR; Q1 indicates the 25th, while Q3 indicates the 75th percentile) and the whiskers indicate Q1-(1.5*IQR) or Q3 + (1.5*IQR). The line within the box represents the median. The y-axis indicates the module eigengene, while the x-axis shows each group.

  5. Supplementary Figure 5 Module preservation between groups.

    This figure shows the module preservation Zsummary statistic for WGCNA analyses run separately on each group in order to show that networks are highly preserved (Zsummary > 10) across groups.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–5

  2. Reporting Summary

  3. Supplementary Table 1

    Statistics for linear mixed effect model analysis of clinical trajectory differences. Sheet 1 (Trajectories_ANOVA_allGrps) shows the F-statistic and p-values for main effects of Age and Group and the Age*Group interaction. Sheets 2–4 are the same F-statistic and p-values for analyses on pairwise group comparisons. Any comparison passing multiple comparison correction at FDR q<0.05 is highlighted in red

  4. Supplementary Table 2

    Statistics for ROI analyses. Sheet 1 called “ROI_ANOVAstats” are the ANOVA statistics from ROI analyses. Sheet 2 called “ROI_PairwiseGroupComparisons” are the statistics for the pairwise group-comparisons for ROI analyses

  5. Supplementary Table 3

    Differential expression analysis results. Sheet 1 called “DE_subtypes_genes” shows results from differential expression analysis at the gene-level. The main model evaluates the effect of group (TD, ASD Good, ASD Poor) with an F-test and corresponding p-value. The FDR q-value is also shown for each gene. No gene passes for a statistically significant of group at FDR q<0.05. All other columns in Sheet 1 show the pairwise group comparisons (t-statistic, p-value, and FDR q per each gene). Sheet 2 called “DE_subtypes_ME” shows results from differentially expression analysis on module eigengenes from the WGCNA analysis. The main model evaluated is the same model as the gene-level DE analysis, testing an effect of group (TD, ASD Good, ASD Poor), and the remaining columns show statistics for each pairwise group comparison. Just like the DE analysis at the gene-level, no modules show statistically significant effects of group surviving FDR q<0.05

  6. Supplementary Table 4

    Statistics from PLS analyses. Sheet 1 (“Subtype_PLS”) shows statistics for the PLS model that included TD and ASD subtypes (ASD Good and ASD Poor) as separate groups. Sheet 2 (“CaseCtrl_PLS”) shows statistics for the PLS model that included TD and ASD as the main group distinction (with no distinction for ASD language outcome subtypes). Sheet 3 (“Subtype_PLS_LV1_moduleCorr”) shows the fMRI-gene co-expression correlations and 95% confidence intervals for each group and each module and indicates which modules are non-zero or not

  7. Supplementary Table 5

    Biological process enrichment results for each module. Each sheet shows the results of biological process enrichment tests for each module from MetaCore GeneGO

  8. Supplementary Table 6

    Lists of genes within each module along with module membership statistics for each module. Each row shows a gene and each column shows the module membership value of that gene to each module. The column labeled “Module” shows the module label assigned to each gene

  9. Supplementary Table 7

    Demographic and clinical behavioral variables summary. This table shows demographic information such as sample size, sex, age, and descriptive statistics (mean with standard deviation in parentheses) for each clinical variable at the intake and outcome assessments

  10. Supplementary Table 8

    ANOVA statistics for cell type surrogate proportion variables estimated with CellCODE. Surrogate proportion variables of different leukocyte cell types were estimated with CellCODE and tested for a group difference with an ANOVA. The table reports the F-statistic and p-value for the group effect from these ANOVAs

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/s41593-018-0281-3