Genetic regulation of OAS1 nonsense-mediated decay underlies association with COVID-19 hospitalization in patients of European and African ancestries

  1. A. Rouf Banday
  2. Megan L. Stanifer
  3. Oscar Florez-Vargas
  4. Olusegun O. Onabajo
  5. Brenen W. Papenberg
  6. Muhammad A. Zahoor
  7. Lisa Mirabello
  8. Timothy J. Ring
  9. Chia-Han Lee
  10. Paul S. Albert
  11. Evangelos Andreakos
  12. Evgeny Arons
  13. Greg Barsh
  14. Leslie G. Biesecker
  15. David L. Boyle
  16. Mark S. Brahier
  17. Andrea Burnett-Hartman
  18. Mary Carrington
  19. Euijin Chang
  20. Pyoeng Gyun Choe
  21. Rex L. Chisholm
  22. Leandro M. Colli
  23. Clifton L. Dalgard
  24. Carolynn M. Dude
  25. Jeff Edberg
  26. Nathan Erdmann
  27. Heather S. Feigelson
  28. Benedito A. Fonseca
  29. Gary S. Firestein
  30. Adam J. Gehring
  31. Cuncai Guo
  32. Michelle Ho
  33. Steven Holland
  34. Amy A. Hutchinson
  35. Hogune Im
  36. Les’Shon Irby
  37. Michael G. Ison
  38. Naima T. Joseph
  39. Hong Bin Kim
  40. Robert J. Kreitman
  41. Bruce R. Korf
  42. Steven M. Lipkin
  43. Siham M. Mahgoub
  44. Iman Mohammed
  45. Guilherme L. Paschoalini
  46. Jennifer A. Pacheco
  47. Michael J. Peluso
  48. Daniel J. Rader
  49. David T. Redden
  50. Marylyn D. Ritchie
  51. Brooke Rosenblum
  52. M. Elizabeth Ross
  53. Hanaisa P. Sant Anna
  54. Sharon A. Savage
  55. Sudha Sharma
  56. Eleni Siouti
  57. Alicia K. Smith
  58. Vasiliki Triantafyllia
  59. Joselin M. Vargas
  60. Jose D. Vargas
  61. Anurag Verma
  62. Vibha Vij
  63. Duane R. Wesemann
  64. Meredith Yeager
  65. Xu Yu
  66. Yu Zhang
  67. Steeve Boulant
  68. Stephen J. Chanock
  69. Jordan J. Feld
  70. Ludmila Prokunina-Olsson

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Abstract

The chr12q24.13 locus encoding OAS1–OAS3 antiviral proteins has been associated with coronavirus disease 2019 (COVID-19) susceptibility. Here, we report genetic, functional and clinical insights into this locus in relation to COVID-19 severity. In our analysis of patients of European ( n  = 2,249) and African ( n  = 835) ancestries with hospitalized versus nonhospitalized COVID-19, the risk of hospitalized disease was associated with a common OAS1 haplotype, which was also associated with reduced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) clearance in a clinical trial with pegIFN-λ1. Bioinformatic analyses and in vitro studies reveal the functional contribution of two associated OAS1 exonic variants comprising the risk haplotype. Derived human-specific alleles rs10774671-A and rs1131454 -A decrease OAS1 protein abundance through allele-specific regulation of splicing and nonsense-mediated decay (NMD). We conclude that decreased OAS1 expression due to a common haplotype contributes to COVID-19 severity. Our results provide insight into molecular mechanisms through which early treatment with interferons could accelerate SARS-CoV-2 clearance and mitigate against severe COVID-19.

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  1. Version published to 10.1038/s41588-022-01113-z
  2. ScreenIT

    SciScore for 10.1101/2021.07.09.21260221: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    RandomizationColocalization and correlation coefficients between OAS1 and Golgin-97 expression were generated with LSM700 Zen software by analyzing randomly imaged fields of view (5-7 fields) containing at least 7 cells from IFNβ-treated wells.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationAuthentication: Cell lines were either used within 6 months after purchase or periodically authenticated by microsatellite fingerprinting (AmpFLSTR Identifiler, ThermoFisher) by the Cancer Genomics Research Laboratory/DCEG/NCI.
    Contamination: All cell lines were regularly tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection kit (Lonza)

    Table 2: Resources

    Antibodies
    SentencesResources
    Fixed cells were incubated with mouse anti-Golgin-97 antibody (1:250 dilution, ThermoFisher, A-21270) for 3 hrs at room temperature, washed, and then stained with anti-rabbit Alexa Fluor 488 (1:500 dilution, ThermoFisher, A21202).
    anti-Golgin-97
    suggested: (Thermo Fisher Scientific Cat# A-21270, RRID:AB_221447)
    Cells were then incubated with rabbit anti-OAS1 antibody (1:100 dilution, ThermoFisher, PA5-82113) overnight, washed, and stained with anti-rabbit Alexa Fluor 680 (1:500 dilution, ThermoFisher, A10043).
    anti-rabbit
    suggested: (Thermo Fisher Scientific Cat# A10043, RRID:AB_2534018)
    Blots were blocked in 2.5% milk in 1% TBS-Tween before staining with rabbit anti-OAS1 antibody (1:200, Thermo Fisher, PA5-82113) and rabbit anti-GAPDH antibody (1:500, Abcam, ab9485).
    anti-OAS1
    suggested: (Thermo Fisher Scientific Cat# PA5-82113, RRID:AB_2789274)
    PA5-82113
    suggested: (Thermo Fisher Scientific Cat# PA5-82113, RRID:AB_2789274)
    anti-GAPDH
    suggested: (Abcam Cat# ab9485, RRID:AB_307275)
    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV-2 infections: SARS-CoV-2 (strain BavPat1) was obtained from the European Virology Archive, amplified in Vero E6 cells, and used at passage 3.
    Vero E6
    suggested: RRID:CVCL_XD71)
    A549-ACE2 cells (2.0×105 cells) were seeded in 12-well plates 24 hrs before transfection.
    A549-ACE2
    suggested: None
    The same procedure was applied to analyze Hi-C data for THP-1 monocytic cell line untreated or treated with INFb for 6 hrs.
    THP-1
    suggested: CLS Cat# 300356/p804_THP-1, RRID:CVCL_0006)
    The A549 and T24 cells were seeded in a 12-well plate at a cell density of 2×105 and transfected after 24 hrs with 200 ng of allele-specific mini-genes using Lipofectamine 3000 transfection reagent (Invitrogen) in 3 biological replicates.
    A549
    suggested: None
    Oxford Nanopore RNA-seq: A549 or HT1376 cells (2×106 per sample) were seeded in T25 flasks overnight.
    HT1376
    suggested: None
    Analysis of nonsense-mediated decay (NMD) of OAS1 isoforms: We downloaded RNA-seq data for HeLa cells (OAS1-p42 expressing) with and without siRNA-mediated knockdown of NMD genes – SMG6 and SMG7 (SRA:PRJNA340370).
    HeLa
    suggested: None
    Western blotting: Cells (Caco2, HT1376, A549, and HBEC) were plated in 6-well plates (
    Caco2
    suggested: None
    Recombinant DNA
    SentencesResources
    Cells were transfected with the indicated GFP or OAS1 plasmids using Lipofectamine 2000.
    OAS1
    suggested: None
    These fragments were cloned in sense orientation in Exontrap vector pET01 (MoBiTec) using XhoI and NotI restriction sites and validated by Sanger sequencing.
    pET01
    suggested: None
    Software and Algorithms
    SentencesResources
    We imputed the whole chromosome 12 based on population-specific reference panels: the Trans-Omics for Precision Medicine (TOPMed) reference panel was used for individuals of European ancestry; the Consortium on Asthma among African-ancestry populations in the Americas (CAAPA) reference panel was used for individuals of African ancestry; the American admixture population from Phase 3 of the 1000 Genomes Project (AMR-1KGP) reference panel was used for individuals of Hispanic-ancestry; and the Genome Asia pilot (GAsP) reference panel was used for individuals of East Asian-ancestry.
    1000 Genomes Project
    suggested: (1000 Genomes Project and AWS, RRID:SCR_008801)
    GAsP
    suggested: (GASP, RRID:SCR_008703)
    Haplotype association analyses using logistic regression were done with PLINK (v1.07), with a custom selection of a reference haplotype.
    PLINK
    suggested: (PLINK, RRID:SCR_001757)
    LD plots were generated with Haploview 4.2.
    Haploview
    suggested: (Haploview, RRID:SCR_003076)
    Colocalization and correlation coefficients between OAS1 and Golgin-97 expression were generated with LSM700 Zen software by analyzing randomly imaged fields of view (5-7 fields) containing at least 7 cells from IFNβ-treated wells.
    Zen
    suggested: None
    Briefly, the raw FASTQ files were aligned with STAR version 7.1.31 to the reference human genome assembly (hg38).
    STAR
    suggested: (STAR, RRID:SCR_004463)
    Analysis of ATAC-seq, ChIP-seq, and Hi-C data of cell lines: The ATAC-seq, H3K27ac ChIP-seq, Hi-C, and RNA-seq raw data for SW780, HT1376, and SCABER bladder cancer cell lines were downloaded from NCBI SRA (ID: PRJNA623018) using the SRA tools.
    ChIP-seq
    suggested: (ChIP-seq, RRID:SCR_001237)
    The output bigwig files were then uploaded to the UCSC genome browser for visualization.
    UCSC genome browser
    suggested: (UCSC Genome Browser, RRID:SCR_005780)
    The chromatin loops in Hi-C data were detected using Hiccups in Juicer tools with default settings.
    Juicer
    suggested: (Juicer, RRID:SCR_017226)
    Analysis of exonic splicing enhancer activity affected by rs1131454 within OAS1 exon 3: The allele-specific 27 bp sequence (GUCAGUUGACUGGC[A/G]GCUAUAAACUA) centered on rs1131454 was used for the prediction of exonic splicing enhancer (ESE)/silencer (ESS) motifs using the Human Splicing Finder (HSF, www.umd.be/HSF3/).
    Human Splicing Finder
    suggested: (Human Splicing Finder, RRID:SCR_005181)
    Final libraries were loaded into MinION Fluidics Module flow cells (Oxford Nanopore, FLO-MIN106D), and sequencing was carried out on GridION MK1 and MinION MK1C instruments (Oxford Nanopore) for 3 days, using default parameters.
    MinION
    suggested: (MinION, RRID:SCR_017985)
    The FASTQ files generated by Nanopore GridION long-read sequencer were trimmed using Porechop (https://github.com/rrwick/Porechop) and aligned to the hg19 genome using Minimap2 (https://github.com/lh3/minimap2) with -ax splice command.
    Porechop
    suggested: (Porechop, RRID:SCR_016967)
    Minimap2
    suggested: (Minimap2, RRID:SCR_018550)
    The FASTQ files were aligned with STAR aligner (https://github.com/alexdobin/STAR) with default settings followed by quantification of isoforms-specific reads for alternative splicing junctions of OAS1 exon 3 with adjacent exons.
    https://github.com/alexdobin/STAR
    suggested: (Hamilton Microlab STAR Automated Liquid Handling, RRID:SCR_019993)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04354259RecruitingInterferon Lambda for Immediate Antiviral Therapy at Diagnos…

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

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  3. Version published to 10.1101/2021.07.09.21260221v1 on medRxiv