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Abstract

A major goal of biomedicine is to understand the function of every gene in the human genome1. Loss-of-function mutations can disrupt both copies of a given gene in humans and phenotypic analysis of such ‘human knockouts’ can provide insight into gene function. Consanguineous unions are more likely to result in offspring carrying homozygous loss-of-function mutations. In Pakistan, consanguinity rates are notably high2. Here we sequence the protein-coding regions of 10,503 adult participants in the Pakistan Risk of Myocardial Infarction Study (PROMIS), designed to understand the determinants of cardiometabolic diseases in individuals from South Asia3. We identified individuals carrying homozygous predicted loss-of-function (pLoF) mutations, and performed phenotypic analysis involving more than 200 biochemical and disease traits. We enumerated 49,138 rare (<1% minor allele frequency) pLoF mutations. These pLoF mutations are estimated to knock out 1,317 genes, each in at least one participant. Homozygosity for pLoF mutations at PLA2G7 was associated with absent enzymatic activity of soluble lipoprotein-associated phospholipase A2; at CYP2F1, with higher plasma interleukin-8 concentrations; at TREH, with lower concentrations of apoB-containing lipoprotein subfractions; at either A3GALT2 or NRG4, with markedly reduced plasma insulin C-peptide concentrations; and at SLC9A3R1, with mediators of calcium and phosphate signalling. Heterozygous deficiency of APOC3 has been shown to protect against coronary heart disease4,5; we identified APOC3 homozygous pLoF carriers in our cohort. We recruited these human knockouts and challenged them with an oral fat load. Compared with family members lacking the mutation, individuals with APOC3 knocked out displayed marked blunting of the usual post-prandial rise in plasma triglycerides. Overall, these observations provide a roadmap for a ‘human knockout project’, a systematic effort to understand the phenotypic consequences of complete disruption of genes in humans.

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Acknowledgements

D.S. is supported by grants from the National Institutes of Health, the Fogarty International, the Wellcome Trust, the British Heart Foundation, and Pfizer. P.N. is supported by the John S. LaDue Memorial Fellowship in Cardiology from Harvard Medical School. H.-H.W. is supported by a grant from the Samsung Medical Center, Korea (SMO116163). S.K. is supported by the Ofer and Shelly Nemirovsky MGH Research Scholar Award and by grants from the National Institutes of Health (R01HL107816), the Donovan Family Foundation, and Fondation Leducq. Exome sequencing was supported by a grant from the NHGRI (5U54HG003067-11) to S.G. and E.S.L. D.G.M. is supported by a grant from the National Institutes of Health (R01GM104371). J.D. holds a British Heart Foundation Chair, European Research Council Senior Investigator Award, and NIHR Senior Investigator Award. The Cardiovascular Epidemiology Unit at the University of Cambridge, which supported the field work and genotyping of PROMIS, is funded by the UK Medical Research Council, British Heart Foundation, and NIHR Cambridge Biomedical Research Centre. In recognition for PROMIS fieldwork and support, we also acknowledge contributions made by the following: M. Z. Ozair, U. Ahmed, A. Hakeem, H. Khalid, K. Shahid, F. Shuja, A. Kazmi, M. Qadir Hameed, N. Khan, S. Khan, A. Ali, M. Ali, S. Ahmed, M. W. Khan, M. R. Khan, A. Ghafoor, M. Alam, R. Ahmed, M. I. Javed, A. Ghaffar, T. B. Mirza, M. Shahid, J. Furqan, M. I. Abbasi, T. Abbas, R. Zulfiqar, M. Wajid, I. Ali, M. Ikhlaq, D. Sheikh, M. Imran, M. Walker, N. Sarwar, S. Venorman, R. Young, A. Butterworth, H. Lombardi, B. Kaur and N. Sheikh. Fieldwork in the PROMIS study has been supported through funds available to investigators at the Center for Non-Communicable Diseases, Pakistan and the University of Cambridge, UK.

Author information

Author notes

    • Danish Saleheen
    •  & Pradeep Natarajan

    These authors contributed equally to this work.

    • Philippe Frossard
    • , John Danesh
    • , Daniel J. Rader
    •  & Sekar Kathiresan

    These authors jointly supervised this work.

Affiliations

  1. Department of Biostatistics and Epidemiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA

    • Danish Saleheen
    •  & Wei Zhao
  2. Center for Non-Communicable Diseases, Karachi, Pakistan

    • Danish Saleheen
    • , Asif Rasheed
    • , Mozzam Zaidi
    • , Maria Samuel
    • , Atif Imran
    • , Faisal Majeed
    • , Madiha Ishaq
    • , Saba Akhtar
    •  & Philippe Frossard
  3. Center for Genomic Medicine, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA

    • Pradeep Natarajan
    •  & Sekar Kathiresan
  4. Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA

    • Pradeep Natarajan
    • , Irina M. Armean
    • , Konrad J. Karczewski
    • , Anne H. O’Donnell-Luria
    • , Kaitlin E. Samocha
    • , Benjamin Weisburd
    • , Namrata Gupta
    • , Daniel G. MacArthur
    • , Stacey Gabriel
    • , Eric S. Lander
    • , Mark J. Daly
    •  & Sekar Kathiresan
  5. Analytic and Translational Genetics Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA

    • Irina M. Armean
    • , Konrad J. Karczewski
    • , Anne H. O’Donnell-Luria
    • , Kaitlin E. Samocha
    • , Benjamin Weisburd
    • , Daniel G. MacArthur
    •  & Mark J. Daly
  6. Institute for Translational Medicine and Therapeutics, Department of Genetics, and Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA

    • Sumeet A. Khetarpal
    • , Kevin Trindade
    • , Megan Mucksavage
    •  & Daniel J. Rader
  7. Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University, Samsung Medical Center, Seoul, Korea

    • Hong-Hee Won
  8. Division of Genetics and Genomics, Boston Children’s Hospital, Boston, Massachusetts, USA

    • Anne H. O’Donnell-Luria
  9. Faisalabad Institute of Cardiology, Faisalabad, Pakistan

    • Shahid Abbas
  10. National Institute of Cardiovascular Disorders, Karachi, Pakistan

    • Nadeem Qamar
    • , Khan Shah Zaman
    • , Zia Yaqoob
    • , Tahir Saghir
    • , Syed Nadeem Hasan Rizvi
    •  & Anis Memon
  11. Punjab Institute of Cardiology, Lahore, Pakistan

    • Nadeem Hayyat Mallick
  12. Karachi Institute of Heart Diseases, Karachi, Pakistan

    • Mohammad Ishaq
    •  & Syed Zahed Rasheed
  13. Red Crescent Institute of Cardiology, Hyderabad, Pakistan

    • Fazal-ur-Rehman Memon
  14. The Civil Hospital, Karachi, Pakistan

    • Khalid Mahmood
  15. Liaquat National Hospital, Karachi, Pakistan

    • Naveeduddin Ahmed
  16. Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    • Ron Do
  17. The Charles Bronfman Institute for Personalized Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    • Ron Do
  18. Children’s Hospital Oakland Research Institute, Oakland, California, USA

    • Ronald M. Krauss
  19. MRC/BHF Cardiovascular Epidemiology Unit, Department of Public Health and Primary Care, University of Cambridge, UK

    • John Danesh
  20. Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK

    • John Danesh
  21. Department of Human Genetics, University of Pennsylvania, USA

    • Daniel J. Rader

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Contributions

Sample recruitment and phenotyping was performed by D.S., P.F., J.D., A.R., M.Z., M.S., A.I., S.A., F.Ma., M.I., S.A., K.T., N.H.M., K.S.Z., N.Q., M.I., S.Z.R., F.Me., K.M., N.A., and R.M.K. D.S., P.F., J.D., and W.Z. performed array-based genotyping and runs-of-homozygosity analyses. Exome sequencing was coordinated by D.S., N.G., S.G., E.S.L., D.J.R., and S.K. P.N., W.Z., H.H.W., and R.D. performed exome-sequencing quality control and association analyses. P.N., I.M.A., K.J.K., A.H.O., B.W., and D.G.M. performed variant annotation. D.S., S.K., and D.J.R. performed confirmatory genotyping and lipoprotein biomarker assays. D.S. and A.R. conducted recall-based studies for the APOC3 knockouts. P.N. and M.J.D. performed bioinformatics simulations. P.N. and K.E.S. performed constraint score analyses. D.S., P.N., and S.K. designed the study and wrote the paper. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Danish Saleheen or Sekar Kathiresan.

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

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