Human knockouts and phenotypic analysis in a cohort with a high rate of consanguinity (original) (raw)

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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

  1. Danish Saleheen and Pradeep Natarajan: These authors contributed equally to this work.
  2. Philippe Frossard, John Danesh, Daniel J. Rader and Sekar Kathiresan: These authors jointly supervised this work.

Authors and 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. Massachusetts General Hospital and Department of Medicine, Center for Genomic 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. Department of Medicine, Analytic and Translational Genetics Unit, 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. Department of Genetics, and Department of Medicine, Institute for Translational Medicine and Therapeutics, 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. Department of Public Health and Primary Care, MRC/BHF Cardiovascular Epidemiology Unit, 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

Authors

  1. Danish Saleheen
  2. Pradeep Natarajan
  3. Irina M. Armean
  4. Wei Zhao
  5. Asif Rasheed
  6. Sumeet A. Khetarpal
  7. Hong-Hee Won
  8. Konrad J. Karczewski
  9. Anne H. O’Donnell-Luria
  10. Kaitlin E. Samocha
  11. Benjamin Weisburd
  12. Namrata Gupta
  13. Mozzam Zaidi
  14. Maria Samuel
  15. Atif Imran
  16. Shahid Abbas
  17. Faisal Majeed
  18. Madiha Ishaq
  19. Saba Akhtar
  20. Kevin Trindade
  21. Megan Mucksavage
  22. Nadeem Qamar
  23. Khan Shah Zaman
  24. Zia Yaqoob
  25. Tahir Saghir
  26. Syed Nadeem Hasan Rizvi
  27. Anis Memon
  28. Nadeem Hayyat Mallick
  29. Mohammad Ishaq
  30. Syed Zahed Rasheed
  31. Fazal-ur-Rehman Memon
  32. Khalid Mahmood
  33. Naveeduddin Ahmed
  34. Ron Do
  35. Ronald M. Krauss
  36. Daniel G. MacArthur
  37. Stacey Gabriel
  38. Eric S. Lander
  39. Mark J. Daly
  40. Philippe Frossard
  41. John Danesh
  42. Daniel J. Rader
  43. Sekar Kathiresan

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.

Corresponding authors

Correspondence toDanish Saleheen or Sekar Kathiresan.

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Competing interests

The authors declare no competing financial interests.

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Extended data figures and tables

Extended Data Figure 1 pLoF mutations are typically seen in very few individuals.

The site-frequency spectrum of synonymous, missense, and high-confidence pLoF mutations is represented. Points represent the proportion of variants within a 1 × 10−4 minor allele frequency bin for each variant category. Lines represent the cumulative proportions of variants categories. The bottom inset highlights that most pLoF variants are often seen in no more than one or two individuals. The top inset highlights that virtually all pLoF mutations are very rare.

Extended Data Figure 2 Intersection of homozygous pLoF genes between PROMIS and other cohorts.

We compared the counts and overlap of unique homozygous pLoF genes in PROMIS with other exome sequenced cohorts.

Extended Data Figure 3 QQ-plot of recessive model pLoF association analysis across phenotypes.

Analyses to determine whether homozygous pLoF carrier status was associated with traits was performed where there were at least two homozygous pLoF carriers phenotyped per trait. The observed versus the expected results from 15,263 associations are displayed here demonstrating an excess of associations beyond a Bonferroni threshold.

Extended Data Figure 4 Carriers of pLoF alleles in CYP2F1 have increased IL-8 concentrations.

Participants who had pLoF mutations in the CYP2F1 gene had higher concentrations of IL-8, whereas heterozygotes had a more modest effect when compared to the rest of the cohort of non-carriers. IL-8 concentration is natural log transformed. Bars represent 1.5× interquartile range beyond the 25th and 75th percentiles.

Extended Data Figure 5 Carriers of pLoF alleles in TREH have decreased concentrations of several lipoprotein subfractions.

Participants who had pLoF mutations in the TREH gene had lower concentrations of several lipoprotein subfractions. Bars represent 1.5× interquartile range beyond the 25th and 75th percentiles.

Extended Data Figure 6 Nondiabetic homozygous pLoF carriers for A3GALT2 have diminished insulin C-peptide concentrations.

Among nondiabetics, those who were homozygous pLoF for A3GALT2 had substantially lower fasting insulin C-peptide concentrations. This observation was not evident in nondiabetic heterozygous pLoF A3GALT2 participants. Insulin C-peptide is natural log transformed. Bars represent 1.5× interquartile range beyond the 25th and 75th percentiles.

Extended Data Figure 7 Example of a second polymorphism in-phase which rescues a putative protein-truncating mutation.

Short-reads that align to genomic positions 65,339,112 to 65,339,132 on chromosome 1 are displayed for one individual with a putative homozygous pLoF genotype in this region. The SNP at position 65,339,122 from G to T is annotated as a nonsense mutation in the JAK1 gene. However, all three homozygotes of this mutation carried a tandem SNP in the same codon (A to G at 65,339,124) thus resulting in a glutamine and effectively rescuing the protein-truncating mutation.

Extended Data Figure 8 Anticipated number of genes knocked out with increasing sample sizes by minimum knockout count.

We simulate the number of genes expected to be knocked out by minimum knockout count per gene at increasing sample sizes. We perform this simulation with and without the observed inbreeding.

Extended Data Figure 9 PROMIS participants have an excess burden of runs of homozygosity compared with other populations.

Consanguinity leads to regions of genomic segments that are identical by descent and can be observed as runs of homozygosity. Using genome-wide array data in 17,744 PROMIS participants and reference samples from the International HapMap3, the burden of runs of homozygosity (minimum 1.5 Mb) per individual was derived and population-specific distributions are displayed, with outliers removed. This highlights the higher median runs of homozygosity burden in PROMIS and the higher proportion of individuals with very high burdens.

Extended Data Figure 10 Down-sampling of synonymous and high confidence pLoF variants to validate simulation.

a, b, We ran simulations to estimate the number of unique, completely knocked out genes at increasing sample sizes. Before applying our model, we first applied this approach to a range of sample sizes below 7,078 for variants that were not under constraint, synonymous variants (a), and for high-confidence null variants (b). At the observed sample size, we did not observe significant selection. We expect that at increasing sample sizes, there may be a subset of genes that will not be tolerated in a homozygous pLoF state. In fact, our estimates are slightly more conservative when comparing outbred simulations with a recent description of >100,000 Icelanders using a more liberal definition for pLoF mutations.

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Saleheen, D., Natarajan, P., Armean, I. et al. Human knockouts and phenotypic analysis in a cohort with a high rate of consanguinity.Nature 544, 235–239 (2017). https://doi.org/10.1038/nature22034

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