title: Human knockouts and phenotypic analysis in a cohort with a high rate of consanguinity.
abstract: A major goal of biomedicine is to understand the function of every gene in the human genome. 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 high. 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 Asia. 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 disease; 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.
authors.affiliation: Department of Biostatistics and Epidemiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA.|Center for Non-Communicable Diseases, Karachi, Pakistan.
authors.forename: Danish
authors.initials: D
authors.lastname: Saleheen
authors.affiliation: Center for Genomic Medicine, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.|Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.
authors.forename: Pradeep
authors.initials: P
authors.lastname: Natarajan
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.|Analytic and Translational Genetics Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Irina M
authors.initials: IM
authors.lastname: Armean
authors.affiliation: Department of Biostatistics and Epidemiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhao
authors.affiliation: Center for Non-Communicable Diseases, Karachi, Pakistan.
authors.forename: Asif
authors.initials: A
authors.lastname: Rasheed
authors.affiliation: 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.
authors.forename: Sumeet A
authors.initials: SA
authors.lastname: Khetarpal
authors.affiliation: Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University, Samsung Medical Center, Seoul, Korea.
authors.forename: Hong-Hee
authors.initials: HH
authors.lastname: Won
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.|Analytic and Translational Genetics Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Konrad J
authors.initials: KJ
authors.lastname: Karczewski
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.|Analytic and Translational Genetics Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.|Division of Genetics and Genomics, Boston Children's Hospital, Boston, Massachusetts, USA.
authors.forename: Anne H
authors.initials: AH
authors.lastname: O'Donnell-Luria
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.|Analytic and Translational Genetics Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Kaitlin E
authors.initials: KE
authors.lastname: Samocha
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.|Analytic and Translational Genetics Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Weisburd
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.
authors.forename: Namrata
authors.initials: N
authors.lastname: Gupta
authors.affiliation: Center for Non-Communicable Diseases, Karachi, Pakistan.
authors.forename: Mozzam
authors.initials: M
authors.lastname: Zaidi
authors.affiliation: Center for Non-Communicable Diseases, Karachi, Pakistan.
authors.forename: Maria
authors.initials: M
authors.lastname: Samuel
authors.affiliation: Center for Non-Communicable Diseases, Karachi, Pakistan.
authors.forename: Atif
authors.initials: A
authors.lastname: Imran
authors.affiliation: Faisalabad Institute of Cardiology, Faisalabad, Pakistan.
authors.forename: Shahid
authors.initials: S
authors.lastname: Abbas
authors.affiliation: Center for Non-Communicable Diseases, Karachi, Pakistan.
authors.forename: Faisal
authors.initials: F
authors.lastname: Majeed
authors.affiliation: Center for Non-Communicable Diseases, Karachi, Pakistan.
authors.forename: Madiha
authors.initials: M
authors.lastname: Ishaq
authors.affiliation: Center for Non-Communicable Diseases, Karachi, Pakistan.
authors.forename: Saba
authors.initials: S
authors.lastname: Akhtar
authors.affiliation: 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.
authors.forename: Kevin
authors.initials: K
authors.lastname: Trindade
authors.affiliation: 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.
authors.forename: Megan
authors.initials: M
authors.lastname: Mucksavage
authors.affiliation: National Institute of Cardiovascular Disorders, Karachi, Pakistan.
authors.forename: Nadeem
authors.initials: N
authors.lastname: Qamar
authors.affiliation: National Institute of Cardiovascular Disorders, Karachi, Pakistan.
authors.forename: Khan Shah
authors.initials: KS
authors.lastname: Zaman
authors.affiliation: National Institute of Cardiovascular Disorders, Karachi, Pakistan.
authors.forename: Zia
authors.initials: Z
authors.lastname: Yaqoob
authors.affiliation: National Institute of Cardiovascular Disorders, Karachi, Pakistan.
authors.forename: Tahir
authors.initials: T
authors.lastname: Saghir
authors.affiliation: National Institute of Cardiovascular Disorders, Karachi, Pakistan.
authors.forename: Syed Nadeem Hasan
authors.initials: SNH
authors.lastname: Rizvi
authors.affiliation: National Institute of Cardiovascular Disorders, Karachi, Pakistan.
authors.forename: Anis
authors.initials: A
authors.lastname: Memon
authors.affiliation: Punjab Institute of Cardiology, Lahore, Pakistan.
authors.forename: Nadeem
authors.initials: N
authors.lastname: Hayyat Mallick
authors.affiliation: Karachi Institute of Heart Diseases, Karachi, Pakistan.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Ishaq
authors.affiliation: Karachi Institute of Heart Diseases, Karachi, Pakistan.
authors.forename: Syed Zahed
authors.initials: SZ
authors.lastname: Rasheed
authors.affiliation: Red Crescent Institute of Cardiology, Hyderabad, Pakistan.
authors.forename: Fazal-Ur-Rehman
authors.initials: FU
authors.lastname: Memon
authors.affiliation: The Civil Hospital, Karachi, Pakistan.
authors.forename: Khalid
authors.initials: K
authors.lastname: Mahmood
authors.affiliation: Liaquat National Hospital, Karachi, Pakistan.
authors.forename: Naveeduddin
authors.initials: N
authors.lastname: Ahmed
authors.affiliation: Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA.|The Charles Bronfman Institute for Personalized Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Ron
authors.initials: R
authors.lastname: Do
authors.affiliation: Children's Hospital Oakland Research Institute, Oakland, California, USA.
authors.forename: Ronald M
authors.initials: RM
authors.lastname: Krauss
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.|Analytic and Translational Genetics Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: MacArthur
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.
authors.forename: Stacey
authors.initials: S
authors.lastname: Gabriel
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.
authors.forename: Eric S
authors.initials: ES
authors.lastname: Lander
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.|Analytic and Translational Genetics Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Daly
authors.affiliation: Center for Non-Communicable Diseases, Karachi, Pakistan.
authors.forename: Philippe
authors.initials: P
authors.lastname: Frossard
authors.affiliation: MRC/BHF Cardiovascular Epidemiology Unit, Department of Public Health and Primary Care, University of Cambridge, UK.|Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
authors.forename: John
authors.initials: J
authors.lastname: Danesh
authors.affiliation: 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.|Department of Human Genetics, University of Pennsylvania, USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Rader
authors.affiliation: Center for Genomic Medicine, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.|Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.
authors.forename: Sekar
authors.initials: S
authors.lastname: Kathiresan
mesh_terms: D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase / Q000172:deficiency / Q000235:genetics; D053305:Apolipoprotein C-III / Q000172:deficiency / Q000235:genetics; D015331:Cohort Studies; D003241:Consanguinity; D003327:Coronary Disease / Q000097:blood / Q000235:genetics; D000072467:Cytochrome P450 Family 2 / Q000235:genetics; D004252:DNA Mutational Analysis; D004041:Dietary Fats / Q000494:pharmacology; D059472:Exome / Q000235:genetics; D005215:Fasting / Q000097:blood; D005260:Female; D017353:Gene Deletion; D005787:Gene Frequency; D005796:Genes / Q000235:genetics*; D056726:Genetic Association Studies / Q000379:methods*; D006720:Homozygote; D006801:Humans; D016209:Interleukin-8 / Q000097:blood; D008297:Male; D008875:Middle Aged; D009203:Myocardial Infarction / Q000097:blood / Q000235:genetics; D020880:Neuregulins / Q000235:genetics; D010154:Pakistan; D010375:Pedigree; D010641:Phenotype; D010750:Phosphoproteins / Q000235:genetics; D019518:Postprandial Period; D022821:RNA Splice Sites / Q000235:genetics; D059386:Reverse Genetics / Q000379:methods; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics; D014280:Triglycerides / Q000097:blood
chemical_list: D053305:Apolipoprotein C-III; C508606:CXCL8 protein, human; D004041:Dietary Fats; D016209:Interleukin-8; D020880:Neuregulins; D010750:Phosphoproteins; D022821:RNA Splice Sites; D017923:Sodium-Hydrogen Exchangers; D014280:Triglycerides; C467747:neuregulin-4; C093376:sodium-hydrogen exchanger regulatory factor; C110332:CYP2F1 protein, human; D000072467:Cytochrome P450 Family 2; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; C526684:PLA2G7 protein, human