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.1 Loss-of-function (LoF) 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 who carry LoF mutations in a homozygous state. In Pakistan, consanguinity rates are notably high.2 Here, we sequenced 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 South Asians.3 We identified individuals carrying predicted LoF (pLoF) mutations in the homozygous state, and performed phenotypic analysis involving >200 biochemical and disease traits. We enumerated 49,138 rare (<1 % minor allele frequency) pLoF mutations. These pLoF mutations are predicted to knock out 1,317 genes in at least one participant. Homozygosity for pLoF mutations at PLAG27 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 signaling. Finally, APOC3 is a gene which retards clearance of plasma triglyceride-rich lipoproteins and where heterozygous deficiency confers protection against coronary heart disease.4,5 In Pakistan, we now observe APOC3 homozygous pLoF carriers; we recalled these knockout humans and challenged with an oral fat load. Compared with wild-type family members, APOC3 knockouts 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.

PI4K2A deficiency in an intellectual disability, epilepsy, myoclonus, akathisia syndrome

“…The probability of loss of function intolerance (pLI) score for PI4K2A according to the Exome Aggregation Consortium (ExAC) is 0.98, indicating high probability that the gene is intolerant to loss‐of‐function variants, even in heterozygous state 7. There are no homozygous loss of function variants reported in PI4K2A in population databases7, 8, 9, 10 or in over 10,500 individuals of Pakistani origin from a study of myocardial infarction 11. Sanger sequencing confirmed the variant and showed that it segregates in homozygous state with the disease (Fig.…”

Settling the score: variant prioritization and Mendelian disease

“…The simplest imaginable approach is to use Sequence Ontology terms to quickly prioritize variants in an ad hoc manner under the assumption that, for example, a variant creating a premature stop codon is typically more damaging than a missense variant. However, this is a poor approach because the average human harbours hundreds of putative loss-of-function alleles in both heterozygous and homozygous states 4,22,34,35 (TABLE 1). Such a simplistic filtering approach is also ill-advised because a stop codon in a poorly conserved gene may be more tolerated than a missense variant in another highly conserved gene.…”

Applied cultural and social studies are needed for a sustainable reduction of genetic disease incidence

“…A proper diagnosis can as such give a relief to the patient and the care giver, since giving the condition a name makes it more "real" (Rich, 2016). Recently, extensive catalogs of knock-out mutations in the human genome have been facilitated by applying the rapidly evolving genome sequencing technology on inbred human populations (Saleheen et al, 2017;Sulem et al, 2015). This resource together with mouse genetic models and the ability to edit the genome in model cell lines using tools like CRISPR/Cas9 has -and will -greatly enhance our understanding of the function of the genes and the proteins they code for and how these functions are related to disease.…”