Three-quarters of human genes have no known associated phenotype. Studying humans knocked out for a gene is an innovative way to understand biology; however such individuals are very rare. To identify human knockouts, we are leveraging high levels of consanguinity in Pakistan. Using whole-exome sequencing (WES) in 10,500 participants, we have already identified 1843 human knockouts for 1317 genes. This program has identified knockouts for genes that were thought to be essential through studies in mice. We are also characterizing novel phenotypic associations and identifying novel biology through provoked physiological studies in knockouts and individuals who are haploinsufficient.
In-collaboration with several institutes, we are rapidly expanding our WES and whole-genome sequencing studies in > 200,000 participants. Because of high levels of consanguinity in Pakistan, efficiency in identifying human knockouts is several folds higher through sequencing studies in Pakistan compared to other outbred populations. We anticipate that once sequencing is completed in all 200,000 participants, we should be able to identify knockouts for more than 8000 unique genes.
This program can also help to prioritize therapeutic pathways that are safe and beneficial for inhibition in humans compared to those that are not. Life-long genetic deficiency of a given gene in human knockouts can provide a mechanism to understand therapeutic modulation of that gene in other humans; providing insights about the safety profile of a drug; mechanism of action; biological pleiotropy; and effective surrogate biomarkers for clinical trials. Hence, deep phenotyping studies in human knockouts can characterize several features of the relationship between therapeutic modulation of a drug target and risk of disease.
All of our study participants are consented for call back. During a call-back, we not only conduct deep phenotyping studies of the proband but we also conduct phenotyping studies of related family members. Because, most participants in our resource are born of first cousin marriages or / and are married to their first cousins, during such call backs, we end up identifying more knockouts or individuals who are haplo-insufficient; hence increasing our sample size for deep phenotyping studies. By also genotyping offsprings of parents who are haplo-insufficient, we are also able to find out if a knockout genotype is compatible with life.
We are able to conduct both hypothesis free phenotyping of knockouts and conduct provoked physiological testing according to the gene knocked out in our study participants. Such provoked physiological testing can include oral fat tolerance studies, oral glucose tolerance studies, lipoprotein kinetic studies, echo-cardiograms and others.