- Predicted loss-of-function (pLoF) mutations disrupted both copies of a given human gene, such as APOC3, and significantly altered or removed protein expression among a patient population of 10,503 Pakistanis who are known for a high rate of consanguinity
- A roadmap for a ‘human knockout project’ is outlined following gene knockout testing of those with homozygous pLoF mutations
- When homozygous pLoF carriers for APOC3 received oral fat, they showed a marked reduction in the usual rise of plasma triglycerides
- For future pLoF testing to be effective, validation must prove loss of gene function, including but not limited to the use targeted assays
- By using multiple patient populations with high degrees of consanguinity, the power of human gene knockout testing increases
Predicted loss-of-function (pLoF) mutations disrupted both copies of a given human gene, such as APOC3, and significantly altered or removed protein expression. Massachusetts General Hospital investigators, including Pradeep Natarajan, MD, director of Preventive Cardiology and Sekar Kathiresan, MD, director of the Center for Genomic Medicine, were among a team of researchers who studied mutation-associated gene knockouts with potential therapeutic impacts and gleaned insights into how to effectively conduct a ‘human knockout project.’
Consanguinity equates to more homozygous pLoF mutations and the Pakistani population has a high consanguinity rate. Researchers sequenced the protein-coding regions of 10,503 adult participants in the Pakistan Risk of Myocardial Infarction Study (PROMIS). From 49,138 rare (<1% minor allele frequency) pLoF mutations, an estimated 1,317 genes were disrupted, each in at least one person. The team screened for 201 distinct phenotypes for individuals with one of 426 genes for which two or more participants had homozygous pLoF mutations. They then analyzed the blood of 84 participants for 1,310 protein biomarkers using a proteomics assay and found highly significant results for 26 gene–trait pairs, including for PLA2G7, CYP2F1, TREH, A3GALT2, NRG4, SLC9A3R1 and APOC3.
In a sub-study, carriers and non-carriers of homozygous pLoF mutations (n=13) of APOC3 in a family were compared for the effect of oral ingestion of 50 g m−2 of fat. APOC3 is known to inhibit fat removal from circulation. Suppression of the gene for the protein lessened or removed its effect. Those with homozygote mutations of APOC3 had significantly lower post-prandial triglyceride levels compared to the non-homozygote group (468.3 mg dl−1 over 6 h versus 1,267.7 mg dl−1 over 6 h).
Other gene-associated functions were also altered or lost in certain understandable ways. Mutations around PLA2G7 and gene knockout, for example, were linked to absent enzymatic activity of soluble lipoprotein-associated phospholipase A2. Also notable were that:
- For CYP2F1, knockout resulted in higher plasma interleukin-8 concentrations
- For TREH, mutation-linked knockout revealed lower concentrations of apoB-containing lipoprotein
- For A3GALT2 or NRG4, plasma insulin C-peptide concentrations substantially reduced
- For SLC9A3R1, mediation of calcium and phosphate signaling was seen
Based on their findings, the researchers stress the importance of recruiting by genotype, for a larger human knockout project. They also recommend:
- Sequencing (deep-coverage) the protein-coding of genomic regions
- Using a broad panel of biochemical and clinical phenotypes and thoroughly evaluating people clinically when a gene is knocked out
- Ensuring an ability to re-contact those with gene knockouts (including family members) for follow-up
- Phenotyping based on hypothesis for certain study subjects
Finally, for future pLoF testing to be effective, the researchers believe that validation, such as through targeted assays, must prove loss of gene function.