Functional analysis of natural PCSK 9 mutants in modern and archaic humans

PCSK 9 is the last member of the proprotein convertases ( PC s) family and its gene is mutated in ~ 2% to 3% of individuals with familial hypercholesterolemia ( FH ). This protein enhances the degradation of the low‐density lipoprotein receptor ( LDLR ) and hence increases the levels of circulating LDL ‐cholesterol ( LDL c). Studies of the underlying mechanism(s) regulating the activity of different mutations in the PCSK 9 gene are ongoing as they enhance our understanding of the biology and clinical relevance of PCSK 9 and its partners. In an attempt to unravel the regulation of PCSK 9 transcription and possibly identify mutation ‘hot spot’ regions with alterations in CpG methylation, we present for the first time the complete methylome profile of the PCSK 9 gene in modern and archaic humanoids. Our data showed that the genomes of modern humans and archaic PCSK 9 exhibit a similar methylation pattern. Next, we defined the mechanistic consequences of three PCSK 9 natural mutations ( PCSK 9‐R96L, ‐R105W, and ‐P174S) and one archaic Denisovan mutation ( PCSK 9‐H449L) using various complementary cellular and in vitro binding assays. Our results showed that the PCSK 9‐H449L is a loss‐of‐function ( LOF ) mutation, likely due to its lower binding affinity to the LDLR . Similarly, PCSK 9‐R96L and ‐R105W are LOF mutations, even though they have been identified in FH patients. The PCSK 9‐R105W mutation leads to a significantly lower autocatalytic processing of pro PCSK 9. PCSK 9‐P174S resulted in a LOF in both extracellular and intracellular pathways. In conclusion, our extensive analyses revealed that all studied mutations result in PCSK 9 LOF , via various mechanisms, leading to lower levels of LDL c.