Functional characterization of hypertriglyceridemia-associated mutations in Lipase Maturation Factor 1 by a...

Lipase maturation factor 1 (LMF1) is a chaperone for lipoprotein lipase (LPL) and critically required for the enzyme to attain lipase activity. LMF1 has been identified as a canonical gene affected in Familial Chylomicronemia Syndrome (FCS) and it has also been extensively analyzed in Multifactorial Chylomicronemia Syndrome (MCS), the polygenic form of severe hypertriglyceridemia (hTG). While recent genetic studies resulted in an explosion in the number of hTG-associated LMF1 variants in different populations, their functional significance remains largely unknown. Here, we present a second-generation LMF1 activity assay allowing the streamlined quantitative functional analysis of LMF1 variants. The assay is based on the reconstitution of lipase maturation in transfected LMF1-deficient cells and fluorescence-based measurement of LPL activity in the culture medium. The use of Gaussia luciferase (GLuc)-LMF1 fusion constructs allows the assessment of LMF1 protein expression and the calculation of LMF1 specific activity. Several variants previously reported as neutral resulted in loss of LMF1 function in the new assay demonstrating increased sensitivity to detect variants of modest effect sizes. Furthermore, the functional analysis of 14 hTG-associated missense mutations identified 12 loss-of-function (LOF) variants. To gain initial insights into the structural determinants of LMF1 function, we analyzed the domain distribution of 73 hTG-associated missense variants and identified Loop C as a critical region in lipase maturation. In conclusion, we developed a simple, non-radioactive and sensitive quantitative assay of LMF1 activity to facilitate the functional analysis of hyperlipidemia-associated genetic variants and structure-function studies.