Identification of a Gain-of-Function LIPC Variant as a Novel Cause of Familial Combined Hypocholesterolemia

Abstract

Background: Atherosclerotic cardiovascular disease is the main cause of mortality worldwide and is strongly influenced by circulating low-density lipoprotein (LDL) cholesterol levels. Only a few genes causally related to plasma LDL cholesterol levels have been identified so far, and only 1 gene, ANGPTL3, has been causally related to combined hypocholesterolemia. Here, our aim was to elucidate the genetic origin of an unexplained combined hypocholesterolemia inherited in 4 generations of a French family.

Methods: Using next-generation sequencing, we identified a novel dominant rare variant in the LIPC gene, encoding for hepatic lipase, which cosegregates with the phenotype. We characterized the impact of this LIPC-E97G variant on circulating lipid and lipoprotein levels in family members using nuclear magnetic resonance-based lipoprotein profiling and lipidomics. To uncover the mechanisms underlying the combined hypocholesterolemia, we used protein homology modeling, measured triglyceride lipase and phospholipase activities in cell culture, and studied the phenotype of APOE*3.Leiden.CETP mice after LIPC-E97G overexpression.

Results: Family members carrying the LIPC-E97G variant had very low circulating levels of LDL cholesterol and high-density lipoprotein cholesterol, LDL particle numbers, and phospholipids. The lysophospholipids/phospholipids ratio was increased in plasma of LIPC-E97G carriers, suggestive of an increased lipolytic activity on phospholipids. In vitro and in vivo studies confirmed that the LIPC-E97G variant specifically increases the phospholipase activity of hepatic lipase through modification of an evolutionarily conserved motif that determines substrate access to the hepatic lipase catalytic site. Mice overexpressing human LIPC-E97G recapitulated the combined hypocholesterolemic phenotype of the family and demonstrated that the increased phospholipase activity promotes catabolism of triglyceride-rich lipoproteins by different extrahepatic tissues but not the liver.

Conclusions: We identified and characterized a novel rare variant in the LIPC gene in a family who presents with dominant familial combined hypocholesterolemia. This gain-of-function variant makes LIPC the second identified gene, after ANGPTL3, causally involved in familial combined hypocholesterolemia. Our mechanistic data highlight the critical role of hepatic lipase phospholipase activity in LDL cholesterol homeostasis and suggest a new LDL clearance mechanism.

Keywords: LIPC protein, human; Lipc protein, mouse; cholesterol, HDL; cholesterol, LDL; phospholipases.

Figure 1.

A novel variant in LIPC cosegregates with familial combined hypocholesterolemia. Pedigree of family with familial combined hypocholesterolemia. Squares indicate male family members; circles, female family members. Slashes indicate deceased individuals. Roman numerals to the left of the pedigree indicate the generation; numerals to the upper left of each symbol indicate the individual family member. Basic lipid parameters of the recruited family members are indicated in the table below the pedigree. Values of total cholesterol, triglyceride, and low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels below the fifth percentile for age and sex are in bold.

Figure 2.

Lipoprotein particle quantification by nuclear magnetic resonance. Log₂-fold changes in plasma lipoprotein particle quantities and diameters as measured by nuclear magnetic resonance and compared with an age-matched control population for family members III.9 (A, adults) and IV.4 and IV.5 (B, children). HDL indicates high-density lipoprotein; HDL-C, high-density lipoprotein cholesterol; L, large; LDL, low-density lipoprotein; M, medium; P, particles; S, small; TG, triglycerides; VLDL, very-low-density lipoprotein; and Z, diameter.

Figure 3.

E97G modifies the structural conformation of HL. A, Global structure of the homology model of hepatic lipase (HL). The N and C termini are on the top left and right, respectively. Dashed circle indicates the zone of particular interest in this study, with the location of the E97 and K276 residues and of the amino acids of the catalytic triad (S168, D194, and H279). B, Closer view of the structural motif (salt bridge and hydrogen bond) involving E97, W99, and K276. Residues of the lid domain are shown in blue. C, Detailed view of the surroundings of the K276 residue in the E97G mutant model (orange) and the HL homology model (purple) The name of some residues is indicated for clarity. Dashed lines represent the interactions (salt bridge, hydrogen bonds) between the various residues. Residues of the lid domain are shown in blue.

Figure 4.

E97G alters hepatic lipase substrate specificity. A and B, Triglyceride (TG) lipase activity and phospholipase A1 (PLA1) activity in medium of heparin-treated immortalized human hepatocytes with overexpression of wild-type LIPC (LIPC-WT), LIPC-E97G, or LIPC-S168G. C and D, Triglyceride lipase activity and PLA1 activity in medium of heparin-treated immortalized human hepatocytes with a wild-type allele, a heterozygous presence of the E97G variant, or a corrected wild-type allele. Each enzymatic activity was corrected for the amount of released hepatic lipase (HL). E, Lipidomics data of plasma of control individuals (n=5) or E97G carriers (n=3). Values are depicted in nanomoles per liter (phosphatidylethanolamine [PE], lysophosphatidylethanolamine [LysoPE], phosphatidylinositol [PI], ceramides [Cer]) or micromoles per liter (fatty acids [FAs], phosphatidylcholine [PC], lysophosphatidylcholine [LysoPC], sphingomyelin [SM]). F, Ratios of plasma FA levels between control individuals (n=5) and E97G carriers (n=3). G, Phospholipid (PL) ratios of lysophospholipids/phospholipids in control individuals (n=5) or family members (n=3). Cell culture data are of 3 independent experiments with a technical duplicate. Statistical significance determined by Mann-Whitney tests. ns Indicates not significant; and RFU, relative fluorescence units. *P<0.05; **P<0.01; ***P<0.001.

Figure 5.

Overexpression of the LIPC-E97G variant markedly lowers LDL-C and HDL-C in APOE*3.Leiden.CETP mice. A, Experimental setup of mice study overexpressing AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV-TBG-LIPC-E97G in APOE*3.Leiden.CETP mice. B, Human hepatic lipase (HL) levels as determined by ELISA in preheparin and postheparin plasma of mice overexpressing low or high doses of AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV-TBG-LIPC-E97G. C and D, Triglyceride (TG) lipase activity and phospholipase A1 (PLA1) activity in plasma of APOE*3.Leiden.CETP mice overexpressing low or high doses of AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV-TBG-LIPC-E97G and after heparin injection. E, Triglycerides (TG), phospholipids, and cholesterol concentrations in plasma of APOE*3.Leiden.CETP mice overexpressing low or high doses of AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV-TBG-LIPC-E97G. F, Cholesterol and triglyceride (TG) concentrations in fast protein liquid chromatography (FPLC)–separated pooled plasma of APOE*3.Leiden.CETP mice overexpressing high doses of AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV-TBG-LIPC-E97G. G through I, Cholesterol (CHOL) and D7-cholesterol (D7-CHOL) levels extracted from liver (G), feces (H), and plasma (I) of APOE*3.Leiden.CETP mice overexpressing high doses of AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV-TBG-LIPC-E97G and injected with D7-cholesterol 3 days before death. n=5 or 6 per group. A 1-way ANOVA with Tukey correction for multiple comparisons was used for statistical analysis, with a P value cutoff at P<0.05. eGFP indicates enhanced green fluorescent protein; and LIPC, lipase C, hepatic type. *P<0.05; **P<0.01; ***P<0.001.

Figure 6.

Overexpression of the LIPC-E97G variant promotes peripheral cholesterol uptake. A and C, Decay of plasma ³H activity (glycerol tri[³H]oleate, hydrolysable; A) and plasma ¹⁴C activity ([¹⁴C]cholesteryl oleate, nonhydrolysable) levels (C) in APOE*3.Leiden.CETP mice overexpressing AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV-TBG-LIPC-E97G injected with very-low-density lipoprotein (VLDL)–like particles. B and D, Decay of plasma ³H activity (glycerol tri[³H]oleate, hydrolysable; B) and plasma ¹⁴C activity ([¹⁴C]cholesteryl oleate, nonhydrolysable) levels (D) in APOE*3.Leiden.CETP mice¹⁴ overexpressing AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV³-TBG-LIPC-E97G injected with radiolabeled murine VLDL. E, Liver ¹⁴C activity (cholesteryl ester, nonhydrolysable; left) and liver ³H activity (triolein, hydrolysable) levels (right) in APOE*3.Leiden.CETP mice overexpressing AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV-TBG-LIPC-E97G injected with VLDL-like particles. F, Liver ¹⁴C activity (cholesteryl ester, nonhydrolysable; left) and liver ³H activity (triolein, hydrolysable levels (right) in APOE*3.Leiden.CETP mice overexpressing AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV-TBG-LIPC-E97G injected with radiolabeled murine VLDL. G and I, Tissue ¹⁴C activity (cholesteryl ester, nonhydrolysable; G) and ³H activity (triolein, hydrolysable; I) levels in APOE*3.Leiden.CETP mice overexpressing AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV-TBG-LIPC-E97G injected with VLDL-like particles. H and J, Tissue ¹⁴C activity (cholesteryl ester, nonhydrolysable; H) and ³H activity (triolein, hydrolysable; J) levels in APOE*3.Leiden.CETP mice overexpressing AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV-TBG-LIPC-E97G injected with radiolabeled murine VLDL. n=8 per group. A 1-way ANOVA with Tukey correction for multiple comparisons was used for statistical analysis, with a P value cutoff at P<0.05. gWAT indicates gonadal white adipose tissue; iBAT, interscapular brown adipose tissue; sBAT, subscapular brown adipose tissue; and sWAT, subcutaneous white adipose tissue; and TGRL‚ triglyceride-rich lipoprotein. *P<0.05, human wild-type (LIPC-WT) vs LIPC-E97G. $P<0.05, enhanced green fluorescent protein (eGFP) vs LIPC-WT. #P<0.05, eGFP vs LIPC-E97G.