Original Articles

Characterization of Three Kindreds With Familial Combined Hypolipidemia Caused by Loss-of-Function Mutations of ANGPTL3Clinical Perspective

Livia Pisciotta, Elda Favari, Lucia Magnolo, Sara Simonelli, Maria Pia Adorni, Raffaella Sallo, Tatiana Fancello, Ivana Zavaroni, Diego Ardigò, Franco Bernini, Laura Calabresi, Guido Franceschini, Patrizia Tarugi, Sebastiano Calandra, Stefano Bertolini
https://doi.org/10.1161/CIRCGENETICS.111.960674
Circulation: Genomic and Precision Medicine. 2012;5:42-50
Originally published February 14, 2012

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Abstract

Background—Angiopoietin-like protein 3 (ANGPTL3) affects lipid metabolism by inhibiting the activity of lipoprotein and endothelial lipases. Angptl3 knockout mice have marked hypolipidemia, and heterozygous carriers of ANGPLT3, loss-of-function mutations were found among individuals in the lowest quartile of plasma triglycerides in population studies. Recently, 4 related individuals with primary hypolipidemia were found to be compound heterozygotes for ANGPTL3 loss-of-function mutations.

Methods and Results—We resequenced ANGPTL3 in 4 members of 3 kindreds originally identified for very low levels of low-density lipoprotein cholesterol and high-density lipoprotein cholesterol (0.97±0.16 and 0.56±0.20 mmol/L, respectively) in whom no mutations of known candidate genes for monogenic hypobetalipoproteinemia and hypoalphalipoproteinemia had been detected. These subjects were found to be homozygous or compound heterozygous for ANGPTL3 loss-of-function mutations (p.G400VfsX5, p.I19LfsX22/p.N147X) associated with the absence of ANGPTL3 in plasma. They had reduced plasma levels of triglyceride-containing lipoproteins and of HDL particles that contained only apolipoprotein A-I and pre-β–high-density lipoprotein. In addition, their apolipoprotein B–depleted sera had a reduced capacity to promote cell cholesterol efflux through the various pathways (ABCA1-, SR-BI–, and ABCG1-mediated efflux); however, these subjects had no clinical evidence of accelerated atherosclerosis. Heterozygous carriers of the ANGPTL3 mutations had low plasma ANGPTL3 and moderately reduced low-density lipoprotein cholesterol (2.52±0.38 mmol/L) but normal plasma high-density lipoprotein cholesterol.

Conclusions—Complete ANGPTL3 deficiency caused by loss-of-function mutations of ANGPTL3 is associated with a recessive hypolipidemia characterized by a reduction of apolipoprotein B and apolipoprotein A-I–containing lipoproteins, changes in subclasses of high-density lipoprotein, and reduced cholesterol efflux potential of serum. Partial ANGPTL3 deficiency is associated only with a moderate reduction of low-density lipoprotein.

Introduction

Monogenic hypobetalipoproteinemias include a heterogeneous group of disorders characterized by reduced plasma levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and apolipoprotein B (apoB) below the fifth percentile of the distribution in the population.1,2 Most prominent among these disorders is familial hypobetalipoproteinemia (FHBL), in which the low LDL-C and apoB phenotype is transmitted as an autosomal dominant trait. Individuals with heterozygous FHBL are identified during population screening for low plasma cholesterol or in clinical settings for the presence of low TC, LDL-C, and apoB levels associated with fatty liver disease.1,2 Approximately 50% of FHBL heterozygotes are carriers of mutations in the APOB gene (apoB-linked FHBL) that result in either truncated apoBs (with reduced capacity to bind lipids and to form lipoproteins) or amino acid substitutions that cause the retention of mutant apoBs along the secretory pathway.1–5

Clinical Perspective on p 50

An FHBL-like phenotype has been reported in individuals carrying loss-of-function (LOF) mutations of PCSK9 (proprotein convertase subtilisin kexin type 9).6,7 LOF mutations of PCSK9 prevent or reduce the PCSK9-mediated degradation of the LDL receptor and consequently are associated with an increased receptor-mediated uptake of LDL by the liver.6,7 This leads to a reduction of plasma LDL-C levels, which in heterozygous carriers ranges from 30% to 70% of control values.8–12

In a large proportion of FHBL individuals, however, no mutations are found in either APOB or PCSK9, which suggests that other genes are involved.1,2,10 Recently, the ANGPTL3 gene, which encodes angiopoietin-like 3 protein (ANGPTL3), has become a novel candidate gene in certain conditions of hypobetalipoproteinemia. The role of ANGPTL3 in lipoprotein metabolism emerged from the discovery of an LOF mutation of Angptl3 in a strain of obese mice (KK/Snk strain) that exhibited a severe recessive hypolipidemia.13 This role was confirmed by the observation that treatment with recombinant ANGPTL3 or adenovirus-mediated overproduction of ANGPTL3 elevated plasma triglycerides and TC in mice,13 and inactivation of Angptl3 was associated with a marked hypolipidemia.14 Subsequent studies showed that ANGPTL3 increased very low-density lipoprotein (VLDL) triglyceride levels by inhibiting lipoprotein lipase activity.14–16 ANGPTL3 was also found to inhibit endothelial lipase (EL) and to contribute to modulate high-density lipoprotein (HDL) metabolism in mice.17,18 Recently, the resequencing of ANPTL3 in participants in the Dallas Heart Study showed that sequence variants, likely to be LOF alleles, were found in the lowest quartile of plasma triglyceride levels.19 Finally, LOF mutations of ANGPTL3 were found to be the cause of a recessive form of combined hypolipidemia characterized by very low levels of triglyceride, LDL-C, and HDL cholesterol (HDL-C) in 4 members of a large family previously ascertained on the basis of low LDL-C.20

During a survey of a cohort of individuals with the clinical diagnosis of possible monogenic hypobetalipoproteinemia not linked to the APOB or PCSK9 genes, we identified some subjects in whom low LDL-C and apoB levels were associated with a marked reduction of plasma HDL-C, which suggested the possibility that they had mutations in ANGPTL3.20 In the present study, we report the clinical, biochemical, and genetic features of 4 subjects belonging to 3 unrelated kindreds who were found to have a complete ANGPTL3 deficiency caused by LOF mutations of the ANGPLT3 gene.

Methods

Study Participants

The subjects investigated in the present study were Italians and had been living for several generations in northern Italy. They were selected from a group of 150 individuals with primary hypobetalipoproteinemia who had been referred to the Lipid Clinic (at the University Hospitals in Genoa, Parma, Milan and Modena) over the last 2 decades. The selection criteria included low LDL-C and apoB associated with a marked reduction of HDL-C (below the fifth percentile) and apolipoprotein A-I (apoA-I; hypobetalipoproteinemia-hypoalphalipoproteinemia) in the absence of mutations in the APOB, MTP, PCSK9, APOA1, LCAT, and ABCA1 genes. A DNA sample was available from all members of the 3 kindreds (designated the DV, MR, and CP kindred, respectively), although plasma/serum was available only from members of 2 kindreds (DV and MR kindreds). Informed consent was obtained from all participants, and the study protocol was approved by the ethics committees of the participating institutions.

Biochemical Analyses

Plasma Lipids

Plasma levels of TC, free cholesterol, HDL-C, and triglycerides were determined by standard techniques (Roche Diagnostics GmbH, Mannheim, Germany). LDL-C was calculated with the Friedewald formula. Plasma apoA-I, apoB, and lipoprotein(a) were measured by nephelometry (Siemens AG Healthcare, Munich, Germany). In some cases, plasma lipoproteins were separated by density gradient ultracentrifugation.21

HDL Subpopulations

Plasma levels of HDL particles that contained only apoA-I (LpA-I) and both apoA-I and apoA-II (LpA-I:A-II) were determined by electroimmunodiffusion.22 HDL subclass distribution according to particle size was determined by nondenaturing polyacrylamide gradient gel electrophoresis (4% to 30%) of the d<1.21 g/mL total plasma lipoprotein fraction. HDLs were divided into small (Ø<8.2 nm), medium (8.2<Ø<8.8 nm), and large (Ø>8.8 nm) particles.23 Plasma content of pre-β-HDL was assessed by nondenaturing 2-dimensional electrophoresis and was expressed as a percentage of total apoA-I.22 Plasma cholesterol esterification rate and plasma lecithin–cholesterol acyltransferase (LCAT) activity were determined as described previously.22 Plasma HDL subpopulations were also investigated in a control group (21 subjects) collected by matching (for age and sex) each carrier of ANGPL3 mutations with 3 healthy subjects randomly selected from a sample of the general population.

Plasma ANGPTL3 Assay

Plasma concentration of ANGPTL3 was determined by ELISA with a monoclonal antibody (AdipoGen, Incheon, Korea). The intra-assay and interassay coefficients of variation were 3.8% and 6.5%, respectively.

Sequence of Candidate Genes of Hypobetalipoproteinemia and Hypoalphalipoproteinemia

Genomic DNA was extracted from peripheral blood by a standard procedure. APOB, MTP, PCSK9 APOA1, ABCA1, and LCAT genes were sequenced as described previously.24,25 The subjects were also genotyped for APOE polymorphism.25

Sequence of ANGPTL3 Gene

The exons of the ANGPTL3 gene were amplified (online-only Data Supplement Table S.1) and sequenced with an automatic sequencer CEQ2000 DNA Analysis System (Beckman Coulter, Fullerton, CA). The mutations were designated according to the Human Genome Variation Society (www.hgvs.org/mutnomen, 2011 version). Screening of ANGPTL3 mutations was performed in 200 individuals (100 males and 100 females) randomly selected from a sample of the general population. Screening of the c.1198+1g>t mutation was conducted by restriction fragment analysis (online-only Data Supplement Methods); the other mutations (c.55delA and c.439_442delAACT) were screened by direct sequencing of exon 1.

In Vitro Expression of ANGPTL3 Minigenes

To investigate the effect of the mutation in the donor splice site of intron 6 (c.1198+1g>t) of the ANGPTL3 gene, we constructed 2 minigenes (mutant and wild-type) by polymerase chain reaction amplification of the appropriate ANGPTL3 region of genomic DNA from the proband of the DV kindred and from a control subject (online-only Data Supplement Methods). Analysis of the transcripts of ANGPTL3 minigenes in COS-1 cells was performed as specified in the online-only Data Supplement Methods.

Cell Culture and Cholesterol Efflux Studies

Efflux studies were performed as described previously26,27 with either J774 macrophages, Fu5AH rat hepatoma cells, or Chinese hamster ovary (CHO) cells expressing human ABCG1 (CHO-GI cells).28,29 J774 cells were cultured in 10% FCS-RPMI, Fu5AH cells in 10% FCS-DMEM, and CHO-GI cells in 10% FCS-DMEM. For efflux studies, cells were labeled with [1,2-3H] cholesterol in the presence of an acetyl-coenzyme A acyltransferase inhibitor (2 μg/mL Sandoz 58035; Sigma-Aldrich, Milano, Italy). J774 cells were treated with or without 0.3 mmol/L cAMP (cpt-AMP; Sigma Aldrich, Milano, Italy) in 0.2% BSA for 18 hours to upregulate ABCA1.27,30,31 Fu5AH cells were pretreated for 2 hours with DMEM containing 0.2% BSA with or without 10 μmol/L BLT (block lipid transfer-1) to inhibit scavenger receptor class B type I (SR-BI).32 The efflux medium was prepared with 2.8% (vol/vol) serum after removal of apoB-containing lipoproteins (apoB-depleted serum)33 or 2% (vol/vol) whole serum (online-only Data Supplement Methods).

Statistical Analysis

The statistical analyses were performed with the PASW 18.0 statistical software package (SPSS Inc, Chicago, IL). Differences in plasma concentration of lipids, apolipoproteins A-I and B, LpA-I, and LpA-I:A-II; in the content of pre-β-HDL; and in HDL size distribution among homozygote/compound heterozygotes, heterozygotes, and control subjects were assessed by ANOVA, and multiple comparisons among means were performed by t test with Bonferroni correction. The statistical significance for linear trends among the 3 groups was evaluated.

Differences in cellular cholesterol efflux between homozygous/compound heterozygous and heterozygous carriers of ANGPTL3 mutation were assessed by general linear model repeated-measures analysis with genotype as the between-subjects factor and familial relationship as the covariate. The relationship between the cholesterol efflux potential of apoB(−) (apoB-depleted) sera and plasma HDL-C concentration was evaluated by Pearson r coefficient.

Results

DV Kindred

The proband (subject I.1 in Figure 1, DV kindred) is a 65-year-old white female who, at the age of 52 years, was referred to the Lipid Clinic for very low plasma lipid levels (TC 1.52 mmol/L, LDL-C 0.98 mmol/L, HDL-C 0.52 mmol/L, triglyceride 0.32 mmol/L, apoA-I 56 mg/dL, and apoB 34 mg/dL). The separation of plasma lipoproteins by density gradient ultracentrifugation showed an almost complete absence of the VLDL peak and a substantial reduction of the LDL and HDL peaks (online-only Data Supplement Figure S.1). The proband had no family history of cardiovascular disease and no clinical manifestations of cardiovascular disease. The ECG stress test was normal, and the carotid intima-media thickness (maximum 0.8 mm) was within the normal range. Because secondary hypolipidemias were excluded, we assumed that the patient had a primary hypobetalipoproteinemia-hypoalphalipoproteinemia. During the 13 years of follow-up, she has been in good health; her plasma lipid profile remained unchanged (Table). A recent ultrasound examination of the carotid arteries showed a moderate increase in maximum intima-media thickness (1.0–1.2 mm). Abdominal ultrasound examination did not reveal the presence of fatty liver.

Figure 1.
Figure 1.

Pedigrees of the 3 kindreds carrying mutations in the ANGPTL3 gene. The probands are indicated by an arrow. Male subjects, □; female subjects, ○. In the DV kindred, the homozygote and the simple heterozygotes are indicated by completely filled or half-filled symbols, respectively. In the MR and CP kindreds, the simple heterozygotes are indicated by symbols with a single-shade pattern (dotted), whereas the compound heterozygotes are indicated by a double-shaded pattern (dotted and gray).

View this table:
Table.

ANGPTL3 Genotype and Plasma Lipid and Apolipoprotein Concentrations in Members of the 3 Kindreds

MR Kindred

The proband is a healthy 59-year-old white male (subject II.1 in Figure 1, MR kindred) who in 2010 was referred to the Lipid Clinic for low TC and HDL-C levels and a family history of premature coronary artery disease (his father had died of a heart attack at the age of 40 years). The proband was a former moderate smoker; his physical examination was negative, and routine laboratory tests were within the normal values with the exception of plasma lipids. The plasma lipid profile revealed a reduction of LDL-C, triglycerides, and HDL-C (below the first and fifth percentiles adjusted for age and sex, respectively; Table). Carotid ultrasound examination showed the presence of fibrous plaques at the left carotid bifurcation with a 25% stenosis. Abdominal ultrasound examination did not reveal any abnormalities. The proband's brother (subject II.2 in Figure 1 and the Table) was healthy and had a lipid profile superimposable to that of the proband. He was a current smoker; the ultrasound carotid examination revealed a moderate increase in intima-media thickness (maximum intima-media thickness 0.9 mm). Abdominal ultrasound examination did not reveal any abnormalities.

CP Kindred

The proband (subject I.1 in Figure 1, CP kindred) was a white female who in 1996, at the age of 85 years, had been admitted to the hospital for an acute respiratory tract infection. She was found to have a marked hypolipidemia (Table), apparently unexplained by the intercurrent pneumonia or other diseases. She died suddenly at the age of 91 years.

Data concerning the other family members of the 3 kindreds are presented in the online-only Data Supplement Results.

Resequencing of ANGPTL3

The resequencing of ANGPTL3 showed that the proband of the DV kindred was homozygous for a g>t transversion in the donor splice site of intron 6 (c.1198+1g>t; Figure 1; online-only Data Supplement Figure S.2). This mutation causes a partial retention of intron 6 in mature mRNA, which leads to a frameshift, with the formation of a premature termination codon (p.G400VfsX5; online-only Data Supplement Results and online-only Data Supplement Figures S.3 and S.4). The proband's offspring were carriers of this mutation. The screening of 200 healthy individuals randomly selected from the Italian general population led to the identification of 1 heterozygous carrier of the c.1198+1g>t mutation. This subject is a 36-year-old female with the following lipid profile: TC 4.42 mmol/L, LDL-C 2.82 mmol/L, HDL-C 1.34 mmol/L, triglyceride 0.56 mmol/L, apoAI 147 mg/dL, and apoB 78 mg/dL; her APOE genotype was ε3ε3.

The proband of the MR kindred and his brother were found to be compound heterozygous for the following mutations: (1) A single-nucleotide deletion in exon 1 (c.55delA) that resulted in a frameshift that led to a truncated protein (p.I19LfsX22; Figure 1; online-only Data Supplement Figure S.5) and (2) a 4-nucleotide deletion in exon 1 (c.439_442delAACT) that resulted in a premature termination codon with the formation of a truncated protein (p.N147X; Figure 1; online-only Data Supplement Figure S.6). The mother and the niece of the proband were heterozygous for the c.55delA mutation, thus confirming that the 2 mutations found in the proband and his brother were on separate alleles (Figure 1).

The proband of the CP kindred was found to be compound heterozygous for the 2 mutations identified in the MR kindred (Figure 1). The proband's son was heterozygous for the c.55delA mutation. To the best of our knowledge, the MR and CP kindreds were unrelated. These mutations were not detected in 200 randomly selected individuals from the general population.

Plasma Levels of ANGPTL3

The plasma ANGPTL3 concentration was determined in 200 healthy subjects (100 males and 100 females) randomly selected from the general population. The values found in these subjects had a skewed distribution (median 289.5 ng/mL, interquartile range 229.2–368.6 ng/mL; values ranged from 119–590 ng/mL). This assay was performed in all subjects in the DV and MR kindreds. ANGPTL3 was undetectable in the plasma of carriers of the 2 mutant ANGPTL3 alleles. In the 2 heterozygotes of the DV kindred, the plasma ANGPTL3 level was 87 ng/mL in subject II.1 and 76 ng/mL in subject II.2. In the 2 heterozygotes of the MR kindred, the plasma ANGPTL3 level was 175 ng/mL in subject I.1 and 98 ng/mL in subject III.1.

In the following sections, all carriers of the 2 mutant ANGPTL3 alleles are indicated as ANGPTL3(−/−); heterozygous carriers are indicated as ANGPTL3(+/−) and control subjects as ANGPTL3(+/+).

Characterization of Plasma HDL

The main features of plasma HDL, plasma cholesterol esterification rate, and plasma LCAT activity in members of the DV and MR kindreds are shown in online-only Data Supplement Table S.2. More specifically, compared with a group of control subjects, the ANGPTL3(−/−) subjects of these kindreds showed a marked reduction (>50%) of plasma LpA-I levels (Figure 2A), a greatly reduced content of pre-β-HDL (Figure 2B), and an increase of small HDL associated with a reduction of large HDL (Figure 2C). In the ANGPTL3(+/−) subjects, HDL size and HDL size distribution were within the normal ranges; however, in 3 of them, the content of pre-β-HDL was reduced (online-only Data Supplement Table S.2). In the proband of DV kindred, cholesterol esterification rate and LCAT activity were close to the lower limits of the normal range. In the 2 ANGPTL3(−/−) siblings in the MR kindred, cholesterol esterification rate activity was reduced, and LCAT activity was close to the lower limits of the normal range (online-only Data Supplement Table S.2).

Figure 2.
Figure 2.

Plasma concentration of LpA-I and LpA-I:A-II particles (A) and of pre-β-HDL (B) and subclass distribution of plasma HDL according to particle size (C) in control subjects [ANGPTL3(+/+)], in homozygotes or compound heterozygotes [ANGPTL3(−/−)], and in heterozygotes [ANGPTL3(+/−)] for loss-of-function mutations of the ANGPTL3 gene. Plasma content of pre-β-HDL is expressed as percentage of total apoA-I. HDLs were divided into small (Ø<8.2 nm), medium (8.2<Ø<8.8 nm), and large (Ø>8.8 nm) particles. Results are expressed as mean±SD. The statistical analysis was performed by ANOVA, and multiple comparisons among means were performed by t test with Bonferroni correction. *P<0.05, ANGPTL3(−/−) vs ANGPTL3(+/−) and ANGPTL3 (+/+); §P<0.05, ANGPTL3(−/−) vs ANGPTL3(+/−); and †P<0.05, ANGPTL3(−/−) vs ANGPTL3(+/+).

Cholesterol Efflux Capacity of Serum

The capacity of serum from subjects in the DV and MR kindreds to promote cell cholesterol efflux (efflux potential) was tested in different cell models that selectively release cholesterol through distinct pathways.27,34–36 Efflux potential was evaluated with apoB(−) sera (apoB-depleted sera) or whole sera.33,36 The aqueous diffusion process was evaluated in J774 murine macrophages, which, under basal conditions, express low levels of ABCA1, ABCG1, and SR-BI and release membrane cholesterol to extracellular acceptors mainly by aqueous diffusion.26,34 The efflux potential of apoB(−) sera from ANGPTL3(−/−) subjects was significantly lower than that of apoB(−) sera from ANGPTL3(+/−) subjects (6.43±0.82% versus 9.45±0.71%, P=0.05; Figure 3A), which, in turn, was similar to the efflux potential of a standard apoB(−) serum (8.75±0.59%).

Figure 3.
Figure 3.

Cholesterol efflux from J774 murine macrophages to apoB-depleted sera from carriers of ANGPTL3 mutations. A, Cholesterol efflux under basal conditions (aqueous diffusion); B, total cholesterol efflux in cells stimulated with cAMP; C, ABCA1-mediated cholesterol efflux (ie, total efflux minus efflux from unstimulated cells). Cholesterol efflux values of 3 assays for each subject are given as mean±SD.

In a second set of experiments, we measured cholesterol efflux after stimulation of J774 murine macrophages with cAMP, which upregulates the ABCA1 protein.31 Under these conditions, total release of cholesterol occurs mainly via the ABCA1 and aqueous diffusion pathways.35 As shown in Figure 3B, the total cholesterol efflux to apoB(−) sera from ANGPTL3(−/−) subjects was significantly lower than the efflux to apoB(−) sera from ANGPTL3(+/−) subjects (8.14±0.73% versus 13.37±0.63%, P=0.006), which, in turn, was similar to the efflux to a standard apoB(−) serum (14.49±0.56%).

The ABCA1-mediated cholesterol efflux to apoB(−) sera from carriers of mutant alleles showed large interindividual variability. On average, the cholesterol efflux potential of apoB(−) sera from ANGPTL3(−/−) subjects was significantly lower than the efflux to apoB(−) sera from ANGPTL3(+/−) subjects (1.61±0.36% versus 3.98±0.31%, P=0.008; Figure 3C), but for both genotypes it was significantly lower than that of standard apoB(−) serum (6.42±0.55%, P=0.02). Interestingly, the efflux potential of apoB(−) serum from the proband of the DV kindred was extremely low compared with that of the 2 ANGPTL3(−/−) subjects of the MR kindred (Figure 3C). A similar difference in efflux potential was observed between the ANGPTL3(+/−) subjects of the DV kindred and those of the MR kindred (Figure 3C). The low level of efflux potential of sera from the proband of the DV kindred and her daughter could be attributed to the low pre-β-HDL content found in their sera (online-only Data Supplement Table S.2). The results obtained with whole sera confirmed those obtained with apoB(−) sera in all J774 cell models used above (online-only Data Supplement Figure S.7A–C).

Next, we tested apoB(−) sera for their capacity to promote efflux of cholesterol from Fu5AH hepatoma cells, which strongly express SR-BI protein in the plasma membrane. Cells were incubated with sera either in the absence or presence of BLT-1, a specific inhibitor of SR-BI–mediated cholesterol efflux.32 The SR-BI–mediated cholesterol efflux to apoB(−) sera from ANGPTL3(−/−) subjects was much lower than the efflux to apoB(−) sera from ANGPTL3(+/−) subjects (1.42±0.61% versus 4.71±0.53%, P=0.01; Figure 4A). On average, the apoB(−) sera from ANGPTL3(+/−) subjects displayed an efflux potential similar to that of the standard apoB(−) serum (4.49±0.44%); however, among the ANGPTL3(+/−) subjects, apoB(−) sera from subject II.1 of the DV kindred and subject I.1 of the MR kindred showed the highest ability to promote SR-BI–mediated efflux, probably because of their high levels of total HDL-C and large HDL particles (Table; online-only Data Supplement Table S.2). The efflux capacity of whole sera from all mutation carriers to promote SR-BI–mediated efflux was comparable to that observed with apoB(−) sera (online-only Data Supplement Figure S.8A).

Figure 4.
Figure 4.

SR-BI– and ABCG1-mediated cholesterol efflux to apoB-depleted sera of carriers of ANGPTL3 mutations. A, Cholesterol efflux from SR-BI–expressing Fu5AH hepatoma cells incubated either in the presence or absence of the SR-BI inhibitor BLT-1. SR-BI–mediated efflux is calculated as the difference between cholesterol efflux from untreated and BLT-1–treated cells. B, Cholesterol efflux from ABCG1-overexpressing CHO cells (CHO-G1 cells). ABCG1-mediated efflux was calculated as the difference between cholesterol efflux in CHO-G1 cells and in parental CHO-K1 cells. Cholesterol efflux values of 3 assays for each subject are given as mean±SD.

Finally, we determined the capacity of apoB(−) sera to promote ABCG1-mediated cell cholesterol efflux.28,29 ABCG1-mediated cholesterol efflux to apoB(−) sera from ANGPTL3(−/−) subjects was significantly lower than that to apoB(−) sera of ANGPTL3(+/−) subjects (7.17±0.76% versus 11.53±0.66%, P=0.01; Figure 4B). On average, the apoB(−) sera of ANGPTL3(+/−) subjects displayed an ABCG1-mediated efflux potential comparable to that of a standard apoB(−) serum (11.42±0.64%). In contrast to the results of the other efflux pathways, the ABCG1-mediated efflux potential of whole sera was not significantly different among carriers of ANGPTL3 mutations, irrespective of the presence of 1 or 2 mutant alleles (online-only Data Supplement Figure S.8B). This observation would support the notion that apoB-containing lipoproteins (LDL) present in whole serum act as acceptors in the ABCG1-mediated cholesterol efflux pathway.37

Discussion

In the present study, we describe 4 subjects with a plasma lipid profile characterized by (1) a substantial reduction of LDL-C and apoB (hypobetalipoproteinemia) comparable to that observed in subjects with heterozygous FHBL caused by APOB mutations1,2; (2) a marked reduction of HDL-C and apoA-I (on average, 0.56±0.20 mmol/L and 61.5±12.9 mg/dL, respectively; hypoalphalipoproteinemia), comparable to that observed in heterozygotes for missense or nonsense/frameshift mutations of the APOA1 gene (0.59±0.29 mmol/L and 69.2±25.4 mg/dL, respectively)38 and slightly lower than that observed in heterozygotes for mutations in the ABCA1 (0.79±0.22 mmol/L and 88.5±20.0 mg/dL, respectively)39 or LCAT (0.83±0.22 mmol/L and 106.5±19.7 mg/dL, respectively) genes22; and (3) a markedly reduced level of plasma triglyceride-rich lipoproteins (online-only Data Supplement Figure S.9). This peculiar lipid profile, which resembles that found recently in carriers of ANGPTL3 LOF mutations,20 prompted us to resequence the ANGPTL3 gene in the present study subjects, who were shown to be homozygous or compound heterozygous for “null” alleles.

The c.1198+1g>t mutation (DV kindred) had been reported previously by Romeo et al19 (designated 62842495) in heterozygous individuals of African descent and assumed to be deleterious. Here, we demonstrate that in vitro, this mutation generates an abnormal mRNA that contains the 5′ end of intron 6, which is predicted to encode a truncated protein. In addition, c.439_442delAACT (MR kindred) had been reported previously by Romeo et al19 (designated 62836264 in/del) in European and African-American heterozygous individuals. The third mutation (c.55delA; MR and CP kindreds) had not been reported previously. We found that these mutations (either in homozygosity or in compound heterozygosity) were associated with complete ANGPTL3 deficiency. The plasma lipid profile of the ANGPTL3(−/−) subjects in the present study was fairly similar to that of the 4 individuals described by Musunuru et al20 who were compound heterozygotes for LOF mutations (p.S17X/p.E129X). Although markedly reduced, the plasma triglyceride level in the ANGPTL3(−/−) subjects in the present study was slightly higher than that found in S17X/E129X carriers (0.49±0.13 versus 0.23±0.03 mmol/L, P=0.03), a difference that might be related to age, because subjects in the present study were older (65.5±3.8 versus 34.5±10.6 years; online-only Data Supplement Table S.3).

The presence of hypoalphalipoproteinemia in the ANGPTL3(−/−) subjects prompted us to investigate the subclass distribution and functionality of plasma HDL. First, we found a marked reduction in the level of LpA-I combined with a moderate decrease in that of LpA-I:A-II, an observation that may be explained by the increased activity of EL, after the elimination of the inhibitory effect induced by ANGPTL3 (see below). Studies in mice have shown that EL exerts its lipolytic activity more effectively on LpA-I than on LpA-I:A-II particles.40 Second, we observed an increase in small HDL that might be related to a reduction in the cholesterol esterification rate and possibly to reduced transfer of cholesteryl esters mediated by the cholesteryl ester transfer protein. It is likely that the low plasma triglyceride level found in ANGPTL3(−/−) subjects causes a reduced availability of triglyceride to be exchanged with cholesteryl ester during the lipid transfer process mediated by the cholesteryl ester transfer protein. The most striking observation was the reduction of pre-β-HDL in ANGPTL3(−/−) subjects, which was unexpected in the presence of a putative increased activity of EL and reduced cholesterol esterification rate.

Overall, the reduction in plasma HDL accounts for the marked reduction in the capacity of the apoB(−) sera of these individuals to promote the efflux of cholesterol. This concept is supported by the observation that the capacity of apoB(−) sera from all ANGPTL3 mutation carriers (−/− and +/− subjects taken together) to promote cholesterol efflux (total efflux and SR-BI– and ABCG1-mediated efflux) was directly correlated with plasma levels of HDL-C (R2=0.83, P<0.02; R2=0.76, P<0.05; and R2=0.85, P<0.02, respectively).

In view of the low plasma HDL and the reduced cholesterol efflux capacity of sera (a parameter recently found to show an inverse relationship with carotid intima-media thickness and to be a strong predictor of coronary heart disease),36 we would have expected evident manifestations of preclinical/clinical atherosclerosis to be present in the ANGPTL3(−/−) subjects in the present study; however, with the small number of subjects, we were unable to confirm an association between reduced cell cholesterol efflux and accelerated atherosclerosis, probably because of the markedly reduced levels of apoB-containing lipoproteins found in these individuals. It is likely that lifelong exposure to low LDL-C levels might have counterbalanced the “proatherogenic” effect related to low HDL-C. The partial ANGPTL3 deficiency found in ANGPTL3(+/−) subjects was characterized by 2 features. First, in these individuals, the mean level of plasma LDL-C (2.52±0.38 mmol/L) was below the 25th percentile of the population distribution, which resulted in a phenotype of “moderate hypobetalipoproteinemia.” This LDL-C value was slightly but not significantly higher than that reported by Musunuru et al20 in 13 heterozygous carriers of S17X or E129X mutations (1.86±0.58 mmol/L; online-only Data Supplement Table S.3) but was similar to that found in heterozygous carriers of other ANGPTL3 truncations identified in the Dallas Heart Study20 (online-only Data Supplement Table S.4). It should be stressed, however, that the mean LDL-C found in ANGPTL3(+/−) subjects in the present study was not significantly different with respect to control subjects (online-only Data Supplement Figure S.9), thus suggesting that ANGPTL3(+/−) subjects are not easily discernible in population screening for low LDL-C compared with carriers of APOB truncations, whose LDL-C levels are usually well below the fifth percentile.1,2 Second, the mean plasma level of HDL-C in heterozygotes in the present study (1.43±0.45 mmol/L) was unremarkable, as previously shown in heterozygotes for S17X or E129X mutations or in heterozygotes for other LOF mutations of ANGPTL3 identified in the Dallas Heart Study (online-only Data Supplement Tables S.3 and S.4). Collectively, the results of these studies suggest that partial ANGPTL3 deficiency does not affect plasma HDL-C levels and HDL subclass distribution and function.

Although the reduced plasma levels of triglyceride in ANGPTL3 deficiency can be explained by the elimination of the inhibitory effect of ANGPTL3 on lipoprotein lipase activity, the mechanism by which complete or partial ANGPTL3 deficiency lowers plasma LDL-C remains to be established. The recent finding that carriers of S17X and E129X mutations had decreased rate of VLDL and apoB production and an increased fractional catabolic rate for LDL-apoB20 suggests that ANGPTL3 may have a broader range of function in the metabolism of apoB-containing lipoproteins, independent of lipoprotein lipase.

In addition, the mechanism underlying the low HDL-C level found in subjects with complete ANGPTL3 deficiency is not fully understood. The elimination of ANGPTL3-mediated inhibition of lipoprotein lipase activity is expected to increase the rate of lipolysis of VLDL and chylomicrons, with a reduction in plasma triglyceride and a parallel increase in HDL-C levels.41 On the other hand, elimination of the ANGPTL3 inhibitory effect on EL is expected to increase the hydrolysis of HDL phospholipids, leading to a decrease in plasma HDL-C.17,42 Thus, in complete ANGPTL3 deficiency, the level and possibly the characteristics of plasma HDL would be the result of the gain of function of 2 lipolytic pathways (lipoprotein lipase–mediated and EL-mediated pathways) that have opposing effects on plasma HDL-C levels.

Sources of Funding

This work was supported by a grant from the University of Genoa (S.B.), by a grant from the Health Service of Emilia-Romagna Region (DiAL-ER project; I.Z., D.A., F.B., S.C.), and by a grant from Fondazione Cassa di Risparmio di Modena (P.T.).

Disclosures

None.

  • Received May 17, 2011.
  • Accepted October 26, 2011.

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Clinical Perspective

ANGPTL3 (angiopoietin-like protein 3), secreted by the liver and circulating in plasma, is an inhibitor of lipoprotein lipase and endothelial lipase. Lipoprotein lipase functions as a triglyceride lipase on chylomicrons and very low-density lipoprotein (VLDL); endothelial lipase functions as a phospholipase on phospholipid-rich high-density lipoprotein (HDL). In this study, we showed that complete deficiency of ANGPTL3, caused by loss-of-function mutations in the corresponding gene, was associated with low blood levels of triglycerides, low-density lipoprotein (LDL), and HDL, which resulted in the recessive condition designated familial combined hypolipidemia. Partial ANGPTL3 deficiency, as found in heterozygotes for ANGPTL3 mutations, is associated with reduced circulating levels of LDL only. We also demonstrated that serum from subjects with complete ANGPTL3 deficiency has a reduced capacity to promote in vitro cell cholesterol efflux (the first step of reverse cholesterol transport) as the result of the reduced HDL concentration. However, despite low plasma HDL levels, these subjects do not have accelerated atherosclerosis, probably because of their lifelong exposure to low blood levels of VLDL and LDL. These findings suggest that ANGPTL3 might be a target for hypolipidemic drugs. This concept is supported by the observation that ANGPTL3 deficiency reduces plasma levels of VLDL and the atherogenic alterations of blood vessels in apolipoprotein E knockout mice, an animal model of accelerated atherosclerosis. Inhibition of ANGPTL3 with small molecules, antisense oligonucleotides, or monoclonal antibodies might be protective against the accumulation of atherogenic lipoproteins by increasing the catabolism and clearance of triglyceride-rich lipoproteins.

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  • Characterization of Three Kindreds With Familial Combined Hypolipidemia Caused by Loss-of-Function Mutations of ANGPTL3Clinical Perspective
    Livia Pisciotta, Elda Favari, Lucia Magnolo, Sara Simonelli, Maria Pia Adorni, Raffaella Sallo, Tatiana Fancello, Ivana Zavaroni, Diego Ardigò, Franco Bernini, Laura Calabresi, Guido Franceschini, Patrizia Tarugi, Sebastiano Calandra and Stefano Bertolini
    Circulation: Genomic and Precision Medicine. 2012;5:42-50, originally published February 14, 2012
    https://doi.org/10.1161/CIRCGENETICS.111.960674
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