# Synergic combination of the monogenic ANGPTL3 p.H343R variant and a polygenic predisposition in a family with hypobetalipoproteinemia

Manon Levy, Alexandre Janin, Oriane Marmontel, Anthony Fourier, Séverine Nony, Antoine Rimbert, Bertrand Cariou, Sylvie Villar-Fimbel, Sybil Charrière, Philippe Moulin, et al.

► To cite this version:

Manon Levy, Alexandre Janin, Oriane Marmontel, Anthony Fourier, Séverine Nony, et al. Synergic combination of the monogenic ANGPTL3 p.H343R variant and a polygenic predisposition in a family with hypobetalipoproteinemia. Atherosclerosis, 2025, 411, pp.120569. 10.1016/j.atherosclerosis.2025.120569. hal-05361341

HAL Id: hal-05361341

https://hal.science/hal-05361341v1

Submitted on 12 Nov 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Synergic combination of the monogenic ANGPTL3 p.H343R variant and a polygenic predisposition in a family with hypobetalipoproteinemia

Manon Levy<sup>a,b</sup>, Alexandre Janin<sup>c,d</sup>, Oriane Marmontel<sup>d,e</sup>, Anthony Fourier<sup>d</sup>, Séverine Nony<sup>d</sup>, Antoine Rimbert<sup>f</sup>, Bertrand Cariou<sup>f</sup>, Sylvie Villar-Fimbel<sup>a</sup>, Sybil Charrière<sup>a,b,e</sup>, Philippe Moulin<sup>a,b,e</sup>, Mathilde Di Filippo<sup>d,e,*</sup>

<figure><img src="image_2.png" /></figure>

## HIGHLIGHTS

* ANGPTL3 p.H343R variant cosegregated with decreased LDL-C and apoB concentrations.

* p.H343R decreases ANGPTL3 secretion and is considered as a loss of function variant.

* A LDL-C low polygenic risk score suggests a polygenic hypobetalipoproteinemia (HBL).

* HBL results here from the combination of the p.H343R and a polygenic predisposition.

## GRAPHICAL ABSTRACT

### Aim of the study

To investigate whether the ANGPTL3 p.H343R variant causes the hypobetalipoproteinemia (HBL) phenotype in a family.

### Main findings

Carriers of p.H343R had significantly lower LDL-C compared to non-carriers with a similar polygenic background. The variant reduced ANGPTL3 secretion *in vitro*, and its synergistic effect with polygenic predisposition to HBL was confirmed in the UK Biobank.

### Conclusions

The HBL phenotype in this family arises from the synergy between the ANGPTL3 p.H343R loss-of-function variant and a polygenic predisposition.

<figure><img src="image_3.png" /></figure>

atherosclerosis

Levy et al.: Synergic combination of the monogenic ANGPTL3 p.H343R variant and a polygenic predisposition in a family with hypobetalipoproteinemia.

## ABSTRACT

Background and aims: Primary hypobetalipoproteinemia (HBL) is mostly due to a polygenic origin or to monogenic disorders including loss of function (LOF) variants in APOB, much less frequently Angiopoietin-like 3 gene (ANGPTL3). A new heterozygous variant of uncertain significance (VUS), p.H343R missense variant in ANGPTL3 cosegregated with HBL in a family. The aim of the present study was to assess *in vitro* the functionality of this variant and to establish its causality in this family.

<footnote><sup>a</sup> Hospices Civils de Lyon, Fédération d'Endocrinologie, Hopital Louis Pradel, Bron, France</footnote>

<footnote><sup>b</sup> Université Lyon 1 Claude Bernard, Lyon, France</footnote>

<footnote><sup>c</sup> CNRS UMR 5261 - INSERM U1315 - Université de Lyon - Université Claude Bernard Lyon 1 Institut NeuroMyoGène, Lyon, France</footnote>

<footnote><sup>d</sup> Hospices Civils de Lyon, Service de Biochimie et de Biologie Moléculaire, Bron, France</footnote>

<footnote><sup>e</sup> INSERM U1060 CarMen, Oullins, France</footnote>

<footnote><sup>f</sup> Nantes Université, CHU Nantes, CNRS, INSERM, l'institut du thorax, F-44000, Nantes, France</footnote>

<footnote>* Corresponding author. Hospices Civils de Lyon, Service de Biochimie et de Biologie Moléculaire, Centre de Biologie Est, 59 boulevard Pinel, 69500, Bron, France.
E-mail addresses: manon.levy@gmail.com (M. Levy), alexandre.janin01@chu-lyon.fr (A. Janin), oriane.marmontel@chu-lyon.fr (O. Marmontel), anthony.fourier@chu-lyon.fr (A. Fourier), severine.nony@chu-lyon.fr (S. Nony), antoine.rimbert@univ-nantes.fr (A. Rimbert), bertrand.cariou@univ-nantes.fr (B. Cariou), sylvie.fimbel@chu-lyon.fr (S. Villar-Fimbel), sybil.charriere@chu-lyon.fr (S. Charrière), philippe.moulin@chu-lyon.fr (P. Moulin), mathilde.di-filippo@chu-lyon.fr (M. Di Filippo).</footnote>
**Methods:** Targeted next-generation sequencing was performed in the proband to assess monogenic and polygenic origins using an LDL-C-dedicated polygenic risk score (PRS<sub>LDL-C</sub>). The effect of the variant was investigated by assessing the ANGPTL3 protein secretion *in vitro*. A replication study was performed in the UK-biobank.

**Results:** All 8 HBL subjects had PRS<sub>LDL-C</sub> below the first decile, supporting a polygenic HBL. Six family members also carrying the p.H343R variant (n = 6) had a significantly lower LDL-C than the polygenic members non-carriers of the p.H343R (n = 2) (*p*=0.012). *In vitro*, p.H343R variant significantly decreased ANGPTL3 concentration in the medium (−82%, *p*=0.029) and the ratio of secretion (0.28 ± 0.06 vs. 1.00 ± 0.30 *p*=0.029, n = 3) compared to the wild-type. The synergistic combination of the p.H343R ANGPTL3 variant and a polygenic HBL predisposition was confirmed in the UK biobank.

**Conclusions:** This study shows that the novel ANGPTL3-p.H343R variant decreases ANGPTL3 secretion *in vitro* and can now be considered as a LOF variant. The lipid phenotype in this family results from a synergistic combination of the p.H343R ANGPTL3 variant and a polygenic HBL predisposition.

# 1. Introduction

Primary hypobetalipoproteinemia (HBL) refers to a heterogeneous group of inherited lipid disorders defined by low-density lipoprotein cholesterol (LDL-C) or apolipoprotein B (apoB) plasma concentrations below the fifth percentile according to age and sex [1]. Primary HBL is mostly due to a polygenic origin. The cumulative effects of multiple LDL-C-affecting alleles can be calculated at the individual level using an LDL-C-dedicated polygenic risk score (PRS<sub>LDL-C</sub>), based on the effect of several SNPs identified by GWAS [2]. A PRS<sub>LDL-C</sub> below the 10th percentile of a general population is commonly used to identify a polygenic HBL [3]. The monogenic causes include mostly monoallelic APOB-related familial HBL secretion defect (FHBL-SD2) leading to the secretion of a truncated protein in 30-40 % of cases, and more rarely monoallelic loss-of-function (LOF) in *Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9)* leading to an increased clearance of LDL [4]. More recently, LOF variants in *Angiopoietin-like 3 (ANGPTL3)* gene leading to enhanced catabolism of apoB containing lipoprotein have also been identified [5,6]. This etiology of HBL with a semi-dominant inheritance was named familial combined hypolipidemia (FHBL-EC1, OMIM # 605019). The phenotype includes decreased concentrations of LDL-C in case of monoallelic LOF, associated in addition with a concomitant decrease in plasma concentrations of high-density lipoprotein cholesterol (HDL-C) and triglyceride (TG) in case of biallelic inheritance [5]. The variants reported so far are mostly premature truncated variants (PTV) [5].

ANGPTL3, located on chromosome 1, is mostly expressed in the liver [5]. It encodes for a 460-amino-acids protein, which acts as an inhibitor of both lipoprotein lipase (LPL) and endothelial lipase (EL) [7,8]. Some studies have found no significant decrease in LDL-C concentrations in patients carrying a heterozygous LOF variant in *ANGPTL3* [9,10]. Additionally, many authors have shown a less striking but significant decrease in LDL-C and/or TG in heterozygous than in homozygous carriers of LOF variant in *ANGPTL3* [6,11–13]. Moreover, several loci near *ANGPTL3* have also been consistently associated with plasma LDL-C and/or TG concentrations in Genome Wide Association Studies (GWAS) [11,12]. Pharmacological inhibition of ANGPTL3 results in a very marked reduction in plasma triglyceride concentrations, a reduction of LDL-C, apoB and to a lesser extent, a reduction of HDL-C concentrations, providing a powerful new tool for reducing LDL-C in patients with homozygous familial hypercholesterolaemia [13,14].

The genetic analysis performed in our cohort of HBL patients have identified several ANGPTL3 variants: two LOF variants (one missense and one PTV), three heterozygous missense variants and one in frame deletion of one amino acid of uncertain significance (VUS) according to the American College of Medical Genetics and Genomics (ACMG) recommendations [15]. One new family carrying the p.H343R missense variant caught our attention because this VUS was associated with reduced concentrations of LDL-C and apoB over 3 generations.

As the effect of heterozygous ANGPTL3 missense variants on lipid profile remains controversial, we considered that the use of *in vitro* tests was required to consistently establish the causal link between the variant and the phenotype as much as the PRS<sub>LDL-C</sub> was low in this pedigree. Therefore, the aim of the present study was to assess the functionality of the p.H343R ANGPTL3 missense variant *in vitro*.

# 2. Materials and methods

## 2.1. Studied population

Patients were referred to the lipid clinic of Hospices Civils de Lyon, France, for assessment of an abnormal lipid profile (see Supplemental Material).

## 2.2. Lipid profile and ANGPTL3 concentration

Total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and apolipoproteins B concentrations were determined using commercial kits on an Architect C16000 autoanalyser (Abbott Diagnostics; NortH Chicago, IL, USA). LDL-C concentrations were calculated using the Friedewald formula or determined after ultracentrifugation as previously described [16] and were compared to percentiles in the French population [17,18]. When pretreatment value was not available (I.2), untreated LDL-C was estimated using correction factors for statins medication [19]. The multiple of median (MoM) of the LDL-C (LDL-MoM) were calculated as previously described for age and sex adjustment [20].

ANGPTL3 plasma concentration was assessed in the proband by ELISA method using a commercially available kit (Cat. No. DANL30; Bio-Techne, Minneapolis, MN, USA).

## 2.3. Genetic analysis

After genomic deoxyribonucleic acid (DNA) extraction, ANGPTL3 (NM_014495.4) as well as APOB and PCSK9 genes were sequenced with Next Generation Sequencing (NGS) Illumina technology using the DсиSeq panel and analyzed as previously described in all family members [21]. The ANGPTL3 variant was confirmed by Sanger sequencing using the primers mentioned in Supplemental Table 1.

Interpretation of the variants were performed as previously described [21].

The PRS<sub>LDL-C</sub> was calculated using a compilation of 12 single nucleotide polymorphisms (SNPs) associated to the LDL-C level, according to the method described by Talmud et al. [22]. As all family members carry at least one ε2 allele, to assess the polygenic contribution independently of APOE genotype, a modified PRS, PRS<sub>LDL-CnoAPOE</sub> was calculated using the same compilation of SNPs with the exception of rs429358 and rs7412 in APOE. Patients with a PRS < the 10th percentile of controls (score<0.593 and < 0.704 for PRS<sub>LDL-C</sub> [23] and PRS<sub>LDL-CnoAPOE</sub> respectively) were considered to have polygenic HBL.

DNA samples from all family members were sent to the National Center of Research in Human Genomics (Centre National de Recherche en Génomique Humaine, CNRGH) for whole genome sequencing (WGS), as previously described [24].

## 2.4. Expression vector construction

The vector construction is detailed in Supplemental Material. Briefly, ANGPTL3 cDNA obtained from total liver cDNA without its termination codon was amplified by polymerase chain reaction. The pcDNA3.1 (−)/Myc-His B vector was linearized and the purified PCR products were
further subcloned into the digested vector and transformed into NEB 10-beta competent *E. coli*.

Site-directed mutagenesis was performed to obtain a *ANGPTL3* mutant expression vector carrying the p.H343R (c.1028A>G) variant (NM_014495.4). We also selected 2 variations from the literature to be used as positive controls for subsequent experiments: p.N173S (c.518A>G, normal secretion but reduced function) and p.E375K (c.1123G>A, reduced secretion) [25,26].

## 2.5. Quantification of ANGPTL3 in cultured cells

HEK293T cells were cultured at 37 °C, 5 % CO₂ and 95 % air in humidified chamber. 200000 cells per well were seeded in 6-wells plate and cultured with 2 ml Dulbecco's Modified Eagle's Medium supplemented with 5 % fetal bovine serum and 1 % penicillin/streptomycin. Cells were transfected with an empty vector or plasmids containing wild-type, p.H343R, p.N173S or p.E375K mutated forms. Transfections were performed in triplicate with Fugene HD transfection reagent (Cat. No. E2311; Promega, Madison, WI, USA) using 3 µl for 2 µg of DNA per well.

After 48h, cell medium was harvested and centrifuged (5000g, 4 °C, 5 min) for collection of the supernatant. Cells were washed in phosphate-buffered saline (PBS) and then incubated on ice in 0.35 ml of 1X RIPA buffer (Cat. No. 9806S; Ozyme, Saint-Cyr-L'Ecole, France) supplemented with protease inhibitor (Cat. No. 11873580001; Roche Applied Science, Penzberg, Germany) for 5 min. Cells were subsequently scraped and harvested, sonicated 3 times 10 s on ice, and centrifuged (14000g, 4 °C, 15 min) for collection of the supernatant. ANGPTL3 concentrations were measured in duplicate and after 3 different transfections. Protein concentrations in the cells' lysate were determined using a BCA protein assay kit (Cat. No. 23225; Pierce, Thermo Fisher Scientific, Carlsbad, CA, USA).

1.5 ml samples were purified from the cells' culture media using HisPur Ni-NTA Purification Kit (Cat. No. 88227, Thermo Fisher Scientific, Carlsbad, CA, USA) and then concentrated to 90 µl on Amicon Ultra 4 10.000MWCO (Cat. No. Millipore UFC801096; Merk, Darmstadt, Germany). ANGPTL3 was quantified in cell lysate and media of each plasmid construction using the Quantikine ELISA Human ANGPTL3 Immunoassay kit in duplicate according to the manufacturer's instructions. Cell lysates were diluted using the diluent from the ELISA kit to 1:4 and media to 1:10 before the assay, except for the empty vector medium that was used pure. The absorbencies were read on an Enspire 2300 analyzer (PerkinElmer; Every, France). Results were normalized to the total protein concentration of the samples. The ratio of ANGPTL3 secretion was calculated by dividing its amount in medium by its amounts in the lysates. For this ratio, the normalization was performed by dividing each ratio to the mean ratio of wild-type conditions.

## 2.6. Cross-validation in the UK biobank

The UK Biobank study, described in detail previously is a population-based prospective cohort in the United Kingdom which included over 500,000 individuals aged between 40 and 69 years were included from 2006 to 2010 [27]. This research was conducted using the UK Biobank resource under application number 49823. Of 502,366 participants, we excluded individuals on lipid lowering treatments and with missing genetic data or plasma lipid measurements. We screened for carriers of the *ANGPTL3* p.H343R variant using whole exome sequencing (WES) data. Full details of the WES in the UK Biobank have been reported previously [28]. In short, WES was performed using IDT xGen Exome Research Panel v1.0, targeting 38 997 831 bases in 19 396 genes. Exomes were captured including 100 bp flanking regions.

The PRS<sub>LDL-C</sub> and PRS<sub>LDL-CnOAPOE</sub> were calculated as previously described. The 12 SNPs genotypes were extracted from Axiom Genotyping Array for the UK Biobank participants using bgen files with plink2 [29]. Variants were imputed using the Haplotype Reference

Consortium and UK10K + 1000 Genomes reference panels. When mentioned LDL-cholesterol values were corrected for age and sex.

Genetic and phenotypic data were combined and processed using RStudio (v.2022.02.1) with different R-packages and statistical comparisons between groups were performed with a one-side *t*-test.

## 2.7. Statistical analysis

Data were analyzed using a non-parametric Mann-Whitney two-tailed test. All statistical analysis were performed on Excel 2016 (Microsoft Corporation, Redmond, USA), GraphPad Prism 10.0 (GraphPad Software, San Diego, USA) and SPSS. Given our a priori hypothesis that carriers of the deleterious variant would have lower LDL-C concentrations than non-carriers, statistical comparisons were performed using a one-tailed *t*-test, which provides greater power for detecting differences in the predicted direction. The differences were considered to be statistically significant with *p* < 0.05.

# 3. Results

## 3.1. Clinical and biological data

The proband was a 36-year-old woman with a ten-year history of type 2 diabetes and no history of cardiovascular disease. Her lipid profile was consistent with a primary HBL (Fig. 1 and Supplemental Table 2). To clarify the origin of the hypocholesterolemic phenotype, a segregation study was conducted in the family. Seric lipid analyses revealed that a HBL phenotype was present in multiple family members and followed an apparent autosomal dominant mode of inheritance (Fig. 1, Supplemental Table 2).

## 3.2. PRS and APOE genotype

All subjects (n = 8, I.1, I.2, II.1, II.2, III.1, III.3, III.4, III.6, see Supplemental Table 2) with low LDL-C concentrations had a PRS<sub>LDL-C</sub> below the first decile, supporting a polygenic HBL. All family members carry at least one ε2 allele, and 5 of them were ε2/ε2 (II.1, II.2, III.1, III.3, III.4). The PRS<sub>LDL-CnOAPOE</sub> was below the 10th percentile in 5 subjects (I.1, II.2, III.3, III.4 and III.5).

## 3.3. Identification of an ANGPTL3 variant

The NGS revealed that the proband was heterozygous for the *ANGPTL3* c.1028G>A variant leading to the p.H343R missense. This variant affects a moderately conserved amino acid, located in the globular protein fibrin homology domain. It was predicted to be benign and without effect on splicing *in silico*. The frequency of this variant is 0.021 % according to the GnomAD database. This variant has already been described in a normolipidemic control in the literature [30] and in our laboratory in a patient on lipid-lowering treatment with severe hypertriglyceridaemia, in a context of alcohol consumption precluding a consistent assessment of his lipid phenotype. Measurements of plasma concentration of ANGPTL3 was available in the proband and showed normal concentration (ie 121 ng/ml; normal interval range: 46–157 ng/ml) as observed in the literature in several heterozygous carriers of *ANGPTL3* LOF [5]. Interestingly, a p.Y344S *ANGPTL3* missense variant affecting the next amino acid had been reported in the literature. This variant interferes with ANGPTL3 secretion and heterozygotes carriers had also a decreased plasma concentration of both LDL-C and TG without significant decrease in ANGPTL3 concentration versus non-carriers [25].

This variant was classified as a VUS according to the ACMG criteria (PM2, PP1, PP4, BP4). She did not carry any *APOB* or *PCSK9* causative variant. Lipid profiles and genetic analysis of the other family members showed that the p.H343R variant was present only in individuals with a polygenic HBL (PRS<sub>LDL-C</sub><10th percentile) and LDL-C below the 5th
<figure><img src="image_4.png" /><figcaption>Fig. 1. Pedigree of the family carrying the p.H343R variant.</figcaption></figure>

The probands (P) is indicated by an arrow. Roman numerals to the left of the pedigree indicate the generation; numerals to the left of each symbol indicate the individual family member. Squares indicate male family members; circles, female family members.

+ Subject I2 was treated by atorvastatin 20 mg; ++ Estimated untreated LDL-C values

LDL-C percentile ranges are given according to age and sex [17,18] (first percentile not determined in the reference) [17]; * Concentrations below the laboratory reference for age.

PRS<sub>LDL-C</sub> percentiles are determined in the MONALISA and FGR French cohorts as described in Ref. [38] (unpublished data).

percentile (I.2, II.1, II.2, III.1, III.3, III.4, III.6). Their median LDL-C (0.37 MoM IQR 0.22) is significantly lower than that found in the two subjects who did not carry the p.H343R variant and who had also a low PRS<sub>LDL-C</sub> (I.1 and III.5 median 0.63 MoM IQR:0.16 *p*=0.036) (Fig. 1, Supplemental Table 2). When we consider PRS<sub>LDL-C</sub><0.10th percentile, the median LDL-C (0.31 IQR:0.16) is non-significantly lower in the three carriers of the p.H343R (II.2, III.3, III.4) vs. the two non-carriers (I.1 and III.5 median 0.63MoM IQR:0.16 *p*=0.1).

Whole genome sequencing of the entire family revealed no other variant that could be responsible for the lipid phenotype. Further details about their clinical and biological characteristics are provided in Fig. 1

and Supplemental Table 2.

### 3.4. Effect on ANGPTL3 secretion

ELISA measurements showed similar ANGPTL3 concentrations in cell lysates for p.H343R variant (−37 %, *p*=0.4), as well as for controls, p.N173S (−37 %, *p*=0.1) and p. E375K (−20 %, *p*=0.4) compared to the wild-type condition (see Fig. 2 and Supplemental Table 3).

p.H343R variant significantly decreased ANGPTL3 concentration in the medium (−82 %, *p*=0.029) compared to the wild-type condition. ANGPTL3 was absent in cell medium of ANGPTL3-p.E375K, which was

<figure><img src="image_5.png" /><figcaption>Fig. 2. p.H343R ANGPTL3 secretion in transfected HEK293T cells</figcaption></figure>

ANGPTL3 concentrations were measured in the cell lysates (A) and in the medium (B) in duplicate and after 3 different transfections. Results were normalized to the total protein concentration of the samples. The ANGPTL3 media/lysate ratio was calculated (C).

Data were analyzed using a non-parametric Mann-Whitney two-tailed test.

Empty vector (EV, used as negative control), wild type construct (WT) and different variants constructs. p.N173S has normal secretion but reduced function, p.E375K has reduced secretion) [25,26]. p.H343R is the variant of interest

Panel (A), no difference was significant.

Results were shown in Supplemental Table 3.
used as a control for reduced secretion and was non-significantly
decreased in cell medium of p.N173S variant (−60 %, *p*=0.10), the
control for normal secretion but reduced function.

p.H343R variant significantly reduced the ratio of ANGPTL3 secre-
tion (0.28±0.06 compared to the wild-type condition (1.00±0.30,
p=0.029). The ratio of ANGPTL3-p.N173S secretion was similar to the
wild-type condition (0.68 ± 0.22, p=0.34).

3.5. *Cross-validation in the UK biobank*

In the UK Biobank we found 60 carriers of the p.H343R ANGPTL3
variant (heterozygous, HR in Fig. 3).

LDL-C of carriers was not significantly different from non-carriers (HH in Fig. 3; n = 318,534) (3.66 ± 0.093 vs. 3.73 ± 0.001 mmol/L, *p*=0.24, Fig. 3A). This observation is also true for normalized values (*p*=0.25) (Supplemental Fig. 1A).

The control subjects (HOM_REF, HH) with a PRS<sub>LDL-CnoAPOE</sub><10th percentile exhibited a mean difference of 0.31 mmol/L when compared to controls subjects with PRS<sub>LDL-CnoAPOE</sub>>10th percentile (*p* < 0.001) (3.45 ± 0.004 vs. 3.76 ± 0.002 mmol/L respectively, Fig. 3B–Supplemental Table 4). In comparison, carriers of the **ANGPTL3** p.H343R (HR), with a PRS<sub>LDL-CnoAPOE</sub><10th percentile, exhibited a mean difference of 0.76 mmol/L (more than twice bigger than in the non-carriers group) when compared to carriers with PRS<sub>LDL-CnoAPOE</sub>>10th percentile (*p* < 0.001) (3.75 ± 0.1 vs. 2.98 ± 0.16 mmol/L respectively, see normalized LDL-C in Supplemental Fig. 1B). Moreover, carriers of the **ANGPTL3** p.H343R (HR) with a PRS<sub>LDL-CnoAPOE</sub><10th percentile, exhibited a mean difference of 0.47 mmol/L when compared to controls subjects (HH), with PRS<sub>LDL-CnoAPOE</sub><10th percentile (*p*=0.01) (3.45 ± 0.004 vs. 2.98 ± 0.16 mmol/L respectively, Fig. 3B). These observations suggest a synergistic interaction between the variant ANGPTL3 p.H343R with the PRS-LDL-C.

4. Discussion

Genetic analysis of a family referred for HBL revealed that both the
heterozygous missense p.H343R variant of ANGPTL3 and a low PRS<sub>LDL-C</sub>, segregated with reduced LDL-C and apoB concentrations suggestive of
the combination of a monogenic and a polygenic disorder (Fig. 4).

A monogenic origin is found in only 15–50 % of people with primary HBL [3,31]. The remaining forms may be due to polygenic origins. The use of PRS<sub>LDL-C</sub> in recent studies suggests that up to 50 % of primary HBL could be due to a polygenic origin [3,23]. In this family, members with HBL phenotype had low PRS<sub>LDL-C</sub>, but it is unusual for a polygenic form to co-segregate with a phenotype over three generations and to present such low levels of LDLc. Interestingly, among subjects with a low PRS<sub>LDL-C</sub>, those carrying the p.H343R ANGPTL3 had significantly lower LDL-C concentration than non-carriers. Therefore, *in vitro* studies were conducted to determine whether this ANGPTL3 p.H343R missense variant causes a LOF.

We showed that ANGPTL3 secretion was substantially reduced for the ANGPTL3-p.H343R (−82 %) construct compared to the wild type form with a magnitude similar to that found with other variants that associate with low plasma TG or LDL-C concentration [25,26]. This variant can therefore be considered as a LOF. The effect of ANGPTL3 LOF variants on LDL-C varies among studies and its molecular mechanisms of action are not fully determined. ANGPTL3-deficiency leads to an increased activity of LPL and EL [7,8], but these lipases have no cholesterase function. We could not detect any activity attributable to recombinant wild-type ANGPTL3 under our assay conditions [32] (see Supplemental Material). Specifically, no direct inhibition of LPL activity was observed in the cell culture media using this recombinant wild-type ANGPTL3. This lack of detectable effect may be related to interference from the vector tag, insufficient ANGPTL3 concentration despite concentration steps, possible loss of protein function during concentration procedures, and/or the use of human VLDL as the TG substrate.

It has also been suggested that the decrease in LDL-C might result from a lower hepatic secretion of its precursor, very-low density lipoprotein (VLDL) particles [33]. According to this hypothesis, ANGPTL3-deficiency may be responsible for a reduced supply of free fatty acids from the circulation into the liver, leading to a reduced lipidation of VLDL particles [33]. Additionally, Burks et al. showed that ANGPTL3 deficiency in HepG2 cells impairs lipoprotein production, decreasing ApoB and TG rich lipoprotein secretion and increasing fatty acid oxidation, without hepatic lipid accumulation. ANGPTL3 also produces adaptive changes in hepatic lipid metabolism by upregulating several genes involved in the lipid metabolism [34].

The co-occurrence of an ANGPTL3 LOF and a polygenic HBL leading

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** LDL-C according to ANGPTL3 genotype and PRS in the UK Biobank</figcaption></figure>

Plasma LDL-C concentrations according to carriers of the ANGPTL3 gene variant and the PRS<sub>LDL-CnoAPOE</sub> at the 10th percentile cutoff. Shown are the mean LDL-C concentrations according to the genotype. The box plots give the median levels (middle horizontal line in each box), the interquartile ranges (delineated by the top and bottom of each box), and outliers falling below the 5th percentile or above the 95th percentile (points below or above the vertical lines, respectively). The mean comparisons between groups were performed using a one side *t*-test.
**Aim of the study**

To investigate whether the
**ANGPTL3** p.H343R variant causes
the hypobetalipoproteinemia (HBL)
phenotype in a family.

**Main findings**

Carriers of p.H343R had
significantly lower LDL-C compared
to non-carriers with a similar
polygenic background. The variant
reduced ANGPTL3 secretion *in vitro*,
and its synergistic effect with
polygenic predisposition to HBL was
confirmed in the UK Biobank.

**Conclusions**

The HBL phenotype in this family
arises from the synergy between
the **ANGPTL3** p.H343R loss-of-
function variant and a polygenic
predisposition.

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Graphical abstract.</figcaption></figure>

to a severe phenotype in this family, whereas this missense variant alone
was not associated with a significant decrease of LDL-C in the UKBB
raises the question of the need for additional factors to reveal HBL in
heterozygous ANGPTL3 LOF carriers. Indeed, the decrease in LDL
cholesterol concentration is also moderate or inconstant in heterozygous
carriers of ANGPTL3 PTV LOF [14,35], in general population, as well as
in HBL families [36,37]. Importantly, the same trend was observed in
the UK Biobank using the 165-SNP PRS [38], although it did not reach
statistical significance, likely due to limited statistical power as the score
could not be calculated for all participants because of missing
sequencing data for some polymorphisms (data not shown). Our finding
highlights the interest of calculating the PRS and suggests a synergic
combination of the ANGPTL3 p.H343R and a polygenic HBL indepen-
dent of APOE genotype. Interestingly, genes used in the PRS calculation
are mainly involved in lipid synthesis or VLDL secretion (CELSR2,
SLC22A1), LDL uptake (APOB, LDLR, PCSK9, myosin regulatory light
chain interacting protein (MYLIP)) and sterol efflux (ABCG8). These
mechanisms may therefore be complementary and thus potentiate the
effect of ANGPTL3 LOF.

Determining the causal variation in patients with primary HBL is an
important issue, firstly, to establish the diagnosis by the molecular
characterization of the defect, but also because the risk of hepatic
steatosis depends on the gene involved. Indeed, pathogenic APOB vari-
ants increase the risk of liver steatosis in both HBL and in general
population [35,39]. Conversely, neither carriers of ANGPTL3 LOF vari-
ants nor polygenic HBL have an increased risk of liver disease [23,40].

**5. Limitation**

Our study was based on a family study with a limited number of
family members. In addition, due to the low frequency of the variant in
the general population, only 60 heterozygous carriers were identified in
the UK-biobank. Of note, the LDL-C determination, either using Frie-
dewald equation (family members II.1, II.2 and III.1) or measured by a
direct method (UK-Biobank) hampers our study. Indeed, according to

the literature [18] and observations for III.3 and III.4, the results for
carriers of p.H343R who are ε2/ε2 APOE homozygotes were probably
overestimated. Other limitations included the use of HEK cells instead of
hepatic cell lines (such as HepG2) and the lack of ANGPTL3 activity
data, since decreased activity could contribute to lower LDL-C levels
even in the presence of normal plasma ANGPTL3 concentrations.

In conclusion, the functional *in vitro* study of the p.H343R hetero-
zygous ANGPTL3 missense variant, found in family members with LDL-C
and apoB concentrations below the fifth percentile supports the causal
involvement of the p.H343R ANGPTL3 variant, with a crucial additional
contribution of a polygenic predisposition. This observation shows the
complexity of the diagnosis when occurs a cosegregation between a
monogenic and a polygenic disorder.

**Author contribution**

Conceptualization: AJ, AF, AR, MDF.

Data curation: ML, OM, AR, MDF.

Formal analysis: ML, SN, CB, AR.

Funding acquisition: BC.

Investigation: SVF, ML, PM.

Methodology: AJ, AF, AR, MDF.

Project administration: MDF.

Resources: MDF.

Software: AR.

Supervision: PM, MDF.

Validation: ML, AJ, AR, OM, MDF.

Visualization: ML, MDF.

Writing original draft: ML, AJ, OM, BC, SC, PM, MDF.

Approved the version to be published, agreed to be accountable for
all aspects of the work: all authors.

**Financial support**

Whole genome sequencing was supported by the French national
project CHOPIN (Cholesterol Personalized Innovation) funded by the
Agence Nationale de la Recherche (ANR-16-RHUS-0007).

Declaration of competing interest

The authors declare the following financial interests/personal re-
relationships which may be considered as potential competing interests:
Bertrand Cariou reports financial support was provided by Agence
Nationale de la Recherche (ANR-16-RHUS-0007). If there are other
authors, they declare that they have no known competing financial in-
terests or personal relationships that could have appeared to influence
the work reported in this paper.

Acknowledgements

The authors warmly thank the members of the family, nurses of endocrinology unit for recruiting the patients, and laboratory technicians for their assistance, especially Mrs Caroline BOUVEYRON.

The present research was conducted using the UK Biobank resource under the application number 49823. We are most grateful to the Bio- informatics Core Facility of Nantes BiRD, member of Biogenouen, Institut Français de Bioinformatique (IFB) (ANR-11-INBS-0013) for the use of its resources and for its technical support.

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.atherosclerosis.2025.120569.

References

[1] Schonfeld G, Lin X, Yue P. Familial hypobetalipoproteinemia: genetics and metabolism. Cell Mol Life Sci CMLS 2005;62:1372-8. https://doi.org/10.1007/s00180-005-4473-0.

[2] Futema M, Bourbon M, Williams M, Humphries SE. Clinical utility of the polygenic LDL-C SNP score in familial hypercholesterolemia. Atherosclerosis 2018;277: 457-63. https://doi.org/10.1016/j.atherosclerosis.2018.06.006.

[3] Balder J-W, Rimbert A, Zhang X, Viel M, Kanninga R, van Dijk F, Lansberg P, Sinke R, Kuivenhoven JA. Genetics, lifestyle, and low-density lipoprotein cholesterol in young and apparently healthy women. Circulation 2018;137: 820-31. https://doi.org/10.1161/CIRCULATIONAHA.117.032479.

[4] Bredefeld C, Hussain M, Averna M, Black D, Brin M, Burnett J, Charrière S, Cuerq C, Davidson N, Deckelbaum R, Goldberg I, Granot E, Hegele R, Ishibashi S, Karmally W, Levy E, Moulin P, Okazaki H, Poinsot P, Rader D, Takahashi M, Tarugi P, Traber M, Di Filippo M, Peretti N. Guidance for the diagnosis and treatment of hypolipidemia disorders. J Clin Lipidol 2022;16:797-812. https://doi.org/10.1016/j.jacl.2022.08.009.

[5] Minicocci I, Santini S, Cantisani V, Stitziel N, Kathiresan S, Arroyo JA, Martí G, Pisciotta L, Noto D, Cefalù AB, Maranghi M, Labbadia G, Pigna G, Pannozzo F, Ceci F, Ciociola E, Bertolini S, Calandra S, Tarugi P, Averna M, Arca M. Clinical characteristics and plasma lipids in subjects with familial combined hypolipidemia: a pooled analysis. J Lipid Res 2013;54:3481-90. https://doi.org/10.1194/jlr.P039875.

[6] Musunuru K, Pirruccello JP, Do R, Peloso GM, Guiducci C, Sougnez C, Garimella KV, Fisher S, Abreu J, Barry AJ, Fennell T, Banks E, Ambrogio L, Cibulskis K, Kernytsky A, Gonzalez E, Rudzicz N, Engert JC, DePristo MA, Daly MJ, Cohen JC, Hobbs HH, Altshuler D, Schonfeld G, Gabriel SB, Yue P, Kathiresan S. Exome sequencing, ANGPTL3 mutations, and familial combined hypolipidemia. N Engl J Med 2010;363:2220-7. https://doi.org/10.1056/NEJMoa1002926.

[7] Shimizugawa T, Ono M, Shimamura M, Yoshida K, Ando Y, Koishi R, Ueda K, Inaba T, Minekura H, Kohama T, Furukawa H. ANGPTL3 decreases very low density lipoprotein triglyceride clearance by inhibition of lipoprotein lipase. J Biol Chem 2002;277:33742-8. https://doi.org/10.1074/jbc.M203215200.

[8] Shimamura M, Matsuda M, Yasumo H, Okazaki M, Fujimoto K, Kono K, Shimizugawa T, Ando Y, Koishi R, Kohama T, Sakai N, Kotani K, Komuro R, Ishida T, Hirata K, Yamashita S, Furukawa H, Shimomura I. Angiopoietin-like Protein3 regulates plasma HDL cholesterol through suppression of endothelial lipase. Arterioscler Thromb Vasc Biol 2007;27:366-72. https://doi.org/10.1161/01.ATV.0000252827.516268.99.

[9] Robciuc MR, Maranghi M, Lahikainen A, Rader D, Bensadoun A, Öörni K, Metso J, Minicocci I, Ciociola E, Ceci F, Montali A, Arca M, Ehnholm C, Jauhiainen M. Angptl3 deficiency is associated with increased insulin sensitivity, lipoprotein lipase activity, and decreased serum free fatty acids. Arterioscler Thromb Vasc Biol 2013;33:1706-13. https://doi.org/10.1161/ATVBAHA.113.301397.

[10] Minicocci I, Montali A, Robciuc MR, Quagliarini F, Censi V, Labbadia G, Gabiati C, Pigna G, Sepe ML, Pannozzo F, Lütjohann D, Fazio S, Jauhiainen M, Ehnholm C, Arca M. Mutations in the ANGPTL3 gene and familial combined hypolipidemia: a

clinical and biochemical characterization. J Clin Endocrinol Metab 2012;97: E1266-75. https://doi.org/10.1210/jc.2012-1298.

[11] Consortium Engage, Surakka I, Horikoshi M, Mägi R, Sarin A-P, Mahajan A, Lagou V, Marullo L, Ferreira T, Miraglio B, Timonen S, Kettunen J, Pirinen M, Karjalainen J, Thorleifsson G, Hägg S, Hottenga J-J, Isaacs A, Ladenvall C, Beekman M, Esko T, Ried JS, Nelson CP, Willenborg C, Gustafsson S, Westra H-J, Blades M, de Craen AJM, de Geus EJ, Deelen J, Grallert H, Hamsten A, Havulinna AS, Hengstenberg C, Houwing-Duistermaat JJ, Hyppönen E, Karssen LC, Lehtimäki T, Lyssenko V, Magnusson PKE, Mihailov E, Müller-Nurasyid M, Pindi J-P, Pedersen NL, Penninx BWJH, Polera M, Pers TH, Peters A, Rung J, Smit JH, Steinthorsdottir V, Tobin MD, Tsernikova N, van Leeuwen EM, Viikari JS, Willemes SM, Willemens G, Schunkert H, Erdmann J, Samani NJ, Käprio J, Lind L, Gieger C, Metspalu A, Slagboom PE, Groop L, van Duijn CM, Eriksson JG, Jula A, Salomaa V, Boomsma DI, Power C, Raitakari OT, Ingelsson E, Järvelin M-R, Thorsteinsdottir U, Franke L, Ikonen E, Kallioniemi O, Pietäinen V, Lindgren CM, Stefansson K, Palotie A, McCarthy MI, Morris AP, Prokopenko I, Ripatti S. The impact of low-frequency and rare variants on lipid levels. Nat Genet 2015;47: 589-97. https://doi.org/10.1038/ng.3300.

[12] Global lipids genetics consortium, discovery and refinement of loci associated with lipid levels. Nat Genet 2013;45:1274-83. https://doi.org/10.1038/ng.2797.

[13] Nurmohamed NS, Navar AM, Kastelein JJP. New and emerging therapies for reduction of LDL-cholesterol and apolipoprotein B. J Am Coll Cardiol 2021;77: 1564-75. https://doi.org/10.1016/j.jacc.2020.11.079.

[14] Dewey FE, Gusarova V, Dunbar RL, O'Dushlaine C, Schurmann M, Gottesman O, McCarthy S, Van Hout CTV, Bruse S, Dansky HM, Leader JB, Murray MF, Ritchie MD, Kirchner HL, Habegger L, Lopez A, Penn J, Zhao A, Shao W, Stahl N, Murphy AJ, Hamon S, Bouzelmat A, Zhang R, Shumel B, Pordy R, Gipe D, Herman GA, Sheu WHH, Lee I-T, Liang K-W, Guo X, Rotter JI, Chen Y-DI, Kraus WE, Shah SH, Damrauer S, Small A, Rader DJ, Wulff AB, Nordestgaard BG, Tybjærg-Hansen A, van den Hoek AM, Princen HMG, Ledbetter DH, Carey DJ, Overton JD, Reid JG, Sasiela WJ, Banerjee P, Shuldiner AR, Borecki IB, Teslovich TM, Yancopoulos GD, Mellis SJ, Gromada J, Baras A. Genetic and pharmacologic inactivation of ANGPTL3 and cardiovascular disease. N Engl J Med 2017;377:211-21. https://doi.org/10.1056/NEJMoa1612790.

[15] Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E, Voelkerding K, Rehm HL, ACMG Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American college of medical genetics and genomics and the association for molecular pathology. Genet Med Off J Am Coll Med Genet 2015;17:405-24. https://doi.org/10.1038/gim.2015.30.

[16] Michenaud L, Martré N, Rimbert A, Marmontel O, Charrière S, Gibert C, Bouveiron C, Mammi J, Cariou B, Moulin P, Di Filippo M. Evaluation of biochemical algorithms to screen dysbetalipoproteinemia in ε2ε2 and rare APOE variants carriers. Clin Chem Lab Med 2024. https://doi.org/10.1515/cclm-2024-0587.

[17] Mellerio H, Alberti C, Druet C, Capelier F, Mercat J, Josserand E, Vol S, Tichet J, Lévý-Marchal C. New modeling of reference values of cardiovascular risk factors in children aged 7 to 20 years. Pediatrics 2012;129:e1020-9. https://doi.org/10.1542/peds.2011-0449.

[18] Wargny M, Goronflot T, Rimbert A, Boursier J, Kab S, Henny J, Lainé A, Leux C, Smati S, Hadjadj S, Le May C, Goldberg M, Zins M, Cariou B. Primary hypocholesterolemia is associated with an increased risk of hepatic complications in the general population. J Hepatol 2024;80:846-57. https://doi.org/10.1016/j.jhep.2024.01.030.

[19] Haralambos K, Whatley SD, Edwards R, Gingell R, Townsend D, Ashfield-Watt P, Lansberg P, Datta DBN, McDowell IFW. Clinical experience of scoring criteria for familial hypercholesterolaemia (FH) genetic testing in Wales. Atherosclerosis 2015;240:190-6. https://doi.org/10.1016/j.atherosclerosis.2015.03.003.

[20] Marmontel O, Abou-Khalil Y, Bluteau O, Cariou B, Carreau V, Charrière S, Divry E, Gallo A, Moulin P, Paillard F, Peretti N, Rabès J, Varret M, Carrié A, Di Filippo M. Additive effect of APOE rare variants on the phenotype of familial hypercholesterolemia. Arterioscler Thromb Vasc Biol 2023;43:e270-8. https://doi.org/10.1161/ATVBAHA.123.319146.

[21] Marmontel O, Rollat-Farnier PA, Wozny A-S, Charrière S, Vanhoye X, Simonet T, Chatron N, Collin-Chavagnac D, Nony S, Dumont S, Mahl M, Jacobs C, Janin A, Causy C, Poinsot P, Tauveron I, Bardel C, Millat G, Peretti N, Moulin P, Marçais C, Di Filippo M. Development of a new expanded next-generation sequencing panel for genetic diseases involved in dyslipidemia. Clin Genet 2020;98:589-94. https://doi.org/10.1111/cge.13832.

[22] Talmud PJ, Shah S, Whittall R, Futema M, Howard P, Cooper JA, Harrison SC, Li K, Drenos F, Karpe F, Neil HAW, Descamps OS, Langenberg C, Lench N, Kivimaki M, Whittaker J, Hingorani AD, Kumari M, Humphries SE. Use of low-density lipoprotein cholesterol gene score to distinguish patients with polygenic and monogenic familial hypercholesterolaemia: a case-control study. Lancet 2013;381: 1293-301. https://doi.org/10.1016/S0140-6736(12)62127-8.

[23] Rimbart A, Vanhoye X, Coulibaly D, Marrec M, Pichelin M, Charrière S, Vanhoye X, Simonet T, Valéro R, Wargny M, Carrié A, Lindenbaum P, Deleuze J-F, Genin E, Redon R, Rollat-Farnier PA, Goxe D, Degraef G, Marmontel O, Divry E, Bigot-Corbel E, Moulin P, Cariou B, Di Filippo M. Phenotypic differences between polygenic and monogenic hypobetalipoproteinemia. Arterioscler Thromb Vasc Biol 2021;41: e63-71. https://doi.org/10.1161/ATVBAHA.120.315491.

[24] Dijk W, Di Filippo M, Kooijman S, Van Eenige R, Rimbert A, Caillaud A, Thedrez A, Arnaud L, Pronk A, Garçon D, Kotin T, Lindenbaum P, Ozcariz Garcia E, Pais De Barros J-P, Duvillard L, Si-Tayeb K, Amigo N, Le Questel J-Y, Rensen PCN, Le May C, Moulin P, Cariou B. Identification of a gain-of-function LIPC variant as a
novel cause of familial combined hypocholesterolemia. Circulation 2022;146: 724-39. https://doi.org/10.1161/CIRCULATIONAHA.121.057978.

[25] Wang X, Wang D, Shan Z. Clinical and genetic analysis of a family diagnosed with familial hypobetalipoproteinemia in which the proband was diagnosed with diabetes mellitus. Atherosclerosis 2015;239:552-6. https://doi.org/10.1016/j. atherosclerosis.2015.02.031.

[26] Romeo S, Yin W, Kozlitina J, Pennacchio LA, Boerwinkle E, Hobbs HH, Cohen JC. Rare loss-of-function mutations in ANGPTL family members contribute to plasma triglyceride levels in humans. J Clin Investig 2008;JCI37118. https://doi.org/ 10.1172/JCI37118.

[27] Sudlow C, Gallacher J, Allen N, Beral V, Burton P, Danesh J, Downey P, Elliott P, Green J, Landray M, Liu B, Matthews P, Ong G, Pell J, Silman A, Young A, Sprosen T, Peakman T, Collins R. UK biobank: an open access resource for identifying the causes of a wide range of complex diseases of middle and old age. PLoS Med 2015;12:e1001779. https://doi.org/10.1371/journal.pmed.1001779.

[28] Van Hout CV, Tachmazidou I, Backman JD, Hoffman JD, Liu D, Pandey AK, Gonzaga-Jauregui C, Khalid S, Ye B, Banerjee N, Li AH, O'Dushlaine C, Marcketta A, Staples J, Schurmann C, Hawes A, Maxwell E, Barnard L, Lopez A, Penn J, Habegger L, Blumenfeld AL, Bai X, O'Keeffe S, Yadav A, Praveen K, Jones M, Salerno WJ, Chung WK, Surakka I, Willer CJ, Hveem K, Leader JB, Carey DJ, Ledbetter DH, Geisinger-Regeneron DiscovEHR Collaboration, Cardon L, Yancopoulos GD, Economides A, Coppola G, Shuldiner AR, Balasubramanian S, Cantor M, Genetics Center Regeneron, Nelson MR, Whittaker J, Reid JG, Marchini J, Overton JD, Scott RA, Abecasis GR, Yerges-Armstrong L, Baras A. Exome sequencing and characterization of 49,960 individuals in the UK biobank. Nature 2020;586:749-56. https://doi.org/10.1038/ s41586-020-2853-0.

[29] Chang CC, Chow CC, Tellier LC, Vattikuti S, Purcell SM, Lee JJ. Second-generation PLINK: rising to the challenge of larger and richer datasets. GigaScience 2015;4:7. https://doi.org/10.1186/s13742-015-0047-8.

[30] Bea AM, Franco-Marín E, Marco-Benedí V, Jarauta E, Gracia-Rubio I, Cenarro A, Civeira F, Lamiquiz-Moneo I. ANGPTL3 gene variants in subjects with familial combined hyperlipidemia. Sci Rep 2021;11:7002. https://doi.org/10.1038/ s41598-021-86384-y.

[31] Blanco-Vaca F, Martin-Campos JM, Beteta-Vicente Á, Canyelles M, Martínez S, Roig R, Farré N, Julve J, Tondo M. Molecular analysis of APOB, SAR1B, ANGPTL3, and MTTP in patients with primary hypocholesterolemia in a clinical laboratory setting: evidence supporting polygenicity in mutation-negative patients. Atherosclerosis 2019;283:52-60. https://doi.org/10.1016/j. atherosclerosis.2019.01.036.

[32] Di Filippo M, Marçais C, Charrière S, Marmontel O, Broyer M, Delay M, Merlin M, Nollace A, Valéro R, Lagarde M, Pruneta-Deloche V, Moulin P, Sassolas A. Post-heparin LPL activity measurement using VLDL as a substrate: a new robust method

for routine assessment of plasma triglyceride lipolysis defects. PLoS One 2014;9: e96482. https://doi.org/10.1371/journal.pone.0096482.

[33] Tikka A, Jauhiainen M. The role of ANGPTL3 in controlling lipoprotein metabolism. Endocrine 2016;52:187-93. https://doi.org/10.1007/s12020-015- 0838-9.

[34] Burks KH, Xie Y, Gildea M, Jung I-H, Mukherjee S, Lee P, Pudupakkam U, Wagoner R, Patel V, Santana K, Alisio A, Goldberg IJ, Finck BN, Fisher EA, Davidson NO, Stitziel NO. ANGPTL3 deficiency impairs lipoprotein production and produces adaptive changes in hepatic lipid metabolism. J Lipid Res 2024;65: 100500. https://doi.org/10.1016/j.jlr.2024.100500.

[35] Di Costanzo A, Pirona I, Buonaiuto S, D'Erasmo L, Bini S, Tramontano D, Covino S, Maiorca C, Minicocci I, Sponziello M, Baratta F, Commodari D, Colonna V, Via A, Arca M. Effect of cholesterol-lowering variants in ANGPTL3 and APOB genes on liver disease. J Am Coll Cardiol 2024;84:1767-70. https://doi.org/10.1016/j. jacc.2024.08.013.

[36] Musunuru K, Pirruccello JP, Do R, Peloso GM, Guiducci C, Sougnez C, Garimella KV, Fisher S, Abreu J, Barry AJ, Fennell T, Banks E, Ambrogio L, Cibulskis K, Kernytsky A, Gonzalez E, Rudzicz N, Engert JC, DePristo MA, Daly MJ, Cohen JC, Hobbs HH, Altshuler D, Schonfeld G, Gabriel SB, Yue P, Kathiresan S. Exome sequencing, ANGPTL3 mutations, and familial combined hypolipidemia. N Engl J Med 2010;363:2220-7. https://doi.org/10.1056/NEJMoa1002926.

[37] Minicocci I, Santini S, Cantisani V, Stitziel N, Kathiresan S, Arroyo JA, Martí G, Pisciotta L, Noto D, Cefalù AB, Maranghi M, Labbadia G, Pigna G, Pannozzo F, Ceci F, Ciociola E, Bertolini S, Calandra S, Tarugi P, Averna M, Arca M. Clinical characteristics and plasma lipids in subjects with familial combined hypolipidemia: a pooled analysis. J Lipid Res 2013;54:3481-90. https://doi.org/10.1194/jlr. P039875.

[38] Vanhoye X, Bardel C, Rimbert A, Moulin P, Rollat-Farnier P, Muntaner M, Marmontel O, Dumont S, Charrière S, Cornélis F, Ducluzeau P, Fonteille A, Nobecourt E, Peretti N, Schillo F, Wargny M, Cariou B, Meirhaeghe A, Di Filippo M. A new 165-SNP low-density lipoprotein cholesterol polygenic risk score based on next generation sequencing outperforms previously published scores in routine diagnostics of familial hypercholesterolemia. Transl Res 2022. https://doi.org/ 10.1016/j.trsl.2022.12.002.

[39] Smati S, Wargny M, Boursier J, Moulin P, Di Filippo M, Cariou B. Prevalence of liver steatosis and fibrosis in adults with primary hypobetalipoproteinemia: results from the HYPOCHOL study. Clin Gastroenterol Hepatol Off Clin Pract J Am Gastroenterol Assoc 2024;S1542-3565(24). https://doi.org/10.1016/j. cgh.2024.06.031. 00606-2.

[40] D'Erasmo L, Di Martino M, Neufeld T, Fraum TJ, Kang CJ, Burks KH, Di Costanzo A, Minicocci I, Bini S, Maranghi M, Pigna G, Labbadia G, Zheng J, Fierro D, Montali A, Ceci F, Catalano C, Davidson NO, Lucisano G, Nicolucci A, Arca M, Stitziel NO. ANGPTL3 deficiency and risk of hepatic steatosis. Circulation 2023;148:1479-89. https://doi.org/10.1161/CIRCULATIONAHA.123.065866.