INTRODUCTION

Apolipoprotein (apo) A-V is a key regulator of plasma triglyceride (TG) levels [1]. Biallelic loss-of-function (LOF) variants in the APOA5 gene are a well-documented cause of familial chylomicronemia syndrome (FCS) [2], which is characterized by severely compromised plasma lipolysis resulting in pathogenic elevation predominantly of intestinally-derived chylomicrons, refractory hypertriglyceridemia (HTG), characteristic physical findings, abdominal pain with failure to thrive, and high lifetime risk of pancreatitis. The consequences of heterozygosity – that is, a monoallelic pathogenic variant of APOA5 – is less well appreciated. We recently found that the phenotype associated with monoallelic variants in APOA5 is highly variable both within and between patients over time, associated with normal TG, mild-to-moderate and severe HTG phenotypes, with secondary factors playing an important modulatory role [3▪]. This was perhaps counterintuitive to preconceived assumptions that heterozygosity for LOF variants in APOA5 would be associated with an intermediate HTG phenotype, following an incorrect analogy with familial hypercholesterolemia. Here, we synthesize this new understanding with previous information to provide an up-to-date characterization of APOA5 variants in HTG. 

Box 1:

no caption available

EXPRESSION AND PHYSIOLOGICAL ROLES OF APOLIPOPROTEIN A5 IN LIPOPROTEIN METABOLISM

Apo A-V is one of the first human proteins identified using a primordial artificial intelligence approach leveraging comparative DNA sequence analysis [4]. It was initially determined to be expressed almost exclusively in the liver and secreted with TG-rich lipoproteins, primarily very-low density lipoprotein (VLDL) [4]. Subsequently, expression of apo A-V in the small intestine was detected [5]. Intestine-derived apo A-V is presumed to circulate in plasma with lipoproteins of intestinal origin, i.e., chylomicrons. Interestingly, apo A-V of hepatic origin is also found in the bile, technically making it an exocrine secretion that may reach the intestinal lumen to possible exert effects there [6]. Despite its very low absolute plasma concentration relative to other apolipoproteins (i.e. <1 μg/ml) [7], apo A-V is a potent regulator of plasma TG concentrations [8].

Apo A-V reduces plasma TG concentrations via multiple mechanisms. First, apo A-V plays an intracellular role whereby it interferes with hepatic synthesis of VLDL particles, averting their secretion into the circulation, by associating with cellular membrane components and various lipid species within hepatocytes [9–11]. Second, apo A-V is bound to TG-rich lipoproteins (TGRLs) in plasma [7] and directly enhances the activity of lipoprotein lipase (LPL) to clear TG from circulation, although this also depends on the concurrent presence of apo C-II [7,11,12] and glycosylphosphatidylinositol anchored high density lipoprotein binding protein 1 (GPIHBP1) [13–15]. Specifically, this function of apo A-V is most likely due to its ability to bind to and/or interact with heparan sulfate proteoglycans (HSPGs) [13] and GPIHBP1 [14,15] on endothelial cell surfaces, which enhance the association of apo A-V-containing lipoproteins with endothelial cell surface features associated with LPL. GPIHBP1 is a major capillary lumen binding site and anchor for LPL [16] and is also the platform upon which LPL-mediated lipolysis occurs [17,18]. Third, there is some evidence that apo A-V mediates hepatic uptake of TRL remnants through interaction with members of the low-density lipoprotein (LDL) receptor family [19].

Recently, a novel role for apo A-V was described by which it indirectly enhances LPL activity by competing with LPL for binding to a unique inhibitory epitope present in the ANGPTL3/8 complex, thereby suppressing the LPL-inhibitory effects of the complex [20,21▪▪]. Additionally, this function of apo A-V plays a role in regulating selective tissue uptake of circulating TG in the fed versus fasted state [20,21▪▪,22–26]. Specifically, in the fed state, increased insulin signaling induces the expression of ANGPTL8 in liver and adipose tissue while downregulating hepatic apo A-V production and adipose ANGPTL4 expression. The net effect is an increase in circulating ANGPTL3/8 complex uninhibited by apo A-V, which suppresses LPL activity in oxidative tissues such as skeletal muscle. TG hydrolysis in adipose tissue in turn increases because ANGPTL8 expressed in adipose tissue complexes with ANGPTL4, and the ANGPTL4/8 complex has reduced LPL-inhibitory activity compared to ANGPTL4 alone. Additionally, binding of the ANGPTL4/8 complex to adipose tissue LPL blocks interaction with circulating ANGPTL3/8 and ANGPTL4, which results in most circulating TG in the fed state being hydrolyzed in adipose tissue, where adipocytes take up the released fatty acids and store them for energy. The opposite occurs in the fasted state. Finally, recent tantalizing experimental data suggest that the modulating influence of ANGPTL4/8 includes effects that link the coagulation system with lipolysis [27▪], an interaction that requires further exploration. These functions are summarized in Table 1.

Table 1 - Known and putative roles of apolipoprotein A-V in regulating plasma triglyceride levels Location of action Description Intracellular, within hepatocytes Interferes with hepatic synthesis of very low-density lipoprotein (VLDL) particles, averting their secretion into circulation. May also lead to hepatic lipid accumulation Plasma, bound to circulating triglyceride-rich lipoproteins Directly enhances the activity of lipoprotein lipase (LPL) to clear triglycerides from circulation by strengthening the association of apo A-V-containing lipoproteins with endothelial cell surface features associated with LPL Mediates hepatic uptake of triglyceride-rich lipoprotein remnants via interaction with members of the low-density lipoprotein (LDL) receptor family Indirectly enhances LPL activity by competing with LPL for binding to a unique LPL-inhibitory epitope present in the angiopoietin-like protein 3/8 (ANGPTL3/8) complex. This functionality also plays a role in fed versus fasted state selective tissue uptake of circulating triglycerides

Thus, integrating the above multiple effects suggests that apo A-V promotes lipolysis and thus its plasma levels should be inversely related to TG levels. However, it has long been appreciated that there is a somewhat counterintuitive positive correlation between plasma levels of apo A-V and TG [28]. This is the same direction of correlation seen between TG and apo C-III, an inhibitor of LPL activity that counteracts the effect of apo A-V [29]. One possible explanation for this direct correlation is that apo A-V circulates on TGRL and thus within the macro-biochemical context, is positively correlated with TG levels. However, at the micro-biochemical regional level in close proximity to LPL, such as the endothelial cell or adipocyte, apo A-V enhances lipolysis, eventually resulting in TG reduction, but not at an immediate or sufficiently large scale as to be reflected in total plasma TG concentration, whose decline would be delayed and reactive, adhering to a slower time course.

PROTEIN STRUCTURE OF APO A-V

After cleavage of the 23 amino acid signal peptide, the mature apo A-V protein consists of an ∼39 kDa protein composed of 343 amino acids [4,30]. It is secreted as a component of high density lipoprotein (HDL), VLDL and chylomicrons [4,9,30]. Apo A-V has two coiled-coil domains and a large α-helical content with most recent predictions of structure, indicating an α-helical content ∼35% in the lipid-free state and increasing to ∼45% upon association with lipid, corresponding to elongation and stabilization of α-helix segments [31].

Apo A-V has several main functional domains. First, the N-terminal region spanning residues 1 to 146 of the mature protein is the likely hydrophilic domain of the protein with the α-helices in this region adopting a water-soluble helix bundle configuration [31]. Second, the C-terminal region spanning residues 295–343 of mature apo A-V is highly hydrophobic [10,30,31] and has lipid binding properties [30–33]. Finally, the central intervening region between the terminal regions spanning residues 147–294 contains a string of residues associated with enhancement of LPL activity by interacting with GPIHBP1. Specifically, a positively charged region spanning residues 186–227 is involved in binding to HSPG [13], LDL receptor family members [19] and GPIHBP1 [18]. There is strong evidence that this positively charged region on apo A-V and the acidic domain of GPIHBP1 are both required for interaction [14], which likely facilitates the LPL-enhancing function. Thus, enhancement of efficient LPL-mediated lipolysis of TGRLs requires coordination between GPIHBP1, apo A-V and LPL [30]. Another region of note within the central intervening region of apo A-V is the hydrophobic region spanning residues 161 to 181 preceding the positively charged region; this region has been implicated in enabling apo A-V to bind to the surface of intracellular lipid droplets [34]. This may explain the function of apo A-V to reduce hepatic VLDL secretion, although this may also result in concurrent hepatic lipid accumulation [30].

Currently, the exact region of apo A-V associated with its ability to bind the ANGPTL3/8 complex is unknown [20,21▪▪], although some inferences might be drawn based on properties of the apo A-V-interacting epitope of the ANGPTL3/8 complex. Specifically, it seems that the apo A-V-interacting epitope is a hydrophilic leucine zipper-like motif [21▪▪]. Thus, apo A-V might interact with this motif either through its N-terminal domain, which is hydrophilic [31], or via residues in the intervening region between its terminal domains [32], since the C-terminal region of apo A-V is highly hydrophobic [10,31] and might thus be unlikely to be involved directly in this interaction.

GENOMIC STRUCTURE OF APOA5

The gene encoding apo A-V in humans, namely APOA5, is located on chromosome 11q23.3 within the apolipoprotein gene cluster that also includes APOA1, APOC3, and APOA4[2,4]. The APOA5 gene is composed of four exons and three introns spanning roughly 3.05 kb on the reverse strand. As mentioned above, the gene is expressed primarily in the liver and secondarily in the small intestine.

BIALLELIC APOA5 VARIANTS AND FAMILIAL CHYLOMICRONEMIA SYNDROME

APOA5 is one of the five canonical genes in which presence of biallelic LOF variants underlies an extremely rare – prevalence of ∼1–10 in a million [35] – condition known as familial chylomicronemia syndrome (FCS) [36]. FCS is characterized by sustained, refractory severe HTG due to essentially complete loss of LPL-mediated lipolysis of TGRLs, leading to excessive accumulation of chylomicrons in circulation [37]. Clinically, this manifests as severe HTG that is resistant to treatment, potentially resulting in several other systemic manifestations such as lipemia retinalis, eruptive xanthomatosis, hepatosplenomegaly, abdominal and acute pancreatitis, which can be fatal [1,36].

Loss of LPL-mediated lipolytic activity is caused primarily by biallelic LOF variants (i.e. homozygous or compound heterozygous) in one of the following genes; LPL in 70–80% of all cases, with the remaining 20–30% of cases caused by variants in APOA5, APOC2, GPIHBP1 or LMF1 genes [1,2,36]. These genes comprise those encoding lipoprotein lipase (LPL gene), and its four essential co-factors, apo A-V (APOA5 gene), apo C-II (APOC2 gene), glycosylphosphatidylinositol anchored high density lipoprotein binding protein 1 (GPIHBP1 gene), and lipase maturation factor 1 (LMF1 gene), respectively. Occasional FCS cases are digenic, with affected individuals having single heterozygous variants in two of these genes. In depth discussion of the diagnosis and management of FCS is beyond the scope of this review, and is covered elsewhere [1,37].

PHENOTYPIC IMPACT OF HETEROZYGOUS RARE PATHOGENIC VARIANTS IN APOA5

The phenotype associated with biallelic LOF variants in the FCS genes is well understood. However, despite being much more common, the phenotype associated with monoallelic or heterozygous LOF variants in these genes is less well appreciated. We previously showed that ∼3–4% of the general population with a normal lipid profile are heterozygous for rare pathogenic variants in one of the five FCS genes [38]. Furthermore, heterozygosity for rare variants in these genes is statistically over-represented in mild-to-moderate and severe HTG cohorts, at three- and fivefold enrichment respectively [38,39]. However, the heterozygous state is not 100% causal for HTG – it merely predisposes to increased risk of HTG. In addition, we recently showed that heterozygosity for rare LOF variants in both LPL and APOA5 is associated with highly variable TG levels both within and between patients over time, associated at times with normal TG, mild-to-moderate and severe HTG phenotypes [3▪,40▪].

A CURATED ASSEMBLY OF APOA5 VARIANTS

With respect to definite or likely disease-causing variants of APOA5, to the best of our knowledge, there are currently at least 118 unique rare variants reported in the literature and/or databases as being associated or with suggestive evidence of possible associations with phenotypes such as FCS and HTG, but also with atherosclerotic cardiovascular disease (ASCVD). We have summarized the coding sequence variants resulting in amino acid changes alongside the relative positions of the major functional domains of apo A-V in Fig. 1. We have also summarized outlined the nucleic acid changes for noncoding region variants in Fig. 2. Finally, we have curated and annotated some details of these variants in Table 2. Full details and notes related to molecular defect (where applicable) and exact citations are in Table 1, Supplemental Digital Content, https://links.lww.com/COL/A28.

FIGURE 1:

Map of reported APOA5 coding sequence variants. Boxes represent functional domains of the apo A-V peptide: Black represents the signal peptide, purple represents the N-terminal hydrophilic domain, magenta represents the lipid droplet binding domain, orange represents the positively charged GPIHBP1-interacting domain, and the yellow represents the C-terminal hydrophobic domain (lipid binding domain). Axis numbering represents the amino acid residue number in the primary structure of the newly synthesized apo A-V peptide and the specific residues indicated represent the first and last residues of the domains they share color with. All variants are color-coded according to pathogenicity classification according to ACMG guidelines: red indicates pathogenic or likely pathogenic, orange indicates a variant of uncertain significance (VUS), and green indicates benign or likely benign.

FIGURE 2:

Map of reported APOA5 noncoding variants. Gene map of APOA5 annotated with variants discovered in the regulatory regions [5’ and 3’ untranslated regions (UTRs), promoter region, etc.], splice donor and acceptor sites, and introns. Numbering underneath boxes indicates exons. Major structural features are color-coded. Black boxes indicate untranslated sequences, blue boxes indicate sequences coding for the apo A-V signal peptide, and green boxes indicate sequences coding for the mature protein. Variants are presented using nucleic acid changes. All variants are color-coded according to pathogenicity classification according to ACMG guidelines: Red indicates pathogenic or likely pathogenic, orange indicates a variant of uncertain significance (VUS), and green indicates benign or likely benign.

Table 2 - APOA5 variants reported in literature and/or clinical testing Variant type Nucleotide change Amino acid changea,b ACMG classificationc Regulatory c.-1464T>C N/A VUS Regulatory c.-1131T>C N/A Benign Splicing c.-33+1G>A N/A Likely pathogenic Regulatory c.-3A>G N/A Benign Gross deletion c.16_39del p.Ala6_Ala13del Likely pathogenic Splicing c.49+1G>A N/A Likely pathogenic Splicing c.49+5G>C N/A Likely pathogenic Splicing c.50-1G>A N/A Likely Pathogenic Missense c.56C>G p.Ser19Trp VUS Small deletion c.58delG p.Ala20Profs∗37 Likely pathogenic Small duplication c.73_76dup p.Gly26Glufs∗37 Likely pathogenic Missense c.77G>T p.Gly26Val VUS Small deletion c.77delG p.Gly26Alafs∗31 Likely pathogenic Missense c.104G>A p.Ser35Asn VUS Small deletion c.109delG p.Asp37Thrfs∗20 Likely pathogenic Small deletion c.117_120del p.Arg40Trpfs∗16 Likely pathogenic Missense c.119G>T p.Arg40Met Likely benign Small deletion c.138del p.Gln46Hisfs∗11 Likely pathogenic Missense c.154G>A p.Glu52Lys VUS Nonsense c.154G>T p.Glu52Ter Likely pathogenic Splicing c.161+3G>C N/A Likely pathogenic Splicing c.161+5G>C N/A Likely pathogenic Splicing c.162-43A>G N/A VUS Missense c.197A>G p.Asn66Ser VUS Small deletion c.211delC p.Leu71Trpfs∗4 Likely pathogenic Missense c.278G>A p.Arg93Gln VUS Missense c.280C>T p.Arg94Trp VUS Nonsense c.283C>T p.Gln95Ter Likely pathogenic Nonsense c.289C>T p.Gln97Ter Likely pathogenic Nonsense c.292G>T p.Glu98Ter Likely pathogenic Missense c.295G>A p.Glu99Lys VUS Small deletion c.295_297delGAG p.Glu99del VUS Small deletion c.305_307del p.Glu102del VUS Missense c.313G>T p.Ala105Ser VUS Small insertion c.326_327insC p.Tyr110Leufs∗158 Likely pathogenic Missense c.331A>G p.Met111Val VUS Missense c.346G>C p.Glu116Gln VUS Nonsense c.346G>T p.Glu116Ter Likely pathogenic Missense c.352G>A p.Val118Met VUS Missense c.377G>A p.Arg126Gln VUS Missense c.398C>G p.Thr133Arg VUS Nonsense c.415C>T p.Gln139Ter Likely pathogenic Small deletion c.427delC p.Arg143Alafs∗57 Likely pathogenic Missense c.434A>G p.Gln145Arg VUS Missense c.436G>A p.Glu146Lys VUS Nonsense c.442C>T p.Gln148Ter Likely pathogenic Small deletion c.447_450delGCAG p.Glu149Aspfs∗50 Likely pathogenic Small insertion-deletion c.447delGinsCTC p.Glu149Aspfs∗52 Likely pathogenic Missense c.457G>A p.Val153Met Benign Nonsense c.466G>T p.Glu156Ter Likely pathogenic Missense c.473C>T p.Thr158Ile VUS Missense c.482A>G p.Gln161Arg VUS Missense c.482A>T p.Gln161Leu VUS Missense c.494G>A p.Gly165Asp VUS Missense c.494G>C p.Gly165Ala VUS Small duplication c.494dup p.Val166Argfs∗102 Likely pathogenic Missense c.518T>C p.Leu173Pro VUS Small duplication c.550dup p.Thr184Asnfs∗84 Likely pathogenic Missense c.551C>G p.Thr184Ser VUS Missense c.553G>T p.Gly185Cys VUS Missense c.563A>G p.Lys188Arg VUS Missense c.578C>T p.Pro193Leu VUS Small deletion c.579_592del14 p.Tyr194Glyfs∗69 Likely pathogenic Missense c.589A>G p.Ser197Gly VUS Small deletion c.593_606del14 p.Leu198Argfs∗65 Likely pathogenic Missense c.610C>T p.Arg204Cys VUS Small deletion c.614_624del11 p.His205Profs∗59 Likely pathogenic Missense c.640G>C p.Ala214Pro VUS Missense c.644C>T p.Pro215Leu Likely benign Small duplication c.653_654dup p.Ala219Profs∗79 Likely pathogenic Small deletion c.654delC p.Ala219Profs∗78 Likely pathogenic Missense c.655G>C p.Ala219Pro VUS Missense c.659G>T p.Ser220Ile VUS Missense c.665C>T p.Ala222Val VUS Missense c.667C>T p.Arg223Cys VUS Nonsense c.685C>T p.Gln229Ter Likely pathogenic Missense c.694T>C p.Ser232Pro VUS Small deletion c.694_705del12 p.Ser232_Leu235del Likely pathogenic Missense c.697C>T p.Arg233Trp VUS Small deletion c.724delC p.Leu242Cysfs∗55 Likely pathogenic Missense c.725T>C p.Leu242Pro VUS Missense c.733C>T p.Arg245Cys VUS Missense c.756G>C p.Gln252His VUS Missense c.758T>C p.Leu253Pro Likely pathogenic Missense c.763G>A p.Glu255Lys VUS Missense c.764A>G p.Glu255Gly Benign Nonsense c.775A>T p.Arg259Ter Likely pathogenic Small deletion c.795del p.Thr266Leufs∗31 Likely pathogenic Missense c.811G>T p.Gly271Cys VUS Missense c.815C>A p.Pro272Gln VUS Nonsense c.823C>T p.Gln275Ter Likely pathogenic Missense c.830T>C p.Leu277Pro VUS Missense c.844C>A p.Arg282Ser VUS Missense c.844C>T p.Arg282Cys VUS Nonsense c.847C>T p.Gln283Ter Likely pathogenic Missense c.875C>T p.Thr292Ile VUS Nonsense c.883C>T p.Gln295Ter Likely pathogenic Missense c.887T>G p.Ile296Arg VUS Small deletion c.888delA p.Ile296Metfs∗42 Likely pathogenic Missense c.902G>C p.Arg301Pro VUS Nonsense c.913C>T p.Gln305Ter Likely pathogenic Small deletion c.926_928delAGG p.Glu309del VUS Nonsense c.937C>T p.Gln313Ter Likely pathogenic Missense c.941T>G p.Leu314Arg VUS Missense c.944C>T p.Ala315Val VUS Missense c.956C>T p.Pro319Leu VUS Missense c.962A>T p.His321Leu Benign Missense c.972C>G p.Phe324Leu VUS Small deletion c.980_981delAG p.Glu327Valfs∗19 Likely pathogenic Small deletion c.990_993delAACA p.Asp332Valfs∗5 Likely pathogenic Small deletion c.995_998delACAG p.Asp332Valfs∗5 Likely pathogenic Gross insertion c.999insGGCAAGG
TTGTGAGCAAGCT
GCAGGCCC p.Ser333Argfs∗5