PCSK9 Gene Participates in the Development of Primary Dyslipidemias

Abstract

Dyslipidemias are a group of diseases, which are characterized by abnormal blood concentrations of cholesterol, triglycerides and/or low-density lipoprotein-cholesterol (LDL-c). Dyslipidemia is a determinant condition for the progress of an atherosclerotic plaque formation. The resulting atherogenicity is due to at least two mechanisms: first, to the accumulation in the plasma of lipid particles that have the capacity to alter the function of the endothelium and deposit at the atheromatous plaque, and second, at an insufficient concentration of multifactorial type of high density lipoprotein-cholesterol (HDL-c), whose function is to protect against the development of atherosclerosis. Its highest prevalence is encountered among individuals with diabetes, hypertension or overweight. Hyperlipidemia is one of the main predisposing factors for the development of cardiovascular disease. Hyperlipidemia can be the result of a genetic condition, the secondary expression of a primary process or the consequence of exogenous factors (food, cultural, socio-economic, etc.), all of which lead to the elevation of plasma lipid levels. The objective of this study was to carry out an analysis of the genes involved in the development of dyslipidemias that lead to cardiovascular disease with special emphasis on the proprotein convertase subtilin/kexin type 9 (PCSK9) gene. The PCSK9 gene participates in the development of primary dyslipidemias, mainly familial hypercholesterolemia, currently the pharmacological treatment of choice to reduce LDL-c are statins, however, it has been observed that these have been insufficient to eliminate cardiovascular risk, especially in subjects with primary forms of hypercholesterolemia related to genetic mutations, or statin intolerance.

Introduction

Cardiovascular diseases (CVDs) are considered the leading cause of death in Mexico and worldwide [1,2]. According to the WHO website, in 2016 alone, CVDs produced an estimate of 17.9 million deaths [2]. Strikingly, 82.0% of the 16 million deaths due to non-communicable diseases that occur in people under 70 years of age, take place in low and middle income countries. And in turn, 37.0% of such events find their origins in CVDs [1]. In particular, atherosclerotic CVDs, namely ischemic heart disease and cerebrovascular disease, account for the greater part of CVD mortality, even if the trend has been decreasing throughout the last decades [3, 4, 5]. Amid the principal risk factors for CVD, we can list a sedentary lifestyle, excessive consumption of saturated fats, smoking, diabetes mellitus (DM) and high blood pressure [6]. Although hypertension is, globally, the risk factor with the largest attributable risk for CVD mortality [3], it is well known that hypertension and dyslipidemia act in a synergistic way on the pathophysiology of atherosclerotic CVDs [7]. In fact, dyslipidemia is regarded as a prerequisite for the maturation of an endothelial primary lesion into an atheromatous plaque [8,9]. Thus, the detection and treatment of plasmatic lipid alterations are key to the prevention and management of chronic non-communicable diseases.

Dyslipidemias are defined as a set of diseases caused by abnormal concentrations of blood lipoproteins. They are considered metabolic disorders that are largely conditioned by environmental factors, lifestyles, metabolic problems associated with obesity, insulin resistance and DM. Yet, some forms of dyslipidemias appear to be more frequent in direct relatives of dyslipidemic individuals, compared with the general population [10]. The most common dyslipidemias are characterized by low levels of cholesterol bound to high-density lipoprotein (HDL-c) and elevation of tri-glyceride (TG) levels. Interestingly, the ratio between TGs and HDL-c can be an indicator of the presence of resistance to insulin [11]. Cholesterol transported by low-density lipoproteins (LDL-c) seem to play a crucial role in the development of atherosclerotic diseases. Evidence points at a strong correlation between LDL-c hypercholesterolemia and atherosclerotic CVDs [12,13]. Atherogenicity is due to at least two mechanisms: first, to the accumulation in the plasma of particles that have the ability to alter the function of the endothelium and can be deposited at the atheromatous plaques, and second, to an insufficient concentration of particles that protect against the development of atherosclerosis [12, 13, 14].

Dyslipidemias can be classified according to the criteria established by the WHO and divided into primary and secondary (see Table 1). Primary dyslipidemias are those that occur due to genetic conditions affecting apolipoproteins, their receptors or enzymes implicated in lipid metabolism [10,15]. On the other hand, secondary dyslipidemias are produced by acquired alterations in the function of some of these components owing to the type of diet, associated pathologies or drug consumption [12,13]. The present review aims to analyze the contribution of genetic factors on the origins of primary dyslipidemias, with special focus on the proprotein convertase subtilin/ kexin type 9 (PCSK9) gene.

Table 1.

Classification of hyperlipoproteinemias based on the criteria established by the World Health Organization.

Electrophoresis	Lipoproteins	Lipids	Diagnosis
chylomicron band at the origin	fasting chylomicronemia	triglycerides; cholesterol	familial hyperchylomicronemia (type I)
β band increased	LDL increased	cholesterol	isolated or severe hypercholesterolemia (type IIA)
pre β band; β increased	VLDL; LDL increased	cholesterol; triglycerides	combined hyperlipidemia (type IIB)
β floating band	β-VLDL (residual chylomicrons; IDL)	triglycerides; cholesterol	hyperlipidemia mixed (type III)
pre β band increased	VLDL	triglycerides	isolated or severe hypertriglyceridemia (type IV)
band of chylomicrons; pre β increased	chylomicrons; VLDL	triglycerides; cholesterol	hypertriglycerdemia (type V)

LDL: low-density lipoprotein; VLDL: very-low-density lipoprotein; IDL: intermediate density lipoprotein.

PCSK9 Biology Overview. In humans, the PCSK9 gene is located on the short arm of chromosome 1 at a locus correlated with familial hypercholesterolemia (FH), a highly prevalent form of autosomal dominant hypercholesterolemia (ADH) [16,17]. The PCSK9 gene is a serine protease that promotes the internalization and later lysosomal degradation of the LDL receptor (LDLr), for the most part, in the liver tissue [18,19]. The principal action of PCSK9 is too direct LDLr toward lysosomal degradation, thereby reducing LDLr expression at the cell surface and increasing plasma LDL-c levels, carriers of loss-of-function PCSK9 variants have lower plasma LDL-c levels, PCSK9 might also regulate other receptors such as APOE2R, CD36, and very low-density lipoprotein receptor (VLDLr) gene [20]. As a consequence, LDL-c does not clear optimally, which ultimately manifests as an increase in plasmatic LDL-c levels [21]. Lung and liver stand as the main sources of PCSK9 [22], where it is synthesized to be later secreted into the bloodstream [23,24]. Because of its role in cholesterol metabolism, PCSK9 rapidly enticed the attention as a therapeutic target [25,26]. Currently, PCSK9 inhibitors are recommended, besides statins, in order to lower LDL-c levels in both primary and secondary prevention regimes for individuals with high risk of atherosclerotic CVDs [27]. Regarding diagnosis and prognosis, PCSK9 levels tend to be higher in coronary artery disease (CAD) patients than in healthy controls, when considering confounding factors [28]. In that same line, it has been suggested that PCSK9 plasma levels could be a strong predictor of coronary arteries calcification [29]. In patients with suspected acute coronary syndrome, PCSK9 levels appeared elevated only when vascular lesions were confirmed, assessed by coronary angiography [30]. And what is more, the severity of CAD correlates robustly with PCSK9 concentrations, in a model incorporating lipid or inflammation indices as mediator variables [28]. Thus, in addition to its involvement on the LDLr lifecycle, PCSK9 also seems to play an important role controlling inflammatory status and, therefore, atherosclerosis risk [31].

PCSK9 Mutations Related to Familial Hyperlipidemia. As expected, alterations in PCSK9 gene are related to dyslipidemias and atherosclerotic CVDs. In a general fashion, gain-of-function mutations of PCSK9 lead to high levels of circulating LDL-c and enhanced risk of CVD [32], whereas nonsense loss-of-function mutations have the opposite effect [33, 34, 35]. In 2003, a pioneering study by Abifadel et al. [36] identified two missense mutations of the PCSK9 gene that were linked to ADH in French families, in which alterations at LDLr and APOB genes were previously discarded. Since that seminal study, a number of subsequent genetic and clinical investigations have associated a defective PCSK9 gene with several forms of FH, characterized by a dominant inheritance pattern. First, a variant was identified in an American Utah kindred, which was produced by a single nucleotide exchange at the seventh exon of the PCSK9 gene sequence that resulted in the missense D374Y substitution [37]. This was motivated by a previous report that unveiled a linkage between FH and the chromosome 1p32 locus, where the PCSK9 gene is located [38]. In a parallel manner, the Utah D374Y along with the N157K substitutions were found in a sample of Norwegian FH patients [39]. Consistent with these reports, the D374Y mutation was later found in English families, correlating with a serious dyslipidemic profile [40]. Remarkably, at that same codon, a point mutation led to the D374H substitution in two Portuguese FH patients [41]. Later on, four more mutations were encountered in the French population, three of them in highly conserved residues located in the catalytic domain of PCSK9. However, only R218S could be properly linked to ADH, according to DNA family members samples as well as to the clinical history of the family [42]. Notably, the S127R mutation, originally described in the French population [35], was also observed in South African families, which exhibited a FH phenotype [43]. And in New Zealand, two variants (D129G, A168E) were associated with FH and family history of CVD. Intriguingly, both S127R and D129G forms were found unable to be secreted by HuH7 cells, suggesting that these mutated forms of PCSK9 might bind to LDLr at the intracellular compartment [43]. Furthermore, 24 PCSK9 variants were reported as specific for the Japanese population, with some of them segregated to either low or high LDL-c groups [34].

As depicted in Figure 1 of the study by Hopkins et al. [32], there are a variety of regions implicated in missense gain-of-function mutations of PCSK9, each of them expressing a different degree of severity in LDL-c dysregulation. A quick inspection of such a figure allows one to acknowledge that defects in exons 2 and 7 produce the stronger effects [32]. Notwithstanding the wide heterogeneity of missense mutations, they all seem to share common clinical manifestations, encompassing tendon xanthomas, premature myocardial infarction and stroke [44]. Recently, in addition to missense mutations, copy number variations of the PCSK9 gene have been recently associated with FH. The duplication of the whole gene conducted to the highest PCSK9 plasma concentration ever reported, being nearly 5000 ng/mL in one of the cases, accompanied by a pronounced dyslipidemia [45]. And even in normolipidemic subjects, a polymorphism affecting the 3’ untranslated region of the PCSK9 gene, correlate with lower circulating HDL-c, interestingly, with no effect over LDL-c [46].

PCSK9 and Familial Hyperlipidemia Genetic Heterogeneity. As a cause of ADH, PCSK9 defects remain relatively rare compared with mutations affecting LDLr or apolipoprotein B100 (apoB-100). For the most part, FH cases are attributed to altered variants of LDLr, followed far behind by APOB, PCSK9 as well as other genes. Roughly, such proportion is essentially similar across different populations including those found in Mexico and Latin America [47,48], US and Canada [49], Portugal [41], Gran Canaria Island in Spain [50], Lebanon [51] and Taiwan [52], just to quote a few examples. But though uncommon, the ample diversity of PCSK9 mutants and polymorphic forms could be a determinant for the differences found in cholesterol metabolisms across populations, even in a more dynamic manner than LDLr or apo-B100 [44]. Of note, a more aggressive form of ADH is related to PCSK9 compared with either defective LDLr or apoB-100, reflecting as comparatively increased levels of both total plasma cholesterol and LDL-c, and possibly, premature development of atherosclerotic CVDs [32,40,41]. Even more pronounced dyslipidemias derive from mutations affecting multiple genes. Indeed, individuals carrying double mutations in both LDLr and PCSK9 display a more severe dyslipidemic phenotype than simple heterozygotes for mutated forms of LDLr, resembling the phenotype resulting from the homozygous LDLr founders [53,54]. In contrast, a leucine in-frame insertion in PCSK9 exon 1 seems to partially counteract LDLr defects [51]. And not surprisingly, homozygous carriers of PCSK9 gain-of-function mutations show larger elevations of LDL-c plasma levels, in comparison with heterozygotes [54].

Animal Models of Familial Hyperlipidemia Carrying Altered Types of PCSK9. Of paramount importance for the study of dyslipidemias, genetically engineered animal models have been shown to be able to recapitulate in some respects the phenotype found in FH human patients, when PCSK9 is altered. For instance, when human PCSK9 carrying the D374Y Utah mutation was stably expressed in either mice or hamsters, animals clearly developed hypercholesterolemia followed by atherosclerotic lesions in the aorta and its branches [55,56]. Using a similar approach, overexpression of the mutated murine D377Y-PCSK9 led to decreased hepatic LDLr, hyperlipidemia, vascular calcification and collagen deposition at atheromatous plaques [57]. In opposition, PCSK9 knock-out mice developed 74.0% less aortic cholesterol accumulation than their wild type counterparts after 12 months of western diet exposure. Moreover, PCSK9 knock-out seemed to prevent, in great measure, atherosclerosis due to the lack of apolipoprotein E [58].

Very similar outcomes were obtained in studies performed in swine models. Transgenic Yucatan minipigs carrying human PCSK9-D374Y exhibited reduced hepatic LDLr receptors, accompanied by severe hypercholesterolemia that mostly affected the LDL-c fraction [59]. Under a high-fat, high-cholesterol regime, transgenic pigs presented with rapidly progressing atheromatous plaques in aorta and iliofemoral arteries, with pathological characteristics mimicking human atheromas [59]. Likewise, Ossabaw minipigs expressing primate PCSK9-D374Y and subjected to an atherogenic diet scheme showed early atherosclerosis and endothelial dysfunction at aorta, coronary and renal arteries, as a consequence of a pronounced dyslipidemia [60,61].

Candidate Genes with Active Participation in the Development of Primary Dyslipidemias. The most common genetic disorder of HDL-c is familial hypoalphalipoproteinemia (FHA) (HDL-c levels between 20 and 40 mg/ dL) and a family history of low HDL-c levels in at least one first-degree relative. The metabolic etiology in many cases appears to be accelerated catabolism of HDL and its apolipoproteins, and some subjects, but not all, are characterized by small, lipid-poor HDL particles and defective lipid efflux. Familial hypoalphalipoproteinemia was previously considered to be a dominant disorder due to mutations in the ABCA1 gene in some families and of unknown genes in other families. Several monogenic disorders of extremely low HDL-c levels have also been described. Although these monogenic causes are rare, and together, they may explain only a small portion (1.0%) of low HDL-c cases in the general population, they have demonstrated that extremely low HDL-c levels influence multiple organs, and thus, the clinical significance of HDL deficiency extends beyond cardiovascular risk [62, 63, 64].

APOB Gene. ApoB-100 is a component of LDL located at 2p24-p23; the APOB gene is made up of 29 exons and encodes two main isoforms of ApoB (see Table 2), ApoB-48 and ApoB-100. When ApoB is damaged, LDL-c cannot bind to LDLr, and in consequence, LDL-c levels remain elevated in the bloodstream [65]. In contrast with PCSK9, there is a limited but important number of mutations in ApoB-100 that can lead to FH. Of these, the R3500Q is the most important [66]. In Europe, only 2.5% of the FH cases are due to ApoB defects [67]. For the eastern population, the R3500Trp variant is the most common [68].

Table 2.

Genes involved in the development of dyslipidemias.

Gene	Locus	Phenotype
PCSK9	1p32	related to autosomal dominant HAD and elevation of blood cholesterol levels
APOB	2p24-23	increase in blood cholesterol
LDLRAP1/ARH	1p36-35	related to autosomal recessive HAR and accumulation of LDL receptor in cell membranes
APOE	19q13.32	associated with hyperlipoproteinemia type 3, Alzheimer’s disease, lipoproteic glomerulopathy and familial hypercholesterolemia
LDLr	19p13.1-13.3	associated with familial hypercholesterolemia
ABCG5 and ABCG8	2p21	both genes are related to the appearance of sitosterolemia

HAD: dominant hypercholesterolemia; HAR: recessive hypercholesterolemia; LDL: low-density lipoprotein.

LDLRAP1/ARH (autosomal recessive hypercholesterolemia) Gene. In opposition to LDLr, APOB and PCSK9, the LDL receptor gene adapted to protein 1 (LDLRAP1) is responsible for a type of hypercholesterolemia inherited following an autosomal recessive pattern. For these reasons, LDLRAP1 is also known as the ARH gene, [36]. The LDLRAP1 gene is located at chromosome 1p36-35 [69], made up of nine exons that encode a protein of 308 amino acids. In the ARH, the internalization of the ligand-receptor complex (APOB-LDLr) is not carried out, which produces LDLr accumulating at the cell membrane. Despite the aforementioned, it is much less frequent to find cases of FH compared with ADH, the number of cases reported to date does not exceed 100 [70]. These cases have been found in Lebanese, Mexican, Japanese, Indian, English, Turkish, American and Syrian populations [71,72].

APOE Gene. As ApoB, apolipoprotein E (ApoE) is also a structural component of LDL. The ApoE gene is located at chromosome 19q13.32, made up of four exons. It has been found that damage to apolipoprotein B may be associated with hyperlipoproteinemia type 3, Alzheimer’s disease, lipoprotein glomerulopathy and FH, causing, in the latter, an excessive deposit of cholesterol in the tissues, due to the binding, internalization and catabolism of lipo-proteins, behaving as a ligand of the LDL receptor in liver tissues, the best-known mutation being Leu167del [72, 73, 74].

LDLr Gene. The LDLr is located on the short arm of chromosome 19 (p13.1-13.3) and consists of 18 exons and 17 introns [75]. Point mutations present in this gene can affect the functionality of the developed protein, however, mutations can occur that affect the promoter of the gene, preventing it from being transcribed, and thereby interrupting the synthesis of the protein; other mutations include substitutions and those that affect the cytoplasmic domain of the receptor, thus preventing your internalization. Mutations of the LDLr gene associated with problems such as FH are divided into five classes, if the synthesis of LDLr, its transport, its union, its internalization or its recycling does not work correctly, there will be an accumulation of cholesterol in the blood, facilitating the formation of atheromatous plaques, xantales, tendinous xanthomas and corneal arches [76, 77, 78, 79, 80].

ABCG8 and ABCG5 Genes. The ABCG8 and ABCG5 genes, each consisting of 13 exons, are located on chromosome 2p21; both genes are related to the appearance of sitosterolemia, which is a rare autosomal recessive disorder characterized by intestinal hyperabsorption of all sterols, including cholesterol and plant and shellfish sterols, and impaired ability to excrete sterols into bile. Patients frequently develop tendon and tuberous xanthomas, accelerated atherosclerosis, and premature CAD. They have identified multiple mutations in the ABCG8 gene and mutations in the ABCG5 gene in patients with sitosterolemia. The ABCG5 and ABCG8 genes normally cooperate to limit intestinal absorption and to promote biliary excretion of sterols, and mutated forms of these transporters predispose to sterol accumulation, FH and atherosclerosis [81,83].

ABCA1 Gene. The ABCA1 gene belongs to a group of genes called the ATP-binding cassette family located at 9q31. It moves phospholipids and cholesterol across the cell membrane for the formation ofHDL-c, and has an important role in the initial phase of reverse cholesterol transport. Mutations in this gene have been associated with Tangier disease, an autosomal-recessive disorder characterized by deposition of cholesterol esters in organs, and familial HDL deficiency, low cellular cholesterol efflux due to mutant ABCA1 that leads to reduced apolipoprotein A-I stability and rapid catabolism of HDL-c [84, 85, 86, 87].

PCSK9 Inhibitor Therapy. Proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitors are promising therapies that inhibit the degradation of LDL receptors in the hepatocyte and thus increase LDL-c uptake from the blood. Among the various monoclonal antibodies developed against PCSK9, two stand out: evolocumab and alirocumab, these have been approved for clinical use, both fully human monoclonal antibodies are administered subcutaneously. Three large randomized, double-blind, placebo-controlled studies have provided cardiovascular results evaluating PCSK9 therapy with inhibitors, these studies are: FOURIER trial, SPIRE-1 and SPIRE-2 trials, and ODYSSEY Outcomes trial. The PCSK9 inhibitors are now proven to be valid additions to the clinicians’ armamentarium for the treatment of dyslipidemia. These drugs reduce plasma LDL-c level by approximately 60.0%, significantly reduce the risk of major vascular events and have no adverse effects except for injection-site reactions. These therapeutics could offer the opportunity to intervene earlier and more easily to treat dyslipidemia and potentially to largely eradicate coronary disease [88].