PCSK9 GENE PARTICIPATES IN THE DEVELOPMENT
OF PRIMARY DYSLIPIDEMIAS
Matías-Pérez D1, Pérez-Santiago AD1, Sánchez Medina MA1, Alpuche Osorno JJ2, García-Montalvo IA1
*Corresponding Author: Dr. Iván A. García-Montalvo, Division of Postgraduate Studies and Research,
Tecnológico Nacional de México/Instituto Tecnológico de Oaxaca, Oaxaca City, Oaxaca, México. Av.
Víctor Bravo Ahuja No. 125, Esq. Calzada Tecnológico Oaxaca, Oaxaca. Tel./Fax: +52-951-501-5016.
E-mail: ivan.garcia@itoaxaca.edu.mx
page: 5
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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 [3-5]. 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 [12-14].
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.
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 [33-35]. 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 [62-64].
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].
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 tis- sues, 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
[72-74].
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 [76-80].
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 [84-87].
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].
Final Considerations. As final considerations we
can mention that the PCSK9 gene has an important role
in the development of primary dyslipidemias, mainly FH,
the main risk factor for disease is LDL-c. Currently, the
treatment pharmacological by 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 intolerant to statins, and
new pharmacological therapies have drawn attention to
the inhibition of this gene. It is necessary to continue researching
even more because PSCK9 is not the only gene
that has participation in these pathologies, which is why
it requires integration and collaboration between medical
specialists, geneticists and molecular biologists, being
essential for adequate advice to people at risk for any
pathology, always taking care of the ethical aspects that
these studies involve.
Acknowledgments. Authors’ contributions: I.A.
García-Montalvo, A.D. Pérez-Santiago, J.J. Alpuche
Osorno, M.A Sánchez Medina and D. Matías-Pérez participated
in the concept of study, design, writing and critical
review of the manuscript. This manuscript is a review
article that addresses the role played by the PCSK9 gene
in primary dyslipidemia, as well as the description of other
genes in the pathology.
Declaration of Interest. The authors report no conflicts
of interest. The authors alone are responsible for the
content and writing of this article.
Ethical Responsibilities. Protection of people and
animals. The authors declare that no experiments have
been conducted on humans or animals for this research.
Confidentiality of the Data. The authors declare that
patient data does not appear in this article.
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