PROPROTEIN CONVERTASE SUBTILISIN–KEXIN TYPE 9 GAIN-OF-FUNCTION VARIANTS
DNA linkage mapping in families from Utah and France in whom elevated LDL cholesterol segregated in an autosomal dominant pattern – without any detectable mutations in either the LDLR or APOB genes – indicated that a stretch of chromosome 1p36 cosegregated perfectly with hypercholesterolemia [1,2]. Among the genes in this region was NARC1 (neural apoptosis regulated convertase 1, alias PCSK9), a recently identified serine protease related to bacterial subtilisin and yeast kexin . DNA sequencing of the 12 exons of PCSK9 in affected members of the three French families identified two distinct rare heterozygous missense mutations, namely p.S127R and p.F216L, which were both absent from normolipidemic family members and the general population . The mutation in the Utah kindred was found to be PCSK9 p.D374Y . A GOF mechanism was suspected to underlie the elevated LDL cholesterol levels in carriers, and this was soon confirmed when overexpression of Pcsk9 in murine liver produced hypercholesterolemia by reducing LDL receptor numbers and activity .
PCSK9 GOF variants comprise the third most common cause of autosomal dominant familial hypercholesterolemia, accounting for less than 1% of familial hypercholesterolemia cases in most clinical cohorts. In contrast, LDLR LOF mutations and APOB receptor binding defective mutations account for 80–90% and 5–10% of cases, respectively, in most familial hypercholesterolemia cohorts . More than 30 disease-causing PCSK9 GOF variants have been reported ; the vast majority of these are missense variants, affect all coding regions except for exon 3 and are scattered throughout all domains of the PCSK9 protein. All are ultrarare variants with allele frequencies less than 0.1% (hereafter used interchangeably with ‘mutations’) in the general population. Among reported GOF and LOF mutations, there are no overlapping positions in which an amino acid has been altered: the two classes of variants appear to be mutually exclusive in this respect.
GAIN-OF-FUNCTION VARIANT MECHANISMS
Functional studies of human GOF variants indicate that although enhanced degradation of the LDL receptor is the final common pathway resulting in hypercholesterolemia, different GOF variants achieve this end via distinct mechanisms (Table 1). For instance, a c.332C > A transversion in the 5′UTR produces a 2.5-fold increase in transcriptional activity, leading to an increase in PCSK9 levels and thus increased LDL receptor degradation  and ultimately elevated LDL cholesterol levels. Apart from this regulatory variant, the remainder of reported GOF variants have post-translational consequences and occur throughout all domains of the protein (Fig. 1b).
Prodomain mutations are excellent examples of the mechanistic diversity that results in increased LDL receptor degradation, as there is a mixture of intracellular and extracellular effects. For instance, the introduction of a tyrosine-sulfation site by the p.D35Y variant leads to enhanced intracellular PCSK9 activity, causing increased LDL receptor degradation; although there is no change in autocatalytic activity, the Asp to Tyr amino acid change may modify the secondary structure of the prosegment, resulting in an increased affinity for intracellular LDL receptors . Interestingly, decreases in the autocatalytic cleavage of the prosegment from proPCSK9 have also been demonstrated to increase the intracellular degradation of LDL receptors through a dominant negative mechanism .
In contrast, the well known p.S127R and p.D129G mutations occur near the autocatalytic processing site and produce a substantial decrease in zymogen cleavage activity [11,12]. In particular, p.S127R decreases autocatalytic cleavage up to 66% . Unprocessed fragments act in a dominant negative fashion [14,20], with increased intracellular LDL receptor degradation [11,16,20]. Specifically for the p.S127R mutant, the structural change reportedly increased the affinity for the extracellular domain of the LDL receptor by five-fold .
Increases in receptor degradation are also achieved through extracellular actions, as is the case for p.L108R and p.D129N: these mutations do not produce abnormal cleavage or protein misfolding, but instead appear to increase the stability of the interaction between PCSK9 and the LDL receptor outside the cell [10,14].
Distinct from the prodomain, mutations in the catalytic domain share very similar mechanisms and effects . Furin cleavage within the Golgi inactivates PCSK9 , rendering it less effective in regulating LDL receptor levels ; therefore, variants that lead to complete or partial resistance to furin processing result in an increase in active PCSK9 molecules and LDL receptor degradation. Mutations that completely abolish furin cleavage include p.R215H  and p.R218S [12,17], whereas p.F216L [13,22] and p.D374Y [12,15,22] induce partial or complete resistance to furin cleavage, respectively.
p.D374Y is considered to be the most active GOF mutant [16,19], given that this position is responsible for PCSK9 binding to the epidermal growth factor-like domain of the LDL receptor . With the change in protein structure, the increased stability of the PCSK9–LDL receptor interaction produces a 10-fold increase in receptor degradation [12,16] and a 61% decrease in LDL internalization . In a transgenic minipig, liver-specific expression of the human p.D374Y variant resulted in decreased hepatic LDL receptor levels, impaired LDL cholesterol clearance, severe hypercholesterolemia, and spontaneous development of atherosclerotic lesions seen with noninvasive imaging . Functional assays on another prominent variant, p.D374H, have demonstrated the same mechanisms as p.D374Y, but to a slightly lesser extent: His produces an 8-fold increase in LDL receptor affinity , compared with the 25-fold increase produced by Tyr [13,16,17].
Less is known about the GOF mechanisms for variants residing within the C-terminal. The p.N425S mutation disrupts the hinge region (residues 422–439) of the protein and may have structural consequences that influence the interaction of the mutant PCSK9 with extracellular LDL receptors . Another mutation, p.R469W, although not within the hinge region but in the M1 module of the C-terminal Cys-His-rich domain (residues 453–531) , also affects the PCSK9–LDL receptor interaction through an unclear mechanism . Both mutations increase external degradation of LDL receptors, so it is possible that other C-terminal variants may lead to improper folding and more stable extracellular interactions [24▪▪].
GAIN-OF-FUNCTION VARIANTS AND CARDIOVASCULAR DISEASE
Association of PCSK9 GOF variants with premature cardiovascular disease (CVD), particularly coronary heart disease (CHD), has been derived largely from observational cohort studies and anecdotally from family studies . The cumulative prevalence of these ultrarare variants is so low that even by next-generation sequencing of thousands of individuals, they do not register as causes of early CHD [26▪]. This contrasts with the strong association of rare LDLR familial hypercholesterolemia mutations with early CHD found in samples of several thousand cases [26▪], which is not surprising as LDLR LOF mutations are perhaps 50–100 times more common than PCSK9 GOF mutations in familial hypercholesterolemia . Interestingly, double heterozygotes for PCSK9 GOF and LDLR LOF mutations have a lipid phenotype that is intermediate between typical homozygous and heterozygous familial hypercholesterolemia . Finally, the first compound heterozygote for two PCSK9 GOF mutations, an 11-year-old Portuguese girl, had a phenotype that was milder than typical homozygous familial hypercholesterolemia, with untreated LDL cholesterol of 6.0 mmol/l that fell to 2.3 mmol/l with low-dose atorvastatin [28▪].
Some compelling evidence linking heterozygous PCSK9 GOF variants to premature CHD risk came from a recent cohort study [29▪▪]. Among 164 heterozygotes for 16 different PCSK9 GOF variants from 12 centers in eight countries, the authors found that untreated LDL cholesterol levels were higher than those in patients either with LDLR-defective or LDLR-negative forms of heterozygous familial hypercholesterolemia or with familial defective apo B [29▪▪]. Furthermore, 33% of heterozygotes for PCSK9 GOF variants had CHD, with mean age-of-onset of 49 ± 13 years. A few studied patients with PCSK9 GOF variants responded well – 73% reduction in LDL cholesterol – to treatment with the PCSK9 mAb alirocumab [29▪▪].
PROPROTEIN CONVERTASE SUBTILISIN–KEXIN TYPE 9 LOSS-OF-FUNCTION VARIANTS
PCSK9 LOF mutations reported after sequencing of the PCSK9 gene in individuals with extremes of LDL cholesterol from the Dallas Heart Study revealed relatively common heterozygous nonsense mutations in African-American individuals with hypobetalipoproteinemia . Corroboration of the impact of PCSK9 deficiency was first seen with an animal model: the livers of Pcsk9−/− null mice had increased expression of the LDL receptor but not mRNA and showed a reduction in plasma cholesterol levels by 48% compared with wild-type littermates [31,32]. Currently, more than 20 likely and possible disease-causing LOF variants in PCSK9 have been reported, of which the vast majority are missense variants . They are scattered throughout all domains of the PCSK9 protein, affecting all coding regions. Although most are ultrarare, with allele frequencies less than 0.1% in the population, a few LOF variants that will be discussed further, such as p.R46L, p.Y142X, and p.C679X, can actually be considered ‘uncommon polymorphisms’ with allele frequencies of 1–5% in certain subpopulations [30,33–35].
LOSS-OF-FUNCTION VARIANT MECHANISMS
Although the main outcome is reduced PCSK9 secretion, LOF mutations act through many mechanisms (Table 2); a distinct difference between LOF and GOF mechanisms is that the majority of LOF variants disrupt earlier processes in the PCSK9 life cycle (Fig. 1c).
For the relatively common p.R46L variant occurring early in the prodomain, the precise mechanism that leads to the reduction of PCSK9 is still incompletely understood; however, the variant is associated with reduced phosphorylation, which may cause avid proteolytic degradation of PCSK9 . Alternatively, this variant may alter the interaction of PCSK9 and the LDL receptor: protein products with this variant have been shown to have ∼50% decreased affinity for the LDL receptor .
Of the remaining prodomain LOF variants, many share similar mechanisms in which post-translational modifications affect the wild-type functionality of PCSK9. The in-frame deletion mutant p.R97del alters an alpha-helix in the prodomain, disrupting the autocatalytic processing of proPCSK9 and preventing secretion of the mature molecule . Similarly, the mutation p.G106R impairs autocatalytic cleavage and results in a complete inability to leave the endoplasmic reticulum [11,12]. In a family with hypobetalipoproteinemia, an individual with an LDL cholesterol level of 16 mg/dl (0.4 mmol/l) was a carrier for two PCSK9 mutations: p.R104C and p.V114A. Although the individual mechanism for each variant was not defined, phenotypically they behaved in a codominant or recessive manner, whereas together in-vitro they acted in a dominant negative manner, with severely impaired autocatalytic activity and inability to leave the endoplasmic reticulum .
Another putative mechanism for PCSK9 LOF variants is nonsense-mediated mRNA decay (NMD). For instance, the p.A68fsL82X truncation is predicted to induce NMD. Although this has not been functionally validated, such a mechanism would explain the decrease in PCSK9 protein products . An additional truncation mutation, p.Y142X, occurs early in the transcript, and the absence of any in-vitro expression of the construct is consistent with NMD as well .
Requiring a separate mechanistic category, mutations that directly affect the autocatalytic cleavage site at position 152 of the prosegment can have pronounced effects. For instance, the p.Q152H mutant from a hypocholesterolemic French–Canadian family was shown to act as a dominant negative allele: autocatalytic processing was abolished, and the mutant remained in the endoplasmic reticulum . In addition, the secretion of the wild-type form of PCSK9 was reduced by 50% . For a different LOF variant at the same amino acid residue, namely p.Q152A, movement of PCSK9 from the endoplasmic reticulum into the Golgi still occurred – possibly due to a ‘relaxed specificity pocket’ – but its interaction with LDL receptors is yet to be examined . The apparent difference in mechanistic readouts from different variants at the same amino acid position underscores the diversity and complexity of mechanistic action of PCSK9 mutations.
Among LOF variants within the catalytic domain, there have been several reported mechanisms of impaired function. For instance, the p.N157K variant disrupts an alpha-helix domain that is important in the binding of the LDL receptor . This disruption causes a decrease in receptor affinity, leading to an increase of both cell-surface LDL receptors and the internalization of LDL cholesterol [11,43].
The remaining catalytic variants are consistently unable to secrete PCSK9. The p.G236S mutant undergoes normal autocatalytic cleavage, but cannot leave the endoplasmic reticulum due to abnormal folding . Similarly, p.L253F and p.N354I are unable to leave the endoplasmic reticulum; however, in these instances, the compromised function is due to decreased autocatalytic activity [15,35]. Specifically for p.L253F, the inability to leave the endoplasmic reticulum is ultimately associated with a 30% reduction in LDL cholesterol levels in heterozygotes .
Interestingly, it has been shown that the p.R237W variant leads to an increase in surface LDL receptors and LDL cholesterol internalization, but the exact mechanism is unclear; abnormal autocatalytic cleavage has been ruled out . Similarly, p.H391N is another reported LOF variant, but the precise mechanism(s) of action that produce impaired PCSK9 function has/have not yet been elucidated.
The hinge region separating the catalytic domain from the C-terminal is important for the processing and folding of PCSK9. The early termination codon introduced by p.W428X, although not functionally validated, likely produces a misfolded protein that cannot be secreted from the cell. This mechanism has been shown for another hinge region variant, p.R434W, which disrupts cell secretion due to a decrease in autocatalytic activity of the proPCSK9 prosegment  and misfolding in the endoplasmic reticulum .
Two functionally validated C-terminal variants have almost identical intracellular consequences: p.S462P and p.C679X are retained in the endoplasmic reticulum due to misfolding  and conformational changes [12,15,22]. The final C-terminal variant, p.A443T, has a distinct mechanism of action compared with neighboring mutations. The Ala to Thr amino acid change introduces a novel O-glycosylation site that favors furin-mediated cleavage of the protein , producing more inactive PCSK9 molecules that regulate cell-surface LDL receptor levels less effectively  than their active counterparts.
LOSS-OF-FUNCTION VARIANTS AND CARDIOVASCULAR DISEASE
The apparent good health observed in rare individuals with two PCSK9 LOF nonsense alleles and very low LDL cholesterol levels has allayed fears about possible deleterious consequences of virtually complete suppression of the protein's expression [46,47]. Furthermore, it is interesting that the presence of the heterozygous PCSK9 p.R46L LOF mutation in ∼3% of 582 patients with heterozygous familial hypercholesterolemia was associated with noticeably reduced LDL cholesterol levels and CHD risk compared to those with heterozygous familial hypercholesterolemia and wild-type PCSK9 .
In a now classic example of the so-called Mendelian randomization approach, investigators from the aforementioned Dallas Heart Study examined the relationship between PCSK9 sequence variations that reduce LDL cholesterol and CHD events in the Atherosclerosis Risk in Communities study . Of 3363 African-American patients, 2.6% carried a heterozygous nonsense variant in PCSK9 – either p.Y142X or p.C679X . Carriers had 28% reduced LDL cholesterol and 88% reduced CHD risk over a 15-year period . No functional assessments were performed, but as they were nonsense mutations, p.Y142X and p.C679X were assumed to be LOF variants. Of the 9524 white patients, 3.2% had the PCSK9 p.R46L missense variant that was associated with 15% reduced LDL cholesterol and 47% reduced CHD risk . In a meta-analysis of three cohorts comprising more than 66 000 individuals, heterozygotes for the p.R46L allele had a 12% reduction in LDL cholesterol and a 28% decrease in CHD risk . It is of interest that the three different mutations – p.Y142X, p.C679X, and p.R46L – impart a complete or partial loss of function to PCSK9 through different mechanisms. The final common biochemical phenotype is reduced LDL cholesterol: the degree of CHD risk reduction observed was proportional to the degree of LDL cholesterol reduction, irrespective of the particular LOF mechanism at the cellular level. The genetic epidemiology observations indicated the lifelong CVD benefit of genetically-determined low levels of LDL cholesterol. These were the crucial genetic epidemiological observations that inspired the development of PCSK9 inhibitors .
In aggregate, these findings indicate a much greater degree of subtlety and complexity underlying the clinical concept of ‘loss-of-function’ or ‘gain-of-function’ as applied to rare variants of PCSK9. However, despite the apparent underlying mechanistic heterogeneity, the final common pathway when PCSK9 secretion or structure is altered still appears to be the level of LDL cholesterol. Furthermore, the totality of observational and Mendelian randomization data would indicate that the resultant LDL cholesterol is the driving force behind lifelong susceptibility or resistance to CVD risk. However, as human mega-databases linking DNA sequencing information with long-term CVD outcomes are acquired across millions or tens of millions or individuals, it might one day be possible to test whether individual PCSK9 variants with differing GOF or LOF mechanisms of action have an incremental effect on CVD risk over and above their effects on LDL cholesterol levels.
We thank Dr Nabil Seidah for his valuable comments.
Financial support and sponsorship
R.A.H. is supported by the Jacob J. Wolfe Distinguished Medical Research Chair, the Martha G. Blackburn Chair in Cardiovascular Research, the Edith Schulich Vinet Chair in Human Genetics, and operating grants from the Canadian Institutes for Health Research (Foundation Award), the Heart and Stroke Foundation of Ontario (T-000353) and Genome Canada through Genome Quebec (award 4530).
Conflicts of interest
R.A.H. has received honoraria for membership on advisory boards and speakers’ bureaus for Aegerion, Amgen, Merck, Pfizer, Sanofi, and Valeant.
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Keywords:Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved.
PCSK9; hyperlipidemia; hypolipidemia; LDL cholesterol; vascular disease risk