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Novel loss-of-function PCSK9 variant is associated with low plasma LDL cholesterol in a French-Canadian family and with impaired processing and secretion in cell culture.

Familial hypercholesterolemia (FH) [6] is characterized by high circulating concentrations of LDL particles and increases the risk of cardiovascular disease (1). Mutations in the LDLR [7] (low density lipoprotein receptor), APOB [apolipoprotein B (including Ag(x) antigen)], and PCSK9 (proprotein convertase subtilisin/kexin 9) genes are linked with FH1, FH2, and FH3, respectively (1, 2). Recently, 2 other loci for hypercholesterolemia have been identified on chromosome 16q22.1 (FH4) and 8q24.22, although the cognate genes have not yet been defined (3, 4). The LDL receptor (LDLR) clears LDL particles from the circulation, whereas apolipoprotein B100 is the protein component of the LDL particle that interacts with the LDLR (5). PCSK9 is a secreted glycoprotein (6) that interacts with the LDLR and mediates its lysosome-dependent degradation (7-10). Largely produced in the liver and the intestine (6), PCSK9 is synthesized in the endoplasmic reticulum (ER) as a preproprotein of692 amino acid residues (6). As a prerequisite for its exit from the ER, PCSK9 autocatalytically cleaves its prodomain at [VFAQ.sub.152] [down arrow] SIP (10, 11). The approximately 14-kDa propeptide and the approximately 62-kDa PCSK9 then form a heterodimer, which transits through the Golgi apparatus, is secreted, and interacts with the LDLR (6, 10, 12).

The gene encoding PCSK9 is highly polymorphic. Two categories of PCSK9 sequence variants produce mild to moderate (and opposing) phenotypes. Gain-of-function sequence variants cause a reduction in the LDLR that leads to hypercholesterolemia (13) or to autosomal dominant hypercholesterolemia in cases of severe phenotypic variants (2, 14). PCSK9 loss-of-function sequence variants decrease LDLR degradation, thereby reducing LDL cholesterol (LDLC) concentrations (15-17). Cell culture and animal studies have established that the LDLR is the downstream effector for PCSK9 gain-of-function and loss-of-function activities at the protein level (10, 18-20). Longitudinal population studies have shown significant reductions in the risk of coronaryartery disease in carriers of loss-of-function PCSK9 variants (88% and 47% for PCSK9-C679X and -R46L heterozygotes, respectively) (21).

The characterization of naturally occurring gain-of-function and loss-of-function human PCSK9 variants has increased our understanding of the cell biology and function of this secreted glycoprotein. This work has included identification of the amino acid residues important for PCSK9 autocatalytic processing, secretion, and biological activity--information that provides insight into the mechanism by which PCSK9 mediates LDLR degradation. The mechanisms of many PCSK9 variants remain unknown, however. In this study, we identified a novel PCSK9 sequence variant in a white French-Canadian family that is associated with low circulating LDLC concentrations. We carried out cell culture studies to characterize the molecular mechanism behind this loss-of-function PCSK9 phenotype.

Study Participants and Methods


After obtaining written informed consent, we collected blood samples from all study participants after a 12-h fast and made clinical measurements according to study protocols approved by the ethics committees of the Ottawa Hospital Research Institute and Clinical Research Institute of Montreal. We obtained blood samples from 15 participants recruited to the Clinical Research Institute of Montreal. The comparison group consisted of 210 individuals recruited to the Ottawa Hospital Lipid Clinic. Participants underwent anthropometric measurements, including height and weight measurements. Body mass index was calculated as the weight in kilograms divided by the square of the height in meters.


Blood was collected into EDTA-containing Vacutainer tubes (BD), and plasma and blood leukocytes were obtained by centrifuging blood samples at 1560g for 10 min at 22[degrees]C. Serum for lipid measurements was obtained by collecting blood into BD SST[TM] Vacutainer tubes, allowing the blood sample to clot at room temperature for 20 min, and centrifuging the sample at 1560g for 10 min at 22[degrees]C. Total cholesterol and triglycerides were measured with enzymatic methods on an Ortho Clinical Diagnostics Vitros 250 analyzer. HDL cholesterol was measured by a direct enzymatic method (Beckman Coulter) on the Synchron LX20 Pro analyzer (Beckman Coulter); the LDLC concentration was calculated with the Friedewald equation.


Genomic DNA was isolated from blood leukocytes with the QIAamp DNA Blood Kit (Qiagen). The primers and PCR conditions used for amplifying individual PCSK9 exons were as described by Abifadel et al. (2). Standard DNA-sequencing services were carried out by Bio Basic.


The plasma PCSK9 concentration was quantified with a human PCSK9 ELISA from CycLex. This assay has an intraassay CV of 1.5%-2.6% and an interassay CV of 2.9%-7.1%. All samples were quantified 4 times.


The cDNA of human PCSK9 was cloned into the pIRES2-EGFP vector with a C-terminal V5 tag, as previously described (6). Mutations were introduced by site-directed mutagenesis, also as described (22). The mouse anti-V5 IgG used for immunoprecipitation and immunoblotting of V5-tagged recombinant PCSK9 was obtained from Invitrogen. The anti-PCSK9 antibody used for immunoblotting (anti-IB PCSK9 Ab) was produced by recombinant PCSK9 vaccination (7). The rabbit anti-human LDLR IgG and the mouse anti-transferrin receptor IgG were from Cedarlane. Secondary antimouse and antirabbit IgGs were from Amersham/GE Healthcare Life Sciences.


HuH7 cells were grown at 37[degrees]C in Dulbecco's modified Eagle medium containing 100 mL/L fetal bovine serum and 28 mg/L gentamicin. At 24 h after plating, we transiently transfected 3 x [10.sup.5] cells with cDNA encoding human PCSK9 (pIRES2) according to the standard protocol for Effectene[R] (Qiagen) (6). Media from cultures of HuH7 cells transiently transfected with cDNA encoding human PCSK9 or a nonrelevant DNA control were collected 48 h later in the presence of a Complete Mini Protease Inhibitor Cocktail (Roche) and a phosphatase inhibitor (200 [micro]mol/L sodium orthovanadate) and centrifuged at 13 000gfor 3 min. Cell lysis was carried out in 1 x RIPA buffer (50 mmol/L Tris, pH 7.6, 150 mmol/L NaCl, 10 mL/L NP-40, 5 g/L deoxycholate, 1 g/L SDS) in the presence of the inhibitors mentioned above. The protein concentrations of total cell lysates were measured with the Bradford dye-binding method (Bio-Rad Protein Assay Kit; Bio-Rad Laboratories).


Proteins were electrophoresed through a 7% NuPAGE Tris-acetate gel (Invitrogen), electroblotted onto nitrocellulose, and immunoblotted according to a standard protocol. The primary anti-PCSK9 antibody used for immunoblotting (anti-IB PCSK9 Ab-03) (7) was raised in rabbits against recombinant PCSK9 amino acid residues 31-454 and used at a dilution of 1 part in 2000. The primary anti-V5 antibody (Invitrogen) was used at the same dilution, and the secondary antibody was used at a dilution of 1 part in 5000. Immunoblots were revealed by chemiluminescence with Western Lightning Plus (PerkinElmer) on X-Omat film (Kodak). The Chemigenius 2XE imager and GeneTools software (Syngene) were used for densitometric quantification of signals.


The results of quantification of secreted PCSK9 by ELISA were expressed as the mean and SE (n = 4, human plasma;n = 3,spentmedia).LDLR was quantified via immunoblotting followed by densitometry analysis (n = 3). Representative immunoblots are provided (see below). The unpaired Student f-test was used for statistical analyses of differences. P values <0.05 were considered statistically significant.



We identified an individual in a French-Canadian family with a very low circulating PCSK9 concentration (Fig. 1; see Table 1 in the Data Supplement that accompanies the online version of this article at http://www., compared with concentrations in a general white Canadian population (n = 210; 68.5 [micro]g/L vs 326.9 [micro]g/L), as well as low LDLC concentrations (14th percentile, adjusted for age and sex). We sequenced the 12 PCSK9 exons and the exon-intron boundaries in this individual. She carried a missense mutation at base pair 456 (G [right arrow] C) in exon 3 that yielded a proPCSK9 amino acid substitution (Q152H) at the P1 site of autocatalytic cleavage (Fig. 2A). We recruited members of the family and sequenced their PCSK9 exons and exon-intron boundaries (Fig. 1; see Table 1 in the online Data Supplement). This table also shows the fasting plasma lipid concentrations and PCSK9 concentrations for these individuals, as well as the mean values for the 210 white Canadians. The data for additional characteristics (age, sex, glucose concentration, body mass index) are also presented in Table 1 in the online Data Supplement.

Four individuals in the pedigree carried the Q152H mutation (Fig. 1; see Table 1 in the online Data Supplement). Their LDLC concentrations were 64.4% (III.2), 68.8% (III.3), 25.7% (II.4), and 33.1% (II.9) lower than those of unrelated individuals (see Table 1 in the online Data Supplement). These individuals' circulating PCSK9 concentrations were 84.3% (III.2), 87.3% (III.3), 79.0% (II.4), and 64.7% (II.9) lower than those of the general white Canadian population (see Table 1 in the online Data Supplement). Although the Q152H carriers and the general population had mean plasma triglyceride and HDL cholesterol concentrations that did not differ significantly [triglycerides, 135.8 mg/dL and 138.2 mg/dL (1.53 mmol/L vs 1.56 mmol/L), respectively (P = 0.94); HDL cholesterol, 54.7 mg/dL and 46.4 mg/dL (1.42 mmol/L vs 1.20 mmol/L), respectively (P = 0.14)], Q152H carriers had significantly lower plasma concentrations of total cholesterol and LDLC than the general population [154.6 mg/dL vs 213.6 mg/dL (4.00 mmol/L vs 5.53 mmol/L) (P = 0.0069) and 72.8 mg/dL vs 140.1 mg/dL (1.91 mmol/L vs 3.63 mmol/L) (P = 0.0031), respectively]. After adjustment for age and sex, the plasma LDLC concentrations for 3 of the 4 Q152H carriers were below the fifth percentile, and the proband was at the 14th percentile (Fig. 1; see Table 1 in the online Data Supplement). Noncarriers of the Q152H mutation within the family had LDLC concentrations that ranged from the fifth percentile to the 90th percentile, after adjustment for age and sex (Fig. 1; see Table 1 in the online Data Supplement).


Sequencing of the 12 PCSK9 exons in this family also revealed that 6 members (II.2, II.5, II.7, II.8, III.5, III.6; Fig. 1) carried a Leu insertion (c.43_44insCTG and denoted L10ins) within a stretch of 9 Leu residues in the signal peptide for PCSK9. This insertion was associated with lower LDLC concentrations in a white population (17). Two other members (II.6, III.4) carried both the L10ins and the R46L sequence variants within the PCSK9 propeptide, which are also associated with PCSK9 loss of function (21) (Fig. 1; see Table 1 in the online Data Supplement). In fact, several population studies have found that the risk of cardiovascular disease is decreased by approximately 50% for PCSK9-R46L heterozygotes, the LDLC concentrations of which are reduced by approximately 14% on average, compared with age- and sex-matched controls (21, 23, 24). The PCSK9-L10ins variant is associated with an approximately 14% reduction in LDLC concentrations in white populations (17), but it is not associated with a significant lowering in the LDLC concentration in individuals of African descent (25). Three individuals (II.2, III.2, III.3) also carried the PCSK9-I474V variant, which has been found in several other populations. The I474V variant is not associated with any changes in the LDLC concentration (25, 26). The fact that several members of this family carried multiple loss-of-function PCSK9 variants may indicate that these variants may be more frequent in some French-Canadian cohorts than in the general population.


Fig. 2A depicts the domain structure of preproPCSK9, its sites of posttranslational modifications (sulfation at Y38, phosphorylation at S47 and S688, and glycosylation at N533), the residues surrounding the site of prosegment cleavage, the catalytic residues (D186, H226, S386), and the oxyanion hole residue (N317). Secreted PCSK9 can interact with the LDLR and enter with it into the endocytic recycling pathway, thereby decreasing the rate of LDLR recycling and increasing lysosome-dependent LDLR degradation (7-9, 27).

To compare the clinical lipoprotein profiles and plasma PCSK9 measurements of individuals carrying the novel PCSK9-Q152H variant, we investigated the biosynthesis and secretion ofPCSK9 and its effect on LDLR degradation. Western blotting results for V5-tagged PCSK9 (Fig. 2B) show the distribution of proPCSK9 and intracellular PCSK9 (PCSK9 in panel B) in total cell lysates from liver HuH7 cells transiently transfected with wild-type (WT) PCSK9 or PCSK9-Q152H (lanes 1 and 2, respectively). The Q152H amino acid substitution at position 152 greatly reduced the ability of proPCSK9 to undergo autocatalytic cleavage, compared with WT (lane 2). Immunoprecipitation and subsequent immunoblotting procedures revealed secretion of PCSK9-WT into the medium but detected no PCSK9-Q152H secretion into the medium (Fig. 2B, lanes 3 and 4, respectively). This reduced proprotein processing and loss of secretion produced LDLR concentrations (Fig. 2C) that were not significantly higher (relative concentration, approximately 1.4) than those in mock-transfected control cells (relative concentration, 1; P = 0.25); however, these LDLR concentrations were significantly higher (relative concentration, 1.4) than those in cells transfected with PCSK9-WT (relative concentration, approximately 0.4; P = 0.03). The inset in Fig. 2C shows representative immunoblots for the LDLR and the transferrin receptor control.



In addition to blocking cleavage of the proPCSK9 zymogen, the Q152H variant also affected PCSK9-WT processing and secretion (Fig. 3, A and B, respectively), and this effect protected LDLR from degradation in cell culture (Fig. 3C). Fig. 3A shows equal amounts of PCSK9 secreted from HuH7 cells transfected with 250 ng of either PCSK9-WT (untagged) or PCSK9-WT-V5 (V5-tagged) cDNA (lanes 1 and 2, respectively). Lanes 1 and 2 of Fig. 3B show the intracellular processing of proPCSK9 to PCSK9. In contrast, PCSK9-Q152H-V5 was not secreted (Fig. 3A, lane 3). Cotransfection of PCSK9-WT with increasing amounts of PCSK9WT-V5 was recompensed with increased PCSK9 secretion (Fig. 3A, lanes 4-6), and both proforms were processed as in single transfections (compare lanes 1 and 2 with lanes 3-5 in Fig. 3B). In contrast, cotransfection of PCSK9-WT with increasing amounts of PCSK9Q152H-V5 significantly decreased PCSK9-WT secretion (Fig. 3A, lanes 7-9), even at the highest ratio of WT cDNA to Q152H cDNA (4:1) (Fig. 3A; compare lanes 1 and 7; P = 0.002). Cotransfection of equal amounts of PCSK9-Q152H-V5 with PCSK9-WT significantly decreased PCSK9 secretion by 78% and 90% (Fig. 3A; compare lane 1 or 6 with lane 9; P = 0.0002 and 0.0004, respectively), whereas immunoblotting showed that the intracellularly processed form of PCSK9-WT was reduced (Fig. 3B; compare iPCSK9-no tag lane 1 or 6 with lane 9). Lanes 1-9 of Fig. 3C show a representative immunoblot of LDLR in HuH7 cells transiently transfected with PCSK9-WT, PCSK9-WT-V5, or PCSK9-Q152H-V5. As expected, the LDLR concentration decreased with cotransfection of increasing amounts of PCSK9-WT-V5 with PCSK9-WT (Fig. 3C, lanes 4-6); however, lanes 7-9 of Fig. 3C show that cotransfection of increasing amounts of PCSK9-Q152H-V5 protected the LDLR from degradation by PCSK9-WT. Transfections with 250 ng of PCSK9-WT significantly decreased LDLR concentrations relative to those for control cells (Fig. 3C, lane 1 vs lane 3; P = 0.05); however, cotransfection in the presence of either 125 ng or 250 ng of Q152H (Fig. 3C, lanes 8 and 9) significantly increased the relative LDLR concentration to 0.94 ( P = 0.03) and 1.1 (P = 0.02), respectively, compared with PCSK9-WT (0.79; lane 1). This approximately 40%-50% increase in relative LDLR concentration from that expected for WT can be attributed at least partially to the decrease in secreted PCSK9-WT in the presence of the Q152H variant. In Fig. 1 in the online Data Supplement, we demonstrate the linearityofPCSK9 secretion for the amounts of PCSK9-WT cDNA (untagged) and PCSK9-WT-V5 cDNA used (see above). Fig. 2 in the online Data Supplement shows that endogenous concentrations of intracellular PCSK9 were decreased upon overproduction of PCSK9-Q152H, compared with mock-transfected cells, and there was a corresponding significant upregulation of LDLR in these cells (P = 0.02). Fig. 3 in the online Data Supplement illustrates the cotransfection of our plasmids by immunocytochemistry.



The loss-of-function PCSK9-Q152H variant is the first described in a white Canadian population to have such a profound effect on plasma cholesterol concentrations. Several other loss-of-function PCSK9 variants with strong phenotypic effects on the LDLC concentration have been described for other populations. Two such variants occur in carriers of African descent, and both are nonsense variants. One of the variants occurs in the prodomain of PCSK9. No PCSK9 is produced, owing to an early truncation (PCSK9-Y142X). The other variant is a C-terminal nonsense mutation (PCSK9-C679X) that causes the retention of autocatalytically cleaved PCSK9 in the ER (16, 21). The LDLC concentrations in carriers of these variants range from the first percentile to the 50th percentile, with a mean lowering in the LDLC concentration of 40%, after adjustment for age- and sex-matched controls (16). The plasma PCSK9 concentrations in carriers of the C679X and Y142X variants are approximately 60% lower than those in their control population (28). The third variant, a compound mutation (R104C/V114A) found in a French family and associated with familial hypobetalipoproteinemia, exhibits a dominant negative effect on PCSK9 secretion (29). In Fig. 3A, we show that cotransfection of equal amounts of PCSK9-Q152H-V5 and nontagged PCSK9-WT decreased WT secretion by approximately 80% (from 175 [micro]g/L to 35 [micro]g/L). This decrease was not a general effect of cotransfection, because transfection of equal amounts of PCSK9-WT-V5 and nontagged PCSK9-WT (Fig. 3A, lane 6) increased the concentration of secreted PCSK9 to 275 [micro]g/L. Therefore, in the ex vivo cell culture conditions of cotransfection, our PCSK9-Q152H variant does have a dominant negative effect on PCSK9-WT secretion. Whether this effect also occurs in vivo is not known; however, persons carrying this variant have 79% less circulating PCSK9 than unrelated noncarriers. If this effect does occur in vivo, we expect that such an effect would amplify the loss-of-function phenotype of the Q152H variant.

Conversely, there are some gain-of-function PCSK9 variants--S127R and D127G--that display decreased autocatalytic cleavage and secretion but are associated with hypercholesterolemic phenotypes (10, 30). In vitro binding assays have shown 5-fold increased binding of the PCSK9-S127R variant to the LDLR compared with WT, a result that could partly account for its gain of function (31). Alternatively, these variants may mediate intracellular LDLR degradation more efficiently. This pathway has been described by Poirier et al. (32), although the relative contributions of the intracellular and extracellular degradation routes with respect to PCSK9-mediated LDLR degradation are not fully understood.

Several studies have reported a positive correlation between the plasma PCSK9 concentration and the LDLC concentration in general populations (28, 33-36). Other studies have documented variable changes in plasma PCSK9 for carriers of PCSK9 loss-of-function and gain-of-function variants compared with control individuals (noncarriers of a particular PCSK9 variant) (34, 36, 37). In fact, circulating PCSK9 may differentially affect plasma LDLC concentrations, depending on whether an individual carries a PCSK9 variant that alters its LDLR-degrading activity and depending on the mode of action of that particular PCSK9 mutation and/or variant. Overall, this variability means that the plasma PCSK9 concentration as measured by ELISA does not necessarily predict the LDLC concentration, because the LDLC concentration can, in some instances, be strongly influenced by the mode of action of a PCSK9 variant (36). For instance, carriers of the gain-of-functionPCSK9-D374Y variant, which is associated with autosomal dominant hypercholesterolemia and very high LDLC concentrations, have lower plasma PCSK9 concentrations than the general population (37), a finding that conflicts with the reports of a positive correlation between the plasma PCSK9 concentration and the LDLC concentration (28, 33-36). The mode of action of the PCSK9-D374Y variant has been well studied, however. It binds 10 times better to the liver LDLR than PCSK9-WT, thereby decreasing the plasma PCSK9 concentrations in D374Y carriers compared with noncarriers and augmenting LDLR degradation (38). On the other hand, plasma PCSK9 concentrations are also reduced in carriers of the loss-of-function PCSK9-R46L variant, which is associated with reduced LDLC concentrations (24, 36, 37), although the reason for this observation is less clear. Our studies with HEK293 cell cultures showed no change in the rate of PCSK9-R46L secretion (data not shown). Our previous studies with cell cultures have shown that the propeptide of the R46L variant displays a greater susceptibility to cleavage, which may affect the half-life of the circulating PCSK9 complex (39). Others have shown that the PCSK9-R46L variant binds less strongly to the LDLR, although how this binding would affect the plasma PCSK9 concentration is unclear (38). The primary mode of action (or inaction) of the PCSK9-Q152H mutation identified and characterized in this study is clear, however. The protein is not secreted, and therefore plasma concentrations are low, protecting the liver LDLRs from PCSK9-mediated LDLR degradation (Figs. 1 and 2).

In a previous report, we described our investigation of the effect of amino acid substitutions around the autocatalytic cleavage site of PCSK9 ([VFAQ.sub.152] [down arrow] SIP) (10). We showed that PCSK9 tolerates an Ala substitution for Glu at P1 and that its relative processing and secretion are approximately 80% of PCSK9-WT (10). In the present study, we have shown that the naturally occurring amino acid substitution of His for Glu prevents autocatalytic cleavage by PCSK9 in the ER, thereby precluding PCSK9 secretion (Figs. 2 and 3). Consequently, this PCSK9 variant no longer affects the downregulation of cell surface LDLR through the endosomal/lysosomal pathway, a major route for PCSK9-mediated LDLR degradation (40). Our cell biology findings are consistent with the findings of our human studies, in which individuals carrying the PCSK9-Q152H mutation showed very low plasma PCSK9 concentrations (>79% reduction compared with the general Canadian population) and consequently low concentrations of circulating LDLC owing to the upregulation of liver LDLR. This report is the first of a loss-of-function mutation in PCSK9 within the white Canadian population that displays such a profound effect on cholesterolemia. It also reinforces the suggestion that lowering the PCSK9 concentration by blocking PCSK9 synthesis, processing, or secretion could be an effective therapeutic strategy to complement current lipid-lowering drugs.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: J. Davignon, Quebec Consortium on Drug Discovery (CQDM).

Consultant or Advisory Role: J. Davignon, Abbott/Solvay, Astra-Zeneca, Ascasi, Cortria, Genzyme, McCain, Merck, Pfizer, and Roche.

Stock Ownership: None declared.

Honoraria: J. Davignon, Abbott/Solvay, AstraZeneca, Ascasi, Cortria, Genzyme, McCain, Merck, Pfizer, and Roche.

Research Funding: J. Mayne, Canadian Institutes of Health Research (CIHR) (CTP 82946 and MOP 102741), and the Heart and Stroke Foundation of Ontario; T.C. Ooi, the Heart and Stroke Foundation of Ontario; J. Davignon, Merck Canada, Pfizer, and AstraZeneca; N.G. Seidah, Canadian Institutes of Health Research (CIHR) (CTP 82946 and MOP 102741) and the Richard and Edith Strauss Foundation; M. Mbikay, Canadian Institutes of Health Research (CIHR) (CTP 82946 and MOP 102741), the Richard and Edith Strauss Foundation, and the Heart and Stroke Foundation of Ontario; M. Chretien, Canadian Institutes of Health Research (CIHR) (CTP 82946 and MOP 102741), the Richard and Edith Strauss Foundation, the Fondation Jean-Louis Levesque, and the Heart and Stroke Foundation of Ontario.

Expert Testimony: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Acknowledgments: We are indebted to the family members who participated in this study, as well as to the persons recruited from the general Canadian population. The authors thank Pavel Milman for his assistance with immunocytochemistry.


(1.) Austin MA, Hutter CM, Zimmern RL, Humphries SE. Genetic causes of monogenic heterozygous familial hypercholesterolemia: a HuGE prevalence review. Am J Epidemiol 2004;160:407-20.

(2.) Abifadel M, Varret M, Rabes JP, Allard D, Ouguerram K, Devillers M, et al. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat Genet 2003;34:154-6.

(3.) Marques-Pinheiro A, Marduel M, Rabes JP, Devillers M, Villeger L, Allard D, et al. A fourth locus for autosomal dominant hypercholesterolemia maps at 16q22.1. Eur J Hum Genet 2010;18: 1236-42.

(4.) Cenarro A, Garcia-Otin AL, Tejedor MT, Solanas M, Jarauta E, Junquera C, et al. A presumptive new locus for autosomal dominant hypercholesterolemia mapping to 8q24.22. Clin Genet 2011; 79:475-81.

(5.) Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science 1986;232:34-47.

(6.) Seidah NG, Benjannet S, Wickham L, Marcinkiewicz J, Jasmin SB, Stifani S, et al. The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation. Proc Natl Acad Sci U S A 2003;100:928-33.

(7.) Nassoury N, Blasiole DA, Tebon Oler A, Benjannet S, Hamelin J, Poupon V, et al. The cellular trafficking of the secretory proprotein convertase PCSK9 and its dependence on the LDLR. Traffic 2007;8:718-32.

(8.) Zhang DW, Lagace TA, Garuti R, Zhao Z, Mc Donald M, Horton JD, et al. Binding of proprotein convertase subtilisin/kexin type 9 to epidermal growth factor-like repeat A of low density lipoprotein receptor decreases receptor recycling and increases degradation. J Biol Chem 2007;282: 18602-12.

(9.) Lagace TA, Curtis DE, Garuti R, McNutt MC, Park SW, Prather HB, et al. Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice. J Clin Invest 2006; 116:2995-3005.

(10.) Benjannet S, Rhainds D, Essalmani R, Mayne J, Wickham L, Jin W, et al. NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol. J Biol Chem 2004;279:48865-75.

(11.) Naureckiene S, Ma L, Sreekumar K, Purandare U, Lo CF, Huang Y, et al. Functional characterization of Narc 1, a novel proteinase related to proteinase K. Arch Biochem Biophys 2003;420:55-67.

(12.) Benjannet S, Rhainds D, Hamelin J, Nassoury N, Seidah NG. The proprotein convertase (PC) PCSK9 is inactivated by furin and/or PC5/6A: functional consequences of natural mutations and posttranslational modifications. J Biol Chem 2006; 281:30561-72.

(13.) Allard D, Amsellem S, Abifadel M, Trillard M, Devillers M, Luc G, et al. Novel mutations of the PCSK9 gene cause variable phenotype of autosomal dominant hypercholesterolemia. Hum Mutat 2005;26:497.

(14.) Timms KM, Wagner S, Samuels ME, Forbey K, Goldfine H, Jammulapati S, et al. A mutation in PCSK9 causing autosomal-dominant hypercholesterolemia in a Utah pedigree. Hum Genet 2004; 114:349-53.

(15.) Berge KE, Ose L, Leren TP. Missense mutations in the PCSK9 gene are associated with hypocholesterolemia and possibly increased response to statin therapy. Arterioscler Thromb Vasc Biol 2006; 26:1094-100.

(16.) Cohen J, Pertsemlidis A, Kotowski IK, Graham R, Garcia CK, Hobbs HH. Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9. Nat Genet 2005;37:161-5.

(17.) Yue P, Averna M, Lin X, Schonfeld G. The c.43_44insCTG variation in PCSK9 is associated with low plasma LDL-cholesterol in a Caucasian population. Hum Mutat 2006;27:460-6.

(18.) Maxwell KN, Breslow JL. Adenoviral-mediated expression of Pcsk9 in mice results in a low-density lipoprotein receptor knockout phenotype. Proc Natl Acad Sci U S A 2004;101:7100-5.

(19.) Rashid S, Curtis DE, Garuti R, Anderson NN, Bashmakov Y, Ho YK, et al. Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9. Proc Natl Acad Sci U S A 2005;102: 5374-9.

(20.) Zaid A, Roubtsova A, Essalmani R, Marcinkiewicz J, Chamberland A, Hamelin J, et al. Proprotein convertase subtilisin/kexin type 9 (PCSK9): hepatocyte-specific low-density lipoprotein receptor degradation and critical role in mouse liver regeneration. Hepatology 2008;48:646-54.

(21.) Cohen JC, Boerwinkle E, MosleyTH Jr, Hobbs HH. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N Engl J Med 2006;354:1264-72.

(22.) Elagoz A, Benjannet S, Mammarbassi A, Wickham L, Seidah NG. Biosynthesis and cellular trafficking of the convertase SKI-1/S1P: Ectodomain shedding requires SKI-1 activity. J Biol Chem 2002;277:11265-75.

(23.) Scartezini M, Hubbart C, Whittall RA, Cooper JA, Neil AH, Humphries SE. The PCSK9 gene R46L variant is associated with lower plasma lipid levels and cardiovascular risk in healthy U.K. men. Clin Sci (Lond) 2007;113:435-41.

(24.) Guella I, Asselta R, Ardissino D, Merlini PA, Peyvandi F, Kathiresan S, et al. Effects of PCSK9 genetic variants on plasma LDL cholesterol levels and risk of premature myocardial infarction in the Italian population. J Lipid Res 2010;51:3342-9.

(25.) Kotowski IK, Pertsemlidis A, Luke A, Cooper RS, Vega GL, Cohen JC, Hobbs HH. A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol. Am J Hum Genet 2006;78:410-22.

(26.) Miyake Y, Kimura R, Kokubo Y, Okayama A, Tomoike H, Yamamura T, Miyata T. Genetic variants in PCSK9 in the Japanese population: rare genetic variants in PCSK9 might collectively contribute to plasma LDL cholesterol levels in the general population. Atherosclerosis 2008;196: 29-36.

(27.) Qian YW, Schmidt RJ, Zhang Y, Chu S, Lin A, Wang H, et al. Secreted proprotein convertase subtilisin/kexin-type 9 downregulates low-density lipoprotein receptor through receptor-mediated endocytosis. J Lipid Res 2007;48:1488-98.

(28.) Lakoski SG, Lagace TA, Cohen JC, Horton JD, Hobbs HH. Genetic and metabolic determinants of plasma PCSK9 levels. J Clin Endocrinol Metab 2009;94:2537-43.

(29.) Cariou B, Ouguerram K, Zair Y, Guerois R, Langhi C, Kourimate S, et al. PCSK9 dominant negative mutant results in increased LDL catabolic rate and familial hypobetalipoproteinemia. Arterioscler Thromb Vasc Biol 2009;29:2191-7.

(30.) Homer VM, Marais AD, Charlton F, Laurie AD, Hurndell N, Scott R, et al. Identification and characterization of two non-secreted PCSK9 mutants associated with familial hypercholesterolemia in cohorts from New Zealand and South Africa. Atherosclerosis 2008;196:659-66.

(31.) Cunningham D, Danley DE, Geoghegan KF, Grif for MC, Hawkins JL, Subashi TA, et al. Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia. Nat Struct Mol Biol 2007;14:413-9.

(32.) Poirier S, Mayer G, Poupon V, McPherson PS, Desjardins R, Ly K, et al. Dissection of the endogenous cellular pathways of PCSK9-induced low density lipoprotein receptor degradation: evidence for an intracellular route. J Biol Chem 2009;284:28856-64.

(33.) Baass A, Dubuc G, Tremblay M, Delvin EE, O'Loughlin J, Levy E, et al. Plasma PCSK9 is associated with age, sex, and multiple metabolic markers in a population-based sample of children and adolescents. Clin Chem 2009;55:1637-45.

(34.) Dubuc G, Tremblay M, Pare G, Jacques H, Hamelin J, Benjannet S, et al. A new method for measurement of total plasma PCSK9: clinical applications. J Lipid Res 2010;51:140-9.

(35.) Lambert G, Ancellin N, Charlton F, Comas D, Pilot J, Keech A, et al. Plasma PCSK9 concentrations correlate with LDL and total cholesterol in diabetic patients and are decreased by fenofibrate treatment. Clin Chem 2008;54:1038-45.

(36.) Mayne J, Raymond A, Chaplin A, Cousins M, Kaefer N, Gyamera-Acheampong C, et al. Plasma PCSK9 levels correlate with cholesterol in men but not in women. Biochem Biophys Res Com mun 2007;361:451-6.

(37.) Humphries SE, Neely RD, Whittall RA, Troutt JS, Konrad RJ, Scartezini M, et al. Healthy individuals carrying the PCSK9 p.R46L variant and familial hypercholesterolemia patients carrying PCSK9 p.D374Y exhibit lower plasma concentrations of PCSK9. Clin Chem 2009;55:2153-61.

(38.) Fisher TS, Lo Surdo P, Pandit S, Mattu M, Santoro JC, Wisniewski D, et al. Effects of pH and low density lipoprotein (LDL) on PCSK9-dependent LDL receptor regulation. J Biol Chem 2007;282: 20502-12.

(39.) Dewpura T, Raymond A, Hamelin J, Seidah NG, Mbikay M, Chretien M, Mayne J. PCSK9 is phosphorylated by a Golgi casein kinase-like kinase ex vivo and circulates as a phosphoprotein in humans. FEBS J 2008;275:3480-93.

(40.) Ni YG, Condra JH, Orsatti L, Shen X, Di Marco S, Pandit S, et al. Aproprotein convertase subtilisin-like/kexin type 9 (PCSK9) C-terminal domain antibody antigen-binding fragment inhibits PCSK9 internalization and restores low density lipoprotein uptake. J Biol Chem 2010;285:12882-91.

Janice Mayne, [1] * Thilina Dewpura, [1] Angela Raymond, [1] Lise Bernier, [2] Marion Cousins, [3] Teik Chye Ooi, [3] Jean Davignon, [2] Nabil G. Seidah, [4] Majambu Mbikay, [5] and Michel Chretien [1,5] *

[1] Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Ontario, Canada; [2] Hyperlipidemia and Atherosclerosis Research Group, Clinical Research Institute of Montreal, Montreal, Quebec, Canada; [3] Clinical Research Laboratory, Division of Endocrinology and Metabolism, Department of Medicine, The Ottawa Hospital, University of Ottawa, Ottawa, Ontario, Canada; [4] Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec, Canada; [5] Chronic Disease Program, Ottawa Hospital Research Institute, The Ottawa Hospital, University of Ottawa, Ottawa, Ontario, Canada.

* Address correspondence to: J.M. at Ottawa Institute of Systems Biology, University of Ottawa, 451 Smyth Rd., Roger Guindon Hall 4510E, Ottawa, Ontario, K1H 8ME Canada. Fax 613-562-5655; e-mail M.C. at Chronic Disease Program, Ottawa Hospital Research Institute, The Ottawa Hospital, University of Ottawa, Ottawa, Ontario KIY 4E9, Canada. Fax 613-761-4920; e-mail

Received March 11, 2011; accepted July 22, 2011.

Previously published online at DOI: 10.1373/clinchem.2011.165191

[6] Nonstandard abbreviations: FH, familial hypercholesterolemia; LDLR, LDL receptor; ER, endoplasmic reticulum; LDLC, LDL cholesterol; anti-IB PCSK9 Ab, anti-PCSK9 antibody used for immunoblotting; WT, wild type.

[7] Human genes: LDLR, low density lipoprotein receptor; APOB, apolipoprotein B; PCSK9, proprotein convertase subtilisin/kexin type 9.
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Title Annotation:Molecular Diagnostics and Genetics
Author:Mayne, Janice; Dewpura, Thilina; Raymond, Angela; Bernier, Lise; Cousins, Marion; Ooi, Teik Chye; Da
Publication:Clinical Chemistry
Date:Oct 1, 2011
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