Plasma PCSK9 levels correlate with cholesterol in men but not in women

Biochemical and Biophysical Research Communications 361 (2007) 451–456 www.elsevier.com/locate/ybbrc

Plasma PCSK9 levels correlate with cholesterol in men but not in women Janice Mayne a,*, Angela Raymond a, Anna Chaplin b, Marion Cousins b, Nadine Kaefer a, Charles Gyamera-Acheampong a, Nabil G. Seidah c, Majambu Mbikay a, Michel Chre´tien a, Teik Chye Ooi a,b a

Hormones, Growth and Development Program, Ottawa Health Research Institute, The Ottawa Hospital, University of Ottawa, Ottawa, Ont., Canada b Clinical Research Laboratory, Division of Endocrinology and Metabolism, Department of Medicine, The Ottawa Hospital, University of Ottawa, Ont., Canada c Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Que., Canada Received 29 June 2007 Available online 18 July 2007

Abstract Proprotein convertase subtilisin kexin-like 9 (PCSK9) is a secreted glycoprotein that negatively regulates low density lipoprotein receptor (LDLR) levels. Several single nucleotide polymorphisms (SNPs) in PCSK9 have been linked to autosomal dominant hypercholesterolemia (ADH). Conversely, hypocholesterolemia associates with both ‘loss of function’ nonsense and missense SNPs in PCSK9. We examined the association of plasma PCSK9 with lipoprotein parameters in 182 normolipidemics. For men (n = 98) plasma PCSK9 averaged 6.08 ± 1.96 lg/ml and Spearman analysis revealed significant correlation between it and total cholesterol (TC), LDLC, and TC/ high density lipoprotein (HDLC) (r = 0.276, 0.282, and 0.228, respectively). For women (n = 84) plasma PCSK9 averaged 6.46 ± 1.99 lg/ml having no correlation with TC, LDLC or TC/HDLC. The ratio of plasma PCSK9/LDLC increased in men carrying ‘loss of function’ PCSK9 variations. Our results suggest a gender difference in PCSK9 regulation and function with PCSK9 correlated to TC and LDLC in men but not women.  2007 Elsevier Inc. All rights reserved. Keywords: Plasma PCSK9; Loss of function; Hypocholesterolemia; Hypercholesterolemia; Single nucleotide polymorphisms

Proprotein convertase subtilisin kexin-like 9 (PCSK9) is a member of the mammalian serine proprotein convertase (PC) family that is responsible for the proteolytic maturation of secretory proteins including neuropeptides, pro-hormones, cytokines, growth factors, receptors, cell surface proteins, and serum proteins [1,2]. PCSK9 is the first PC family member to be implicated in a dominant phenotype, namely ADH [3], characterized by an increase in LDLC and premature atherosclerosis [4]. Several SNPs in PCSK9 associate with ADH, classified as familial hypercholesterolemia 3 (FH3): S127R within the prodomain of PCSK9, and F216L and D374Y within the catalytic domain of PCSK9 [3,5]. Although the exact mechanism is unknown, and to date

*

Corresponding author. Fax: +1 613 761 4355. E-mail address: [email protected] (J. Mayne).

0006-291X/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.07.029

no physiological substrates have been identified for PCSK9, we do know that PCSK9 inhibits LDLC clearance by degrading LDLR. We do not know if this degradation is direct or indirect, but evidence suggests that it takes place in an acidic endosomal or lysosomal compartment, preventing normal recycling of the LDLR [6–8]. Studies have shown that D374Y PCSK9 binds 10· more strongly to the LDLR at the cell surface of hepatocytes than wildtype PCSK9 [9] causing decreased LDLR recycling and increased degradation. PCSK9 null mice have a hypocholesterolemic phenotype [10], and several nonsense and missense variants in PCSK9 have been reported to associate with hypocholesterolemia in humans [11–16]. The effect of these in PCSK9-dependent LDLR degradation is unclear but they may affect parameters such as PCSK9 secretion, stability and/or PCSK9:LDLR affinity. PCSK9 SNPs that result in ‘gain of function’ increase degradation of LDLR and predispose

452

J. Mayne et al. / Biochemical and Biophysical Research Communications 361 (2007) 451–456

carriers to hypercholesterolemia, whereas nonsense and missense PCSK9 SNPs that result in ‘loss of function’ decrease degradation of the LDLR and associate with hypocholesterolemia [17]. These studies, along with those that have shown decreased risk of coronary artery disease (CAD) for ‘loss of function’ carriers (88% and 47% for C679X and R46L heterozygotes, respectively) have generated great interest in the development of PCSK9 inhibitors that could augment current dyslipidemic therapies [15]. To date the regulation and function of PCSK9 have been studied largely using cell culture and animal models. The objective of this study—the first of its kind—was to determine if plasma PCSK9 can be used to study its regulation in humans, serve as a reflection of cholesterol homeostasis and/or as an indicator of PCSK9 SNPs. We examined the relationship between PCSK9 plasma levels and lipoprotein parameters in 182 normolipidemics and the effect of ‘loss of function’ PCSK9 SNPs. Data indicates that a PCSK9 assay can be used to study its regulation. Materials and methods Constructs and antibodies. The cDNA of human PCSK9 was cloned into the pIRES2-EGFP with a C-terminal V5 tag as described [2]. The anti-PCSK9 antibody used for immunoblotting (anti-IB PCSK9 Ab) was produced by recombinant PCSK9 vaccination [18]. The antibody used for immunoprecipitation (anti-native PCSK9 Ab) was raised in rabbits by DNA vaccination of the mammalian expression vector pIRES2-EGFP into which the cDNA for human PCSK9 had been inserted [19]. Immunoprecipitation and immunoblotting. Immunoprecipitations and immunoblotting were carried out following standard protocols with antinative PCSK9 Ab (1:500) or preimmune sera (1:500) and 25 ll of ProteinA agarose (Sigma–Aldrich) overnight at 4 C. For immunoblotting, the primary anti-IB PCSK9 Ab was used at 1/1000. Immunoblots were revealed by chemiluminescence using Western Lightening Plus (Perkin– Elmer) on X-OMAT film (Kodak). For the plasma PCSK9 assay we immunoprecipitated 25 ll of plasma using excess anti-native PCSK9 Ab. The PCSK9 signal was quantified by densitometry using Syngene’s Chemigenius 2XE imager and GeneTool software. Concentration was determined in comparison to a known amount of recombinant PCSK9 protein. All samples were quantified a minimum of two times. Intra-assay coefficient of variability (CV) was 6.39%. Interassay CV was 8.73%. Subjects and sample handling. We obtained blood samples from 182 participants recruited by The Ottawa Hospital Lipid Clinic following a 12 h fast. All subjects gave informed written consent and the ethics committee approved the study protocols. Blood was collected into EDTAvacutainer tubes and centrifuged at 3000 rpm for 10 min at 22 C to obtain plasma and blood leukocytes. To obtain serum for lipid measurements, blood was collected into SST-vacutainer tubes, allowed to clot at room temperature for 20 min and centrifuged at 3000 rpm for 10 min at 22 C. TC and TG were measured using enzymatic methods on an Ortho Clinical Diagnostics Vitros 250. HDLC was measured using a direct enzymatic method (Beckman Coulter) on the Synchron LX20PRO analyzer (Beckman Coulter) and LDLC was calculated by the Friedewald equation. Genotyping. Genomic DNA was isolated from blood leukocytes using QIAamp DNA Blood Kit (Qiagen Sciences, MD). Primer sequences and polymerase chain reaction (PCR) for amplification of the individual exons of the PCSK9 gene were as per Abifadel et al. [3]. Standard DNAsequencing reactions using version 3.1 of the Big Dye Terminator cycle sequencing kit were analyzed on an Applied Biosystems 3730 DNA Analyzer (Applied Biosystems, Foster City, CA). Statistical analysis. Results are expressed as means ± SD except where indicated. Lipid parameters between men and women were analyzed by the Mann–Whitney U test. Spearman correlation coefficients (r) were

determined to assess the relationship between different parameters. Data were analyzed using SAS/PC software and significance defined as p < 0.05.

Results The anti-native PCSK9 antibody specifically recognizes PCSK9 from human plasma We tested the specificity of the anti-native PCSK9 antibody for reactivity to plasma PCSK9 by immunoprecipitation followed by immunoblotting (Fig. 1). A specific band at 60 kDa was immunoprecipitated with the anti-native PCSK9 antibody (lane 2) that was absent with preimmune serum (lane 1). In preabsorption studies, preincubation of the immunoblotting antibody with recombinant V5 tagged-PCSK9 effectively competed out this signal (data not shown). Gender dichotomy in the correlation between plasma PCSK9 and lipoprotein parameters We collected plasma from 182 normolipidemic individuals defined as the 5th–95th percentile for TC, TG, HDLC, and LDLC, adjusted for age and sex [20], and determined their level of plasma PCSK9. Fig. 2 is a histogram of the distribution of plasma PCSK9 levels while the inset shows the determination of its concentration for one individual. Table 1 gives the means ± SD for age, body mass index (BMI), lipid, and plasma PCSK9 measurements of all participants, and gender sub-categories. Neither plasma PCSK9 nor TC was significantly different between men and women. TG and HDLC means were significantly higher and lower, respectively for our male versus female participants, a documented trend [21]. Male (n = 98) plasma PCSK9 levels ranged from 0.56 to 13.39 lg/ml with a mean of 6.08 ± 1.96 lg/ml and female (n = 84) plasma PCSK9 levels ranged from 0.42 to 12.33 lg/ml with a mean of 6.46 ± 1.99 lg/ml. For all 182 participants Spearman analysis showed no correlation between PCSK9 and lipoprotein parameters (Table 2). Analyses of gender sub-categories showed a positive correlation between PCSK9 and TC (r = 0.276, p = 0.006), LDLC (r = 0.282, p = 0.005), and TC/HDLC (r = 0.228, p = 0.024) in men (Table 2 and Fig. 3A–C, respectively) while no such correlation was observed in women (Table 2 and Fig. 3D–F).

Fig. 1. Specificity of anti-native PCSK9 antibody. Plasma PCSK9 was immunoprecipitated with preimmune sera (lane 1) or anti-native PCSK9 Ab (lane 2) and immunoblotted with the anti-IB PCSK9 Ab as described in Materials and methods.

J. Mayne et al. / Biochemical and Biophysical Research Communications 361 (2007) 451–456 Frequency Distribution PCSK9 50

Men

40

Women

Densitometry Value(X105)

Total

45

Freq uency (n)

35 30 25 20

Standard Curve 8 6 4 unknown

2

r 2= 0.9431 0

50 100 150 200 rPCSK9 (ng)

15 10 5 0

0-

1

12

23

3-

4

4-

5

56

67

7-

8

8-

9

9-

10

10

-1

1

11

-1

2

12

1 - 1 3- 1 3 4

Plasma PCSK9 Concentration ( ug/ml)

Fig. 2. Histogram of the distribution of PCSK9 and standard curve for determining PCSK9 concentration in plasma assay. Twenty-five microliter of human plasma was immunoprecipitated with anti-native PCSK9 Ab (1/ 500) as described in Materials and methods. The immunoblot was probed with the anti-IB PCSK9 Ab. The PCSK9 signal intensity was measured by densitometry and its concentration determined against known concentrations (lg/ml) of recombinant protein (inset).

Plasma PCSK9 as an indicator of ‘loss of function’ PCSK9 SNPs To examine the possibility that plasma PCSK9 levels may be an indicator of possible PCSK9 SNPs, we measured it in individuals below the 5th percentile for TC and LDLC. We genotyped PCSK9 exons of three individ-

453

uals with PCSK9 levels significantly lower (defined as greater than one SD from the mean) than the normolipidemics. Proband 1, a male, was a compound heterozygote for the reported nonsense and missense SNPs resulting in C679X and A443T variations in the C-terminal region of PCSK9 (Fig. 4; CX/AT). We observed a 46.3% reduction in plasma PCSK9 and 80% reduction in LDLC in comparison with normolipidemics (Fig. 4A and B). Previous reports show the C679X and A443T PCSK9 variations associate with 28% and 2% reduction in LDLC, respectively [13,15]. Proband 2 (female; Fig. 4; IV/L10), a compound heterozygote for the I474V and c.43–44insCTG (L10) PCSK9 variations, had a 41.2% reduction in plasma PCSK9 and 47% reduction in LDLC in comparison with normolipidemics (Fig. 4D and E). The L10 variation, located in the signal peptide domain of PCSK9, is associated with a reported 21% reduction in LDLC levels [11]. The I474V PCSK9 SNP was reported to lower LDLC by 8% in one study [22] but was contradicted by a second study [12]. Proband 3, a male, had a 33.5% reduction in plasma PCSK9 and 70% reduction in LDLC compared with normolipidemics (Fig. 4A and B; RL/L10, AV). He was a compound heterozygote for the R46L and A53V/ L10 PCSK9 variations located within its prosegment (R46L and A53V). Only the R46L PCSK9 variation is associated with reduced LDLC levels (21%) [23]. Four members of proband 3’s family subsequently participated in the study and are shown in Fig. 4A–B and D–E: father’s genotype RL, sister’s genotype L10/AV, nieces’ genotypes L10/AV and RL. The effect of the R46L PCSK9 variation in lowering LDLC was more pronounced in the male proband and father than in his female relatives, while LDLC

Table 1 Clinical characteristics and fasting plasma lipid and PCSK9 levels

Age (years) BMI (kg/m2) PCSK9 (lg/ml) Total cholesterol Triglycerides LDL cholesterol HDL cholesterol TC/HDLC

All subjects (n = 182)

Men (n = 98)

Women (n = 84)

53 ± 13 27.1 ± 4.3 6.25 ± 1.97 5.57 ± 0.85 1.47 ± 0.59 3.67 ± 0.78 1.23 ± 0.29 4.77 ± 1.32

56 ± 12 27.4 ± 3.8 6.08 ± 1.96 5.64 ± 0.76 1.66 ± 0.66 3.77 ± 0.67 1.12 ± 0.25 5.27 ± 1.33

49 ± 14* 26.7 ± 4.7 6.46 ± 1.99 5.49 ± 0.94 1.26 ± 0.41*** 3.56 ± 0.89* 1.37 ± 0.28*** 4.18 ± 1.05***

Data are means ± SD in mmol/L except where noted. Male and female comparisons by Mann–Whitney U test *p < 0.05,

**p

< 0.001,

***p

< 0.0001.

Table 2 Relationship between plasma PCSK9 and baseline lipid levels PCSK9 vs.

All subjects (n = 182) r

Total cholesterol Triglycerides LDL cholesterol HDL cholesterol TC/HDLC

0.130 0.051 0.084 0.078 0.073

Men (n = 98) p 0.080 0.498 0.261 0.298 0.329

r

Women (n = 84) p

0.276 0.169 0.282 0.089 0.228

Spearman correlation coefficient (r) *p < 0.05. Numbers in bold show significant correlation.

0.006* 0.096 0.005* 0.382 0.024*

r

p 0.009 0.025 0.070 0.168 0.025

0.937 0.823 0.524 0.127 0.823

454

J. Mayne et al. / Biochemical and Biophysical Research Communications 361 (2007) 451–456

Women

r = 0.009 p = 0.937

LDLC (m mol/L)

Relationship between plasma PCSK9 and LDL Cholesterol 6.5 5.5 4.5 3.5 2.5 1.5

B

Men

r = 0.282 p = 0.005

E

Women

T C/HDLC

Comparison of PCSK9 between Normolipidemics and ‘Loss of Function’ PCSK9 SNP Carriers 8

8.0

Men

r = 0.228 p = 0.024

F

Women

2

2

4 6 8 10 12 plasma PCSK9 (ug/ml)

Fig. 3. Relationship between plasma PCSK9 and TC and LDLC. Plasma PCSK9, TC, and LDLC were measured in men (A and B, respectively) and women (C and D, respectively) and Spearman correlation (r) and significance (p) determined as described in Materials and methods.

B

Discussion The novel findings of this study were (i) the correlation and (ii) the gender dichotomy observed for plasma PCSK9 and cholesterol levels. Gender sub-analyses showed that in men, plasma PCSK9 is positively correlated with TC, LDLC and, to a lesser degree, TC/HDLC (Table 2 and Fig. 3). The correlation observed for men between plasma PCSK9 and TC is responsible for the weaker and non-significant (r = 0.130, p = 0.080) trend between plasma PCSK9 and TC in all participants (Table 2). Likewise, it is the LDLC component of TC that is influencing the correlation between plasma PCSK9 and TC/HDLC in men since no correlation exists between plasma PCSK9 and HDLC or TG (Table 2). The r2 value for the plasma PCSK9 and LDLC correlation in men is 0.08 suggesting that PCSK9 is responsible for approximately 8% of the variability in TC and LDLC levels in this group. Only one other study has suggested gender as a factor in PCSK9 physiology. This study documented a difference in the correlation of the E670G PCSK9 variation with an increased incidence of CAD in men versus women [24].

E

Women

2 1 Comparison of PCSK9/LDLC between Normolipidemics and ‘Loss of Function’ PCSK9 SNP Carriers 6 5 4 3 2 1

C

WT

levels in female relatives heterozygous for the L10 PCSK9 variation was variable: for the proband’s sister LDLC was 15% higher and for the niece 60% lower than the female average. This variability has been noted in previous studies [14,15]. Interestingly, plasma PCSK9/LDLC ratio was noticeably increased in male carriers of ‘loss of function’ PCSK9 SNPs but not in female carriers of such SNPs (Fig. 4C and F).

Men

5 4 3

r = 0.025 p = 0.823

4.0 2 4 6 8 10 12 plasma PCSK9 (ug/ml)

Women

Comparison of LDLC between Normolipidemics and ‘Loss of Function’ PCSK9 SNP Carriers

6.0

2.0

D

Men

4

6

C

A

6

r =-0.070 p = 0.524

Relationship between plasma PCSK9 and TC/HDLC 10.0

plasma PCSK9 (μg/ml)

r = 0.276 D p = 0.006

LDLC ( mmol/L)

8.0 7.0 6.0 5.0 4.0 3.0

Men

PCSK9/LDLC

TC ( mmol/L )

Relationship between plasma PCSK9 and Total Cholesterol A

Men

F

P3

P3F

P1

RL/ L10,AV

RL

CX/AT

Women

P3S P3N P3N WT L10,AV RL L10,AV

P2 IV/ L10

Fig. 4. Comparison of plasma PCSK9 in normolipidemics and ‘loss of function’ PCSK9 SNP carriers. Plasma PCSK9, LDLC, and plasma PCSK9/LDLC were compared between normolipidemics and ‘loss of function’ PCSK9 SNPs carriers (A–C, respectively, for men and D–F, respectively, for women), where P1, Proband 1; P2, Proband 2; P3, Proband 3; P3F, Proband 3’s father; P3N, Proband 3’s niece; and P3S, Proband 3’s sister. PCSK9 genotypes are WT, wildtype; AV, A53V; AT, A443T; CX, C679X; IV, I474V; L10, c.43–44insCTG; and RL, R46L.

The explanation for these gender differences is unknown. However, we do know that estrogen augments LDLR levels, while androgens attenuate this effect [25,26]. The effect(s) of these hormones on PCSK9 transcription and/ or translation have not been studied. Sub-analyses of our female participants into pre- (<50 years) and post-menopausal (>50 years) did not change our previous analyses. We do not know, however, how many women in our study are undergoing hormone replacement therapy or are taking oral contraceptives. Indeed, if estrogen and/or androgen affect PCSK9, our values may be skewed by such therapies. Our studies demonstrate that mean plasma PCSK9 levels in men (6.08 lg/ml) and women (6.46 lg/ml) do not differ significantly. Using an indirect ELISA method one other study measured plasma PCSK9 at 150 ng/ml [9]. The reason for this discrepancy is not known, but may be due to differences in antibody specificity. We used DNA vaccination of full length human PCSK9 to generate our antibody for assay. Thus, the antibody is very specific for full length, native PCSK9. PCSK9 is modified during secretion and peptide antibodies may not react to the same extent as one directed against properly folded, full-length PCSK9.

J. Mayne et al. / Biochemical and Biophysical Research Communications 361 (2007) 451–456

In our study, one individual carried the C679X ‘loss of function’ PCSK9 variation that in cell culture prevents PCSK9 secretion [27]. Indeed, this individual has approximately one-half the average circulating level of PCSK9 (compare 3.25 versus 6.08 lg/ml, respectively), indicating that plasma PCSK9 reflects its level of hepatic biosynthesis and secretion. Therefore, plasma PCSK9 and/or the ratio of plasma PCSK9/LDLC may allow us to identify those individuals who carry a PCSK9 SNP affecting their lipoprotein homeostasis. We would predict that those who carry a PCSK9 SNP associated with ‘loss of function’, and that are significantly expressing that phenotype, will have higher values of plasma PCSK9/LDLC than the norm, whereas those that carry PCSK9 SNPs associated with ‘gain of function’ might be expected to have sub-normal values. We base the latter prediction on cell culture studies of ‘gain of function’ PCSK9 SNPs that show increased affinity and LDLR-dependent up-take of PCSK9 [9,28]. In conclusion, plasma PCSK9 may serve a diagnostic or prognostic purpose and may be useful in the clinical assessment of PCSK9 inhibitors [10,29]. Acknowledgments This work was supported by a Canadian Stroke Network Grant (to M.C., M.M., T.C.O., and J.M.), a University Medical Research Fund from the University of Ottawa (to T.C.O.) and CIHR Grant # MOP 36496 (to N.G.S. and M.M.). We thank Nicolas Stewart for critically reading this manuscript and JoAnn McDonald for her excellent secretarial assistance. References [1] N.G. Seidah, A. Prat, Precursor convertases in the secretory pathway, cytosol and extracellular milieu, Essays Biochem. 38 (2002) 79–94. [2] N.G. Seidah, S. Benjannet, L. Wickham, J. Marcinkiewicz, S.B. Jasmin, S. Stifani, A. Basak, A. Prat, M. Chretien, The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation, Proc. Natl. Acad. Sci. USA 100 (2003) 928–933. [3] M. Abifadel, M. Varret, J.P. Rabes, D. Allard, K. Ouguerram, M. Devillers, C. Cruaud, S. Benjannet, L. Wickham, D. Erlich, A. Derre, L. Villeger, M. Farnier, I. Beucler, E. Bruckert, J. Chambaz, B. Chanu, J.M. Lecerf, G. Luc, P. Moulin, J. Weissenbach, A. Prat, M. Krempf, C. Junien, N.G. Seidah, C. Boileau, Mutations in PCSK9 cause autosomal dominant hypercholesterolemia, Nat. Genet. 34 (2003) 154–156. [4] M.A. Austin, C.M. Hutter, R.L. Zimmern, S.E. Humphries, Genetic causes of monogenic heterozygous familial hypercholesterolemia: a HuGE prevalence review, Am. J. Epidemiol. 160 (2004) 407–420. [5] K.M. Timms, S. Wagner, M.E. Samuels, K. Forbey, H. Goldfine, S. Jammulapati, M.H. Skolnick, P.N. Hopkins, S.C. Hunt, D.M. Shattuck, A mutation in PCSK9 causing autosomal-dominant hypercholesterolemia in a Utah pedigree, Hum. Genet. 114 (2004) 349–353. [6] D.W. Zhang, T.A. Lagace, R. Garuti, Z. Zhao, M. McDonald, J.D. Horton, J.C. Cohen, H.H. Hobbs, Binding of PCSK9 to EGF-A repeat of LDL receptor decreases receptor recycling and increases degradation, J. Biol. Chem. 282 (2007) 18602–18612. [7] N. Nassoury, D.A. Blasiole, A. Tebon Oler, S. Benjannet, J. Hamelin, V. Poupon, P.S. McPherson, A.D. Attie, A. Prat, N.G. Seidah, The

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20] [21]

[22]

[23]

[24]

455

cellular trafficking of the secretory proprotein convertase PCSK9 and its dependence on the LDLR, Traffic 6 (2007) 718–732. S. Benjannet, D. Rhainds, R. Essalmani, J. Mayne, L. Wickham, W. Jin, M.C. Asselin, J. Hamelin, M. Varret, D. Allard, M. Trillard, M. Abifadel, A. Tebon, A.D. Attie, D.J. Rader, C. Boileau, L. Brissette, M. Chretien, A. Prat, N.G. Seidah, NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol, J. Biol. Chem. 279 (2004) 48865–48875. T.A. Lagace, D.E. Curtis, R. Garuti, M.C. McNutt, S.W. Park, H.B. Prather, N.N. Anderson, Y.K. Ho, R.E. Hammer, J.D. Horton, Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice, J. Clin. Invest. 116 (2006) 2995– 3005. S. Rashid, D.E. Curtis, R. Garuti, N.N. Anderson, Y. Bashmakov, Y.K. Ho, R.E. Hammer, Y.A. Moon, J.D. Horton, Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9, Proc. Natl. Acad. Sci. USA 102 (2005) 5374–5379. P. Yue, M. Averna, X. Lin, G. Schonfeld, The c.43_44insCTG variation in PCSK9 is associated with low plasma LDL-cholesterol in a Caucasian population, Hum. Mutat. 27 (2006) 460–466. Y. Miyake, R. Kimura, Y. Kokubo, A. Okayama, H. Tomoike, T. Yamamura, T. Miyata, 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 (2007). Available online 21 February 2007. I.K. Kotowski, A. Pertsemlidis, A. Luke, R.S. Cooper, G.L. Vega, J.C. Cohen, H.H. Hobbs, A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol, Am. J. Hum. Genet. 78 (2006) 410–422. T. Fasano, A.B. Cefalu, E. Di Leo, D. Noto, D. Pollaccia, L. Bocchi, V. Valenti, R. Bonardi, O. Guardamagna, M. Averna, P. Tarugi, A novel loss of function mutation of PCSK9 gene in white subjects with low-plasma low-density lipoprotein cholesterol, Arterioscler. Thromb. Vasc. Biol. 27 (2007) 677–681. J. Cohen, A. Pertsemlidis, I.K. Kotowski, R. Graham, C.K. Garcia, H.H. Hobbs, Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9, Nat. Genet. 37 (2005) 161–165. K.E. Berge, L. Ose, T.P. Leren, Missense mutations in the PCSK9 gene are associated with hypocholesterolemia and possibly increased response to statin therapy, Arterioscler. Thromb. Vasc. Biol. 26 (2006) 1094–1100. J. Cameron, O.L. Holla, T. Ranheim, M.A. Kulseth, K.E. Berge, T.P. Leren, Effect of mutations in the PCSK9 gene on the cell surface LDL receptors, Hum. Mol. Genet. 15 (2006) 1551–1558. S. Benjannet, D. Rhainds, J. Hamelin, N. Nassoury, N.G. Seidah, The proprotein convertase (PC) PCSK9 is inactivated by Furin and/ or PC5/6A: functional consequences of natural mutations and posttranslational modifications, J. Biol. Chem. 281 (2006) 30561–30572. P.S. Chowdhury, M. Gallo, I. Pastan, Generation of high titer antisera in rabbits by DNA immunization, J. Immunol. Methods 249 (2001) 147–154. B.M. Rifkind, The lipid research clinics population studies data report, The Prevelence Study (1980) NIH 80-1527. Plasma lipid distributions in selected North American populations: the Lipid Research Clinics Program Prevalence Study. The Lipid Research Clinics Program Epidemiology Committee, Circulation 60 (1979) 427–439. K. Shioji, T. Mannami, Y. Kokubo, N. Inamoto, S. Takagi, Y. Goto, H. Nonogi, N. Iwai, Genetic variants in PCSK9 affect the cholesterol level in Japanese, J. Hum. Genet. 49 (2004) 109–114. J.C. Cohen, E. Boerwinkle, T.H. Mosley Jr., H.H. Hobbs, Sequence variations in PCSK9, low LDL, and protection against coronary heart disease, N. Engl. J. Med. 354 (2006) 1264–1272. D. Evans, F.U. Beil, The E670G SNP in the PCSK9 gene is associated with polygenic hypercholesterolemia in men but not in women, BMC Med. Genet. 7 (2006) 66.

456

J. Mayne et al. / Biochemical and Biophysical Research Communications 361 (2007) 451–456

[25] P.M. Smith, A. Cowan, B.A. White, The low-density lipoprotein receptor is regulated by estrogen and forms a functional complex with the estrogen-regulated protein ezrin in pituitary GH3 somatolactotropes, Endocrinology 145 (2004) 3075–3083. [26] G.E. Croston, L.B. Milan, K.B. Marschke, M. Reichman, M.R. Briggs, Androgen receptor-mediated antagonism of estrogen-dependent low density lipoprotein receptor transcription in cultured hepatocytes, Endocrinology 138 (1997) 3779–3786. [27] Z. Zhao, Y. Tuakli-Wosornu, T.A. Lagace, L. Kinch, N.V. Grishin, J.D. Horton, J.C. Cohen, H.H. Hobbs, Molecular characterization of

loss-of-function mutations in PCSK9 and identification of a compound heterozygote, Am. J. Hum. Genet. 79 (2006) 514–523. [28] D. Cunningham, D.E. Danley, K.F. Geoghegan, M.C. Griffor, J.L. Hawkins, T.A. Subashi, A.H. Varghese, M.J. Ammirati, J.S. Culp, L.R. Hoth, M.N. Mansour, K.M. McGrath, A.P. Seddon, S. Shenolikar, K.J. Stutzman-Engwall, L.C. Warren, D. Xia, X. Qiu, Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia, Nat. Struct. Mol. Biol. 14 (2007) 413–419. [29] M.S. Brown, J.L. Goldstein, Biomedicine. Lowering LDL-not only how low, but how long? Science 311 (2006) 1721–1723.