Plasma levels of PCSK9 and phenotypic variability in familial hypercholesterolemia

The extent of hypercholesterolemia varies considerably in patients with familial hypercholesterolemia (FH). We hypothesized that the variability of the FH phenotype might be partly explained by variation in proprotein convertase subtilisin kexin type 9 (PCSK9) activity. Individuals between 18 and 53 years of age who had been tested for a pathogenic LDLR or APOB mutation were eligible. Mutation carriers with a LDL-C level below the 75th percentile (called “FH low”) were selected, as well as those with LDL-C above the 90th percentile (called “FH high”). Relatives who tested negative for the mutation were the “controls.” PCSK9 plasma levels were assessed in 267 individuals who did not receive cholesterol-lowering treatment at the time of the study. Mean PCSK9 plasma levels (95% CI) were lower in the FH-low group compared with the FH-high group [152 (137–167) ng/ml vs. 186 (165–207) ng/ml, P= 0.010] and the control group [177 (164–190) ng/ml, P= 0.013]. Mean PCSK9 levels did not statistically differ between the FH-high and control groups (P= 0.50). Plasma PCSK9 levels are positively associated with LDL-C levels in FH patients and might contribute to the phenotypic severity in this disorder. Therefore, the results of pharmaceutical inhibition of PCSK9 in FH patients are eagerly awaited. The extent of hypercholesterolemia varies considerably in patients with familial hypercholesterolemia (FH). We hypothesized that the variability of the FH phenotype might be partly explained by variation in proprotein convertase subtilisin kexin type 9 (PCSK9) activity. Individuals between 18 and 53 years of age who had been tested for a pathogenic LDLR or APOB mutation were eligible. Mutation carriers with a LDL-C level below the 75th percentile (called “FH low”) were selected, as well as those with LDL-C above the 90th percentile (called “FH high”). Relatives who tested negative for the mutation were the “controls.” PCSK9 plasma levels were assessed in 267 individuals who did not receive cholesterol-lowering treatment at the time of the study. Mean PCSK9 plasma levels (95% CI) were lower in the FH-low group compared with the FH-high group [152 (137–167) ng/ml vs. 186 (165–207) ng/ml, P= 0.010] and the control group [177 (164–190) ng/ml, P= 0.013]. Mean PCSK9 levels did not statistically differ between the FH-high and control groups (P= 0.50). Plasma PCSK9 levels are positively associated with LDL-C levels in FH patients and might contribute to the phenotypic severity in this disorder. Therefore, the results of pharmaceutical inhibition of PCSK9 in FH patients are eagerly awaited. Familial hypercholesterolemia (FH, MIM #143890) is a frequent autosomal codominant disorder of lipoprotein metabolism. It is clinically characterized by elevated levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), the presence of tendon xanthomas, and premature atherosclerosis. Defects in genes that code for proteins involved in the hepatic clearance of LDL-C underlie this hereditary disorder (1Goldstein J.L. Hobbs H.H. Brown M.S. Familial hypercholesterolemia.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th edition. McGraw-Hill, New York2001: 2863-2913Google Scholar). In fact, more than a 1,000 different mutations in the genes coding for the LDL-receptor (LDLR, MIM +606945), apolipoprotein B (APOB, MIM +107730), and proprotein convertase subtilisin/kexin type 9 (PCSK9, MIM +607786) are now known to cause FH (1Goldstein J.L. Hobbs H.H. Brown M.S. Familial hypercholesterolemia.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th edition. McGraw-Hill, New York2001: 2863-2913Google Scholar–2Innerarity T.L. Weisgraber K.H. Arnold K.S. Mahley R.W. Krauss R.M. Vega G.L. Grundy S.M. Familial defective apolipoprotein B-100: low density lipoproteins with abnormal receptor binding.Proc. Natl. Acad. Sci. USA. 1987; 84: 6919-6923Crossref PubMed Scopus (369) Google Scholar, 3Abifadel M. Varret M. Rabes J.P. Allard D. Ouguerram K. Devillers M. Cruaud C. Benjannet S. Wickham L. Erlich D. et al.Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.Nat. Genet. 2003; 34: 154-156Crossref PubMed Scopus (2168) Google Scholar). If left untreated, the risk of cardiovascular disease is severely increased (4Huijgen R. Vissers M.N. Defesche J.C. Lansberg P.J. Kastelein J.J. Hutten B.A. Familial hypercholesterolemia: current treatment and advances in management.Expert Rev. Cardiovasc. Ther. 2008; 6: 567-581Crossref PubMed Scopus (64) Google Scholar), but the prognosis of FH can be improved substantially with cholesterol-lowering treatment (5Versmissen J. Oosterveer D.M. Yazdanpanah M. Defesche J.C. Basart D.C. Liem A.H. Heeringa J. Witteman J.C. Lansberg P.J. Kastelein J.J. et al.Efficacy of statins in familial hypercholesterolaemia: a long term cohort study.BMJ. 2008; 337: a2423Crossref PubMed Scopus (548) Google Scholar). The identification of a mutation that underlies FH in a particular kindred enables genetic testing of family members for the presence of the same mutation and makes it possible to initiate effective medical management before the cardiovascular consequences of FH become clinically manifest (4Huijgen R. Vissers M.N. Defesche J.C. Lansberg P.J. Kastelein J.J. Hutten B.A. Familial hypercholesterolemia: current treatment and advances in management.Expert Rev. Cardiovasc. Ther. 2008; 6: 567-581Crossref PubMed Scopus (64) Google Scholar). This notion has led to the implementation of a nationwide genetic cascade screening program for FH in the Netherlands, and since 1994, approximately 20,000 individuals with FH have been found and treated (6Umans-Eckenhausen M.A. Defesche J.C. Sijbrands E.J. Scheerder R.L. Kastelein J.J. Review of first 5 years of screening for familial hypercholesterolaemia in the Netherlands.Lancet. 2001; 357: 165-168Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). However, molecularly diagnosed FH patients do not always exhibit a hypercholesterolemic phenotype. In fact, 15% of the heterozygous mutation carriers identified by our national screening program show pretreatment LDL-C levels below the 75th percentile for age and gender (7Huijgen R. Kindt I. Verhoeven S.B. Sijbrands E.J. Vissers M.N. Kastelein J.J. Hutten B.A. Two years after molecular diagnosis of familial hypercholesterolemia: majority on cholesterol-lowering treatment but a minority reaches treatment goal.PLoS ONE. 2010; 5: e9220Crossref PubMed Scopus (108) Google Scholar). The reasons why some individuals with a confirmed FH genotype lack the hypercholesterolemia phenotype are largely unknown. We hypothesized that such nonpenetrance of an FH mutation could, in part, be explained by variation in PCSK9 activity. PCSK9 is a natural inhibitor of the LDLR: it binds to the hepatic LDLR and thereby directs it toward lysosomal degradation rather than to recycling to the cell membrane (8Lambert G. Charlton F. Rye K.A. Piper D.E. Molecular basis of PCSK9 function.Atherosclerosis. 2009; 203: 1-7Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 9Abifadel M. Rabes J.P. Devillers M. Munnich A. Erlich D. Junien C. Varret M. Boileau C. Mutations and polymorphisms in the proprotein convertase subtilisin kexin 9 (PCSK9) gene in cholesterol metabolism and disease.Hum. Mutat. 2009; 30: 520-529Crossref PubMed Scopus (204) Google Scholar). The presence of a specific gain-of-function mutation in PCSK9 aggravates the hypercholesterolemia phenotype exerted by a concurrent pathogenic LDLR mutation (10Abifadel M. Rabes J.P. Jambart S. Halaby G. Gannage-Yared M.H. Sarkis A. Beaino G. Varret M. Salem N. Corbani S. et al.The molecular basis of familial hypercholesterolemia in Lebanon: spectrum of LDLR mutations and role of PCSK9 as a modifier gene.Hum. Mutat. 2009; 30: E682-E691Crossref PubMed Scopus (75) Google Scholar, 11Pisciotta L. Priore O.C. Cefalu A.B. Noto D. Bellocchio A. Fresa R. Cantafora A. Patel D. Averna M. Tarugi P. et al.Additive effect of mutations in LDLR and PCSK9 genes on the phenotype of familial hypercholesterolemia.Atherosclerosis. 2006; 186: 433-440Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Conversely, low activity of PCSK9 could lead to increased presence of LDLR at the hepatic cell surface and, consequently, to increased clearance of plasma LDL-C. This would theoretically reduce the extent of cholesterol elevation caused by an LDLR mutation. To test this hypothesis, we measured plasma PCSK9 levels in individuals who underwent DNA testing for genetic FH (12Huijgen R. Vissers M.N. Kindt I. Trip M.D. de Groot E. Kastelein J.J. Hutten B.A. Assessment of carotid atherosclerosis in normocholesterolemic individuals with proven mutations in the low-density lipoprotein receptor or apolipoprotein B genes.Circ. Cardiovasc. Genet. 2011; 4: 413-417Crossref PubMed Scopus (36) Google Scholar). The study population derived from participants of a previous single center cross-sectional study, described in detail elsewhere (12Huijgen R. Vissers M.N. Kindt I. Trip M.D. de Groot E. Kastelein J.J. Hutten B.A. Assessment of carotid atherosclerosis in normocholesterolemic individuals with proven mutations in the low-density lipoprotein receptor or apolipoprotein B genes.Circ. Cardiovasc. Genet. 2011; 4: 413-417Crossref PubMed Scopus (36) Google Scholar). In short, we recruited individuals from the database of the national screening program for autosomal dominant hypercholesterolemia. Men and women between 18 and 55 years of age were eligible for the original study if they were genetically tested for the specific pathogenic (13Huijgen R. Kindt I. Fouchier S.W. Defesche J.C. Hutten B.A. Kastelein J.J. Vissers M.N. Functionality of sequence variants in the genes coding for the low-density lipoprotein receptor and apolipoprotein B in individuals with inherited hypercholesterolemia.Hum. Mutat. 2010; 31: 752-760Crossref PubMed Scopus (39) Google Scholar) LDLR or APOB mutation residing within their family between January 2007 and January 2010 and if they had a known lipid profile. Individuals were excluded if they were unable to participate within 18 months after the genetic test. Individuals using cholesterol-lowering medication before genetic testing and probands, who were primarily clinically diagnosed, were excluded. Individuals from whom we failed to obtain sufficient plasma, required for PCSK9 measurements, were also excluded. Individuals who had been identified with a pathogenic mutation were categorized based on their untreated LDL-C level at genetic diagnosis. Mutation carriers with a LDL-C level below the age- and sex-specific 75th percentile were referred to as “FH low”; those with LDL-C above the 90th percentile were referred to as “FH high.” A third group, consisting of first-degree relatives negative for the familial LDLR or APOB mutation, was referred to as “control.” The selected individuals who consented made a single study visit to the Academic Medical Center in Amsterdam within 18 months after the genetic test. The study was approved by the local Ethics Committee. Blood samples were obtained for analysis of lipid measures and spare plasma between 8 AM and 10 AM after an overnight fast. These samples were collected in 7 ml EDTA Vacutainer® (K3E 15% 0.084 ml; BD Vacutainer Systems, Plymouth, UK) venous blood collection tubes using standard phlebotomy practices. Immediately after collection, tubes were gently inverted five times, and then centrifuged at 1,500–2,000 g for 15 min. The supernatant plasma was centrifuged again in similar fashion. The plasma was transferred into 2 ml freezer vials in 0.5 ml aliquots. The samples were frozen at −80°C and shipped on dry ice. PCSK9 concentrations were measured in triplicate using the CY-8079 ELISA kit (Cyclex, Nagano, Japan), according to the manufacturer's protocol. The medical history was recorded and physical examination performed according to a standardized procedure (13Huijgen R. Kindt I. Fouchier S.W. Defesche J.C. Hutten B.A. Kastelein J.J. Vissers M.N. Functionality of sequence variants in the genes coding for the low-density lipoprotein receptor and apolipoprotein B in individuals with inherited hypercholesterolemia.Hum. Mutat. 2010; 31: 752-760Crossref PubMed Scopus (39) Google Scholar). Carotid arteries were examined with ultrasound to assess intima-media thickness (cIMT), using methodology previously described in detail (14de Groot E. Hovingh G.K. Wiegman A. Duriez P. Smit A.J. Fruchart J.C. Kastelein J.J. Measurement of arterial wall thickness as a surrogate marker for atherosclerosis.Circulation. 2004; 109: III33-III38PubMed Google Scholar). Differences in demographic and clinical characteristics among the three predefined groups (FH low, FH high, and control) were evaluated using linear or logistic regression analysis. Linear regression analysis was applied to evaluate the association between PCSK9 and patient characteristics, LDL-C, or cIMT, and to assess differences in plasma PCSK9 levels among the three predefined groups. Multivariable regression models were applied to adjust for potential confounders. Inclusion in a final model was determined by backward stepwise elimination. All analyses were performed using the generalized estimating equations (GEE) method to account for correlations within families. The exchangeable correlation structure was used for these models. The main study outcome pertains to the individuals who remained untreated until the study visit. For transparency, we also analyzed the entire population of participants, including individuals who initiated statin treatment after genetic diagnosis (see the supplementary data). Variables with a skewed distribution were log-transformed before statistical analyses. A P-value < 0.05 was considered statistically significant. Data were analyzed with SPSS for Windows 16.0.2 (Chicago, IL). Among the screened population, 2,016 individuals met inclusion criteria for the original study. Recruitment was discontinued when a sufficient number of individuals with and without genetic FH were enrolled. A total of 421 individuals provided to participate in the original study. these 421 were for the of PCSK9 individuals without FH were excluded they were than 53 and for we did not have spare plasma to The between genetic testing was carriers were more treated with statins after diagnosis than were and those from the FH-high group had initiated statin treatment more than the individuals from the FH-low group (see supplementary In 267 individuals were untreated at the time of the study visit. characteristics of the untreated participants, into the three are in LDL-C levels were between the individuals from the FH-low and control levels were in the FH-high the of the three of the left and carotid arteries was in the FH-high group than in the FH-low group and the control of the untreated study vs. FH vs. FH vs. FH blood genetic FH study for blood and percentile LDL-C for age and for blood and in a percentile LDL-C for age and of the effect of statin treatment on PCSK9 levels our main study outcome was based on the untreated The untreated individuals from the FH-low group had lower PCSK9 levels [152 (137–167) compared with untreated individuals from the FH-high group 186 (165–207) ng/ml, P= 0.010] and (164–190) ng/ml, P= supplementary PCSK9 levels are associated with patient characteristics, were not among the three predefined groups. Therefore, we for these characteristics and blood by of a linear regression analysis. PCSK9 levels (95% CI) were lower for the individuals from the FH-low group compared with the FH patients from the FH-high group ng/ml vs. ng/ml, P= For the entire cohort of treated and untreated individuals characteristics, supplementary PCSK9 plasma levels were again lower in the FH-low group compared with the FH-high group (P= and the control group (P= and levels did not statistically differ between the FH-high and control groups (P= PCSK9 plasma levels (95% CI) were ng/ml, ng/ml, and ng/ml for the and FH-high We also associated plasma PCSK9 levels with LDL-C levels at the study visit for the untreated individuals with genetic The analysis that the percentile LDL-C for mutation and PCSK9 levels were the that remained associated with LDL-C levels after backward A analysis for association between PCSK9 and LDL-C plasma levels between the different mutation was However, this did not lead to to the number of individuals in mutation not low LDL-C levels in untreated individuals with genetic FH were primarily in those who a LDLR or APOB gene mutation that is associated with hypercholesterolemia in those who had low plasma levels of between LDL-C levels at the study visit and clinical characteristics in the untreated participants with genetic to to to percentile LDL-C for age and for untreated individuals a specific FH or mutation in to the severity of that specific mutation, as previously described in detail (see to to to to of regression percentile LDL-C for age and for untreated individuals a specific FH or mutation in to the severity of that specific mutation, as previously described in detail (see R. Kindt I. Fouchier S.W. Defesche J.C. Hutten B.A. Kastelein J.J. Vissers M.N. Functionality of sequence variants in the genes coding for the low-density lipoprotein receptor and apolipoprotein B in individuals with inherited hypercholesterolemia.Hum. Mutat. 2010; 31: 752-760Crossref PubMed Scopus (39) Google Scholar). in a of regression the association between and clinical characteristics in the untreated individuals without In the regression PCSK9 levels remained statistically associated with after backward elimination. In the of the lipid did not statistically associated in the plasma PCSK9 levels were positively associated with after for the lipid and cardiovascular risk between and clinical characteristics for untreated to to to to to to blood to to to to before to to of regression before analysis. in a of regression In the study, PCSK9 levels were measured in a cross-sectional study of individuals who had genetic with or without severely elevated LDL-C and that PCSK9 levels were lower in normocholesterolemic FH patients than in the groups. PCSK9 levels were associated with LDL-C levels groups. a would be that low plasma PCSK9 activity might lead to lower LDL-C levels in heterozygous To our this study is the first to plasma levels of PCSK9 between FH patients with and FH patients without severely elevated LDL-C However, groups have on the effect of genetic variation in the PCSK9 gene on the phenotype of (10Abifadel M. Rabes J.P. Jambart S. Halaby G. Gannage-Yared M.H. Sarkis A. Beaino G. Varret M. Salem N. Corbani S. et al.The molecular basis of familial hypercholesterolemia in Lebanon: spectrum of LDLR mutations and role of PCSK9 as a modifier gene.Hum. Mutat. 2009; 30: E682-E691Crossref PubMed Scopus (75) Google Scholar, 11Pisciotta L. Priore O.C. Cefalu A.B. Noto D. Bellocchio A. Fresa R. Cantafora A. Patel D. Averna M. Tarugi P. et al.Additive effect of mutations in LDLR and PCSK9 genes on the phenotype of familial hypercholesterolemia.Atherosclerosis. 2006; 186: 433-440Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, J. mutation in the PCSK9 gene has on the levels of total cholesterol in familial hypercholesterolemia 2010; PubMed Scopus Google Scholar) and that individuals who pathogenic mutations in PCSK9 and LDLR had LDL-C levels than did their relatives with mutation (10Abifadel M. Rabes J.P. Jambart S. Halaby G. Gannage-Yared M.H. Sarkis A. Beaino G. Varret M. Salem N. Corbani S. et al.The molecular basis of familial hypercholesterolemia in Lebanon: spectrum of LDLR mutations and role of PCSK9 as a modifier gene.Hum. Mutat. 2009; 30: E682-E691Crossref PubMed Scopus (75) Google Scholar). Conversely, and the effect of a PCSK9 mutation, in FH J. mutation in the PCSK9 gene has on the levels of total cholesterol in familial hypercholesterolemia 2010; PubMed Scopus Google Scholar). FH patients they identified the mutation in who had lower levels than did those without the mutation. These results of the association between genetic variation in PCSK9 and LDL-C levels our as we that FH patients with low levels of PCSK9 also have low LDL-C A is this variation in PCSK9 mutations in PCSK9 might be a J. mutation in the PCSK9 gene has on the levels of total cholesterol in familial hypercholesterolemia 2010; PubMed Scopus Google Scholar). We PCSK9 in a cohort of heterozygous FH patients who were selected for low LDL-C and J. mutation in the PCSK9 gene has on the levels of total cholesterol in familial hypercholesterolemia 2010; PubMed Scopus Google Scholar), we found the in PCSK9 in of those patients R. K. de Defesche J.C. Kastelein J.J. Hovingh G.K. Fouchier S.W. variation in and in carriers of pathogenic autosomal dominant hypercholesterolemic mutations with low LDL-C Mutat. PubMed Scopus Google Scholar). genetic variation in PCSK9 contribute to a FH but the explained low (10Abifadel M. Rabes J.P. Jambart S. Halaby G. Gannage-Yared M.H. Sarkis A. Beaino G. Varret M. Salem N. Corbani S. et al.The molecular basis of familial hypercholesterolemia in Lebanon: spectrum of LDLR mutations and role of PCSK9 as a modifier gene.Hum. Mutat. 2009; 30: E682-E691Crossref PubMed Scopus (75) Google Scholar, J. mutation in the PCSK9 gene has on the levels of total cholesterol in familial hypercholesterolemia 2010; PubMed Scopus Google Scholar). In to genetic plasma levels of of a has been identified A. G. M. J. E. J. M. Plasma PCSK9 is associated with and in a of and 2009; PubMed Scopus Google Scholar). We also that PCSK9 levels are associated with more carotid of lipid we that plasma levels of PCSK9 were positively associated with in patients with disease treated with a low in a control study in the to New et These with the that PCSK9 activity is associated with lower LDL-C levels and a risk of disease J.C. E. Hobbs H.H. in low and J. 2006; PubMed Scopus Google Scholar), the inhibition of PCSK9 as a of In fact, are in L. S. S. L. R.M. et proprotein convertase type 9 (PCSK9) PCSK9 and low density lipoprotein 2010; Full Text Full Text PDF PubMed Scopus Google N. N. M. C. J. A PCSK9 and LDLR in and in ONE. 2010; 5: PubMed Scopus Google Scholar, A. R.M. inhibition of proprotein convertase subtilisin/kexin type 9 in Full Text Full Text PDF PubMed Scopus Google Scholar, M. A. A. D. K. R. et PCSK9 plasma cholesterol in and cholesterol in Natl. Acad. Sci. USA. 2008; PubMed Scopus Google Scholar, of the between PCSK9 and the low-density lipoprotein J. 2009; PubMed Scopus Google Scholar, J.C. Piper D.E. D. C. J. J. et proprotein convertase subtilisin/kexin type 9 cholesterol in and Natl. Acad. Sci. USA. 2009; PubMed Scopus Google Scholar). of our study this is an study, and a between low plasma PCSK9 levels and lack of a hypercholesterolemia phenotype be a number of participants initiated statin treatment between the genetic FH diagnosis and study visit. statin treatment results in increased PCSK9 this the of our G. I. M.A. G. a in PCSK9 and correlation with 2010; Full Text Full Text PDF PubMed Scopus Google G. A. J. L. A. the gene the proprotein convertase in familial 2004; PubMed Scopus Google Scholar, G. levels of proprotein convertase subtilisin/kexin type 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). we could that PCSK9 levels were lower in FH patients with low LDL-C levels than in those with in treated and untreated This the notion that the differences in plasma PCSK9 levels between groups are not to the effect of statin our cohort of FH patients of carriers of a of pathogenic LDLR and APOB a we were unable to on the effect of specific of LDLR The PCSK9 plasma levels remained a of plasma LDL-C after for the percentile of LDL-C by specific the severity of the FH mutation be the for the association that we between plasma PCSK9 levels and LDL-C In with our plasma PCSK9 levels were to be and positively associated with LDL-C levels in a cohort of FH from single LDLR mutation et data). In plasma PCSK9 levels contribute to low LDL-C levels in FH Therefore, the results of pharmaceutical inhibition of PCSK9 in FH patients are eagerly awaited. The study participants, as well as for with familial hypercholesterolemia carotid intima-media thickness of proprotein convertase subtilisin kexin type 9 total cholesterol