Severe Hypercholesterolemia in Four British Families With the D374Y Mutation in the PCSK9 Gene
Long-Term Follow-Up and Treatment Response
Objective— Analysis of long-term (30 years) clinical history and response to treatment of 13 patients with the D374Y mutation of PCSK9 (PCSK9 patients) from 4 unrelated white British families compared with 36 white British patients with heterozygous familial hypercholesterolemia attributable to 3 specific mutations in the low-density lipoprotein (LDL) receptor gene (LDLR) known to cause severe phenotype.
Methods and Results— The PCSK9 patients, when compared with the LDLR patients, were younger at presentation (20.8±14.7 versus 30.2±15.7 years; P=0.003), had higher pretreatment serum cholesterol levels (13.6±2.9 versus 9.6±1.6 mmol/L; P=0.004) that remained higher during treatment with simvastatin (10.1±3.0 versus 6.5±0.9 mmol/L; P=0.006), atorvastatin (9.6±2.9 versus 6.4±1.0 mmol/L; P=0.006), or current lipid-lowering therapy, including LDL apheresis and partial ileal bypass in 2 PCSK9 patients (7.0±1.6 versus 5.4±1.0 mmol/L; P=0.001), and were affected >10 years earlier by premature coronary artery disease (35.2±4.8 versus 46.8±8.9 years; P=0.002). LDL from PCSK9 patients competed significantly less well for binding to fibroblast LDL receptors than LDL from either controls or LDLR patients.
Conclusions— These British PCSK9 patients with the D374Y mutation have an unpredictably severe clinical phenotype, which may be a unique feature for this cohort, and requires early and aggressive lipid-lowering management to prevent cardiovascular complications.
Familial hypercholesterolemia (FH) is a common autosomal dominant disorder caused by mutations in the low-density lipoprotein (LDL) receptor gene (LDLR) leading to defective catabolism of plasma LDL by the liver and characterized by elevated levels of LDL cholesterol, tendon xanthomas, and excessive deposition of cholesterol in the arterial wall, causing premature atherosclerosis.1 An almost identical clinical syndrome to FH, called familial defective apolipoprotein B (apoB), can occur as a result of a dominantly inherited mutation of the ApoB gene, which encodes the ligand for the LDL receptor, causing impaired catabolism of LDL.2 Recently, heterozygous missense variants in a gene named PCSK9 (protein convertase subtilisin/kexin9) have been described to cosegregate with hypercholesterolemia in families of European origin.3–6
PCSK9 encodes a putative protease, which is a member of the subtilisin-like protein convertase family.7,8 Its physiological role has not yet been elucidated, but there is substantial evidence that it is involved in cholesterol homeostasis.9–12 PCSK9 is responsive to sterols and is a putative sterol regulatory element-binding protein (SREBP) target in mice.11,12 Adenovirus-mediated overexpression of wild-type PCSK9 in mice led to severe hypercholesterolemia by decreasing the amount of LDL receptor protein in the liver without reducing LDL receptor mRNA levels.13 However, similar overexpression of 2 naturally occurring PCSK9 missense mutants decreased hepatic LDL receptor protein content to the same extent as wild type.14 Further evidence that PCSK9 is involved in normal cholesterol metabolism comes from 2 recent observations: decreased plasma LDL cholesterol and apoB and increased LDL receptor protein in PCSK9 knockout mice,15 and 40% reduction in LDL cholesterol levels in human subjects heterozygous for nonsense mutation in PCSK9.16 These studies suggest that PCSK9 might function to reduce LDL receptor protein levels in liver but offer no convincing explanation for how missense mutations in PCSK9 cause hypercholesterolemia.
We have shown recently that expression of the missense D374Y and S127R variants (but not wild-type or F216L variant, nor a catalytically inactive mutant S386A) of PCSK9 in stably transfected rat liver cells increases secretion of apoB100-containing lipoproteins by 2- to 4-fold.6 Our findings are consistent with lipoprotein turnover data in 2 French patients heterozygous for the S127R mutation of PCSK9, which showed a 3-fold increase in very low-density lipoprotein–apoB production rate, with a 2-fold increase in LDL–apoB production rate, along with 30% reduction in fractional clearance of LDL compared with controls;17 increased content of apoB in triglyceride-rich lipoproteins in these patients has also been reported.18 Based on studies published so far, it appears that, unlike FH attributable to mutations in LDLR, in which the catabolic defect is the leading cause of hypercholesterolemia, missense (gain-of-function) mutations in PCSK9 may cause severe hypercholesterolemia by a dual mechanism: decreased LDL receptor activity and apoB100 overproduction. Further data on this important question are needed.
Only 10 families with autosomal dominant hypercholesterolemia attributable to PCSK9 missense mutations have been described in the literature to date.3–6 However, there have been no published reports on long-term follow-up of patients with this condition that elucidate the natural history of the disease and their response to lipid-lowering treatment compared with “classical” FH patients. In this article, we describe 4 unrelated white British families comprising 13 affected individuals with the D374Y mutation of PCSK9 who have been followed up for up to 30 years. We compare the clinical characteristics and response to lipid-lowering treatment in these patients with 3 well-characterized groups of British whites with heterozygous FH attributable to 3 specific mutations in LDLR known to cause a severe phenotype. In addition, we investigated whether LDL from patients with mutations in PCSK9 was able to compete effectively with labelled LDL from healthy volunteers for binding to skin fibroblast LDL receptors.
Materials and Methods
Patients With Mutation of PCSK9
Four index patients with autosomal dominant FH from unrelated white British families were referred to the Hammersmith Hospital Lipid Clinic because of difficulty in achieving target serum cholesterol concentrations on lipid-lowering medication. They have been followed up regularly by us for 12 to 30 years. After the 4 index patients were identified to be heterozygous for D374Y in PCSK9, screening of available family members identified 13 affected individuals, comprising 5 men and 8 women (mean age 20.8±14.7 years at presentation; range 22 months to 57 years). The basic clinical characteristics of the 12 individuals from the first 3 families have been described briefly.6 The nuclear family 4 (Figure 1D) has only 1 affected member (index patient II,1), whose details are presented in the online supplement (available at http://atvb.ahajournals.org).
Patients With Mutation of LDLR
Patients Involved in the Retrospective Response to Treatment Study
Thirty-six British whites with “classical” heterozygous FH attributable to 1 of 3 types of LDL receptor mutation were selected from the Hammersmith Hospital Lipid Clinic database. They comprised 17 women and 19 men, with a mean age at presentation of 30.2±15.7 years (range 5 to 65 years). These patients were selected solely because they were carriers of well-characterized mutations known to cause a severe phenotype in heterozygous individuals. The 3 groups comprised patients with (1) a single amino acid substitution in exon 4 (n=14); (2) a point mutation in the 3′ splice site of exon 3 that results in exclusion of exon 3 from the mRNA (n=8); and (3) a premature stop codon that results in undetectable amounts of LDLR protein (n=14).
Patients Involved in Competition for Binding of LDL to Normal Skin Fibroblast LDL Receptors and in the Assessment of LDL Particle Size
Three groups of patients took part in these studies: (1) all living patients with mutations in the PCSK9 shown in Figure 1, excluding 1 6-year-old girl (family 3; patient III,3) and 1 patient with partial ileal bypass (family 1; patient III,1). This PCSK9 group comprised 5 men and 4 women, with a mean age of 43.1±16.8 years, mean on-treatment serum LDL cholesterol at the time of study 5.4±3.1 mmol/L, triglyceride 1.40±1mmol/L, and total apoB 132.0±41.4 mg/dL; (2) 4 patients with different LDLR mutations (D200G; W66G; deletion/frameshift in exon 9; deletion of exons 2 to 6); mean age 51.1±19.0 years, mean on-treatment serum LDL cholesterol at the time of study 3.1±0.7 mmol/L, triglyceride 0.9±0.4 mmol/L, and total apoB 81.5±15.1 mg/dL; (3) 3 patients with familial defective apoB3500 (mean age 60.1±16.0 years, mean on-treatment LDL cholesterol 4.2±1.6 mmol/L, triglyceride 1.0±0.3 mmol/L, and total apoB 124.0±6.6 mg/dL). At the time of the study, all patients were on treatment consisting of statins with or without ezetimibe. A control group comprised 5 normolipidemic volunteers (mean age 38.1±12.7 years, mean serum LDL cholesterol 3.1±0.6, triglyceride 1.2±0.2, and total apoB 69.5±8.5 mg/dL), who were not on any lipid-modifying drugs.
None of the patients were obese or had diabetes mellitus, hypothyroidism, or any other conditions known to influence cholesterol levels or to require treatment with medication (other than lipid-lowering drugs) affecting serum lipid levels.
Ethics research committee approval was obtained for this study, and all subjects gave informed written consent.
Study Protocol and Methods
Fasting venous blood samples were obtained for measurement of standard serum lipid parameters using automated enzymatic assays; LDL cholesterol was calculated using the Friedewald formula.
Retrospective Assessment of Treatment Response
The information about response to treatment with statins was analyzed after careful retrospective assessment of medical case notes of all affected individuals. The serum total cholesterol values on treatment for each patient have been derived as a mean of 2 measurements ≥3 months apart after the patient had been on treatment with the same statin and dose for ≥3 months and when no physiological or other pathological causes that might have interfered with lipid levels had been identified.
Clinical Characteristics of Patients With the D374Y Mutation in PCSK9 and FH Patients With Defined LDL Receptor Mutations Involved in Retrospective Analysis of Treatment Response
The main clinical characteristics of patients with D374Y mutation of PCSK9 (PCSK9 patients) and the 3 groups of “classical” FH patients with severe LDLR mutations (LDLR patients) are summarized in Table I (available online at http://atvb.ahajournals.org). Of the 13 affected PCSK9 individuals, 6 had premature coronary heart disease (CHD) and 8 had tendon xanthomas; 13 of the LDLR patients had CHD, and 18 had tendon xanthomas. In all affected individuals, D374Y was on an allele of PCSK9 with the same haplotype (please see online supplement).
Despite the much younger age at presentation of the PCSK9 patients compared with the 36 LDLR patients (20.8±14.7 versus 30.2±15.7 years; P=0.003), they had significantly higher serum total cholesterol concentrations (13.6±2.9 versus 9.6±1.6 mmol/L; P=0.004) and were affected at a much earlier age by premature CHD (35.3±4.8 versus 46.8±8.9 years; P=0.002). Although within the normal range, mean fasting serum triglyceride was significantly higher in the PCSK9 group (1.7±0.7 versus 1.08±0.57 mmol/L; P=0.002); high-density lipoprotein (HDL) cholesterol levels (1.2±0.4 versus 1.2±0.27; P=NS) were similar in the 2 groups.
Figure 1 shows the pedigrees of the 4 PCSK9 families and summarizes the response to lipid-lowering management in each index patient over a long period of time. Because this is a disease for which the molecular basis was unraveled only recently and about which very limited clinical information is available, we describe in some detail the long-term medical history of the index patients and their affected relatives who have been followed up for up to 30 years (please see online supplement).
Comparison of Response to Treatment Between Patients With PCSK9 and LDL Receptor Mutations
Figure 2 shows the response to treatment in the PCSK9 patients and the 3 groups of LDLR patients. Because there were no significant differences between the 3 LDLR patient groups in either pretreatment serum total cholesterol levels, or in absolute or percentage decreases in total serum cholesterol during treatment with simvastatin and atorvastatin, the LDLR patients have been analyzed as a single group. Mean serum total cholesterol concentrations remained significantly higher in the PCSK9 patients during treatment with either simvastatin (10.1±3 versus 6.5±0.9 mmol/L; P=0.006) or atorvastatin (9.6±2.9 versus 6.4±1.0 mmol/L; P=0.006; Figure 2A), despite the fact that the PCSK9 and LDLR groups received similar mean doses of simvastatin (48 versus 37.1 mg daily) or atorvastatin (64.0 versus 58.8 mg daily). Furthermore, 2 of the PCSK9 patients required, in addition to treatment with statins, a partial ileal bypass in 1 instance (family 1; patient III,1) and long-term LDL apheresis in the other (family 3; patient II,1) to improve serum cholesterol levels. However, although a trend was observed, the percent reduction in total cholesterol during treatment was not significantly lower in the PCSK9 group than in the 3 groups of LDLR patients (Figure 2B).
When serum total cholesterol levels were compared while all patients were on their current lipid-lowering therapy (Figure 2C), comprising statins plus ezetimibe or bile acid sequestrants, again, the values in the PCSK9 patients remained higher despite their younger age (7.0±1.6 versus 5.4±1.0 mmol/L; P=0.001); similarly, the total cholesterol to HDL cholesterol ratio remained significantly higher (5.4±0.95 versus 4.3±1.5; P=0.046) in the PCSK9 group. All the PCSK9 patients were on maximal doses of atorvastatin (80 mg) or rosuvastatin (40 mg in all adults and 10 mg in a minor) plus ezetimibe (10 mg daily); 2 patients, as already mentioned, had, in addition, partial ileal bypass and chronic treatment with LDL apheresis. The statin doses of the LDLR patients were substantially smaller, varying between 20 and 80 mg daily for atorvastatin (mean 54.1±23.1 mg) and between 10 and 40 mg daily for rosuvastatin (mean 25.0±21.1 mg); not all patients in this group were on concomitant treatment with ezetimibe or bile acid sequestrants. Despite the smaller statin doses, the LDLR FH patients achieved lower total cholesterol levels, and there was no need for additional interventions.
LDL Binding and Competition Assays
To assess whether LDL from PCSK9 D374Y patients was able to bind to the LDL receptor with the same affinity as LDL from normolipidemic individuals, we determined its ability to compete for binding of normolipidemic 125I-labelled LDL to human skin fibroblasts in culture. In preliminary experiments with 2 different preparations of labelled LDL from a normolipidemic donor, we found that competition by LDL from a PCSK9 patient was significantly impaired compared with LDL from a normolipidemic donor (Figure 3A). To determine whether this impaired binding was common to all PCSK9 patients, LDL samples from 9 PCSK9 patients were then compared with LDL from 5 healthy volunteers, 4 LDLR patients who were on similar treatment regimes to the PCSK9 patients, and 3 patients with heterozygous familial defective apoB100 (FDB; Figure 3B). As expected, LDL from patients with FDB showed the weakest ability to compete and bind to the LDL receptors. LDL from the LDLR patients and the controls competed equally well for binding, whereas LDL from the PCSK9 patients did indeed show significantly impaired competition for binding when compared with LDL from either normolipidemic controls or LDLR patients on similar lipid-lowering treatment to the PCSK9 patients. Thus, PCSK9 LDL appears to have reduced affinity for binding to the LDL receptor.
To investigate the underlying reason for this, we determined the composition of the LDL and found that the mean cholesterol:protein ratio of PCSK9 LDL was significantly lower than that of the control subjects (Figure 3C, left). As expected, LDLR and FDB LDL had higher cholesterol:protein ratios than normal LDL. The triglyceride contents of the various LDL samples were not significantly different (data not shown), and thus the ratio of cholesterol plus triglyceride to protein was lower in PCSK9 LDL than normal LDL or LDL from LDLR patients (Figure 3C, right). This suggests that LDL particles, isolated in the density range 1.020 to 1.050 g/mL, from PCSK9 patients were smaller than LDL from normolipemic individuals or LDLR and FDB patients. However, there was no significant difference in whole plasma LDL particle size measured by nondenaturing gradient gel electrophoresis (data not shown).
In this article, we describe 4 unrelated white British families comprising 13 individuals with severe autosomal dominant hypercholesterolemia attributable to the D374Y variant of the PCSK9 gene. When these patients are compared with typical heterozygous FH patients with known mutations in LDLR, even those selected as having null mutations, this group of PCSK9 patients seems to be more severely affected in that their pretreatment serum total cholesterol concentrations were higher, and levels on treatment with statins of total cholesterol and total cholesterol to HDL cholesterol ratio remained higher. Possibly as a consequence of these 2 phenotypic features, the PCSK9 patients with the D374Y variant developed premature CHD >10 years earlier compared with the group of heterozygous carriers of severe mutations in LDLR. Unusually, 2 women 30 and 31 years of age died of premature CHD without any other risk factors,6 and the 22-month-old son of 1 of them had total cholesterol of 13.4 mmol/L (family 2; III,2). In the extensive experience of managing FH patients in the Lipid Clinics at Hammersmith and Aintree Hospitals, these features are uncommon for a heterozygous carrier of a gene affecting cholesterol metabolism.
The mean serum total cholesterol level of the white British carriers of the D374Y mutation is higher not only when compared with the group of LDLR FH patients attending the same Lipid Clinic but also when compared those British patients with clinical diagnosis for definite FH with FH patients selected to have null mutations or for being CHD positive or to FH patients with “refractory” hypercholesterolemia, as summarized in Table II (available online at http://atvb.ahajournals.org). Our PCSK9 patients were significantly younger than any other group of LDLR FH patients, thus the higher mean serum cholesterol cannot be explained by age. This severity of pretreatment levels of serum total cholesterol in heterozygous carriers of the D374Y variant of the PCSK9 gene has not been reported previously by other investigators, and it may be a specific feature of this cohort of 13 British individuals. However, it is important to note that 2 Norwegian patients5 with the same mutation, 19 and 41 years of age, had total serum cholesterol levels of 13.6 mmol/L. Furthermore, the affected members of the Utah family whose “off-treatment” cholesterol levels were reported were stated to be on very strict low-fat diets at the time.19 This may explain, at least in part, why they do not appear to be as severely hypercholesterolemic as a group as our British patients with the same mutation, whose serum cholesterol concentrations were measured before dietary advice had been provided. Indeed, like the severely affected British and Norwegian patients, there are family members in the Utah pedigree reported to have total serum cholesterol levels >11 mmol/L at 9 and 16 years of age.
In contrast, the French families carrying the F216L or S127R variants do not appear to have very severe hypercholesterolemia.3 Thus, it is likely that in patients with mutations in the PCSK9 gene, as is the case with patients with FH attributable to mutations in LDLR,1,20 the nature of the molecular defect has an impact on the severity of hypercholesterolemia. It is also possible that heterozygotes with the same missense D374Y mutation in the PCSK9 gene can have different phenotypic expression, as already observed in FH patients with the same LDL receptor mutation,21 and that a combination of environmental and genetic factors promotes the unusual severity of hypercholesterolemia in the British pedigrees.
Long-term management of the 13 patients described in this article has proved to be difficult. Despite maximal lipid-lowering medication, none of the patients have so far achieved currently acceptable “target” cholesterol levels.22 It is of interest that in some, but not all, PCSK9 patients, the addition of ezetimibe led to pronounced reduction in serum cholesterol, as did a very strict low-cholesterol low-fat diet; these effects are most probably attributable to drastically reduced absorption of cholesterol in the small intestine and not directly related to LDL receptor activity. Because there is no published data yet on long-term management and response to treatment in patients with PCSK9 mutations, we cannot compare our observations with other cohorts of patients with this particular mutation.
The mechanism underlying this severe autosomal dominant hypercholesterolemia associated with the D374Y mutation in PCSK9 in this group of British patients is not yet understood, nor is it clear why they should be so difficult to manage clinically compared with other FH patients. It could be speculated that the dual pathophysiological mechanism involved, namely decreased LDL receptor protein and increased apoB100 secretion, documented to occur in in vitro and in vivo studies, may aggravate the phenotype. The lipid-lowering action of statins is via inhibition of 3-hydroxy-3-methylglutaryl–coenzyme A, leading to reduced cholesterol biosynthesis and cellular cholesterol levels, which activates SREBP-2 and leads to transcriptional activation not only of the LDLR, which results in increased LDL receptor activity and lowering of plasma LDL, but also of PCSK9, which appears to have the opposing effect of reducing LDL receptor protein.12,14,15 Thus, treatment of PCSK9 patients with statins will presumably also increase the activity of the dominant-negative mutant form, further attenuating the beneficial effects of statin treatment and not allowing them to achieve target serum cholesterol levels22 even when maximal statin doses or a combination of lipid-lowering drugs are implemented, as observed in this study.
Our results indicate that LDL from PCSK9 patients have impaired capacity to compete for binding to normal fibroblast LDL receptors when compared with LDL from LDLR patients on similar lipid-lowering therapy and also with control subjects. The LDL from LDLR patients behaved similarly to those of control subjects, suggesting that the lipid-lowering medication is not responsible for the difference observed between PCSK9 and LDLR patients. The abnormal composition of PCSK9 LDL suggests that the conformation of apoB on the surface of the particles may be different from that of normal LDL and contribute to poor binding affinity. Our observations provide further evidence for the complexity of the phenotype in PCSK9 patients and raises the question as to the possible involvement of impaired LDL binding in addition to overproduction of apoB6 in causing their hypercholesterolemia.
The strengths of the study presented here include the fact that all the patients are British whites who have been attending our Lipid Clinics for many years and have received similar management. The PCSK9 patients all carry the same D374Y variant of PCSK9 on the same haplotype, suggesting that they share a common ancestor, whereas the LDLR patients were purposely selected to carry specific mutations in LDLR known to cause a severe hypercholesterolemic phenotype. Although the conclusions are limited by the relatively small number of patients and the retrospective nature of the study, the results clearly show that this group of British patients with the D374Y mutation of the PCSK9 have an unpredictably severe clinical phenotype, which requires early and aggressive lipid-lowering management to prevent cardiovascular complications. Currently available lipid-lowering drugs do not achieve adequate control of their hypercholesterolemia. The decreased plasma cholesterol levels and hypersensitivity to statins observed in mice lacking Pcsk915 suggests that in the future, compounds that lead to inhibition of PCSK9 activity may have synergistic if not additive effects when combined with statins. Adequate management of this disorder is expected to improve the prognosis of these patients, as has been shown for those with more typical heterozygous FH.19
This study was funded in part by the British Heart Foundation (PG/03/020/15126). We are indebted to the patients and their families for their willing cooperation, to Dr D. Wile (University Hospital, Aintree, Liverpool) for facilitating access to his patient, and to Professor Gilbert Thompson, who was involved in the past care of some of the patients.
- Received May 26, 2005.
- Accepted September 23, 2005.
Goldstein J, Hobbs H, Brown M. Familial hypercholesterolemia. In: Valle D, Scriver CR, Beaudet A, Sly WS, Childs B, Kinzler KW, Volgestein B, eds. The Metabolic and Molecular Bases of Inherited Disease. 8th ed. New York, NY: McGraw Hill; 2001: 2863–2913.
Innerarity TL, Mahley RW, Weisgraber KH, Bersot TP, Krauss RM, Vega GL, Grundy SM, Friedl W, Davignon J, McCarthy BJ. Familial defective apolipoprotein B-100: a mutation of apolipoprotein B that causes hypercholesterolemia. J Lipid Res. 1990; 31: 1337–1349.
Abifadel M, Varret M, Rabes JP, Allard D, Ouguerram K, Devillers M, Cruaud C, Benjannet S, Wickham L, Erlich D, Derre A, Villeger L, Farnier M, Beucler I, Bruckert E, Chambaz J, Chanu B, Lecerf JM, Luc G, Moulin P, Weissenbach J, Prat A, Krempf M, Junien C, Seidah NG, Boileau C. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat Genet. 2003; 34: 154–156.
Sun XM, Eden ER, Tosi I, Neuwirth CK, Wile D, Naoumova RP, Soutar AK. Evidence for effect of mutant PCSK9 on apolipoprotein B secretion as the cause of unusually severe dominant hypercholesterolemia. Hum Mol Genet. 2005; 14: 1161–1169.
Naureckiene S, Ma L, Sreekumar K, Purandare U, Frederick LC, Huang Y, Chiang LW, Grenier JM, Ozenberger BA, Steven JJ, Kennedy JD, DiStefano PS, Wood A, Bingham B. Functional characterization of NARC 1, a novel proteinase related to proteinase K. Arch Biochem Biophys. 2003; 420: 55–67.
Seidah NG, Benjannet S, Wickham L, Marcinkiewicz J, Jasmin SB, Stifani S, Basak A, Prat A, Chretien M. 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: 928–933.
Horton JD, Shah NA, Warrington JA, Anderson NN, Park SW, Brown MS, Goldstein JL. Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes. Proc Natl Acad Sci U S A. 2003; 100: 12027–12032.
Maxwell KN, Soccio RE, Duncan EM, Sehayek E, Breslow JL. Novel putative SREBP and LXR target genes identified by microarray analysis in liver of cholesterol-fed mice. J Lipid Res. 2003; 44: 2109–2119.
Dubuc G, Chamberland A, Wassef H, Davignon J, Seidah NG, Bernier L, Prat A. Statins upregulate PCSK9, the gene encoding the proprotein convertase neural apoptosis-regulated convertase-1 implicated in familial hypercholesterolemia. Arterioscler Thromb Vasc Biol. 2004; 24: 1454–1459.
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: 7100–7105.
Park SW, Moon YA, Horton JD. Post-transcriptional regulation of low density lipoprotein receptor protein by proprotein convertase subtilisin/kexin type 9a in mouse liver. J Biol Chem. 2004; 279: 50630–50638.
Rashid S, Curtis DE, Garuti R, Anderson NN, Bashmakov Y, Ho YK, Hammer RE, Moon YA, Horton JD. Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9. Proc Natl Acad Sci U S A. 2005; 102: 5374–5379.
Ouguerram K, Chetiveaux M, Zair Y, Costet P, Abifadel M, Varret M, Boileau C, Magot T, Krempf M. Apolipoprotein B100 metabolism in autosomal-dominant hypercholesterolemia related to mutations in PCSK9. Arterioscler Thromb Vasc Biol. 2004; 24: 1448–1453.
Lalanne F, Lambert G, Amar MJ, Chetiveaux M, Zair Y, Jarnoux AL, Ouguerram K, Friburg J, Seidah NG, Brewer HB Jr, Krempf M, Costet P. Wild-type PCSK9 inhibits LDL clearance but does not affect apoB-containing lipoprotein production in mouse and cultured cells. J Lipid Res. 2005; 46: 1312–1319.
Haddad L, Day IN, Hunt S, Williams RR, Humphries SE, Hopkins PN. Evidence for a third genetic locus causing familial hypercholesterolemia. A non-LDLR, non-APOB kindred. J Lipid Res. 1999; 40: 1113–1122.
Sun XM, Patel DD, Bhatnagar D, Knight BL, Soutar AK. Characterization of a splice-site mutation in the gene for the LDL receptor associated with an unpredictably severe clinical phenotype in English patients with heterozygous FH. Arterioscler Thromb Vasc Biol. 1995; 15: 219–227.
Pimstone SN, Sun XM, du SC, Frohlich JJ, Hayden MR, Soutar AK. Phenotypic variation in heterozygous familial hypercholesterolemia: a comparison of Chinese patients with the same or similar mutations in the LDL receptor gene in China or Canada. Arterioscler Thromb Vasc Biol. 1998; 18: 309–315.
De Backer G, Ambrosioni E, Borch-Johnsen K, Brotons C, Cifkova R, Dallongeville J, Ebrahim S, Faergeman O, Graham I, Mancia G, Manger C, V, Orth-Gomer K, Perk J, Pyorala K, Rodicio JL, Sans S, Sansoy V, Sechtem U, Silber S, Thomsen T, Wood D. European guidelines on cardiovascular disease prevention in clinical practice. Third Joint Task Force of European and Other Societies on Cardiovascular Disease Prevention in Clinical Practice. Eur Heart J. 2003; 24: 1601–1610.