Autosomal Dominant Hypercholesterolemia Research Articles

An Antibody against the C-Terminal Domain of PCSK9 Lowers LDL Cholesterol Levels In Vivo

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with autosomal dominant hypercholesterolemia, a state of elevated levels of LDL (low-density lipoprotein) cholesterol. Autosomal dominant hypercholesterolemia can result in severe implications such as stroke and coronary heart disease. The inhibition of PCSK9 function by therapeutic antibodies that block interaction of PCSK9 with the epidermal growth factor-like repeat A domain of LDL receptor (LDLR) was shown to successfully lower LDL cholesterol levels in clinical studies. Here we present data on the identification, structural and biophysical characterization and in vitro and in vivo pharmacology of a PCSK9 antibody (mAb1). The X-ray structure shows that mAb1 binds the module 1 of the C-terminal domain (CTD) of PCSK9. It blocks access to an area bearing several naturally occurring gain-of-function and loss-of-function mutations. Although the antibody does not inhibit binding of PCSK9 to epidermal growth factor-like repeat A, it partially reverses PCSK9-induced reduction of the LDLR and LDL cholesterol uptake in a cellular assay. mAb1 is also effective in lowering serum levels of LDL cholesterol in cynomolgus monkeys in vivo. Complete loss of PCSK9 is associated with insufficient liver regeneration and increased risk of hepatitis C infections. Blocking of the CTD is sufficient to partially inhibit PCSK9 function. Antibodies binding the CTD of PCSK9 may thus be advantageous in patients that do not tolerate complete inhibition of PCSK9.

Apoe P.Leu167Del Mutation in Familial Hypercholesterolemia

Autosomal dominant hypercholesterolemia (ADH) is caused by mutations in the low density lipoprotein receptor (LDLR), its ligand apoB (APOB) or proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. Yet DNA sequencing does not identify mutations in these genes in a significant number of cases with a phenotype of ADH, suggesting that ADH has multiple genetic etiologies.

Inherited lipemic splenomegaly and the spectrum of apolipoprotein E p.Leu167del mutation phenotypic variation

We review disorders associated with splenomegaly and dyslipidemia with an emphasis on the APOE p.Leu167del mutation. Recent studies suggest that this rare mutation may present more often without splenomegaly in patients with familial combined hyperlipidemia or autosomal dominant hypercholesterolemia. We supplement the literature review by reporting a new kindred. We reviewed our 3405-patient lipid clinic database to identify persons with dyslipidemia and splenomegaly. Identified patients were evaluated for relevant disorders, including genetic testing for a 3-base pair deletion in APOE that causes deletion of leucine at position 167 of apolipoprotein E. We identified 4 patients with splenomegaly and dyslipidemia, one of whom had a heterozygous APOE p.Leu167del mutation. This proband is a 76-year-old man with a history of splenomegaly first noted at age 13 and subsequent diagnosis of hypertriglyceridemia, low high-density lipoprotein cholesterol, leukopenia, and thrombocytopenia in his third decade. He never required splenectomy, and his splenic enlargement regressed over time with medical management of his hypertriglyceridemia. The APOE p.Leu167del mutation was also found in the proband's son and granddaughter, neither of whom has splenomegaly or marked dyslipidemia. Splenomegaly in association with dyslipidemia may indicate the presence of an underlying disorder. We discuss possible causes, review the literature relating to the rare APOE p.Leu167del mutation, and present a 3-generation kindred with variable phenotypic expression of this mutation. Severity of expression may depend on genotype, sex, or effective medical management of dyslipidemia or a combination of these factors. Aggressive lipid treatment may improve or prevent splenomegaly among patients with this disorder.

Effects of RG7652, a fully human mAb against proprotein convertase subtilisin/kexin type 9, on LDL-c: a Phase I, randomised, double-blind, placebo-controlled, single- and multiple-dose study

Purpose: Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates hepatic LDL receptors, playing a key role in cholesterol metabolism. Gain of function genetic mutations cause autosomal dominant hypercholesterolaemia, while nonsense mutations lower LDL-c and the risk of coronary events. RG7652 (MPSK3169A) is a fully human IgG1 monoclonal antibody (mAb) directed against PCSK9. This first-in-human study evaluated safety, tolerability, pharmacokinetics, pharmacodynamics, and efficacy in healthy individuals with elevated LDL-c. Methods: This Phase I study tested single and multiple doses of RG7652 and placebo given subcutaneously to 80 healthy volunteers with elevated serum LDL-c concentrations. Subjects entered six ascending, single-dose (SD) and four multiple-dose (MD) cohorts allocated 6 active:2 placebo. Two MD cohorts included atorvastatin therapy (40 mg daily) prior to administration of study drug. Following cohort safety reviews, escalation to the next higher dose cohort continued. The primary efficacy endpoint was change in LDL-c 2 weeks after final dose of study drug, with safety follow-up occurring during Weeks 8-16. Results: Eighty subjects (mean age 45 years, range 19-64; 48% male) with mean LDL-c of 162 mg/dL (4.2 mmol/L) were randomised. RG7652 reduced mean LDL-c by as much as 90 mg/dL (60%) from baseline, with a dose-dependent effect that appeared to saturate at the highest doses. The average reduction in LDL-c at Day 15 in SD and Day 36 in MD was >40 mg/dL in all cohorts other than the lowest dose. RG7652 had similar LDL-c lowering effects and kinetics when added to atorvastatin. No dose-limiting safety effects were identified and no subjects discontinued study drug for adverse events (AEs). Thirty-seven AEs, all mild, were attributed to study drug: 27 in 14 RG7652 subjects; 10 in 6 placebo subjects. Conclusions: The anti-PCSK9 mAb, RG7652, safely decreased LDL-c in healthy volunteers with elevated LDL-c as monotherapy and in combination with atorvastatin. The results support further testing of RG7652 in dyslipidaemic patients.

GW24-e2907 Effects of RG7652, a fully human mAb against proprotein convertase subtilisin/kexin type 9, on LDL-c: a Phase I, randomised, double-blind, placebo-controlled, single- and multiple-dose study

ObjectivesProprotein convertase subtilisin/kexin type 9 (PCSK9) regulates hepatic LDL receptors, playing a key role in cholesterol metabolism. Gain of function genetic mutations cause autosomal dominant hypercholesterolaemia, while nonsense mutations lower...

GW24-e3589 A novel mutation 1587_1588 del2 of the low-density lipoprotein receptor gene associated with familial hypercholesterolemia in a Chinese family

ObjectivesFamilial hypercholesterolemia (FH) is an autosomal dominant hypercholesterolemia caused by mutation in the low density lipoprotein receptor gene (LDLR). The identification of the causative mutation provides definitive diagnosis so that...

Identification and biochemical analysis of a novel APOB mutation that causes autosomal dominant hypercholesterolemia.

Patients with autosomal dominant hypercholesterolemia (ADH) have a high risk of developing cardiovascular disease that can be effectively treated using statin drugs. Molecular diagnosis and family cascade screening is recommended for early identification of individuals at risk, but up to 40% of families have no mutation detected in known genes. This study combined linkage analysis and exome sequencing to identify a novel variant in exon 3 of APOB (Arg50Trp). Mass spectrometry established that low-density lipoprotein (LDL) containing Arg50Trp APOB accumulates in the circulation of affected individuals, suggesting defective hepatic uptake. Previously reported mutations in APOB causing ADH have been located in exon 26. This is the first report of a mutation outside this region causing this phenotype, therefore, more extensive screening of this large and highly polymorphic gene may be necessary in ADH families. This is now feasible due to the high capacity of recently available sequencing platforms.

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PCSK9 - A New and Potent Approach to Lowering Cholesterol

A quantum leap in managing difficult hypercholesterolemia may be close at hand. Unfortunately, hypercholesterolemia and high cardiovascular (CV) risk have become universal problems throughout the world. Al-Waili et al. in their case report on a mutation in the proprotein convertase subtilisin kexin type 9 serine protease (PCSK9) gene in Omani Arab patients with autosomal dominant familial hypercholesterolemia (ADFH), offered the first description of such a mutation in an Arab population and added emphasis to the possible importance of a new therapeutic approach that appears to be on the horizon.1

Abstract 5: A Novel APOE p.Leu166del Mutation in Autosomal Dominant Familial Hypercholesterolemia

Autosomal dominant hypercholesterolemia (ADH) is caused by mutations in the low density lipoprotein receptor (LDLR), its ligand apoB (APOB) or in proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. Yet DNA sequencing does not identify mutations in these genes in a significant number of cases with a phenotype of ADH, suggesting that ADH has multiple genetic etiologies. Through a combination of clinical examination, biochemical analysis, candidate gene sequencing, and next-generation exome sequencing, we identified an in-frame three base-pair deletion in apolipoprotein E (APOE, c.496_498delCTC) resulting in a novel Leu166del mutation. The proband presented with an acute myocardial infarction at age 43, requiring urgent coronary revascularization. He has extensive tendinous xanthomas and xanthelasmas, elevated levels of total cholesterol (11.2 mmol/L, 457 mg/dL)), LDL-C (9.69 mmol/L, 374 mg/dL), normal HDL-C (1.62 mmol/L, 63 mg/dL) and triglycerides levels (1.13 mmol/L, 100 mg/dL). A HPLC lipoprotein profile showed selective increase in LDL-C. One sister is heterozygous for the APOE Leu166del mutation and has an LDL-C of 5.3 mmol/L (180 mg/L). Another sister, with an LDL-C of 3.8 mmol/L (120 mg/dL) was not affected. DNA sequencing was performed for the LDLR, PCSK9, LDL-R adapter protein-1 (LDLRAP1) and exon 26 of the APOB genes no causal mutations were identified. We then performed exome sequencing on three individuals from the family. Approximately 54,000 variants were identified. We removed variants unlikely to be causal in the LDL-C genome-wide association studies, variants with a minor allele frequency &gt;5%, synonymous or intronic variants and variants that did not fit a dominant model of transmission. We identified 49 missense mutations, 1 frameshift and 2 in-frame deletions in 52 genes. Using data derived from exome chip genotypes for association with LDL-C, the strongest evidence of association was found for the APOE gene. The Leu166del mutation is predicted to alter the protein structure of Apo E near the α-helix within the receptor binding domain. This report confirms that ADH can be caused by mutations within the APOE gene and represents the 4th loci causing ADH.

Characterization of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Trafficking Reveals a Novel Lysosomal Targeting Mechanism via Amyloid Precursor-like Protein 2 (APLP2)

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low density lipoprotein receptor protein levels by diverting it to lysosomes. Monoclonal antibody therapeutics aimed to neutralize PCSK9 have been shown to successfully lower serum LDL levels; however, we previously found that such therapeutic antibodies are subject to PCSK9-mediated clearance. In this study, we discovered that PCSK9 interacts via its C-terminal domain directly and in a pH-dependent manner with amyloid precursor protein as well as its closely related family member, amyloid precursor protein-like protein 2. Furthermore, we determined that amyloid precursor protein-like protein-2, but not amyloid precursor protein, is involved in mediating postendocytic delivery of PCSK9 to lysosomes and is therefore important for PCSK9 function. Based on our data, we propose a model for a lysosomal transport complex by which a soluble protein can target another protein for degradation from the luminal side of the membrane by bridging it to a lysosomally targeted transmembrane protein.

A DNA Microarray for the Detection of Point Mutations and Copy Number Variation Causing Familial Hypercholesterolemia in Europe

To facilitate genetic cascade screening for familial hypercholesterolemia (FH) in Europe, two versions (7 and 9) of a DNA microarray were developed to detect the most frequent point mutations in the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), and proprotein convertase subtilisin/kexin 9 (PCSK9) genes. The design of these microarrays is based on LIPOchip, version 4, which detects 191 LDLR and APOB mutations identified in Spanish patients with FH. A major improvement of LIPOchip, versions 7 and 9, is the ability to detect copy number variation (deletions or duplications of entire exons) in LDLR, thus abolishing the need to perform multiplex ligase-dependent probe amplification in patients with FH. The aim of this study was to validate a tool capable of detecting point mutations and copy number variations simultaneously and to evaluate its use and the newly developed software for analysis in clinical practice by reanalysis of several patients with known mutations causing FH. With the help of these validations, several aspects were analyzed, improved, and implemented in a newer version, which was evaluated through an internal validation.

More on Cholesterol Trafficking in the Body!

More on Cholesterol Trafficking in the Body!

Low Density Lipoprotein Binds to Proprotein Convertase Subtilisin/Kexin Type-9 (PCSK9) in Human Plasma and Inhibits PCSK9-mediated Low Density Lipoprotein Receptor Degradation

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a secreted protein that binds to the epidermal growth factor-like-A domain of the low density lipoprotein receptor (LDLR) and mediates LDLR degradation in liver. Gain-of-function mutations in PCSK9 are associated with autosomal dominant hypercholesterolemia in humans. Size-exclusion chromatography of human plasma has shown PCSK9 to be partly associated with undefined high molecular weight complexes within the LDL size range. We used density gradient centrifugation to isolate LDL in plasma pooled from 5 normolipidemic subjects and report that >40% of total PCSK9 was associated with LDL. Binding of fluorophore-labeled recombinant PCSK9 to isolated LDL in vitro was saturable with a K(D) ∼ 325 nM. This interaction was competed >95% by excess unlabeled PCSK9, and competition binding curves were consistent with a one-site binding model. An N-terminal region of the PCSK9 prodomain (amino acids 31-52) was required for binding to LDL in vitro. LDL dose-dependently inhibited binding and degradation of cell surface LDLRs by exogenous PCSK9 in HuH7 cells. LDL also inhibited PCSK9 binding to mutant LDLRs defective at binding LDL. These data suggest that association of PCSK9 with LDL particles in plasma lowers the ability of PCSK9 to bind to cell surface LDLRs, thereby blunting PCSK9-mediated LDLR degradation.

Autosomal Dominant Hypercholesterolemia: Needs for Early Diagnosis and Cascade Screening in the Tunisian Population

Autosomal dominant hypercholesterolemia (ADH) is characterized by an isolated elevation of plasmatic low-density lipoprotein (LDL), which predisposes to premature coronary artery disease (CAD) and early death. ADH is largely due to mutations in the low-density lipoprotein receptor gene (LDLR), the apolipoprotein B-100 gene (APOB), or the proprotein convertase subtilisin/kexin type 9 (PCSK9). Early diagnosis and initiation of treatment can modify the disease progression and its outcomes. Therefore, cascade screening protocol with a combination of plasmatic lipid measurements and DNA testing is used to identify relatives of index cases with a clinical diagnosis of ADH. In Tunisia, an attenuated phenotypic expression of ADH was previously reported, indicating that the establishment of a special screening protocol is necessary for this population.

Potential of Proprotein Convertase Subtilisin/Kexin Type 9 Based Therapeutics

The link between proprotein convertase subtilisin/kexin type 9 (PCSK9) and cholesterol metabolism was established only in 2003 when genetic mapping and positional cloning in patients with autosomal dominant hypercholesterolemia in which linkage to the loci coding for the LDL receptor and apolipoprotein B had been excluded identified the genetic defect missense as mutations in PCSK9, a protein/enzyme previously unknown to be related to lipid metabolism. Laboratory-based investigations confirmed that these were gain-of-function mutations. Further studies in cohorts with low LDL cholesterol (LDLc) levels from large epidemiological cardiovascular studies reported that loss-of-function mutations in PCSK9 were associated with protection from cardiovascular disease. An additional critical observation provided evidence that the interaction of PCSK9 and the LDL receptor was through circulating, not intracellular, PCSK9, which bound to the receptor, and then mediated the recycling of the LDL receptor. These findings established PSCK9 as a potential therapeutic target and resulted in biopharmaceutical companies developing interventions designed to lower LDLc levels. Clinical development programs for monoclonal antibodies against PCSK9 have advanced rapidly with completion of comprehensive phase 1 and 2 trials with both REGN727/SAR236553 (REGN727) and AMG 145, clearly demonstrating substantial reductions in LDLc levels in patients receiving diet alone, low, moderate, and high doses of statins, or statin combined with ezetimibe, and both heterozygous familial hypercholesterolemia and nonfamilial hypercholesterolemia subjects. Concomitant and parallel reductions in the levels of apolipoprotein B and its related lipoproteins, and small but significant increases in HDL cholesterol levels were seen as anticipated. An unanticipated and robust decrease in lipoprotein(a) levels was also noted. Although these trials have been relatively short term, no significant safety issues or target organs of interest have emerged. Larger and much longer phase 3 trials are now in progress to assess the long-term tolerability, safety, and impact on cardiovascular disease events of these very effective LDLc lowering compounds.

Spectrum of mutations and phenotypic expression in patients with autosomal dominant hypercholesterolemia identified in Italy

ObjectiveTo determine the spectrum of gene mutations and the genotype–phenotype correlations in patients with Autosomal Dominant Hypercholesterolemia (ADH) identified in Italy. MethodsThe resequencing of LDLR, PCSK9 genes and a selected region of APOB gene were conducted in 1018 index subjects clinically heterozygous ADH and in 52 patients clinically homozygous ADH. The analysis was also extended to 1008 family members of mutation positive subjects. ResultsMutations were detected in 832 individuals: 97.4% with LDLR mutations, 2.2% with APOB mutations and 0.36% with PCSK9 mutations. Among the patients with homozygous ADH, 51 were carriers of LDLR mutations and one was an LDLR/PCSK9 double heterozygote. We identified 237 LDLR mutations (45 not previously reported), 4 APOB and 3 PCSK9 mutations. The phenotypic characterization of 1769 LDLR mutation carriers (ADH-1) revealed that in both sexes independent predictors of the presence of tendon xanthomas were age, the quintiles of LDL cholesterol, the presence of coronary heart disease (CHD) and of receptor negative mutations. Independent predictors of CHD were male gender, age, the presence of arterial hypertension, smoking, tendon xanthomas, the scalar increase of LDL cholesterol and the scalar decrease of HDL cholesterol. We identified 13 LDLR mutation clusters, which allowed us to compare the phenotypic impact of different mutations. The LDL cholesterol raising potential of these mutations was found to vary over a wide range. ConclusionsThis study confirms the genetic and allelic heterogeneity of ADH and underscores that the variability in phenotypic expression of ADH-1 is greatly affected by the type of LDLR mutation.

Mutation in the PCSK9 Gene in Omani Arab Subjects with Autosomal Dominant Hypercholesterolemia and its Effect on PCSK9 Protein Structure

Proprotein convertase subtilisin/kexin type (PCSK9) is a crucial protein in LDL cholesterol (LDL-C) metabolism by virtue of its pivotal role in the degradation of the LDL receptor. Mutations in the PCSK9 gene have previously been found to segregate with autosomal dominant familial hypercholesterolemia (ADFH). In this study, DNA sequencing of the 12 exons of the PCSK9 gene has been performed for two patients with a clinical diagnosis of familial hypercholesterolemia where mutation in the LDL-receptor gene hasn't been excluded. One missense mutation was detected in the exon 9 PCSK9 gene in the two ADFH patients. The patients were found to be heterozygote for Ile474Val (SNP rs562556). Using an array of in silico tools, we have investigated the effect of the above mutation on different structural levels of the PCSK9 protein. Although, the mutation has already been reported in the literature for other populations, to the best of our knowledge this is the first report of a mutation in the PCSK9 gene from the Arab population, including the Omani population.

Autosomal Dominant Hypercholesterolemia: Needs for Early Diagnosis and Cascade Screening in the Tunisian Population

Autosomal dominant hypercholesterolemia (ADH) is characterized by an isolated elevation of plasmatic lowdensity lipoprotein (LDL), which predisposes to premature coronary artery disease (CAD) and early death. ADH is largely due to mutations in the low-density lipoprotein receptor gene (LDLR), the apolipoprotein B-100 gene (APOB), or the proprotein convertase subtilisin/kexin type 9 (PCSK9). Early diagnosis and initiation of treatment can modify the disease progression and its outcomes. Therefore, cascade screening protocol with a combination of plasmatic lipid measurements and DNA testing is used to identify relatives of index cases with a clinical diagnosis of ADH. In Tunisia, an attenuated phenotypic expression of ADH was previously reported, indicating that the establishment of a special screening protocol is necessary for this population. Keywords: Autosomal dominant hypercholesterolemia, Screening protocol, Molecular default

PCSK9 inhibition and LDL cholesterol lowering: the biology of an attractive therapeutic target and critical review of the latest clinical trials

PCSK9 is a serine protease expressed in the liver and the intestine. Gain-of-function mutations in PCSK9 are associated with autosomal dominant hypercholesterolemia, independently of mutations in apoB-100 or the LDL receptor (LDLR). Wild-type PCSK9 binds to the extracellular domain of the LDLR at the surface of hepatocytes, is internalized with the receptor and redirects it to lysosomes where the complex is degraded. Conversely, loss-of-function mutations in PCSK9 are associated with lower levels of LDL cholesterol (LDL-C) and reduced cardiovascular risk. Inhibition of PCSK9 binding to the LDLR with human monoclonal antibodies or reduction of PCSK9 expression with siRNA are the two main strategies to reduce LDL-C, which are actively pursued in the clinical development phases. In this article, the current evidence for PCSK9 as a target for reducing LDL-C and results of Phase I and II clinical trials with anti-PCSK9 therapeutic strategies are reviewed.