Sara Hamon

Characterization of Autosomal Dominant Hypercholesterolemia Caused by PCSK9 Gain of Function Mutations and its Specific Treatment with Alirocumab, a PCSK9 Monoclonal Antibody

Background—Patients with PCSK9 gene gain of function (GOF) mutations have a rare form of autosomal dominant hypercholesterolemia. However, data examining their clinical characteristics and geographic distribution are lacking. Furthermore, no randomized treatment study in this population has been reported. Methods and Results—We compiled clinical characteristics of PCSK9 GOF mutation carriers in a multinational retrospective, cross-sectional, observational study. We then performed a randomized placebo-phase, double-blind study of alirocumab 150 mg administered subcutaneously every 2 weeks to 13 patients representing 4 different PCSK9 GOF mutations with low-density lipoprotein cholesterol (LDL-C) ≥70 mg/dL on their current lipid-lowering therapies at baseline. Observational study: among 164 patients, 16 different PCSK9 GOF mutations distributed throughout the gene were associated with varying severity of untreated LDL-C levels. Coronary artery disease was common (33%; average age of onset, 49.4 years), and untreated LDL-C concentrations were higher compared with matched carriers of mutations in the LDLR (n=2126) or apolipoprotein B (n=470) genes. Intervention study: in PCSK9 GOF mutation patients randomly assigned to receive alirocumab, mean percent reduction in LDL-C at 2 weeks was 62.5% (P<0.0001) from baseline, 53.7% compared with placebo-treated PCSK9 GOF mutation patients (P=0.0009; primary end point). After all subjects received 8 weeks of alirocumab treatment, LDL-C was reduced by 73% from baseline (P<0.0001). Conclusions—PCSK9 GOF mutation carriers have elevated LDL-C levels and are at high risk of premature cardiovascular disease. Alirocumab, a PCSK9 antibody, markedly lowers LDL-C levels and seems to be well tolerated in these patients. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique Identifier: NCT01604824.

Effect of PCSK9 Inhibition by Alirocumab on Lipoprotein Particle Concentrations Determined by Nuclear Magnetic Resonance Spectroscopy

Background In patients with discordance between low‐density lipoprotein (LDL) cholesterol and LDL particle (LDL‐P) concentrations, cardiovascular risk more closely correlates with LDL−P. Methods and Results We investigated the effect of alirocumab, a fully human monoclonal antibody to proprotein convertase subtilisin/kexin type 9, on lipoprotein particle concentration and size in hypercholesterolemic patients, using nuclear magnetic resonance spectroscopy. Plasma samples were collected from patients receiving alirocumab 150 mg every 2 weeks (n=26) or placebo (n=31) during a phase II, double‐blind, placebo‐controlled trial in patients (LDL cholesterol ≥100 mg/dL) on a stable atorvastatin dose. In this post hoc analysis, percentage change in concentrations of LDL−P, very‐low‐density lipoprotein particles, and high‐density lipoprotein particles from baseline to week 12 was determined by nuclear magnetic resonance. Alirocumab significantly reduced mean concentrations of total LDL‐P (−63.3% versus −1.0% with placebo) and large (−71.3% versus −21.8%) and small (−54.0% versus +17.8%) LDL‐P subfractions and total very‐low‐density lipoprotein particle concentrations (−36.4% versus +33.4%; all P<0.01). Total high‐density lipoprotein particles increased with alirocumab (+11.2% versus +1.4% with placebo; P<0.01). There were greater increases in large (44.6%) versus medium (17.7%) or small high‐density lipoprotein particles (2.8%) with alirocumab. LDL‐P size remained relatively unchanged in both groups; however, very‐low‐density and high‐density lipoprotein particle sizes increased to a significantly greater extent with alirocumab. Conclusions Alirocumab significantly reduced LDL‐C and LDL‐P concentrations in hypercholesterolemic patients receiving stable atorvastatin therapy. These findings may be of particular relevance to patients with discordant LDL‐C and LDL‐P concentrations. Clinical Trial Registration URL: https://clinicaltrials.gov. Unique identifier: NCT01288443.

Risk Alleles Associated with Neovascularization in a Pachychoroid Phenotype

Macular neovascularization is most commonly associated with age-related macular degeneration (AMD) but also may occur in a number of other contexts including pachychoroid spectrum disease, uveitis, myopia, and macular dystrophies. Phenotypically, neovascularization occurring in pachychoroid disease is distinguished from neovascular AMD by the relatively younger age of pachychoroid patients, absence or paucity of drusen, thicker choroids than those seen in AMD, and a more insidious onset. However, neovascular tissue in pachychoroid eyes seems to resemble that of AMD in constitution, multimodal imaging appearance, traversal of tissue boundaries, and response to antiangiogenic therapy. Therefore, it is likely that pachychoroid neovasculopathy shares mechanistic factors with neovascular AMD. The sequence of events that culminate in macular neovascularization remains unclear, but the last decade has seen increasing focus on the role of susceptibility alleles for neovascular AMD, particularly those related to the complement pathway. The purpose of this case-control study was to determine whether 12 candidate DNA single nucleotide polymorphisms (SNPs) known to confer susceptibility to neovascular AMD might also relate to the risk of neovascularization in patients with a pachychoroid phenotype (Supplementary Table S1, available at www.aaojournal.org). White subjects aged more than 50 years were recruited and assigned consecutively to 4 phenotyped cohorts: (A) neovascular AMD (quality control), (B) pachychoroid phenotype with type 1 neovascularization, (C) pachychoroid spectrum without neovascularization, and (D) subjects without evidence of pachychoroid disease, AMD, or neovascularization (control). Patients with other macular or retinal vascular disease, uveitis, or prior photodynamic therapy were excluded. Phenotypic classifications were based on clinical features and multimodal imaging performed as part of the ongoing care of each patient. All patients recruited into either of the pachychoroid groups (B or C) had at least 1 prior episode of central serous chorioretinopathy (neurosensory detachment or pathognomonic descending tracks on fundus autofluorescence) in addition to pachychoroid features. Type 1 neovascularization was defined anatomically as previously described. In pachychoroid eyes, shallow irregular pigment epithelial detachments were interpreted as neovascularized. Type 1 neovascularization was attributed to pachychoroid disease (group B) rather than AMD (group A) if drusen were absent or minimally present (no greater than Age-Related Eye Disease Study category 1) and if pachychoroid features were seen in the choroid. Type 1 neovascularization was attributed to AMD (group A) rather than pachychoroid disease (group B) in patients with typical findings of AMD, such as drusen and retinal pigment epithelium changes in the absence of pachychoroid features. Patients who exhibited overlapping features of AMD and pachychoroid disease were excluded. Buccal mucosal swabs (SK-1, Isohelix Ltd., Harrietsham, Kent, UK) from each subject were analyzed using the RetnaGene AMD Laboratory Developed Test, which surveys a panel of 12 SNPs (Supplementary Table S1, available at www.aaojournal.org). Data were analyzed using R version 3.0.2 (http://www.R-project.org). Genetic testing and analysis did not begin until phenotyping and recruitment were complete. For each polymorphism, intergroup comparisons were performed using the Fisher exact test with a Bonferroni adjustment. Risk allele odds ratios (ORs) were also calculated. Age and phenotypic comparisons were made using the Student 2-tailed t test in Excel 2013 (Microsoft Corporation, Redmond, WA). A total of 201 subjects were recruited, 51 in group A and 50 in each of groups B to D. Demographic profiles are summarized in Supplementary Table S2 (available at www.aaojournal.org). Subjects with neovascularization (AþB) were older than those without neovascularization (CþD) (P 1⁄4 8.5 10 ). Pachychoroid eyes (BþC) had thicker subfoveal choroids than normal eyes (D) (P 1⁄4 6.1 10 ), and eyes with neovascular AMD (A) had thinner choroids than normal eyes (D) (P 1⁄4 0.017). In group D (controls), none of the polymorphisms deviated from the HardyeWeinberg equilibrium and pairwise estimates of linkage disequilibrium were <0.80 for each pair of SNPs, suggesting that they are generally independent and that the cohort was outbred and not subject to confounding selection pressures. Table 1 summarizes risk allele frequency for each SNP in each subject group A to D, with intergroup comparisons by Fisher exact test and ORs with confidence intervals. When comparing subjects with neovascular AMD with normal subjects (A vs. D), ORs and confidence intervals for 8 of 12 risk alleles were >1. A similar pattern of ORs was seen when comparing neovascular AMD with non-neovascular pachychoroid (A vs. C). The pattern of ORs comparing pachychoroid neovasculopathy with non-neovascular pachychoroid (B vs. C) was similar in direction but smaller in magnitude. Comparison of non-neovascular pachychoroid with controls (C vs. D) showed ORs 1. In the comparison of neovascular AMD with pachychoroid neovasculopathy (A vs. B), the only SNP that showed a significant difference between the 2 groups was rs2274700 in CFH. With respect to the 12 SNPs tested, we found that the genetic profile of patients developing neovascularization in the context of pachychoroid disease resembles that of patients with neovascular AMD and that a number of risk alleles were found with greater frequency in the 2 neovascular cohorts than in non-neovascular pachychoroid or normal subjects. Risk alleles in ARMS2 and CFH were found with greater frequency in patients with pachychoroid neovasculopathy than in those with non-neovascular pachychoroid, suggesting a genetic basis for the phenotypic distinction between these entities. However, the implications of these differences at the tissue level are

EFFICACY OF ALIROCUMAB IN 1,191 PATIENTS WITH A WIDE SPECTRUM OF MUTATIONS IN GENES CAUSATIVE FOR FAMILIAL HYPERCHOLESTEROLEMIA

Methods: From 6 clinical trials of alirocumab (one Phase 2, five Phase 3), 1191 patients with elevated LDL-C and phenotypic FH (including 758 treated with alirocumab) were sequenced for mutations using the SEQPRO LIPO platform in LDL receptor (LDLR), apolipoprotein B (APOB), PCSK9 (PCSK9), LDL receptor adaptor protein 1 (LDLRAP1), and signal-transducing adaptor protein 1 (STAP1) genes. New mutations were confirmed by Sanger sequencing and MLPA analysis in case of large gene rearrangements in the original DNA samples.