Familial hypercholesterolaemia (FH) is characterized by increased circulating low-density lipoprotein (LDL) cholesterol leading to premature atherosclerosis and coronary heart disease. Although FH is usually caused by mutations in LDLR, mutations in APOB and PCSK9 also cause FH but only a few mutations have been reported, APOB p.R3527Q being the most common. However, 30–80% of clinical FH patients do not present an identifiable mutation in any of the described genes. To identify the genetic cause of the hypercholesterolaemia in 65 patients without mutations in LDLR, PCSK9 or in fragments of exon 26 and 29 of APOB currently analysed, we performed whole sequencing of APOB by pyrosequencing. A total of 10 putative mutations in APOB were identified. Flow cytometry with fluorescently labelled LDL from patients and relatives showed that p.Arg1164Thr (exon 22) and p.Gln4494del (exon 29) presented a 40% decrease in internalization in lymphocytes and HepG2 cells, very similar to APOB3527. The proliferation assays with U937 cells showed reduced growth for both cases. The variant p.Tyr1247Cys was found to be neutral and other three alterations were considered polymorphisms. Our results emphasize the need to study the whole APOB in routine protocols to improve patient identification and cardiovascular risk assessment.

Objective: Homozygous familial hypercholesterolemia is a rare disease usually caused by LDLR (low-density lipoprotein receptor) mutations. Homozygous familial hypercholesterolemia is characterized by markedly elevated LDL-C (low-density lipoprotein cholesterol) levels and an extremely high risk of premature atherosclerotic cardiovascular disease. A phase 2, proof-of-concept study (NCT02265952) demonstrated that evinacumab, a fully human monoclonal antibody to ANGPTL3 (angiopoietin-like 3 protein), reduced LDL-C levels in 9 patients with genotypically confirmed homozygous familial hypercholesterolemia and was well tolerated. The aim of this study was to analyze the effects of evinacumab on LDLR activity in lymphocytes purified from patients in the proof-of-concept study. Approach and Results: LDLR activity was assessed in patient lymphocytes before and after treatment with evinacumab and versus lymphocytes carrying wild-type LDLR, and also in an LDLR-defective Chinese hamster ovary cell line (CHO- ldl A7) transfected with plasmids encoding the LDLR variants. Overall mean peak reduction in LDL-C with evinacumab was −58±18%, occurring between Week 4 and Week 12. Mutations identified in the 9 patients were shown to be pathogenic, with loss of LDLR activity versus wild type. Two of the LDLR variants, p.(Cys681*) and p.(Ala627Profs*38), were class 2 type mutations that are retained in the endoplasmic reticulum. Six variants were class 3 type mutations with impaired LDL-C binding activity: p.(Trp87Gly), occurring in 2 patients, p.(Gln254Pro), p.(Ser177Leu), p.(Gly335Val), and p.(Ser306Leu). Evinacumab had no effect on LDLR activity. Conclusions: These results suggest that evinacumab is effective for lowering LDL-C in patients with homozygous familial hypercholesterolemia, and the inhibition of ANGPTL3 in humans lowers LDL-C in a mechanism independent of the LDLR.