Identificación de variantes genéticas asociadas a hipercolesterolemia familiar en niños y adolescentes de la Región del Biobío, Chile / Identification of genetic variants associated with familial hypercholesterolemia in Chilean children and adolescents

Background: Familial hypercholesterolemia (FH) is commonly associated with mutations in-LDL receptor (LDLR), apolipoprotein B (APOB) and proprotein convertase subtilisin/kexin type 9 (PCSK9). Aim: To identify genetic variants associated with FH in a population of children and adolescents with hypercholesterolemia or a family history of-demonstrated early CVD. Material and Methods: Clinical and biochemical parameters were evaluated, and nine genes related to FH were sequenced namely LDLR, APOB, PCSK9, LDLRAP1, LIPA, APOE, ABCG5, ABCG8 and STAP1, in 55 children and adolescents aged 1 to 18 years old, from non-consanguineous families. Results: Mutations associated with FH were found in 17 children and adolescents, corresponding to p.Asp47Asn, duplication of exons 13-15 and p.Ser326Cys of the LDLR gene; p.Glu204* and Ile268Met of the APOE gene. Thirteen patients were heterozygous, two homozygous, two compound heterozygous, and one double heterozygous. Conclusions: Children and adolescents carrying mutations associated with FH were found by selective screening, which constitutes the first stage in the identification of genetic variants in our country.

Phenotypic characterization and predictive analysis of p.Asp47Asn LDL receptor mutation associated with Familial Hypercholesterolemia in a Chilean population.

BACKGROUND: Familial hypercholesterolemia (FH) is an inherited disorder mainly caused by mutations in the LDL receptor (LDL-R) and characterized by elevation of low-density lipoprotein cholesterol (LDL-C) levels and premature cardiovascular disease. OBJECTIVE: In this study, we evaluated the clinical phenotype of the p.Asp47Asn, described as an uncertain pathogenic variant, and its effect on the structure of LDL-R and ligand interactions with apolipoproteins. METHODS: 27 children and adolescents with suspected FH diagnosis were recruited from a pediatric endocrinology outpatient clinic. Blood samples were collected after 12 h fasting for lipid profile analysis. DNA sequencing was performed for six FH-related genes by Ion Torrent PGM platform and copy number variation by MLPA. For index cases, a familial cascade screening was done restricted to the same mutation found in the index case. In silico analysis were developed to evaluate the binding capacity of LDL-R to apolipoproteins B100 and E. RESULTS: Lipid profile in children and adolescents demonstrated higher LDL-C levels in p.Asp47Asn carriers compared to the wild type genotype. In silico analysis predicted a reduction in the binding capacity of the ligand-binding modules LA1-2 of p.Asp47Asn LDL-R for ApoB100 and ApoE, which was not produced by local structural changes or folding defects but as a consequence of a decreased apparent affinity for both apolipoproteins. CONCLUSION: The clinical phenotype and the structural effects of p.Asp47Asn LDL-R mutation suggest that this variant associates to FH.

[Identification of genetic variants associated with familial hypercholesterolemia in Chilean children and adolescents]. / Identificación de variantes genéticas asociadas a hipercolesterolemia familiar en niños y adolescentes de la Región del Biobío, Chile.

BACKGROUND: Familial hypercholesterolemia (FH) is commonly associated with mutations in-LDL receptor (LDLR), apolipoprotein B (APOB) and proprotein convertase subtilisin/kexin type 9 (PCSK9). AIM: To identify genetic variants associated with FH in a population of children and adolescents with hypercholesterolemia or a family history of-demonstrated early CVD. MATERIAL AND METHODS: Clinical and biochemical parameters were evaluated, and nine genes related to FH were sequenced namely LDLR, APOB, PCSK9, LDLRAP1, LIPA, APOE, ABCG5, ABCG8 and STAP1, in 55 children and adolescents aged 1 to 18 years old, from non-consanguineous families. RESULTS: Mutations associated with FH were found in 17 children and adolescents, corresponding to p.Asp47Asn, duplication of exons 13-15 and p.Ser326Cys of the LDLR gene; p.Glu204* and Ile268Met of the APOE gene. Thirteen patients were heterozygous, two homozygous, two compound heterozygous, and one double heterozygous. CONCLUSIONS: Children and adolescents carrying mutations associated with FH were found by selective screening, which constitutes the first stage in the identification of genetic variants in our country.

Pharmacological inhibition of the F1 -ATPase/P2Y1 pathway suppresses the effect of apolipoprotein A1 on endothelial nitric oxide synthesis and vasorelaxation.

AIM: The contribution of apolipoprotein A1 (APOA1), the major apolipoprotein of high-density lipoprotein (HDL), to endothelium-dependent vasodilatation is unclear, and there is little information regarding endothelial receptors involved in this effect. Ecto-F1 -ATPase is a receptor for APOA1, and its activity in endothelial cells is coupled to adenosine diphosphate (ADP)-sensitive P2Y receptors (P2Y ADP receptors). Ecto-F1 -ATPase is involved in APOA1-mediated cell proliferation and HDL transcytosis. Here, we investigated the effect of lipid-free APOA1 and the involvement of ecto-F1 -ATPase and P2Y ADP receptors on nitric oxide (NO) synthesis and the regulation of vascular tone. METHOD: Nitric oxide synthesis was assessed in human endothelial cells from umbilical veins (HUVECs) and isolated mouse aortas. Changes in vascular tone were evaluated by isometric force measurements in isolated human umbilical and placental veins and by assessing femoral artery blood flow in conscious mice. RESULTS: Physiological concentrations of lipid-free APOA1 enhanced endothelial NO synthesis, which was abolished by inhibitors of endothelial nitric oxide synthase (eNOS) and of the ecto-F1 -ATPase/P2Y1 axis. Accordingly, APOA1 inhibited vasoconstriction induced by thromboxane A2 receptor agonist and increased femoral artery blood flow in mice. These effects were blunted by inhibitors of eNOS, ecto-F1 -ATPase and P2Y1 receptor. CONCLUSIONS: Using a pharmacological approach, we thus found that APOA1 promotes endothelial NO production and thereby controls vascular tone in a process that requires activation of the ecto-F1 -ATPase/P2Y1 pathway by APOA1. Pharmacological targeting of this pathway with respect to vascular diseases should be explored.

Apolipoprotein A-I enhances proliferation of human endothelial progenitor cells and promotes angiogenesis through the cell surface ATP synthase.

BACKGROUND: Human endothelial progenitor cells (hEPC) correspond to a subtype of stem cells which, in the presence of angiogenic stimuli, can be mobilized from bone marrow to circulation and then recruited to the damaged endothelium, where they differentiate into mature endothelial cells. High-density lipoproteins (HDL) increase the level and functionality (proliferation, migration, differentiation, angiogenesis capacity) of circulating hEPC; however, the contribution of receptors for HDL and/or apolipoprotein A-I (apoA-I), the main HDL apolipoprotein, in these effects is still unclear. On mature endothelial cells, the cell surface F1-ATP synthase has been previously characterized as a high affinity receptor of apoA-I, whereas the scavenger receptor SR-BI mainly binds with fully lipidated HDL and displays a poor affinity for lipid-free apoA-I. Furthermore, it was shown that apoA-I binding to surface ATP synthase on mature endothelial cells promotes cell proliferation, whereas inhibits apoptosis. In this work, we aimed to determine the effect of apoA-I in the proliferation and the angiogenic capacity of early hEPC, and the contribution of the cell surface ATP synthase in these events. RESULTS: We first evidenced that early hEPC express the ATP synthase at the surface of nonpermeabilized cells, where it is not colocalized with MitoTracker, a mitochondria marker. ApoA-I (50 µg/mL) increases hEPC proliferation (+14.5%, p<0.001) and potentiates the effect of hEPC on a cellular model of angiogenesis, with an increase of +31% (p<0.01) in branch point counting and in tubule length. These effects of apoA-I were totally reversed in the presence of ATP synthase inhibitors, such as IF1 or oligomycin, whereas the inhibition of the HDL receptor, SR-BI, partially inhibits these events. CONCLUSIONS: These results provide the first evidence that surface ATP synthase is expressed on early hEPC, where it mediates apoA-I effects in hEPC proliferation and in angiogenesis. This knowledge could be helpful for future investigations focused on the regulation of the number and functionality of these cells and in the development of new therapies for the treatment of diseases, such as cardiovascular disease.