Archives of Biochemistry and Biophysics 696 (2020) 108655 Available online 29 October 2020 0003-9861/© 2020 Elsevier Inc. All rights reserved. Identifcation and characterization of a rare variant in apolipoprotein A-IV, p.(V336M), and evaluation of HDL functionality in a Greek cohort with extreme HDL cholesterol levels Angeliki Chroni a , Loukianos Rallidis b , Despoina Vassou c , Christina Gkolfnopoulou a , Paraskevi Papakosta f, g , Maria I. Zervou d , George N. Goulielmos d , Estela Kiouri b , Danae Pappa e , Elias Eliopoulos e , Dimitris Kardassis f, g, * a Institute of Biosciences and Applications, National Center for Scientifc Research “Demokritos”, Agia Paraskevi, Athens, Greece b Second Department of Cardiology, “Attikon” Hospital, and School of Medicine, National and Kapodistrian University of Athens, Athens, Greece c Genomics Facility, Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece d Section of Molecular Pathology and Human Genetics, Department of Internal Medicine, School of Medicine, University of Crete, Heraklion, Greece e Laboratory of Genetics, Department of Biotechnology, Agricultural University of Athens, Athens, Greece f Department of Basic Medical Sciences, University of Crete Medical School, Heraklion, Greece g Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece A R T I C L E INFO Keywords: High density lipoprotein Single nucleotide polymorphisms HDL functionality Apolipoprotein A-IV Coronary artery disease Genetic heterogeneity Mutations ABSTRACT High-Density Lipoprotein cholesterol (HDL-C) levels do not correlate well with Coronary Artery Disease (CAD) risk, while HDL functionality affects atherogenesis and is a better prognostic marker for CAD. Often, the extreme HDL-C levels have a multigenic origin. Here, we searched for single-nucleotide polymorphisms (SNPs) in ten genes of HDL metabolism in a Greek cohort with very low (<10th percentile, n = 13) or very high (>90th percentile, n = 21) HDL-C. We also evaluated the association between HDL-C levels, HDL functionality (anti- oxidant capacity) and CAD in the subjects of this cohort. Individuals with low HDL-C levels had higher tri- glyceride levels, lower apoA-I levels, decreased HDL anti-oxidant capacity and higher incidence of CAD compared with individuals with control or high HDL-C levels. With next generation sequencing we identifed 18 exonic SNPs in 6 genes of HDL metabolism and for selected amino acid changes we performed computer-aided structural analysis and modeling. A previously uncharacterized rare apolipoprotein A-IV variant, apoA-IV [V336M], present in a subject with low HDL-C (14 mg/dL) and CAD, was expressed in recombinant form and structurally and functionally characterized. ApoA-IV [V336M] had similar α-helical content to WT apoA-IV but displayed a small thermodynamic stabilization by chemical unfolding analysis. ApoA-IV [V336M] was able to associate with phospholipids but presented reduced kinetics compared to WT apoA-IV. Overall, we identifed a rare apoA-IV variant in a subject with low HDL levels and CAD with altered biophysical and phospholipid binding properties and showed that subjects with very low HDL-C presented with HDL dysfunction and higher incidence of CAD in a Greek cohort. 1. Introduction There is strong evidence from numerous epidemiological studies that the plasma levels of high-density lipoprotein cholesterol (HDL-C) are inversely associated with the risk for cardiovascular disease (CVD) which is the leading cause of death in Western societies [1]. Further- more, low levels of HDL-C are frequently found in patients with meta- bolic diseases such as type 2 diabetes (T2D) and metabolic syndrome, or chronic infammatory diseases, and these clinical phenotypes are major risk factors for CVD [2]. The molecular and genetic basis of extreme HDL-C phenotypes in humans is still poorly understood. Mutations in the three key genes of HDL metabolism, apolipoprotein A-I (APOA1), lecithin-cholesterol acyltransferase (LCAT) and ATP-binding cassette transporter A1 (ABCA1), are rare and explain the extreme HDL-C levels in only a few percent of the individuals in the general population [3–5]. Furthermore, * Corresponding author. Department of Basic Medical Sciences, University of Crete Medical School, Heraklion, Greece. E-mail address: kardasis@imbb.forth.gr (D. Kardassis). Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi https://doi.org/10.1016/j.abb.2020.108655 Received 2 September 2020; Received in revised form 20 October 2020; Accepted 24 October 2020