Familial hypercholesterolemia (FH) is the most common monogenic disease (1), with more than 90% of affected individuals carrying variants in the LDL receptor gene (LDLR, NM_000527) (2). A smaller percentage, approximately 5 to 8% of FH cases, results from variants in the apolipoprotein B-100 gene (APOB, NM_000384), (3) and less than 1% result from gain-of-function variants in the PCSK9 gene (PCSK9, NM_174936) (4). LDL receptors, located on the plasma membrane, mediate the clearance of LDL from the circulation. Apolipoprotein B-100 (apo B-100) serves as the primary ligand for LDL binding to its receptor. Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation of LDL receptors, thereby reducing their availability on the cell surface. Variants in the genes encoding LDLR, APOB, or PCSK9 lead to reduced LDL clearance and consequent hypercholesterolemia (5). The heterozygous form of FH (HeFH) affects approximately 35 million individuals worldwide (6). It predisposes to the development of early atherosclerosis and clinical manifestations of coronary artery disease (CAD), such as angina pectoris and myocardial infarction (5).

In individuals with FH, treatment with statins or other cholesterol-lowering agents, such as ezetimibe or PCSK9 inhibitors, reduces the incidence of CAD (7). Moderate-to high-intensity statin therapy has been associated with a 44% reduction in the risk of CAD and mortality in these patients (8). Nonetheless, reducing the residual cardiovascular risk and improving CAD prevention in FH remains a significant challenge. In this context, the anti-atherosclerotic properties of the HDL fraction offer an array of possibilities that have been scarcely explored.

HDL plays a pivotal role in cholesterol homeostasis, acting as a recipient of cholesterol from apo B-containing lipoproteins and facilitating the formation of cholesteryl esters, a more stable form of cholesterol. This esterification process, which occurs primarily in the HDL fraction, is catalyzed by the enzyme lecithin–cholesterol acyltransferase (LCAT), using apolipoprotein A-I (apo A-I)—the main apolipoprotein of HDL—as a cofactor (9). Cholesteryl ester transfer protein (CETP) mediates the bidirectional exchange of cholesteryl esters and triglycerides between HDL and apo B-containing lipoproteins (10) between the apo B-containing lipoproteins and HDL. Our group developed an in vitro assay to quantify the transfer of radiolabeled unesterified and esterified cholesterol from a protein-free lipid nanoemulsion, mimicking the structure of LDL, to the HDL fraction by incubating it with whole plasma. Using this method, we previously showed that reduced cholesterol transfer to HDL was associated with the presence of CAD or with established cardiovascular risk factors (11, 12, 13).

HDL also takes up cholesterol from peripheral cells for excretion into the bile via the so-called reverse cholesterol transport (RCT) pathway (14) . In vitro, studies have shown that reduced cholesterol efflux from macrophages to HDL is associated with atherogenesis, supporting the role of cholesterol transfer from both plasma lipoproteins and peripheral cells to HDL as a natural anti-atherogenic defense mechanism (15). In addition to its role in cholesterol metabolism, HDL exerts several other protective functions, including antioxidative, anti-inflammatory, vasodilatory, anti-apoptotic, and anti-thrombotic activities—largely attributed to its diverse protein cargo (16).

Building on these findings, our group previously reported that individuals with HeFH have reduced unesterified cholesterol (UC) transfer to HDL (17). This indicates that plasma LDL buildup might impair HDL metabolism and weaken its anti-atherogenic functions. Based on these observations, we hypothesize that impaired cholesterol transfer to HDL could contribute to the faster development of atherosclerosis seen in FH. Qualitative abnormalities in HDL particles have been described in these patients; as reviewed by Ganjali et al. (18) previous studies report triglyceride and sphingomyelin enrichment, decreased cholesterol efflux capacity, and impaired anti-inflammatory and antioxidant functions of HDL in FH.

Our study aims to expand this research by evaluating a contemporary, genetically confirmed HeFH cohort, previously treated with statins, in which subclinical CAD was assessed using coronary computed tomography, while simultaneously examining HDL functionality (including cholesterol transfer, antioxidant capacity, subfractions) as well as plasma levels of CETP, LCAT, and paraoxonase-1 (PON-1) activity. This approach aims to clarify whether HDL-related metrics are associated with subclinical CAD in HeFH.