Genetic and Metabolic Determinants of Elevated Lipoprotein (a) – KRINGLE2

Epidemiological (cohorts), biochemical (liquid chromatography tandem mass spectrometry), genetic (WES and GWAS) as well as cell culture techniques.

We have shown that apo(a) size is proportional to the relative reduction in Lp(a) induced by PCSK9 inhibitors (Blanchard 2022) and that the reduction in Lp(a) levels associated with the E2 isoform of apoliprotein E is not affected by the transition to a dysbetalipoproteinaemia phenotype of homozygous carriers of this isoform (Chemello 2022).

We also showed by genetically investigating a large family from Reunion Island that the hyper Lp(a) phenotype of this family members is causatively associated with an apo(a) allele containing 21 Kringle 4 domains as well as a deleterious combination of SNPs within the LPA locus, known to be associated with an increase in LPA gene expression. We also demonstrate that this specific allele is associated with an extreme genetic risk score for cardiovascular diseases (Coassin 2022).

We have shown that among four miRs candidates identified in silico by sequence complementarity, only miR 455-5p was able to lower LPA gene and apo(a) protein expression. This has been shown using a cellular clone over expressing human apo(a) cDNA. We have also generated hiPSCs from a samples obtained from an hyper Lp(a) patient from the family described above (Coassin 2022). Two clones have been successfully differentiated into hepatocyte like cells (HLCs). These cells are functional and secrete human apo(a) unlike those generated from a random individual with normal Lp(a) levels. This has been shown when the cells were grown in 2D as well as in 3D as organoids.

1. V Blanchard et al. (2022) “The size of apolipoprotein (a) is an independent determinant of the reduction in lipoprotein (a) induced by PCSK9 inhibitors.” Cardiovascular Research 118, 2103-2111.

2. S Coassin et al. (2022) “Genome-wide Characterization of a Highly Penetrant Form of Hyperlipoprotein(a)emia Associated with Genetically Elevated Cardiovascular Risk.” Circulation Genomic and Precision Medicine 15, e003489.

3. K Chemello et al. (2022) “Genetic and mechanistic insights into the modulation of circulating Lipoprotein (a) concentration by apolipoprotein E isoforms” Current Atherosclerosis Reports 24, 399-405.

4. Patent - EP21305421.6 filed by Inserm Transfert on 01 April 2021. Strategy of hepatic differentiation of iPS cells in 3D using Biomimesys.

One in five individuals displays elevated lipoprotein (a) [Lp(a)], a highly atherogenic lipoprotein resembling low-density lipoproteins (LDL). Pathophysiological, epidemiological and genetic studies demonstrate that when circulating Lp(a) levels are high (above 125 nmol/L), cardiovascular event rates sharply increase.
The major structural difference between Lp(a) and LDL is that Lp(a) contains a large signature protein, apolipoprotein (a) [apo(a)]. Apo(a) is extremely polymorphic in size as it contains 1 to more than 40 kringle-IV2 (KIV2) domains giving origin to more than 40 isoforms in humans. The size of apo(a) is inversely correlated with the circulating levels of Lp(a), but the exact molecular and metabolic pathways regulating Lp(a) plasma concentrations have not been clearly established yet. The goal of the present research project is to decipher these pathways.

(1) For instance, the molecular mechanisms governing Lp(a) production are poorly understood. Short RNAs (miRs) control gene expression and have been shown to modulate lipoproteins homeostasis. miRs work by inducing RNA silencing and thereby reduce target genes expression. We will investigate the influence of these regulatory elements on the expression of the gene encoding apo(a).
(2) In the population, Lp(a) levels can vary by up to 100-fold in carriers of identical apo(a) isoforms. We have recruited a large family in which several individuals present extremely high Lp(a) levels. We will determine the genetic causes on their apo(a) gene that are responsible for their extreme Lp(a) plasma concentrations leading to premature cardiovascular events.
(3) Lp(a) levels are resistant to lifestyle changes and lipid lowering drugs such as statins, which poses a real challenge for clinical management. A novel class of lipid lowering agents, the PCSK9 inhibitors induce a 30% reduction in circulating Lp(a) levels. We will investigate the mechanisms by which PCSK9 inhibitors modulate Lp(a) plasma levels and the influence of the size of apo(a) on the response of patients to these novel therapies.
(4) The gene encoding apolipoprotein E (apoE) is the only gene besides apo(a) and pcsk9 to have a significant association with circulating Lp(a) levels. Humans display three major apoE isoforms (e2/e3/e4) that differ by the presence of different amino acids at position 112 and 158. Carriers of the e2 allele display much lower Lp(a) than non-e2 carriers. We will study the pathway by which this particular apoE isoform lowers Lp(a) in humans.

Taken together these studies have profound implications in terms of deciphering the genetic and metabolic pathways regulating plasma Lp(a) levels, and pave the way to enhanced diagnosis and therapeutics approaches for patients at high risk of Lp(a)-induced cardiovascular diseases.