Genetic functional studies of low density lipoprotein- cholesterol (LDL-C) associated variants and the genetic spectrum of familial hypercholesterolemia in different ethnic groups

Since the end of the 20th century, cardiovascular disease has been the major cause of death worldwide. Hypercholesterolemia is major risk factor for CVD. Family-based linkage analyses in patients with familial hypercholesterolemia (FH) have revealed the major loci that are involved in cholesterol hemostasis. These include low density lipoprotein receptor (LDLR), proprotein convertase subtilisin/kexin type-9 protein (PCSK9), and apolipoprotein B (APOB). However, these discovered genes only explain disease etiology in a proportion of patients with the clinical phenotype, and roughly 60% of patients who are diagnosed with FH are negative for any FH-causative mutation. This suggests that (1) there are undetected new mutations in known genes, (2) that disease-causing mutations might occur in unidentified gene(s), (3) disease may occur as a result of an accumulation of common small-effect LDL-C raising variants. My project has three main aims. First, the identification of mutations in patients clinically diagnosed with FH in two ethnic groups: UK (n=69) and Iran (n=16). Several novel mutations were identified in LDLR [UK: p.(Cys392Tyr), p.(Tyr553Ser) and p.(Tyr553*); Iran: p.(Leu479Gln) and p.(Glu668*)] and PCSK9 [p.(Arg357Cys)] using next generation sequencing technology. The second aim was to investigate the functional role explaining the Genome-Wide Association (GWAS) LDLR “hit” Single Nucleotide Polymorphism (SNP) rs6511720. This SNP and one other in strong linkage disequilibrium (LD) (rs57217136) were both found to act as a cis-regulatory element, where the sequence around the rare alleles of the SNPs is a target for proteins that enhance gene transcription. The third aim was studying the Annexin A2 (ANXA2) gene. AnxA2 recently was found to be involved in the LDL-R pathway through PCSK9. The study aimed to identify and determine the functional role of common SNPs that are associated with LDL-C. I showed that the ANXA2 common SNP rs17845226 (V98L) minor allele, was associated with significantly higher levels of LDL-C and a higher risk of CHD in a large prospective study of healthy UK men (NPHSII). The SNP shows strong LD with SNPs in the intragenic region, and I showed that the minor alleles of rs17191344 and rs11633032 are targets for proteins that repress gene transcription and the subsequent lower levels of AnxA2 protein means that there will be higher levels of PSCK9-mediated degradation of the LDL-R and this will lead to an increase in LDL-C levels. Finally, I identified that rs116928563 in the 3'UTR of ANXA2 was a potential site for a micro-RNA (miRNA-155*) binding, however, the study failed to result in any definite conclusions about the influence of miRNA-155 in ANXA2 expression and LDL-C levels. However, it gives insight into how a SNP also may affect gene expression at the post-transcription level by creating or destroying micro-RNA binding sites

Identifying low density lipoprotein cholesterol associated variants in the Annexin A2 (ANXA2) gene.

BACKGROUND AND AIMS: Annexin-A2 (AnxA2) is an endogenous inhibitor of proprotein convertase subtilisin/kexin type-9 (PCSK9). The repeat-one (R1) domain of AnxA2 binds to PCSK9, blocking its ability to promote degradation of low-density lipoprotein cholesterol-receptors (LDL-R) and thereby regulate low-density lipoprotein cholesterol (LDL-C) levels. Here we identify variants in ANXA2 influencing LDL-C levels and we determine the molecular mechanisms of their effects. RESULTS: The ANXA2 single nucleotide polymorphism (SNP) genotype-phenotype association was examined using the Second-Northwick-Park Heart Study (NPHSII) (n∼2700) and the UCL-LSHTM-Edinburgh-Bristol (UCLEB) consortium (n∼14,600). The ANXA2-R1 domain coding-SNP rs17845226 (V98L) associated with LDL-C, homozygotes for the minor allele having ≈18.8% higher levels of LDL-C (p = 0.004), and higher risk of coronary heart disease (CHD) (p = 0.04). The SNP is in modest linkage disequilibrium (r(2) > 0.5) with two intergenic SNPs, rs17191344 and rs11633032. Both SNPs showed allele-specific protein binding, and the minor alleles caused significant reduction in reporter gene expression (≈18%, p < 0.001). In the expression quantitative trait loci (eQTL) study, minor allele homozygotes have significantly lower levels of ANXA2-mRNA expression (p = 1.36 × 10(-05)). CONCLUSIONS: Both rs11633032 and rs17191344 SNPs are functional variants, where the minor alleles create repressor-binding protein sites for transcription factors that contribute to reduced ANXA2 gene expression. Lower AnxA2 levels could increase plasma levels of PCSK9 and thus increase LDL-C levels and risk of CHD. This supports, for the first time in humans, previous observations in mouse models that changes in the levels of AnxA2 directly influence plasma LDL-C levels, and thus implicate this protein as a potential therapeutic target for LDL-C lowering

Identification of the Functional Variant(s) that Explain the Low-Density Lipoprotein Receptor (<i>LDLR</i>) GWAS SNP rs6511720 Association with Lower LDL-C and Risk of CHD

<div><p>Background</p><p>The Low-Density Lipoprotein Receptor (<i>LDLR</i>) SNP rs6511720 (G>T), located in intron-1 of the gene, has been identified in genome-wide association studies (GWAS) as being associated with lower plasma levels of LDL-C and a lower risk of coronary heart disease (CHD). Whether or not rs6511720 is itself functional or a marker for a functional variant elsewhere in the gene is not known.</p><p>Methods</p><p>The association of <i>LDLR</i> SNP rs6511720 with incidence of CHD and levels of LDL-C was determined by reference to CARDIoGRAM, C4D and Global lipids genetics consortium (GLGC) data. SNP annotation databases were used to identify possible SNP function and prioritization. Luciferase reporter assays in the liver cell line Huh7 were used to measure the effect of variant genotype on gene expression. Electrophoretic Mobility Shift Assays (EMSAs) were used to identify the Transcription Factors (TFs) involved in gene expression regulation.</p><p>Results</p><p>The phenotype-genotype analysis showed that the rs6511720 minor allele is associated with lower level of LDL-C [beta = -0.2209, p = 3.85 x10^-262], and lower risk of CHD [log (OR) = 0.1155, p = 1.04 x10^-7]. Rs6511720 is in complete linkage. Rs6511720 is in complete linkage disequilibrium (LD) with three intron-1 SNPs (rs141787760, rs60173709, rs57217136). Luciferase reporter assays in Huh7 cells showed that the rare alleles of both rs6511720 and rs57217136 caused a significant increase in <i>LDLR</i> expression compared to the common alleles (+29% and +24%, respectively). Multiplex Competitor-EMSAs (MC-EMSA) identified that the transcription factor serum response element (SRE) binds to rs6511720, while retinoic acid receptor (RAR) and signal transducer and activator of transcription 1 (STAT1) bind to rs57217136.</p><p>Conclusion</p><p>Both <i>LDLR</i> rs6511720 and rs57217136 are functional variants. Both these minor alleles create enhancer-binding protein sites for TFs and may contribute to increased <i>LDLR</i> expression, which is consequently associated with reduced LDL-C levels and 12% lower CHD risk.</p></div

DNA binding properties of <i>LDLR</i> intron-1 SNPs.

<p>Conventional EMSA analysis of the <i>LDLR</i> intron-1 SNPs (rs6511720, rs141687760, rs60173709, and rs57217136). Binding of SREBP1 was used as the control (lane 1 and 2). The lanes with a labeled probe showed a specific band indicated by arrows, while when the unlabeled probe was added the band disappeared. These four SNPs have allele-specific binding, indicated by arrows. (-) = deletion and (*) = minor allele.</p

<i>LDLR</i> luciferase constructs and SNP luciferase activity in Huh7 cell line.

<p>A) Schematic presentation of LDLR<b>-</b>luciferase-construct (promoter only) and LDLR<b>-</b>luciferase-enhancer-constructs. The constructs were transfected into Huh7 cells. B) Results of luciferase reporter assays showing relative expression of LDLR-luciferase-enhancer constructs of <i>LDLR</i> SNPs relative to the LDLR-luciferase (no enhancer) construct. (-) = deletion and (*) = minor allele.</p

Genome-wide maps of chromatin state of LDLR intron-1.

<p>Schematic presentation of the LDLR intron-1 chromatin status (<a href="https://genome-euro.ucsc.edu" target="_blank">https://genome-euro.ucsc.edu</a>). The area of interest in intron-1 is highlighted in light-blue color. Promoter/ Enhancer histone marker of seven cell lines (GM12878, H1-hESC, HSMM, HUVEC, K562, NHEK, and NHLF). FAIRE: formaldehyde assisted isolation of regulatory elements.</p