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