Genetic variants associated with cardiovascular diseases and related risk factors highlight novel potential therapeutic approaches

Despite significant progress in diagnosis, medical treatment and drug development, cardiovascular diseases (CVD) remain a leading cause of death. Atherosclerosis, the main driver of most CVD, develops in response to the biologic effects of underlying risk factors, including dyslipidemia, type 2 diabetes (T2D), obesity and non-alcoholic liver disease (NAFLD) [1, 2]. Current lipid-lowering therapies cannot eliminate the cardiovascular risk, likely due to lack of influence or detrimental effects on major risk factors beyond low-density lipoprotein cholesterol (LDL-C) [3, 4*]. Hence, identification of new genes or pathways that can be targeted for the treatment of atherosclerosis without increasing, and preferably while decreasing other metabolic risk factors, is of great significance in the effort to reduce CVD burden. Here, we discuss novel potential therapeutic approaches identified through large data-driven genetic discovery efforts combined with follow-up functional analyses aimed at translating genetic discoveries into new biological insights.

Genome-wide association studies (GWAS) screen the genome for associations between genetic variants and biological traits or diseases. Over the past decade, GWAS uncovered more than 150 genetic risk loci for coronary artery disease (CAD) [5], and over 300 loci associated with plasma lipid traits [6]. Nevertheless, the molecular and physiological functions of many of these genetic variants as well as their potential as therapeutic targets remain unclear. We recently reported findings of GWAS aimed at identifying novel potential therapeutic targets that may reduce the risk of CVD without increasing the risk of liver disease, diabetes, or other metabolic disorders [7**]. Analysis of liver-related blood traits in approximately 70,000 participants of the Nord-Trøndelag Health Study revealed 9 variants predicted to result in loss-of-function of the protein that were associated with one or more liver-related blood traits. Among these, ZNF529:p.K405X (rs1376217616) was found to be associated with decreased LDL-C without being associated with elevated liver enzymes or non-fasting glucose. In follow-up functional studies, we found that silencing of ZNF529 in human hepatoma cells led to enhanced LDL uptake via induction of the LDL receptor (LDLR). These studies indicate a therapeutic potential for lowering LDL-C by inhibition of ZNF529, which may reduce the cardiovascular risk without increasing other metabolic risk factors. Elucidating the mechanisms by which ZNF529 regulates hepatic LDLR to lower LDL-C without causing liver damage or hyperglycemia warrants further research. However, the use of animal models for such studies is limited as ZNF529 does not have a homolog in rodents.

Recent GWAS followed by comprehensive functional studies utilizing novel mouse models uncovered a potential therapeutic target for hypercholesterolemia and atherosclerosis. A common noncoding variant (rs1997243, 11 kb upstream of G protein-coupled receptor 146, GPR146) was found to be associated with hypercholesterolemia and had strong linkage disequilibrium with a coding variant of GPR146 (rs11761941: GPR146:p.Gly11Glu) [6, 8**, 9]. By studying mice with whole-body, liver- and adipose tissue-specific deficiency of GPR146, Yu et al. [8**] elucidated the mechanisms by which GPR146 regulates lipid metabolism. GPR146 was found to induce hepatic sterol regulatory element binding protein 2 (SREBP2) via activation of the extracellular signal-regulated kinase (ERK) signaling pathway, leading to increased very low-density lipoprotein (VLDL) secretion, thus regulating circulating LDL-C and triglyceride levels. Indeed, GPR146 deficiency was found to reduce both plasma cholesterol and triglycerides in wild-type mice. Studies in Ldlr knockout mice showed that loss of GPR146 reduces hypercholesterolemia and atherosclerosis independent of LDLR activity. Han et al. [9], confirmed reduced circulating cholesterol in Gpr146 knockout mice and provided further genetic evidence that GPR146 regulates cholesterol levels also in humans. Altogether, these intriguing findings indicate a therapeutic potential of GPR146 inhibition for the treatment of dyslipidemia and CVD. Further research is warranted to identify agonist and antagonist ligands of GPR146. Moreover, considering that GPR146 was found to regulate hepatic VLDL secretion, the impact of GPR146 inhibition on other metabolic disorders, particularly NAFLD, should be investigated.

GWAS identified variants near the gene ILRUN (inflammation and lipid regulator with ubiquitin-associated-like and NBR1-like domains) at chromosome 6p21 to be associated with plasma lipid traits and CAD [5, 10**]. Recently, Bi et al [10**] identified ILRUN as a novel regulator of lipid and lipoprotein metabolism. The newly generated Ilrun knockout mice presented lowered circulating cholesterol largely due to decreased high-density lipoprotein cholesterol (HDL-C) via attenuation of hepatic apolipoprotein A-I production and subsequent HDL formation. Loss of Ilrun was also found to lower plasma non-HDL-C by reducing hepatic VLDL production. Furthermore, Ilrun knockout mice showed increased expression of hepatic proliferator-activated receptor alpha (PPARα) and ILRUN was found to interact with nuclear PPARα via its ubiquitin-associated-like domain. Interestingly, when challenged with Western-type diet, Ilrun knockout mice showed reduced body weight, liver/body weight ratio, hepatic cholesterol and triglycerides. However, Ilrun deficiency caused impaired glucose tolerance due to attenuated glucose-stimulated insulin secretion. The therapeutic potential of ILRUN inhibition for treating dyslipidemia, atherosclerosis and NAFLD warrants further research using appropriate animal models. Considering the observed glucose intolerance in Ilrun knockout mice, glucose homoeostasis should be carefully evaluated in such studies.

Beyond identifying new genes, GWAS may reveal novel metabolic pathways implicated in disease pathology. Accumulating evidence indicates lower circulating glycine as a common denominator in CVD and related metabolic disorders including dyslipidemia, T2D and NAFLD [11–15]. Wittemans et al. [13**] performed a meta-analysis of GWAS to investigate the causality and mechanisms of the association between glycine and cardiometabolic diseases. They identified 22 novel genetic loci associated with glycine levels and constructed genetic scores for glycine. Among 88,800 coronary heart disease (CHD) cases and 485,266 controls, glycine was found to be genetically associated with lower CHD risk. This was confirmed using observational analyses in 11,147 participants including 2,053 CHD cases. Beyond indicating a causal role for glycine in CVD, these studies suggest a therapeutic potential for glycine. We previously reported that among all amino acids, glycine has the most prominent anti-atherogenic effects attenuating macrophage foam cell formation [14]. Recently, we found that genes driving glycine biosynthesis are suppressed in humans and mice with NAFLD. We applied genetic and dietary approaches to limit glycine availability that resulted in exacerbated hyperlipidemia and NAFLD. We further explored glycine-based compounds as potential therapies for NAFLD and identified a tripeptide (Gly-Gly-L-Leu, DT-109) that lowered NAFLD symptoms by enhancing fatty acid oxidation and glutathione synthesis while modulating the gut microbiome [15**]. Overall, GWAS, observational and functional studies indicate a causal role and therapeutic potential for glycine in cardiometabolic diseases, the latter warrants further clinical evaluation.

FURTHER RECOMMENDED READING.

Additional papers of interest combining genetic discovery efforts with follow-up functional analyses that identified novel genes associated with CVD risk or circuiting lipid traits.

• Li Z, Votava JA, Zajac GJM, et al. Integrating Mouse and Human Genetic Data to Move beyond GWAS and Identify Causal Genes in Cholesterol Metabolism. Cell Metab. 2020;31:741–754.e5.

• Zhang YY, Fu ZY, Wei J, et al. A LIMA1 variant promotes low plasma LDL cholesterol and decreases intestinal cholesterol absorption. Science. 2018;360:1087–1092.

• Aherrahrou R, Guo L, Nagraj VP, et al. Genetic Regulation of Atherosclerosis-Relevant Phenotypes in Human Vascular Smooth Muscle Cells. Circ Res. 2020;127:1552–1565.