Genetic associations with carotid intima-media thickness link to atherosclerosis with sex-specific effects in sub-Saharan Africans

Atherosclerosis precedes the onset of clinical manifestations of cardiovascular diseases (CVDs). We used carotid intima-media thickness (cIMT) to investigate genetic susceptibility to atherosclerosis in 7894 unrelated adults (3963 women, 3931 men; 40 to 60 years) resident in four sub-Saharan African countries. cIMT was measured by ultrasound and genotyping was performed on the H3Africa SNP Array. Two new African-specific genome-wide significant loci for mean-max cIMT, SIRPA (p = 4.7E-08), and FBXL17 (p = 2.5E-08), were identified. Sex-stratified analysis revealed associations with one male-specific locus, SNX29 (p = 6.3E-09), and two female-specific loci, LARP6 (p = 2.4E-09) and PROK1 (p = 1.0E-08). We replicate previous cIMT associations with different lead SNPs in linkage disequilibrium with SNPs primarily identified in European populations. Our study find significant enrichment for genes involved in oestrogen response from female-specific signals. The genes identified show biological relevance to atherosclerosis and/or CVDs, sex-differences and transferability of signals from non-African studies.

Supplementary Note 1

F-box and leucine rich repeat protein 17 (FBXL17)
Our study identified rs552690895 (p = 2.5E-08) in FBXL17 in the combined set. F-box and leucine rich repeat protein 17 (FBXL17) is characterized by an approximately 40-amino acid F-box motif. SCF complexes are formed by SKP1 (S-phase kinase-associated protein 1), cullin (CUL1), and F-box proteins, and act as protein-ubiquitin ligases. F-box proteins interact with SKP1 through the F-box, and they interact with ubiquitination targets through other protein interaction domains (1). Evidence suggest that the SCF is a key complex in the ubiquitineproteasome system (UPS) that is involved in 70.0-90.0% of protein degradation processes (2). It has been found that protein degradation by the UPS play a central role in cardiovascular physiology and disease: from endothelial function, the cell cycle, atherosclerosis, myocardial ischaemia, cardiac hypertrophy, inherited cardiomyopathies, and heart failure (3)(4)(5)(6)(7). A GWAS in Lithuanian families found that variants in FBXL17 were associated with coronary heart diseases (8). Other reports on genetic associations for FBXL17 include studies for educational attainment and mathematical ability (9), intelligence (10), and pulse pressure (11).

Signal regulatory protein alpha (SIRPA)
Our combined analysis identified also genetic association of rs6045318 (p = 4.7E-08) with cIMT in the SIRPA gene. Signal regulatory protein alpha (SIRPA) is a regulatory membrane glycoprotein from the SIRP family, which inhibits the cytoskeleton-intensive process of phagocytosis by the macrophage. SIRPA is activate cancer cells (through high expression of CD47) and upregulated SIRPA inhibits macrophage-mediated destruction. This mediation of phagocytosis and polarization of macrophages is important in the pathophysiology of atherosclerosis (12). There is evidence that SIRPA is involved in discrete stages of cardiovascular cell lineage differentiation (13) and that defects in the gene (knock out) reduces atherosclerosis in mice (14). SIRPA expression has been found as a signature of inflamed atherosclerotic plaque (15). Previous reports on GWAS studies associate SIRPA with blood protein (16,17), and the percentage of basophil in granulocytes (18).

Sorting nexin 29 (SNX29)
On the chromosome 16, rs147978408 (p = 6.3E-09) was the top cIMT associated variant in SNX29 for the male-specific analysis. The sorting nexin (SNX) family is a diverse group of cytoplasmic-and membrane-associated phosphoinositide-binding proteins that play pivotal roles in the regulation of protein trafficking. SNX gene variants are associated with CVDs, and dysfunction of the SNX pathway is involved in several forms of cardiovascular disease (CVD) (19). In a study of genes that regulate smooth muscle cell differentiation and disease risk, SNX29 was involved in pathways for occlusion of blood vessels and atherosclerosis (20). Ito and collaborators identified sex-dependent differentially methylated regions close to SNX29 in mouse liver and found that this methylation status was influenced by testosterone and contributed to sex-dimorphic chromatin decondensation (21). This might explain the sex-specific effect observed in our study. A study in children with sickle cell disease, identified SNX29 variants as suggestive of association with systolic blood pressure (22). Also, variants in SNX29 were found in suggestive association with subcutaneous adipose tissue in women (Sung et al. 2016). In patients with pulmonary arterial hypertension, SNX29 variation was reported for differential responses to vasodilator treatment (24). Further GWAS analysis stratified by hypertensive status showed that the association was driven by the hypertensive group (effect three times higher in hypertensives compared to the nonhypertensives), therefore demonstrating that the association of SNX29 with cIMT might be mediated by the vascular remodeling caused by hypertension. GWA studies reported SNX29 variants for association with educational attainment, mathematical ability and cognitive function measurement (Lee et al. 2018), intelligence (10,25), bone mineral density (26), and smoking (Liu et al. 2019).

Mitogen-activated protein kinase kinase kinase 7 (MAP3K7)
In the male-specific analysis, we found rs284509 (p=5.3E-08) in MAP3K7 region on chromosome 6 to be associated with cIMT. Mitogen-activated protein kinase kinase kinase 7 (MAP3K7) also called TAK1 encodes a serine/threonine protein kinase family member, with a central role as regulator of cell death. Because of its role in kinase pathway, and regulation of transforming growth factor beta (TGF-b), MAP3K7 plays a role in growth inhibition in vascular smooth muscle cells and can be atheroprotective or atherogenic (28). More biological evidence of the contribution of MAP3K7 to atherosclerosis is through its regulation by micro-RNAs (29,30). In a study of women receiving hormone replacement therapy, variants in MAP4K4, a gene targeting MAP3K7 (31) were associated to cIMT (32). The sex-specific association observed might be related to the fact that MAP3K7IP3 (located on the X chromosome), which is known to form a ternary complex with MAP3K7 in response to inflammatory stimuli, has shown sex-differential expression in ischemic stroke (33,34). In a study on expression of androgen-modulated micro-RNAs, it has been reported that MAP3K7 was a target of mmu-miR-467h and mmu-miR-669i in the angiogenesis and transforming growth factor beta receptor signalling pathways (35). Despite biological relevance to atherosclerosis, GWA studies reported variants in gene region to be associated with cancer progression (36), anti-TNF response in rheumatoid arthritis (37), attention deficit hyperactivity disorder (38), adolescent idiopathic scoliosis , and sporadic amyotrophic lateral sclerosis (Xie et al. 2014). Our study is the first to report MAP3K7 association with a CVD phenotype.

La-related protein 6 (LARP6)
LARP6 (La-related protein 6) is a ribonucleoprotein domain family member 6. Studies showed that it has a role in collagen regulation by targeting mRNA encoding Type I collagen Sukhanov and collaborators found that IGF1R deficiency downregulated collagen mRNA-binding protein LARP6 and vascular collagen, and showed an atheroprotective effect (45,46). Collagen is a hallmark of atherosclerotic plaque stability, thus alteration of the collagen balance may lead to an instability of atherosclerotic lesions, and therefore promote plaque formation and rupture (15,47). In the Taiwanese population, the LARP6 locus was found to be associated with coronary artery disease (48). In European ancestry populations, LARP6 was found associated with insulin measurement (49). However, Mendelian randomization for cIMT found that despite the limited effects of proinsulin-increasing SNP scores on cIMT, proinsulin was unlikely to have causal effects on cIMT (50). Myocardial gene expression in non-ischemic human heart failure found LARP6 to be differentially expressed between men and women (1.36 fold) (51). The female-specific effect of the loci may find its explanation in the enhancer function of rs78172571 in high LD with rs150840489 (the top SNP associated in our female-specific) on THAP10 gene (FDR = 2.03E-17) known to be regulated by oestrogen.

Prokineticin 1 (PROK1)
Prokineticin 1 (PROK1), also called endocrine gland derived vascular endothelial growth factor (EG-VEGF), is a specific placental angiogenic factor which play a role in the control of normal (e.g endometrial decidualization) and pathological placental angiogenesis (52). It is involved in pathologies such as recurrent pregnancy loss, gestational trophoblastic diseases, foetal growth restriction, and preeclampsia (53)(54)(55)(56)(57). The gene is known to be predominantly expressed in the steroidogenic glands, such as ovary, testis, and adrenal cortex, and is often complementary to the expression of vascular endothelial growth factor (VEGF), suggesting that these molecules function in a coordinated manner. The function and particular pattern of this gene's activity might explain why we identified the locus only in our female-specific analysis. Our study is the first to report PROK1 for any trait in a GWAS.

Caldesmon 1 (CALD1)
Our gene-based analysis identified caldesmon 1 significantly associated with cIMT in our combined set led by rs7781307 (p = 2.1E-06) on 7q33. Caldesmon 1 is calmodulin binding protein encoding for a calmodulin-and actin-binding protein that play a major role in the regulation of smooth muscle contraction, cell migration and cell invasion (58). CALD1 was identified as key gene in the "regulation of actin cytoskeleton" module from protein-protein interaction network resulting from a bioinformatics analysis of key pathways and genes in advanced coronary atherosclerosis (59). A study screening for keys genes for abdominal aortic aneurysm found that CALD1 was leading a KEGG enrichment signal pathways (Vascular smooth muscle contraction) of differential expressed genes (DEGs) (60). Underexpression of CALD1 was found to be a key feature of calcification of vascular smooth muscle cells from atherosclerotic plaque (15,61,62). Additionally, studies on epigenetic modifications reported CALD1 to exhibit differential methylation in atherosclerosis (63)(64)(65) FLT4 or Vascular endothelial growth factor receptor 3 (VEGFR3) is a major signalling protein involved in angiogenesis, vasculogenesis and maintenance of the endothelium. By acting as receptor to VEGFC and VEGFD, it plays an essential role in lymphangiogenesis in adults and in the development cardiovascular system during embryonic phase. Defect and/or downregulation of VEGR3 was found to lead to cardiovascular failure in embryonic stage and to higher mortality after myocardial infarction in mice models (70,71). Biological studies have highlighted the role of FLT4 in atherosclerosis in major pathological processes. The gene has been reported to be involved in plaque instability by two process: the mediation of monocytes/macrophages apoptosis and consequently alteration plaque stability (72) ; and the modulation of vascular remodelling and shear stress resulting in plaques haemorrhages and calcification in carotids (73)(74)(75). Our study is the first to report association of FLT4 locus (rs112967731, p = 5.7E-07, female-specific) with cIMT or any cardiovascular phenotype in GWAS studies. Previous studies reported the locus for association with folic acid measurement (76) and blood protein measurements (16,17).