Alopecia areata is a common hair loss disease characterized by patchy, non-scarring hair loss [18]. The etiology and pathogenesis of AA are unclear, and it is an autoimmune disease with a combination of genetic, immune and environmental factors. AA has a strong genetic basis, and familial AA cases have a poorer prognosis compared to disseminated cases, as shown by a tendency for frequent disease recurrence and resistance to treatment [19–20]. Genetic findings suggest that AA has a polygenic structure, and some studies have shown associations between AA and genes; the genes that have been identified are mainly immune-related genes, some of which are also involved in hair-related and other genes [21–22]. Research on the genetic aspects of AA is still insufficient and further studies are needed to improve treatment and prognosis. We first merged the GSE45512, GSE58573, GSE68801, and GSE74761 datasets and screened for AA associated differential genes and obtained 852 DEGs including 429 up-regulated and 423 down-regulated DEGs. Then, Mendelian randomization study was conducted using the eQTL database as an exposure factor and the AA GWAS data as an outcome factor, yielding 182 candidate genes associated with AA, including 81 high-risk candidate genes and 101 low-risk candidate genes. The DEGs were taken and intersected with the candidate genes to obtain four causal genes, including two up-regulated genes, GIMAP6 and ALOX15, and two down-regulated genes, GALNT6 and HEG1. Mendelian randomization analyses of these four causal genes and AA yielded the results of GIMAP6 and AA (IVW OR: 1.868; 95% CI: 1.175–2.970, p = 0.008), ALOX15 and AA (IVW OR: 1.616; 95% CI: 1.167–2.239, p = 0.004), GALNT6 and AA (IVW OR: 0.710; 95% CI: 0.533–0.945, p = 0.019), and HEG1 and AA (IVW OR: 0.648; 95% CI: 0.431–0.975, p = 0.037). Subsequently, we performed a sensitivity test on the results obtained by Mendelian randomization, which illustrated the reliability of the results of the Mendelian randomization analyses of the four causal genes with AA in this study by heterogeneity analysis and pleiotropy analysis. The causal genes were analyzed by GO enrichment and were mainly clustered in phosphatidylethanolamine biosynthetic process, endothelial cell morphogenesis, lipoxygenase pathway, ventricular trabecula myocardium morphogenesis, pericardium development, engulfment of apoptotic cell, and acetylgalactosaminyltransferase activity. The above findings suggest the involvement of lipoxygenases, bioactive peptides, endothelial cell development, and apoptosis in the pathogenesis of AA, which is consistent with previous studies [23–26]. KEGG pathway analysis revealed that the four causal genes were significantly enriched in the pathways of linoleic acid, ferroptosis, arachidonic acid metabolism, and adhesion junction, suggesting that these signaling pathways may play important roles in AA. It has been shown that linoleic acid induces the growth of human hair follicle dermal papilla cells by activating Wnt/β-catenin signaling [27]. Linoleic acid signaling pathway is likely to be an important research direction for the prevention and treatment of AA. Next, we conducted GSEA enrichment analysis on four causal genes, revealing that the high expression groups of genes GIMAP6 and ALOX15 are active in cytokine receptor interactions, intestinal immune network for IgA production, and hematopoietic cell lineage, while the low expression groups of genes GALNT6 and HEG1 are active in the hematopoietic cell lineage and intestinal immune network for IgA production. Suggests that AA may be related to hematopoietic cells and intestinal immunity. Arachidonic acid 15-lipoxygenase (ALOX15), a member of the lipoxygenase family of enzymes, and lipoxygenase (LOX), which converts polyunsaturated fatty acids, such as linoleic acid and arachidonic acid, into reactive lipid metabolites, thereby affecting cellular structure, metabolism, and signaling, and is involved in pathophysiological processes of a variety of immune and inflammatory diseases [28]. Lipoxygenase and linoleic acid were also found to play important roles in AA in our GO and KEGG enrichment results described above. The human ALOX15 gene is located in a cluster of genes on the short arm of chromosome 17 and is widely present in eosinophils, macrophages, bronchial epithelial cells, and skin[29]. It has been shown that ALOX15 and its related products are expressed at high levels in many pathological human tissues and organs, which consequently cause inflammation, oxidative stress and iron death [30]. ALOX15 has a pro-inflammatory effect, and it has been found that ALOX15 metabolites can stimulate the expression of IL-6 and TNF-α in a dose-dependent manner [31], and it promotes the production of inflammatory factors through the activation of mitogen-activated protein kinase, protein kinase C and other pathways [32]. ALOX 15 has also been associated with oxidative stimulation in humans. During cellular metabolism, the lipid oxidase ALOX 15 interacts with GSH-Px to regulate cellular redox status and apoptotic pathways. The expression of ALOX15 increases the secretion of reactive oxygen species and free radicals, which promotes the oxidative stress process and apoptosis in cells [33–34]. Thus, ALOX15 may promote AA by stimulating cytokine expression and inducing an inflammatory cascade response as well as modulating cellular redox status and apoptotic pathways. ALOX15 inhibitors may provide new research directions for the treatment of AA and its complications. GIMAP is an immune-associated protein that is expressed in lymphocytes and regulates signaling and cell development within the immune system [35]. The human GIMAP gene family consists of seven functional members that are clustered in the 293 kb region of human chromosome 7 [36], and the GIMAP gene family is expressed in human lymphocytes and vascular endothelial cells [37]. GIMAP6 is a T cell-associated cytoplasmic protein, and GIMAP6 plays a role in regulating immune function by controlling cell death and T cell activation [38]. It has been found that GIMAP6 knockdown accelerates T cell activation [39], suggesting that GIMAP6 can act as an anti-apoptotic protein and a negative regulator of T cell activation. GIMAP6 possesses GTP hydrolyzing activity and GIMAP is genetically correlated with GTP-binding proteins to regulate intracellular processes that are critical for cellular function [40]. GIMAP6 also controls cell survival and autophagy, which contributes to immune cell function as well as cytokine and immunoglobulin release [41]. Defects in GIMAP6 affect autophagy, which is defective and leads to malfunctioning of various metabolic activities in the body by affecting lipids and basic metabolic circuits [42]. Thus, GIMAP6 may be involved in the process of AA by regulating T cell activation, controlling cell survival and autophagy. GALNT6 is an enzyme that mediates the initiation step of mucin-type O-glycosylation, is a member of a family of 20 polypeptides of GALNTs [43]. Mislocalization and dysregulation of GALNTs expression were found to lead to aberrant glycosylation in cancer cells, and aberrant glycosylation affects many cellular properties, including cell proliferation, differentiation, migration, apoptosis, and immune response [44]. GALNT6 is one of the key enzymes catalyzing mucin-type O-glycosylation, which is involved in a variety of diseases such as breast, colorectal, lung adenocarcinoma, and ovarian cancer [45–48]. Studies have shown that GALNT6 is a tumor-promoting gene in breast cancer, and GALNT6 activates the PI3K/Akt signaling pathway, which is involved in promoting migration and invasion of breast cancer cells [49]. Meanwhile, GALNT6 knockdown increased the expression of IL-1b, IL-6, TNF-α and IL-18 in pancreatic ductal adenocarcinoma cells, and increased the release of inflammatory mediators, GALNT6 also promoted apoptosis through signaling pathways such as NF-κB [50]. It has been shown that GALNT6 enhances the invasive behavior of ovarian cancer cells by regulating EGFR activity, and GALNT6 knockdown decreases EGFR phosphorylation, whereas GALNT6 overexpression increases phosphorylation [48]. Therefore, GALNT6 may be involved in the process of AA by regulating the glycosylation process, NF-κB signaling pathway, and EGFR activity, leading to aberrant cell differentiation, impaired growth signaling, and dysregulation of inflammatory factors. HEG1 is closely related to angiogenesis and embryonic development, and is a single transmembrane mucin like glycoprotein rich in endothelial cells. It is mainly expressed in endothelial cells and is involved in angiogenesis, vascular integrity, and embryonic development [51]. The HEG1 cytoplasmic structural domain is associated with signaling molecules for cell proliferation [52], and HEG1 can regulate the expression of genes related to cell proliferation, survival, and differentiation through activation of Wnt signaling by β-linker proteins and induction of epithelial-mesenchymal transition [53]. HEG1 is primarily involved in cardiac development and endothelial cell junction formation [54], plasma HEG1 protein levels are negatively correlated with a number of cardiovascular risk factors, and reduced HEG1 protein levels are associated with increased body mass index, hypercholesterolemia, and hypertension [55]. HEG1 is enriched in multiple metabolism-related pathways, including glucose metabolism, lipid metabolism, and nucleotide metabolism signaling. When HEG1 expression is reduced, it affects various intracellular signaling networks. HEG1 expression is down-regulated in lung adenocarcinomas and correlates with poor prognosis [56], suggesting that lack of HEG1 expression leads to enhanced proliferative activity of cancer cells. Therefore, HEG1 may be involved in the process of AA by participating in vascular endothelial cell generation and regulating the Wnt/β-catenin signaling pathway in cell proliferation and differentiation.
Our results showed that inflammatory and immune effects are indispensable in the development and progression of AA. Therefore, we further performed immune infiltration analysis on samples from the experimental group of AA and the normal control group to investigate the involvement of various immune cell subtypes in AA. The data showed a significant difference between naive B cells, CD8 + T cells, γ-δ T cells, M1 macrophages, and M2 macrophages in the normal and experimental groups, and these immune cells infiltrated more in AA, suggesting that these immune cells play a role in AA. It has been shown that CD8 + T cells are the predominant pathogenic cell type in AA, which confirms the findings of our study in agreement with it [57]. Therefore, CD8 + T cells have the potential to be a new approach for the prevention and treatment of AA. Afterwards, we validated the four causal genes selected and used the GSE80342 dataset as the validation group. We found that there were significant differences between the GALNT6 and HEG1 genes in the validation group, and their expression levels in the AA experimental group were lower than those in the normal control group. This is consistent with the results of our mendelian randomization study. Unfortunately, the genes GIMAP6 and ALOX15 did not differ significantly in the validation group, which may be related to the small sample size of the validation group, and a larger sample size is needed for validation.Understanding the genetic variation in AA helps to understand the mechanisms that lead to its development and helps to identify susceptible individuals. The GALNT6 and HEG1 AA associated genes that we have worked out can be used as a starting point for creating knockout and transgenic mice for further research. Human genetics research has provided opportunities for the development of new drugs for many diseases, and this study hopes to provide new research directions for the pathogenesis and clinical treatment of AA. In the future, larger samples as well as basic clinical studies will be needed to validate the current findings.