New Insights into Risk Genes and Their Candidates in Multiple Sclerosis

Oligodendrocytes are central nervous system glial cells that wrap neuronal axons with their differentiated myelin membranes as biological insulators. There has recently been an emerging concept that multiple sclerosis could be triggered and promoted by various risk genes that appear likely to contribute to the degeneration of oligodendrocytes. Despite the known involvement of vitamin D, immunity, and inflammatory cytokines in disease progression, the common causes and key genetic mechanisms remain unknown. Herein, we focus on recently identified risk factors and risk genes in the background of multiple sclerosis and discuss their relationships.


Introduction
The myelin sheath is formed as a multilamellar membrane structure through the spiral wrapping of neuronal axons that act as insulators [1][2][3][4]. The transmission of each action potential on a limited membrane region is significantly promoted by the resulting saltatory conduction. Electrical signals are quickly derived to adjacent or distant neuronal cells and neuronal networks. If the myelin is damaged, however, fast signal transmission is not achieved, which causes defective neuronal function. This phenomenon is typically observed in demyelinating states. One well-known demyelinating disease is multiple sclerosis (MS) of the central nervous system (CNS). It is thought that MS is often caused by an abnormal autoimmune reaction in the CNS.
First defined by the National Multiple Sclerosis Society (in the United States) in 1996, MS is a chronic inflammatory disease that is characterized by demyelination mainly in the brain and, in turn, axonal degeneration [5]. The prevalence of MS is higher in Caucasians in the United States and Europe. The incidence rate is more than 100 patients per 100,000 population members in some areas of Northern Europe [6]. In Japan, the prevalence was estimated to be 1 to 5 patients per 100,000 population members, but this number has reportedly increased to 14 to 18 over a single decade [7]. The incidence of MS is increasing in both developed and developing countries [8]. The average age of onset of MS is middle-age and the disease is approximately twice as common in women than in men [9,10].
It is unclear why the number of MS patients has increased recently across countries and regions. There are various risks and possible reasons for the development of the disease, including smoking, vitamin D deficiency, obesity, and Epstein-Barr virus, which is a type of herpes virus [11]. MS also has genetic factors, as first-degree relatives and identical twins have a 25% chance of being affected [12]. The major histocompatibility complex (MHC) HLA-DRB1*15:01 allele was the first factor identified as a risk factor for MS [13]. Subsequent studies have shown that interleukin (IL) 2Rα and IL7R are also genetic factors [14].
Neurol. Int. 2023, 15 25 MS symptoms likely depend on the tissues and regions where demyelination occurs. Some of the most common symptoms are optic neuritis and brainstem and spinal cord syndromes. Early clinical symptoms usually recover, but relapses are often followed by sequelae [15]. The onset is related to the location and size of the lesion, and even small lesions in the symptomatic zone are likely to cause symptoms, with magnetic resonance imaging showing typical "Dawson's fingers" with periventricular lesions [16].
This narrative review will focus on the previously reported major risk factors of MS. We describe the disease and the possible therapeutic signaling pathways related to the risk factors as well as risk gene products in MS. We have selected the references using inclusion criteria that focus on reviews of MS and original papers. We have also included large-scale meta-analyses of original genetic studies.

MS and Environmental Factors (Vitamin D)
Epidemiological studies have shown that there are racial differences in developing MS, with a minimum prevalence at the equator and an increase with northern or southern latitudes [17]. Vitamin D is produced primarily by the action of ultraviolet B rays on the skin. This is supported by circumstantial evidence suggesting environmental factors of vitamin D deficiency due to a lack of sunlight as a predisposing factor for MS [18]. Indeed, vitamin D deficiency has been suggested as a possible cause of MS and/or contributor to the progression of MS, but it likely has limited pathological effects [19]. It has been reported that people with blood 25-OH-D levels of 40 ng/mL or higher have a 62% lower risk of MS than those with levels below 25 ng/mL [20], suggesting that having normal vitamin D levels reduces the risk of MS [9]. Serum 25-OH-D is a metabolite of vitamin D used to assess vitamin D levels in vivo (Figure 1) [20]. Adil et al. reported levels of vitamin D bioavailability and adipose tissue-secreted hormones such as adiponectin and leptin [21]. MS risk correlated with a genetic predisposition to the body mass index (BMI) but anticorrelated with the 25-OH-D level. Leptin and adiponectin have no effect on the increased risk of MS due to lowered vitamin D levels. Vitamin D supplementation modestly reverses the effect of obesity on MS [22]. In support of this study, Michaela et al. examined the association between 52 risk variants identified through genome-wide association studies (GWASs) and disease severity in MS and found that they were not associated with MS severity in terms of cohort, gender, age of onset, and HLA-DRB1*15:01 allele [21].
Scazzone et al. investigated the effects of vitamin D-related genes on MS susceptibility. Of the 12 vitamin D gene product pathways investigated, the most studied was the vitamin D receptor and the least studied were other vitamin D-related gene products. Scazzone et al. reported that it is not clear whether these mutations directly affect the risk of MS [23]. When vitamin D supplementation is used as a treatment, no statistically significant differences were found and its effectiveness could not be demonstrated [19,24]. Despite the lack of significant difference, vitamin D alters the transcriptome profile of macrophages and microglia. In addition, vitamin D activates T cells (Figure 1) [25]. MS risk and genetic abnormalities in vitamin D metabolism have been reported in several cases, with genetic abnormalities for CYP27B1 in the cytochrome P450 family gene products, which is a regulator of calcitriol synthesis, influencing MS risk [26].
Genetic polymorphisms in the gene-encoding molecules involved in vitamin D homeostasis are associated with vitamin D deficiency. However, Klotho, which is coded as a protein with vitamin D metabolism, has no genotypic frequencies that differ between MS patients and controls [27]. This finding means that the role of Klotho does not involve genetic susceptibility to MS.
Several studies have strengthened the candidacy of the environmental factors between vitamin D and the most major risk gene HLA-DRB1*15:01 allele. Vitamin D deficiency is also reported to be an important MS disease pathogenesis.  [23]. When vitamin D supplementation is used as a treatment, no statistically significant differences were found and its effectiveness could not be demonstrated [19,24]. Despite the lack of significant difference, vitamin D alters the transcriptome profile of macrophages and microglia. In addition, vitamin D activates T cells ( Figure 1) [25]. MS risk and genetic abnormalities in vitamin D metabolism have been reported in several cases, with genetic abnormalities for CYP27B1 in the cytochrome P450 family gene products, which is a regulator of calcitriol synthesis, influencing MS risk [26].
Genetic polymorphisms in the gene-encoding molecules involved in vitamin D homeostasis are associated with vitamin D deficiency. However, Klotho, which is coded as a protein with vitamin D metabolism, has no genotypic frequencies that differ between MS patients and controls [27]. This finding means that the role of Klotho does not involve genetic susceptibility to MS.

MS and Environmental Factors (Immunity)
CNS fibers are covered with myelin sheaths whose composition contains abundant lipids and proteins. In MS, demyelinating plaques are involved in an immune response triggered by T cells. Proteolipid protein (PLP), myelin basic protein (MBP), and myelin oligodendrocyte glycoprotein (MOG) proteins have been well-studied as self-antigens involved in demyelination in MS [28]. The autoimmune disease against MOG is called MOG antibody (MOG-IgG)-associated disease [29]. The clinical features are considered to reflect a unique disease with a different etiology from MS and optic neuritis, related to aquaporin-4 (AQP-4)-IgG [30,31].
Immune responses to myelin-associated glycoprotein (MAG) have been primarily implicated in the development of MS. Increased MAG-recognizing T and B cells in MS patients have been observed [32]. However, the MAG peptide itself did not elicit disease-specific T and B cell responses, suggesting that this is secondary to demyelination rather than an attack on MAG by immune responses [33]. In addition to MAG proteins, environmental factors such as viral infection trigger demyelination somewhat. Then, differentiation into CD4-positive T cells and Th1-type cells results in one of the key events in the early stages of MS ( Figure 1) [34].
Most of the more than 100 mutations in MS reported to date are related to the human leukocyte antigen (HLA) and the immune system, supporting the idea that MS is an immune disease. However, these mutations account for only 25% of heritability, leading to the new concept of "phantom heritability." Sawcer et al. proposed insufficient nonredundant unnecessary sufficient (INUS), which describes the plurality of causation when a mutation cannot be found [35].
Experimental autoimmune encephalomyelitis (EAE) is a typical mouse model of MS and has also been the basis for its etiology and therapeutic development with regard to induced CNS inflammation. A few researchers have put forth that the debate should not be focused on EAE, arguing that the phenotype is weak [36]. Microglia, macrophages, and dendritic cells, which are potent antigen-presenting cells, have been reported to be increased in EAE mice [37]. Microglia and macrophages are present in MS lesions, myelin proteins MBP, and PLP as well as the minor myelin proteins [38]. Faber et al. compared gene expression in opticospinal EAE (OSE) and MOG EAE models. They demonstrated a more extensive enrichment of human MS risk genes among transcripts differentially expressed in OSE than in MOG EAE [39].
When Li et al. analyzed the transcriptional profiling data in the human brain in MS, 133 known and unknown genes were identified [40]. They included genes encoding a number of extracellular matrixes, such as collagen, signal-triggering receptor, and molecules involved in immune-related pathways and phosphatidylinositol-3 kinase (PI3K)-Akt pathways. Among them, four major extracellular and transmembrane proteins, IL17A, IL2, CD44, and IGF1, and 16 extracellular proteins interacting with IL17A have been associated with MS pathogenesis. Additionally, Del-1, which is an interacting protein with IL17A that may be associated with MS progression and relapse, has been identified as a probable biomarker.
Regulatory T cell (Treg) alteration has also been implicated in the pathogenesis of MS. X-linked forkhead box P3 (FoxP3) plays a crucial role in the development and stability of Tregs. However, FoxP3 and vitamin D3 did not have any association with MS [41].
The humoral immune response to Epstein-Barr virus nuclear antigen 1 (EBNA-1)specific immunoglobulin γ (IgG) titers in families with MS was determined as a result of investigating the role of specific genetic loci on the antiviral IgG titers. The EBNA-1 IgG gradient being the highest in MS patients and the lowest in biologically unrelated spouses indicates a genetic contribution to EBNA-1 IgG levels that is only partially explained by HLA-DRB1*15:01 carriership [42].
Although it was previously known that non-coding RNAs (ncRNAs) create transcription noise, they are also now believed to be regulators of immune responses. Dysregulation of ncRNAs is one of the underlying mechanisms of immune disorders such as MS. Several studies have reported the aberrant expression of ncRNAs in the sera or blood cells of MS patients [43][44][45]. The results of these studies propose different classes of ncRNAs (long non-coding RNAs, microRNAs, and circular RNAs) as diagnostic or predictive markers in MS [46].
Demyelinating plaques are related to the autoimmune response in MS. The production of inflammatory cytokines caused by the immune response, such as PLP, MBP, and MOG, revealed MS as a chronic inflammatory disease.

Gene Risk and Signaling Pathway
Environmental cues associated with the increased risk of developing MS have been established, and over 200 risk loci with moderate to subtle effects have been described.
To dissect the influence of genetic predisposition and environmental factors, Florian et al. investigated the peripheral immune signatures of 61 monozygotic twin pairs discordant for MS. They revealed an inflammatory shift in a monocyte cluster of twins with MS, coupled with the emergence of a population of naive helper T cells that have a transient response IL2 as MS-related immune alterations [47]. The research on genetically identical (monozygotic) twins shows that the concordance rate for MS is approximately 30%. This indicates that genetic and environmental factors interact with MS. Baranzini et al. examined DNA methylation and gene expression across the genome in three monozygotic twins discordant for MS; however, there were no consistent differences in DNA sequence [48]. It is surprising that the environment strongly indicated epigenetic modifications to germline susceptibility based on studies of adoptees, half-siblings, and avuncular pairs. The fact that complete explanations for disease heritability were unachieved after whole-genome association studies warrants consideration of all the factors contributing to disease risk, such as genetic, epigenetic, and environmental factors [49].
As many as 200 single-nucleotide polymorphisms (SNPs) are associated with MS risk (Table 1) [50][51][52][53]. Gresle et al. analyzed MS risk expression quantitative trait loci associations for 129 distinct genes in MS patients [54]. They identified the MS risk SNPs, rs2256814 Myelin transcription factor 1 (MYT1) in CD4 cells and rs12087340 RF00136 in monocyte cells. IL7 receptor (IL7R) is a member of the type I cytokine receptor family and is a primary pleiotropic receptor in immune cells (Figure 1). Two GWASs of MS reported that three SNPs outside of the MHC region were associated with MS: rs6897032 within the IL7R gene and two SNPs (rs2104286 and rs12722489) in the IL2R gene [14,55]. Omraninava et al. revealed that the IL7RA gene rs6897932 SNP decreases MS susceptibility ( Figure 1) [56]. Infection with the herpes virus and Mycoplasma pneumonia create grounds for MS. The T allele in the IFNγ gene (+874) and the genotypes of AA and AG at the TNFα gene (-308) at position−308 were considered potential risk factors for MS ( Figure 1) [57]. Despite GWASs explaining that there are common SNPs associated with various diseases, known common variants only account for part of the estimated heritability of common complex diseases. Nadia et al. identified the rare functional variants analyzed within a large Italian MS multiplex family with five affected members [58]. Another recent study showed that up to 5% of MS inheritability may be accounted for by rare variations in the gene coding sequence, with four novel low genes driving MS risk independently of common variant signals [59]. Based on the research of a large cohort of Italian individuals, researchers identified three SNPs (rs4267364, rs8070463, rs67919208) that were involved in the regulation of TBK1 Binding Protein 1 (TBKBP1) and prioritized them as functionally relevant in MS [60]. Recent GWAS research in MS that has analyzed up to 47,000 MS patients and 68,000 healthy controls has determined more than 200 non-MHC genome-wide associations. The results show that immune cells, such as T cells, B cells, and monocytes, have susceptible gene specificity [61]. The International Multiple Sclerosis Consortium analyzed the large-scale GWAS data of 47,000 MS patients and 68,000 healthy controls and established a reference genetic map of MS. Their findings demonstrate the enrichment of MS genes in these brainresident immune cells, suggesting that they may have a role in targeting an autoimmune process to the CNS, although MS is most likely initially triggered by a perturbation in peripheral immune responses [52]. The Janus kinase and signal transducer and activator of the transcription (JAK/STAT) pathway is essential for both innate and acquired immunity. It has also been reported to be associated with several neuroinflammatory diseases (Figure 2) [62]. In EAE mice, Th1 cells produce interferon-gamma (IFNγ) via STAT4 and inflammatory macrophages, which promote macrophage activation. Similarly, Th17 produces granulocyte-macrophage colony-stimulating factor (GM-CSF) in the CNS and promotes macrophage polarization to inflammation via JAK/STAT5 (Figure 2) [63]. be associated with several neuroinflammatory diseases (Figure 2) [62 cells produce interferon-gamma (IFNγ) via STAT4 and inflamm which promote macrophage activation. Similarly, Th17 produces phage colony-stimulating factor (GM-CSF) in the CNS and promotes zation to inflammation via JAK/STAT5 (Figure 2) [63]. A comprehensive analysis of genes in the brain of MS patients h levels of immune cell populations and decreased ones of endothelial Treg cells in MS lesions [64]. Toll-like receptors (TLRs) have a variet axonal pathway formation and dorsoventral patterning in the CNS. T pathogen-associated molecular patterns (PAMPs), have been id A comprehensive analysis of genes in the brain of MS patients has shown increased levels of immune cell populations and decreased ones of endothelial cells, Th1 cells, and Treg cells in MS lesions [64]. Toll-like receptors (TLRs) have a variety of roles, including axonal pathway formation and dorsoventral patterning in the CNS. TLR ligands, such as pathogen-associated molecular patterns (PAMPs), have been identified as T cell promoters in MS. In particular, TLR2 expression is high in MS lesions and TLR2 activation induces the expression of pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α, which are implicated in exacerbated inflammation (Figure 3) [65]. The HLA signal in the Italian population maps to a glycoprotein involved in dendritic cell (DC) maturation, such as TNFSF14 gene encoding LIGHT. Miriam et al. reported that the TNFSF14 intronic SNP rs1077667 was the main MS-associated variant in the region. That means that the intronic variant rs1077667 alters the expression of TNFSF14 in DCs, which may play a role in MS pathogenesis [66]. A variant in TNHSH13B, encoding the cytokine and drug target B-cell activating factor (BAFF), was associated with upregulated humoral immunity through increased levels of soluble BAFF, B lymphocytes, and immunoglobulins in MS [67]. Leptin (LEP) and leptin receptor (LEPR) overexpression are related to MS activity and progression, and peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC1A) is able to affect the reactive oxygen species production in the pathogenesis of MS. LEP rs7799039 and LEPR rs1137101 genetic variants modify the serum LEP levels and PGC1A rs8192678 alters the PGC1A activity. Ivana et al. revealed that the PGC1A rs8192678 minor allele had an increased risk for the occurrence of MS, and LEP rs7799039 affected the LEP gene expression in relapsing-remitting patients [68]. sion, and peroxisome proliferator-activated receptor gamma co-activator 1-alph (PGC1A) is able to affect the reactive oxygen species production in the pathogenesis MS. LEP rs7799039 and LEPR rs1137101 genetic variants modify the serum LEP levels an PGC1A rs8192678 alters the PGC1A activity. Ivana et al. revealed that the PGC1 rs8192678 minor allele had an increased risk for the occurrence of MS, and LEP rs779903 affected the LEP gene expression in relapsing-remitting patients [68]. Furthermore, in relapsing MS, reduced suppression of cytokine signaling-3 (SOCS expression in the CNS and immune cells may induce LEP-mediated overexpression pro-inflammatory cytokines ( Figure 3) [69]. Pattern recognition receptors, which are tri gered by both microbe-associated molecular patterns and damage-associated molecul patterns, have been reported to regulate innate immune responses in MS and an EA model. Pattern recognition receptor signaling promotes inflammatory-producing cyt kine production in CNS autoimmune diseases (Figure 3) [70]. NF-κB is involved in a wid Figure 3. Interaction of some receptors with their cognate ligands induces the expression of proinflammatory cytokines in immune cells. Pathogen-associated molecular patterns interaction with TLRs, SOCS3 activation by cytokine receptors, microbe-associated molecular patterns or damageassociated molecular patterns binding to pattern recognition receptors, and/or activation through NF-κB are involved in the regulation of the expression of inflammatory cytokines, which are responsible for MS, in immune cells.
Furthermore, in relapsing MS, reduced suppression of cytokine signaling-3 (SOCS3) expression in the CNS and immune cells may induce LEP-mediated overexpression of proinflammatory cytokines (Figure 3) [69]. Pattern recognition receptors, which are triggered by both microbe-associated molecular patterns and damage-associated molecular patterns, have been reported to regulate innate immune responses in MS and an EAE model. Pattern recognition receptor signaling promotes inflammatory-producing cytokine production in CNS autoimmune diseases (Figure 3) [70]. NF-κB is involved in a wide range of vital processes, including inflammation, cell proliferation, and differentiation. Abnormal NF-κB activation has been reported to be closely associated with the development of MS and EAE [71].
In MS, the altered Foxp3-E2 variant-associated inhibitory activity of Treg cells is associated with defective signaling via IL-2 and glycolysis, which modulates Treg cell induction and function in autoimmunity [72]. The expression of vascular endothelial growth factors and matrix metallopeptidases involved in angiogenesis is increased in MS. These genes are also involved in basement membrane degradation and blood-brain barrier disruption, which allows immune cells to infiltrate the CNS in EAE and MS (Figure 4) [73]. Programmed cell death 1 (PD-1) is known as an immune checkpoint that is associated with several autoimmune diseases. Research on the frequency of PD-1 genotypes and alleles in MS patients shows that PD-1 gene polymorphisms may be associated with MS [74]. Phosphorylation of receptor-interacting protein kinase 1 (RIPK1) in astrocytes and microglia triggers a detrimental neuroinflammatory program that contributes to the neurodegenerative environment in MS (Figure 4) [75].
Programmed cell death 1 (PD-1) is known as an immune checkpoint that is associated with several autoimmune diseases. Research on the frequency of PD-1 genotypes and alleles in MS patients shows that PD-1 gene polymorphisms may be associated with MS [74]. Phosphorylation of receptor-interacting protein kinase 1 (RIPK1) in astrocytes and microglia triggers a detrimental neuroinflammatory program that contributes to the neurodegenerative environment in MS (Figure 4) [75]. Risk genes have been well studied by meta-analyses and many SNPs have been identified. MYT1, IL2R, IL7R, IFNγ, and TNFα, among others, are considered to be the major risk genes in MS. The related major signaling in MS is the JAK/STAT pathway.

Conclusions and Perspective
We have examined and discussed the genetic risks in the background of MS. The major risks include (1) the genes related to vitamin D deficiency, (2) the genes involved in the immune response, and (3) the genes responsible for inflammatory cytokines and the related signaling molecules. Nucleotide sequence analyses with advancing technologies have clarified that there are an increasing number of other possible categories of risk genes besides these in MS. In the future, molecules related to these risk gene products may be promising therapeutic target candidates. Risk genes have been well studied by meta-analyses and many SNPs have been identified. MYT1, IL2R, IL7R, IFNγ, and TNFα, among others, are considered to be the major risk genes in MS. The related major signaling in MS is the JAK/STAT pathway.

Conclusions and Perspective
We have examined and discussed the genetic risks in the background of MS. The major risks include (1) the genes related to vitamin D deficiency, (2) the genes involved in the immune response, and (3) the genes responsible for inflammatory cytokines and the related signaling molecules. Nucleotide sequence analyses with advancing technologies have clarified that there are an increasing number of other possible categories of risk genes besides these in MS. In the future, molecules related to these risk gene products may be promising therapeutic target candidates.