N⁶-methyladenosine (m⁶A), the most abundant RNA modification, refers to the methylation at the N⁶ position of adenosine mainly located in the RRACH sequence (R=A or G, H=A, C, or U) and is a dynamic and reversible process, in which the addition, removal, and recognition of methyl is responsible by methyltransferases, demethylases, and m⁶A RNA binding proteins, respectively (1,2). m⁶A can participate in various physiological functions, such as tissue development, heat shock response, DNA damage response, and circadian clock control. It can also participate in pathological process by regulating the expression or structure of genes due to its involvement in all aspects of RNA metabolism, including RNA processing, nuclear export, stability, translation, and degradation (3-17).

A proper m⁶A level is necessary for sustaining normal bioprocesses, which mainly relies on the appropriate expression and function of methyltransferases and demethylases. m⁶A can be found not only in mRNAs, but also in non-coding RNAs, such as microRNAs (miRNAs/miRs) and long non-coding RNAs (lncRNAs). The fate of m⁶A-modified RNA is dependent on the protein that binds to it. By recognizing and binding to target mRNAs in an m⁶A-dependent manner, YTH m⁶A-binding protein 1 (YTHDF1) facilitates translation initiation and protein synthesis (8), while YTH m⁶A-binding protein 2 (YTHDF2) enhances the degradation of target mRNA (7,18). YTH m⁶A-binding protein 3 (YTHDF3) interacts with YTHDF1 or YTHDF2 to promote mRNA translation or increase mRNA degradation (19,20). YTH domain-containing 1 (YTHDC1) and YTH domain-containing 2 (YTHDC2) promotes the translocation of m⁶A-modified mRNA from the nucleus to the cytoplasm and elevates the translation efficiency of m⁶A-modified mRNA, respectively (21-25). Insulin like growth factor 2 mRNA binding protein (IGF2BP)1, IGF2BP2 and IGF2BP3 enhance mRNA stability (26). Heterogeneous nuclear ribonucleoprotein (HNRNP)C and HNRNPG regulate the alternative splicing of mRNAs in an m⁶A-dependent manner (11,27). HNRNPA2B1 promotes the maturation of miRNAs by recognizing m⁶A on pri-miRNAs and interacting with DROSHA and DDGCR8(28). m⁶A modification on lncRNAs can influence the interaction of lncRNAs with RNA binding proteins through an ‘m⁶A switch’ mechanism or the interaction between lncRNAs and miRNAs, which may lead to alterations in the gene expression of target RNAs (11,29). Thus, the genetic variant that influences m⁶A, such as m⁶A-associated single nucleotide polymorphism (m⁶A-SNP) may be related to various pathological processes.

m⁶A-SNP, which is in close proximity to or in the methylation site, results in the gain or loss of the m⁶A methylation site due to the alteration of the nucleotide (30). It is generally acknowledged that SNPs in various regions of genes, such as the regulatory and coding regions may affect the expression, structure, or function of genes through disparate patterns. For example, SNPs in the regulatory region, including the promoter region, 5'-untranslated region (UTR), and 3'-UTR can influence the binding of transcription factors or miRNAs, and in turn alter the expression of genes (31-33). The coding region consists of exons and introns. Non-synonymous coding SNPs alter the composition of amino acids of the protein that the gene encodes and affect the structure and/or function of the protein (34-36). Although synonymous coding SNPs do not modify the amino acid sequence of the protein, they exert an effect on mRNA conformation, protein folding, and subcellular localization (37-39). SNPs in introns play a crucial role in regulating the functions of genes by affecting the activity of the splice site (40). The function of m⁶A-SNP is not confined to the aforementioned layers, as the base substitution of m⁶A-SNP causes the gain or loss of the methylation site. Furthermore, previous studies have demonstrated that m⁶A-SNPs have a stronger association with diseases or clinical manifestations than non-m⁶A-SNPs (41-43). Therefore, it is valuable to explore the role of m⁶A-SNPs in the occurrence, progression, treatment, and prognosis of diseases. This may prove to be helpful in identifying susceptible individuals, determining patients' survival, clarifying disease pathogenesis, discovering new treatment targets, and improving patients' prognosis. The present review summarizes recent findings on m⁶A-SNPs.

The development of the majority of diseases is attributed to the interaction of genetic and environmental factors. Genome-wide association studies (GWAS) have identified numerous disease-associated genetic variants and revolutionized the understanding of the genetic architecture of diseases. However, a major challenge that needs to be combatted is the identification of functional or causative variants among those disease-associated genetic variants.

A number of studies have identified some genes related to various diseases or traits based on the combined analysis of GWAS and other public data (Table I) (41-57). Firstly, disease-associated m⁶A-SNPs were selected from GWAS according to the m6Avar database. Secondly, on the basis of expression quantitative trait loci (eQTL) datasets, those m⁶A-SNPs with eQTL signals were selected. Thirdly, the expression of the corresponding genes harboring m⁶A-SNPs that exhibited eQTL signals was further evaluated by means of expression datasets and differently expressed genes were ascertained. The base change of m⁶A-SNP caused by germline variants modulates the m⁶A level, alters the binding of protein and regulatory motifs, and affects the expression of genes, and is consequently linked to the development of diseases (Table II). For instance, rs3818978 is located in the 5'-UTR of MRPS21 and is predicted to change the binding of 33 protein and four regulatory motifs. Furthermore, rs3818978 is associated with the expression of MRPS21 and ADAMTSL4 in the aorta and with the plasma levels of seven proteins, which are enriched in the extracellular region and cytoplasmic vesicle. ADAMTSL4 has been reported to be associated with arterial fragility. Thus, rs3818978 may play a critical role in the occurrence of spontaneous coronary artery dissection (44). rs4829 in the 3'-UTR of TOM1L1 is near the m⁶A modification site, according to sequence-based RNA adenosine methylation site predictor (SRAMP) and can interact with polyadenylate-binding protein cytoplasmic 1, which is considered to participate in the occurrence of breast cancer induced by small nucleolar RNA host gene 14 (SNHG14). Therefore, rs4829 may regulate the expression of TOM1L1 to be involved in the development of breast cancer by altering the m⁶A modification level and protein binding (45).

Mo et al (58) jointly analyzed GWAS data of multiple sclerosis (MS) with eQTL data from four studies using summary data-based mendelian randomization (SMR) and found that the expression of 29 genes was significantly associated with MS (P_SMR<5x10^-6). Among the SNPs in these genes, m⁶A-SNP rs923829 in methyltransferase-like protein (METTL)21B was not only associated with the risk of MS (P=1.35x10^-10), but was associated with the expression of METTL21B in 37 tissues (58). Moreover, the association of rs923829 with the expression of METTL21B was confirmed in 40 unrelated Chinese Han individuals. These results suggested the critical role of m⁶A-SNP rs923829 in the development of MS by regulating the expression of METTL21B (58).

Following an integrated analysis of GWAS data, m⁶A peaks of HeLa and HepG2 epithelial cell lines, and expression data, five genes harboring m⁶A-SNPs including C6orf47 and SNAPC4 were identified to be involved in inflammatory bowel disease (IBD) (59). Subsequent research indicated that the total m⁶A modification levels and the expression of METTL3 and YTHDF1 were enhanced in HCT116 cells treated with IFNγ, and cytokine levels were upregulated in the intestinal mucosa of patients with IBD (59). Moreover, the overexpression of METTL3 and the interference of YTHDH1 led to the altered expression of C6orf47 and SNAPC4, respectively. Therefore, m⁶A modification played key roles in the occurrence of IBD (59).

Ruan et al (60) identified acyl-CoA synthetase medium chain family member 5 (ACSM5) as a candidate gene for thyroid cancer by jointly analyzing GWAS data, thyroid eQTL data and m⁶A-SNPs. ACSM5 was found to be downregulated in thyroid cancer tissues, which was associated with the poor prognosis of patients with thyroid cancer. According to prediction by SRAMP, m⁶A modification sites with very high confidence all contained an SNP site, the base change of which resulted in the loss of m⁶A modification sites (60). Furthermore, the knockdown of METTL3 decreased the m⁶A modification level and the expression of ACSM5. Hence, it was suggested that m⁶A-SNPs participated in disease development and progression by affecting expression of ACSM5(60).

The combined analysis of the m6Avar database and eQTLs associated with sepsis has revealed 15,720 m⁶A-cis-eQTLs in 1,321 genes. Among these genes, 17 genes were enriched in platelet degranulation process, a typical biomarker of sepsis, and 12 genes gathered in the pathway of Staphylococcus aureus infection, the most common pathogenic bacterium in sepsis, which suggested that m⁶A-SNPs played key roles in sepsis (61).

Both the mining of GWAS and the combined analysis of the m6Avar database with eQTLs have facilitated the identification of functional variants and causal genes associated with various diseases, and have helped to provide new direction for future studies on disease pathogenesis. Moreover, a relatively broad significance threshold was adopted to analyze the association between m⁶A-SNP and diseases, which avoided missing valuable information. However, there are some limitations in the aforementioned studies. Firstly, the associations between m⁶A-SNPs and diseases were not validated in additional independent studies. Secondly, the inconsistency of samples used in eQTL analysis and in the analysis of differentially expressed genes might have some degree of influence on the results due to the tissue specificity of gene expression. Thirdly, whether m⁶A-SNPs affected m⁶A modification levels and the expression of corresponding genes were not examined experimentally. Recently, some studies on the effects of m⁶A SNP and the underlying mechanisms were conducted, as described below.

The A allele of rs5746136 in superoxide dismutase 2 (SOD2) was previously found to be associated with a reduced risk of bladder cancer. A mechanistic analysis demonstrated that the transition of base from G to A led to an increased m⁶A modification level of SOD2 and increased the binding of HNRNPC with SOD2 followed by the upregulation of SOD2. The overexpression of SOD2 inhibited the proliferation, migration, and invasion of bladder cancer cells, which suggested that SOD2 functioned as tumor suppressor gene for bladder cancer (62).

Another study demonstrated that the transversion of the C to the G allele of rs3088107 resulted in a decreased m⁶A modification level of ring finger protein, transmembrane 2 (RNFT2) in 293T cells and reduced the expression of RNFT2 in bladder cancer cells (57). Moreover, the G allele of rs3088107 inhibited the proliferation and migration of bladder cancer cells compared to the C allele of rs3088107(57).

The A allele of rs1800241 in ankyrin repeat and LEM domain containing 1 (ANKLE1) has been linked to a decreased risk of colorectal cancer, which may be attributed to the enhanced expression of ANKLE1 by elevating the m⁶A modification level of ANKLE1 mediated by METTL3, METTL14, or WTAP and increasing binding of YTHDF1 with ANKLE1. Furthermore, the overexpression of ANKLE1[A] repressed the proliferation of colorectal cancer cells and promoted genomic stability more effectively than the overexpression of ANKLE1[G] (63).

Individuals carrying the exportin 1 (XPO1) gene rs3087898 T allele were previously shown to be more susceptible to coeliac disease (CD) than those carrying the rs3087898 C allele (64). The XPO1 mRNA transcript with the T allele was shown to have a higher m⁶A modification level and higher translation efficiency by increasing the binding of YTHDF1 with the XPO1 mRNA than transcription with the C allele. Subsequent experiments indicated that the allele-specific increase of XPO1 activated the NF-κB signaling pathway to facilitate the development of CD (64).

The GT genotype of rs142933486 in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta (PIK3CB) was previously found to predicted the poor survival of patients with pancreatic ductal adenocarcinoma (PDAC) (65). Cell biology experiments revealed that the overexpression of the T allele reduced the m⁶A modification level of PIK3CB, decreased the binding of YTHDF2 with PIK3CB, and in turn, increased the mRNA stability and expression of PIK3CB. Consistently, a higher PIK3CB expression was associated with the poor prognosis of patients with PDAC, particularly in patients with PTEN deficiency. Further analyses demonstrated that the overexpression of PIK3CB[T] activated the AKT signaling pathway to promote the proliferation and migration of PTEN-deficient PDAC cells, with a more prominent effect than the overexpression of PIK3CB[G] (65).

The aforementioned studies have indicated that the base change of m⁶A-SNPs leads to alterations in m⁶A modification levels, a conversion from genetics to epigenetics, and the expression variation of corresponding genes, which is dependent on the RNA binding proteins that recognize m⁶A methylation. Furthermore, the expression variation of genes affects the biological behavior of cells, which explains the association of m⁶A-SNPs with the risk or prognosis of diseases. In particular, in bladder cancer, colorectal cancer, CD, and PDAC, the overexpression of a specific allele alone can significantly modify the function of corresponding genes. All these findings may provide an experimental foundation for the development of novel therapeutic strategies. For example, XPO1 and PIK3CB have the potential to function as therapeutic targets for CD and PDAC, respectively.

In conclusion, m⁶A-SNPs play a pivotal role in all stages of diseases. In the future, the identification of m⁶A-SNPs as disease biomarkers and ascertaining the functions of these m⁶A-SNPs may prove beneficial, as it may help to identify susceptible individuals in a timely manner. It may also clarify the role of corresponding genes in the occurrence and progression of diseases, and may thus aid the development of novel treatment strategies, ultimately improving the survival of patients.