1.1. Methods to Detect Methylation Level in DNA Sequence

Epigenetics refers to inheritance variations in DNA chromosomes without changing the DNA sequence, further regulating gene expression. One of the most extensively studied epigenetic modifications is DNA methylation, which is the covalent modification of cytosine. This modification offered a molecular mechanism by which cells develop and proliferate.

Recent advancements in DNA detection proved their role in tumor detection and cancer-curing. These applications require modifications of profiling approaches. Some of these detection methods are discussed below [4].

1.1.2. Biological Identification Method to Determine Methylation Level in DNA

Utilization of enzymes to detect DNA methylation is also possible as these methods offer more specificity (Figure 1). A pair of isoschizomers and nonisoschizomers is used to cleave the required identical sequences but has different sensitivity to the methylation level. SmaI, Not I, Hpa II, and BstU I are methylation-sensitive restriction enzymes that separate DNA pairs. They only cut nonmethylated DNA and do not influence methylated DNA. The results of this method are based on revealing nonmethylated DNA locations but targeting enzyme sites. The only drawback is that this technique covers small genome levels due to limited CpG sites [4].

The second technique that comes under biological identification is enzyme-related techniques. The restriction-landmark genomic scanning (RLGS) approach is the first example where Not I restriction enzyme digested DNA samples. This digestion will give the idea of methylated sites. Electrophoresis is run after separating the base pair with restriction enzymes, which analyzes various CpG sites’ methylation statuses. The limitation of this process is the reliability of the results. An enhanced enzymatic digestion technique that produces more precise results is the methylation-sensitive restriction-endonuclease PCR/southern (MS-RE-PCR) method [12].

This technique uses restriction enzymes Msp I and Hpa II to treat the DNA samples and detect 5-CCGG-3 sequences. From these restriction enzymes, Msp I could not clip nonmethylated cytosine in the DNA sample and only separated methylated C in 5-CmCGG-3. Following this procedure, PCR was used to detect the level of methylated cytosine. Another method utilizes a pair of fluorophores, and an extinguisher is designated beside the molecular beam radius. The fluorophore signals intensify, indicating the concentration of methylation when 5-methylcytosine fragments are generated in the probe by the Dam MTase catalyst. However, the high cost of this process limits its widespread use [13].

Zinc finger proteins (ZF), MBD, and anti-5-methylcytosine IgG1 antibodies are employed in the biodependence reaction to assess the methylation levels in a sample. This method relies on the attachment of methylated DNA sequences to MBD proteins. To detect 5-methylcytosine in the genome, microarray (MBD-chip) or sequencing technologies such as MBDCap-seq and MethylCap-seq are utilized [6].

2.1. Atopic Dermatitis (AD)

In AD, epidermal barrier dysfunction and immune deregulation are confirmed. Apart from this, gene regulation also plays a crucial role in disease progression. Different gene methylation profiling is evidence of disease progression via methylation alterations.

Due to lower DNMT1 mRNA levels in PBMCs of AD patients with high blood IgE levels, it has been suggested that DNMT1 inhibition and subsequent hypomethylation may play a role in this subset of AD patients. However, there has been limited research on the dysregulation of DNA methylation in AD [17]. In one study by Liang et al., it was found that monocytes from 10 AD patients showed global hypomethylation compared to healthy controls. They also discovered that the promoter of the Fc epsilon receptor 1 gamma (FCER1G), which correlated with gene expression, was hypomethylated in AD patients [18]. Additionally, when control monocytes were treated with the DNMT inhibitor azacytidine, hypomethylation occurred, leading to increased FcRI transcription and surface expression. In a related study, the role of thymic stromal lymphopoietin (TSLP) in disease progression was explored. The researchers observed that azacytidine treatment of KCs caused changes in skin lesions, including hypomethylation of the TSLP promoter, as well as an increase in TSLP mRNA and protein expression [19].

Another scientist compared regular control patients’ DNA methylation patterns (Figure 2) with diseased patients and concluded that DNA methylation changes were found in whole blood for the genes involved in nucleotide-binding oligomerization and pyrin domain–containing receptors (NLRPs), which are known to alter innate immunity via inducing NF-κB activation. In addition to these multifarious investigations, DNA methylation at diverse CpG sites indicates amplified SIRL-1 expression in monocytes.

When tested, the scientists determined the methylation level in genes involved in disease progression, such as S100A2, S100A7, S100A8, S100A9, and S100A15. Among these genes, methylation changes were only found for S100A5, which was hypermethylated, as shown in Table 1. In KCs, the expressions of KRT6A and KRT6B and, in innate immune cells, the level of OAS1, OAS2, and OAS3 escalated. OAS2 shows decreased methylation [37].

Another study claimed that approximately 25 genes were hypermethylated and 10 were hypomethylated in CD4+ CLA+ T cells presented in AD patients compared to control [38]. Demethylations of the VSTM1 promoter gene can also lead to AD as it influences the expression of SIRL-1 in monocytes. Production of reactive oxygen species is limited by the SIRL-1, which is related to the pathogenesis of AD [22]. In addition, another finding reported differential methylation at CpG sites of the KIF3 A gene, which gives an idea of the impact of DNA methylation on AD development [39].

2.2. Eczema

Eczema herpeticum (EH) is a worsening state of AD in which HSV-1 exposure is a prime factor for disease progression (Figure 3). Apart from this, ADEH+ patients represent the severity of the disease because the action of interferon is attenuated and the serum level of IgE is elevated. The exact mechanism is still unclear. Many studies have suggested the role of DNA methylation in disease development that participated in autoimmune disorders [23].

The filaggrin gene (FLG) protects the skin by creating a barrier to reduce water loss from the skin. The expression of FLG was decreased, leading to an increase in eczema, as shown in Table 1. CpG (cg18593727) and CpGs in IL4 (cg23943829) and in IL13 (cg04303330) were differently methylated when tested in the laboratory in ADEH+ patients.

2.3. Androgenetic Alopecia (AGA)

AGA is the baldness due to the androgen receptor gene due to an excessive response to androgens. Androgen receptor activation causes the growth phase or anagen phase of the usual hair growth cycle to be shortened. In cases of AGA, excessive activation leads to follicular shrinkage through a gradually shortened anagen phase, resulting in thinner and shorter hair follicles that may not even eventually reach the epidermis [40].

One study performed by Martinez-Jacobo determined the presence of higher AR methylation levels in occipital follicles. This may lead to protection of hair follicles against hair loss and miniaturization [24]. DNMT1 is crucial in establishing androgenic alopecia as it is required to regulate methylation patterns. Reduced expression of DNAMT1 can trigger baldness, which suggests a gene role in the progression of androgenic alopecia [24].

2.4. Systemic Lupus Erythematosus (SLE)

T and B cell overstimulation can lead to SLE. SLE is an autoimmune disease that causes organ damage. Along with environmental factors, genetics also play a role, but the molecular mechanisms are still unclear [28].

Most recently, altered DNA methylation profiles of various genes have been addressed. The gene for killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4) was found to be demethylated when tested [41]. Apart from this, increased expression of serine/threonine protein phosphatase 2A (PP2A) is found in T cells from SLE patients where this reduction in methylation of sites containing CpG in the proximal promoter of the PP2A gene is the factor that causes the elevation in gene expression [25]. After comparison with normal control patients, it was annotated that women with SLE, as well as those with rheumatoid arthritis, have DNA methylation in the proximal promoter of the estrogen receptor 1 (ESR1) gene, which codes for the extensively expressed estrogen receptor α [17, 42].

Proinflammatory effector cytokines and IL-2 are not expressed by T cells from SLE patients due to increased DNA methylation of IL2 regulatory domains. In contrast to prior research, T cells from SLE patients showed enhanced DNA methylation of Foxp3 gene regulatory regions [27, 28, 43]. SLE patients’ site-specific DNA methylation in the CD8 gene cluster leads to decreased activity of the CD8 coreceptor, as shown in Table 1. This alteration will cause an increase in DN T cells responsible for revealing proinflammatory effector cytokines containing IFN-γ, IL-17F, IL-12, IL-18, and others.

Overall, these investigations contribute to the role of modulation in DNA methylation levels in T cell subset commitment and lineage-specific cytokine expression. Moving further towards lymphocytes, global methylation is decreased in SLE patients [25].

2.5. Psoriasis

Psoriasis affects 125 million individuals worldwide. Hyperproliferative KCs and invading immune cells are characteristics of the chronic and recurring and inflammatory skin condition psoriasis. Even though the exact pathogenesis is still unclear, many studies have introduced the role of genetic factors in psoriasis [33]. A research confirmed that the level of DNA methylation in skin lesions is elevated compared to that of normal skin.

DNA methylation alterations are located in the promoter and first gene exon, which regulate the transcription process [30]. Compared to DNase I hypersensitivity sites, non-DNase I hypersensitive sites had a higher methylation level [30]. There are differences in the methylation of CG05590156, which is present in the TRIO gene that codes for the GEF (guanine nucleotide exchange factor) of the RHOA and RAC1 GTPases and serves a number of biological functions. Additionally, the psoriasis candidate genes CYP2S1 (CG19430423) and EIF2C2 (CG00288598) were shown to have varying amounts of methylation [30].

The methylation level also controls the gene expression of S100Aug, a gene responsible for cell proliferation and immune mechanism [44]. Furthermore, it was demonstrated that 12 other genes, including TRIM22, OAS2, MAN1C1, EIF2C2, ECE1, CYP2S1, C10orf99, and DLGAP4, had negative correlations with the degree of methylation, whereas the expression of three genes, GDPD3, TRIM14, and CCND1, correlated favorably with the degree of methylation [31]. More than 33% methylation difference has been observed in genes Wnt7B, NFATc1, CELSR2, and FZD7. In terms of methylation patterns of skin on different body areas, methylation in the abdomen and back is lower than in the limbs [45].

Patients with psoriasis have hypomethylated human leukocyte antigen (HLA)-DMA and G protein-coupled receptor 128 (GPR128) genes, but they have hypermethylated tenascin-XB (TNXB) and macrophage stimulating 1 receptor (MST1R) genes [32]. PBMCs contain dendritic cells, phagocytes, monocytes, lymphocytes, and a few other cell types. According to a study, psoriasis patients’ PBMCs express more DNMT1 than those from the healthy control group [46]. Compared to healthy patients, individuals with psoriasis had hypermethylated CD4+ T cell chromosomes at high methylation levels. Other hypermethylated genes are PPAPDC3, TP73, and FANK1 [33].

2.6. Systemic Sclerosis (SSc)

SSc includes problems with internal organs, blood vessels, the digestive tract, and skin, which is tighter and more challenging. SSc is also known as scleroderma. The primary factors of disease development include early endothelial injury, an inflammatory infiltration, and a subsequent fibrotic response despite the complicated pathophysiology of scleroderma [47].

Ying Luo determined the level of DNA methylation in African American patients. He found that of 3.8 million CpG sites, approximately 1180 CpGs are differentially methylated in diseased patients. DLX5, a gene responsible for cell proliferation, was found to be hypermethylated in skin fibroblast [48]. TMEM140 and MCG12916 are also in a hypermethylated form in the same patients where DLX5 and TMEM140 showed expression, and MCG12916 is downregulated in samples of the same patients. The findings of this study showed that, with the exception of CpG islands, where differentially methylated CpGs (DMCs) were hypermethylated, other regions displayed notable CpG hypomethylation [34]. Additionally, peripheral blood mononuclear cells from individuals with SSc demonstrated hypomethylation of the IFN regulatory factor 7 (IRF7) promoter [49].

Another study tested the methylation pattern in juvenile patients, and the results suggested a global reduction in methylation levels in CD4+ T cells. This further suggests that this may lead to reduced expression of DNMT1 [50]. Changes in methylation patterns were unique in both cases, where differentially methylated gene count is more in localized scleroderma (jLS) compared to juvenile-onset systemic sclerosis (jSSc) [51]. Regarding methylation level, most genes are hypermethylated in blood samples. ARRB1 is hypomethylated in jLS patients, a gene belonging to the arrestin gene family [36]. TEAD3 is hypermethylated, a gene responsible for cell proliferation and contact activities [52]. The ITCH gene is also hypermethylated and is only seen in jLS patients. The function of this gene is to regulate immune function [53]. Differential methylation of ACTN1 and CAPN8 in jLS patients was also discovered [54]. Both jSSc and jLS patients had higher levels of DNA methylation than controls at CpG sites in the FGFR2 5 ^′-UTR, while jLS patients’ levels exceeded those of jSSc patients’ [28].