DNA methylation is a biochemical process of methyl group binding with the cytosine ring carbon at position 5 to form 5 methylcytosine (5-mC). In mammals, DNA methylation occurs preferentially in CpG dinucleotides located throughout the whole gene either as single dinucleotide or concentrated into CpG-islands in vicinity of gene promoters. Hypermethylation of the promoters is an indicator of dense heterochromatin conformation, which blocks the binding of TF to DNA and leads to inactivation of gene transcription. The lowlevel methylation of promoters (hypomethylation) is associated with open chromatin conformation and active transcription of the gene (Eden and Cedar, 1994). DNA methylation is a reversible process, which could therefore be considered as a therapeutic target.

Recent studies confirmed a global DNA hypomethylation in T-cells and monocytes of RA patients compared to healthy individuals (de Andres et al., 2015). Genome-wide analysis of DNA methylation by microarrays revealed its alterations in B-cells on the early stages of RA in patients who have not yet received treatment compared to healthy donors (Glossop et al., 2016).

Cribbs et al. (2014) analyzed an aberrant function of regulatory T cells (Treg) in RA patients and found a specific region in the promoter of the CTLA-4 (−658 CpG), which was hypermethylated in comparison with healthy controls. DNA hypermethylation prevents binding of the nuclear factor of activated T cells (NF-AT) with cytoplasmic one, called NF-ATc2, which leads to decrease of CTLA-4 expression. As a consequence, Treg cells were unable to induce expression and activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO), which in turn resulted in a failure to activate the immunomodulatory kynurenine pathway (Cribbs et al., 2014). Furthermore, treatment with methotrexate induced DNA hypomethylation of FoxP3 locus in Treg. This results in the gene

upregulation with consequent increase of CTLA-4 concentration and normalization of Treg function in RA. These studies clearly illustrate how aberrant DNA methylation can affect cell functions and how epigenetic mechanisms can be used in therapy (Cribbs A.P. et al., 2015). To determine differentially methylated regions as potential epigenetic risk factors and markers of RA predispositions, Liu et al. (2013) performed epigenomewide association study. Using Illumina 450k microarrays they examined more than 485,000 CpG sites in peripheral blood of 354 RA patients and 337 healthy donors. As a result, 10 differentially methylated CpG sites were identified. All of them are localized on 6p12.1 and form two separate clusters within the locus also containing the genes of the major histocompatibility complex (MHC) that is known as the risk locus of RA (Raychaudhuri et al., 2012). This confirms the role of DNA methylation as an additional mechanism determining susceptibility to RA. Importantly, the heterogeneity of cell population isolated from a whole blood may cause diverse methylation profile. Thus, this factor should be taken into account in bioinformatic analysis to reduce possible biases.

Some of these results were confirmed by other studies. Aberrant DNA methylation was detected in peripheral blood mononuclear cells (PBMCs) of RA patients. For example, van Steenbergen et al. (2014) demonstrated that cg23325723 site was significantly associated with RA (p = 0.026) in PBMCs. Four other CpG sites (cg16609995, cg19555708, cg19321684, and cg25949002) demonstrated similar different methylation in PBMCs comparing to control samples, which was not, however, statistically significant.

Other studies have shown abnormal methylation of one cytosine in the IL-6 promoter in RA PBMCs associated with reduction of its transcription (Nile et al., 2008). At the same time the loss of cytosine methylation in the IL-10 promoter correlates with higher expression of IL-10 in such cells (Chen et al., 2011).

LRPAP1 gene is expressed in PBMCs and encodes the chaperone of low density lipoprotein receptor-related protein 1, that affects the activity of transforming growth factor beta (TGF-β) (Kolker et al., 2012). It was found that 4 CpGdinucleotides in exon 7 of LRPAP1 were hypermethylated in patients who demonstrated no response to the therapy by TNF inhibitors (etanercept) compared to responders. The locus of cg04857395 overlaps structures involved in alternative splicing: the region associated with trimethylation of histone H3 at lysine 36 (H3K36me3) and the binding site of CCCTC-binding factor, which is a methyl-sensitive transcriptional repressor (Lev Maor et al., 2015).

An important point to consider in epigenetic studies of PBMCs is the effect of cell heterogeneity. If the experimental data are not normalized according to the proportion of the cells of different types in the fraction of PBMCs, the differentially methylated regions (DMRs) in certain cell types could be missed.

DNA methylation in peripheral blood mononuclear cells was recently described by Zhu et al. (2019). DNA methylation profiling and gene expression profiling were measured in patients with RA and in healthy controls. Differentially methylated sites and genes identified an interferon inducible gene interaction network. The significance of PARP9 gene methylation and its associated change in the expression in the pathogenesis of RA was demonstrated. In addition, its ability to positively regulate IL2, which stimulates various cells of the immune response, has been revealed (Zhu et al., 2019).

Epigenetic regulation of immune cells can be crucial for the development and maintenance of autoimmune diseases, such as RA. Julià et al. (2017) investigated the methylation patterns of B lymphocytes in patients with RA and systemic lupus erythematosus. Differentially methylated in patients and in the control group CpG sites were located in the CD1C, TNFSF10, PARVG, NID1, DHRS12, ITPK1, ACSF3, and TNFRSF13C genes and two intergenic regions (10p12.31). Differential methylation of these genes was also reproduced in the cohort of patients with SLE. This indicates similar patterns of epigenetic changes in B-lymphocytes in these two autoimmune diseases (Julià et al., 2017).

### ABERRATIONS OF DNA METHYLATION IN SYNOVIAL FIBROBLASTS IN RA

Rheumatoid arthritis synovial fibroblasts (RASFs) have a unique, non-random methylation pattern - methylome, which is specifically reorganized during the disease progression and varies depending on the joint localization. A precise mechanism of the methylome changing remains still unclear, but the overall pattern of differential methylation corresponds to the aggressive phenotype acquisition in SF results in the development of the disease. Earlier studies of epigenetic changes in RASFs demonstrated an abnormal expression of retroviral sequences LINE-1, associated with loss of silencing of these mobile elements as a result of hypomethylation (Neidhart et al., 2000). Global DNA hypomethylation is observed in many hyperproliferating tissues and is associated with a relative lack of methyl groups' donor S-adenosylmethionine (SAM). SAM is required to restore the DNA methylation after cell division, as well as for polyamines recycling. Increased cell proliferation leads to increased polyamines processing, competing with DNA methylation (Brooks, 2012). Interestingly, the key enzymes that are involved in the polyamine synthesis are encoded in the X chromosome and an elevated level of polyamines is found in many autoimmune diseases including RA (Furumitsu et al., 1993). Thus, high level of polyamines is associated not only with DNA hypomethylation, but also with an increased risk of RA development in women.

Nakano et al. (2013a) have shown that DNA hypomethylation increases expression of numerous genes: growth factors/ receptors, extracellular matrix proteins, adhesion molecules, and matrix degrading enzymes, etc. Expression of the DNA methyltransferase-1 (DNMT1) is reduced on protein level in RASFs comparing to the osteoarthritis synovial fibroblasts (OASF), particularly when stimulated with cytokines or growth factors. However, DNMT1A transcripts levels are similar in both of these cell types. Additionally, transcription of DNMT1 could be reduced by stimulation of IL-1 (Nakano et al., 2013a).

A number of differentially methylated loci were reported in RASFs. For example, hypomethylation of CXCL12

(chemokine C-X-C motif ligand 12) promoter is associated with the gene's upregulation and accumulation of the protein in the joints of RA patients that contributes to chronic inflammation. TBX5 regulates expression of proinflammatory cytokines and chemokines in SF including CXCL12 chemokine, which is the downstream effector of the same pathway. Hypomethylation of TBX5 (T-box transcription factor 5) increases its own expression as well as CXCL12 expression in RASFs. Treatment of SF cell culture with 5-aza-20 -deoxycitidine (5-aza-dC) to achieve DNA demethylation induces hypomethylation of promoter region and subsequent re-activation of CXCL12 expression (Karouzakis et al., 2014).

Comprehensive analysis of DNA methylation in RASFs by Nakano et al. (2013b) identified 1 859 differently methylated loci. Some of the hypomethylated loci that were critically important for RA pathogenesis were located in the genes CHI3L1, CASP1, STAT3, MAP3K5, MEFV, and WISP3. Conversely, genes TGFBR2 and FOXO1 were hypermethylated. As shown by analyzing regulatory pathways, the aberrantly methylated genes were involved in cell migration, adhesion, transendothelial penetration and interactions in the extracellular matrix (Nakano et al., 2013b).

Analyzed patterns of DNA methylation suggest its aberrations do not occur randomly, but are specifically related to regulatory pathways involved in RA pathogenesis. Interestingly, RASFs DNA methylation patterns are altering during disease development and progression from early to chronic stage (Whitaker et al., 2013).

A recent study reported genome differently methylated sites that are localized in CpG-islands regions in promoters of RA patients with different clinical symptoms and age of manifestation (Karouzakis et al., 2018). Specifically methylated CpG-islands were found on every stage of the disease. Significant hypermethylation of CpG-islands was revealed in the promoters of peptidase M20 containing domain-1 gene (PM20D1), SHROOM1 and engrailed-1 homeobox protein (EN1) at very early RASFs compared to normal SFs. SHROOM1 gene is involved in the development of nervous tissue and the rearrangement of microtubules during cell division. The chondrocytes of knee joints in early RA and transient arthritis patients significantly differ in SHROOM1 methylation making it a valuable biomarker for early diagnostics of the disease (Bonin et al., 2016).

Another set of genes with hypermethylated promoters has been identified in patients with chronic disease: microfibrillarassociated protein 2 (MFAP2), discoidin domain receptor (DDR1) tyrosine kinase and the major histocompatibility complex HLA-C. Several identified CpG-islands were specifically hypermethylated in the SFs of very early and/or chronic RA. The MFAP2 binds TGFβ and the members of bone morphogenetic protein (BMP) family, thus regulates release and activation of these factors involved in the development of arthritis (Weinbaum et al., 2008). Tyrosine kinase DDR1 binds collagens and can regulate various cellular processes such as cell migration, invasion, and proliferation (Juskaite et al., 2017). This indicates promising targets for further functional experiments that

could explain the phenotypic changes in RASFs and their invasive behavior.

To identify novel therapeutic targets for RA treatment, the analysis of the methylome was recently suggested along with other data on RASFs. Whitaker et al. (2015) have combined findings from genome-wide association studies and analysis of differential gene expression and DNA methylation analysis in RASFs and OASFs. As a result, a number of genes were chosen as prominent candidates for further investigation relevant for RA pathogenesis: ELMO1, LBH, and PTPN11, which are directly involved in the pathogenesis of RA and may be used as therapeutic targets.

ELMO1 encodes a protein involved in cytoskeleton reorganization, which is crucial for phagocytosis of apoptotic cells and cell motility. ELMO1 promoter is hypermethylated in RASFs, and its knockdown suppresses the RASFs migration and invasion by reducing the activation of RAC1 GTPase (Whitaker et al., 2015). These results demonstrate how the integration of datasets from genome-wide methylation and gene expression analyses allows identifying proteins with previously unknown critical role in RA development. Such a complex "omics" approach can be extended from studying only promoter regions of the genes to enhancers, silencers and other regulatory sequences with almost unknown effect of methylation. The application of such approaches led to discovery of novel differentially methylated loci in the RASFs.

Previously it was thought a protein encoded by LBH contributes only in embryonic development. However, LBH promotor was found to be hypomethylated in RASFs as well as its enhancer. The gene knockdown affected the transcriptome including pathways that control cell growth and proliferation in RASFs (Ekwall et al., 2015). Interestingly, its enhancer domain contains single nucleotide polymorphism (SNP) rs906868 associated with RA. The combination of the SNP genotype and methylation affects the activity of enhancer and, consequently, expression of LBH (Hammaker et al., 2016).

PTPN11 encodes the tyrosine phosphatase SHP2 and is upregulated in RASFs (Stanford et al., 2013). Analysis of the PTPN11 enhancer in RASFs revealed hypermethylation, which increased the sensitivity of cells to glucocorticoids and their aggressiveness. This not only explained the mechanism of action of PTPN11 in RASFs but also demonstrated SHP2 as a potential therapeutic target in RA. The results were confirmed on mouse models of arthritis (Whitaker et al., 2016).

Summing up, analysis of the methylome in RASFs contributes to understanding of RA pathogenesis. Not only local cytokine environment but other factors can potentially affect DNA methylation pattern and participate in establishing a stable phenotype of RASFs. Identification of these environmental factors could shed light on the predisposition to RA development and progression. In addition, studies of RASFs used the novel "omics" technologies – include not only methylome but also the other types of epigenetic markers – will help discover novel molecular factors in the RA pathogenesis and determine potential therapeutic targets. Considering the role of differentially methylated genes from the pathway perspective, several cascades were reported to be usually disturbed in very

early RASFs and chronic RASFs in comparison with normal SFs but exhibit no changes in the SFs in transient arthritis. These regulatory pathways include cadherin, integrin, WNT signaling of cell adhesion, components of the actin cytoskeleton and antigen presentation.

#### HISTONE MODIFICATIONS IN IMMUNE CELLS IN RA

Histone modifications are important epigenetic marks that affect gene expression and determine phenotype of cells. Histone proteins are bound to DNA and regulate the accessibility of gene promoters for transcription factors. The basic functional unit of chromatin is the nucleosome. It contains 147 base pairs of DNA, which are wrapped around a histone octamer that consists of two copies each of histones H2A, H2B, H3, and H4. The epigenetic landscape of histones can be modified by numerous mechanisms including acetylation, methylation, citrullination, phosphorylation, ubiquitinylation, and sumoylation (Tessarz and Kouzarides, 2014). Some histone marks are associated with an open chromatin structure. This makes chromatin accessible to transcription factors and can significantly increase gene expression. These include histones lysine residues acetylation (H3K9, H3K14, H4K5, and H4K16) and methylation (H2BK5, H3K4, H3K36, and H3K79); phosphorylation of histone H3 threonine 3 (H3T3) and serines (H3S10 and H3S28), and as well as histone 4 serine 1 (H4S1) and H2BK120 ubiquitinylation. On the other hand, repressive histone marks that correlate with heterochromatin state and gene repression include methylation of H3K9, H3K27, and H4K20; ubiquitination of H2AK119; and sumoylation of H2AK126, H2BK6, and H2BK7 (Araki and Mimura, 2016). Most studies have focused on the acetylation and methylation of histones, although citrullinated histones in RASFs has also been reported (Wang et al., 2016).

Different types of histone modifications are colocalized throughout the genome in order to stabilize active or repressed chromatin states. This complicates analysis of RA-specific alterations of histone marks. The expression of histonemodifying enzymes (e.g., histone deacetylases and HDAC) could be studied instead. The interest to the expression of HDAC in RA arose after reports on its inhibitors, which were considered as novel and promising therapeutic strategy of inflammatory diseases. One of the studies revealed activation of HDAC in PBMCs of RA patients compared to healthy controls (Toussirot et al., 2013). Controversial data were also obtained for HDAC activity in the synovial tissues of RA patients. Presumably, HDAC activity depends strongly to the disease progression and therapy. This may explain discrepancies in the measurements among different cohorts of patients whereas the changes that were identified in larger and clearly defined groups got lost.

#### HISTONE MODIFICATIONS IN RASFs

Similarly as in immune cells, direct studies of histone modifications in synovial fibroblasts are quite rare. Huber et al. (2007) demonstrated an overall increase of acetylation associated with reduced HDAC activity as a result of decreased HDAC1 and HDAC2 gene expression in the synovial tissue of RA patients. Suppression of HDAC1 and HDAC2 suggests the balance between histone acetylases (HAT) and HDAC activity shifts toward hyperacetylation in RA synovial tissues. However, another study demonstrated that HDAC activity and HDAC1 expression were upregulated in RA synovial tissue (Kawabata et al., 2010). Not all members of the HDAC family have pro-inflammatory effect. For example, HDAC5 demonstrates anti-inflammatory functions in SFs. That indicates the applicability of specific rather than general HDAC inhibitors for the RA treatment (Angiolilli et al., 2016). Recently, suppression of HDAC3 expression was found to be as effective in suppressing pro-inflammatory factors in the RASFs as general inhibition of HDAC, which makes HDAC3 a promising candidate for targeted therapy (Angiolilli et al., 2017).

#### Abnormal Modifications of Histones Are Involved in the Activation of RASFs

H3K27 specific histone methyltransferase (HMT) – the enhancer of zeste homolog 2 (EZH2) – is highly expressed in RASFs as a result of TNFα induction of nuclear factor kappa B (NF-KB) and mitogen-activated protein kinase (MAPK) pathways. The expression of the secreted frizzled-related protein 1 (SFRP1) – a EZH2 target gene – is increased under the active histone marks (H3K4me3 and H3K27me3) in its promoter that leads to the Wnt (wingless-type MMTV integration site signaling)-pathway inhibition in RASFs (Trenkmann et al., 2011).

Transcription factor T-box transcription factor 5 (TBX5) is overexpressed in RASFs and active histone marks – including H4K4me3 and histone acetylation – are widely represented in its promoter. The overexpression of TBX5 affects expression of 790 genes including IL-8, chemokine C-X-C motif ligand 12 (CXCL12) and chemokine C-C motif ligand 20 (CCL20) confirming its role as an inductor and regulator of chemokines important in RA development (Karouzakis et al., 2014).

Ai et al. (2018) performed the comprehensive study, describing histone modification, open chromatin, RNA expression, and genome-wide DNA methylation in synovial fibroblasts in RA patients. To determine complex multidimensional interactions in the epigenetic regulation of RA, an integrative analysis was performed using a new method for detecting genomic regions with similar profiles. In addition to the known pathological pathways that are activated in RA, the authors found novel pathway activated in RA that was previously known to be associated with Huntington's disease (Ai et al., 2018).

In the other paper (Webster et al., 2018) epigenetic changes in RASF in 79 pairs of discordant on RA monozygotic twins were revealed. An epigenetic signature has been shown to indicate the association of stress response pathways and RA pathogenesis. It is noteworthy that potential epigenetic disruption of multiple RUNX3 transcription factor binding sites was proposed to be associated with disease development.

### Histones Modifications Affect Matrix Metalloproteinase Genes Regulation

Araki et al. (2015) reported significantly higher levels of the activating trimethylation mark H3K4me3 in promoters of MMP-1, MMP-3, MMP-9, and MMP-13 along with reduced of the repressive modification H3K27me3 in promoters of MMP-1 and MMP-9 in RASFs. Furthermore, the elevated level of histone H4 acetylation was associated with upregulation of MMP-1 (Maciejewska-Rodrigues et al., 2010).

Tryptophan-aspartate (WD) repeat-containing protein 5 (WDR5) is a major subunit of the proteins bound with SET1 (COMPASS) or COMPASS-like complexes that catalyze H3K4 methylation. WDR5 knockdown reduces the level of H3K4me3 marks as well as the abundance of MMP-1, MMP-3, MMP-9, and MMP-13 in RASFs. IL-6 and soluble IL-6 receptor α (sIL-6Rα) induce the expression of MMP-1, MMP-3, and MMP-13 but not of MMP-9: It has been shown that IL-6-induced signal transducer and activator of transcription 3 (STAT3) binds to MMP-1, MMP-3, and MMP-13 promoters but not with that of MMP-9. High expression of IL-6 was associated with high level of histone H3 acetylation (H3ac) of the IL-6 promoter in RASFs (Wada et al., 2014).

#### microRNAs AS AN EPIGENETIC FACTOR ASSOCIATED WITH THE DEVELOPMENT OF RA

MicroRNAs (miRNAs) are small non-coding RNAs of 17–25 nt that regulate gene expression by either repressing the translation or causing degradation of multiple target mRNAs (Fabian et al., 2010). miRNAs play an important role in many biological processes including the development of the immune system and the subsequent regulation of immunity both innate and acquired (Chen et al., 2016). Currently more than 100 miRNAs have been identified that could potentially affect the molecular pathways in immune cells development and their functions regulation (Baulina et al., 2016).

#### ABERRATIONS OF microRNA EXPRESSION IN RA

Aberrant miRNA regulation occurs in various cells and tissues in RA (Tavasolian et al., 2018). The role of miRNA in the inflammatory process includes both control of cytokine production and protection of cartilage tissue by regulating catabolic activity, proliferation and resistance to apoptosis.

The increased production of IL-17 by T helper cells (Th17) in the synovial fluid may suppress miRNA-23b and so enhance expression of (TGF)-β-activated kinase 1/MAP3K7 binding protein and Iκβ kinase α contributing to inflammation. miRNA-21 promotes the differentiation of Th2 and Treg cells and is also associated with the regulation of Treg apoptosis (Salehi et al., 2015). Pro-inflammatory phenotype of Treg is determined by an aberrant miRNA-146a expression; thus its reduction in RA patients inversely correlated with disease activity and expression of its direct target gene STAT1 (Zhou et al., 2015).

A number of studies have described miRNAs that modulate inflammatory or catabolic functions of RASFs thereby contributing to the development of the aggressive phenotype in RA (Vicente et al., 2016).

miRNA-146 and miRNA-155 were first described as abnormally expressed in synovial tissue, RASFs and synovial fluid of patients with RA and still remain the best characterized candidates. miRNA-155 is upregulated in RASFs, where – in addition to the pro-inflammatory activity – it regulates destructive processes due to the matrix metalloproteinases MMP-1 and MMP-3 expression repressing (Long et al., 2013). Similarly, miRNA-146a express increasingly in RASFs, despite a known role as a negative regulator of inflammation in immune cells (Vicente et al., 2016).

Increased expression in RASFs has also been determined for a number of other transcripts: miRNA-203 (Stanczyk et al., 2011), miRNA-221 (Yang and Yang, 2015), miRNA-663 (Miao et al., 2015a), miRNA-222 and miRNA-323- 3p (Pandis et al., 2012). Besides their role in immune processes, these microRNAs are involved in oncogenesis by regulating cell invasiveness and migration in different types of tumors.

Earlier reports show miRNA-203 overexpression associated with hypomethylation of MMP1 and IL-6 gene promoters induces these proteins production. Regulation by DNA methylation was also observed in mir-203. Interestingly, this miRNA high-level expression in RASFs is observed with its own promoter hypomethylation (Stanczyk et al., 2011).

Suppression of miRNA-221 inhibits the production of pro-inflammatory cytokines by fibroblasts, causes cell apoptosis and reduces their migration and invasion of RASFs (Yang and Yang, 2015).

miRNA-663 regulates proliferation of RASFs and production of IL-6 via inhibition of the tumor suppressor APC thereby affecting the Wnt signaling pathway (Miao et al., 2015a).

Overexpression of miRNA-124a in RASFs may disrupt the cell cycle and lead to inhibition of cell proliferation through repression of its target genes CDK-2 and MCP-1. Additionally, it was shown that miRNA-124a expression is regulated by methylation of the gene from which it is transcribed. Demethylation of the miRNA-124a gene by 5-aza-dC reduces RASF proliferation and expression of TNF-α (Zhou et al., 2016). Regulation by DNA methylation was also observed in mir-203. Its high expression in RASFs is associated with the promoter hypomethylation (Stanczyk et al., 2011).

The similar effect on RASFs proliferation has miRNA-34a∗ (Niederer et al., 2012). It regulates genes of the apoptosis inhibitor XIAP. Furthermore, direct correlation of miRNA-21, miRNA-25, and miRNA-124a from peripheral blood cells with estradiol level in plasma was described in women with RA. The effect of estradiol on miRNA-124a is particularly interesting, as this miRNA has an effect on synovial proliferation (Singh et al., 2013). Decreased expression of both miRNA-124a and miRNA-34a∗ as well as the others: miRNA-152, miRNA-375, and miRNA-22 in RASFs contributes to RA development (Vicente et al., 2016).

miRNA-152 and miRNA-375 directly target DNMT1. Thus, the increase of their expression leads to activation of Wnt-signaling pathway (Miao et al., 2015b).

Cyr61 expression is increased by miRNA-22 suppression, affecting various genes involved in different processes: angiogenesis, inflammation, matrix structure reorganization, IL-6 production with subsequent differentiation of Th17 and synovial hyperplasia. Earlier data show that the reduced expression of miRNA-22 is caused by p53 mutation that is frequent in RASFs (Lin et al., 2014).

The loss of miRNA-10a-5p expression in RASFs upregulates target gene TBX5 thereby promoting the production of TLR3, MMP-13 and a number of pro-inflammatory cytokines (Hussain et al., 2018). Suppressed expression of miRNA-10a may cause the activation of NF-κB and enhancing the release of proinflammatory cytokines TNF-α, IL-1β, IL-6, and IL-8, chemokine MCP-1 and matrix metalloproteinases MMP-1 and MMP-13 (Mu et al., 2016).

Deregulations of expression affect entire miRNA clusters. However, the expression of individual miRNAs of a cluster may be altered differentially. For example, miRNA-18a is upregulated in the miRNA-17-92 cluster, which plays an important role in the regulation of apoptosis in the RASFs while miRNA-19a/b, miRNA-20a and miRNA-30a-3p are downregulated. Such an effect may be caused by the different target genes affection and different signal pathways regulation (Vicente et al., 2016).

The RA associated changes of miRNA expression were also confirmed in other cells of the joint capsule. For example, miRNA-323-3p is involved in the regulation of Wnt and cadherin signaling pathways, and its overexpression in chondrocytes causes degradation of the cartilaginous matrix and promotes bone erosion. Similar effect was demonstrated for miRNA-140, which contributes to reduction of joint destruction via suppression of ADAMSTS5. Upregulation of some miRNAs (miRNA-30a, miRNA-204, miRNA-211, miRNA-320, and miRNA-335) is associated with suppression of osteoblast differentiation via RUNX2 regulation (Moran-Moguel et al., 2018).

### CIRCULATING miRNAs AS POTENTIAL MARKERS OF RA DEVELOPMENT

In addition to aberrant miRNAs expression in the joint area, they were also detected in blood: plasma, serum, and various blood cells (**Table 1**). In one of the first studies of miRNA expression in RA a significant difference between the 26 microRNAs expression patterns was demonstrated in patients compared to healthy donors. Three of those, namely miRNA-24, miRNA-26a, and miRNA-125a-5p have been proposed as a potential diagnostic panel with sensitivity and specificity of 78.4 and 92.3%, respectively (Murata et al., 2013).

Nevertheless, estimation of two other miRNAs expression level is considered more prominent at the moment. miRNA-146a and miRNA-155 are overexpressed both in whole blood and PBMCs of RA patients (Mookherjee and El-Gabalawy, 2013).

Many miRNAs that are aberrantly expressed in synovial tissues in RA are also deregulated in peripheral blood. However, these changes are not always similar. The concentrations of miRNA-16, miRNA-132, and miRNA-223 were significantly lower in the synovial fluid than in plasma of patients with RA, and no correlation between them was found (Murata et al., 2010). Elevated level of miRNA-125b was observed in both serum and synovial tissues of RA patients (Zhang B. et al., 2017).

While miRNA-146a is overexpressed in synovial tissue of RA patients comparing with healthy donors, a reduction of circulating miR-146a was reported in the peripheral blood (Wang et al., 2012). Besides, miRNA-146a from PBMCs does not demonstrate any correlation with disease activity. That is in contrast with the same miRNA quantified in serum. Thus, the miRNAs obtained from different types of samples may have different prognostic value or not have it at all (Ayeldeen et al., 2018).

Changes in the expression of miRNAs may be associated with the RA therapy (**Table 1**). Specifically, miRNA-146a, miRNA-155, and miRNA-16 levels were decreased in serum at the early RA stages after treatment with disease-modifying antirheumatic drugs (DMARDs) (Filková et al., 2014). In contrast the quantity of miRNA-16-5p, miRNA-23-3p, miRNA125b-5p, miRNA-126-3p, miRNA-146a-5p, and miRNA-223-3p in plasma was significantly elevated after combined anti-TNF-α/DMARD therapy (Castro-Villegas et al., 2015).

### miRNAs Genes Polymorphism Associated With RA

miRNAs not only regulate gene expression in RA but also are themselves subject to regulation by various factors. As in protein-coding genes, polymorphism of miRNA genes or their target genes may be associated with predisposition to RA. The number of studies has reported a significant role of some nucleotide polymorphisms located in the genes of miRNA-146a and miRNA-499 precursors (Ayeldeen et al., 2018). For example, the importance of polymorphism rs3746444 in miRNA-499 was confirmed for the RA development among the patients of Caucasian race. Genotypes TC and CC and allele C at this SNP in miRNA-499 were characterized as independent risk factors of joint erosion in RA patients. The frequency of GG genotype of rs2910164 in miRNA-146a is significantly higher in patients with RA compared to healthy donors (Ayeldeen et al., 2018). Another study that included 200 RA patients and 120 healthy donors has demonstrated the correlation of SNP rs22928323 in miRNA-149 with RA development but no association with further clinical characteristics (Xiao et al., 2015).

#### DNA Methylation of miRNA Genes

Similarly to protein-coding genes, DNA methylation is an important mechanism of miRNA regulation. It was demonstrated

TABLE 1 | Potential diagnostic and prognostic markers of RA among miRNAs.

that miRNA-124a and miRNA-203 are controlled by DNA methylation of respective genes in RASFs. In vitro 5-azacitidine treatment leads to DNA demethylation and transcriptional re-activation (Stanczyk et al., 2011; Zhou et al., 2016). de la Rica et al. (2013) provided additional evidence of such epigenetic regulation in the whole genome by profiling of the methylome and analysis of miRNA and mRNA expression in RASFs in parallel. Expression of 11 miRNAs was reduced in RA samples comparing to control osteoarthritis samples and was associated with hypermethylated genes thereof. In contrast, four other miRNAs were upregulated upon hypomethylation of CpG sites in vicinity of their genes (de la Rica et al., 2013).

#### Long Non-coding and Circular RNAs in RA

Several long non-coding RNAs (lncRNAs) were deregulated in RA by miRNAs. The expression of the most characterized lncRNA HOTAIR (HOX transcript antisense RNA) was significantly reduced in chondrocytes that were pretreated with lipopolysaccharide in order to suppress the inflammatory process. This transcript is able to suppress mir-138-mediated synthesis of NF-κB, since miRNA-138 is a HOTAIR direct target (Zhang H. et al., 2017).

Expression of the other lncRNA ZFAS1 (zinc finger antisense 1) is increased in RA synovial tissue compared to healthy donors and can enhance migration and invasion of RASFs by directly affecting miRNA-27a (Ye et al., 2018).

lncRNA GAPLINC (Gastric Adenocarcinoma Predictive Long Intergenic Non-coding RNA) is overexpressed in RASFs and regulates their proliferation, migration and pro-inflammatory cytokine production by operating as a sponge for miRNA-382-5p, and miRNA-575 (Mo et al., 2018).

A possible effect of circular RNAs (circRNAs) on miRNAs in RA was revealed, too. Presumably, these transcripts

complementarily bind to miRNAs thereby preventing their interaction with target genes (Zheng et al., 2017).

#### SUMMARY AND FUTURE DIRECTIONS

In the last few decades, many studies have shown that epigenetic mechanisms are involved in the regulation of all biological processes in the body from impregnation to death. These functional mechanisms are involved in genome reorganization, control of gametogenesis and early embryogenesis, and play an important role in cell differentiation. Changes in DNA methylation and posttranslational modifications of histones are key epigenetic events contributing to the reorganization of chromatin into euchromatin, heterochromatin, and regions of nuclear compartmentalization, which allow to regulate gene expression by forcing them to be consistently switched on and off for the normal development of a multicellular organism. Epigenetic changes may form and appear over a long period of time, for example, during the training and organization of memory (Moosavi and Motevalizadeh Ardekani, 2016). Aberrations of epigenetic modifications can cause the development of congenital defects, hereditary diseases and multifactorial diseases including malignant tumors in different periods of life. DNA methylation, histone modifications, expression of proteins that generate or remove epigenetic marks, and ncRNAs affect inflammatory and matrix-degrading pathways and could be changed in RA. Epigenetic mechanisms play an important role in the pathogenesis of the disease.

Unlike genetic lesions, epigenetic alterations are reversible and could be modulated by diet, drugs and other environmental factors. This epigenetic flexibility suggests strategies for prevention and therapy of diseases with confirmed pathogenic role of epigenetic factors (Pashayan et al., 2016).

Therapeutic targeting of epigenetic mechanisms can be a successful approach in the treatment of chronic inflammatory diseases. Significant efforts have already been made to develop drugs that able to restore or alter the epigenetic mechanisms. DNA methyltransferase inhibitors (DNMT), 5-azacitidine (Vidaza) and 5-Aza-20deoxy-5-azacitidine (Decitabine) are already being used to treat inflammatory conditions in pancreatitis therapy. Two types of HDAC inhibitors (HDACi) are used for treatment: pan-inhibitors with broad spectrum of action and specific inhibitors that target a specific class of HDAC enzyme. To date, the Food and Drug Administration (FDA) has approved four HDACi: Vorinostat, Romidepsin, Panobinostat, and Belinostat. These products have the minimal side effects and are mainly used for the hematological tumors treatment (Samanta et al., 2017).

Moreover epigenetic alterations are a source of diagnostic and prognostic markers. The gradual changes of epigenetic marks that may be caused by environmental factors can result in both the development and progression of pathological conditions. DNA methylation marks are the

best characterized, its role is comprehensively studied in cancer development. The existence of loci and genes with differential methylation patterns, varying, respectively, the stage of the disease, was also revealed in patients during RA progression (Ai et al., 2015). However, the plasticity of the epigenome is a complication for the researches. The prognostic markers panels consisted of epigenetically modified genes could differs individually due to the variability of environmental factors affecting patients. Smoking is the most common environmental risk factor in RA development and its severity determination. In addition to directly affecting lung tissues, smoking alters the expression of sirtuins (SIRT), the proteins of the deacetylase families that are involved in modification of histone and non-histone proteins. SIRT maintain the integrity of the genome during the cellular response to stress by using epigenetic mechanisms, and are therefore key molecules in the body's adaptation process. The change of expression of SIRT1 and SIRT6 in RASFs was demonstrated (Engler et al., 2016).

In contrast to genetic aberrations that are persist throughout life, epigenetic changes may vary in different cell populations as well as in the same cell depending on conditions and developmental stage. Since the whole blood cells, T- and B-lymphocyte populations, and SFs exhibit different DNA methylation patterns, the analysis of epigenetic markers in a mixed cell population may hamper in the correct evaluation of the epigenetic panel (Liu et al., 2013; Glossop et al., 2016).

Similarly, miRNA profiles differ across individuals, cell populations and may be affected by concomitant diseases (Huang et al., 2011). Thus, it is important to consider the extracted cell type during the development of RA epigenetic markers panel. Individual patient therapeutic response could also be predicted with the epigenetic panel use. Currently, a large number of drugs for RA treatment is undergoing preclinical and clinical trials, however, the existing data linked the epigenetic changes and response to therapy is limited. Thus, intensive investigations in this field are critically needed. International cooperation will enable the access to larger patient cohorts thereby improving the quality of studies. However, when planning international consortia, it is important to consider the differences in ethnic composition that may be reflected in epigenetic variations (Rawlings-Goss et al., 2014). Some differences that were related to race and nationality of patients were demonstrated even for genetic markers.

Nevertheless, despite of the complications, investigation of epigenetic markers is undoubtedly a great achievement of molecular biology and molecular medicine. Epigenetic changes are the earliest factors that are associated with the development of the disease before its clinical manifestation. They can be used for prevention and monitoring of patient condition and also are the earliest to reflect the effect of the drug at the cellular level, before any systemic response of organism manifested as certain symptoms. Moreover, some of epigenetic markers, such

as changes in circulating miRNAs level in plasma, may be more accessible for evaluation than the other molecular markers.

#### AUTHOR CONTRIBUTIONS

MN, DZ, AL, and AZ contributed conception the review. MN, IB, DM, EK, EA, MB, and AD wrote sections of the

#### REFERENCES

manuscript. All authors contributed to manuscript revision, read and approved the submitted version.

#### FUNDING

This research was supported by Ministry of Education and Science of the Russian Federation (Agreement 14.605.21.0003, unique project ID RFMEFI60518X0003).

influence on gene expression. Arthritis Rheum. 43, 2634–2647. doi: 10.1002/ 1529-0131(200012)43:12<2634::aid-anr3>3.0.co;2-1

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Nemtsova, Zaletaev, Bure, Mikhaylenko, Kuznetsova, Alekseeva, Beloukhova, Deviatkin, Lukashev and Zamyatnin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Transposable Elements and Their Epigenetic Regulation in Mental Disorders: Current Evidence in the Field

#### Błazej Misiak ˙ 1 \*, Laura Ricceri2 and Maria M. Sa¸ siadek1

1 Department of Genetics, Wrocław Medical University, Wrocław, Poland, 2 Centre for Behavioural Sciences and Mental Health, Istituto Superiore di Sanità, Rome, Italy

Transposable elements (TEs) are highly repetitive DNA sequences in the human genome that are the relics of previous retrotransposition events. Although the majority of TEs are transcriptionally inactive due to acquired mutations or epigenetic processes, around 8% of TEs exert transcriptional activity. It has been found that TEs contribute to somatic mosaicism that accounts for functional specification of various brain cells. Indeed, autonomous retrotransposition of long interspersed element-1 (LINE-1) sequences has been reported in the neural rat progenitor cells from the hippocampus, the human fetal brain and the human embryonic stem cells. Moreover, expression of TEs has been found to regulate immune-inflammatory responses, conditioning immunity against exogenous infections. Therefore, aberrant epigenetic regulation and expression of TEs emerged as a potential mechanism underlying the development of various mental disorders, including autism spectrum disorders (ASD), schizophrenia, bipolar disorder, major depression, and Alzheimer's disease (AD). Consequently, some studies revealed that expression of some sequences of human endogenous retroviruses (HERVs) appears only in a certain group of patients with mental disorders (especially those with schizophrenia, bipolar disorder, and ASD) but not in healthy controls. In addition, it has been found that expression of HERVs might be related to subclinical inflammation observed in mental disorders. In this article, we provide an overview of detrimental effects of transposition on the brain development and immune mechanisms with relevance to mental disorders. We show that transposition is not the only mechanism, explaining the way TEs might shape the phenotype of mental disorders. Other mechanisms include the regulation of gene expression and the impact on genomic stability. Next, we review current evidence from studies investigating expression and epigenetic regulation of specific TEs in various mental disorders. Most consistently, these studies indicate altered expression of HERVs and methylation of LINE-1 sequences in patients with ASD, schizophrenia, and mood disorders. However, the contribution of TEs to the etiology of AD is poorly documented. Future studies should further investigate the mechanisms linking epigenetic processes, specific TEs and the phenotype of mental disorders to disentangle causal associations.

Keywords: retrotransposon, DNA methylation, LINE-1, Alu, SINE, SVA

Edited by: Yun Liu, Fudan University, China

#### Reviewed by:

Nicole Grandi, University of Cagliari, Italy Apiwat Mutirangura, Chulalongkorn University, Thailand

> \*Correspondence: Błazej Misiak ˙<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Epigenomics and Epigenetics, a section of the journal Frontiers in Genetics

> Received: 10 April 2019 Accepted: 04 June 2019 Published: 25 June 2019

#### Citation:

Misiak B, Ricceri L and Sa¸ siadek MM (2019) Transposable Elements and Their Epigenetic Regulation in Mental Disorders: Current Evidence in the Field. Front. Genet. 10:580. doi: 10.3389/fgene.2019.00580

### INTRODUCTION

fgene-10-00580 June 21, 2019 Time: 16:38 # 2

Mental disorders represent complex phenotypes and are the leading causes of global disease burden (Vigo et al., 2016). The phenotype complexity of mental disorders manifests in symptomatic and biological overlap, impeding a diagnostic process that is based on a clinical consensus without a crucial role of biological markers. Heritability rates of mental disorders are high, exceeding 80% in twin studies of schizophrenia and bipolar disorder (Cardno and Gottesman, 2000; McGuffin et al., 2003; Misiak et al., 2016). However, monogenic determinants with high penetrance rates have not been identified so far, and the concept of major mental disorders as the polygenic phenotypes prevails in the research approaches. Consequently, a paradigm shift toward investigating polygenic signatures, gene × environment (G × E) interactions and epigenetic mechanisms has been widely observed in the recent years.

The term 'epigenetics' refers to a number of reversible mechanisms that impacts gene expression without altering DNA sequence, and include DNA methylation and hydroxymethylation at the CpG islands, histone modifications as well as the regulation by microRNA species. It is now increasingly being recognized that the brain development is a complex process during which there is an increased sensitivity to the regulatory effects of epigenetic mechanisms (Nagy and Turecki, 2012). In light of existing evidence, major mental disorders, especially schizophrenia and autism spectrum disorders (ASD), are perceived as the neurodevelopmental disorders, occurring due to the effects of various genetic and environmental factors that affect critical periods of the brain development (Meredith, 2015).

Transposable elements (TEs) are the highly repetitive DNA sequences that constitute more than 50% of the human genome and contain about 52% of all CpG dinucleotides (Cordaux and Batzer, 2009; Su et al., 2012). Therefore, methylation at TEs is believed to serve as a proxy measure of global DNA methylation. Some TEs share similarity to exogenous viral agents and thus they are called endogenous retroviruses (Griffiths, 2001). Only about 7% of TEs are transcriptionally active (Oja et al., 2008). It has been estimated that approximately 0.27% of human genetic diseases are caused by retrotransposition (Callinan and Batzer, 2006).

Less is known about the contribution of TEs to the etiology of mental disorders. However, there is accumulating evidence that retrotransposition plays an important role in shaping somatic mosaicism that accounts for functional specification of brain cells (Baillie et al., 2011; Poduri et al., 2013). For instance, it has been reported that the transposition of long interspersed element (LINE)-1 sequences may play a role in differentiation of neurons during the brain development (Muotri et al., 2010).  Therefore, aberrant epigenetic regulation of TEs has been hypothesized to play an important role in the development of mental disorders. In this article, we provide an overview of transposition processes with relevance to major psychiatric disorders. Next, we review human and animal model studies investigating expression and epigenetic regulation of TEs in various mental disorders. Finally, we provide a summary of evidence with future directions and potential translation of findings to personalized precision medicine.

### BRIEF OVERVIEW OF TEs IN THE HUMAN GENOME – CLASSIFICATION AND NOMENCLATURE

Classification of TEs in the human genome was shown in **Figure 1**. A detailed description of the structure and function of various TEs can be found elsewhere (Darby and Sabunciyan, 2014). All TEs can be divided into two subgroups – type I TEs (retrotransposons) and type II TEs (DNA transposons). Type I TEs can be divided into two subgroups – long terminal repeat (LTR) elements, represented by the human endogenous retroviruses (HERVs) and non-LTR sequences that include LINEs, short interspersed elements (SINEs) and processed pseudogenes (Dewannieux and Heidmann, 2005). Retrotransposons act via RNA intermediates that are converted to DNA sequences before transposition (reverse transcription) (Munoz-Lopez and Garcia-Perez, 2010). Type II TEs encode enzymes required for insertion and excision, enabling direct transposition processes without the use of RNA intermediates (Pray, 2008b). Some TEs are autonomous and encode all enzymes that are necessary for transposition, while the rest of them require a transcriptional activity of other transposons. Type II TEs have lost a transposition activity (Darby and Sabunciyan, 2014).

The HERV sequences have likely existed as exogenous infectious factors; however, they have lost this activity due to acquired mutations (Bannert and Kurth, 2006). These TEs constitute 8% of the human genome and contain genes that are conservative for all retroviruses, including the gag, pro, pol, and env genes (Lander et al., 2001; Vargiu et al., 2016). The gag gene encodes proteins that build up matrix, capsid and nucleocapsid. Pro and pol encode protease, reverse transcriptase and integrase. In turn, the env gene is expressed to surface and transmembrane proteins. The HERV sequences in the human genome represent three classes of retroviruses: class I (e.g., HERV-E, HERV-W, HERV-FRD, and HERV-H), class II (e.g., HERV-K), and class III (e.g., HERV-L). This classification is based on the similarity to exogenous retroviruses. The HERV-K sequences are the youngest and exert the highest transcriptional activity. The HERV sequences can provide promoters, enhancers, repressors, poly-A signals and alternative splicing sites for human transcripts (Vargiu et al., 2016).

The LINEs that represent non-LTR elements, possess an autonomous retrotransposition activity and include LINE-1 and LINE-2 sequences. These sequences make up approximately 21% of the human genome (Lander et al., 2001; Schumann et al., 2010). The LINE-1 sequences contain their own promoters and encode two open reading frame proteins – ORF1 that is an RNA-binding protein and ORF2 with endonuclease and reverse transcriptase activities. They are the most abundant sequences from the LINE family, making up 18% of the human genome (Lander et al., 2001). The majority of LINE-1 sequences are transcriptionally inactive. The LINE-2 sequences in the human

genome are highly defective and can encode either one or two ORF proteins (Darby and Sabunciyan, 2014).

The SINEs are active and non-autonomous TEs, represented by the Alu and the Mammalian wide Interspersed Repeat (MIR) elements (11 and 3% of the human genome, respectively). The Alu sequences were named based on sharing a common cleavage site for the AluI restriction enzyme (Houck et al., 1979). The Alu sequences are active but require the reverse transcriptase that is encoded by LINE-1 sequences (Pray, 2008a). In turn, the MIR elements are inactive. It has recently been shown that SINEs may form more complex sequences that are classified as the SVA retrotransposons. The SVA sequences have been formed by coupling the SINEs, a variable number of tandem repeats and the Alu retrotransposons. The SVAs also require the LINE-1 expression for mobilization. These sequences contribute to about 0.1% of the human genome and are the most active group of retrotransposons (Ostertag et al., 2003; Wang et al., 2005).

In turn, pseudogenes are DNA sequences that are related to real genes but they have lost at least some protein-coding abilities. It has been found that mRNA of pseudogenes can be reverse transcribed by the proteins encoded by LINE-1 sequences and transferred into other regions of the genome, creating processed pseudogenes. It has been estimated that the human genome consists of over 7,800 pseudogenes (Zhang et al., 2003). In case of integration close to active promoters, processed pseudogenes can be further transcribed. As listed by Kazazian (2014), they share the following characteristics: (1) their sequences are similar to the transcribed part of the parent gene; (2) they lack all or most introns; (3) they contain a poly-A tail attached to the 30 -most transcribed nucleotide; and (4) they are flanked at their 50 and 30 terminals by target site duplications of 5–20 nucleotides.

Finally, little is known about type II TEs (DNA transposons) that have never been active in the human genome. Type II TEs include the hAT, MuDR, piggyBac, and Tc1/mariner sequences (Munoz-Lopez and Garcia-Perez, 2010). These transposons do not act via RNA intermediates and encode enzymes that enable their mobilization. Due to their inactivity their causal role in the etiology of human diseases is less likely (Darby and Sabunciyan, 2014).

### INSIGHTS INTO POTENTIAL MECHANISMS UNDERLYING THE ROLE OF TEs IN MENTAL DISORDERS

A recent review of human monogenic diseases that occur due to retrotransposition suggests that only the transposition of LINE-1, Alu, and SVA sequences might be deleterious, underlying the development of monogenic diseases (Kaer and Speek, 2013). Retrotransposition might affect various gene regions via altering their sequence or influencing expression activity. For instance, the Alu sequences contain several stop codons that may result in a truncated protein (Mighell et al., 1997). This mechanism has been discovered in patients with hemophilia B caused by transferring the Alu-Ya5 element into a protein coding region of the factor IX gene (Vidaud et al., 1993). In case of transposition into promoter regions, these sequences might impact gene expression. Another scenario originates from sequence homology that can promote homologous recombination, leading to insertions and deletions. Finally, the SVA tandems can mobilize exons, contributing to complex rearrangements.

However, the effects of alterations in DNA sequence triggered by retrotransposition have not been found to underlie the

development of common mental disorders. In the majority of studies of patients with mental disorders (reviewed in detail below), altered expression and/or epigenetic regulation of retrotransposons have been reported. There are several epigenetic processes that act as defense mechanism against retrotransposition, including DNA methylation, histone modifications, small RNA-mediated regulation and posttranscriptional silencing by DICER and siRNA (Lapp and Hunter, 2016). Indeed, the majority of TEs in the human genome are hypermethylated (Pray, 2008b). Although DNA methylation acts as a defense mechanism, it cannot be excluded that hypermethylation of newly inserted TEs can lead to further changes in chromatin conformation, triggering changes in the expression of adjacent genes. It is most likely that retrotransposition occurs during early development when epigenetic marks are removed (Darby and Sabunciyan, 2014). There are also some well characterized histone modifications, including trimethylation of lysine 9 and lysine 27 at histone H3 (H3K9me3 and H3K27me3, respectively), which lead to heterochromatin formation and transcriptional silencing of TEs (Day et al., 2010; Baker et al., 2012).

It should be noted that only a small subset of TEs has been reported to be involved in retrotransposition. For instance, only 30–60 LINE-1 sequences in diploid cells are capable of retrotransposition (Sassaman et al., 1997). In addition, the majority of LINE-1 sequences are methylated to a certain degree. It has been found that LINE-1 methylation might impact gene expression via specific mechanisms [for review see (Kitkumthorn and Mutirangura, 2011)]. Firstly, LINE-1 sequences may produce unique RNA transcripts that act beyond the LINE-1 location. Alternatively, the reverse LINE-1 promoter can transcribe unique DNA sequences beyond the 50 end of LINE-1. The second scenario is that intragenic LINE-1 RNAs decrease the expression of host gene via the nuclear RNAinduced silencing complexes. Indeed, it has been found that the Argonaute-2 (AGO2) protein targets intronic LINE-1 premRNA complexes leading to down-regulation of gene expression in cancer cells (Aporntewan et al., 2011).

Global DNA hypomethylation that progresses with aging has been associated with genomic instability (Jung and Pfeifer, 2015). Hypomethylated genome regions are prone to accumulate various types of DNA lesions that include oxidative damage, depurination, depyrimidation and pathologic endogenous double-strand breaks (Mutirangura, 2019). The latter ones are now believed to act as intermediate products that drive genomic instability (Mutirangura, 2019). Accumulating evidence indicates that methylation of TEs might protect against genomic instability processes. For instance, it has been demonstrated that Alu siRNA increases Alu methylation levels, lowers endogenous DNA damage and increases DNA resistance to DNA damaging agents (Patchsung et al., 2018). Similarly, LINE-1 hypomethylation may contribute to genomic instability via interactions with the ATM gene expression (Kitkumthorn and Mutirangura, 2011). However, the contribution of a reduction in the Alu methylation to genomic instability might be greater than that of LINE-1 or HERV sequences (Jintaridth and Mutirangura, 2010).

It remains largely unknown how changes in the expression of TEs might contribute to the development of mental disorders. It has been hypothesized that the presence of TEs in the human genome provides immunity against several infectious agents. Indeed, the mechanisms that contributed to HERV insertions are analogous to those used for replication by exogenous retroviruses (Grandi and Tramontano, 2018). Therefore, changes in the expression of TEs, e.g., via epigenetic processes, might impact immune responses and make the host more liable to exogenous infections. There is evidence that HERV-derived peptides may interact with innate immunity via various mechanisms. For instance, HERV proteins are able to interact with pattern recognition receptors (PRRs) that play a pivotal role in antiviral responses (Hurst and Magiorkinis, 2015). Emerging evidence indicates that exogenous viruses, including herpesviruses and influenza virus, might modulate the expression of HERV sequences. This mechanism might play a protective role and has been reviewed in detail by Grandi and Tramontano (2018). In brief, HERV transcripts might interact with homologous RNA from exogenous retroviruses, leading to the formation of molecules that are recognized by PRRs, acting as innate immunity sensors. The similarity of HERV proteins to those exogenous retroviruses allow them to compete with cellular receptors. This similarity might also trigger complementation events that impair formation of viral particles after cellular infection. On the other site, HERV proteins may suppress innate immunity. It has been reported that HERV-K proteins inhibit the activation of T cells (Morozov et al., 2013) as well as reduce the levels of interleukin-6 and Toll-like Receptor 7 (Laska et al., 2017).

### TRANSPOSABLE ELEMENTS AND THEIR EPIGENETIC REGULATION IN MENTAL DISORDERS

As mentioned above, expression of TEs might play an important role in shaping immune responses against exogenous infections. Aberrant immune-inflammatory responses have been reported in several mental disorders. Also, a number of exogenous infections have been found to impact a risk of mental disorders. Below, we review studies investigating TEs and their epigenetic regulation in specific mental disorders, starting from the rationale of these studies that is based on the contribution of immuneinflammatory processes. A summary of human studies was provided in **Table 1**.

## Autism Spectrum Disorders (ASD)

Overexpression of HERV-H has been observed in peripheral blood mononuclear cells (PBMC) of children with ASD (Balestrieri et al., 2012, 2016). Similar findings have also been observed in two different ASD mouse models – inbred BTBR T+tf/J mice and CD-1 outbred mice prenatally exposed to valproic acid. In both of these mouse models, the expression of several endogenous retrovirus (ERV) families (ETnI, ETnII-α, ETnII-β, ETnII-γ, MusD, and IAP) was significantly higher in comparison with corresponding controls (Cipriani et al., 2018). Interestingly, the studies in mouse models provide additional

(Continued)

#### TABLE 1 | Continued

fgene-10-00580 June 21, 2019 Time: 16:38 # 6

AD, Alzheimer's disease; ASD, autism-spectrum disorder; BD, bipolar disorder; COBRA, combined bisulfite restriction analysis; CSF, cerebrospinal fluid; FES, firstepisode schizophrenia; FESaff, first-episode schizoaffective disorder; H3K9me3, histone H3 lysine 9 trimethylation; HCs, healthy controls; MeCP2, methyl CpG binding protein 2; MDD, major depressive disorder; MIP, methamphetamine-induced psychosis; MS-HRM, methylation-sensitive high-resolution melting; PBMCs, peripheral blood mononuclear cells; RT-PCR, real-time polymerase chain reaction; SCZ, schizophrenia; SCZaff, schizoaffective disorder; qPCR, quantitative polymerase chain reaction.

information on the potential use of ERV sequences as biomarkers: (i) a higher expression of ERV was observed both in the peripheral blood mononuclear cells and the brain, suggesting that altered profile of peripheral ERV sequences may reflect similar alterations at the brain level; (ii) ERV overexpression in ASD mouse models is detectable from prenatal stage till the adulthood and (iii) ERV overexpression in ASD mouse models is also accompanied by increased expression of pro-inflammatory

cytokines and Toll-like receptors. Furthermore, a subsequent study in one of the models (mice prenatally exposed to valproic acid) provided evidence that higher levels of ERVs are also detectable in the offspring (second and third generations) of those mice exposed prenatally to valproic acid (Tartaglione et al., 2018).

Also LINE-1 retrotransposons have been associated with ASD (Shpyleva et al., 2018; Suarez et al., 2018). The levels of LINE-1 ORF1 and ORF2 transcripts have been investigated in four brain regions of patients with idiopathic autism (the frontal cortex, anterior cingulate, auditory cortex, and cerebellum). Elevated LINE-1 expression together with lower binding affinity of repressive MeCP2 protein and histone H3K9me3 to LINE-1 sequences was observed only in the cerebellum, suggesting a lessening of epigenetic repression and consequently an increase in chromatin accessibility. Interestingly, the increase in LINE-1 expression was also inversely correlated with glutathione redox status, consistent with reports indicating that LINE-1 expression is increased under pro-oxidant conditions (Shpyleva et al., 2018). The overexpression of LINE-1 within a single brain region is suggestive of a mosaicism-like impact of retrotransposons and definitively needs further investigation. In partial agreement with the findings of increased LINE-1 expression in ASD, data concerning LINE-1 methylation status in lymphoblastoid peripheral cells have provided evidence of reduced methylation in a subgroup of patients with severe language impairment (Tangsuwansri et al., 2018).

It has also been shown that the Alu sequence, the most abundant of all TEs in the human genome, deserves further research in ASD (Saeliw et al., 2018). Indeed, this study investigated the Alu methylation and expression in lymphoblastoid peripheral cells from ASD patients. Although the global methylation of Alu subfamilies was not significantly different between ASD and control group, when ASD samples were divided according to phenotypic subgroups, methylation patterns of the AluS subfamily were different from those in relative controls in two of the ASD subgroups, and within one of the subgroup (mild phenotype), the Alu expression was correlated with methylation status. Despite the limited sample size (particularly of subgroups), these data suggest that classification of ASD patients in phenotypic subgroups may represent a useful tool in investigating associations of TEs with the highly heterogeneous ASD diagnostic construct.

#### Schizophrenia-Spectrum Disorders

It has been clearly demonstrated that winter-spring seasonality of birth as well as prenatal and postnatal infections increase a risk of developing schizophrenia (McGrath and Welham, 1999; Davies et al., 2003; Khandaker et al., 2013). Moreover, the largest genome-wide association study revealed that variation within the HLA genes is strongly associated with schizophrenia susceptibility (Ripke et al., 2014). Finally, schizophrenia patients present with several indices of subclinical inflammation in terms of pro-inflammatory cytokine profiles (Miller et al., 2011; Frydecka et al., 2018), alterations of lymphocyte counts (Miller et al., 2013; Karpinski et al., 2016, 2018 ` ) and elevated levels of C-reactive protein (CRP) (Fernandes et al., 2016). On the basis of a meta-analysis, Arias et al. (2012) found a higher prevalence of infections with several agents, including Human Herpesvirus (HHV) 2, Borna Disease Virus, Chlamydia pneumoniae, Chlamydia psittaci, and Toxoplasma gondii in patients with schizophrenia compared to healthy controls.

Accumulating evidence indicates altered expression of HERV sequences in patients with schizophrenia. Karlsson et al. (2002) found nucleotide sequences homologous to those of the HERV-W pol gene in the cerebrospinal fluid (CSF) of 28.6% of firstepisode schizophrenia patients and in 5% of patients with chronic schizophrenia. These sequences were not detected in the CSF of individuals with non-inflammatory neurological diseases and healthy controls. Increased levels of HERV-Wrelated gag and pol transcripts and a higher prevalence of the gag and pol antigenemia in peripheral blood from patients with schizophrenia compared to healthy controls have been reported by several studies (Karlsson et al., 2004; Huang et al., 2006; Perron et al., 2008; Yao et al., 2008). The study by Perron et al. (2008) also revealed significantly higher rates of positive HERV-W env antigenemia in patients with schizophrenia than in healthy controls. The HERV-W gag and env antigenemia has been also associated with subclinical inflammation in terms of elevated levels of CRP and pro-inflammatory cytokines (Perron et al., 2008; Melbourne et al., 2018). Interestingly, Huang et al. (2011) found that overexpression of the HERV-W env in the human U251 glioma cells up-regulated a number of schizophreniaassociated genes, including those that encode brain-derived neurotrophic factor, neurotrophic tyrosine kinase receptor type 2 and the dopamine D3 receptor as well as increased the phosphorylation of cyclic adenosine monophosphate response element-binding protein. In this study, mRNA of the HERV-W env gene was detected in plasma from 42 out of 118 recent-onset schizophrenia patients but not in healthy controls. There is also evidence that the expression of HERV-W env induces calcium influx and down-regulates the DISC1 gene expression in the human neuroblastoma cells (Chen et al., 2018). Interestingly, expression level of the HERV-W gag protein has been found to be decreased in the cingulate gyrus and the hippocampus of patients with schizophrenia (Weis et al., 2007). However, a recent analysis of RNA-seq data in the human post mortem brain samples revealed increased transcription of HERV, especially HERV-W and HERV-H elements, in the anterior cingulate cortex, hippocampus and orbitofrontal cortex of patients with schizophrenia and bipolar disorder (Li et al., 2019). Interestingly, the HERV sequences within the ERVWE1 gene (7q21.2) exhibited the highest levels of transcription across all brain regions examined in this analysis. The env gene in this locus encodes syncytin-1, expressed at high levels in the human placenta (Blond et al., 2000; Mi et al., 2000). However, altered expression of this gene has been reported in the areas of active demyelination in patients with multiple sclerosis (Mameli et al., 2007). At this point, it should be noted that myelin alterations are widely observed in patients with schizophrenia (Mighdoll et al., 2015). Although initial results regarding expression of the HERV-W sequences in schizophrenia patients are promising, caution should be taken on the way these results are being interpreted. Indeed, the majority of studies in this field analyzed the overall expression of HERV-W sequences

without investigating specific HERV-W loci. Moreover, no conclusive association between the HERV-W expression and other human pathologies has been documented so far [for review see (Grandi and Tramontano, 2017)].

Less is known about other families of HERVs in patients with schizophrenia. Frank et al. (2005) found overrepresentation of the HERV-K(HML2) group in brain samples of patients with schizophrenia and bipolar disorder. Our group also tested peripheral blood methylation levels of HERV-K sequences in first-episode and multi-episode schizophrenia patients (Mak et al., 2019). We found significantly lower levels of HERV-K methylation in first-episode schizophrenia patients compared to healthy controls. These alterations were not observed in multiepisode schizophrenia patients. Moreover, we did not find an association between HERV-K methylation levels and the deficit schizophrenia subtype that refers to a subgroup of patients with enduring and persistent negative symptoms. However, we found a significant positive correlation between the dosage of antipsychotics and HERV-K methylation levels in multi-episode schizophrenia patients. These findings imply that the HERV-K methylation might normalize in the course of schizophrenia. It is also likely that antipsychotic drugs might impact methylation and expression of HERV-K sequences. In contrast to our findings, Diem et al. (2012) found no significant effects of valproic acid, haloperidol, risperidone and clozapine on the HERV-K expression levels in the human brain cell lines. However, valproic acid was found to strongly up-regulate expression of HERV-W and ERV9 elements.

Some studies also investigated methylation status and expression levels of non-LTR sequences in patients with schizophrenia. Bundo et al. (2014) demonstrated increased LINE-1 retrotransposition in neurons from the prefrontal cortex of patients with schizophrenia, especially in the genes involved in synaptic functions. These findings were confirmed in induced pluripotent cells from patients with 22q11 deletion syndrome as well as in a mouse model of schizophrenia (maternal immune activation paradigm). In agreement with these results, a significant increase in the number of intragenic LINE-1 insertions has been observed in the dorsolateral prefrontal cortex of patients with schizophrenia compared to healthy controls (Doyle et al., 2017). Over-representation of these insertions appeared within the gene ontologies called "cell projection" and "postsynaptic membrane," suggesting their role in the brain development. In some studies, LINE-1 methylation was tested in peripheral blood leukocytes of patients with schizophrenia, providing mixed findings (Misiak et al., 2015; Li et al., 2018; Fachim et al., 2019; Kalayasiri et al., 2019). The study by our group revealed lower LINE-1 methylation only in patients with first-episode schizophrenia and a positive history of childhood trauma. Among various childhood adversities, emotional trauma was most strongly associated with the LINE-1 methylation status. These results are in agreement with a previous study, showing that the LINE-1 methylation might be involved in resilience and susceptibility to develop post-traumatic stress disorder (Rusiecki et al., 2012). Moreover, increased expression of LINE-1 in response to stress has been reported in various cell lines (Li and Schmid, 2001; Capomaccio et al., 2010). Lower LINE-1 methylation levels in patients with schizophrenia and bipolar disorder were also reported by Li et al. (2018). Other studies revealed hypermethylation of LINE-1 sequences in patients with first-episode psychosis, paranoid schizophrenia and methamphetamine-induced paranoia (Fachim et al., 2019; Kalayasiri et al., 2019).

### Mood Disorders

A recent systematic review indicates that prenatal infections might impact the risk of bipolar disorder (Marangoni et al., 2016). However, this observation is based on a lower number of studies compared to studies addressing the impact of prenatal infections on schizophrenia risk. There is evidence that influenza infection during pregnancy is associated with a fourfold increase in the risk of bipolar disorder in the offspring (Parboosing et al., 2013). Another study demonstrated that prenatal flu exposure increases the risk of bipolar disorder with psychotic features (Canetta et al., 2014). However, no association was found between prenatal infections with HHV-1, HHV-2, Cytomegalovirus or Toxoplasma gondii and bipolar disorder risk (Mortensen et al., 2011). Maternal infections in the second trimester might also contribute to the development of depressive symptoms in the adolescent offspring (Murphy et al., 2017). However, the impact of specific infectious agents has not been tested so far.

Although all major mental disorders are characterized by co-existing subclinical inflammation, some differences, regarding specific pro-inflammatory markers can be indicated (Goldsmith et al., 2016; Misiak et al., 2019). Therefore, it might be hypothesized that the mechanisms leading to subclinical inflammation in bipolar disorder, major depression and schizophrenia-spectrum disorders are different. However, studies investigating expression of TEs do not support this hypothesis. For instance, over-expression of HERV-K sequences has been reported in brain samples of patients with bipolar disorder and schizophrenia (Frank et al., 2005). Similarly, decreased expression of the HERV-W gag protein has been reported in the cingulate gyrus and hippocampus of patients with schizophrenia, bipolar disorder, and major depression (Weis et al., 2007). Finally, hypomethylation of LINE-1 elements in peripheral blood has been observed in patients with bipolar disorder and schizophrenia (Li et al., 2018). Some differences have been detected with respect to the expression of HERV-W sequences. Indeed, Perron et al. (2012) found elevated transcription levels of the HERV-W env sequence in the peripheral blood of patients with bipolar disorder and schizophrenia compared to healthy controls. Expression levels of the HERV-W env sequence were also significantly higher in patients with bipolar disorder than in those with schizophrenia.

#### Alzheimer's Disease

There is a general consensus that aging processes are associated with progressive loss of global DNA methylation and site-specific DNA hypermethylation (Jung and Pfeifer, 2015). Similarly, TEs are subjected to profound epigenetic modifications during aging that appear in the context of organismal and cellular senescence (Cardelli, 2018). For instance, age-related loss of Alu and HERV-K methylation has been well-documented (Bollati et al., 2009;

Jintaridth and Mutirangura, 2010; Gentilini et al., 2013). Moreover, it has been found that the expression of HERV-H, HERV-K and HERV-W changes during the lifespan with distinct patterns (Balestrieri et al., 2015). Importantly, the study by Gentilini et al. (2013) demonstrated that age-related loss of Alu methylation was less apparent in the offspring of centenarians, suggesting the effects of genetic factors associated with longevity. In turn, studies investigating changes of LINE-1 methylation have provided mixed findings (Bollati et al., 2009; Talens et al., 2012; Cho et al., 2015). Finally, there is evidence that chromatin of Alu, SVA, and LINE-1 becomes relatively more open in senescent cells (De Cecco et al., 2013).

Age-related changes in epigenetic modifications of TEs have provided basis for investigating alterations of these processes in Alzheimer's disease. In a single study of earlyonset Alzheimer's disease family, it has been reported that large genomic rearrangements might affect the presenilin-1 gene via the mechanisms involving recombination stimulated by the Alu sequence (Hiltunen et al., 2000). However, subsequent studies have not provided compelling evidence regarding the contribution of TEs to the etiology of Alzheimer's disease. There is only one study, showing increased LINE-1 methylation in peripheral blood leukocytes of patients with Alzheimer's disease, especially those with better cognitive performance, compared to healthy controls (Bollati et al., 2011). However, the authors did not find significant between-group differences in the levels of Alu and SAT-α methylation. Other studies did not confirm these findings regarding LINE-1 methylation (Hernández et al., 2014; Protasova et al., 2017). Alterations of other TEs in patients with Alzheimer's disease have not been tested so far.

### SUMMARY OF EVIDENCE AND FUTURE DIRECTIONS

Although specific retrotransposition events that may account for mental disorders in the manner observed in case of Mendelian diseases have not been identified so far, accumulating evidence indicates the involvement of altered expression and epigenetic regulation of TEs in the pathophysiology of schizophrenia, mood disorders and ASD. Most consistently, previous studies indicate altered expression of HERVs and methylation of LINE-1 sequences. However, specific findings are similar in patients with various mental disorders and thus their use as biomarkers is largely limited. Moreover, the direction of causality is yet to be determined. For instance, it cannot be excluded that altered expression of HERV appears as a consequence of other epigenetic dysregulations that are widely observed in mental disorders. Additionally, severe mental disorders, including schizophrenia and mood disorders, are associated with high prevalence rates of somatic comorbidities, including autoimmune diseases, type 2 diabetes and cardiovascular diseases that have also been associated with altered epigenetic regulation of TEs (Cash et al., 2011; De Hert et al., 2011; Misiak et al., 2013; Nestler et al., 2016; Zhao et al., 2018). Interestingly, there are studies showing that the expression of various HERV sequences appears in a certain subgroup of patients with schizophrenia but not in healthy controls. These findings are consistent with previous studies, showing that immune alterations can be observed only in a subgroup of patients characterized by poor response to treatment and support the concept of psychosis subtypes (Frydecka et al., 2015; Mondelli et al., 2015; Fillman et al., 2016). Other clinical correlates of subclinical inflammation in schizophrenia include, i.e., more severe cognitive deficits (Misiak et al., 2017b), persistent negative symptoms (Goldsmith et al., 2018) and certain neurostructural abnormalities (Najjar and Pearlman, 2015). However, so far studies investigating expression and epigenetic regulation of TEs in schizophrenia have been based on relatively small samples without comprehensive clinical assessment. Similarly, studies investigating the expression of TEs in patients with bipolar disorder did not control for mood status and a severity of psychopathological symptoms.

Another important point is that causal inferences between TEs and mental disorders cannot be established. Firstly, it remains unknown what are the critical periods when alterations in epigenetic regulation and expression of TEs appear. Therefore, future studies should examine epigenetic processes that regulate expression of TEs in patients at early stages of mental disorders or individuals from clinical high risk groups. This is particularly important since several lifestyle characteristics that are highly prevalent among patients with mental disorders, e.g., cigarette smoking and poor dietary habits, might impact TEs per se (Miglino et al., 2014; Miousse et al., 2015). Secondly, the role of HERVs in shaping innate immunity also remains problematic with respect to understanding causal associations. On one side, expression of HERVs might condition resistance to exogenous infections; on the other, exogenous retroviruses have been found to impact the expression of HERVs. Therefore, it remains unknown whether altered expression profiles of HERVs in mental disorders represent cause or consequence of exogenous infections. Future studies should necessarily examine the biological nature and the extent of associations between immune alterations in mental disorders and expression of various TEs.

Finally, more global concordance patterns of different TEs expression in mental disorders are yet to be examined: this could provide further insight into specificity of methylation patterns across different TEs and provide additional information of their use as potential biomarkers. At this point, it is important to note that similar DNA methylation patterns have been described in brain samples and peripheral blood leukocytes of patients with schizophrenia (Van Den Oord et al., 2016).

Another direction for the field is to disentangle the effects of stressful life events on epigenetic regulation of TEs expression. Early-life stress is a known risk factor for mood and psychotic disorders as well as correlates with a number of biological dysregulations in adults (Misiak et al., 2017a; Bielawski et al., 2019; Jaworska-Andryszewska and Rybakowski, 2019). Acute stress has been found to increase the levels of H3K9me3 as well as decrease the levels of H3K9me1 and H3K27me3 in the dentate gyrus and the CA1 layer of the hippocampus in rats (Milne et al., 2009). In turn, chronic restraint stress for 21 days mildly increased the levels of H3Kme4 and reduced the levels of H3K9me3 in the dentate gyrus. Treatment with

fluoxetine reversed changes in the levels of H3K9me3 during chronic restraint stress. More specifically, the same group found that acute stress had increased H3K9me3 enrichment at SINEs (Baker et al., 2012). In turn, our group found lower methylation of LINE-1 sequences in peripheral blood leukocytes of patients with first-episode schizophrenia reporting a positive history of childhood trauma (Misiak et al., 2015). In light of these findings, future studies should further examine the effects of stress on the expression of TEs in patients from various clinical groups and preclinical studies could contribute to this aim.

### AUTHOR CONTRIBUTIONS

BM and MS conceived the concept of this article. BM wrote the Sections "Introduction", "Insights Into Potential

### REFERENCES

Mechanisms Underlying the Role of TEs in Mental Disorders", "Schizophrenia-Spectrum Disorders" and "Mood Disorders". LR wrote the Section "Autism Spectrum Disorders (ASD)". MS prepared the Sections "Brief Overview of TEs in the Human Genome – Classification and Nomenclature" and "Alzheimer's Disease". BM, LR, and MS prepared the Section "Summary of Evidence and Future Directions". All authors contributed to the manuscript revision, read, and approved the submitted version.

#### FUNDING

This work was supported by the statutory project funded by the Wrocław Medical University, Wrocław, Poland (Task Number: ST-A.290.17.032).

stressors. Mutat. Res. Rev. Mutat. Res. 765, 19–39. doi: 10.1016/j.mrrev.2015. 05.003

and longitudinal data on monozygotic twin pairs. Aging Cell 11, 694–703. doi: 10.1111/j.1474-9726.2012.00835.x

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Misiak, Ricceri and Sa¸siadek. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Epigenetic IVD Tests for Personalized Precision Medicine in Cancer

*Jesús Beltrán-García1,2,3, Rebeca Osca-Verdegal2,3, Salvador Mena-Mollá3,4\* and José Luis García-Giménez1,2,3,4\**

*1 Center for Biomedical Network Research on Rare Diseases (CIBERER), Institute of Health Carlos III, Valencia, Spain, 2 INCLIVA Biomedical Research Institute, Valencia, Spain, 3 Department of Physiology, School of Medicine and Dentistry, Universitat de València (UV), Valencia, Spain, 4 EpiDisease S.L. Spin-Off of CIBERER (ISCIII), Valencia, Spain*

Epigenetic alterations play a key role in the initiation and progression of cancer. Therefore, it is possible to use epigenetic marks as biomarkers for predictive and precision medicine in cancer. Precision medicine is poised to impact clinical practice, patients, and healthcare systems. The objective of this review is to provide an overview of the epigenetic testing landscape in cancer by examining commercially available epigeneticbased *in vitro* diagnostic tests for colon, breast, cervical, glioblastoma, lung cancers, and for cancers of unknown origin. We compile current commercial epigenetic tests based on epigenetic biomarkers (i.e., DNA methylation, miRNAs, and histones) that can actually be implemented into clinical practice.

#### *Edited by:*

*Yun Liu, Fudan University, China*

#### *Reviewed by:*

*Jorg Tost, Institut de Biologie François Jacob, Commissariat à l'Energie Atomique et aux Energies Alternatives, France Beisi Xu, St. Jude Children's Research Hospital, United States*

#### *\*Correspondence:*

*José Luis García-Giménez<EMAIL_ADDRESS>Salvador Mena-Mollá<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Epigenomics and Epigenetics, a section of the journal Frontiers in Genetics*

*Received: 29 March 2019 Accepted: 13 June 2019 Published: 28 June 2019*

#### *Citation:*

*Beltrán-García J, Osca-Verdegal R, Mena-Mollá S and García-Giménez JL (2019) Epigenetic IVD Tests for Personalized Precision Medicine in Cancer. Front. Genet. 10:621. doi: 10.3389/fgene.2019.00621*

Keywords: precision medicine, epigenetic biomarker, In Vitro Diagnostic (IVD), DNA methylation, miRNA, cfDNA, circulating nucleosomes

### INTRODUCTION

Epigenetics, a breakthrough discipline in biomedicine, aims to improve precision medicine by discovering new epigenetic mechanisms and providing new epigenetic biomarkers, therapeutic targets, and epigenetic drugs with potential uses in clinical practice.

Most human diseases have complex multifactorial pathologies that result from a pathogenic polymorphism in human genes, besides epigenetic mechanisms, which can modulate the expression of functional genes. Currently, several IVD molecular-based tests contribute to the development of precision oncology, which already offers viable alternatives for cancer diagnostics and prognostics. The Food and Drug Administration (FDA) lists several IVD tests that have been cleared and approved for diagnostics, which can be consulted by searching *Nucleic Acid-Based Test* (Food and Drug Administration, 2019a) and *List of Cleared or Approved Companion Diagnostic Devices (In Vitro and Imaging Tools)* (Food and Drug Administration, 2019b).

For a given phenotype, there is a causal contribution of genetic mutations, copy number variations, epigenetic control, and altered transcription programs and altered complex metabolic inputs. The contribution of the aforementioned factors renders the use of different approaches necessary to understand the physiopathology of complex and multifactorial diseases. In line with this, epigenetic biomarkers can help early diagnosis, disease progression monitoring, disease outcome prediction, selection and stratification of patients by risk, prediction of future comorbidities, and even the evaluation of the positive or negative effects of therapeutic interventions in specific patient subsets. Among others, DNA methylation and microRNAs are markedly more stable than RNA and proteins, which renders the use of these biomarkers more practical and viable in clinical settings (Faruq and Vecchione, 2015; Hashimoto et al., 2016; García-Giménez et al., 2017b). In particular, DNA methylation, microRNAs, and post-translational modifications of histones offer high stability

**68**

in biofluids and in samples with a compromised quality, such as formalin-fixed paraffin embedded (FFPE). Other advantages of epigenetic biomarkers over genetic or protein-based biomarkers are as follows: 1) their dynamic nature; 2) they provide information about the gene function; 3) they inform about the specific genetic programs that alter during disease; and 4) most techniques to analyze epigenetic biomarkers (i.e., RT-qPCR) have already been introduced into clinical laboratories. Therefore, epigenetics has a tremendous potential to improve predictive and precision medicine.

Precision Medicine was defined by the National Research Council's Toward Precision Medicine in 2008 as: "The tailoring of medical treatment to the individual characteristics of each patient … to classify individuals into subpopulations that differ in their susceptibility to a particular disease or their response to a specific treatment. Preventative or therapeutic interventions can then be concentrated on those who will benefit, sparing expense and side effects for those who will not" (Ginsburg and Phillips, 2018). Therefore, precision medicine has started to use potential epigenetic biomarkers in clinical settings.

We recently defined an epigenetic biomarker as "any epigenetic mark or altered epigenetic mechanism which generally serves to evaluate health or disease status and is particularly stable and reproducible during sample processing." An ideal biomarker can be measured in body fluids (i.e., plasma, serum, saliva, semen, urine, etc.) or primary tissue samples (fresh tissue, cells, single cell isolated, fine-needle aspirates, FFPE, etc.). However, for clinical settings, minimal invasive procedures are preferable. In line with this, human plasma as a source of miRNAs and circulating cell-free DNA (cfDNA) is, therefore, the best option. An ideal epigenetic biomarker for precision medicine applications may cover at least one of the following properties: i) predicts the risk of future disease development (risk); ii) defines a disease (detection); iii) reveals information about the natural history of the disease; iv) predicts the outcome of disease (prognostic); v) responds to therapy (predictive); vi) monitors responses to therapy or medication (therapy monitoring); vii) allows to simultaneously make a diagnosis and perform targeted therapy (theragnosis) (García-Giménez et al., 2017b).

To achieve the precision medicine goals, the current challenge is knowing how to obtain a reliable useful biomarker for clinical routine because, for this purpose, the new biomarker requires high accuracy and robustness (Li et al., 2010; Diamandis, 2012) and costeffectiveness. It is noteworthy that less than 1% of the biomarkers obtained in biomedical research is finally implemented into the clinical laboratory (Kern, 2012), with an even lower percentage for epigenetic biomarkers. This low percentage of commercialized IVD tests based on epigenetic biomarkers suggests that the precision medicine ecosystem formed by distinct stakeholders (i.e., patients, providers, payers, and regulators) may increase their knowledge about the impact of epigenetic biomarkers on precision medicine, and might also work together to successfully implement this breakthrough technology in clinical practice.

A number of precision medicine applications are contributing to health care improvements by allowing the precise diagnosis of diseases or by identifying specific disease subsets or stages, and by also improving personalized treatments. Specifically, for cancer, which remains the second leading cause of death worldwide, early detection, the identification of cancer subtypes, and the selection of appropriate therapies are crucial to increase the survival of cancer patients. However, the identification of new tumor biomarkers, especially those based on epigenetic biomarkers with the capability to identify tumor origin or cancer subsets, advances in assay technologies, and the development of sophisticated analytical software techniques (i.e., machine learning and artificial intelligence), will help to improve precision medicine in cancer (Ahlquist, 2018).

### TECHNOLOGIES FOR EPIGENETIC BIOMARKER ANALYSES IN CLINICAL LABORATORIES

Given the prevalence of the DNA methylation alterations at specific genes under a variety of human disease conditions, a promising future is coming for the DNA methylation analysis as an epigenetic biomarker. In fact, DNA methylation is the best-studied epigenetic modification since it was discovered. In addition, miRNAs have attracted a great deal of interest in clinical research for their role in gene regulation, tissue signaling and cellular homeostasis, their high stability in practically all types of biospecimens, and the relatively easy way by which to measure miRNAs in a wide array of biospecimens. Histone variants and histone post-translational modifications are other potential markers that can be analyzed in a wide array of biospecimens for clinical settings.

Therefore, it is not surprising that most current commercial *in vitro* diagnostic tests are based on either the analysis of DNA methylation of specific genes or the measurement of the relative expression of microRNAs, which can be easily measured by RT-qPCR-based methods (i.e., methyLight, methyl-specific PCR, and methylation-sensitive high-resolution melting) and pyrosequencing technologies (García-Giménez et al., 2017a).

There are other assays based on high-throughput analyses to simultaneously measure several CpG sites. This is, for example, the case of the EPICUP® assay, which is based on using human methylation array Beadchip 450K (Illumina). In the following section, we provide details of a selection of current IVD tests based on epigenetic biomarkers that are currently being commercialized for *in vitro* diagnostic in cancer (**Table 1**).

### *IN VITRO* DIAGNOSTIC TESTS BASED ON EPIGENETIC BIOMARKERS

#### Epigenetic-Based IVD Test for Colorectal Cancer

Colorectal cancer (CRC) (MIM 11 4500) is the third most frequent cancer in men and the second most frequent cancer in women worldwide, and accounts for nearly 10% of cancers (Ferlay et al., 2015). CRC is the second leading cause of death by cancer. Five-year survival rates range from more than 90% for stage I to less than 10% for stage IV CRC (Siegel et al., 2012). CRC is characterized by slow progression from detectable precancerous lesions and has a good prognosis when patients are diagnosed

TABLE 1 | Commercially available Epigenetic IVD tests with the potential of improving precision medicine in cancer.

*cfDNA, circulating cell-free DNA; FFPE, formalin-fixed, paraffin-embedded; Sn, Sensitivity; Sp, specificity; NA, data not available.*

in early stages. Non-invasive fecal immunochemical test (FIT) for hemoglobin detection in stools is the most widely used test, but its sensitivity is relatively low in detecting early stage I CRC (53%) and advanced adenomas (≥1.0 cm) (27%) (Morikawa et al., 2005). Therefore, the potential for reducing the burden of CRC by early detection is significant, and efforts are currently being made to develop CRC screening tests and to improve the adherence rates of participation for screening because people scarcely comply with currently available methods (Issa and Noureddine, 2017). The selection of appropriate therapies for CRC patients is also a clinical need. Among the therapies proposed for CRC, anti-epidermal growth factor receptor (EGFR) mAb therapy is not indicated for carriers of RAS mutations [approximately 50% of patients with metastatic CRC because the mutations in the *RAS* gene (mainly in exons 2, 3, and 4 of *KRAS* and *NRAS*) make metastatic CRC patients non responders to anti-EGFRs mAB treatment] (Boleij et al., 2016). So, the identification of additional biomarkers to allow clinicians to select those patients who could benefit by the established therapies is needed.

#### The Cologuard® Stool DNA-Based Test

The first FDA-approved DNA methylation assay for general CRC screening for average-risk adults older than 50 years was Cologuard® (Exact Sciences Corp., Madison, WI). The Cologuard® IVD test is a multitarget stool deoxyribonucleic acid (MT-sDNA) screening test based on the analysis of the methylation levels of genes N-Myc downstream-regulated gene 4 (*NDRG4*) and bone morphogenetic protein 3 (*BMP3*), a mutation in the *KRAS* gene (exon 2, codons 12, 13, using ß-actin as the reference gene), and a non-DNA immunochemical assay for human hemoglobin that allows the precise detection of colon neoplasia (Imperiale et al., 2014). The methylation analysis of *NDRG4* and *BMP3* using *ACTB* (ß-actin) as the reference gene is performed according to the method described by Zou et al. (2012), while fecal hemoglobin biomarker values are obtained by the analytical method described by Lidgard et al. (2013). Cologuard® uses a composite score algorithm that is incorporated into the multitarget stool DNA analytic device software as described by Imperiale et al. (2014).

Cologuard® sensitivity and specificity for CRC detection in a study performed with 9,989 subjects was 92.3% and 86.6%, respectively (Imperiale et al., 2014). Although the Cologuard® test sensitivity was higher than FIT (which measures the presence of blood in the colon in fewer fecal samples) for detecting CRC (92% vs. 74%, p = 0.015), specificity was lower than that shown by the FIT ((87% vs. 95%) (Imperiale et al., 2014). Moreover, the Cologuard® test detected less than half largely advanced adenomas (precancerous lesions), but performs better than the FIT. In fact, the sensitivity for detecting advanced precancerous lesions was 42.4% with DNA testing and 23.8% with the FIT (P < 0.001). These results reinforce the potential of the Cologuard® test as an alternative for surveillance colonoscopy (van Lanschot et al., 2017). However, its high cost and difficult sample pretreatment and management for each analysis type are considered disadvantages for its rapid implementation into clinical routine. Accordingly, the results obtained with the Cologuard® test are delivered to the healthcare provider within 2 weeks from receiving the stool sample.

Despite these disadvantages, both the US Food and Drug Administration and the US Preventive Services Task Force (USPSTF) include the Cologuard® test in their screening exam recommendations (Lin et al., 2016).

#### The Epi proColon® 2.0 Test

The Epi proColon® test (Epigenomics AG, Berlin, Germany) was designed to minimize invasive tests and to increase the adherence rates of the participation of those people screened for CRC. The Epi proColon® test uses peripheral blood samples to analyze the methylation status of the *SEPT9* gene. Septins are essential proteins during cell division, and *SEPT9* hypermethylation has been proposed as a key factor in CRC (Song and Li, 2015). The original assay was designed to extract DNA from 5 ml of plasma samples, bisulfite conversion of DNA, and its purification by a particle-based bis-DNA purification method to improve the recovery of bisulfite-treated DNA, the quantification of converted DNA by real-time PCR, and the subsequent measurement of *SEPT9* methylation, and *ACTB* (ß-actin) as a reference gene, by real-time PCR in a Lightcycler LC480 system (Roche Applied Science) and the Quantitect Multiplex PCR mastermix (Qiagen) (DeVos et al., 2009). Epi proColon® 2.0 (Epigenomics Inc., Germany) was approved by the FDA in 2016 as the first blood test intended for early CRC detection. In a large clinical trial using 1,544 plasma samples from the PRESEPT study cohort (ClinicalTrials.gov, Trial Registration ID: NCT00855348), Epi proColon® demonstrated high sensitivity, which ranged from 77.0% to 81.4%, and specificity from 77.9% to 92.1% (Potter et al., 2014). However, some studies have shown some flaws in the use of Epi proColon® to diagnose CRC, such as its lower sensitivity for stage I than for stages II, III, or IV (Jin et al., 2015). A large multicenter prospective study using blood samples from 53 CRC cases and from 1,457 subjects without CRC from the PRESEPT cohort (ClinicalTrials.gov, Trial Registration ID: NCT00855348) showed low sensitivity (48.2%) for detecting CRC and very low sensitivity (11.2%) for identifying advanced adenoma, with 91.5% specificity (Church et al., 2014). One noteworthy result was that the positive detection rate of the *SEPT9* methylation assay increased exponentially as colorectal lesions became more severe and with more advanced CRC stages (Song et al., 2018), although a negative result does not guarantee absence of cancer.

The results obtained by Song et al. (2018) and He et al. (2018) suggest that the methylation status of *SEPT9* could be applied to CRC stage, size, invasion depth, future risk assessment, metastasis, disease progression monitoring, and therapeutic effect evaluation. A possible flaw of this test is that Epi proColon® detected the methylated status of the same region of the *SEPT9* gene in some patients affected by other cancers (i.e., prostate, breast, lung or other diseases, hypertension, hyperlipidemia, diverticulitis, chronic gastritis, or cardiovascular) and according to their age (Ørntoft et al., 2015). Indeed, the Epi proColon® test was positive in 72 (42%) of 173 patients with other cancers and positive in 33 of 191 patients (17%) with other diseases. In addition, an active clinical trial was run to evaluate the potential of the Epi proColon® test for detecting hepatocellular carcinoma among cirrhotic patients (ClinicalTrials.gov, Trial Registration ID: NCT03311152). These scenarios suggest the potential of this test to diagnose other cancers, such as breast cancer, as demonstrated by Shen et al. (2018), but also the inconvenience of the positive results given for cancer patients who were negative for CRC.

It is worth mentioning that colonoscopy remains the universal gold standard method for CRC diagnostics. In Europe and Asia Pacific, only the use of fecal occult blood test (gFOBT) or quantitative FIT for non-invasive screening is still recommended. However, Chinese guidelines have recently recommended using the test as a complement to other diagnostic approaches, like the guaiac-based gFOBT. In the United States, Epi proColon® is not intended to replace the CRC screening tests recommended by clinical guidelines (i.e., colonoscopy, sigmoidoscopy, and gFOBT), but the Epi proColon® test was FDA-approved for CRC screening those patients unwilling or unable to be screened by recommended methods following guidelines.

#### The EarlyTect® Colorectal Cancer Assay

The EarlyTect® CRC test (Genomictree Inc. Daejeon, South Korea) has recently received CE-IVD certification for the diagnosis of CRC. The EarlyTect™-GI Syndecan2 Methylation Assay is an IVD assay that uses cfDNA isolated from 0.5 ml of serum to analyze the methylation status of *SDC2* (Syndecan-2).

Previous studies have demonstrated the potential of the analysis of the methylation status of the *SDC2* gene for the early diagnosis of CRC. For example, the studies performed by Mitchell et al. (2016) showed lower sensitivity (59%), but relatively good specificity (84%), of methylation-specific PCR assays (probebased MethyLight assays) for *SDC2* in the early detection of CRC. At this point, it is worth mentioning that the amplicon selected to study the methylation status of this gene slightly differed (420 bp downstream to the CpG proposed by Oh et al., 2017).

More recent studies performed by Oh et al. (2017), which evaluated the methylation analysis of *SDC2* to detect CRC using isolated DNA from stool samples, demonstrated a good sensitivity of 90.0% for detecting CRC and 33.3% for small polyps, with a specificity of 90.9%. Furthermore, these authors demonstrated that the *SDC2* methylation level was linked to cancer severity in CRC patients in stages I to IV (n = 50). Similarly, Niu et al. (2017) evaluated the methylation levels of the *SDC2* gene in 497 stool samples and found sensitivities of 81.1% and 58.2% for detecting CRC (n = 196) and adenoma (≥1 cm) (n = 122), respectively, with 93.3% specificity. These results were comparable to that observed by Park et al. (2018), who found that the *SDC2* gene methylation analysis performed with methyl-specific PCR in bowel lavage fluid collected during colonoscopy could detect CRC and precancerous lesions. In this study, *SDC2* methylation was positive in 100% of villous adenoma, high-grade dysplasia, and hyperplastic polyp biopsies in 88.9% of tubular adenoma samples and in 0% of normal mucosal samples. These findings indicate the potential of *SDC2* methylation as a biomarker for early CRC detection with a sensitivity of 80% and a specificity of 88.9%.

The clinical validation of *SDC2* methylation in serum DNA from the CRC patients (n = 131) in stages I to IV (stage I, 26; II, 57; III, 36; IV, 12) and from healthy individuals (n = 125) by quantitative methylation-specific PCR using the methylationspecific TaqMan probe demonstrated 87% sensitivity [114/141; 95% confidence interval (CI), 80.0% to 92.3%] and 95.2% specificity (10/125; 95%CI, 89.8% to 98.2%) (Oh et al., 2013). The sensitivity of the patients in stage I was particularly high with 92%, which suggests the potential utility of this test for early CRC detection and identification of precancerous lesions, such as polyps.

A recent observational clinical trial conducted with the EarlyTect® CRC test (ClinicalTrials.gov, Trial Registration ID: NCT03146520) was designed to validate the clinical performance of the *EarlyTect*® *Colon Cancer test* in stool DNA to detect CRC in a case-control study with 634 participants (Dae Han et al., 2019). Of the 585 evaluated subjects, 245 had CRC, 44 had various sized adenomatous polyps, and 245 obtained negative colonoscopy results. The EarlyTect® CRC test gave an overall sensitivity of 90.2% with area under the curve (AUC) of 0.902 in detecting CRC (0–IV) not associated with tumor stage, and a specificity of 90.2%. The sensitivity for detecting early stages (0-II) was 89.1% (114/128). The EarlyTect® CRC test also detected 66.7% (2/3) and 24.4% (10/41) of advanced and non-advanced adenomas, respectively (Dae Han et al., 2019).

Genomictree Inc. has performed experiments to evaluate the cross-reactivity of the EarlyTect® CRC test in an interim clinical validation with stool DNA from 50 CRC patients (stage I, 10; II, 16; III, 14; IV, 10), 14 irritable bowel syndrome (no colonoscopy was performed), 4 with acute colitis, 11 Crohn's disease (colonoscopy was performed), 14 ulcerative enteritis (colonoscopy was performed), and 50 healthy subjects (endoscopy was not performed). In this study, the sensitivity was 90.0% (45/50) and specificity was 90.9% (5/55). The methylation positivity for *SDC2* was observed in 14.3% (2/14) of the irritable bowel syndrome patients, 25.0% (1/4) of the acute colitis patients, and 35.7% (5/14) of the ulcerative colitis patients, while no Crohn's disease case was positive for the EarlyTect® assay. Notably, sensitivity was 84.6% (22/26) in CRC in stages I and II, which suggests the potential applicability of this test for colorectal detection testing using stool DNA.

#### miRPreDX-31-3p

The miRpredX-31-3p kit (IntegraGen S.A., France) is a CE-IVD marked theranostic test intended to identify patients with metastatic CRC who can benefit from anti-EGFR (epidermal growth factor receptor) therapy. The miRpredX-31-3p kit quantifies relative miR-31-3p levels by RT-qPCR from the total RNA extracted from FFPE samples in primary tumors of patients with metastatic CRC, using a cutoff value of 1.36 for the miR-31-3p expression level to define patients as being low or high expressers of this miRNA (Ramon et al., 2018).

miRpredX-31-3p predicts the potential clinical benefits associated with first-line anti-EGFR (epidermal growth factor receptor) therapy compared with anti-vascular endothelial growth factor receptor (VEGF) therapy or when second or further lines of treatment with anti-EGFR mAB therapy is more beneficial *versus* chemotherapy alone for multiple patient outcomes (Laurent-Puig et al., 2015). Specifically, on one hand, a low miR-31-3p expression in affected tissue is associated with a 12-month survival advantage and a 40% reduced risk of death when using anti-EGFR (cetuximab) therapy *versus* anti-VEGF (bevacizumab) therapy in patients with metastatic CRC. On the other hand, those patients expressing high miR-31-3p levels displayed no differences in outcomes when treated with either anti-EGFR or anti-VEGF therapy (Laurent-Puig et al., 2015; Laurent-Puig et al., 2017). Furthermore, the miR-31-3p expression was evaluated for its potential as a predictive biomarker for anti-EGFR mAb therapy in the patients without mutations in *KRAS* with operable colorectal liver metastases (Pugh et al., 2017).

In an interventional clinical trial in 1,808 subjects (ClinicalTrials.gov, Trial Registration ID: NCT03362684), the predictive potential of the miR-31-3p expression level was studied for the prognostic of patient outcomes, as was the predictive value of the benefit of anti-EGFR therapy (cetuximab) in stage III CRC patients (the patients enrolled in the PETACC-8 Study) (Taieb et al., 2014). The results obtained from this clinical trial demonstrated that patients with the RAS/BRAF wild type who showed low miR-31-3p expression when tumors were treated with cetuximab plus FOLFOX-4 presented improved disease-free survival, overall survival, and survival after recurrence compared with the patients treated with FOLFOX-4 alone.

More recently with logistic regression models, including the miR-31-3p expression level adjusted for potential confounding factors, Laurent-Puig et al. (2019) validate the use of miR-31-3p to differentiate RAS wt metastatic CRC patient outcomes from patients treated with anti-EGFR mAb or anti-VEGF mAb therapy. Those patients with low miR-31-3p levels showed better outcomes when treated with cetuximab compared with bevacizumab.

The miRpredX-31-3p kit was developed on the basis of a standardized RT-qPCR assay for miRNA detection. Several extraction kits (miRNeasy FFPE kit (Qiagen), AllPrep DNA/RNA FFPE Kit (Qiagen), QIAsymphony RNA kit (Qiagen), and Maxwell 16 LEV RNA FFPE kit (Promega)) have been tested to evaluate the efficiency of miRNA extraction from five formalin-fixed, paraffinembedded (FFPE) 5-mm-thick slides. In addition, the analytical sensitivity and specificity, assay robustness, reproducibility, and accuracy of miR-31-3p detection were also demonstrated in different quantitative PCR systems like ABI 7900HT®, ABI StepOne+®, and ABI QS5® (Applied Biosystems) and LightCycler® 480 (Roche) (Ramon et al., 2018). These results demonstrated the good versatility of the miRpredX-31-3p assay and its feasibility for being easily implemented into clinical diagnostic laboratories. The time to perform the assay was not as long after total RNA was isolated from FFPE tumor samples because the assay is based on a simple RT-qPCR reaction (reverse transcription and subsequent real-time PCR). Hence the miRpredX-31-3p assay can analyze up to 12 samples and provide the results in 1 day (see version 8 of the mirpredx instructions manual).

#### The Nu.Q™ Colorectal Cancer Screening Triage Test

NuQ® tests (Volition SA; Namur, Belgium) are intended for diagnosing CRC by analyzing different nucleosome characteristics, including the DNA methylation of DNA bound to nucleosomes, post-translational modifications in histones and histone variants, and the detection of cell-free nucleosomes, although the company is developing a new test based on these biomarkers. NuQ® tests are based on Enzyme-linked immunosorbent assay (ELISA) technology and require only one drop of blood from patients (a 10-μl sample).

The most advanced test is the Nu.Q™ Colorectal Cancer Screening Triage Test, which consists in combining different NuQ® previously CE-IVD marked tests. One of them is the NuQ®X test, which detects the 5-methylcytosine levels present in DNA bound to cell-free circulating nucleosomes.

In a validation study performed by Holdenrieder et al. (2014), serum samples were used in two independent cohorts of subjects: i) 90 subjects, including CRC patients (n = 24), benign colorectal diseases (BCD) (n = 10), and healthy controls (n = 56); ii) 113 subjects, including CRC patients (n = 49), BCD (n = 26), and healthy controls (n = 38). Holdenrieder et al. (2014) used the Nu.Q®X test to evaluate its differential diagnostic performance. Their study showed that the circulating methylated DNA levels significantly lowered in CRC and BCD compared with the healthy controls (p < 0.05), although no difference was found between BCD and CRC. The AUC on the receiver operating characteristic curve was 0.78, and sensitivity was 33% at 95% specificity for CRC and BCD compared to HC, with a sensitivity of 75% at 70% specificity for CRC compared to HC.

Beltrán-Garcia et al. Epigenetic IVD Tests in Cancer

To improve both the sensitivity and specificity of the assays, two new tests were designed: the Nu.Q®T test and the NuQ®V test. Both obtained the CE-IVD mark. The Nu.Q®T test was designed for the diagnostic of CRC by detecting total free circulating nucleosomes (cell-free nucleosome). Nu.Q®V focused on detecting CRC by analyzing histone variants. Finally, they were included in a NuQ® test based on the same nucleosomics ELISA technology (Holdenrieder et al., 2014).

Rahier et al. (2017) used the Nu.Q® assay to evaluate the levels of 12 epitopes [including nucleosome-associated histone modifications: H4K20me3 (mAb), H4PanAc (mAb), pH2AX (mAb), H3K9Me3 (pAb), H2AK119Ub (mAb), H3K9Ac (mAb), and H3K27Ac (mAb); nucleosome-associated DNA modification: 5mC (mAb); nucleosome containing histone variants: H2AZ (mAb); nucleosome-protein adducts: HMGB1 (mAb) and EZH2 (mAb); and finally a conserved nucleosome epitope as reference of total nucleosome content] in the sera of 58 individuals referred for endoscopic CRC detection [patients with CRC (n = 23), patients with pre-cancerous lesions (polyps) (n = 16), and healthy controls (n = 19)]. The multivariate analysis defined a panel of four age-adjusted cf-nucleosomes that provided an AUC of 0.97 for the CRC discrimination of healthy controls with high sensitivity in initial stages (sensitivity of 75% and 86% and specificity of 90% for stages I and II, respectively). The second combination of four cf-nucleosome biomarkers provided an AUC of 0.72 for the identification of patients with pre-cancerous lesions (polyps) (n = 16) in healthy subjects (Rahier et al., 2017).

The Nu.Q™ Colorectal Cancer Screening Triage Test, which is based on the previous described Nu.Q® tests, was evaluated in blinded serum samples from 1,961 FIT-positive individuals. In a set of samples "training set" (n = 1,907), the Nu.Q™ Colorectal Cancer Screening Triage test had the potential to identify a subset of 477 subjects in which colonoscopy was applied and could be avoided. Moreover, the test detected 96.6% of CRCs and 88.5% of highrisk adenomas. The results were corroborated in the "validation set" of samples (n = 1,961), which gave a sensitivity of 91.2% for CRC and 83.0% for high-risk adenoma. Ii was noteworthy that the sensitivity for "screen relevant neoplasia" (considering patients with CRC and high-risk adenomas) was about 86% compared with the 80% obtained with positive FIT and a cutoff value of 200 ng/ml. The results of this large cohort evaluation were promising as the Nu.Q™ Colorectal Cancer Screening Test can reduce unnecessary colonoscopies by 20%, while maintaining sensitivity for CRC close to 90% (Marielle et al., 2017).

The Volition Company announced that the new Nu.Q™ assay would have the potential to detect 81% of CRCs with a specificity of 78% in a cohort of 4,800 CRC patients. Furthermore, the new Nu.Q™ assay detected up to 67% of high-risk adenomas with a specificity of 80% in a cohort of 530 symptomatic patients and initial stage I cancers with a sensitivity of 74% and a specificity of 90% in a pilot study of 58 asymptomatic patients. However, we were unable to find any published results or any registered clinical trial results of this study apart from the company's published interim results.

The Volition Company is developing new-generation Nu.Q assays for other intended uses, such as pancreatic cancer. In fact

the Nu.Q assay was also evaluated for the diagnostic of pancreatic cancer. By using a combination of carbohydrate antigen 19-9 (CA 19-9) levels with a panel of four cf-nucleosome markers, Bauden et al. (2015) obtained an AUC of 0.98 with an overall sensitivity of 92% at a 90% specificity to detect pancreatic cancer in serum samples from a cohort of 59 subjects [including patients with resectable pancreatic cancer (n = 25), patients with benign pancreatic disease (n 0), and healthy individuals (n = 24)].

#### An Epigenetic-Based IVD Test for Breast Cancer

Breast cancer (MIM 114480) is the most commonly diagnosed cancer in women (Torre et al., 2017) and the leading cause of death from cancer in women worldwide (Torre et al., 2016). It is noteworthy that breast cancer can also affect men and, consequently, around 2,670 new cases of invasive breast cancer are expected to be diagnosed in men in 2019. About 20% of breast cancers worldwide are due to environmental or lifestyle risk factors, such as alcohol abuse, excess body weight and fat, and a sedentary lifestyle (Danaei et al., 2005). In addition, screening with the mammography technique has demonstrated its ability to detect breast cancer in early stages, which reduces the mortality risk and increases treatment success (Lauby-Secretan et al., 2015). As a result, new methods that contribute to early diagnosis, the identification of specific subtypes, and the selection of patients who can benefit from specific therapies will increase patient survival for this cancer.

Breast cancer mortality rates are higher than those for any other cancer and account for 25% of cancer cases and 15% of cancer-related deaths (Ferlay et al., 2015). Breast cancer mortality also depends on the cancer subtype. Breast cancer presents several classifications depending on different aspects. It can be classified according to their histological origin, cell differentiation degree, stage, the presence or absence of certain hormone receptors [i.e., hormonal receptors, like estrogen receptor (ER), and progesterone receptor (PR); and the ERBB2 receptor], and molecular subtype (i.e., luminal A, luminal B, HER2, basal-like subtype, normal-like subtype, and Claudin-low subtype).

 Enhancing breast cancer survival and outcome by early detection remains one of the main breast cancer priorities according to the World Health Organization (WHO). Therefore, several efforts are being made by the research community to provide not only new drugs and therapies to treat breast cancer, but to also identify new biomarkers to help implement precision medicine into the clinical management of breast cancer patients (Low et al., 2018). Breast cancer treatment depends partially on the disease state and the breast cancer subtype. Generally speaking, the commonest treatments are targeted therapy, hormonal therapy, radiation therapy, surgery, and chemotherapy, although immunotherapy is being increasingly utilized. Fortunately, the therapeutic options for breast cancer patients are further improved thanks to the use of biomarkers and the implementation of precision medicine (Meisel et al., 2018), in which epigenetic biomarkers can further improve the battery of *in vitro* assays to manage breast cancer.

#### The Therascreen PITX2 RGQ PCR Kit

The *Therascreen PITX2 RGQ PCR kit* (Qiagen, Germany) is a methylation-based CE-IVD marked assay that predicts the response of lymph node-positive, ER-positive, and HER2-negative high-risk breast cancer patients. The test differentiates between the patients more likely to respond to anthracyclines chemotherapy (Aubele et al., 2017), and it obtained the CE-IVD mark in 2018.

The methylation analysis of *PITX2* (a promoter of transcription factor 2 of the pituitary homeobox) demonstrates a high correlation with other diagnostic techniques, has the predictive and prognostic capability for patient identification, and supports clinicians by being the most effective therapy option. *PITX2* methylation has attracted the attention of clinicians for not only breast cancer (Widschwendter et al., 2004; Aubele et al., 2017), but also for other tumor types. Continuous scientific evidence indicates the potential of the *PITX2* methylation analysis to predict breast cancer outcomes in lymph node-positive, ER-positive, and HER2-negative breast cancer patients to adjuvant anthracyclinebased chemotherapy. Therefore, these clinical observations reinforce the idea of using *PITX2* methylation status to support clinicians as the most effective therapy option (Hartmann et al., 2009; Absmaier et al., 2018).

Hartmann et al. (2009) showed that the *PITX2* DNA methylation improved the prediction by using only clinical factors like tumor stage, grade, or age in a cohort of >200 patients. *PITX2* plays an essential role in the disease pathogenesis. In fact, tumors with a hypermethylated *PITX2* status correlate with poorer survival (overall survival and reduced metastasis-free survival), and also with resistance to treatment. In addition, *PITX2* methylation has been associated with the response to adjuvant chemotherapy (Absmaier et al., 2018; Sheng et al., 2017).

Absmaier et al. (2018) explored the validity of this new predictive candidate biomarker in a retrospective exploratory study. To do so, these authors determined the *PITX2* DNA methylation status in non-metastatic TNBC patients treated with adjuvant chemotherapy with anthracycline by a molecular analysis of breast cancer tissues. Univariate and multivariate analyses demonstrated the statistically independent predictive value of *PITX2* DNA methylation. The authors concluded that for those patients with non-metastatic TNBC, the selective determination of the *PITX2* DNA methylation status can serve as a cancer biomarker to predict responses to anthracycline-based adjuvant chemotherapy (Absmaier et al., 2018).

Schriker et al. (2018) performed a clinical study to analyze the performance of the *PITX2* DNA methylation assay compared to microarray technology. These authors concluded that the performance of the *Therascreen PITX2 RGQ PCR* assay showed high reliability and robustness to predict the outcome of patients with high-risk breast cancer to anthracycline-based chemotherapy. In this study, three CpGs from the *PITX2* promoter 2 gene (*PITX2P2*; 4q25) contained in the methylation array (Maier et al., 2007) were selected, and the appropriate Taqman probes were designed to cover these three CpGs in the Therascreen PITX2 RGQ assay (Schricker et al., 2018).

The *Therascreen PITX2 RGQ PCR assay*, developed by Perkins et al. (2018) in conjunction with the Therawis Diagnostics Company, analyzes the methylation status of the *PITX2* gene obtained from the DNA of FFPE biospecimens. *PITX2* methylation is assessed by methylation-specific real-time PCR and exploits the quantitative PCR (qPCR) oligonucleotide hydrolysis principle of two TaqMan probes labeled with different fluorescent dies (FAM™ for fully methylated and HEX™ for fully unmethylated DNA) in combination with methylation nonspecific primers to measure the methylation status of the target sequences of PITX2 gene promoter 2 in bisulfite-treated DNA. The *Therascreen PITX2 RGQ PCR kit* (Qiagen, Catalog no. 873211) has been currently CE-IVD marked and is commercially available (Aubele et al., 2017; Schricker et al., 2018). It runs in the real-time Rotor-Gene Q MDx thermal cycler (Qiagen) or a Rotor-Gene Q MDx 5plex HRM instrument (Qiagen). The percentage of the methylation ratio (PMR = 100/ (1 + 2exp(CtFAM(methylated) − CtHEX(unmethylated))]) is calculated by the Rotor-Gene AssayManager® software with a Gamma Plug-in plus a kit-specific *PITX2* Assay Profile for automated analyses and quality control, including all the validity criteria. Detailed information about the method is described by Schricker et al. (2018) and Maier et al. (2007). The *Therascreeen PITX2 RGQ PCR assay* can be easily adopted in clinical laboratories that already run other Therascreen assays commercialized by Qiagen. The complete workflow is streamlined throughput for a medium sample with highly reliable and robust readouts and can be performed in a time of 2 working days (Perkins et al., 2018).

#### The Epigenetic-Based IVD Test for Cervical Cancer

Cervical cancer (MIM 603956) is the fourth most frequent cancer in women, with an estimation of 570,000 new cases in 2018, which represents 6.6% of all female cancers. Cervical cancer is the fourth commonest cause of death from cancer in women (Vu et al., 2018), which is approximately 8% of the total deaths from cancer. Furthermore, as cervical cancer has no shown symptoms in its early stages, early identification of cervical precancerous lesions is of critical importance (Gradíssimo and Burk, 2017).