Interaction between Long Noncoding RNAs and Syncytin-1/Syncytin-2 Genes and Transcripts: How Noncoding RNAs May Affect Pregnancy in Patients with Systemic Lupus Erythematosus

Background: Patients with systemic lupus erythematosus (SLE) often suffer from obstetric complications not necessarily associated with the antiphospholipid syndrome. These events may potentially result from the reduced placental synthesis of the fusogenic proteins syncytin-1 and syncytin-2, observed in women with pregnancy-related disorders. SLE patients have an aberrant noncoding (nc)RNA signature that may in turn dysregulate the expression of syncytin-1 and syncytin-2 during placentation. The aim of this research is to computationally evaluate and characterize the interaction between syncytin-1 and syncytin-2 genes and human ncRNAs and to discuss the potential implications for SLE pregnancy adverse outcomes. Methods: The FASTA sequences of the syncytin-1 and syncytin-2 genes were used as inputs to the Ensembl.org library to find any alignments with human ncRNA genes and their transcripts, which were characterized for their tissue expression, regulatory activity on adjacent genes, biological pathways, and potential association with human disease. Results: BLASTN analysis revealed a total of 100 hits with human long ncRNAs (lncRNAs) for the syncytin-1 and syncytin-2 genes, with median alignment scores of 151 and 66.7, respectively. Only lncRNAs TP53TG1, TTTY14, and ENSG00000273328 were reported to be expressed in placental tissue. Dysregulated expression of lncRNAs TP53TG1, LINC01239, and LINC01320 found in this analysis has previously been described in SLE patients as well as in women with a high-risk pregnancy. In addition, some of the genes adjacent to lncRNAs aligned with syncytin-1 or syncytin-2 in a regulatory region might increase the risk of pregnancy complications or SLE. Conclusions: This is the first computational study showing alignments between syncytin-1 and syncytin-2 genes and human lncRNAs. Whether this mechanism affects syncytiotrophoblast morphogenesis in SLE females is unknown and requires further investigation.


Introduction
Systemic lupus erythematosus (SLE) is a prototypical autoimmune connective tissue disease that mainly affects women of childbearing age. The worldwide prevalence is reported to range from 36.7 to 366.6 per 100,000 individuals, with various differences according to gender and ethnicity [1]. The pathogenesis is multifactorial: genetics and environmental factors such as ultraviolet light and infections can trigger the disease by chronically activating both innate and adaptive immune responses. The immunological scenario is based on the activation of dendritic cells, neutrophil granulocytes, B and T lymphocytes, and the complement system, leading to mechanisms such as NETosis, autoantibody release, immune complex formation, and type I interferon (IFN) production [2]. SLE is a multiorgan disease with both constitutional and organ-specific manifestations. The latter include renal, hematological, neuropsychiatric, mucocutaneous, musculoskeletal, Int. J. Mol. Sci. 2023, 24, 2259 2 of 18 and serosal symptoms [3]. According to a meta-analysis conducted in the United States with more than 26,000 patients, the overall mortality risk is almost threefold-increased in SLE compared to the general population [4]. The main causes of death are attributed to renal failure, infections, and heart disease [1]. In women of childbearing age, SLE is commonly associated with obstetric complications such as fetal growth restriction, spontaneous abortion, preeclampsia, cesarean section, and preterm delivery. These events can occur in 12% to nearly 40% of SLE women and are dependent on age, disease activity, concomitant medications, autoantibody titers, including antiphospholipid antibodies (aPLs), and type I IFN activity [5]. Underlying mechanisms may encompass both local vasculopathy and inflammation due to trophoblastic antigen recognition. In particular, positivity of aPLs is a risk factor for antiphospholipid syndrome (APS), an autoimmune disorder characterized by pregnancy morbidity and recurrent venous and/or arterial thrombosis. It is estimated that approximately 40% of SLE patients have circulating aPLs, although APS occurs in less than 40% of aPL-positive SLE patients [6]. According to one study, aPL-negative SLE women have twice the risk of obstetric complications such as perinatal death and preterm delivery compared with the general population, suggesting the existence of additional pathogenic pathways [7].
Placental development is controlled by several mediators, two of which are represented by the fusogenic glycoproteins syncytin-1 and syncytin-2. As the name suggests, the main role of syncytins is to induce the fusion of single nucleated cytotrophoblast cells into syncytiotrophoblast, which divides maternal and fetal tissues and has a crucial function in fetal protection and nutrition [8]. Both syncytin-1 and syncytin-2 are involved in the homeostasis, differentiation, proliferation, and survival of syncytiotrophoblast and have additional immunomodulatory properties that induce maternofetal tolerance thanks to the immunosuppressive domain (ISD), which counteracts the activation of dendritic cells, T cells, and the production of IFNs [9][10][11]. Syncytin-1 and syncytin-2 are endogenous retroviral proteins encoded by the envelope gene of proviruses belonging to the human endogenous retrovirus (HERV)-W and HERV-FRD families, respectively. HERVs are genetic elements of ancient retroviral infectious origin that constitute approximately 8% of the human genome. After their integration into the nuclear genome, HERVs have been increasingly silenced by inactivating mutations, although some of them may still retain intact open reading frames (ORFs) and consequently reactivate under physiological or pathogenic circumstances [12]. The expression of syncytin-1 and syncytin-2 transcripts and proteins during pregnancy is a fair example of physiological HERV reactivation. This process is tightly regulated by epigenetic mechanisms and is critical for pregnancy outcome.
On the other hand, abnormal reactivation of HERVs has been linked to the occurrence of human diseases, including autoimmune disorders [13]. In SLE patients, dysregulation of HERV-E members has been found to mimic viral infection and further stimulate IFN response and the production of antibodies against nuclear components [14]. This event may be embedded in a more complex scenario involving other noncoding (nc)RNAs that provide epigenetic and posttranscriptional control of coding and noncoding genes. Studies have shown that SLE is associated with a dysregulated signature of microRNAs (miRNAs) and long ncRNAs (lncRNAs) in peripheral blood mononuclear cells (PBMCs) and kidney tissue [15]. It follows that the alteration of the SLE transcriptome profile could be the basis for impaired expression of syncytin-1 and syncytin-2 during pregnancy, providing an alternative pathogenic view to explain obstetric complications in these patients.
The aim of this research is to computationally evaluate and characterize the interaction between syncytin-1 and syncytin-2 genes and human ncRNAs and to discuss the potential implications for SLE pregnancy adverse outcomes. By using the bioinformatic tool QmRLFS-finder, there was a single case in which the nucleotide sequence of a lncRNA complementary to syncytin-1 gene was predicted to form an R-loop. The involved lncRNA was SLC17A6-DT, normally expressed in the brain, muscle, gastrointestinal tract, glands, and testis.
By consulting the GeneCards database, the retrieved lncRNAs appeared to be mostly expressed in the testis and nervous system. Under physiological circumstances, the placental expression was solely reported for the lncRNAs ENSG00000273328 and TP53TG1.
Enrichment analysis showed that the retrieved lncRNAs are mainly involved in cardiac muscle contraction and adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathways, Figure S2. With the exception of the lncRNAs SCAT1, LINC00320, PURPL, and TP53TG1, associated with the risk of certain types of cancer or genetic diseases, no other associations with human disorders were found.

Discussion
The results of this pivotal in silico study show that syncytin-1 and syncytin-2 genes and transcripts are at the center of an intricate epigenetic network involving coding genes and lncRNAs. The latter are ncRNAs with more than 200 nucleotides that have recently attracted the attention of researchers due to their pathogenic potential in diseases such as cancer, neurodegenerative disorders, and autoimmunity [16]. Dysregulation in the lncRNA signature may also be responsible for impairing critical trophoblast cell functions such as proliferation, migration, invasion, and cell cycle progression [17]. More than 50,000 lncRNAs have been discovered in intergenic or intron/exon regions of coding genes of the human genome, but most have not yet been characterized in terms of their biological functions [18]. LncRNAs have been localized to both the nucleus and cytosol. Nuclear lncRNAs play a crucial role in scaffolding and remodeling chromatin and regulating transcription by binding RBPs and DNA and generating R-loops, which are trimeric DNA-RNA hybrids [19]. On the other hand, cytosolic lncRNAs could control the translation and stability of proteins as they can bind to RBPs, mRNAs, or miRNAs [18].
It has been postulated that lncRNAs could override other noncoding transposable elements, including long interspersed nuclear elements (LINEs), short interspersed nuclear elements (SINEs), and HERVs. In turn, HERV-derived solitary long terminal repeats (LTRs) may provide regulatory sequences and control the expression of neighboring lncRNA genes [20].
SLE patients exhibit a dysregulated ncRNA signature that results in the increased expression of transcripts and antigenic proteins derived from HERV members or lncR-NAs [14,15,[21][22][23][24][25][26]. This event may depend on the SLE cytokine milieu, hormones, chemicals or microbial stimuli [27,28]. The overproduction of nucleic acids may foment the type I IFN response and the release of anti-dsDNA antibodies, both of which have been associated with SLE obstetric complications [28][29][30], Figure 1.
In addition, lncRNA transcripts may trigger an epigenetic mechanism to control the expression of other coding and noncoding genes, some of which, such as HERVs, may play important roles in certain life stages such as pregnancy. As mentioned previously, SLE patients exhibit aberrant HERV expression compared to healthy controls [14,31], which in turn may reflect an altered lncRNA transcriptome. Unfortunately, there are no studies investigating the occurrence of dysregulated synthesis of the HERV-derived env proteins syncytin-1 and syncytin-2 in SLE patients. The role of syncytins in APS pathogenesis also remains unknown, although some recent studies have reported an altered lncRNA signature in these patients compared with controls [32,33]. Indeed, lncRNAs may contribute to several steps of APS pathogenesis, including leukocyte activation, immunothrombosis, and impaired embryonic development [34], but whether these events are influenced by the abnormal expression of syncytin genes has not been investigated to date.
The transmembrane glycoprotein syncytin-1 is encoded by the HERV-W provirus ERVWE1 at the 7q21.2 env locus, whereas syncytin-2, which is homologous to syncytin-1, is encoded by an HERV-FRD provirus at locus 6p24.1 [35]. The upregulation of syncytin-1 in villous and extravillous trophoblasts depends on the binding of the transcription factors cAMP-response element-binding protein (CREB), glial cells missing transcription factor 1 (GCM1), and the hypomethylation of a neighboring MaLR solitary LTR. Expression of syncytin-2 is instead restricted to the villous cytotrophoblast and regulated by GCM1 binding and methylation patterns [35]. Therefore, syncytin-1 and syncytin-2 exhibit distinct cellular expression patterns and time-dependent effects as they separately regulate cell cycle phases in trophoblast cells [36]. Normally, the expression of syncytins is directly proportional to gestational age, and a decrease has been associated with pathological conditions such as hypoxia and preeclampsia [37][38][39]. However, it is unknown whether the differential expression of syncytin genes during placentation could be under the epigenetic control of lncRNAs.
lncRNAs play a crucial role in scaffolding and remodeling chromatin and regulating transcription by binding RBPs and DNA and generating R-loops, which are trimeric DNA-RNA hybrids [19]. On the other hand, cytosolic lncRNAs could control the translation and stability of proteins as they can bind to RBPs, mRNAs, or miRNAs [18].
It has been postulated that lncRNAs could override other noncoding transposable elements, including long interspersed nuclear elements (LINEs), short interspersed nuclear elements (SINEs), and HERVs. In turn, HERV-derived solitary long terminal repeats (LTRs) may provide regulatory sequences and control the expression of neighboring lncRNA genes [20]. SLE patients exhibit a dysregulated ncRNA signature that results in the increased expression of transcripts and antigenic proteins derived from HERV members or lncRNAs [14,15,[21][22][23][24][25][26]. This event may depend on the SLE cytokine milieu, hormones, chemicals or microbial stimuli [27,28]. The overproduction of nucleic acids may foment the type I IFN response and the release of anti-dsDNA antibodies, both of which have been associated with SLE obstetric complications [28][29][30], Figure 1. In addition, lncRNA transcripts may trigger an epigenetic mechanism to control the expression of other coding and noncoding genes, some of which, such as HERVs, may Moreover, according to the results of a preclinical study, syncytin-2, but not syncytin-1, might have immunosuppressive effects through its ISD [40]. These results are consistent with the immunopathogenic activity of HERV-W env proteins, which can stimulate both innate and adaptive immunity [35]. The immunogenicity of HERV-W env proteins seems to be most prominent in neuroinflammatory diseases such as multiple sclerosis (MS) [28], while data concerning SLE pathogenesis are still unclear [41,42].
In this analysis, a total of 100 human lncRNA transcripts were predicted to align with the nucleotide sequence of syncytin-1 or syncytin-2. An aberrant lncRNA transcriptome in the endometrium and placental tissue has been described in association with obstetric complications in women without SLE [17,[43][44][45][46][47][48]. When comparing such literature data with the results of this study, a match was found only for the lncRNAs TP53TG1, LINC01320, and LINC00320 [45][46][47][48]. Other lncRNAs retrieved in the present analysis have been associated with an increased risk of endometriosis or X chromosome instability during early embryonic development [49][50][51][52][53]. Among them, the lncRNA TP53TG1 was predicted to align with the nucleotide sequences of both syncytin-1 and syncytin-2 with scores of 140 (ID: 89.4%) and 51.9 (ID: 92.1%), respectively. TP53TG1 can be localized both intracellularly (nuclear and cytosolic localization) and extracellularly in placental tissue and appears to be involved in cell damage that can result from exposure to agents such as ultraviolet radiation [54], which is a crucial triggering factor for SLE. Although the actual role of TP53TG1 in pregnancy is unknown, one study found increased demethylation of this lncRNA gene in the female cadmium-exposed placenta, which may be responsible for suboptimal fetal growth [45].
Indeed, SLE females might have a different transcriptomic signature than non-SLE females with pregnancy adverse outcomes. However, only a few of the lncRNAs found in this computational analysis have been previously reported in the literature as biomarker candidates for SLE risk [55][56][57][58], Table 5. Interestingly, a recent experimental study characterizing the molecular signature of 54 biopsy specimens from lupus nephritis patients demonstrated that the aforementioned lncRNA TP53TG1 inversely correlated with the degree of glomerulosclerosis [55]. Conversely, none of the lncRNAs reported in studies of APS patients correlated with lncRNAs that showed alignment with syncytin-1 or syncytin-2 genes [32,33].
These discrepancies may be due to the different methodologies and selective tissue expressions of lncRNAs. With only one exception, the studies that aimed to characterize the lncRNA transcriptome in SLE patients did not include pregnant women or analyze placental tissue. The lncRNA profile in the placenta of SLE women was characterized only in a recent Chinese RNA-seq study [59]. Samples were collected between 34.9 and 39.7 weeks of gestation; in 10% of cases, fetal weight was below the 10th percentile, and in two cases, the patients had a cesarean section. The results showed a total of 52 dysregulated lncRNAs in the placental tissue of SLE women compared with controls. Again, none of the 52 lncRNAs reported by the authors correlated with the lncRNAs found in the present study. Different methods, the small sample (10 participating SLE patients), the low SLE disease activity, and the absence of severe obstetric complications such as preeclampsia or fetal loss may be the reasons for these conflicting results.
Although tissue expression was not always available, a number of lncRNAs complementary to syncytin-1 and syncytin-2 have been reported to be physiologically expressed in placental or uterine tissue. In the GeneCards database, placental localization has been described for the lncRNAs TP53TG1, TTTY14, and ENSG00000273328. Two of them (TP53TG1 and TTTY14) have been reported to be associated with pregnancy adverse outcomes or gynecological diseases. In addition to the previously mentioned TP53TG1 [45], the Y-linked lncRNA TTTY14, which aligns with the syncytin-1 sequence, is localized in the nucleus and may be abnormally expressed in the endometrium as a phenomenon of male microchimerism in endometriosis and infertility [53]. Therefore, overexpression of TP53TG1 and TTTY14 in the placenta or uterus could be responsible for pregnancy complications. As suggested by the present study, a hypothetical underlying mechanism could be complementation and/or sequestration of syncytin-1 and syncytin-2 genes or transcripts, ultimately leading to inhibition of their translation into functional glycoproteins.
The results of the enrichment analysis for the detected lncRNA genes showed different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways depending on the type of matching syncytin sequence. Specifically, lncRNA genes that had hits with the syncytin-2 sequence were predicted to be involved in the AMPK signaling pathway, which regulates cellular energy homeostasis. AMPK function has been shown to be required during placental differentiation, providing nutrient transport and protection of both maternal and fetal tissues and, consequently, preventing preeclampsia, intrauterine growth restriction, and preterm birth [60]. Conversely, the results of enrichment analysis for lncRNA genes matching the syncytin-1 sequence showed an association with metabolic pathways occurring in the liver leading to nonalcoholic fatty liver disease, which in turn is associated with pregnancy complications [61,62]. In neither case did the KEGG pathways converge with a dysregulated immune response or alterations in autophagy or phagocytosis, which are hallmarks of SLE pathogenesis [63,64]. Table 5. Summary of literature data showing a possible role of lncRNAs aligning with the nucleotide sequence of syncytin-1 and syncytin-2 in the pathogenesis of SLE or pregnancy complications (GWAS: genome-wide association study; LN: lupus nephritis).

TP53TG1
Hypo-expressed in glomerulosclerosis kidney samples according to a molecular signature study of 51 patients with lupus nephritis [55] Demethylated in female cadmium-exposed placenta according to a genome-wide DNA methylation study of placental tissue from 24 women [45] XACT Unknown Hyper-expressed in both human preimplantation embryos and naive human embryonic stem cells; competes with XIST and prevents X chromosome silencing and functional nullisomy during early human development according to a single-cell RNA-sequencing analysis of more than 100 human embryos [49]; XACT loss of heterozygosity potentially affecting the inactivation of the skewed X chromosome and leading to X chromosome instability in human embryonic stem cells revealed by a high-resolution chromosome microarray analysis of 105 human embryos and derived human embryonic stem cells [50] MIR548XHG Unknown Overexpressed in plasma extracellular vesicles from women with endometriosis according to an RNA-sequencing study of 85 patients and 86 controls [51] LINC01239 Associated with incomplete lupus erythematosus according to a GWAS of 335 patients and 236 controls [56]; Upregulated in morning urine samples from 3 LN patients compared to 3 healthy controls [57] Dysregulated in patients with epithelial ovarian cancer and endometriosis according to a ChIP-sequencing and ATAC-sequencing analysis of a large cohort of endometriosis and epithelial ovarian cancer patients [52] TTTY14 Unknown Hyper-expressed in endometrial samples from infertile women as a phenomenon of male microchimerism according to a transcriptomic profiling study of 60 fertile and infertile participants without endometriosis and 60 fertile and infertile participants with endometriosis [53] LINC01320 Associated with the inflammatory proximal tubule histologic subtype observed in kidney samples from patients with glomerulonephritis, including one case of LN, according to a single-cell RNA-sequencing study [58] Upregulated in the endometrium during the implantation window according to an RNA-sequencing analysis of 30 fertile women [48]; Dysregulated in syncytiotrophoblasts, invasive cytotrophoblasts, and endovascular cytotrophoblasts isolated from placental tissue of 4 women with severe preeclampsia and 4 women with uninfected preterm birth according to a global transcriptional profiling study [46] Syncytin-2

Unknown
As above LINC00320 Unknown Upregulated in spontaneous preterm placenta from 20 women compared to spontaneous term placenta from 20 control subjects according to a transcriptomic RNA-sequencing analysis [47]

As above As above
Because lncRNAs can epigenetically regulate transcription of neighboring genes, chromosomal regions near (within 1.00 Mb) lncRNA genes that contained hits for syncytin-1 and syncytin-2 genes in a regulatory region were analyzed. Protein-coding genes located near syncytin-1-matched lncRNA genes were found to be involved in DNA binding and transcription, stress response, anabolic or metabolic pathways, enzyme reactions, and exocrine gland secretion. Among them, the CNR1 and GRIN2B genes, encoding cannabinoid receptor 1 and NMDA-type ionotropic glutamate receptor subunit 2B, respectively, have been associated with the risk of preeclampsia in case-control studies [65,66]. Importantly, a novel association between SLE and the GRIN2B gene was found in a dataset from a genome-wide association study (GWAS) [67].
Instead, genes bordering syncytin-2-matched lncRNAs were found to be involved in metabolism, cell motility, transcription, translation, ubiquitination, immune defense, and nociception. Polymorphic variants of CYLD, GC, MBL2, and ZNF572 genes have been collectively associated with the risk of preterm birth or recurrent late pregnancy loss [68][69][70][71][72], while dysregulated expression of CYP8B1 may be responsible for pregnancy intrahepatic cholestasis in mice [73]. The TCL6 gene has been reported to be overexpressed in the placental tissue of women with preeclampsia, threatened miscarriage, or spontaneous abortion [74,75]. Finally, the neuropeptide receptor FF 2, encoded by the gene NPFFR2, was found to be overexpressed in the placental tissue of women with preeclampsia and closely related to the production of syncytin-1 and syncytin-2 during pregnancy [36]. Importantly, there is evidence that some of the genes listed above may be critical for the pathogenesis of SLE. These genes include CYLD and MBL2, both of which are involved in the innate immune response. CYLD, encoding a deubiquitinase, appears to be overexpressed in kidney samples from patients with SLE glomerulonephritis [76]. On the other hand, more than 30 publications report an association between polymorphic variants of the MBL2 gene, encoding mannose-binding lectin 2, and SLE [77,78]. Mannose-binding lectin activates complement and is thus involved in the clearance of cellular debris and pathogens [79]. Low levels of mannose-binding lectin have been associated with the risk of disease. Indeed, a deficit in its function could be crucial for the loss of immune tolerance and the development of autoimmune phenomena. Associations between protein-coding genes adjacent to lncRNAs of interest and SLE or pregnancy complications are shown in Table 6.
This analysis also showed that the lncRNA SLC17A6-DT contains an alignment to syncytin-1 in an R-loop-forming sequence, but the pathogenic role of this lncRNA in autoimmunity or pregnancy complications remains to be elucidated.
Finally, a total of 27 and 24 interactions with RBPs were predicted for lncRNAs aligned with syncytin-1 and syncytin-2, respectively. The RBPs included NISCH, AEBP2, SUPT5H, CHIC1, and DNAJC5B, which preside over various processes including cell proliferation and malignant transformation, neural crest migration, transcription elongation, and antiviral defense [80][81][82][83][84]. Interestingly, CHIC1, predicted to bind one syncytin-1-complementary lncRNA and six syncytin-2-complementary lncRNAs, was reported to be progressively hypermethylated and consequently hypo-expressed from morula to blastocyst development in an animal experiment under physiological conditions [85]. However, the relationship between CHIC1 and the expression of syncytins during pregnancy in humans has not yet been investigated. Table 6. Results of studies reporting associations between protein-coding genes adjacent to lncRNA genes with hits in the syncytin-1 and syncytin-2 nucleotide sequence and SLE or pregnancy adverse outcomes (ERα: estrogen receptor α; GWAS: genome-wide association study; ISN/RPS: International Society of Nephrology/Renal Pathology Society; LN: lupus nephritis; qPCR: quantitative polymerase chain reaction; qRT-PCR: quantitative real time-polymerase chain reaction; SNPs: single nucleotide polymorphisms). Higher frequency of codon 52 polymorphism in preterm birth cases compared with term controls and association of MBL2 O/O genotype with risk of preterm birth according to a genotyping study of 204 DNA blood samples [70]; Association between MBL2 genotypes leading to MBL deficiency and recurrent late pregnancy loss independent of LAC positivity according to a genotyping study of 75 patients and 104 controls [72] ENSG00000273328 ZNF572 Unknown Upregulated in amniotic fluid supernatant samples from 21 preterm birth patients compared to term birth controls according to a sequencing and qPCR study [71] ENSG00000273328 CYP8B1 Unknown Undergoes ERα-induced downregulation in mice, leading to impaired bile acid biosynthesis and potential risk of intrahepatic cholestasis in pregnancy [73] LINC02318 TCL6 Unknown Overexpressed in 42 placental tissues from women with preeclampsia compared with controls and hypo-expressed in preeclamptic pregnancies with lower urine protein levels, normal blood pressure, and higher newborn weight according to a qRT-PCR study [74];Overexpressed in placental tissue in threatened abortion pregnancy compared with normal pregnancy and in spontaneous abortion pregnancy compared with induced abortion pregnancy according to a qRT-PCR study of 30 women with spontaneous abortion, 30 women with induced abortion, and 30 control subjects with normal pregnancy [75] ENSG00000248567 NPFFR2 Unknown Hyper-expressed in placental tissue during the first trimester and in placental samples from preeclamptic women and indirectly associated with the expression of syncytin-1 and syncytin-2 in human cytotrophoblast cells [36] In summary, this computational study shows that the genes and transcripts of syncytin-1 and syncytin-2 correspond to human lncRNAs, which have both nuclear and cytosolic localization and may be involved in energetic and metabolic pathways. Remarkably, three of the lncRNAs found (TP53TG1, LINC01239, and LINC01320) have been described in studies to be dysregulated in both SLE patients and women with high-risk pregnancies. In addition, protein-coding genes adjacent to the lncRNAs found have been reported to be associated with gynecologic/obstetric complications in nine cases and with SLE risk in three cases. Although no data are currently available to confirm these pivotal findings, the following hypothesis can be made. Abnormal nuclear and cytoplasmic expression of lncRNAs in the syncytiotrophoblast and placental tissue of pregnant women with SLE might prevent the transcription and translation of syncytin-1 and syncytin-2 mRNA. Moreover, the spongy effect of complementary lncRNAs could be thought to locally reduce maternal immune tolerance to embryonic tissues by sequestering syncytin transcripts and preventing surface expression of the ISD. Increased production of ncRNAs may additionally activate sensing platforms in endosomes or cytosol and trigger local inflammation. Another hypothesis could be that syncytin transcripts may align with lncRNA regulatory or R-loop sequences and interfere with transcription of neighboring genes, for which a clear role in obstetric adverse outcomes or SLE has been demonstrated, Figure 2. with SLE might prevent the transcription and translation of syncytin-1 and syncytin-2 mRNA. Moreover, the spongy effect of complementary lncRNAs could be thought to locally reduce maternal immune tolerance to embryonic tissues by sequestering syncytin transcripts and preventing surface expression of the ISD. Increased production of ncRNAs may additionally activate sensing platforms in endosomes or cytosol and trigger local inflammation. Another hypothesis could be that syncytin transcripts may align with lncRNA regulatory or R-loop sequences and interfere with transcription of neighboring genes, for which a clear role in obstetric adverse outcomes or SLE has been demonstrated, Figure 2. This study has several limitations. One is due to its purely computational nature, which indeed requires further confirmation of the results by laboratory experiments on cells and tissues from SLE patients with pregnancy complications. Moreover, this study did not investigate the alignments between human ncRNAs and env genes of other HERV members, such as HERV-F or HERV-K (HML2) proviruses, which may have complementary or opposite functions to syncytins during placentation and embryogenesis [35].
Finally, the wild-type FASTA sequence of syncytin-1 and syncytin-2 genes was used in the present analysis. Therefore, the effects of polymorphic variants, some of which have This study has several limitations. One is due to its purely computational nature, which indeed requires further confirmation of the results by laboratory experiments on cells and tissues from SLE patients with pregnancy complications. Moreover, this study did not investigate the alignments between human ncRNAs and env genes of other HERV members, such as HERV-F or HERV-K (HML2) proviruses, which may have complementary or opposite functions to syncytins during placentation and embryogenesis [35].
Finally, the wild-type FASTA sequence of syncytin-1 and syncytin-2 genes was used in the present analysis. Therefore, the effects of polymorphic variants, some of which have been associated with the risk of pregnancy complications [86], were not examined with respect to alignment with human ncRNAs.

Analysis of Polymorphic Variants of Human ncRNAs and Adjacent Coding Genes and Associated Diseases
GeneCards database (https://www.genecards.org; accessed on 28 December 2022) [88] was used to search for associations between polymorphic variants of complementary ncR-NAs or adjacent coding genes and human diseases. UniProt Atlas (https://www.uniprot. org; accessed on 28 December 2022) [91] was also queried for biological characterization of proteins encoded by genes adjacent to the ncRNAs of interest.

Conclusions
This is the first computational study aimed at evaluating a possible epigenetic perturbation in the syncytin-1 and syncytin-2 pathways triggered by the imbalanced expression of ncRNAs. The results of this pivotal analysis suggest that unfavorable pregnancy outcomes may be due to altered crosstalk between lncRNAs and syncytin-1 and syncytin-2 genes or transcripts, ultimately leading to the decreased production of these HERV-derived env proteins during syncytiotrophoblast formation or affecting the expression of other genes critical for placentation or the immune response. However, it is unclear whether these results may be applicable to SLE women who experience pregnancy complications. Further in vitro or ex vivo experiments should be performed to confirm this hypothesis.