Aberrant expression of human endogenous retroviruses and SETDB1 in adolescents with anorexia nervosa

Aberrant expression of human endogenous retroviruses and SETDB1 in adolescents with anorexia nervosa | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Aberrant expression of human endogenous retroviruses and SETDB1 in adolescents with anorexia nervosa Federico Amianto, Pier Angelo Tovo, Alice Po, Cristina Calvi, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5386207/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Human endogenous retroviruses (HERVs) represent 8% of the human genome. They are remnants of ancient infections of germinal cells. HERVs are no longer infectious, but some retroviral sequences can be activated and their enhanced expressions have been implicated in a number of diseases, including neuropsychiatric disorders. HERV transcription is regulated by TRIM28 and SETDB1, which are directly involved in the regulation of epigenetic processes, in neural cell differentiation, and brain inflammation. HERVs and TRIM28/SETDB1 expressions have not been investigated in patients affected by anorexia nervosa (AN). We assessed, through a PCR real-time Taqman amplification assay, the transcription levels of pol genes of HERV-H and -K, of env genes of Syncytin 1 (SYN1) and SYN2 as well as of TRIM28 and SETDB1 in whole blood of 37 adolescents with AN and in healthy controls (HC) of comparable age. The transcriptional levels of HERV-H-pol and HERV-K-pol as well as of SETDB1 were significantly higher in AN patients as compared with HC, while no differences were observed for SYN1, SYN2, and TRIM28. Over-expressions of HERVs and of SETDB1 in adolescents with AN suggest that they may be main actors in the pathophysiology of AN and open the way to development of novel therapeutic strategies. anorexia nervosa endogenous retroviruses TRIM28 SETDB1 inflammation epigenetics Figures Figure 1 Figure 2 Figure 3 1. Introduction Anorexia nervosa (AN) is a severe psychiatric disorder characterized by significantly low body weight, intense fear of weight gain, and disturbed body perception ( 1 ). Its complications affect all body systems and may have a fatal outcome ( 1 , 2 ). The etiopathogenesis of the disease remains an unsolved enigma and this hampers the development of novel therapeutic interventions. Initially, researchers focused on psychological fragility of anorexic patients, but now they are working on biological correlates of the disease ( 3 ). For instance, recent investigations emphasize the contribution of heritable genetic propensities. Twin-based heritability of AN is estimated to reach 50–60% ( 4 ). Genome-wide association studies (GWAS) in anorexic patients showed a genetic overlap with other psychiatric disorders, identified significant loci, and revealed direct correlations with psychiatric and metabolic components ( 5 , 6 ). Epigenetics is an emerging element through which environmental factors can modulate gene expressions without changing their fundamental structure. Epigenetic processes are implicated in the development of mental-health phenotypes, including eating disorders ( 7 , 8 ). Among other factors increasingly investigated in AN, there are functional and structural brain anomalies ( 9 , 10 ), underlying inflammatory status ( 11 , 12 , 13 ), dysregulation of the immune system with associated autoimmune reactions ( 11 , 14 , 15 , 16 ) and dysbiosis in intestinal microbiota with impact on brain functions ( 17 , 18 ). Human endogenous retroviruses (HERVs) represent about 8% of our genome. They originate from ancestral infections of germinal cells of primates millions of years ago. During evolution, given the continuous mutations, HERVs have lost the capacity to produce infectious particles. Their retroviral structure is however maintained, with three principal genes: group associated antigens (gag), polymerase (pol), and envelope (env), flanked between two regulatory long terminal repeats (LTRs) ( 19 ). Most HERVs are inactive, but some elements are transcribed, and a few encode proteins. HERVs have been coopted for essential biological functions during intrauterine life ( 20 , 21 ). For instance, two envelope proteins, called Syncytin-1 (SYN1)( 22 ) and Syncytin-2 (SYN2) ( 23 ), are crucial for placental syncytiotrophoblast formation and contribute to maternal-fetal tolerance, given their vigorous immunosuppressive properties ( 24 , 25 ). Postnatally, the physiological functions of HERVs are unclear. Among human tissues, HERV expressions are consistently high in the central nervous system ( 26 ). A transcriptional up-regulation of HERVs has been observed in a number of diseases and thus proposed in their etiopathogenesis, particularly in neurological and neuropsychiatric disorders( 26 , 27 , 28 ) and autoimmune diseases ( 27 , 29 , 30 , 31 ). Actually, HERVs can regulate the transactivation of neighboring cellular genes ( 32 , 33 ). Their mRNAs can be retro-transcribed and reintegrated into DNA causing mutations, or, being recognized as non-self by viral receptors, they can trigger a variety of inflammatory and immune reactions( 27 , 32 , 33 , 34 ) including specific and/or cross-reactive antibodies with tissue epitopes ( 35 , 36 , 37 ). Furthermore, there are mutual interactions between HERVs and intestinal microbiota ( 38 ). HERV expression is modulated by environmental factors via epigenetic mechanisms, such as DNA methylation and heterochromatin formation by histone tails modifications. SET domain bifurcated histone lysine methyltransferase 1 (SETDB1), also known as ESET, is a methyltransferase with high specificity for the lysine 9 residue of histone H3 ( 39 ). SETDB1 is recruited by TRIM28, also called KAP1 or TIF1-β, to form a complex with Krüppel associated box domain zinc finger proteins (KRAB‐ZFPs), the largest family of transcriptional regulators in the human genome ( 40 ). Both TRIM28 and SETBD1 represent specific tags for transcriptional modulation of HERV sequences ( 41 , 42 , 43 , 44 , 45 ). Additionally, they regulate the transactivation of thousands of cellular genes( 46 ) and are directly involved in epigenetic processes ( 42 ), such as the differentiation of cell lineages, the regular functions within the brain, and the modulation of the immune response ( 45 , 47 , 48 , 49 , 50 ). Despite all these considerations suggesting a potential role of HERVs, TRIM28 and SETDB1 in development and/or progression of AN, no study explored their expression in affected patients. Therefore, the aims of the current research were to assess the transcription levels of pol genes of HERV-H and HERV-K, the two most widely studied retroviral families ( 19 ), of env genes of SYN1 and SYN2, as well as of TRIM 28 and SETDB1 in whole blood from adolescents with AN and healthy controls of comparable age. 2. Materials and Methods 2.1 Study Populations AN patients were recruited at the Division of Child and Adolescent Neuropsychiatry, University of Turin, Regina Margherita Children’s Hospital, Turin, Italy. All patients received a clinical evaluation by a trained child and adolescent neuropsychiatrist, ensuring they met the diagnostic criteria outlined in DSM-5 for AN ( 1 ). Healthy controls (HC) included asymptomatic children of comparable age who were tested at the same hospital for routine laboratory examinations and whose results were all within the normal reference range. The study participants did not have evidence of any active medical problems, such as infections, cancer, autoimmune disorders, or neurologic diseases. The study protocol was approved by the Ethical Committee of the Azienda Ospedaliera-Universitaria Città della Salute e della Scienza di Torino, Turin, Italy (Code 0003495, 17/12/2021). All subjects enrolled in the study gave their written informed consent. 2.2 Total RNA Extraction Total RNA was extracted using the automated extractor Maxwell with the RNA Blood Kit (Promega, Madison, WI), that includes treatment with DNase during the RNA extraction process. To further exclude any contamination of genomic DNA, RNA extracts were directly amplified without reverse transcription. RNA concentration and purity were assessed by traditional UV spectroscopy with absorbance at 260 and 280 nm (SimpliNano spectrophotometer, Biochrom US, Holliston, MA, USA). The RNAs were stored at − 80°C until use. 2.3 Reverse Transcription Four hundred nanograms of total RNA were reverse-transcribed with 20 µl of buffer 10X, 4.8 µl of MgCl2 25 mM, 2 µl ImpromII (Promega), 1 µl of RNase inhibitor 20U/l, 0.4 µl random hexamers 250 µM (Promega), 2 µl mix dNTPs 100 mM (Promega), and dd-water in a final volume of 20 µl. The reaction mix was carried out in a GeneAmp PCR system 9700 Thermal Cycle (Applied Biosystems, Foster City, CA, USA) under the following conditions: 5 min at 25°C, 60 min at 42°C and 15 min at 70°C for the inactivation of enzyme; the cDNAs were stored at − 20°C until use. 2.4 Transcription levels of pol genes of HERV-H and -K, of env genes of SYN1 and SYN2, as well as of TRIM28 and SETDB1 by real-time PCR assay Relative expression of transcription levels of pol genes of HERV-H, and HERV-K, of env genes of SYN1 and SYN2 as well as of TRIM28 and SETDB1 were achieved as previously described in detail ( 30 , 31 ) using the primers and probes reported in Table 1 . Briefly, 40 ng of cDNA were amplified in a 20 µL total volume reaction, containing 2.5 U goTaQ MaterMix (Promega), 1.25 mmol/L MgCl2, 500 nmol of specific primers, and 200 nmol of specific probes. All the amplifications were run in a 96-well plate at 95°C for 10 min, followed by 45 cycles at 95°C for 15 s and at 60°C for 1 min. Each sample was run in triplicate. Relative expression of target gene transcripts was performed according to the 2 -ΔΔCt method ( 51 ). GAPDH was selected as a reference gene, as it has been shown to have good efficiency and excellent reproducibility with constant expression in human leucocyte samples ( 52 ) and previously used in our studies. After normalization of the PCR result of each target gene with the housekeeping gene, the method includes additional calibration of this value with the median expression of the same gene evaluated in a pool of healthy controls. The results, obtained with the 2 -ΔΔCt method, show the variations of target gene transcripts relative to the standard set of controls. Since we measured Ct for every target in all samples, we argue that our methods were suitable for HERVs, SETDB1 and TRIM28 detection and quantifications. Table 1 Primers and probes used to assess the transcription levels of pol genes of HERV-H and -K, of env genes of Syncytin 1 and Syncytin 2, of TRIM28 and SETDB1, and of GADPH. Name Primer/ Probe Sequence HERV-H pol Forward 5’-TGGACTGTGCTGCCGCAA-3’ Reverse 5’-GAAGSTCATCAATATATTGAATAAGGTGAGA-3’ Probe 6FAM-5’-TTCAGGGACAGCCCTCGTTACTTCAGCCAAGCTC-3’-TAMRA HERV-K pol Forward 5’-CCACTGTAGAGCCTCCTAAACCC-3’ Reverse 5’-TTGGTAGCGGCCACTGATTT-3’ Probe 6FAM-5’-CCCACACCGGTTTTTCTGTTTTCCAAGTTAA-3’-TAMRA Syncytin 1 env Forward 5’-ACTTTGTCTCTTCCAGAATCG-3’ Reverse 5’-GCGGTAGATCTTAGTCTTGG-3’ Probe 6FAM-5’-TGCATCTTGGGCTCCAT-3’-TAMRA Syncytin 2 env Forward 5’-GCCTGCAAATAGTCTTCTTT-3’ Reverse 5’-ATAGGGGCTATTCCCATTAG-3’ Probe 6FAM- 5’-TGATATCCGCCAGAAACCTCCC-3’-TAMRA TRIM28 Forward 5’-GCCTCTGTGTGAGACCTGTGTAGA-3’ Reverse 5’-CCAGTAGAGCGCACAGTATGGT-3’ Probe 6FAM-5’-CGCACCAGCGGGTGAAGTACACC-3’-TAMRA SETDB1 Forward 5’-GCCGTGACTTCATAGAGGAGTATGT-3’ Reverse 5’-GCTGGCCACTCTTGAGCAGTA-3’ Probe 6FAM-5’-TGCCTACCCCAACCGCCCCAT-3’-TAMRA GAPDH Forward 5’-CGAGATCCCTCCAAAATCAA-3’ Reverse 5’-TTCACACCCATGACGAACAT-3’ Probe 6FAM-5’-TCCAACGCAAAGCAATACATGAAC-3’-TAMRA 2.5 Statistical analysis Mann-Whitney test was used to compare the relative transcription levels of HERV-H-pol, of HERV-K-pol, SYN1, and SYN2 as well as of TRIM28 and SETDB1 between patients with AN and healthy controls. Statistical analyses were done using the Prism software (GraphPad Software). In all analyses, p < 0.05 was taken to be statistically significant. 3. Results 3.1 Study populations Thirty-seven adolescents with AN were enrolled in the study; their characteristics are reported in Table 2 . Table 2. Demographics and clinical characteristics of patients affected by anorexia nervosa (AN). [1] IQR: Interquartile range 25-75%. [2] Comorbidities: at least one among major depression (28 cases), polarized anxiety disorder (25 cases), obsessive-compulsive disorder (16 cases), and post-traumatic stress disorder (4 cases). [3] Pharmacotherapy: at least one among selective serotonin reuptake inhibitors (22 cases), atypical antipsychotics (19 cases), or benzodiazepines (16 cases). 3.2 Transcription levels of pol genes of HERV-H and HERV-K in whole blood of patients with anorexia nervosa (AN) and healthy controls (HC). The transcriptional levels of HERV-H-pol and HERV-K-pol were significantly higher in children with AN than in HC (Fig. 1). Figure 1 . Transcription levels of pol genes of HERV-H and HERV-K in whole blood from 37 children with Anorexia Nervosa (AN) and 36 age-matched healthy controls (HC). Medians and IQR 25%-75%: HERV-H- pol : AN 1.33, 1.09–1.53; HC 0.97, 0.84–1.21; HERV-K- pol : AN 1.54, 1.21–1.81; HC 1.00, 0.86–1.12. 2 −ΔΔCt = Relative expression according to the 2 −ΔΔCt method. Circles and squares show the median of three individual measurements, horizontal lines the median values. 3.2 Transcription levels of env genes of Syncytin (SYN) 1 and Syncytyn (SYN) 2 in whole blood of patients with anorexia nervosa (AN) and healthy controls (HC). The median values of RNA levels of SYN1 and SYN2 were similar in subjects with AN and HC (Fig. 2). Figure 2. Transcription levels of en v genes of Syncytin 1 and Syncytin 2 in whole blood from 37 subjects with AN and 26 healthy controls (HC). Medians and IQR 25%-75%: Syncytin 1: AN 1.15, 0.94–1.50; HC 1.02, 0.76–1.29; Syncytin 2: AN 0.95, 0.82–1.15; HC 1.01, 0.80–1.17. 2 −ΔΔCt = Relative expression according to the 2 −ΔΔCt method. Circles and squares show the median of three individual measurements, horizontal lines the median values. 3.3 Transcription levels of TRIM28 and SETDB1 in patients with AN and age-matched HC. As reported in Fig. 3, the median mRNA levels of TRIM28 were comparable in patients with AN vs. HC, while the median transcriptional levels of SETDB1 were significantly higher in AN patients than in HC. Figure 3 . Transcription levels of TRIM28 and SETDB1 in whole blood from 37 children with anorexia nervosa (AN) and 26 age-matched healthy controls (HCs). Medians and IQR 25%-75%: TRIM28 AN 1.03, 0.75–1.38; HC 0.93, 0.77–1.29; SETDB1 AN 1.41, 1.16–1.69; HC 1.02, 0.84–1.19. 2 −ΔΔCt = Relative expression according to the 2 −ΔΔCt method. Circles and squares show the median of three individual measurements, horizontal lines the median values. 4. Discussion Current results show for the first time that patients with AN exhibit significantly higher transcriptional levels of HERV-H-pol and HERV‐K-pol as compared to age-matched HCs, while the RNA levels of SYN1 and SYN2 are comparable between the two groups. The underlying biological mechanisms responsible for the abnormal expressions of retroviral sequences in patients with AN and their clinical meaning remain to be elucidated. TRIM28 and SETDB1 are potent corepressors of retroviruses. Their activation gives rise to DNA methylation and heterochromatin formation ultimately resulting in HERV silencing ( 44 , 45 ). Given the normal expression of TRIM28 and the enhanced transcription of SETDB1, the high transcript levels of HERV-H-pol and HERV-K-pol cannot be ascribed to impaired activation of TRIM28 and SETDB1. Interestingly, SETDB1 overexpression was found in other settings with up-regulation of HERV elements ( 28 , 31 , 53 ). To this purpose, it must be remembered that the protein complex of TRIM28/SETDB1 is essential for maintaining endogenous retroviruses in a silent state in early embryos and pluripotent stem cells ( 41 , 42 , 43 ). In contrast, when these cells differentiate into distinct somatic cells, transcription of retroviral sequences is no longer dependent from such repressors ( 42 , 54 ), which sometimes may act as transcriptional activators rather than as repressors ( 55 , 56 ). This might occur for some HERV sequences also in AN patients. It cannot be overlooked that functional interactions between TRIM28/SETDB1 and single HERVs may derive from post-translational events between the encoded proteins, while we assessed only their transcriptional profiles. Growing data document the increased levels of pro-inflammatory cytokines in anorexic subjects ( 11 , 12 , 57 ). Release of inflammatory cytokines results in the proteasome-driven activation of NF-kB signaling pathway. After its passage into the nucleus, the active isoform of NF-kB binds to specific motifs of HERVs that, along with inflammatory cytokines, lead to their enhanced transactivation ( 58 ). Notably, HERVs are, in turn, able to elicit induction and progression of a variety of inflammatory and immune responses ( 19 , 59 ). Recognition of HERV RNAs by nucleic acid-sensing toll like receptors (TLRs) may lead to activation of the inflammasome ( 33 , 60 ); for instance, HERV-K stimulates NF-kB pathway through TLR8 ( 61 ). The final result may be a vicious circle leading to increasing inflammatory and immune reactions. In this context, it is worth mentioning that enhanced HERV expressions have been associated with a number of autoimmune diseases ( 27 , 29 , 30 , 31 , 62 ). Eating disorders and autoimmune diseases give each other a mutual increased risk ( 14 , 15 ) that could be explained by the high values of HERV RNA levels in both conditions. A number of clinical and experimental studies highlight the impact of intestinal microbiota on brain activities. Potential connections have been evidenced between abnormal composition of the gut microbiota and AN ( 17 , 18 ). There are mutual interactions between intestinal microbiota and endogenous retroviruses ( 38 ). Germ-free mice lose intestinal expression of several retroviruses, while exposure to bacteria and their products can stimulate retroviral transcription ( 63 , 64 ). The dysbiosis of the gut microbiota observed in AN might thus contribute to the HERV over-expression emerged in our patients. SETDB1 is implicated in a large array of biological activities. It contributes to regular cellular homeostasis within the brain, while its alterations have been associated with several neurological and neuropsychiatric disorders ( 65 , 66 , 67 ). Furthermore, through the complex with TRIM28/KRAB-ZFPs, SETDB1 conditions B lineage differentiation, T cell commitment into regulatory phenotypes, or expansion of DCs and T cell priming toward inflammatory effector T cells ( 45 , 48 , 49 , 68 ). The overexpression of SETDB1 in AN patients might thus contribute to direct the differentiation, expansion, and function of DCs, B cells and T cells towards a peculiar reactivity against specific antigens in genetically predisposed subjects. Signs of autism spectrum disorders (ASD) are over-represented in individuals with AN ( 69 , 70 ), and those with co-occurrence of autistic traits have more severe disturbances and poorer prognosis ( 71 ). It is worth mentioning that enhanced expressions of HERV-H-pol, HERV-K-pol, and of SETDB1 were found in autistic children too ( 28 ). On one side, the hyper-activation of same HERV sequences and of SETDB1 in patients affected by both disorders may account for their more compromised mental health and worse evolution. On the other side, they may represent common biomarkers of these neuropsychiatric disorders and support shared pathogenetic roles. Interestingly, inflammatory status ( 11 , 12 , 57 ), immune reactions ( 11 , 12 , 14 , 15 , 16 ), and epigenetic alterations found in AN can be reversed by nutritional rehabilitation and psychotherapeutic or pharmacologic interventions ( 8 , 72 , 73 ). The aberrant HERV activation may change or persist( 53 , 74 ) over the disease course; their dysregulation may thus determine a transient or a persistent challenge for the host. Whether the high expressions of HERVs and of SETDB1 normalize, as patients recover, remains an unsolved dilemma requiring targeted studies in subjects with disease resolution. The incidence of AN has been increasing in the new millennium in Western countries particularly in young women ( 75 , 76 ). However, enhanced rates of AN are emerging also in Asian, Arab, and Pacific regions, following the increasing industrialization, urbanization, and spread of Western lifestyle ( 77 , 78 ). The debate is open on the role of biological versus sociocultural factors to explain this increased frequency of AN. As mentioned, epigenetic processes may elicit eating disorders ( 79 , 80 ). Furthermore, pollution ( 81 ), cigarette smoking ( 82 ), nutritional changes linked to the lifestyle ( 83 ), and variations in intestinal microbiota influence retrovirus expression ( 63 , 64 ). Therefore, environmental factors thought to be implicated in the development of AN could exert their effects via SETDB1- and/or HERV-driven changes in targeted biologic pathways. Given the heightened HERV expression, several therapeutic measures might be adopted in AN patients, such as monoclonal antibodies, cytotoxic T lymphocytes against retroviral antigens, specific anti-RNAs, and antiretroviral treatments ( 84 , 85 , 86 ). HERVs are upregulated in HIV + subjects ( 87 ), and administration of antiretroviral drugs inhibited both HIV viral burden and HERV expression ( 88 ). Combined antiretroviral therapy in patients with amyotrophic lateral sclerosis to contrast the HERV-K overexpression showed a better disease course in those with positive antiviral findings ( 89 ). A novel drug, originally conceived with anti-HIV and anti-inflammatory activity ( 90 ), induced positive effects in a phased II study in patients with ulcerative colitis ( 91 ). NF-KB and inflammatory cytokines stimulate HERV transcription( 58 ) and SETDB1 is degraded by proteasomal pathway ( 92 ). We demonstrated that antiretroviral drugs inhibit proteasome activity ( 93 , 94 ), with consequent downregulation of NF-kB-driven inflammatory cytokine production. The potential therapeutic benefit of antiretroviral drugs may thus derive not only from their specific antiretroviral effects, but also from indirect actions on host cell components. As discussed, alterations in epigenetic mechanisms may favor the development and progression of AN. Abnormal epigenetic changes can be targeted by specific drugs, such as small molecule compounds ( 95 ). In conclusion, activation is usually considered a prerequisite of causality. Our results suggest that HERVs and SETDB1 might be implicated in the pathophysiology of AN and may provide insights towards the development of novel targeted strategies. Declarations Acknowledgments This research received no external funding. The authors thank the nurses and the head nurse of the day hospital and inpatient service of the SCDU Neuropsichiatria Infantile of the OIRM. Conflict of Interest The authors declare no conflict of interest. Author Contributions P-AT, MB, FA designed the study protocol and supervised the project. P-AT, MB, FA and AP drafted the manuscript. AP enrolled patients with AN and collected their clinical data. CC, PM, SG and IG performed laboratory tests. IG and MB analyzed the data and performed statistical analyses. All authors reviewed, edited, and approved the final manuscript. References American Psychiatric Association Diagnostic and Statistical Manual of Mental Disorders, 5th ed. Washington, DC: American Psychiatric Publishing, 2013. Cost J, Krantz MJ, Mehler PS. Medical complications of anorexia nervosa. Cleve Clin J Med 2020;87:361–366. Couzin-Frankel J. Rethinking anorexia. Science 2020;368: 124–127. Yilmaz Z, Hardaway A, Bulik CM. Genetics and epigenetics of eating disorders. 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Endogenous retroviruses promote homeostatic and inflammatory responses to the microbiota. Cell 2021;184: 3794-3811.e19. Schultz DC, Ayyanathan K, Negorev D, Maul GG, Rauscher FJ. SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins. Genes Dev 2002;16:919–932. Sobocińska J, Molenda S, Machnik M, Oleksiewicz U. KRAB-ZFP transcriptional regulators acting as oncogenes and tumor suppressors: An overview. Int J Mol Sci 2021;22: 2212. Rowe HM, Jakobsson J, Mesnard D, Rougemont J, Reynard S, Aktas T, et al. KAP1 controls endogenous retroviruses in embryonic stem cells. Nature 2010;463:237–240. Rowe HM, Kapopoulou A, Corsinotti A, Fasching L, Macfarlan TS, Tarabay Y, et al. TRIM28 repression of retrotransposon-based enhancers is necessary to preserve transcriptional dynamics in embryonic stem cells. Genome Res 2013;23: 452–461. Matsui T, Leung D, Miyashita H, Maksakova IA, Miyachi H, Kimura H, et al. Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET. Nature 2010;464: 927–931. Fukuda K, Shinkai Y. SETDB1-mediated silencing of retroelements. Viruses 2020;12: 596. Adoue V, Binet B, Malbec A, Fourquet J, Romagnoli P, van Meerwijk JPM, et al. The histone methyltransferase SETDB1 controls T helper cell lineage integrity by repressing endogenous retroviruses. Immunity 2019;50:629-644.e8. Groner AC, Meylan S, Ciuffi A, Zangger N, Ambrosini G, Dénervaud N, et al. KRAB-zinc finger proteins and KAP1 can mediate long-range transcriptional repression through heterochromatin spreading. PLoS Genet 2010;6:e1000869. Spyropoulou A, Gargalionis A, Dalagiorgou G, Adamopoulos C, Papavassiliou KA, Lea RW, et al. Role of histone lysine methyltransferases SUV39H1 and SETDB1 in gliomagenesis: Modulation of cell proliferation, migration, and colony formation. Neuromolecular Med 2014;16:70–82. 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NF-κB and IRF1 induce endogenous retrovirus K expression via interferon-stimulated response elements in its 5′ long terminal repeat. J Virol 2016;90:9338–9349. Grandi N, Tramontano E. Human endogenous retroviruses are ancient acquired elements still shaping innate immune responses. Front Immunol 2018;9:2039. Miyake K, Shibata T, Ohto U, Shimizu T, Saitoh SI, Fukui R, et al. Mechanisms controlling nucleic acid-sensing Toll-like receptors. Int Immunol 2018;30:43–51. Dembny P, Newman AG, Singh M, Hinz M, Szczepek M, Krüger C, et al. Human endogenous retrovirus HERV-K(HML-2) RNA causes neurodegeneration through Toll-like receptors. JCI Insight 2020;5:e131093. Grandi N, Tramontano E. HERV envelope proteins: physiological role and pathogenic potential in cancer and autoimmunity. Front Microbiol 2018;9:462. Young GR, Mavrommatis B, Kassiotis G. Microarray analysis reveals global modulation of endogenous retroelement transcription by microbes. Retrovirology 2014;11:59. Panova V, Attig J, Young GR, Stoye JP, Kassiotis G. Antibody-induced internalisation of retroviral envelope glycoproteins is a signal initiation event. PLoS Pathog 2020;16:e1008605. Markouli M, Strepkos D, Chlamydas S, Piperi C. Histone lysine methyltransferase SETDB1 as a novel target for central nervous system diseases. Prog Neurobiol 2021;200:101968. Zhu Y, Sun D, Jakovcevski M, Jiang Y. Epigenetic mechanism of SETDB1 in brain: implications for neuropsychiatric disorders. Transl Psychiatry 2020;10:115. Xu Q, Goldstein J, Wang P, Gadi IK, Labreche H, Rehder C, et al. Chromosomal microarray analysis in clinical evaluation of neurodevelopmental disorders-reporting a novel deletion of SETDB1 and illustration of counseling challenge. Pediatr Res 2016;80:371–381. Chikuma S, Yamanaka S, Nakagawa S, Ueda MT, Hayabuchi H, Tokifuji Y, et al. TRIM28 expression on dendritic cells prevents excessive T cell priming by silencing endogenous retrovirus. J Immunol 2021;206:1528–1539. Westwood H, Tchanturia K. Autism apectrum disorder in anorexia nervosa: An updated literature review. Curr Psychiatry Rep 2017;19:41. Saure E, Laasonen M, Raevuori A. Anorexia nervosa and comorbid autism spectrum disorders. Curr Opin Psychiatry 2021;34:569–575. Zhang R, Birgegård A, Fundín B, Landén M, Thornton LM, Bulik CM, et al. Association of autism diagnosis and polygenic scores with eating disorder severity. Eur Eat Disord Rev 2022;30:442–458. Steiger H, Booij L, Kahan E, McGregor K, Thaler L, Fletcher E, et al. A longitudinal, epigenome-wide study of DNA methylation in anorexia nervosa: results in actively ill, partially weight-restored, long-term remitted and non-eating-disordered women. J Psychiatry Neurosci 2019;44:205–213. Schiele MA, Gottschalk MG, Domschke K. The applied implications of epigenetics in anxiety, affective and stress-related disorders - A review and synthesis on psychosocial stress, psychotherapy and prevention. Clin Psychol Rev 2020;77: 101830. Tovo PA, Garazzino S, Daprà V, Alliaudi C, Silvestro E, Calvi C, et al. Chronic HCV infection is associated with overexpression of human endogenous retroviruses that persists after drug-induced viral clearance. Int J Mol Sci 2020;21:3980. Martínez-González L, Fernández-Villa T, Molina AJ, Delgado-Rodríguez M, Martín V Incidence of anorexia nervosa in women: A systematic review and meta-analysis. Int J Environ Res Public Health 2020;17:3824. van Eeden AE, van Hoeken D, Hendriksen JMT, Hoek HW. Increase in incidence of anorexia nervosa among 10‐ to 14‐year‐old girls: A nationwide study in the Netherlands over four decades. Int J Eat Disord 2023;56:2295–2303. Thomas JJ, Lee S, Becker AE. Updates in the epidemiology of eating disorders in Asia and the Pacific. Curr Opin Psychiatry 2016;29:354–362. Pike KM, Hoek HW, Dunne PE. Cultural trends and eating disorders. Curr Opin Psychiatry 2014;27:436–442. Steiger H, Booij L. Eating disorders, heredity and environmental activation: getting epigenetic concepts into practice. J Clin Med 2020;9:1332. Breithaupt L, Hubel C, Bulik CM. Updates on genome-wide association findings in eating disorders and future application to precision medicine. Curr Neuropharmacol 2018;16:1102–1110. Azébi S, Batsché E, Michel F, Kornobis E, Muchardt C. Expression of endogenous retroviruses reflects increased usage of atypical enhancers in T cells. EMBO J 2019; 38:e101107. Bergallo M, Galliano I, Daprà V, Pirra A, Montanari P, Pavan M, et al. Transcriptional activity of human endogenous retroviruses in response to prenatal exposure of maternal cigarette smoking. Am J Perinatol 2019;36:1060–1065. Pathak R, Feil R. Environmental effects on chromatin repression at imprinted genes and endogenous retroviruses. Curr Opin Chem Biol 2018;45:139–147. Hartung HP, Derfuss T, Cree BA, Sormani MP, Selmaj K, Stutters J, et al. Efficacy and safety of temelimab in multiple sclerosis: Results of a randomized phase 2b and extension study. Mult Scler 2022;28: 429–440. Giménez-Orenga K, Oltra E. Human endogenous retrovirus as therapeutic targets in neurologic disease. Pharmaceuticals 2021;14: 495. Baldwin ET, Götte M, Tchesnokov EP, Arnold E, Hagel M, Nichols C, et al. Human endogenous retrovirus-K (HERV-K) reverse transcriptase (RT) structure and biochemistry reveals remarkable similarities to HIV-1 RT and opportunities for HERV-K–specific inhibition. Proc Nat Acad Sci USA 2022;119:e2200260119. Laderoute MP, Giulivi A, Larocque L, Bellfoy D, Hou Y, Wu HX, et al. The replicative activity of human endogenous retrovirus K102 (HERV-K102) with HIV viremia. AIDS 2007;21: 2417–2424. Tyagi R, Li W, Parades D, Bianchet MA, Nath A. Inhibition of human endogenous retrovirus-K by antiretroviral drugs. Retrovirology 2017;14:21. Garcia-Montojo M, Fathi S, Norato G, Smith BR, Rowe DB, Kiernan MC, et al. Inhibition of HERV-K (HML-2) in amyotrophic lateral sclerosis patients on antiretroviral therapy. J Neurol Sci 2021;423:117358. Vautrin A, Manchon L, Garcel A, Campos N, Lapasset L, Laaref AM, et al. Both anti-inflammatory and antiviral properties of novel drug candidate ABX464 are mediated by modulation of RNA splicing. Sci Rep 2019;9:792. Vermeire S, Solitano V, Peyrin-Biroulet L, Tilg H, Danese S, Ehrlich H, et al. Obefazimod: A first-in-class drug for the treatment of ulcerative colitis. J Crohns Colitis 2023;17:1689–1697. Pedersen G. Development, validation and implementation of an in vitro model for the study of metabolic and immune function in normal and inflamed human colonic epithelium. Dan Med J 2015;62:B4973. Piccinini M, Rinaudo MT, Chiapello N, Ricotti E, Baldovino S, Mostert M, et al. The human 26S proteasome is a target of antiretroviral agents. AIDS 2002;16:693–700. Piccinini M, Rinaudo MT, Anselmino A, Buccinnà B, Ramondetti C, Dematteis A, et al. The HIV protease inhibitors nelfinavir and saquinavir, but not a variety of HIV reverse transcriptase inhibitors, adversely affect human proteasome function. Antivir Ther 2005;10:215–223. Garcia-Martinez L, Zhang Y, Nakata Y, Chan HL, Morey L. Epigenetic mechanisms in breast cancer therapy and resistance. Nat Commun 2021;12:1786. Additional Declarations The authors have declared there is NO conflict of interest to disclose Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5386207","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":399525722,"identity":"0c68bb4c-931e-46f1-b978-7038644f49e5","order_by":0,"name":"Federico 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08:31:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5386207/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5386207/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75502986,"identity":"326b6f65-695c-435f-b586-eb065b2541b8","added_by":"auto","created_at":"2025-02-05 09:20:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":498183,"visible":true,"origin":"","legend":"\u003cp\u003eTranscription levels of \u003cem\u003epol\u003c/em\u003e genes of HERV-H and HERV-K in whole blood from 37 children with Anorexia Nervosa (AN) and 36 age-matched healthy controls (HC).\u003c/p\u003e\n\u003cp\u003eMedians and IQR 25%-75%: HERV-H-\u003cem\u003epol\u003c/em\u003e: AN 1.33, 1.09–1.53; HC 0.97, 0.84–1.21; HERV-K-\u003cem\u003epol\u003c/em\u003e: AN 1.54, 1.21–1.81; HC 1.00, 0.86–1.12. 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e = Relative expression according to the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method. Circles and squares show the median of three individual measurements, horizontal lines the median values.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-5386207/v1/524ff53084f5f638eca269fc.png"},{"id":75502989,"identity":"df03a1a9-35d7-4893-98f4-1adda2afdbec","added_by":"auto","created_at":"2025-02-05 09:20:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":482243,"visible":true,"origin":"","legend":"\u003cp\u003eTranscription levels of en\u003cem\u003ev\u003c/em\u003e genes of Syncytin 1 and Syncytin 2 in whole blood from 37 subjects with AN and 26 healthy controls (HC).\u003c/p\u003e\n\u003cp\u003eMedians and IQR 25%-75%: Syncytin 1: AN 1.15, 0.94–1.50; HC 1.02, 0.76–1.29; Syncytin 2: AN 0.95, 0.82–1.15; HC 1.01, 0.80–1.17. 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e = Relative expression according to the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method. Circles and squares show the median of three individual measurements, horizontal lines the median values.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-5386207/v1/3e5e7d7dbe14423e623f3e12.png"},{"id":75502987,"identity":"ade62fcf-2ec9-418a-9239-22970212fdbc","added_by":"auto","created_at":"2025-02-05 09:20:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":475241,"visible":true,"origin":"","legend":"\u003cp\u003eTranscription levels of TRIM28 and SETDB1 in whole blood from 37 children with anorexia nervosa (AN) and 26 age-matched healthy controls (HCs).\u003c/p\u003e\n\u003cp\u003eMedians and IQR 25%-75%: TRIM28 AN 1.03, 0.75–1.38; HC 0.93, 0.77–1.29; SETDB1 AN 1.41, 1.16–1.69; HC 1.02, 0.84–1.19. 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e = Relative expression according to the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method. Circles and squares show the median of three individual measurements, horizontal lines the median values.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-5386207/v1/ce2cbb63b73d26c42c704ba4.png"},{"id":75504605,"identity":"e6a7a832-3a6a-490e-b21f-2331d6fa6dfc","added_by":"auto","created_at":"2025-02-05 09:28:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2093431,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5386207/v1/9afaf254-3787-481b-9951-71910445f96f.pdf"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Aberrant expression of human endogenous retroviruses and SETDB1 in adolescents with anorexia nervosa","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAnorexia nervosa (AN) is a severe psychiatric disorder characterized by significantly low body weight, intense fear of weight gain, and disturbed body perception (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Its complications affect all body systems and may have a fatal outcome (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The etiopathogenesis of the disease remains an unsolved enigma and this hampers the development of novel therapeutic interventions. Initially, researchers focused on psychological fragility of anorexic patients, but now they are working on biological correlates of the disease (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). For instance, recent investigations emphasize the contribution of heritable genetic propensities. Twin-based heritability of AN is estimated to reach 50\u0026ndash;60% (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Genome-wide association studies (GWAS) in anorexic patients showed a genetic overlap with other psychiatric disorders, identified significant loci, and revealed direct correlations with psychiatric and metabolic components (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Epigenetics is an emerging element through which environmental factors can modulate gene expressions without changing their fundamental structure. Epigenetic processes are implicated in the development of mental-health phenotypes, including eating disorders (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Among other factors increasingly investigated in AN, there are functional and structural brain anomalies (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), underlying inflammatory status (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), dysregulation of the immune system with associated autoimmune reactions (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) and dysbiosis in intestinal microbiota with impact on brain functions (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHuman endogenous retroviruses (HERVs) represent about 8% of our genome. They originate from ancestral infections of germinal cells of primates millions of years ago. During evolution, given the continuous mutations, HERVs have lost the capacity to produce infectious particles. Their retroviral structure is however maintained, with three principal genes: group associated antigens (gag), polymerase (pol), and envelope (env), flanked between two regulatory long terminal repeats (LTRs) (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Most HERVs are inactive, but some elements are transcribed, and a few encode proteins. HERVs have been coopted for essential biological functions during intrauterine life (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). For instance, two envelope proteins, called Syncytin-1 (SYN1)(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) and Syncytin-2 (SYN2) (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), are crucial for placental syncytiotrophoblast formation and contribute to maternal-fetal tolerance, given their vigorous immunosuppressive properties (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Postnatally, the physiological functions of HERVs are unclear. Among human tissues, HERV expressions are consistently high in the central nervous system (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). A transcriptional up-regulation of HERVs has been observed in a number of diseases and thus proposed in their etiopathogenesis, particularly in neurological and neuropsychiatric disorders(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) and autoimmune diseases (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Actually, HERVs can regulate the transactivation of neighboring cellular genes (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Their mRNAs can be retro-transcribed and reintegrated into DNA causing mutations, or, being recognized as non-self by viral receptors, they can trigger a variety of inflammatory and immune reactions(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) including specific and/or cross-reactive antibodies with tissue epitopes (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Furthermore, there are mutual interactions between HERVs and intestinal microbiota (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHERV expression is modulated by environmental factors via epigenetic mechanisms, such as DNA methylation and heterochromatin formation by histone tails modifications. SET domain bifurcated histone lysine methyltransferase 1 (SETDB1), also known as ESET, is a methyltransferase with high specificity for the lysine 9 residue of histone H3 (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). SETDB1 is recruited by TRIM28, also called KAP1 or TIF1-β, to form a complex with Kr\u0026uuml;ppel associated box domain zinc finger proteins (KRAB‐ZFPs), the largest family of transcriptional regulators in the human genome (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Both TRIM28 and SETBD1 represent specific tags for transcriptional modulation of HERV sequences (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Additionally, they regulate the transactivation of thousands of cellular genes(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e) and are directly involved in epigenetic processes (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e), such as the differentiation of cell lineages, the regular functions within the brain, and the modulation of the immune response (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite all these considerations suggesting a potential role of HERVs, TRIM28 and SETDB1 in development and/or progression of AN, no study explored their expression in affected patients. Therefore, the aims of the current research were to assess the transcription levels of \u003cem\u003epol\u003c/em\u003e genes of HERV-H and HERV-K, the two most widely studied retroviral families (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), of \u003cem\u003eenv\u003c/em\u003e genes of SYN1 and SYN2, as well as of TRIM 28 and SETDB1 in whole blood from adolescents with AN and healthy controls of comparable age.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Study Populations\u003c/h2\u003e \u003cp\u003eAN patients were recruited at the Division of Child and Adolescent Neuropsychiatry, University of Turin, Regina Margherita Children\u0026rsquo;s Hospital, Turin, Italy. All patients received a clinical evaluation by a trained child and adolescent neuropsychiatrist, ensuring they met the diagnostic criteria outlined in DSM-5 for AN (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHealthy controls (HC) included asymptomatic children of comparable age who were tested at the same hospital for routine laboratory examinations and whose results were all within the normal reference range. The study participants did not have evidence of any active medical problems, such as infections, cancer, autoimmune disorders, or neurologic diseases.\u003c/p\u003e \u003cp\u003e The study protocol was approved by the Ethical Committee of the Azienda Ospedaliera-Universitaria Citt\u0026agrave; della Salute e della Scienza di Torino, Turin, Italy (Code 0003495, 17/12/2021). All subjects enrolled in the study gave their written informed consent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Total RNA Extraction\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTotal RNA was extracted using the automated extractor Maxwell with the RNA Blood Kit (Promega, Madison, WI), that includes treatment with DNase during the RNA extraction process. To further exclude any contamination of genomic DNA, RNA extracts were directly amplified without reverse transcription. RNA concentration and purity were assessed by traditional UV spectroscopy with absorbance at 260 and 280 nm (SimpliNano spectrophotometer, Biochrom US, Holliston, MA, USA). The RNAs were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until use.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Reverse Transcription\u003c/h2\u003e \u003cp\u003eFour hundred nanograms of total RNA were reverse-transcribed with 20 \u0026micro;l of buffer 10X, 4.8 \u0026micro;l of MgCl2 25 mM, 2 \u0026micro;l ImpromII (Promega), 1 \u0026micro;l of RNase inhibitor 20U/l, 0.4 \u0026micro;l random hexamers 250 \u0026micro;M (Promega), 2 \u0026micro;l mix dNTPs 100 mM (Promega), and dd-water in a final volume of 20 \u0026micro;l. The reaction mix was carried out in a GeneAmp PCR system 9700 Thermal Cycle (Applied Biosystems, Foster City, CA, USA) under the following conditions: 5 min at 25\u0026deg;C, 60 min at 42\u0026deg;C and 15 min at 70\u0026deg;C for the inactivation of enzyme; the cDNAs were stored at \u0026minus;\u0026thinsp;20\u0026deg;C until use.\u003c/p\u003e \u003cp\u003e \u003cem\u003e2.4 Transcription levels of pol genes of HERV-H and -K, of env genes of SYN1 and SYN2, as well as of TRIM28 and SETDB1 by real-time PCR assay\u003c/em\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eRelative expression of transcription levels of pol genes of HERV-H, and HERV-K, of env genes of SYN1 and SYN2 as well as of TRIM28 and SETDB1 were achieved as previously described in detail (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) using the primers and probes reported in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Briefly, 40 ng of cDNA were amplified in a 20 \u0026micro;L total volume reaction, containing 2.5 U goTaQ MaterMix (Promega), 1.25 mmol/L MgCl2, 500 nmol of specific primers, and 200 nmol of specific probes. All the amplifications were run in a 96-well plate at 95\u0026deg;C for 10 min, followed by 45 cycles at 95\u0026deg;C for 15 s and at 60\u0026deg;C for 1 min. Each sample was run in triplicate. Relative expression of target gene transcripts was performed according to the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). GAPDH was selected as a reference gene, as it has been shown to have good efficiency and excellent reproducibility with constant expression in human leucocyte samples (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e) and previously used in our studies. After normalization of the PCR result of each target gene with the housekeeping gene, the method includes additional calibration of this value with the median expression of the same gene evaluated in a pool of healthy controls. The results, obtained with the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method, show the variations of target gene transcripts relative to the standard set of controls. Since we measured Ct for every target in all samples, we argue that our methods were suitable for HERVs, SETDB1 and TRIM28 detection and quantifications.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers and probes used to assess the transcription levels of \u003cem\u003epol\u003c/em\u003e genes of HERV-H and -K, of \u003cem\u003eenv\u003c/em\u003e genes of Syncytin 1 and Syncytin 2, of TRIM28 and SETDB1, and of GADPH.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimer/\u003c/p\u003e \u003cp\u003eProbe\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHERV-H \u003cem\u003epol\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-TGGACTGTGCTGCCGCAA-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-GAAGSTCATCAATATATTGAATAAGGTGAGA-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProbe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6FAM-5\u0026rsquo;-TTCAGGGACAGCCCTCGTTACTTCAGCCAAGCTC-3\u0026rsquo;-TAMRA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHERV-K \u003cem\u003epol\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-CCACTGTAGAGCCTCCTAAACCC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-TTGGTAGCGGCCACTGATTT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProbe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6FAM-5\u0026rsquo;-CCCACACCGGTTTTTCTGTTTTCCAAGTTAA-3\u0026rsquo;-TAMRA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSyncytin 1 \u003cem\u003eenv\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-ACTTTGTCTCTTCCAGAATCG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-GCGGTAGATCTTAGTCTTGG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProbe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6FAM-5\u0026rsquo;-TGCATCTTGGGCTCCAT-3\u0026rsquo;-TAMRA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSyncytin 2 \u003cem\u003eenv\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-GCCTGCAAATAGTCTTCTTT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-ATAGGGGCTATTCCCATTAG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProbe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6FAM- 5\u0026rsquo;-TGATATCCGCCAGAAACCTCCC-3\u0026rsquo;-TAMRA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTRIM28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-GCCTCTGTGTGAGACCTGTGTAGA-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-CCAGTAGAGCGCACAGTATGGT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProbe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6FAM-5\u0026rsquo;-CGCACCAGCGGGTGAAGTACACC-3\u0026rsquo;-TAMRA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSETDB1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-GCCGTGACTTCATAGAGGAGTATGT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-GCTGGCCACTCTTGAGCAGTA-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProbe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6FAM-5\u0026rsquo;-TGCCTACCCCAACCGCCCCAT-3\u0026rsquo;-TAMRA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAPDH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-CGAGATCCCTCCAAAATCAA-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-TTCACACCCATGACGAACAT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProbe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6FAM-5\u0026rsquo;-TCCAACGCAAAGCAATACATGAAC-3\u0026rsquo;-TAMRA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Statistical analysis\u003c/h2\u003e \u003cp\u003eMann-Whitney test was used to compare the relative transcription levels of HERV-H-pol, of HERV-K-pol, SYN1, and SYN2 as well as of TRIM28 and SETDB1 between patients with AN and healthy controls. Statistical analyses were done using the Prism software (GraphPad Software). In all analyses, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was taken to be statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Study populations\u003c/h2\u003e\n \u003cp\u003eThirty-seven adolescents with AN were enrolled in the study; their characteristics are reported in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Demographics and clinical characteristics of patients affected by anorexia nervosa (AN).\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1738319865.png\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cp\u003e[1] IQR: Interquartile range 25-75%.\u003c/p\u003e\n \u003cp\u003e[2] Comorbidities: at least one among major depression (28 cases), polarized anxiety disorder (25 cases), obsessive-compulsive disorder (16 cases), and post-traumatic stress disorder (4 cases).\u003c/p\u003e\n \u003cp\u003e[3] Pharmacotherapy: at least one among selective serotonin reuptake inhibitors (22 cases), atypical antipsychotics (19 cases), or benzodiazepines (16 cases).\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cem\u003e3.2 Transcription levels of pol genes of HERV-H and HERV-K in whole blood of patients with anorexia nervosa (AN) and healthy controls (HC).\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eThe transcriptional levels of HERV-H-pol and HERV-K-pol were significantly higher in children with AN than in HC (Fig.\u0026nbsp;1).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFigure 1\u003c/strong\u003e. Transcription levels of \u003cem\u003epol\u003c/em\u003e genes of HERV-H and HERV-K in whole blood from 37 children with Anorexia Nervosa (AN) and 36 age-matched healthy controls (HC).\u003c/p\u003e\n \u003cp\u003eMedians and IQR 25%-75%: HERV-H-\u003cem\u003epol\u003c/em\u003e: AN 1.33, 1.09\u0026ndash;1.53; HC 0.97, 0.84\u0026ndash;1.21; HERV-K-\u003cem\u003epol\u003c/em\u003e: AN 1.54, 1.21\u0026ndash;1.81; HC 1.00, 0.86\u0026ndash;1.12. 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e = Relative expression according to the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method. Circles and squares show the median of three individual measurements, horizontal lines the median values.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e3.2 Transcription levels of env genes of Syncytin (SYN) 1 and Syncytyn (SYN) 2 in whole blood of patients with anorexia nervosa (AN) and healthy controls (HC).\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eThe median values of RNA levels of SYN1 and SYN2 were similar in subjects with AN and HC (Fig.\u0026nbsp;2).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFigure 2.\u003c/strong\u003e Transcription levels of en\u003cem\u003ev\u003c/em\u003e genes of Syncytin 1 and Syncytin 2 in whole blood from 37 subjects with AN and 26 healthy controls (HC).\u003c/p\u003e\n \u003cp\u003eMedians and IQR 25%-75%: Syncytin 1: AN 1.15, 0.94\u0026ndash;1.50; HC 1.02, 0.76\u0026ndash;1.29; Syncytin 2: AN 0.95, 0.82\u0026ndash;1.15; HC 1.01, 0.80\u0026ndash;1.17. 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e = Relative expression according to the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method. Circles and squares show the median of three individual measurements, horizontal lines the median values.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Transcription levels of TRIM28 and SETDB1 in patients with AN and age-matched HC.\u003c/h2\u003e\n \u003cp\u003eAs reported in Fig.\u0026nbsp;3, the median mRNA levels of TRIM28 were comparable in patients with AN vs. HC, while the median transcriptional levels of SETDB1 were significantly higher in AN patients than in HC.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFigure 3\u003c/strong\u003e. Transcription levels of TRIM28 and SETDB1 in whole blood from 37 children with anorexia nervosa (AN) and 26 age-matched healthy controls (HCs).\u003c/p\u003e\n \u003cp\u003eMedians and IQR 25%-75%: TRIM28 AN 1.03, 0.75\u0026ndash;1.38; HC 0.93, 0.77\u0026ndash;1.29; SETDB1 AN 1.41, 1.16\u0026ndash;1.69; HC 1.02, 0.84\u0026ndash;1.19. 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e = Relative expression according to the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method. Circles and squares show the median of three individual measurements, horizontal lines the median values.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eCurrent results show for the first time that patients with AN exhibit significantly higher transcriptional levels of HERV-H-pol and HERV‐K-pol as compared to age-matched HCs, while the RNA levels of SYN1 and SYN2 are comparable between the two groups. The underlying biological mechanisms responsible for the abnormal expressions of retroviral sequences in patients with AN and their clinical meaning remain to be elucidated. TRIM28 and SETDB1 are potent corepressors of retroviruses. Their activation gives rise to DNA methylation and heterochromatin formation ultimately resulting in HERV silencing (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Given the normal expression of TRIM28 and the enhanced transcription of SETDB1, the high transcript levels of HERV-H-pol and HERV-K-pol cannot be ascribed to impaired activation of TRIM28 and SETDB1. Interestingly, SETDB1 overexpression was found in other settings with up-regulation of HERV elements (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). To this purpose, it must be remembered that the protein complex of TRIM28/SETDB1 is essential for maintaining endogenous retroviruses in a silent state in early embryos and pluripotent stem cells (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). In contrast, when these cells differentiate into distinct somatic cells, transcription of retroviral sequences is no longer dependent from such repressors (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e), which sometimes may act as transcriptional activators rather than as repressors (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). This might occur for some HERV sequences also in AN patients. It cannot be overlooked that functional interactions between TRIM28/SETDB1 and single HERVs may derive from post-translational events between the encoded proteins, while we assessed only their transcriptional profiles.\u003c/p\u003e \u003cp\u003eGrowing data document the increased levels of pro-inflammatory cytokines in anorexic subjects (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). Release of inflammatory cytokines results in the proteasome-driven activation of NF-kB signaling pathway. After its passage into the nucleus, the active isoform of NF-kB binds to specific motifs of HERVs that, along with inflammatory cytokines, lead to their enhanced transactivation (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Notably, HERVs are, in turn, able to elicit induction and progression of a variety of inflammatory and immune responses (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). Recognition of HERV RNAs by nucleic acid-sensing toll like receptors (TLRs) may lead to activation of the inflammasome (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e); for instance, HERV-K stimulates NF-kB pathway through TLR8 (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). The final result may be a vicious circle leading to increasing inflammatory and immune reactions. In this context, it is worth mentioning that enhanced HERV expressions have been associated with a number of autoimmune diseases (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). Eating disorders and autoimmune diseases give each other a mutual increased risk (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) that could be explained by the high values of HERV RNA levels in both conditions.\u003c/p\u003e \u003cp\u003eA number of clinical and experimental studies highlight the impact of intestinal microbiota on brain activities. Potential connections have been evidenced between abnormal composition of the gut microbiota and AN (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). There are mutual interactions between intestinal microbiota and endogenous retroviruses (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Germ-free mice lose intestinal expression of several retroviruses, while exposure to bacteria and their products can stimulate retroviral transcription (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e). The dysbiosis of the gut microbiota observed in AN might thus contribute to the HERV over-expression emerged in our patients.\u003c/p\u003e \u003cp\u003eSETDB1 is implicated in a large array of biological activities. It contributes to regular cellular homeostasis within the brain, while its alterations have been associated with several neurological and neuropsychiatric disorders (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e). Furthermore, through the complex with TRIM28/KRAB-ZFPs, SETDB1 conditions B lineage differentiation, T cell commitment into regulatory phenotypes, or expansion of DCs and T cell priming toward inflammatory effector T cells (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e). The overexpression of SETDB1 in AN patients might thus contribute to direct the differentiation, expansion, and function of DCs, B cells and T cells towards a peculiar reactivity against specific antigens in genetically predisposed subjects.\u003c/p\u003e \u003cp\u003eSigns of autism spectrum disorders (ASD) are over-represented in individuals with AN (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e), and those with co-occurrence of autistic traits have more severe disturbances and poorer prognosis (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e). It is worth mentioning that enhanced expressions of HERV-H-pol, HERV-K-pol, and of SETDB1 were found in autistic children too (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). On one side, the hyper-activation of same HERV sequences and of SETDB1 in patients affected by both disorders may account for their more compromised mental health and worse evolution. On the other side, they may represent common biomarkers of these neuropsychiatric disorders and support shared pathogenetic roles.\u003c/p\u003e \u003cp\u003eInterestingly, inflammatory status (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e), immune reactions (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), and epigenetic alterations found in AN can be reversed by nutritional rehabilitation and psychotherapeutic or pharmacologic interventions (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e). The aberrant HERV activation may change or persist(\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e) over the disease course; their dysregulation may thus determine a transient or a persistent challenge for the host. Whether the high expressions of HERVs and of SETDB1 normalize, as patients recover, remains an unsolved dilemma requiring targeted studies in subjects with disease resolution.\u003c/p\u003e \u003cp\u003eThe incidence of AN has been increasing in the new millennium in Western countries particularly in young women (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e). However, enhanced rates of AN are emerging also in Asian, Arab, and Pacific regions, following the increasing industrialization, urbanization, and spread of Western lifestyle (\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e). The debate is open on the role of biological versus sociocultural factors to explain this increased frequency of AN. As mentioned, epigenetic processes may elicit eating disorders (\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e). Furthermore, pollution (\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e), cigarette smoking (\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e), nutritional changes linked to the lifestyle (\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e), and variations in intestinal microbiota influence retrovirus expression (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e). Therefore, environmental factors thought to be implicated in the development of AN could exert their effects via SETDB1- and/or HERV-driven changes in targeted biologic pathways.\u003c/p\u003e \u003cp\u003eGiven the heightened HERV expression, several therapeutic measures might be adopted in AN patients, such as monoclonal antibodies, cytotoxic T lymphocytes against retroviral antigens, specific anti-RNAs, and antiretroviral treatments (\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e). HERVs are upregulated in HIV\u0026thinsp;+\u0026thinsp;subjects (\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e), and administration of antiretroviral drugs inhibited both HIV viral burden and HERV expression (\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e). Combined antiretroviral therapy in patients with amyotrophic lateral sclerosis to contrast the HERV-K overexpression showed a better disease course in those with positive antiviral findings (\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e). A novel drug, originally conceived with anti-HIV and anti-inflammatory activity (\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e), induced positive effects in a phased II study in patients with ulcerative colitis (\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e). NF-KB and inflammatory cytokines stimulate HERV transcription(\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e) and SETDB1 is degraded by proteasomal pathway (\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e). We demonstrated that antiretroviral drugs inhibit proteasome activity (\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e, \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e), with consequent downregulation of NF-kB-driven inflammatory cytokine production. The potential therapeutic benefit of antiretroviral drugs may thus derive not only from their specific antiretroviral effects, but also from indirect actions on host cell components. As discussed, alterations in epigenetic mechanisms may favor the development and progression of AN. Abnormal epigenetic changes can be targeted by specific drugs, such as small molecule compounds (\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e). In conclusion, activation is usually considered a prerequisite of causality. Our results suggest that HERVs and SETDB1 might be implicated in the pathophysiology of AN and may provide insights towards the development of novel targeted strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no external funding.\u003c/p\u003e\n\u003cp\u003eThe authors thank the nurses and the head nurse of the day hospital and inpatient service of the SCDU Neuropsichiatria Infantile of the OIRM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP-AT, MB, FA designed the study protocol and supervised the project. P-AT, MB, FA and AP drafted the manuscript. AP enrolled patients with AN and collected their clinical data. CC, PM, SG and IG performed laboratory tests. IG and MB analyzed the data and performed statistical analyses. All authors reviewed, edited, and approved the final manuscript.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAmerican Psychiatric Association Diagnostic and Statistical Manual of Mental Disorders, 5th ed. Washington, DC: American Psychiatric Publishing, 2013.\u003c/li\u003e\n \u003cli\u003eCost J, Krantz MJ, Mehler PS. Medical complications of anorexia nervosa. Cleve Clin J Med 2020;87:361–366. \u003c/li\u003e\n \u003cli\u003eCouzin-Frankel J. Rethinking anorexia. Science 2020;368: 124–127. \u003c/li\u003e\n \u003cli\u003eYilmaz Z, Hardaway A, Bulik CM. Genetics and epigenetics of eating disorders. Adv Genomics Genet 2015;5:131-150. \u003c/li\u003e\n \u003cli\u003eBang L, Bahrami S, Hindley G, Smeland OB, Rødevand L, Jaholkowski PP, et al. 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Nat Commun 2021;12:1786. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"translational-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"tp","sideBox":"Learn more about [Translational Psychiatry](http://www.nature.com/tp/)","snPcode":"41398","submissionUrl":"https://mts-tp.nature.com/cgi-bin/main.plex","title":"Translational Psychiatry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"anorexia nervosa, endogenous retroviruses, TRIM28, SETDB1, inflammation, epigenetics","lastPublishedDoi":"10.21203/rs.3.rs-5386207/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5386207/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHuman endogenous retroviruses (HERVs) represent 8% of the human genome. They are remnants of ancient infections of germinal cells. HERVs are no longer infectious, but some retroviral sequences can be activated and their enhanced expressions have been implicated in a number of diseases, including neuropsychiatric disorders. HERV transcription is regulated by TRIM28 and SETDB1, which are directly involved in the regulation of epigenetic processes, in neural cell differentiation, and brain inflammation. HERVs and TRIM28/SETDB1 expressions have not been investigated in patients affected by anorexia nervosa (AN). We assessed, through a PCR real-time Taqman amplification assay, the transcription levels of pol genes of HERV-H and -K, of env genes of Syncytin 1 (SYN1) and SYN2 as well as of TRIM28 and SETDB1 in whole blood of 37 adolescents with AN and in healthy controls (HC) of comparable age. The transcriptional levels of HERV-H-pol and HERV-K-pol as well as of SETDB1 were significantly higher in AN patients as compared with HC, while no differences were observed for SYN1, SYN2, and TRIM28. Over-expressions of HERVs and of SETDB1 in adolescents with AN suggest that they may be main actors in the pathophysiology of AN and open the way to development of novel therapeutic strategies.\u003c/p\u003e","manuscriptTitle":"Aberrant expression of human endogenous retroviruses and SETDB1 in adolescents with anorexia nervosa","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-05 09:20:11","doi":"10.21203/rs.3.rs-5386207/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"translational-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"tp","sideBox":"Learn more about [Translational Psychiatry](http://www.nature.com/tp/)","snPcode":"41398","submissionUrl":"https://mts-tp.nature.com/cgi-bin/main.plex","title":"Translational Psychiatry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bad2700a-b0d4-4a88-b5fe-3f984659247f","owner":[],"postedDate":"February 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-02-05T09:20:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-05 09:20:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5386207","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5386207","identity":"rs-5386207","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}