Identification and functional analysis of circulating extrachromosomal circular DNA in schizophrenia implicate its negative effect on the disorder

Identification and functional analysis of circulating extrachromosomal circular DNA in schizophrenia implicate its negative effect on the disorder | 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 Research Article Identification and functional analysis of circulating extrachromosomal circular DNA in schizophrenia implicate its negative effect on the disorder Xi Xiang, Xiaoguang Pan, Wei Lv, Shanshan Chen, Haoran Zhang, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3287964/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background : Extrachromosomal circular DNA (eccDNA) is a circular DNA molecule derived and free from linear chromosome, its characteristics and potential function in SCZ remain unclear. Methods : Here, we explored the presence of circulating eccDNA in the plasma of chronic SCZ and healthy control using Circle-seq. Then the molecular role of SCZ over-represented eccDNAs was investigated by bioinformatical and experimental analysis. Results : 211 over-represented eccDNAs carrying genic segments (eccGene), including ecc TAOK2 , ecc DNMT3B , ecc SIRT5, ecc JAG1 and so on, were identified in 10 chronic SCZ patients, whereas only 26 over-represented eccGenes were found in 17 healthy people. Human phenotype ontology enrichment analysis upon the 211 SCZ over-represented eccGenes showed that six of them were enriched significantly in the phenotype of progressive intellectual disability. Functional assays of two artificial eccDNAs carrying TAOK2 -intronic sequence showed that they attenuated the TAOK2 mRNA expression in both U-251MG and SH-SY5Y cell lines, which the function was further confirmed by dual-luciferase reporter gene assay. In addition, RNA-seq analysis showed that introduction of the artificial ecc TAOK2 in U-251MG cells resulted in dysregulation of immune-related biological processes. Conclusions : These findings delineate the circulating eccDNAs profile of SCZ and highlight the regulatory function of ecc TAOK2 and its impact on cellular immune processes, underscoring the eccDNA biology and its potential role as a noninvasive biomarker for diagnosis and monitoring of schizophrenia. Molecular Genetics Epigenetics & Genomics Psychiatry Medical Genetics cell-free DNA ecDNA psychiatric disorder noninvasive biomarker rolling circle amplification LAMA plasma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 │ INTRODUCTION EccDNAs are mobile and circular DNA molecules derived and free from linear chromosomes. They have been broadly found in humans 1, 2 , animals 3, 4 and plants 4, 5 . As the eccDNA originally arises from the eukaryotic genome, it can carry functional elements such as intact oncogene 6–9 , truncated gene segment 2, 10, 11 , miRNA gene 10, 12 , enhancer 8, 9, 13, 14 and so on. As such, eccDNA plays a vital role in driving oncogenesis 8 and causing intratumoral heterogeneity 7, 15 . A well-known source of eccDNA is cell apoptosis 16 , and it can trigger the immune response by activation of the cytosolic DNA sensor Sting 16 . Recently, circulating eccDNA has been found in human blood plasma and serum 17–20 . It is identified in the plasma of pregnant women. The fetal-origin eccDNAs show smaller size and lower methylation levels than the maternal ones 17, 19 . Moreover, the tumor-derived eccDNA with a longer length compared to the normal tissue is detectable in the circulation 20 . These features confer eccDNA the great potential to serve as a noninvasive biomarker for the diagnosis and monitoring of human diseases 20–22 . Circulating eccDNA molecules are recognized as circular forms of cell-free DNA (cfDNA) 1, 23 . They may thus share somewhat similar biogenesis, characteristics and function. cfDNA is a short and linear double-stranded DNA fragment (~ 150 bp) released into the bloodstream following cell death 24, 25 . Several studies show a strong association between cfDNA and SCZ. Compared to the healthy people, SCZ patients have a higher level of cfDNA concentration in the plasma and serum 26–28 . Furthermore, methylation analysis reveals that SCZ patients have elevated level of brain-derived cfDNA, suggesting increased brain cell death or injury of the blood-brain barrier (BBB) in SCZ 24 . On the other hand, post-mortem studies indicated a dysregulation of cell apoptosis in the SCZ brain 29–31 , which may give rise to the aberrant release of brain-specific cfDNA into the circulation. In addition, the SCZ-derived circulating cfDNA enables activation of the STING DNA sensor genes in vitro , which might result in upregulation of the pro-inflammatory cytokines in SCZ 32 . These findings on cfDNA implicate an interplay between the circulating eccDNA and SCZ brain. In human plasma, the majority of circulating eccDNAs are less than 1 kb with two predominant peaks at ~ 202 bp and ~ 338 bp 17, 19 . The limited size of eccDNA impedes its ability to carry complete protein-coding genes 10 . Interestingly, eccDNAs are found to be enriched in genic regions 11, 20 and half of them carry genes or gene segments 2 . Moreover, multiple lines of evidence indicate that eccDNA can express RNA and perturb the transcriptome. Henrik D.M. et al. found numerous junction-specific transcripts of detected eccDNA in human muscle tissues 2 . In hypopharyngeal squamous cell carcinoma cell lines, there is a positive correlation between the differential eccDNAs and differentially expressed genes (DEGs) 33 . More importantly, Terresa et al. demonstrated that: 1) Endogenous eccDNAs are associated with RNA polymerases; 2) EccDNAs carrying exonic sequence express functional regulatory si-like RNAs which lead to suppression of host genes from which the eccDNAs were derived; 3) In contrast to the linear DNA, the initiation of eccDNA transcription is dependent on the circular structure but independent of a canonical promoter 10 . These findings suggest that genome-derived eccDNAs are functional through transcribing RNA molecules and therefore, may have the potential to influence the SCZ brain. To investigate the molecular role of circulating eccDNA in SCZ, herein, we explored the characteristics of plasma-derived eccDNAs from 10 chronic SCZ patients and 17 healthy controls, utilizing the Circle-seq approach. EccDNAs carrying gene segments were compared between the SCZ and healthy control groups and a class of SCZ over-represented eccGenes were identified. Among them, we found several genes were reported to be related to the pathogenesis of SCZ. Outward PCR and Sanger sequencing were utilized to confirm the presence of SCZ-related eccGenes in the corresponding samples. To further investigate the association of eccGenes and SCZ, we performed the human phenotype ontology enrichment analysis upon these genes. One gene, TAOK2 , draws our attention as it is one of the SCZ high-risk genes (HRG) 34 and can affect basal dendrite formation and synaptic development 35, 36 . We constructed two artificial eccDNAs carrying TAOK2 -intron segments which were detected in SCZ patients and evaluated their regulatory role in U-251MG and SH-SY5Y cell lines, using qPCR and dual-luciferase reporter gene assay. Finally, we performed RNA-seq analysis to assess the impact of artificial ecc TAOK2 on the transcriptome of U-251MG cells. 2 │ METHODS 2.1 │ Case recruitment and sample processing All inpatients with schizophrenia were from Zigong Fifth People's Hospital. 10 schizophrenics were randomly selected and met the following criteria: 1) Han nationality, aged 18–45; 2) DSM-IV was diagnosed as schizophrenia; 4) The total course of the disease is at least 5 years; 5) He has been receiving a stable dose of oral antipsychotic drugs for at least 3 months before entering the study. 6)All subjects were given written informed consent. All patients were chronic schizophrenics, with an average first-episode age of 20.4 ± 4.00 years and an average course of disease of 13.6 ± 10.4 years. The control group was 18–45 years old and lived in Zigong City. All subjects did not meet the DSM-V diagnostic criteria for the schizophrenic disorder, bipolar disorder, mental retardation, anxiety spectrum disorder, drugs, alcohol, and other psychoactive substances caused by psychiatric disorders, while not accompanied by serious physical diseases. The present study was approved by the Institutional Review Committee of Shaw Hospital affiliated to Zhejiang University School of Medicine (IRB number: 20210205-35). The basic clinical features of these patients and volunteers are listed in table S1. All subjects in this study were male. The average age of patients with schizophrenia is 31.10 ± 4.41 years old, and the average age of the healthy control group is 28.71 ± 5.51 years old. Among the 10 schizophrenics, 3 (30%) drank alcohol and 8 (80%) smoked. Among the 17 healthy controls, 6 (35.3%) drank alcohol and 3 (17.6%) smoked. There were 2 (20%) schizophrenic patients with a family history of psychosis, and 1 (5.9%) healthy control group with a family history of psychosis. The average BMI of the schizophrenic group was 24.44 ± 3.06, and that of the healthy control group was 22.55 ± 2.95. There was no significant difference between the schizophrenia group and the healthy control group in all items ( p -value > 0.05). Plasma samples were separated from peripheral blood and centrifuged at 1,600 g for 10 min at 4°C. Then the plasma portion was centrifuged again at 16,000 g for another 10 min to remove the cells and debris. All plasma samples were stored at -80°C for further processing. 2.2 │ Purification of plasma eccDNA EccDNA was purified from plasma by the Circle-Seq method as following described. 1) cfDNA isolation: About 300 µl of plasma samples in 1.5 ml Eppendorf tubes added with 20 µl proteinase K were incubated at 55°C at 600 rpm vortex (Eppendorf Thermomixer) for 20 min. Then, total cfDNA was extracted from plasma using the MGIEasy Circulating DNA Extraction Kit (MGI-BGI, China) according to the manufacturer’s protocol. cfDNA was eluted in 45 µl RNase-free water and 1 µl of cfDNA was taken for concentration analysis by Qubit Hs DNA dsDNA High Sensitivity assay on Qubit 3.0 Fluorometer (Invitrogen). 2) Removal of cell-free linear DNA: To remove the linear portions of cfDNA and enrich for circular DNA, 40 µl of cfDNA was digested with the 20 units of Plasmid-Safe DNase (PSD, 10,000u/ml, Epicenter) at 37°C for 16 hours in a 50 µl reaction system. The digestion products were recovered with 90 µl WAHTS® DNA clean Beads (Vazyme) and eluted in 24 µl RNase-free water. Rolling circle amplification (RCA): To increase the signal, the enriched traces number of circular DNA (12 µl out of a total of 24 µl) was amplified greatly via RCA and the RCA reaction system involved. The RCA products were recovered with 80 µl WAHTS® DNA clean Beads (Vazyme) and eluted in 100 µl RNase-free water. The RCA products were concentrated by Qubit 3.0. 2.3 │ Library preparation and eccDNA deep sequencing The φ29-amplified DNA samples (0.5 µg) were firstly sonicated into a 300–500 bp size range on the Covaris LE220 (Covaris). Then, 80 ng DNA fragments were end-repaired, A-tailed, and adapter-ligated using the MGIEasy DNA Library Preparation Kit (MGI-BGI, China). The quality control (including size distribution and concentration) of each library was assessed by the Agilent Bioanalyzer 2100 system. Lastly, the constructed library was deep sequenced on the MGI PE150 platform (BGI-QEI, Qingdao, China). 2.4 │ EccDNA assembly by Circle-Map MultiQC (v1.10.1), FastQC (v0.11.3), and Fastp (v0.21.0) were performed for quality control (QC) of raw sequencing data. Clean reads were mapped to the human reference genome (ref. hg38/ mm10) using BWA software. Circle-Map (V1.1.4) software was adapted to call circular DNA from Circle-Seq data based on reads span on junctions. To improve the confidence of detected-eccDNA, multiple filtration steps were performed as we previously described 21 . 2.5 │ Generation in silico eccDNAs for control comparison Based on the weighted average of chromosome length, we generated in silico eccDNAs across the genome by R statistical suite to minimize the effects of mapping and annotation issues. The generated in silico eccDNAs ranged randomly between 150 bp to 850 bp to mimic our detected plasma eccDNAs. 2.6 │ Genomic annotation Genomic annotation data were obtained from Ensembl database assembly GRCh38. Overlaps between detected eccDNA coordinates and annotated genes were identified using bedtools (iintersect-wo). Specially, the eccDNAs with > 60 bp overlap of a certain gene were defined as “eccGene” in the study. 2.7 │ eccDNA validation by PCR and sanger sequencing Outward PCR and Sanger sequencing were conducted to validate specific gene circles. The PCR primers were listed in table S4. Each 20 ul PCR reaction system included 50 µg phi29-amplified DNA products, 500 nM primer, 10 ul NEBNext High-Fidelity 2X PCR Master Mix (NEB), and PCR reaction for 35 cycles. All reactions were performed accompanied by RCA products pooled from 20 healthy controls. The PCR products were tested by agarose (2.5%) gel electrophoresis. The target products were recovered by QIAEX II Gel Extraction Kit for Sanger sequencing. 2.8 │ Construction of artificial eccDNA Two ecc TAOK2 carrying the TAOK2 intronic sequence were constructed by the ligase-assisted minicircle accumulation (LAMA) strategy as previously described 10 . The sequence of the synthetic linear fragments and corresponding PCR primers for artificial eccDNA preparation were displayed in table S5. 2.9 │ Artificial eccDNA transfection and qPCR U-251MG and SH-SY5Y cell lines were used for the functional assay of artificial ecc TAOK2 s in this study. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Lonza) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% GlutaMAX (Gibco), and penicillin-streptomycin (100 units penicillin and 0.1 mg streptomycin/mL) (Thermo Fisher Scientific) in a 37℃ incubator with 5% CO2 atmosphere and maximum humidity. The transfection was conducted using lipofectamine2000 (Invitrogen) in a 24-well plate according to the manufacturer’s instructions. 500 ng synthetic ecc TAOK2 was transfected in each well and triplicates were performed for each group. Cells transfected with an equal amount of pMax-GFP plasmid served as the control group. All the cells were harvested 48 hours after transfection for RNA isolation using TRIZOL reagent (Servicebio) according to the manufacturer’s protocol. 1 µg RNA was input for reverse transcription (RT) and total cDNA was synthesized using the HiScript® III 1st strand cDNA synthesis kit (+ gDNA wiper) (Vazyme). The quantitive PCR was performed by using the ChamQ Universal SYBR qPCR Master Mix (Vazyme). The primers for TAOK2 mRNA detection were hTAOK2-qPCR-F: 5’-caaggaggtgcggttcttac-3’ and hTAOK2-qPCR-R: 5’-ggtcacagctgcgatctctac-3’. GAPDH was used as an internal reference gene and qPCR primers were hGAPDH-qPCR-F: 5’-gtacgtcgtggagtccactg-3’ and hGAPDH-qPCR-R: 5’-gttgtcatggatgaccttggc-3’. 2.10 │ Dual-luciferase reporter gene assay The intronic fragments of the two eccTAOK2 were cloned from the genomic DNA of U-251MG cells using the primers with linker sequence containing the recognition sites of restriction endonuclease XhoI or NotI (table S6). Then the amplified intronic fragment (TAOK2 intron 1 or 8) was inserted in the psiCHECK2 plasmid (Promega) between the XhoI and NotI sites. Sanger sequencing was conducted for the confirmation of successful construction. Then the effects of artificial ecc TAOK2 on the relative expression level of Renilla luciferase were detected by the Dual-luciferase Reporter Assay System (Promega) according to the manufacturer’s instruction. 2.11 │ RNA sequencing and DEGs functional analysis The total RNA of the U-251MG cells after eccTAOK2#1 or pMax-GFP plasmids transfection was extracted using TRIZOL reagent (Servicebio) according to the manufacturer’s protocol. Then the total RNA was treated with gDNA wiper (Vazyme) at 37 ℃ for 10 min to remove the residual genomic DNA. The quality of total RNA was evaluated by agarose gel electrophoresis and a bioanalyzer (Aligent 2100). Only quality intact RNA was sent for RNA sequencing. The mRNA sequencing was conducted by custom service provided by GENE DENOVO (Guangzhou, China) using an MGIseq-2000 sequencing machine (BGI, China). Genes with |Log 2 (FoldChange)| > 0.5 and p -adjust value < 0.05 were identified as DEGs. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed using the online webtools of OmicShare established by GENE DENOVO ( https://www.omicshare.com/tools/ ). 2.12 │ Statistical Analysis All statistical tests were implemented by R-4.1.2 and GraphPad Prism 9. The difference comparison of the two groups was performed by Wilcoxon’s rank-sum test or Student’s t -test. P < 0.05 was considered statistically significant. 3 │ RESULTS 3.1 │ Detection of circulating eccDNA in plasma using Circle-seq strategy We leveraged the Circle-seq approach 2, 37 to purify and decipher the eccDNAs in the plasma derived from 10 chronic SCZ patients and 17 healthy controls. The workflow of the process is illustrated in figure. 1A. Briefly, the overall cfDNA extracted from the plasma was digested by exonuclease V to remove the linear cfDNA. Then the retained circular DNAs were purified and subjected to Rolling Circle Amplification (RCA) by φ29 polymerase to amplify the eccDNA amount. The eccDNA-RCA products were sequenced by Next-Generation-Sequencing (NGS) and all the high-quality reads were input into the Circle-map program for eccDNA calling. In this process, both split and discordant reads were extracted and used for mapping and identification of the start-end loci of eccDNAs. These eccDNAs were annotated and used for subsequent analysis. Especially, given that only the eccDNA carrying a certain length of gene segment can express potential regulatory RNA molecules 10 , the eccDNAs with > 60 bp overlap of a certain gene loci were defined as “eccGenes” in the study. 3.2 │ Features of circulating eccDNA profile in the plasma In this study, we obtained an average of 11.368 million (ranging from 7.01 to 15.06 million) reads (paired-end 150 bp) per healthy control sample and 11.96 million (ranging from 9.35 to 15.31 million) reads per SCZ sample (table S2). In total, an average of 7929 (varying from 3138 to 13191) and 8390 (varying from 2818 to 18531) eccDNA loci were identified in healthy control and SCZ plasma samples, respectively. The absolute eccDNA counts in each sample of the two groups were comparable with no significant difference (Fig. 1 B). To eliminate the bias introduced by sequencing depth, we compared the eccDNA counts per million mapped reads (EPM) and found no significant difference between the two groups (Fig. 1 C). The length of the most eccDNAs was less than 2 kb with four predominant peaks at around 197 bp, 363 bp, 555 bp and 747 bp (Fig. 1 D). Moreover, we found that 98.8% of the plasma-derived eccDNAs was less than 2 kb and only around 1.2% was between 2 kb and 8 kb in length (Fig. 1 E), indicating the shorter size of plasma-derived eccDNA compared to that observed in cancer cells 38, 39 . Consistent with previous reports in cancers 39 , the GC content of circulating eccDNA was higher than that of the average genomic distribution (Fig. 1 F), suggesting the generation of circulating eccDNAs was not random. On the other hand, the generation frequency of eccDNA in each chromosome (percent of eccDNA per Mb length of DNA) of the two groups was comparable with no significant difference (Fig. 1 G). However, the tendency of eccDNA generation in different chromosomes was varying. For instance, the chromosome (Chr) X and Y arose much fewer eccDNAs than that on Chr 19 or 20, suggesting the DNA accessibility level varied from chromosomes. 3.3 │ Determination of differential eccGenes in either SCZ or healthy control EccDNA carrying gene segment showed the potential to transcribe RNAs 2 and produce functional si-like RNA which leads to suppression of the host gene 10 . In this study, we identified and analyzed the differential eccGenes in either the chronic SCZ or the healthy control. In total, 26 differential eccGenes (Wilcoxon’s rank-sum test, p -value < 0.05) were identified in the healthy control, whereas the number in chronic SCZ was 211 (table S3). Figure 2 A showed the existing frequency of the differential eccGenes in the two groups with p- value < 0.03 (table S3). In the chronic SCZ group, several of the differential genes were well-known and associated with the pathogenesis of SCZ, such as DNMT3B 40, 41 , SIRT5 42 , JAG1 43, 44 , TAOK2 45, 46 and so on. We further compared the 211 SCZ over-represented eccGenes with the combination of two reported SCZ high-risk gene (HRG) sets (104 and 67 HRGs) inferred from the worldwide SCZ GWAS data 34, 47 . The TAOK2 gene was identified, whereas there was no overlapped gene between the healthy control-specific eccGenes ( 26 ) and the SCZ HRGs (Fig. 2 B). We then retrieved the reads distribution of ecc TAOK2 found in the samples utilizing the Integrative Genomics Viewer (IGV). 5/10 SCZ patients contained ecc TAOK2 while no healthy control presented the typical ecc TAOK2 reads signal (Fig. 2 C). Notably, three of the five observed ecc TAOK2 in SCZ were derived from the intron-1 of TAOK2 gene, while the other two were from intron-8 (Fig. 2 C). In addition, to verify the existence of SCZ over-represented eccGenes in the corresponding samples, we performed outward PCR to visualize the junction regions of several eccGenes, including 4 ecc TAOK2 , 1 ecc DNMT3B , 2 ecc JAG1 and 2 ecc SIRT5 . Figure 2 D showed that specific PCR bands were detectable in SCZ samples but not in the pool of the healthy control samples. Sanger sequencing results further confirmed the detailed sequence of the junction sites of each eccGene (Fig. 2 E), which were in accordance with the prediction of Circle-map. 3.4 │ Six SCZ over-represented eccGenes were enriched in the phenotype of progressive intellectual disability To evaluate the effects of SCZ over-represented eccGenes on human phenotypes, we conducted the Human Phenotype Ontology (HPO) analysis ( http://www.webgestalt.org/option.php ) upon the 211 SCZ over-represented eccGenes. The top 10 of the phenotypic abnormalities enriched in both groups were displayed in Fig. 3 A. The term “Intellectual disability, progressive” (IDP) was identified with statistical significance of FDR < 0.05 and p -value = 9.13E-6, while no significant term was found upon the healthy control-specific eccGenes (Fig. 3 A, below). Among the SCZ over-represented eccGenes mapped with HPO gene database, six genes were found overlapping to the IDP cluster genes (6 of 48 genes, enrichment ratio = 11.682), including DDB2 , ERCC3 , PTS , UBE3A , UROC1 and XPA (Fig. 3 B). The existing frequency of each of the six genes in the chronic SCZ group was significantly higher compared to the healthy control (Wilcoxon’s rank-sum test, p -value < 0.05) (Fig. 3 C). In addition, we verified the existence of the 6 IDP-related eccGenes in SCZ samples (two eccDNAs in their corresponding SCZ samples for each gene) by using the outward PCR (Fig. 3 D) and Sanger sequencing of the PCR products (Fig. 3 E). 3.5 │Artificial eccDNA containing TAOK2-intron segment attenuated the TAOK2 mRNA level in brain-derived cells EccDNA that contains exonic sequence can produce functional RNA molecules that suppress the host gene expression 10 , but the regulatory function of eccDNA harboring intron sequence remains unknown. In this study, we found the eccDNAs carrying TAOK2 intronic segments were related to SCZ, as it was detected in 50% (five of ten) of the SCZ patients but not in the 17 healthy controls. We speculated that ecc TAOK2 expresses RNA transcripts composed of the tandem TAOK2 -intron sequence which would be processed to be novel si-like RNAs that target the precursor mRNAs (pre-mRNA) of TAOK2 , resulting in disturbance of the pre-mRNA processing and downregulation of the mature mRNAs. To test the regulatory function of ecc TAOK2 , we synthesized two SCZ over-represented eccDNAs carrying the segments of TAOK2 intron 1 and intron 8, respectively. As showcased in Fig. 2 C, ecc TAOK2 #1 contained a 323 bp portion of the intron-8 and ecc TAOK2 #2 contained 370 bp of the intron-1. The two artificial ecc TAOK2 were constructed according to the ligase-assisted mini-circle accumulation (LAMA) protocol 10 , which is conducted by cycles of DNA denaturation, annealing and ligation processes (Fig. 4 A left). The LAMA products were treated with exonuclease to remove the residual linear DNA (Fig. 4 A right) and the purified circular DNA was identified by digestion with a single restriction endonuclease. Digestion with SspI or StuI on the linear A fragment resulted in two shorter DNA bands, while the circular DNA showed only one long band after digestion (Fig. 4 B). Transfection of the two artificial ecc TAOK2 (Fig. 4 C) resulted in down-regulation of the TAOK2 mRNA level in both the SH-SY5Y and U-251MG cell lines (Fig. 4 D-E), which were assessed by the real-time quantitative PCR: the ecc TAOK2 reduced the TAOK2 mRNA level by an average of 27% and 25% in the SH-SY5Y cell line (Fig. 4 D), and 42% and 20% in the U-251MG cell line (Fig. 4 E). To further validate whether ecc TAOK2 produced regulatory RNAs that target to the intronic sequence, renilla luciferase gene containing the full length of eccTAOK2#1 and #2 sequence in the 3’UTR were co-transfected with the artificial ecc TAOK2 for dual-luciferase assays in U-251MG cells (Fig. 4 F). Ecc TAOK2 #1 and ecc TAOK2 #2 repressed the renilla luciferase carrying their intron-origin sequences by 48.5% and 69.1%, respectively (Fig. 4 G). Together these results suggested that the ecc TAOK2 carrying intronic sequence was able to repress TAOK2 mRNA expression through the production of functional regulatory RNAs which may target the intronic portion of pre-mRNA. 3.6 │Artificial eccTAOK2 dysregulated the immune system in U-251MG cells The circular structure- but not the canonical promoter-dependent transcription of eccDNA 10 confers its potential to influence the phenotypes through expressing specific RNA transcripts 2 . But the impact of eccDNA on the transcriptome of brain cells is unclear. To evaluate the effect of SCZ-derived eccDNA on nerve cells, the artificial ecc TAOK2 was transfected in U-251MG cells and we performed the RNA-seq analysis afterward. In this part of the study, a total of 111 DEGs were identified with 46 downregulated genes (Log 2 (FoldChange) < -0.5 and p- adjust value 0.5 and p- adjust value < 0.05) (Fig. 5 A and table S7). GO enrichment analysis of these DEGs using the GO web tool on the OmicShare online platform highlighted the immune-related biological processes (table S8). Figure 5 B showed the top 20 GO terms with q -value < 0.01, including “immune system process” (GO: 0002376, q -value = 0.000026), “cellular response to chemical stimulus” (GO: 0070887, q -value = 0.000029), “response to stress” (GO: 0006950, q -value = 0.000087) and so on (table S5). KEGG analysis showed these DEGs were enriched in two major signaling pathways: “TNF signaling pathway” and “cytokine-cytokine receptor interaction” (Fig. 5 C-D, table S9) (Fig. 5 C presented the top 20 KEGG enriched terms and Fig. 5 D showed the connection-network of these signaling pathways). Consistent with the GO analysis result, both pathways were connected to the immune-related biological processes, including necroptosis, apoptosis, IL-17 signaling pathway, insulin resistance and so on (Fig. 5 D). Taken together, the RNA-seq analysis indicated the introduction of artificial ecc TAOK2 in U-251MG cells dysregulated the immune-related biological processes, suggesting a potential negative effect of eccDNA on the SCZ brain. 4 │DISCUSSION The foremost goal of the SCZ genetic study is to identify an unambiguous biomarker for diagnosis, monitoring, prognosis and therapeutic target for effective treatment of SCZ. Despite many recent studies that have reported the vital role of eccDNA in tumorigenesis and cancer evolution 13, 33, 39, 48, 49 , the relation between eccDNA and SCZ has never been investigated. In the present study, we first characterized the hallmarks of circulating eccDNA in chronic SCZ patients. The bioinformatical analysis identified the SCZ over-represented eccGenes and suggested several of these eccGenes may be related to SCZ. To investigate the biological role of circulating eccGenes, we tested the regulatory function of eccDNA carrying the TAOK2 -intronic sequence as an example. Functional assays indicated that ecc TAOK2 detected in SCZ can attenuate the TAOK2 expression and dysregulate the immune-related biological processes in brain-derived cells. These results highlight the potential role of eccDNA in SCZ, which has not been studied extensively. The basic features of circulating eccDNAs detected in SCZ patients in this work were similar to those found in pregnant women 17 and gout patients 22 . Both the SCZ patients and healthy people showed a predominant peak of eccDNA size at 363 bp and three lower peaks positioning at 197, 555 and 747 bp. The interval of the length peaks implied the nucleosomal origin of these eccDNAs 17, 19 . The primary cluster of eccDNA at around 363 bp suggested that circulating eccDNA was tend to be generated from di-nucleosomal wrapped DNAs. In this study, we used 300 µL plasma for eccDNA purification and an average of around 8000 eccDNAs were identified in either the SCZ or the healthy control. Considering the large volume of plasma in adult males (~ 50 mL/kg body weight 50 ), it is conceivable that there is a substantial amount of eccDNAs circulating in the peripheral blood: an average of around 2.67 million eccDNAs/kg body weight based on our data without considering the DNA losses during purification. Therefore, it is reasonable to infer that the aberrant biogenesis or metabolism of circulating eccDNAs may be linked to any human diseases, including SCZ. In this work, we identified a total of 211 SCZ over-represented eccGenes in 10 SCZ patients, whereas only 26 differential eccGenes were found in the 17 healthy controls. This skewed data between the two groups implied the aberrant biogenesis or metabolism of eccDNA in SCZ patients. Moreover, it has been observed that both the normal tissue and tumor-derived eccDNAs are detectable in circulation 20 . Thus, the SCZ over-represented circulating eccDNAs may mirror the aberrant status of somatic cells, including the brain cells. Several pieces of evidence provided by our data supported this conjecture: 1) The SCZ group enriched more specific eccGenes than the control group; 2) Among these SCZ over-represented eccGenes, many are related to the etiology of SCZ and 3) six of the SCZ over-represented eccGenes were significantly enriched to the phenotype of progressive intellectual disability, which often co-occurs with SCZ 51–55 . These findings underscored the potential linkage between eccDNA and SCZ. To investigate the molecular function of SCZ over-represented eccGenes, we took the ecc TAOK2 as an example. TAOK2 is a gene located in the SCZ-associated 16p11.2 microduplication region 56–59 . Several psychiatric features, such as speech/language impairments, motor/development delay, intelligent disability and microcephaly, have been identified in patients with 16p11.2 microduplication or microdeletion 60 . Among the 27–29 genes found in the 16p11.2 locus, TAOK2 plays a critical role in regulating the neuronal survival and development in the nervous system 35, 36, 61 . It is composed of 19 exons and 18 introns. Intriguingly, the five ecc TAOK2 detected in SCZ appeared in only two introns of this gene: three in the intron 1 and two from the intron 8 (Fig. 2 C). An interesting phenomenon is that the full length of TAOK2 gene showed a very high degree of conservation in sequence in animals such as horse, cow, dog, panda, rat and dolphin, but not in the birds, Sarcopterygii, or fish (figure S1). The situation is different in those genes located near the TAOK2 locus, like SEZ6L2 , MAPK3 and GDPD3 (figure S1). These findings suggested that not only the exons but also the TAOK2 introns, may play an important role in the neurodevelopment of those animals with higher intelligence than the bird or fish. Moreover, our study demonstrated the regulatory function of ecc TAOK2 -intron in repressing the TAOK2 gene expression. Although the function was confirmed by the dual-luciferase assay, whether the ecc TAOK2 transcripts can target the intronic portion of pre-mRNA is still uncertain. Further studies are warranted to elucidate the detailed regulatory mechanism of eccDNA carrying intronic sequence. The immune system is activated when interacting with pathogens and can affect the central nervous system 62 . A growing body of evidence has indicated the etiology of schizophrenia is involved in neuroinflammation and immune dysfunction 63–73 . Consistent with these findings, our data demonstrated that artificial ecc TAOK2 can lead to dysregulation of the cellular immune-related biological processes, including the TNF signaling pathway and cytokine-cytokine receptor interaction. In addition, diverse immune-response processes were connected with the two main signaling pathways (Fig. 5 D). Previous study has reported that eccDNA acts as a potent innate immunostimulant which is dependent on its circularity structure, but independent of the eccDNA sequence 16 . However, our data suggested that the eccDNA may contribute additional effects on the dysregulation of the immune system via expressing RNA and perturbing the transcriptome. Considering there were also tens of thousands of circulating eccDNAs in healthy people, more attention should be paid to the over-represented genic element carried by eccDNA but not its topological structure in case-control studies. Despite these findings, several issues remain for future study: 1) What are the tissue origins of the circulating eccDNAs? 2) Whether the brain-derived eccDNAs can cross the BBB and release into circulation? 3) Whether the circulating cell-free eccDNAs originated from the non-brain cells can cross the BBB and influence the human brain? 4) Dose the extracellular vesicle contain eccDNA and mediate intercellular cross-talk? 5) Whether the eccDNA can serve as biomarker for SCZ diagnosis and monitoring? Previous studies on circulating cfDNA have provided clues to answer some of the questions addressed. Tissue-specific epigenetic markers carried by the cfDNA have been utilized for identification of the tissue-of-origins of cfDNA 24, 74 . It would thus be convenient to investigate the tissue origins of circulating eccDNA by the strategy. It was postulated that the BBB may prevent cell-free DNA from reaching systematic circulation. However, the circulating tumor DNA (ctDNA) is detectable in the plasma of brain tumor patients 75, 76 , suggesting the absence of the BBB present 77 or alteration of the BBB integrity may influence the ctDNA levels in the circulation 78 . There is also a possibility that the eccDNA can be transported across the BBB by delivery of the extracellular vesicles 79, 80 . Therefore, a new methodology needed to be developed for purification and identification of eccDNAs in vesicles. Lastly, we believe that analysis of eccDNAs derived from large-scale samples combined with the promising machine learning will help to identify the reliable eccDNA biomarkers for diagnosis and monitoring of SCZ. 5 │ CONCLUSIONS This study characterized the hallmarks of circulating eccDNA in chronic SCZ and implicated the potential association between the eccDNA carrying genic segment and the pathogenesis of SCZ. As an exemplar, we demonstrated the regulatory function of ecc TAOK2 in brain-derived cells and observed its potent impact on the innate cellular immune system. Further studies on the eccDNA methylation, the function of eccDNAs carrying different genomic segments, the function of eccDNA in vesicles, eccDNA analysis of large-scale clinical samples may help to better understand the mechanisms of SCZ. Declarations ACKNOWLEDGEMENTS We would like to thank all the participants, staff, and volunteers in Zigong Fifth People's Hospital and Sir Run Run Shaw Hospital of Zhejiang University for their time and contributions to the study. CONFLICT OF INTERESTS The authors declare that they have no competing interests DATA AVAILABILITY STATEMENT The circulating eccDNA sequencing data have been deposited in the genome sequence archive of the Beijing Institute of Genomics, National Center for Bioinformation, Chinese Academy of Science. The accession numbers for the eccDNA sequencing data in this study is HRA004251. ORCID Xi Xiang https://orcid.org/0000-0002-5590-7289 Peng Han https://orcid.org/0000-0002-3405-087X Jinsong Tang http://orcid.org/0000-0003-3796-1377 References Paulsen T, Kumar P, Koseoglu MM, Dutta A. Discoveries of Extrachromosomal Circles of DNA in Normal and Tumor Cells. Trends Genet Apr 2018;34(4):270–278. Moller HD, Mohiyuddin M, Prada-Luengo I, et al. Circular DNA elements of chromosomal origin are common in healthy human somatic tissue. Nat Commun Mar 14 2018;9(1):1069. Cohen S, Menut S, Mechali M. Regulated formation of extrachromosomal circular DNA molecules during development in Xenopus laevis. Mol Cell Biol Oct 1999;19(10):6682–6689. Hotta Y, Bassel A. Molecular Size and Circularity of DNA in Cells of Mammals and Higher Plants. Proc Natl Acad Sci U S A Feb 1965;53:356–362. Lanciano S, Carpentier MC, Llauro C, et al. 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Int J Mol Sci Jun 21 2020;21(12). Heidarzadeh M, Gursoy-Ozdemir Y, Kaya M, Eslami Abriz A, Zarebkohan A, Rahbarghazi R, Sokullu E. Exosomal delivery of therapeutic modulators through the blood-brain barrier; promise and pitfalls. Cell Biosci Jul 22 2021;11(1):142. Supplementary Files TableS1DemographicandClinicalcharacteristics.docx Table S1 Demographic and Clinical characteristics of Schizophrenia Patients and Healthy Control Participants. TableS2.xlsx Table S2 Information of the NGS raw data of each sample measured. TableS3.xlsx Table S3 Detailed information of the differential eccGenes in either SCZ or healthy control groups. TableS4.xlsx Table S4 Information of the selected eccGene for PCR validation and the primer design. TableS5.xlsx Table S5 Linear DNA sequence and the matched PCR primers for eccTAOK2#1 and #2 synthesis using LAMA approach. TableS6.xlsx Table S6 Primers for dual-luciferase plasmid construction. TableS7.xlsx Table S7 Expression level of all the detected genes and the DEGs after eccTAOK2#1 transfection in U-251MG cells. TableS8.xlsx Table S8 GO enrichment analysis of the DEGs. TableS9.xlsx Table S9 KEGG enrichment analysis of the DEGs. FigS1.docx Cite Share Download PDF Status: Posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Tang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYLACCQMwxfiAgQdEJ+BXzYOkhdmAeC1QwCYBoQlosWc/e/iFRcEde37p9muVP2QOM/Cz5xgw/NyBxxaevDQLCYNniTPnnCm7IcFzmEGy540BY+8ZfA7LMTOQMDicYHAjJ+2GAVALkGHAzNiGRwv/G7AWe5CWggSgFnuCWiRyjB8AtTBuuJF+jOEAyBYJQlpuvDEDBvLhxJkzcpglG3jSeSTOPCs42ItHC3t/jvFniT+H7fkl0h9+/NljLcffnrzxwU88WoCATRoSHzwGDIw9kIg6gFcDMNI/foBY+ICB4QcBtaNgFIyCUTAiAQC/RU0WkgszdwAAAABJRU5ErkJggg==","orcid":"","institution":"Department of Psychiatry, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Jinsong","middleName":"","lastName":"Tang","suffix":""}],"badges":[],"createdAt":"2023-08-23 06:01:20","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false,"coiExplicitlySet":false},"doi":"10.21203/rs.3.rs-3287964/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3287964/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":42097336,"identity":"864b1cd4-8e10-416e-b40c-5c83151e3fc4","added_by":"auto","created_at":"2023-08-24 16:54:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":712860,"visible":true,"origin":"","legend":"\u003cp\u003eGeneral features of the plasma-derived eccDNAs. (A) Workflow of the study. (B) Detected eccDNA number in the healthy control and chronic SCZ groups. (C) Normalized eccDNA counts: eccDNA number per million mapped reads (EPM) in the two groups. (D) Length distribution of plasma-derived eccDNA in the two groups. (E) Percentage of eccDNA with different length in total detected eccDNAs in each sample of the two groups. (F) GC content of SCZ, healthy control, in silico and their upstream and downstream regions with equivalent length. (G) Normalized density (ratio of EPM) of eccDNA in the 24 human chromosomes.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3287964/v1/daad161dd0a53072bb5f0a09.png"},{"id":42097341,"identity":"38a1c179-3fc7-44dd-8b27-6fd8028ab6c5","added_by":"auto","created_at":"2023-08-24 16:54:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":930755,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of the differential eccGenes in either the SCZ or healthy control group. (A) Detection frequency of differential eccGenes in the two groups (Wilcoxon’s rank-sum test, \u003cem\u003eP\u003c/em\u003e value \u0026lt; 0.03). The row indicates the eccDNA carrying a certain genic segment; The column represents a sample within the given group. Blue block represents the eccGene was detected in the corresponding sample. (B) Comparison of SCZ over-represented eccGenes with the SCZ high-risk gene sets and the healthy-specific eccGenes by GeneVenn. (C) Length and reads coverage presentation of the five ecc\u003cem\u003eTAOK2\u003c/em\u003edetected in SCZ patients. S211, S226, S238, S239, S245 indicate the sample of SCZ patients. S1-S20 indicate the healthy people. Blue line and bars below represent the intron and exon distribution of the \u003cem\u003eTAOK2\u003c/em\u003e gene. (D) PCR assay and gel visualization of the junction sites of several eccGenes by outward PCR. E Sanger sequencing of the junction sites of 8 eccGenes detected in SCZ.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3287964/v1/3ec15470a8400224c5d8663f.png"},{"id":42097340,"identity":"ca075c8c-833c-4240-ae69-f2728db69924","added_by":"auto","created_at":"2023-08-24 16:54:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":879329,"visible":true,"origin":"","legend":"\u003cp\u003eHuman phenotype ontology (HPO) analysis and PCR verification of SCZ over-represented eccGenes. (A) Human phenotype ontology analysis of SCZ over-represented eccGenes (upper) and healthy ctrl-specific eccGenes (lower). (B) Comparison of the SCZ over-represented eccGenes mapped in the HPO database (42 genes) and the IDP-related gene set (48 genes). (C) Detection frequency of the six eccGenes in the two groups. (D) Outward PCR verification and, E Sanger sequencing results of the junction sites of the six eccGenes detected in SCZ samples.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3287964/v1/efc64cbf5f49ff0670da8f7f.png"},{"id":42096881,"identity":"bdbe6c8c-7f28-4b29-a48d-77be5196003f","added_by":"auto","created_at":"2023-08-24 16:46:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":532990,"visible":true,"origin":"","legend":"\u003cp\u003eSynthesis and functional assay of ecc\u003cem\u003eTAOK2\u003c/em\u003e. (A) Schematic of artificial eccDNA synthesis by LAMA approach (Left) and exonuclease V digestion assay of the LAMA products of the two eccTAOK2 (right). (B) Principle of the artificial eccTAOK2 identification by single restriction endonuclease digestion (left) and the results (right). (C) Experiment schedule of the ecc\u003cem\u003eTAOK2\u003c/em\u003e functional assay. (D) qPCR detection of the \u003cem\u003eTAOK2\u003c/em\u003e mRNA level two days after eccTAOK2 transfection in SH-SY5Y and, (E) U-251MG cell lines. (F) Schematic depicting the theoretical mechanism of how the eccTAOK2 produced si-like RNA and repressed the expression of Renilla luciferase (Rluc) by dual-luciferase reporter gene assay. The eccTAOK2 was co-transfected into cells with the psiCHECK2 plasmid containing the \u003cem\u003eTAOK2\u003c/em\u003e intronic sequence downstream the Rluc gene. The eccTAOK2 might be transcribed and processed to form si-like RNAs which lead to downregulation of the Rluc mRNA level by targeting the 3’UTR. (G) Transfection of eccTAOK2 containing either the portion of TAOK2 intron 1 or 8 repressed the expression of \u003cem\u003eTAOK2\u003c/em\u003egene in U-251MG cells.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3287964/v1/8e25b423a81b291efa40e4cb.png"},{"id":42097834,"identity":"be178b4f-f505-423a-b941-72a124ee9bee","added_by":"auto","created_at":"2023-08-24 17:02:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":522165,"visible":true,"origin":"","legend":"\u003cp\u003eTransfection of the artificial eccTAOK2 dysregulated the immune-related biological processes in U-251MG cells. (A) Volcano plot of the differentially expressed genes (DEGs) after artificial eccTAOK2#1 transfection in U-251MG cells. Blue and red spots indicate the down- (\u003cem\u003en\u003c/em\u003e = 46 DEGs) and up-regulated (\u003cem\u003en\u003c/em\u003e = 65 DEGs) DEGs with \u003cem\u003ep-\u003c/em\u003eadjust value \u0026lt; 0.05 and │Log2(FoldChange)│ \u0026gt; 0.5, respectively. (B) Top 20 of the biological processes enriched by GO enrichment analysis upon the 111 DEGs. The y-axis indicates the term of the biological processes and x-axis represents the gene percentage in each category. The number on the right of each bar indicates the enriched gene number in each cluster and the \u003cem\u003ep\u003c/em\u003e-adjust value is in parenthesis. (C) Top 20 KEGG pathways enriched in the 111 DEGs. (D) KEGG enrichment network plot of the enriched signaling pathways.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3287964/v1/0b914e6dd7f8f1f7eb55447f.png"},{"id":42097835,"identity":"51832df6-3cfe-4ad3-99d8-169290e197ab","added_by":"auto","created_at":"2023-08-24 17:02:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2516491,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3287964/v1/9c076ae4-5817-475e-a770-297dbd2fb135.pdf"},{"id":42096871,"identity":"40ff1ccb-6e77-46be-88f6-d679f877033b","added_by":"auto","created_at":"2023-08-24 16:46:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16953,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1 \u003c/strong\u003eDemographic and Clinical characteristics of Schizophrenia Patients and Healthy Control Participants.\u003c/p\u003e","description":"","filename":"TableS1DemographicandClinicalcharacteristics.docx","url":"https://assets-eu.researchsquare.com/files/rs-3287964/v1/06f4a89ef3e73f94478d0d19.docx"},{"id":42096872,"identity":"fd8f4dd7-2d14-4a03-8b09-07a61549232b","added_by":"auto","created_at":"2023-08-24 16:46:02","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13457,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S2 \u003c/strong\u003eInformation of the NGS raw data of each sample measured.\u003c/p\u003e","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3287964/v1/db2835594b50ac6d283433ea.xlsx"},{"id":42096874,"identity":"602fbc13-0508-43ca-bf86-b61d295d0d54","added_by":"auto","created_at":"2023-08-24 16:46:02","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":79371,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S3\u003c/strong\u003e Detailed information of the differential eccGenes in either SCZ or healthy control groups.\u003c/p\u003e","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3287964/v1/cbf645e8c8a13cce760658bd.xlsx"},{"id":42096876,"identity":"8b26ca8e-b4bf-4023-af6a-52659534d598","added_by":"auto","created_at":"2023-08-24 16:46:02","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":18952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S4\u003c/strong\u003e Information of the selected eccGene for PCR validation and the primer design.\u003c/p\u003e","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3287964/v1/b34eb4cd13c81b62eb0404a1.xlsx"},{"id":42097337,"identity":"233279bc-5989-4522-924e-c2fd6ed0f4e8","added_by":"auto","created_at":"2023-08-24 16:54:02","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":12057,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S5\u003c/strong\u003e Linear DNA sequence and the matched PCR primers for eccTAOK2#1 and #2 synthesis using LAMA approach.\u003c/p\u003e","description":"","filename":"TableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3287964/v1/f95b911bbc30999b9bad90eb.xlsx"},{"id":42097338,"identity":"cb4450e6-ec9d-4354-bb0a-a6a99d916bb5","added_by":"auto","created_at":"2023-08-24 16:54:02","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":12973,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S6 \u003c/strong\u003ePrimers for dual-luciferase plasmid construction.\u003c/p\u003e","description":"","filename":"TableS6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3287964/v1/a2ffcda081baa8c94c37da17.xlsx"},{"id":42096883,"identity":"ebbeb9b6-7857-474b-95e6-e74bbcf6dc03","added_by":"auto","created_at":"2023-08-24 16:46:02","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1256709,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S7 \u003c/strong\u003eExpression level of all the detected genes and the DEGs after eccTAOK2#1 transfection in U-251MG cells.\u003c/p\u003e","description":"","filename":"TableS7.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3287964/v1/ffa181679e5485d72ab653cb.xlsx"},{"id":42096884,"identity":"7311675c-8f93-42cf-87c5-372ef547a21f","added_by":"auto","created_at":"2023-08-24 16:46:02","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":242071,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S8 \u003c/strong\u003eGO enrichment analysis of the DEGs.\u003c/p\u003e","description":"","filename":"TableS8.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3287964/v1/4c13e9c7747b4bc8e2721103.xlsx"},{"id":42096879,"identity":"c3da0996-2d0a-4985-9b2a-8080fbd27fbf","added_by":"auto","created_at":"2023-08-24 16:46:02","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":22187,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S9 \u003c/strong\u003eKEGG enrichment analysis of the DEGs.\u003c/p\u003e","description":"","filename":"TableS9.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3287964/v1/06eaf962245ffa7481c2f0a1.xlsx"},{"id":42096885,"identity":"49506727-bfa6-466e-b0ec-fd974018230d","added_by":"auto","created_at":"2023-08-24 16:46:02","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":2112124,"visible":true,"origin":"","legend":"","description":"","filename":"FigS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3287964/v1/9c14b6bbaf574760230d3469.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eIdentification and functional analysis of circulating extrachromosomal circular DNA in schizophrenia implicate its negative effect on the disorder\u003c/p\u003e","fulltext":[{"header":"1 │ INTRODUCTION","content":"\u003cp\u003eEccDNAs are mobile and circular DNA molecules derived and free from linear chromosomes. They have been broadly found in humans \u003csup\u003e1, 2\u003c/sup\u003e, animals \u003csup\u003e3, 4\u003c/sup\u003e and plants \u003csup\u003e4, 5\u003c/sup\u003e. As the eccDNA originally arises from the eukaryotic genome, it can carry functional elements such as intact oncogene \u003csup\u003e6\u0026ndash;9\u003c/sup\u003e, truncated gene segment \u003csup\u003e2, 10, 11\u003c/sup\u003e, miRNA gene \u003csup\u003e10, 12\u003c/sup\u003e, enhancer \u003csup\u003e8, 9, 13, 14\u003c/sup\u003e and so on. As such, eccDNA plays a vital role in driving oncogenesis \u003csup\u003e8\u003c/sup\u003e and causing intratumoral heterogeneity \u003csup\u003e7, 15\u003c/sup\u003e. A well-known source of eccDNA is cell apoptosis \u003csup\u003e16\u003c/sup\u003e, and it can trigger the immune response by activation of the cytosolic DNA sensor Sting \u003csup\u003e16\u003c/sup\u003e. Recently, circulating eccDNA has been found in human blood plasma and serum \u003csup\u003e17\u0026ndash;20\u003c/sup\u003e. It is identified in the plasma of pregnant women. The fetal-origin eccDNAs show smaller size and lower methylation levels than the maternal ones \u003csup\u003e17, 19\u003c/sup\u003e. Moreover, the tumor-derived eccDNA with a longer length compared to the normal tissue is detectable in the circulation \u003csup\u003e20\u003c/sup\u003e. These features confer eccDNA the great potential to serve as a noninvasive biomarker for the diagnosis and monitoring of human diseases \u003csup\u003e20\u0026ndash;22\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCirculating eccDNA molecules are recognized as circular forms of cell-free DNA (cfDNA) \u003csup\u003e1, 23\u003c/sup\u003e. They may thus share somewhat similar biogenesis, characteristics and function. cfDNA is a short and linear double-stranded DNA fragment (~\u0026thinsp;150 bp) released into the bloodstream following cell death \u003csup\u003e24, 25\u003c/sup\u003e. Several studies show a strong association between cfDNA and SCZ. Compared to the healthy people, SCZ patients have a higher level of cfDNA concentration in the plasma and serum \u003csup\u003e26\u0026ndash;28\u003c/sup\u003e. Furthermore, methylation analysis reveals that SCZ patients have elevated level of brain-derived cfDNA, suggesting increased brain cell death or injury of the blood-brain barrier (BBB) in SCZ \u003csup\u003e24\u003c/sup\u003e. On the other hand, \u003cem\u003epost-mortem\u003c/em\u003e studies indicated a dysregulation of cell apoptosis in the SCZ brain \u003csup\u003e29\u0026ndash;31\u003c/sup\u003e, which may give rise to the aberrant release of brain-specific cfDNA into the circulation. In addition, the SCZ-derived circulating cfDNA enables activation of the STING DNA sensor genes \u003cem\u003ein vitro\u003c/em\u003e, which might result in upregulation of the pro-inflammatory cytokines in SCZ \u003csup\u003e32\u003c/sup\u003e. These findings on cfDNA implicate an interplay between the circulating eccDNA and SCZ brain.\u003c/p\u003e \u003cp\u003eIn human plasma, the majority of circulating eccDNAs are less than 1 kb with two predominant peaks at ~\u0026thinsp;202 bp and ~\u0026thinsp;338 bp \u003csup\u003e17, 19\u003c/sup\u003e. The limited size of eccDNA impedes its ability to carry complete protein-coding genes \u003csup\u003e10\u003c/sup\u003e. Interestingly, eccDNAs are found to be enriched in genic regions \u003csup\u003e11, 20\u003c/sup\u003e and half of them carry genes or gene segments \u003csup\u003e2\u003c/sup\u003e. Moreover, multiple lines of evidence indicate that eccDNA can express RNA and perturb the transcriptome. Henrik D.M. et al. found numerous junction-specific transcripts of detected eccDNA in human muscle tissues \u003csup\u003e2\u003c/sup\u003e. In hypopharyngeal squamous cell carcinoma cell lines, there is a positive correlation between the differential eccDNAs and differentially expressed genes (DEGs) \u003csup\u003e33\u003c/sup\u003e. More importantly, Terresa et al. demonstrated that: 1) Endogenous eccDNAs are associated with RNA polymerases; 2) EccDNAs carrying exonic sequence express functional regulatory si-like RNAs which lead to suppression of host genes from which the eccDNAs were derived; 3) In contrast to the linear DNA, the initiation of eccDNA transcription is dependent on the circular structure but independent of a canonical promoter \u003csup\u003e10\u003c/sup\u003e. These findings suggest that genome-derived eccDNAs are functional through transcribing RNA molecules and therefore, may have the potential to influence the SCZ brain.\u003c/p\u003e \u003cp\u003eTo investigate the molecular role of circulating eccDNA in SCZ, herein, we explored the characteristics of plasma-derived eccDNAs from 10 chronic SCZ patients and 17 healthy controls, utilizing the Circle-seq approach. EccDNAs carrying gene segments were compared between the SCZ and healthy control groups and a class of SCZ over-represented eccGenes were identified. Among them, we found several genes were reported to be related to the pathogenesis of SCZ. Outward PCR and Sanger sequencing were utilized to confirm the presence of SCZ-related eccGenes in the corresponding samples. To further investigate the association of eccGenes and SCZ, we performed the human phenotype ontology enrichment analysis upon these genes. One gene, \u003cem\u003eTAOK2\u003c/em\u003e, draws our attention as it is one of the SCZ high-risk genes (HRG) \u003csup\u003e34\u003c/sup\u003e and can affect basal dendrite formation and synaptic development \u003csup\u003e35, 36\u003c/sup\u003e. We constructed two artificial eccDNAs carrying \u003cem\u003eTAOK2\u003c/em\u003e-intron segments which were detected in SCZ patients and evaluated their regulatory role in U-251MG and SH-SY5Y cell lines, using qPCR and dual-luciferase reporter gene assay. Finally, we performed RNA-seq analysis to assess the impact of artificial ecc\u003cem\u003eTAOK2\u003c/em\u003e on the transcriptome of U-251MG cells.\u003c/p\u003e"},{"header":"2 │ METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 │ Case recruitment and sample processing\u003c/h2\u003e \u003cp\u003eAll inpatients with schizophrenia were from Zigong Fifth People's Hospital. 10 schizophrenics were randomly selected and met the following criteria: 1) Han nationality, aged 18\u0026ndash;45; 2) DSM-IV was diagnosed as schizophrenia; 4) The total course of the disease is at least 5 years; 5) He has been receiving a stable dose of oral antipsychotic drugs for at least 3 months before entering the study. 6)All subjects were given written informed consent. All patients were chronic schizophrenics, with an average first-episode age of 20.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.00 years and an average course of disease of 13.6\u0026thinsp;\u0026plusmn;\u0026thinsp;10.4 years. The control group was 18\u0026ndash;45 years old and lived in Zigong City. All subjects did not meet the DSM-V diagnostic criteria for the schizophrenic disorder, bipolar disorder, mental retardation, anxiety spectrum disorder, drugs, alcohol, and other psychoactive substances caused by psychiatric disorders, while not accompanied by serious physical diseases.\u003c/p\u003e \u003cp\u003e The present study was approved by the Institutional Review Committee of Shaw Hospital affiliated to Zhejiang University School of Medicine (IRB number: 20210205-35). The basic clinical features of these patients and volunteers are listed in table S1. All subjects in this study were male. The average age of patients with schizophrenia is 31.10\u0026thinsp;\u0026plusmn;\u0026thinsp;4.41 years old, and the average age of the healthy control group is 28.71\u0026thinsp;\u0026plusmn;\u0026thinsp;5.51 years old. Among the 10 schizophrenics, 3 (30%) drank alcohol and 8 (80%) smoked. Among the 17 healthy controls, 6 (35.3%) drank alcohol and 3 (17.6%) smoked. There were 2 (20%) schizophrenic patients with a family history of psychosis, and 1 (5.9%) healthy control group with a family history of psychosis. The average BMI of the schizophrenic group was 24.44\u0026thinsp;\u0026plusmn;\u0026thinsp;3.06, and that of the healthy control group was 22.55\u0026thinsp;\u0026plusmn;\u0026thinsp;2.95. There was no significant difference between the schizophrenia group and the healthy control group in all items (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Plasma samples were separated from peripheral blood and centrifuged at 1,600 \u003cem\u003eg\u003c/em\u003e for 10 min at 4\u0026deg;C. Then the plasma portion was centrifuged again at 16,000 \u003cem\u003eg\u003c/em\u003e for another 10 min to remove the cells and debris. All plasma samples were stored at -80\u0026deg;C for further processing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 │ Purification of plasma eccDNA\u003c/h2\u003e \u003cp\u003eEccDNA was purified from plasma by the Circle-Seq method as following described. 1) cfDNA isolation: About 300 \u0026micro;l of plasma samples in 1.5 ml Eppendorf tubes added with 20 \u0026micro;l proteinase K were incubated at 55\u0026deg;C at 600 rpm vortex (Eppendorf Thermomixer) for 20 min. Then, total cfDNA was extracted from plasma using the MGIEasy Circulating DNA Extraction Kit (MGI-BGI, China) according to the manufacturer\u0026rsquo;s protocol. cfDNA was eluted in 45 \u0026micro;l RNase-free water and 1 \u0026micro;l of cfDNA was taken for concentration analysis by Qubit Hs DNA dsDNA High Sensitivity assay on Qubit 3.0 Fluorometer (Invitrogen). 2) Removal of cell-free linear DNA: To remove the linear portions of cfDNA and enrich for circular DNA, 40 \u0026micro;l of cfDNA was digested with the 20 units of Plasmid-Safe DNase (PSD, 10,000u/ml, Epicenter) at 37\u0026deg;C for 16 hours in a 50 \u0026micro;l reaction system. The digestion products were recovered with 90 \u0026micro;l WAHTS\u0026reg; DNA clean Beads (Vazyme) and eluted in 24 \u0026micro;l RNase-free water. Rolling circle amplification (RCA): To increase the signal, the enriched traces number of circular DNA (12 \u0026micro;l out of a total of 24 \u0026micro;l) was amplified greatly via RCA and the RCA reaction system involved. The RCA products were recovered with 80 \u0026micro;l WAHTS\u0026reg; DNA clean Beads (Vazyme) and eluted in 100 \u0026micro;l RNase-free water. The RCA products were concentrated by Qubit 3.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 │ Library preparation and eccDNA deep sequencing\u003c/h2\u003e \u003cp\u003eThe φ29-amplified DNA samples (0.5 \u0026micro;g) were firstly sonicated into a 300\u0026ndash;500 bp size range on the Covaris LE220 (Covaris). Then, 80 ng DNA fragments were end-repaired, A-tailed, and adapter-ligated using the MGIEasy DNA Library Preparation Kit (MGI-BGI, China). The quality control (including size distribution and concentration) of each library was assessed by the Agilent Bioanalyzer 2100 system. Lastly, the constructed library was deep sequenced on the MGI PE150 platform (BGI-QEI, Qingdao, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 │ EccDNA assembly by Circle-Map\u003c/h2\u003e \u003cp\u003eMultiQC (v1.10.1), FastQC (v0.11.3), and Fastp (v0.21.0) were performed for quality control (QC) of raw sequencing data. Clean reads were mapped to the human reference genome (ref. hg38/ mm10) using BWA software. Circle-Map (V1.1.4) software was adapted to call circular DNA from Circle-Seq data based on reads span on junctions. To improve the confidence of detected-eccDNA, multiple filtration steps were performed as we previously described \u003csup\u003e21\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 │ Generation in silico eccDNAs for control comparison\u003c/h2\u003e \u003cp\u003eBased on the weighted average of chromosome length, we generated in silico eccDNAs across the genome by R statistical suite to minimize the effects of mapping and annotation issues. The generated \u003cem\u003ein silico\u003c/em\u003e eccDNAs ranged randomly between 150 bp to 850 bp to mimic our detected plasma eccDNAs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 │ Genomic annotation\u003c/h2\u003e \u003cp\u003eGenomic annotation data were obtained from Ensembl database assembly GRCh38. Overlaps between detected eccDNA coordinates and annotated genes were identified using bedtools (iintersect-wo). Specially, the eccDNAs with \u0026gt;\u0026thinsp;60 bp overlap of a certain gene were defined as \u0026ldquo;eccGene\u0026rdquo; in the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 │ eccDNA validation by PCR and sanger sequencing\u003c/h2\u003e \u003cp\u003eOutward PCR and Sanger sequencing were conducted to validate specific gene circles. The PCR primers were listed in table S4. Each 20 ul PCR reaction system included 50 \u0026micro;g phi29-amplified DNA products, 500 nM primer, 10 ul NEBNext High-Fidelity 2X PCR Master Mix (NEB), and PCR reaction for 35 cycles. All reactions were performed accompanied by RCA products pooled from 20 healthy controls. The PCR products were tested by agarose (2.5%) gel electrophoresis. The target products were recovered by QIAEX II Gel Extraction Kit for \u003cem\u003eSanger\u003c/em\u003e sequencing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 │ Construction of artificial eccDNA\u003c/h2\u003e \u003cp\u003eTwo ecc\u003cem\u003eTAOK2\u003c/em\u003e carrying the TAOK2 intronic sequence were constructed by the ligase-assisted minicircle accumulation (LAMA) strategy as previously described \u003csup\u003e10\u003c/sup\u003e. The sequence of the synthetic linear fragments and corresponding PCR primers for artificial eccDNA preparation were displayed in table S5.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 │ Artificial eccDNA transfection and qPCR\u003c/h2\u003e \u003cp\u003eU-251MG and SH-SY5Y cell lines were used for the functional assay of artificial ecc\u003cem\u003eTAOK2\u003c/em\u003es in this study. The cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) (Lonza) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% GlutaMAX (Gibco), and penicillin-streptomycin (100 units penicillin and 0.1 mg streptomycin/mL) (Thermo Fisher Scientific) in a 37℃ incubator with 5% CO2 atmosphere and maximum humidity. The transfection was conducted using lipofectamine2000 (Invitrogen) in a 24-well plate according to the manufacturer\u0026rsquo;s instructions. 500 ng synthetic ecc\u003cem\u003eTAOK2\u003c/em\u003e was transfected in each well and triplicates were performed for each group. Cells transfected with an equal amount of pMax-GFP plasmid served as the control group. All the cells were harvested 48 hours after transfection for RNA isolation using TRIZOL reagent (Servicebio) according to the manufacturer\u0026rsquo;s protocol. 1 \u0026micro;g RNA was input for reverse transcription (RT) and total cDNA was synthesized using the HiScript\u0026reg; III 1st strand cDNA synthesis kit (+\u0026thinsp;gDNA wiper) (Vazyme). The quantitive PCR was performed by using the ChamQ Universal SYBR qPCR Master Mix (Vazyme). The primers for \u003cem\u003eTAOK2\u003c/em\u003e mRNA detection were hTAOK2-qPCR-F: 5\u0026rsquo;-caaggaggtgcggttcttac-3\u0026rsquo; and hTAOK2-qPCR-R: 5\u0026rsquo;-ggtcacagctgcgatctctac-3\u0026rsquo;. GAPDH was used as an internal reference gene and qPCR primers were hGAPDH-qPCR-F: 5\u0026rsquo;-gtacgtcgtggagtccactg-3\u0026rsquo; and hGAPDH-qPCR-R: 5\u0026rsquo;-gttgtcatggatgaccttggc-3\u0026rsquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 │ Dual-luciferase reporter gene assay\u003c/h2\u003e \u003cp\u003eThe intronic fragments of the two eccTAOK2 were cloned from the genomic DNA of U-251MG cells using the primers with linker sequence containing the recognition sites of restriction endonuclease XhoI or NotI (table S6). Then the amplified intronic fragment (TAOK2 intron 1 or 8) was inserted in the psiCHECK2 plasmid (Promega) between the XhoI and NotI sites. Sanger sequencing was conducted for the confirmation of successful construction. Then the effects of artificial ecc\u003cem\u003eTAOK2\u003c/em\u003e on the relative expression level of Renilla luciferase were detected by the Dual-luciferase Reporter Assay System (Promega) according to the manufacturer\u0026rsquo;s instruction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 │ RNA sequencing and DEGs functional analysis\u003c/h2\u003e \u003cp\u003eThe total RNA of the U-251MG cells after eccTAOK2#1 or pMax-GFP plasmids transfection was extracted using TRIZOL reagent (Servicebio) according to the manufacturer\u0026rsquo;s protocol. Then the total RNA was treated with gDNA wiper (Vazyme) at 37 ℃ for 10 min to remove the residual genomic DNA. The quality of total RNA was evaluated by agarose gel electrophoresis and a bioanalyzer (Aligent 2100). Only quality intact RNA was sent for RNA sequencing. The mRNA sequencing was conducted by custom service provided by GENE DENOVO (Guangzhou, China) using an MGIseq-2000 sequencing machine (BGI, China). Genes with |Log\u003csub\u003e2\u003c/sub\u003e(FoldChange)| \u0026gt; 0.5 and \u003cem\u003ep\u003c/em\u003e-adjust value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were identified as DEGs. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed using the online webtools of OmicShare established by GENE DENOVO (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.omicshare.com/tools/\u003c/span\u003e\u003cspan address=\"https://www.omicshare.com/tools/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 │ Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll statistical tests were implemented by R-4.1.2 and GraphPad Prism 9. The difference comparison of the two groups was performed by Wilcoxon\u0026rsquo;s rank-sum test or Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 │ RESULTS","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 │ Detection of circulating eccDNA in plasma using Circle-seq strategy\u003c/h2\u003e \u003cp\u003eWe leveraged the Circle-seq approach \u003csup\u003e2, 37\u003c/sup\u003e to purify and decipher the eccDNAs in the plasma derived from 10 chronic SCZ patients and 17 healthy controls. The workflow of the process is illustrated in figure. 1A. Briefly, the overall cfDNA extracted from the plasma was digested by exonuclease V to remove the linear cfDNA. Then the retained circular DNAs were purified and subjected to Rolling Circle Amplification (RCA) by φ29 polymerase to amplify the eccDNA amount. The eccDNA-RCA products were sequenced by Next-Generation-Sequencing (NGS) and all the high-quality reads were input into the Circle-map program for eccDNA calling. In this process, both split and discordant reads were extracted and used for mapping and identification of the start-end loci of eccDNAs. These eccDNAs were annotated and used for subsequent analysis. Especially, given that only the eccDNA carrying a certain length of gene segment can express potential regulatory RNA molecules \u003csup\u003e10\u003c/sup\u003e, the eccDNAs with \u0026gt;\u0026thinsp;60 bp overlap of a certain gene loci were defined as \u0026ldquo;eccGenes\u0026rdquo; in the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2 │ Features of circulating eccDNA profile in the plasma\u003c/h2\u003e \u003cp\u003eIn this study, we obtained an average of 11.368\u0026nbsp;million (ranging from 7.01 to 15.06\u0026nbsp;million) reads (paired-end 150 bp) per healthy control sample and 11.96\u0026nbsp;million (ranging from 9.35 to 15.31\u0026nbsp;million) reads per SCZ sample (table S2). In total, an average of 7929 (varying from 3138 to 13191) and 8390 (varying from 2818 to 18531) eccDNA loci were identified in healthy control and SCZ plasma samples, respectively. The absolute eccDNA counts in each sample of the two groups were comparable with no significant difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). To eliminate the bias introduced by sequencing depth, we compared the eccDNA counts per million mapped reads (EPM) and found no significant difference between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The length of the most eccDNAs was less than 2 kb with four predominant peaks at around 197 bp, 363 bp, 555 bp and 747 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Moreover, we found that 98.8% of the plasma-derived eccDNAs was less than 2 kb and only around 1.2% was between 2 kb and 8 kb in length (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), indicating the shorter size of plasma-derived eccDNA compared to that observed in cancer cells \u003csup\u003e38, 39\u003c/sup\u003e. Consistent with previous reports in cancers \u003csup\u003e39\u003c/sup\u003e, the GC content of circulating eccDNA was higher than that of the average genomic distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), suggesting the generation of circulating eccDNAs was not random. On the other hand, the generation frequency of eccDNA in each chromosome (percent of eccDNA per Mb length of DNA) of the two groups was comparable with no significant difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). However, the tendency of eccDNA generation in different chromosomes was varying. For instance, the chromosome (Chr) X and Y arose much fewer eccDNAs than that on Chr 19 or 20, suggesting the DNA accessibility level varied from chromosomes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3 │ Determination of differential eccGenes in either SCZ or healthy control\u003c/h2\u003e \u003cp\u003eEccDNA carrying gene segment showed the potential to transcribe RNAs \u003csup\u003e2\u003c/sup\u003e and produce functional si-like RNA which leads to suppression of the host gene \u003csup\u003e10\u003c/sup\u003e. In this study, we identified and analyzed the differential eccGenes in either the chronic SCZ or the healthy control. In total, 26 differential eccGenes (Wilcoxon\u0026rsquo;s rank-sum test, \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were identified in the healthy control, whereas the number in chronic SCZ was 211 (table S3). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA showed the existing frequency of the differential eccGenes in the two groups with \u003cem\u003ep-\u003c/em\u003evalue\u0026thinsp;\u0026lt;\u0026thinsp;0.03 (table S3). In the chronic SCZ group, several of the differential genes were well-known and associated with the pathogenesis of SCZ, such as \u003cem\u003eDNMT3B\u003c/em\u003e \u003csup\u003e40, 41\u003c/sup\u003e, \u003cem\u003eSIRT5\u003c/em\u003e \u003csup\u003e42\u003c/sup\u003e, \u003cem\u003eJAG1\u003c/em\u003e \u003csup\u003e43, 44\u003c/sup\u003e, \u003cem\u003eTAOK2\u003c/em\u003e \u003csup\u003e45, 46\u003c/sup\u003e and so on.\u003c/p\u003e \u003cp\u003eWe further compared the 211 SCZ over-represented eccGenes with the combination of two reported SCZ high-risk gene (HRG) sets (104 and 67 HRGs) inferred from the worldwide SCZ GWAS data \u003csup\u003e34, 47\u003c/sup\u003e. The \u003cem\u003eTAOK2\u003c/em\u003e gene was identified, whereas there was no overlapped gene between the healthy control-specific eccGenes (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) and the SCZ HRGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). We then retrieved the reads distribution of ecc\u003cem\u003eTAOK2\u003c/em\u003e found in the samples utilizing the Integrative Genomics Viewer (IGV). 5/10 SCZ patients contained ecc\u003cem\u003eTAOK2\u003c/em\u003e while no healthy control presented the typical ecc\u003cem\u003eTAOK2\u003c/em\u003e reads signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Notably, three of the five observed ecc\u003cem\u003eTAOK2\u003c/em\u003e in SCZ were derived from the intron-1 of \u003cem\u003eTAOK2\u003c/em\u003e gene, while the other two were from intron-8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In addition, to verify the existence of SCZ over-represented eccGenes in the corresponding samples, we performed outward PCR to visualize the junction regions of several eccGenes, including 4 ecc\u003cem\u003eTAOK2\u003c/em\u003e, 1 ecc\u003cem\u003eDNMT3B\u003c/em\u003e, 2 ecc\u003cem\u003eJAG1\u003c/em\u003e and 2 ecc\u003cem\u003eSIRT5\u003c/em\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD showed that specific PCR bands were detectable in SCZ samples but not in the pool of the healthy control samples. Sanger sequencing results further confirmed the detailed sequence of the junction sites of each eccGene (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), which were in accordance with the prediction of Circle-map.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4 │ Six SCZ over-represented eccGenes were enriched in the phenotype of progressive intellectual disability\u003c/h2\u003e \u003cp\u003eTo evaluate the effects of SCZ over-represented eccGenes on human phenotypes, we conducted the Human Phenotype Ontology (HPO) analysis (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.webgestalt.org/option.php\u003c/span\u003e\u003cspan address=\"http://www.webgestalt.org/option.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) upon the 211 SCZ over-represented eccGenes. The top 10 of the phenotypic abnormalities enriched in both groups were displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. The term \u0026ldquo;Intellectual disability, progressive\u0026rdquo; (IDP) was identified with statistical significance of \u003cem\u003eFDR\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;=\u0026thinsp;9.13E-6, while no significant term was found upon the healthy control-specific eccGenes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, below). Among the SCZ over-represented eccGenes mapped with HPO gene database, six genes were found overlapping to the IDP cluster genes (6 of 48 genes, enrichment ratio\u0026thinsp;=\u0026thinsp;11.682), including \u003cem\u003eDDB2\u003c/em\u003e, \u003cem\u003eERCC3\u003c/em\u003e, \u003cem\u003ePTS\u003c/em\u003e, \u003cem\u003eUBE3A\u003c/em\u003e, \u003cem\u003eUROC1\u003c/em\u003e and \u003cem\u003eXPA\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The existing frequency of each of the six genes in the chronic SCZ group was significantly higher compared to the healthy control (Wilcoxon\u0026rsquo;s rank-sum test, \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In addition, we verified the existence of the 6 IDP-related eccGenes in SCZ samples (two eccDNAs in their corresponding SCZ samples for each gene) by using the outward PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) and Sanger sequencing of the PCR products (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.5 │Artificial eccDNA containing TAOK2-intron segment attenuated the TAOK2 mRNA level in brain-derived cells\u003c/h2\u003e \u003cp\u003eEccDNA that contains exonic sequence can produce functional RNA molecules that suppress the host gene expression \u003csup\u003e10\u003c/sup\u003e, but the regulatory function of eccDNA harboring intron sequence remains unknown. In this study, we found the eccDNAs carrying \u003cem\u003eTAOK2\u003c/em\u003e intronic segments were related to SCZ, as it was detected in 50% (five of ten) of the SCZ patients but not in the 17 healthy controls. We speculated that ecc\u003cem\u003eTAOK2\u003c/em\u003e expresses RNA transcripts composed of the tandem \u003cem\u003eTAOK2\u003c/em\u003e-intron sequence which would be processed to be novel si-like RNAs that target the precursor mRNAs (pre-mRNA) of \u003cem\u003eTAOK2\u003c/em\u003e, resulting in disturbance of the pre-mRNA processing and downregulation of the mature mRNAs.\u003c/p\u003e \u003cp\u003eTo test the regulatory function of ecc\u003cem\u003eTAOK2\u003c/em\u003e, we synthesized two SCZ over-represented eccDNAs carrying the segments of \u003cem\u003eTAOK2\u003c/em\u003e intron 1 and intron 8, respectively. As showcased in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, ecc\u003cem\u003eTAOK2\u003c/em\u003e#1 contained a 323 bp portion of the intron-8 and ecc\u003cem\u003eTAOK2\u003c/em\u003e#2 contained 370 bp of the intron-1. The two artificial ecc\u003cem\u003eTAOK2\u003c/em\u003e were constructed according to the ligase-assisted mini-circle accumulation (LAMA) protocol \u003csup\u003e10\u003c/sup\u003e, which is conducted by cycles of DNA denaturation, annealing and ligation processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA left). The LAMA products were treated with exonuclease to remove the residual linear DNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA right) and the purified circular DNA was identified by digestion with a single restriction endonuclease. Digestion with SspI or StuI on the linear A fragment resulted in two shorter DNA bands, while the circular DNA showed only one long band after digestion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eTransfection of the two artificial ecc\u003cem\u003eTAOK2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) resulted in down-regulation of the \u003cem\u003eTAOK2\u003c/em\u003e mRNA level in both the SH-SY5Y and U-251MG cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E), which were assessed by the real-time quantitative PCR: the ecc\u003cem\u003eTAOK2\u003c/em\u003e reduced the \u003cem\u003eTAOK2\u003c/em\u003e mRNA level by an average of 27% and 25% in the SH-SY5Y cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), and 42% and 20% in the U-251MG cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). To further validate whether ecc\u003cem\u003eTAOK2\u003c/em\u003e produced regulatory RNAs that target to the intronic sequence, renilla luciferase gene containing the full length of eccTAOK2#1 and #2 sequence in the 3\u0026rsquo;UTR were co-transfected with the artificial ecc\u003cem\u003eTAOK2\u003c/em\u003e for dual-luciferase assays in U-251MG cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Ecc\u003cem\u003eTAOK2\u003c/em\u003e#1 \u003cem\u003eand\u003c/em\u003e ecc\u003cem\u003eTAOK2\u003c/em\u003e#2 repressed the renilla luciferase carrying their intron-origin sequences by 48.5% and 69.1%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Together these results suggested that the ecc\u003cem\u003eTAOK2\u003c/em\u003e carrying intronic sequence was able to repress \u003cem\u003eTAOK2\u003c/em\u003e mRNA expression through the production of functional regulatory RNAs which may target the intronic portion of pre-mRNA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.6 │Artificial eccTAOK2 dysregulated the immune system in U-251MG cells\u003c/h2\u003e \u003cp\u003eThe circular structure- but not the canonical promoter-dependent transcription of eccDNA \u003csup\u003e10\u003c/sup\u003e confers its potential to influence the phenotypes through expressing specific RNA transcripts \u003csup\u003e2\u003c/sup\u003e. But the impact of eccDNA on the transcriptome of brain cells is unclear. To evaluate the effect of SCZ-derived eccDNA on nerve cells, the artificial ecc\u003cem\u003eTAOK2\u003c/em\u003e was transfected in U-251MG cells and we performed the RNA-seq analysis afterward. In this part of the study, a total of 111 DEGs were identified with 46 downregulated genes (Log\u003csub\u003e2\u003c/sub\u003e(FoldChange) \u0026lt; -0.5 and \u003cem\u003ep-\u003c/em\u003eadjust value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 65 upregulated genes (Log\u003csub\u003e2\u003c/sub\u003e(FoldChange)\u0026thinsp;\u0026gt;\u0026thinsp;0.5 and \u003cem\u003ep-\u003c/em\u003eadjust value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and table S7). GO enrichment analysis of these DEGs using the GO web tool on the OmicShare online platform highlighted the immune-related biological processes (table S8). Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB showed the top 20 GO terms with \u003cem\u003eq\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01, including \u0026ldquo;immune system process\u0026rdquo; (GO: 0002376, \u003cem\u003eq\u003c/em\u003e-value\u0026thinsp;=\u0026thinsp;0.000026), \u0026ldquo;cellular response to chemical stimulus\u0026rdquo; (GO: 0070887, \u003cem\u003eq\u003c/em\u003e-value\u0026thinsp;=\u0026thinsp;0.000029), \u0026ldquo;response to stress\u0026rdquo; (GO: 0006950, \u003cem\u003eq\u003c/em\u003e-value\u0026thinsp;=\u0026thinsp;0.000087) and so on (table S5). KEGG analysis showed these DEGs were enriched in two major signaling pathways: \u0026ldquo;TNF signaling pathway\u0026rdquo; and \u0026ldquo;cytokine-cytokine receptor interaction\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D, table S9) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC presented the top 20 KEGG enriched terms and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD showed the connection-network of these signaling pathways). Consistent with the GO analysis result, both pathways were connected to the immune-related biological processes, including necroptosis, apoptosis, IL-17 signaling pathway, insulin resistance and so on (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Taken together, the RNA-seq analysis indicated the introduction of artificial ecc\u003cem\u003eTAOK2\u003c/em\u003e in U-251MG cells dysregulated the immune-related biological processes, suggesting a potential negative effect of eccDNA on the SCZ brain.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 │DISCUSSION","content":"\u003cp\u003eThe foremost goal of the SCZ genetic study is to identify an unambiguous biomarker for diagnosis, monitoring, prognosis and therapeutic target for effective treatment of SCZ. Despite many recent studies that have reported the vital role of eccDNA in tumorigenesis and cancer evolution \u003csup\u003e13, 33, 39, 48, 49\u003c/sup\u003e, the relation between eccDNA and SCZ has never been investigated. In the present study, we first characterized the hallmarks of circulating eccDNA in chronic SCZ patients. The bioinformatical analysis identified the SCZ over-represented eccGenes and suggested several of these eccGenes may be related to SCZ. To investigate the biological role of circulating eccGenes, we tested the regulatory function of eccDNA carrying the \u003cem\u003eTAOK2\u003c/em\u003e-intronic sequence as an example. Functional assays indicated that ecc\u003cem\u003eTAOK2\u003c/em\u003e detected in SCZ can attenuate the \u003cem\u003eTAOK2\u003c/em\u003e expression and dysregulate the immune-related biological processes in brain-derived cells. These results highlight the potential role of eccDNA in SCZ, which has not been studied extensively.\u003c/p\u003e \u003cp\u003eThe basic features of circulating eccDNAs detected in SCZ patients in this work were similar to those found in pregnant women \u003csup\u003e17\u003c/sup\u003e and gout patients \u003csup\u003e22\u003c/sup\u003e. Both the SCZ patients and healthy people showed a predominant peak of eccDNA size at 363 bp and three lower peaks positioning at 197, 555 and 747 bp. The interval of the length peaks implied the nucleosomal origin of these eccDNAs \u003csup\u003e17, 19\u003c/sup\u003e. The primary cluster of eccDNA at around 363 bp suggested that circulating eccDNA was tend to be generated from di-nucleosomal wrapped DNAs. In this study, we used 300 \u0026micro;L plasma for eccDNA purification and an average of around 8000 eccDNAs were identified in either the SCZ or the healthy control. Considering the large volume of plasma in adult males (~\u0026thinsp;50 mL/kg body weight \u003csup\u003e50\u003c/sup\u003e), it is conceivable that there is a substantial amount of eccDNAs circulating in the peripheral blood: an average of around 2.67\u0026nbsp;million eccDNAs/kg body weight based on our data without considering the DNA losses during purification. Therefore, it is reasonable to infer that the aberrant biogenesis or metabolism of circulating eccDNAs may be linked to any human diseases, including SCZ.\u003c/p\u003e \u003cp\u003eIn this work, we identified a total of 211 SCZ over-represented eccGenes in 10 SCZ patients, whereas only 26 differential eccGenes were found in the 17 healthy controls. This skewed data between the two groups implied the aberrant biogenesis or metabolism of eccDNA in SCZ patients. Moreover, it has been observed that both the normal tissue and tumor-derived eccDNAs are detectable in circulation \u003csup\u003e20\u003c/sup\u003e. Thus, the SCZ over-represented circulating eccDNAs may mirror the aberrant status of somatic cells, including the brain cells. Several pieces of evidence provided by our data supported this conjecture: 1) The SCZ group enriched more specific eccGenes than the control group; 2) Among these SCZ over-represented eccGenes, many are related to the etiology of SCZ and 3) six of the SCZ over-represented eccGenes were significantly enriched to the phenotype of progressive intellectual disability, which often co-occurs with SCZ \u003csup\u003e51\u0026ndash;55\u003c/sup\u003e. These findings underscored the potential linkage between eccDNA and SCZ.\u003c/p\u003e \u003cp\u003eTo investigate the molecular function of SCZ over-represented eccGenes, we took the ecc\u003cem\u003eTAOK2\u003c/em\u003e as an example. \u003cem\u003eTAOK2\u003c/em\u003e is a gene located in the SCZ-associated 16p11.2 microduplication region \u003csup\u003e56\u0026ndash;59\u003c/sup\u003e. Several psychiatric features, such as speech/language impairments, motor/development delay, intelligent disability and microcephaly, have been identified in patients with 16p11.2 microduplication or microdeletion \u003csup\u003e60\u003c/sup\u003e. Among the 27\u0026ndash;29 genes found in the 16p11.2 locus, \u003cem\u003eTAOK2\u003c/em\u003e plays a critical role in regulating the neuronal survival and development in the nervous system \u003csup\u003e35, 36, 61\u003c/sup\u003e. It is composed of 19 exons and 18 introns. Intriguingly, the five ecc\u003cem\u003eTAOK2\u003c/em\u003e detected in SCZ appeared in only two introns of this gene: three in the intron 1 and two from the intron 8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). An interesting phenomenon is that the full length of \u003cem\u003eTAOK2\u003c/em\u003e gene showed a very high degree of conservation in sequence in animals such as horse, cow, dog, panda, rat and dolphin, but not in the birds, Sarcopterygii, or fish (figure S1). The situation is different in those genes located near the \u003cem\u003eTAOK2\u003c/em\u003e locus, like \u003cem\u003eSEZ6L2\u003c/em\u003e, \u003cem\u003eMAPK3\u003c/em\u003e and \u003cem\u003eGDPD3\u003c/em\u003e (figure S1). These findings suggested that not only the exons but also the \u003cem\u003eTAOK2\u003c/em\u003e introns, may play an important role in the neurodevelopment of those animals with higher intelligence than the bird or fish. Moreover, our study demonstrated the regulatory function of ecc\u003cem\u003eTAOK2\u003c/em\u003e-intron in repressing the \u003cem\u003eTAOK2\u003c/em\u003e gene expression. Although the function was confirmed by the dual-luciferase assay, whether the ecc\u003cem\u003eTAOK2\u003c/em\u003e transcripts can target the intronic portion of pre-mRNA is still uncertain. Further studies are warranted to elucidate the detailed regulatory mechanism of eccDNA carrying intronic sequence.\u003c/p\u003e \u003cp\u003eThe immune system is activated when interacting with pathogens and can affect the central nervous system \u003csup\u003e62\u003c/sup\u003e. A growing body of evidence has indicated the etiology of schizophrenia is involved in neuroinflammation and immune dysfunction \u003csup\u003e63\u0026ndash;73\u003c/sup\u003e. Consistent with these findings, our data demonstrated that artificial ecc\u003cem\u003eTAOK2\u003c/em\u003e can lead to dysregulation of the cellular immune-related biological processes, including the TNF signaling pathway and cytokine-cytokine receptor interaction. In addition, diverse immune-response processes were connected with the two main signaling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Previous study has reported that eccDNA acts as a potent innate immunostimulant which is dependent on its circularity structure, but independent of the eccDNA sequence \u003csup\u003e16\u003c/sup\u003e. However, our data suggested that the eccDNA may contribute additional effects on the dysregulation of the immune system via expressing RNA and perturbing the transcriptome. Considering there were also tens of thousands of circulating eccDNAs in healthy people, more attention should be paid to the over-represented genic element carried by eccDNA but not its topological structure in case-control studies.\u003c/p\u003e \u003cp\u003eDespite these findings, several issues remain for future study: 1) What are the tissue origins of the circulating eccDNAs? 2) Whether the brain-derived eccDNAs can cross the BBB and release into circulation? 3) Whether the circulating cell-free eccDNAs originated from the non-brain cells can cross the BBB and influence the human brain? 4) Dose the extracellular vesicle contain eccDNA and mediate intercellular cross-talk? 5) Whether the eccDNA can serve as biomarker for SCZ diagnosis and monitoring? Previous studies on circulating cfDNA have provided clues to answer some of the questions addressed. Tissue-specific epigenetic markers carried by the cfDNA have been utilized for identification of the tissue-of-origins of cfDNA \u003csup\u003e24, 74\u003c/sup\u003e. It would thus be convenient to investigate the tissue origins of circulating eccDNA by the strategy. It was postulated that the BBB may prevent cell-free DNA from reaching systematic circulation. However, the circulating tumor DNA (ctDNA) is detectable in the plasma of brain tumor patients \u003csup\u003e75, 76\u003c/sup\u003e, suggesting the absence of the BBB present \u003csup\u003e77\u003c/sup\u003e or alteration of the BBB integrity may influence the ctDNA levels in the circulation \u003csup\u003e78\u003c/sup\u003e. There is also a possibility that the eccDNA can be transported across the BBB by delivery of the extracellular vesicles \u003csup\u003e79, 80\u003c/sup\u003e. Therefore, a new methodology needed to be developed for purification and identification of eccDNAs in vesicles. Lastly, we believe that analysis of eccDNAs derived from large-scale samples combined with the promising machine learning will help to identify the reliable eccDNA biomarkers for diagnosis and monitoring of SCZ.\u003c/p\u003e"},{"header":"5 │ CONCLUSIONS","content":"\u003cp\u003eThis study characterized the hallmarks of circulating eccDNA in chronic SCZ and implicated the potential association between the eccDNA carrying genic segment and the pathogenesis of SCZ. As an exemplar, we demonstrated the regulatory function of ecc\u003cem\u003eTAOK2\u003c/em\u003e in brain-derived cells and observed its potent impact on the innate cellular immune system. Further studies on the eccDNA methylation, the function of eccDNAs carrying different genomic segments, the function of eccDNA in vesicles, eccDNA analysis of large-scale clinical samples may help to better understand the mechanisms of SCZ.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank all the participants, staff, and volunteers in\u0026nbsp;Zigong Fifth People's Hospital\u0026nbsp;and\u0026nbsp;Sir Run Run Shaw Hospital of Zhejiang University for their time and contributions to the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe circulating eccDNA sequencing data have been deposited in the genome sequence archive of the Beijing Institute of Genomics, National Center for Bioinformation, Chinese Academy of Science. The accession numbers for the eccDNA sequencing data in this study is HRA004251.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eORCID\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eXi Xiang\u003c/em\u003e https://orcid.org/0000-0002-5590-7289\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePeng Han\u003c/em\u003e https://orcid.org/0000-0002-3405-087X\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eJinsong Tang\u003c/em\u003e \u003ca href=\"http://orcid.org/0000-0003-3796-1377\"\u003ehttp://orcid.org/0000-0003-3796-1377\u003c/a\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003ePaulsen T, Kumar P, Koseoglu MM, Dutta A. Discoveries of Extrachromosomal Circles of DNA in Normal and Tumor Cells. Trends Genet Apr 2018;34(4):270\u0026ndash;278.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMoller HD, Mohiyuddin M, Prada-Luengo I, et al. 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Neurooncol Adv Jan-Dec 2020;2(1):vdaa016.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBanks WA, Sharma P, Bullock KM, Hansen KM, Ludwig N, Whiteside TL. Transport of Extracellular Vesicles across the Blood-Brain Barrier: Brain Pharmacokinetics and Effects of Inflammation. Int J Mol Sci Jun 21 2020;21(12).\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHeidarzadeh M, Gursoy-Ozdemir Y, Kaya M, Eslami Abriz A, Zarebkohan A, Rahbarghazi R, Sokullu E. Exosomal delivery of therapeutic modulators through the blood-brain barrier; promise and pitfalls. Cell Biosci Jul 22 2021;11(1):142.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"The Seventh Affiliated Hospital of Sun Yat-sen University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"cell-free DNA, ecDNA, psychiatric disorder, noninvasive biomarker, rolling circle amplification, LAMA, plasma","lastPublishedDoi":"10.21203/rs.3.rs-3287964/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3287964/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Extrachromosomal circular DNA (eccDNA) is a circular DNA molecule derived and free from linear chromosome, its characteristics and potential function in SCZ remain unclear.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Here, we explored the presence of circulating eccDNA in the plasma of chronic SCZ and healthy control using Circle-seq. Then the molecular role of SCZ over-represented eccDNAs was investigated by bioinformatical and experimental analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: 211 over-represented eccDNAs carrying genic segments (eccGene), including ecc\u003cem\u003eTAOK2\u003c/em\u003e, ecc\u003cem\u003eDNMT3B\u003c/em\u003e, ecc\u003cem\u003eSIRT5,\u003c/em\u003e ecc\u003cem\u003eJAG1\u003c/em\u003e and so on, were identified in 10 chronic SCZ patients, whereas only 26 over-represented eccGenes were found in 17 healthy people. Human phenotype ontology enrichment analysis upon the 211 SCZ over-represented eccGenes showed that six of them were enriched significantly in the phenotype of progressive intellectual disability. Functional assays of two artificial eccDNAs carrying \u003cem\u003eTAOK2\u003c/em\u003e-intronic sequence showed that they attenuated the \u003cem\u003eTAOK2\u003c/em\u003e mRNA expression in both U-251MG and SH-SY5Y cell lines, which the function was further confirmed by dual-luciferase reporter gene assay. In addition, RNA-seq analysis showed that introduction of the artificial ecc\u003cem\u003eTAOK2\u003c/em\u003e in U-251MG cells resulted in dysregulation of immune-related biological processes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: These findings delineate the circulating eccDNAs profile of SCZ and highlight the regulatory function of ecc\u003cem\u003eTAOK2 \u003c/em\u003eand its impact on cellular immune processes, underscoring the eccDNA biology and its potential role as a noninvasive biomarker for diagnosis and monitoring of schizophrenia.\u003c/p\u003e","manuscriptTitle":"Identification and functional analysis of circulating extrachromosomal circular DNA in schizophrenia implicate its negative effect on the disorder","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-24 16:45:57","doi":"10.21203/rs.3.rs-3287964/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7163794a-87fe-46a7-b7d7-48c25bc380a0","owner":[],"postedDate":"August 24th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":24224044,"name":"Molecular Genetics"},{"id":24224045,"name":"Epigenetics \u0026 Genomics"},{"id":24224046,"name":"Psychiatry"},{"id":24224047,"name":"Medical Genetics"}],"tags":[],"updatedAt":"2023-08-24T16:45:57+00:00","versionOfRecord":[],"versionCreatedAt":"2023-08-24 16:45:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3287964","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3287964","identity":"rs-3287964","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}