LAUSR

Syndrome (Zheng in Mandarin Chinese) is the induction of disease attributes in the theory of traditional Chinese medicine (TCM). This is often used to explain why patients with the same disease show different symptoms. Cold and heat represent the two basic tendencies of a syndrome; therefore, the identification of cold and heat syndromes is key to the diagnosis and treatment of rheumatoid arthritis (RA) according to TCM. However, a traditional thinking model of cold and heat syndromes identifies a reliance on the clinical manifestations, which is inevitably affected by the subjectivity of doctors. It is important to identify objective biomarkers to improve accuracy in the identification of cold and heat syndromes of RA. Previous studies have identified differences in gene expression and function between cold and heat syndromes of RA, suggesting that different syndromes possess a unique biological basis.1,2 However, the biological basis is large and complex; therefor constructing a “disease– syndrome– biomaker” network using modern technology to preserve the characteristics of TCM and also improve the objectivity and applicability in syndrome identification is challenging DNA hydroxymethylation is an epigenetic marker that has attracted increasing attention recently. As a bridge between methylation and demethylation, DNA hydroxymethylation drives demethylation, indicating the reactivation of gene transcriptional expression.3 More importantly, DNA hydroxymethylation is sensitive to environmental stimuli, the state of DNA hydroxymethylation modification is able to change under different environmental stimuli, resulting in phenotypic differences.4 This regulatory mechanism is consistent with the concept of TCM, whereby “environmental differences can break the balance between cold and heat, causing differences in external clinical symptoms in patients with RA”, inferring that it is feasible to explore the epigenetic biomarkers of cold and heat syndromes of RA from DNA hydroxymethylation and gene transcription. The aim of our study was to identify epigenetic biomarkers of cold and heat syndromes of RA by combining DNA hydroxymethylation sequencing and transcriptome sequencing. Our study was approved through ethical review by the First Affiliated Hospital of Yunnan University of Traditional Chinese Medicine (ethical batch number: the scientific research [2020] Ethical Review [005]02). The study was conducted as follows. Ten patients with RA cold syndrome and 10 with heat syndrome were recruited according to the Guideline for Diagnosis and Treatment of Rheumatoid Arthritis Based on TCM Syndromes.5 We also included 10 healthy participants. The general situation of these groups is shown in Table 1. Next, 10 mL peripheral venous blood was extracted from each participant to isolate peripheral blood mononuclear cells; DNA and RNA were further extracted. Hydroxymethylated DNA fragments were obtained through DNA hydroxymethylation immunoprecipitation (hMeDIP), and mRNA was obtained through enzyme digestion. The Illumina highthroughput sequencing platform was used to generate the library, in addition to hMeDIP sequencing (hMeDIPseq) and mRNA sequencing (mRNAseq). Bioinformatics analysis was used to compare the changes in DNA hydroxymethylation and transcriptional expression between cold and heat syndrome genes. Remarkably altered genes at DNA hydroxymethylation were used in Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway enrichment. Genes significantly altered at both the DNA hydroxymethylation and transcription expression levels were identified as epigenetic biomarkers of cold and heat syndromes of RA and the correlation was preliminarily analyzed. Results demonstrated that genomes with significant hydroxymethylation alteration tended to have a higher hydroxymethylation level (Figure S1A). Among 754 differential genes screened, 737 genes showed upregulation (logFC ≥ 1, P < 10−4), involved in 32 pathways including the Wnt signaling pathway, T helper type 17 cell differentiation pathway, and neuroactive ligandreceptor interaction (Figure S1B). The expression of 17 genes was downregulated (logFC ≤ −1, P < 10−4) and related to African trypanosomiasis, Malaria, and Axonal guidance pathways (Figure S1C). There were 125 genes with significant hydroxymethylated changes from patients with heat syndrome (Figure S1D). Among which, 64 genes indicated upregulated expression (logFC ≥ 1, P < 10−4) and enriched Arginine and proline metabolism (Figure S1E). The remaining 61 genes with