A Pilot Study on DNA Methylation in Pediatric HAdV-7-induced Sepsis

Human adenovirus (HAdV) is one of the most common respiratory pathogens affecting children. HAdV infection has high morbidity and mortality, and it may lead to severe complications and long-term pulmonary sequelae. However, the pathogenesis of pediatric HAdV-7-induced sepsis remains unclear. The analysis of DNA methylation proles in peripheral blood is attracting increasing attention as an effective method for investigating the pathogenesis of various diseases and identifying biomarkers of disease progression. Here, we performed reduced representation bisulte sequencing to analyze DNA methylation in peripheral blood samples collected from 11 children with HAdV-7-induced sepsis and 5 healthy children. The Metilene software was used to analyze differential methylation in the two groups. We also performed functional enrichment analysis of the genes with differentially methylated regions (DMRs). We detected 1,138 DMRs between the two groups. Additionally, 122 DMRs were detected between the HAdV-7-induced sepsis survivor and non-survivor groups. After screening based on biological and clinical signicance, we found that a group of genes (KCNQ1OT1, KPNB1, GRB10, HOXA5, HOXA4, and BCL9L) with differential methylation played an essential role in Wnt signaling. Additionally, genes related to the Wnt/β-catenin signaling pathway, such as MEG3, GNAS-AS1, and GNAS, exhibited differential methylation in the survivor and non-survivor groups. Our data suggest that specic patterns of DNA methylation are associated with the occurrence and progression of HAdV-7-induced sepsis. Wnt signaling was also affected by the changes in methylation. Thus, we identied potential biomarkers and therapeutic targets for pediatric HAdV-7-induced sepsis.


Introduction
Human adenovirus (HAdV), a non-enveloped virus with a linear double-stranded DNA genome, belongs to the genus Mastadenovirus and family Adenoviridae [1]. HAdV infections account for at least 5-10% of all respiratory infections in children [2]. Human adenovirus 7 (HAdV-7) has been implicated in epidemics of severe lower respiratory tract infections, and it is among the viruses that are the most strongly associated with respiratory disease outbreaks in children aged less than 5 years worldwide [2]. HAdV-7 infection is usually mild and self-limiting in most immunocompetent patients [3]. However, the incidence of HAdV-7related severe or life-threatening infection in children is increasing. Further, severe adenoviral infections in children can be complicated by acute respiratory distress syndrome (ARDS), respiratory failure, and sepsis [2,4].
Although the role of HAdV-7-induced sepsis in lung injury remains poorly understood, it is considered to be related to immune system activation in the host [5]. In particular, the innate immune response is considered to contribute signi cantly to lung tissue damage caused by HAdV-7-induced sepsis [5]. Although the innate immune response induced by HAdVs is well documented, the mechanism underlying sepsis and in ammatory injury following HAdV-7 infection remains poorly understood.
Epigenetic changes, including DNA methylation, histone modi cation, and RNA regulation, signi cantly affect gene expression during disease development.
DNA methylation is one of the most stable epigenetic changes, and its relevance for prognosis and early diagnosis has been demonstrated in multiple diseases [6][7][8]. In epigenetic studies, blood is a readily accessible tissue source that frequently exhibits changes in response to diseases and environmental exposure to infectious agents [9]. Although the absolute magnitude of DNA methylation at speci c loci differs in different tissues, disease-related interindividual changes in DNA methylation are maintained across tissues within an individual [10]. There is ample evidence linking DNA methylation to immune activation and immune tolerance in humans [11,12]. However, to date, only a limited number of studies have explored DNA methylation in children with HAdV-7-induced sepsis.
In this study, we aimed to identify characteristic DNA methylation patterns and related signaling pathways in children with HAdV-7-induced sepsis using whole blood samples. The results of this study provide insights into the pathogenesis and progression of HAdV-7-induced sepsis and may help identify more effective therapeutic targets.

Research objective
This was a retrospective cohort study conducted at the pediatric intensive care unit (PICU) of Guangzhou Women and Children's Medical Center from April 2018 to April 2020. The inclusion criteria for children with HAdV-7-induced sepsis were: (1) age less than 18 years and greater than 30 days; (2) con rmed diagnosis of severe pneumonia; (3) con rmed diagnosis of HAdV-7 infection, based on polymerase chain reaction (PCR) using a throat swab or in-depth sputum sample; (4) a Sequential Organ Failure Assessment score ≥ 5 points, or a Murray Lung Injury Score > 2 points. Eleven children with HAdV-7-induced sepsis were included. During the same period, ve healthy children who had undergone physical examination in the same hospital were included as controls.
Whole blood samples were collected from the patients at admission to the PICU, while those from healthy children were collected after physical examination.
All blood samples were stored at − 80°C. The study was approved by the ethics committee of Guangzhou Women and Children's Medical Center (Approval no.: 57301). Informed consent was obtained from the parents of the children before study initiation.

Data collection
Data on gender, age, clinical diagnosis, and treatment process were collected for each patient with HAdV-7-induced sepsis. However, gene and age data were not collected from the controls.

Construction of an RRBS library
Genomic DNA was rst isolated from the peripheral blood samples using the Qiagen Blood Mini Kit (Qiagen, Hilden, Germany), and then subjected to agarose gel electrophoresis. The details of RRBS library construction have been reported previously [13]. First, 1 µg of DNA was digested using 100 U of MspI restriction enzyme (New England BioLabs, Ipswich, MA, USA) at 37°C for 16 h. Methylated adapter ligation was performed following blunt ending and dATP addition. Adapter-ligated DNA fragments of 160-350 bp, excised from the 2% agarose gels, were used for the ampli cation of MspI-digested DNA by PCR. Bisul te modi cation of the extracted DNA was performed using the Zymo EZ DNA Methylation-Gold KitTM (ZYMO Research, Irvine, CA, USA) according to the manufacturer's instructions. After bisul te treatment, a methylated cytosine was retained as "C", whereas a non-methylated cytosine was replaced with "U." During PCR ampli cation, the "U" bases were replaced with "T" bases. PCR ampli cation for the generation of the nal libraries was performed using the JumpStart™ Taq DNA Polymerase (Sigma-Aldrich, USA). The cycling conditions were as follows: 13 cycles at 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s. After the analysis using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA) and quanti cation using qPCR, the RRBS libraries were sequenced using the Illumina X Ten platform with 150 bp of paired-end reads.

Data analysis
After the raw sequencing data were processed using the Illumina base-calling pipeline, reads containing more than 30% "N"s or with low-quality values (< 20) in over 10% of the sequence were eliminated. Concurrently, adapter contamination was eliminated using Cutadapt (version 1.9) [14]. The corresponding annotation les were acquired from the UCSC Genome Database, and the clean reads were aligned to the reference genome using BSMAP (version 2.73) [15].
Only the uniquely aligned sequences containing the MspI restriction enzyme digestion site at the end of the read sequences were considered for further analysis. The commonly covered CpG sites with sequencing depths ≥ 5× were selected in each group as candidate sites to conduct global principal component analysis. After bisul te treatment, the cytosines were read as "T" if unmethylated, or as "C" if methylated, and CpG site methylation was identi ed.
The methylation level of cytosines was de ned as the ratio of "C" counts to the total "C" and "T" counts in the sequenced reads. Metilene (version 0.2-7) [16] was used to identify the DMRs between the two groups based on the following criteria: distance between two neighboring candidate CpG sites ≤ 300 bp, ≥ 5 CpG sites, methylation level difference > 0.1, and Q-value < 0.05 obtained using the Benjamini-Hochberg method. Gene promoters were de ned as the regions 2-kb upstream and 0.5-kb downstream of the transcriptional start site. Differentially methylated genes were identi ed based on the presence of one or more DMRs with > 50% sequence overlap with the promoter or gene body. The gene set pathway annotation was retrieved from the gene ontology (GO) database (http://geneontology.org/page/go-database) [17]. Functional enrichment analysis was performed using the hypergeometric test. Additionally, the Benjamini-Hochberg method was used to control the false discovery rate (FDR) with a threshold of 0.05.

DMR selection
The DMRs located in the promoter regions were sorted based on low to high FDR. The 20 DMRs with the lowest FDR were selected for the analysis. Table 1 summarizes the clinical characteristics of the 11 pediatric patients with HAdV-7-induced sepsis, which included ve deceased patients. The mean age was 22.3 ± 25.9 months, and the mean weight was 10.8 ± 2.9 kg. Females accounted for 36.4% of the patient population. All patients were on ventilator support and received vasoactive drugs, 63.6% received extracorporeal membrane oxygenation, and 54.5% received high-frequency ventilation. The mean duration of hospitalization was 27 ± 14 days, and the duration of PICU stay was 21 ± 15 days. The median length of stay in the hospital before admission to the PICU was 7 days. The mean PaO2/FiO2 (P/F) index was 74.2 ± 30.9. In patients with HAdV-7-induced sepsis, 45.5% mortality was recorded. The reduction of the P/F index (< 150) indicated that all patients suffered from ARDS and sepsis.

Regions and genes with differential methylation
The DNA methylation level in blood was compared between patients with HAdV-7-induced sepsis and healthy controls as well as between surviving and nonsurviving patients with HAdV-7-induced sepsis ( Table 2). A total of 1,138 DMRs were identi ed following FDR correction for multiple testing. Among these, 241 DMRs showed hypomethylation, and 897 showed hypermethylation between the HAdV7-induced sepsis group and the control group. Moreover, 122 DMRs were identi ed between the HAdV7-induced sepsis survivor and non-survivor groups, with 89 DMRs being hypomethylated and 33 being hypermethylated.

Chromosomal distribution in regions with differential methylation
The chromosomal distribution of DMRs was further analyzed. The DMRs that distinguished patients with HAdV-induced sepsis from healthy controls were primarily located on chromosomes 19, 1, 17, and 2. The DMRs between surviving and non-surviving patients with sepsis were primarily located on chromosomes 19, 20, 17, and 9 (Table 4).

GO enrichment analysis
The comparison between controls and patients with HAdV-7-induced sepsis revealed that genes associated with the α-β T cell receptor complex, apoptosis, and nitrogen compound metabolism contained regions of hypermethylation (Fig. 2a), whereas those associated with serotonin secretion from platelets, signal transduction, and creatine kinase activity contained regions of hypomethylation (Fig. 2b). The comparison between the survivor and non-survivor groups showed that genes associated with autophagy and endosomal transport contained hypermethylated regions (Fig. 2c), whereas those associated with the cell cycle, p53 binding, p53-mediated signal transduction, and nucleosomes contained hypomethylated regions (Fig. 2d).

Key site screening
The DMRs in the promoters between the HAdV-7-induced sepsis and control groups were sorted based on low to high FDR, and the 20 DMRs with lowest FDR were selected for analysis. Among the 44 genes corresponding to the selected DMRs, 6 (13.6%) (CD3D, IRGM, CD3/CD3G, KPNB1, HELZ2, and TNFRSF6B) were found to encode sepsis-related proteins. The GenBank database (https://www.ncbi.nlm.nih.gov/) was referred to for gene function information The expression of the selected DMRs correlated with that of KCNQ1OT1, KPNB1, GRB10, HOXA5, HOXA4, and BCL9L.
The DMRs in the promoters between the HAdV-7-induced sepsis survivor and non-survivor groups were also sorted based on low to high FDR, and 20 DMRs with the lowest FDR were selected for analysis. The expression of the selected DMRs correlated with that of MEG3, KCNQ1OT1, HOXA4, GNAS-AS1, and GNAS.

Discussion
To our knowledge, this study is the rst to investigate epigenome-wide DNA methylation in children with HAdV-7-induced sepsis and healthy control children.
The DNA methylation status in surviving and non-surviving patients was also compared. Our analysis of 11 children with HAdV-7-induced sepsis and 5 healthy controls led to the identi cation of 1,138 DMRs related to HAdV-7-sepsis occurrence and 122 DMRs related to sepsis progression. The functional enrichment analysis of 239 genes with DMRs located in the promoter region indicated the involvement of these genes in apoptosis, T cell activation, serotonin secretion from platelets, and autophagy. Since abnormalities in these functions have been reported in sepsis [18][19][20][21], our data are reliable.
In this study, 20 DMRs located in gene promoters and selected based on the FDR value could be used to distinguish pediatric patients with HAdV-7-induced sepsis from healthy controls and surviving patients from non-surviving patients. Of the affected genes, multiple genes, including KCNQ1OT1, KPNB1, GRB10, HOXA5, HOXA4, BCL9L, MEG3, GNAS-AS1, and GNAS, were associated with the Wnt/β-catenin signaling pathway. The Wnt/β-catenin signaling pathway plays a vital role in cell migration, genetic stability, and apoptosis, and it has also been linked to developmental diseases and cancer. As Wnt/β-catenin signaling affects many other signaling cascades, its dysfunction may lead to deleterious effects. A previous single-center study on three neonates with sepsis and three healthy neonates [22] and an analysis on 134 patients [23] (68 patients of sepsis and 66 controls) reported that genes associated with the Wnt/β-catenin signaling pathway undergo methylation changes in response to sepsis. However, these studies reported different sepsis-inducing pathogens. Our ndings suggest that the Wnt/β-catenin signaling pathway plays an essential role in the pathogenesis and progression of HAdV-7-induced sepsis.
The relationship between viral infection and the Wnt/β-catenin signaling pathway has been described previously. Currently, increasing evidence has shown the importance of this pathway in the replication, latency, and pathogenicity of multiple viruses, including human immunode ciency virus [24], hepatitis C virus [25], and cytomegalovirus [26]. However, to date, only limited information has been published on the effect of HAdV-7 infection on Wnt/β-catenin signaling. Our study is the rst to present evidence on this association. We found that the levels of methylation in KCNQ1OT1, KPNB1, and GRB10 were higher in children. In contrast, the levels of methylation in HOXA5, HOXA4, and BCL9L were lower in patients than in controls.
The multiple differentially methylated genes identi ed in our study share established functional relationships with the Wnt/β-catenin pathway. The long noncoding RNA of KCNQ1OT1 directly interacts with β-catenin, inhibits its degradation, and upregulates Wnt/β-catenin signaling [27]. KPNB1 encodes a transport factor in nuclear membrane that might assist β-catenin transport across the nuclear membrane via Wnt signaling [28]. GRB10 is a multi-module adapter protein known to interact with various transmembrane tyrosine kinase receptors and with the low-density lipoprotein receptor, a component of the Wnt cell surface receptor. Wnt-induced phosphorylation of low-density lipoprotein receptor-related protein 6 might in uence the degradation of β-catenin, which is a key regulatory step in the Wnt pathway [29]. HOXA5 is a critical regulator of stem cell differentiation; it contributes the development of the respiratory system and inhibits Wnt/β-catenin signaling [30]. HOXA4 is a transcription factor that inhibits Wnt signaling by suppressing the protein expression of β-catenin, cyclin D1, c-Myc, and surviving [31]. BCL9L is a component of the β-catenin degradation complex, and the activity of this complex is inhibited by the activation of Wnt signaling [32]. We found that the methylation level of GNAS-AS1 was higher in surviving than in non-surviving patients with HAdV-7-induced sepsis. Conversely, the methylation levels of MEG3, KCNQ1OT1, and HOXA4 were lower in survivors than in non-survivors. Previous studies have shown that MEG3 interacts with miR4261 to relieve the inhibition of Dickkopf-2 and block Wnt/β-catenin signal transduction [33]. GNAS-AS1 regulates Wnt/β-catenin signaling by mediating β-catenin expression [34]. GNAS is a critical component of the cell membrane receptor pathway and can activate Wnt/β-catenin signaling [35]. However, in this study, GNAS was found to contain both hypermethylated and hypomethylated regions. While further studies are required to con rm the expression level of GNAS in response to sepsis, adequate literature on this topic remains unavailable. Our ndings suggest that the regulation of Wnt/βcatenin signaling may play a role in the occurrence and development of sepsis caused by HAdV-7 in children.

Conclusions
In this study, we used RRBS to evaluate the abnormal DNA methylation patterns in whole blood samples from children with HAdV-7-induced sepsis. Our ndings indicate that the changes in the DNA methylation patterns in peripheral blood cells are related to the pathogenesis and progression of HAdV-7induced sepsis. In particular, genes associated with Wnt/β-catenin signaling and the epigenetic changes in these genes may play a role in this disease.
Our study has some limitations. First, we used whole blood samples, with the proportion of immune cells differing between samples. Therefore, our analysis may be more informative with respect to speci c immune cell alterations. Second, the relationship between DNA methylation and gene expression levels was not explored. Third, the sample size was small, and the results should be con rmed in studies with a larger sample size as well as in studies with animal models and gene knockout experiments. Nevertheless, our ndings provide evidence that the analysis of DNA methylation is a suitable method for evaluating the pathogenesis of pediatric HAdV-7-induced sepsis and may help develop new prognostic and therapeutic tools.

Declarations
Funding This study was supported by the Internal Fund of the Internal Medicine Department of Guangzhou Women and Children's Medical Center (NKE-PRE-2019-016).

Con icts of interest
None.
Availability of data and material The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.