Major methylation alterations on the CpG markers of inflammatory immune associated genes after IVIG treatment in Kawasaki disease
© The Author(s). 2016
Published: 12 August 2016
Kawasaki disease (KD) is an autoimmune disease preferentially attacking children younger than five years worldwide. So far, the principal treatment to KD is the administration of Intravenous immunoglobulin (IVIG). Although DNA methylation plays important regulation roles in diseases, few studies investigated the regulation roles of DNA methylation in KD.
In this study, we focused not only on the DNA methylation alterations resulted from KD onset but also on DNA methylation alterations resulted from IVIG administration. To do so, we investigated the effects of KD’s onset and IVIG administration on CpG marker’s methylation alterations.
We first found that DNA methylation alterations reflecting disease onset or IVIG administration are contributed mainly by the CpG markers on autosomes. In addition, we also demonstrated that some CpG markers carry methylation alteration among samples, forcing the expression abundance of the downstream genes to be also altered and negatively correlated with methylation profile. Finally, compared with KD’s onset, IVIG administration brings stronger impact on methylation alteration. And, such alterations were conducted mainly by hyper-methylating CpG markers, forcing the corresponding genes to keep lower expression levels. Moreover, the genes regulated by the altered CpG markers with IVIG administration are enriched in the pathways associated with inflammatory immune response.
In summary, our result provides researchers with another way into the regulation mechanism through which IVIG represses excessive inflammatory responses.
Kawasaki disease (KD), also called mucocutaneous lymph node syndrome, is a childhood systemic vasculitis. It was first described by Dr. Tomisaku Kawasaki in 1967 . KD mainly affects small to mediums sized vessels, with the development of coronary artery aneurysms being the most severe complication of KD. If left untreated, coronary artery lesions may develop in up to 25 % of patients with KD, which increases the risk of coronary artery aneurysm formation, coronary artery thrombosis, myocardial infarction or even sudden death [2, 3]. Current treatment guidelines for KD include intravenous immunoglobulin (IVIG) therapy during the acute phase. IVIG is given within the first ten days of fever and it may reduce the risk of coronary artery formation to 2–4 % . In addition to treating KD, IVIG is also applied in treating patients with primary immune deficiency or acute infection by maintaining adequate level of IgG in circulation system and enhancing immune ability. However, in KD patients, IVIG represses excessive inflammation rather than enhances immune ability. Actually, there is still a debate on the precise mechanism by which IVIG represses immune response and that’s why we are curious about the methylation alteration after IVIG administration.
The exact pathogenesis of KD is still a matter of debate, although existing research points to both environmental and genetic factors. KD appears to be associated with ethnicity, and is more prevalent in Asian children . Retrospective studies have also found that children whose parents or siblings were attacked by KD are at a higher risk of developing the disease [6, 7]. Genetic polymorphisms have also been identified in patients with KD, most of which have been associated with B cell and T cell immunity [8–10]. Apart from genetic factors, KD tends to occur in seasonal clusters suggesting an infectious or transmissible triggering agent .
DNA methylation plays an important role in regulation of gene expression through establishing and maintaining the DNA methylation status in gene promoters . In mammals, within promoter regions, when the methyl group is chemically bounded to the cytosine of the CpG di-nucleotide (also called CpG marker), the expression abundance of the downstream gene is usually decreased. Therefore, by hypo- or hyper-methylating the CpG markers at the promoter regions, DNA methylation performs regulation abilities in diseases, especially when the downstream genes are functionally related to diseases [13–15].
In our previous study, we investigated the regulation roles of DNA methylation in KD by comparing the CpG markers’ methylation profiles between control and KD patients [16, 17]. We concluded that the CpG markers in FCGR2A promoter are hypo-methylated in KD patients, increasing the expression abundance of FCGR2A. In this study, we focused not only on the DNA methylation alterations resulted from KD onset but also on DNA methylation alterations resulted from IVIG administration. To do so, we collected DNA sample from control subjects, KD patients before IVIG treatment and KD patients after IVIG treatment. By bisulfite conversion of DNA samples and microarray detection of methylation profile, we investigated the effects of KD’s onset and IVIG administration on CpG marker’s methylation alterations.
Collection of subjects and clinical DNA samples
In Kaohsiung Chang Gung memorial Hospital, Taiwan, we enrolled 4 febrile control subjects (FC), 7 KD patients at acute phase (KD1) and 7 KD patients three weeks after IVIG treatment (KD3). Here, FC denotes febrile control, including the patients with fever but not diagnosed as KD by a physician. We collected whole blood samples from the subjects based on the IRB approvals by the Chang Gung Memorial Hospital (No.101-0680A3). Each KD3 subject was treated with IVIG (2 gm/kg) over a 12-hour period. Blood cells were collected and subjected to DNA extraction followed by bisulfite conversion of genomic DNA samples.
Profiling of CpG methylation
We used Illumina HumanMethylation27 BeadChip to perform the genome-wide screening of DNA methylation patterns. HumanMethylation27 BeadChip was designed to detect methylation patterns of 27,578 CpG markers, spanning human genome. Detailed information of HumanMethylation27 BeadChip is available via the following URL: http://support.illumina.com/array/array_kits/infinium_humanmethylation27_beadchip_kit.html. For each HumanMethylation27 BeadChip assay, 200 ng of bisulfite-converted genomic DNA was applied according to the manufacturer’s instructions. For each CpG markers, HumanMethylation27 BeadChip designs two probes to distinguish cytosine (originally methylated) from thymine (originally un-methylated so that converted into uracil by bisulfite treatment and converted into thymine by reverse PCR reaction). By detecting the relative intensities of the probes for cytosine and thymine, methylation percentages (named β values) are determinable.
Results and discussion
Summary of significantly altered CpG markers on chromosomes
Major alterations in DNA methylation patterns caused by IVIG treatment
Results of pathway enrichment analysis on the CpG marker related genes
The results of pathway enrichment analysis. We did pathway enrichment analysis with Partek on the genes located by the CpG markers significantly altered in KD3 vs FC or KD3 vs KD1 comparisons. Among the significant pathways (p < 0.05), four are co-enriched in the two comparisons
p-value: KD3 vs FC
p-value: KD3 vs KD1
Hematopoietic cell lineage
Cytokine-cytokine receptor interaction
Chemokine signaling pathway
Jak-STAT signaling pathway
Table 1 demonstrates that the significant pathways shared by the two comparisons are Hematopoietic cell lineage, Cytokine-cytokine receptor interaction, Chemokine signaling pathway and Jak-STAT signaling pathway. Through the hematopoietic cell lineage pathway, hematopoietic stem cell differentiates into different blood cells, including T cell, NK cell, basophil, macrophage, B cell and so on, responding to diverse stimulus or necessity . Therefore, hematopoietic cell lineage plays important roles in immune response. Cytokine are soluble proteins, secreted by donor cells responding to stimulus and transported by circulation system to target cells. Through the cytokine-cytokine receptor interaction pathway, cytokines bind to their receptors on the target cell membrane . Next, through the Jak-STAT signaling pathway, the external signals initiated by cytokines can be passed down into the target cells, triggering immune activities and activating inflammation responses . Similar to cytokine, chemokine also plays positive roles in up-regulating inflammatory immune responses . Therefore, the pathways altered by IVIG administration are immune-related. They play either up-stream or down-stream roles in regulating inflammatory immune response.
Actually, there is still a debate on the precise mechanism by which IVIG represses immune response, although several possible mechanisms have been proposed [24–28]. Our results of pathway enrichment analysis shows that IVIG administration performs the ability of repressing harmful inflammation through hypo- or hyper-methylating the CpG markers whose down-stream genes are involved inflammatory immune response. Such result provides the researchers with another way into the regulation mechanism through which IVIG represses excessive inflammatory responses. Although our result provides researchers with another way into the regulation mechanism through which IVIG represses excessive inflammatory responses, we can not exclude the involvement of aspirin. Salicylic acid is the major compound of aspirin and is usually administrated with IVIG for alleviation of excessive inflammatory immune response. Therefore, the effect of salicylic acid on methylation alteration needs more attentions.
Methylation alteration between samples
Altered change of gene expression between samples
It is a consensus that hypermethylation at gene promoter decreases the expressions of the downstream gene. However, not every hypermethylation brings down-regulation of genes. Such cases can also be observed in our data. Actually, gene expression undergoes several regulation mechanisms, including acetylation of chromatin , methylation at CpG marker, the binding of transcription factor on promoter , miRNA regulation  and so on. Therefore, gene expression abundance is an overall result of all regulation mechanisms. So far, it is difficult to monitor all regulation mechanisms simultaneously so that only methylation at CpG marker is considered in this study.
We first found that DNA methylation alterations reflecting disease onset or IVIG administration are contributed mainly by autosomes; sex chromosomes only contribute little. In addition, some markers carry methylation alteration between samples. As a result, their downstream genes are also altered and are negatively correlated with methylation profile. Finally, IVIG administration brings stronger impact on methylation alteration than KD onsets does. And, such alterations were conducted mainly by hyper-methylating CpG markers. Moreover, the genes whose promoter CpG markers are altered with IVIG administration are enriched in the pathways associated with inflammatory immune response.
We thank the Genomics & Proteomics Core Laboratory, Department of Medical Research, Kaohsiung Chang Gung Memorial Hospital for technical supports.
Publication of this article has been funded by the grants from the Ministry of Science and Technology, Taiwan (MOST 102-2314-B-182-053-MY3) and Chang Gung Memorial Hospital (CMRPG8E0021, CMRPG8E0031, CMRPF6E0041, CMRPG8E0051, CMRPG8E0061, CLRPG8D0112 and CMRPG8D0032). This article has been published as part of BMC Medical Genomics Volume 9 Supplement 1, 2016. Selected articles from the 5th Translational Bioinformatics Conference (TBC 2015): medical genomics. The full contents of the supplement are available online https://bmcmedgenomics.biomedcentral.com/articles/supplements/volume-9-supplement-1.
Availability of data and materials
The raw data is available via https://mega.nz/#!Zog3XCBY!-MnOyi5d5fFG3PlFqkkF3nYIvfPqC-hWjIlxwMR9tO0.
SCL and WCC conducted data analysis. MMG, YHH, HCK, FCH, HRY were responsible for clinical sample collection and experiments. HCK supervised this work. SCL, YHH and WCC wrote the manuscript. All authors read and approved the final manuscript.
The authors declared that they have no competing interests.
Consent for publication
The subjects contributing clinical samples to this study (or their guardians) fully understand the purposes of this study and signed on the informed consent voluntarily.
Ethics approval and consent to participate
This study design was reviewed and approved by the IRB of the Chang Gung Memorial Hospital (No.101-0680A3).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Burns JC, Glode MP. Kawasaki syndrome. Lancet. 2004;364(9433):533–44.View ArticlePubMedGoogle Scholar
- Burns JC, Shike H, Gordon JB, Malhotra A, Schoenwetter M, Kawasaki T. Sequelae of Kawasaki disease in adolescents and young adults. J Am Coll Cardiol. 1996;28(1):253–7.View ArticlePubMedGoogle Scholar
- Ko TM, Kuo HC, Chang JS, Chen SP, Liu YM, Chen HW, Tsai FJ, Lee YC, Chen CH, Wu JY, et al. CXCL10/IP-10 is a biomarker and mediator for Kawasaki disease. Circ Res. 2015;116(5):876–83.View ArticlePubMedGoogle Scholar
- Oates-Whitehead RM, Baumer JH, Haines L, Love S, Maconochie IK, Gupta A, Roman K, Dua JS, Flynn I. Intravenous immunoglobulin for the treatment of Kawasaki disease in children. Cochrane Database Syst Rev. 2003;4:CD004000.PubMedGoogle Scholar
- Holman RC, Belay ED, Christensen KY, Folkema AM, Steiner CA, Schonberger LB. Hospitalizations for Kawasaki syndrome among children in the United States, 1997-2007. Pediatr Infect Dis J. 2010;29(6):483–8.PubMedGoogle Scholar
- Uehara R, Yashiro M, Nakamura Y, Yanagawa H. Clinical features of patients with Kawasaki disease whose parents had the same disease. Arch Pediatr Adolesc Med. 2004;158(12):1166–9.View ArticlePubMedGoogle Scholar
- Uehara R, Yashiro M, Nakamura Y, Yanagawa H. Kawasaki disease in parents and children. Acta Paediatr. 2003;92(6):694–7.View ArticlePubMedGoogle Scholar
- Lee YC, Kuo HC, Chang JS, Chang LY, Huang LM, Chen MR, Liang CD, Chi H, Huang FY, Lee ML, et al. Two new susceptibility loci for Kawasaki disease identified through genome-wide association analysis. Nat Genet. 2012;44(5):522–5.View ArticlePubMedGoogle Scholar
- Kuo HC, Onouchi Y, Hsu YW, Chen WC, Huang JD, Huang YH, Yang YL, Chao MC, Yu HR, Juan YS, et al. Polymorphisms of transforming growth factor-beta signaling pathway and Kawasaki disease in the Taiwanese population. J Hum Genet. 2011;56(12):840–5.View ArticlePubMedGoogle Scholar
- Onouchi Y, Gunji T, Burns JC, Shimizu C, Newburger JW, Yashiro M, Nakamura Y, Yanagawa H, Wakui K, Fukushima Y, et al. ITPKC functional polymorphism associated with Kawasaki disease susceptibility and formation of coronary artery aneurysms. Nat Genet. 2008;40(1):35–42.View ArticlePubMedGoogle Scholar
- Huang WC, Huang LM, Chang IS, Chang LY, Chiang BL, Chen PJ, Wu MH, Lue HC, Lee CY. Epidemiologic features of Kawasaki disease in Taiwan, 2003-2006. Pediatrics. 2009;123(3):e401–5.View ArticlePubMedGoogle Scholar
- Zhang Z, Zhang R. Epigenetics in autoimmune diseases: Pathogenesis and prospects for therapy. Autoimmun Rev. 2015;14(10):854–63.Google Scholar
- Tsai KW, Hu LY, Wu CW, Li SC, Lai CH, Kao HW, Fang WL, Lin WC. Epigenetic regulation of miR-196b expression in gastric cancer. Genes Chromosomes Cancer. 2010;49(11):969–80.View ArticlePubMedGoogle Scholar
- Tsai KW, Liao YL, Wu CW, Hu LY, Li SC, Chan WC, Ho MR, Lai CH, Kao HW, Fang WL, et al. Aberrant hypermethylation of miR-9 genes in gastric cancer. Epigenetics. 2011;6(10):1189–97.View ArticlePubMedPubMed CentralGoogle Scholar
- Coupland KG, Kim WS, Halliday GM, Hallupp M, Dobson-Stone C, Kwok J. Effect of PSEN1 mutations on MAPT methylation in early-onset Alzheimer’s disease. Curr Alzheimer Res. 2015;12(8):745–51.Google Scholar
- Kuo HC, Chang JC, Yu HR, Wang CL, Lee CP, Huang LT, Yang KD. Identification of an association between genomic hypomethylation of FCGR2A and susceptibility to Kawasaki disease and intravenous immunoglobulin resistance by DNA methylation array. Arthritis Rheumatol. 2015;67(3):828–36.View ArticlePubMedGoogle Scholar
- Kuo HC, Hsu YW, Wu MS, Woon PY, Wong HS, Tsai LJ, Lin RK, Klahan S, Hsieh KS, Chang WC. FCGR2A Promoter Methylation and Risks for Intravenous Immunoglobulin Treatment Responses in Kawasaki Disease. Mediators Inflamm. 2015;2015:564625.View ArticlePubMedPubMed CentralGoogle Scholar
- Orange JS, Hossny EM, Weiler CR, Ballow M, Berger M, Bonilla FA, Buckley R, Chinen J, El-Gamal Y, Mazer BD, et al. Use of intravenous immunoglobulin in human disease: a review of evidence by members of the Primary Immunodeficiency Committee of the American Academy of Allergy, Asthma and Immunology. J Allergy Clin Immunol. 2006;117(4 Suppl):S525–53.View ArticlePubMedGoogle Scholar
- Li SC, Wang FS, Yang YL, Tiao MM, Chuang JH, Huang YH. Microarray Study of Pathway Analysis Expression Profile Associated with MicroRNA-29a with Regard to Murine Cholestatic Liver Injuries. Int J Mol Sci. 2016, 17(3).Google Scholar
- Shapiro-Shelef M, Calame K. Regulation of plasma-cell development. Nat Rev Immunol. 2005;5(3):230–42.View ArticlePubMedGoogle Scholar
- Pestka S, Krause CD, Sarkar D, Walter MR, Shi Y, Fisher PB. Interleukin-10 and related cytokines and receptors. Annu Rev Immunol. 2004;22:929–79.View ArticlePubMedGoogle Scholar
- Kisseleva T, Bhattacharya S, Braunstein J, Schindler CW. Signaling through the JAK/STAT pathway, recent advances and future challenges. Gene. 2002;285(1-2):1–24.View ArticlePubMedGoogle Scholar
- Thelen M, Stein JV. How chemokines invite leukocytes to dance. Nat Immunol. 2008;9(9):953–9.View ArticlePubMedGoogle Scholar
- Viard I, Wehrli P, Bullani R, Schneider P, Holler N, Salomon D, Hunziker T, Saurat JH, Tschopp J, French LE. Inhibition of toxic epidermal necrolysis by blockade of CD95 with human intravenous immunoglobulin. Science. 1998;282(5388):490–3.View ArticlePubMedGoogle Scholar
- Maverakis E, Kim K, Shimoda M, Gershwin ME, Patel F, Wilken R, Raychaudhuri S, Ruhaak LR, Lebrilla CB. Glycans in the immune system and The Altered Glycan Theory of Autoimmunity: a critical review. J Autoimmun. 2015;57:1–13.View ArticlePubMedGoogle Scholar
- Garcia-Garcia E, Rosales C. Signal transduction during Fc receptor-mediated phagocytosis. J Leukoc Biol. 2002;72(6):1092–108.PubMedGoogle Scholar
- Nimmerjahn F, Ravetch JV. The antiinflammatory activity of IgG: the intravenous IgG paradox. J Exp Med. 2007;204(1):11–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Clynes R. Immune complexes as therapy for autoimmunity. J Clin Invest. 2005;115(1):25–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Khor CC, Davila S, Breunis WB, Lee YC, Shimizu C, Wright VJ, Yeung RS, Tan DE, Sim KS, Wang JJ, et al. Genome-wide association study identifies FCGR2A as a susceptibility locus for Kawasaki disease. Nat Genet. 2011;43(12):1241–6.View ArticlePubMedGoogle Scholar
- Onouchi Y, Ozaki K, Burns JC, Shimizu C, Terai M, Hamada H, Honda T, Suzuki H, Suenaga T, Takeuchi T, et al. A genome-wide association study identifies three new risk loci for Kawasaki disease. Nat Genet. 2012;44(5):517–21.View ArticlePubMedGoogle Scholar
- Wang Z, Young RL, Xue H, Wagner GP. Transcriptomic analysis of avian digits reveals conserved and derived digit identities in birds. Nature. 2011;477(7366):583–6.View ArticlePubMedGoogle Scholar
- Tsai KW, Chang B, Pan CT, Lin WC, Chen TW, Li SC. Evaluation and application of the strand-specific protocol for next-generation sequencing. Biomed Res Int. 2015;2015:182389.PubMedPubMed CentralGoogle Scholar
- Annalisa I, Robert S. The role of linker histone H1 modifications in the regulation of gene expression and chromatin dynamics. Biochim Biophys Acta. 2015;1859(3):486–95.Google Scholar
- Metzger CS, Koutsimpelas D, Brieger J. Transcriptional regulation of the VEGF gene in dependence of individual genomic variations. Cytokine. 2015;76(2):519–26.Google Scholar
- Pan CT, Tsai KW, Hung TM, Lin WC, Pan CY, Yu HR, Li SC. miRSeq: A User-Friendly Standalone Toolkit for Sequencing Quality Evaluation and miRNA Profiling. Biomed Res Int. 2014;2014:462135.PubMedPubMed CentralGoogle Scholar