Next-generation sequencing identifies micro-RNA–based biomarker panel for Kawasaki disease
Authors: Ho-Chang Kuo, Kai-Sheng Hsieh, Mindy Ming-Huey Guo, Ken-Pen Weng, Luo-Ping Ger, Wen-Ching Chan, Sung-Chou Li
Journal: Journal of Allergy and Clinical Immunology (2016)

Kawasaki disease (KD) is an autoimmune disease preferentially attacking children younger than 5 years. Symptoms of KD include fever for at least 5 days, oral mucosal inflammation, nonsupportive conjunctivitis, lymphadenopathy, edema of the extremities, and a polymorphous rash. If left untreated, almost 20% to 25% of the patients with KD may develop severe coronary artery aneurysms, making KD the leading cause of acquired heart disease in young children in developed countries.1Burns J.C. Glodé M.P. Kawasaki syndrome.Lancet. 2004; 364: 533-544Abstract Full Text Full Text PDF PubMed Scopus (777) Google Scholar The successful detection of KD within the first 10 days of fever onset followed by treatment with high-dose intravenous immunoglobulin can greatly reduce the risk of severe coronary artery lesions.1Burns J.C. Glodé M.P. Kawasaki syndrome.Lancet. 2004; 364: 533-544Abstract Full Text Full Text PDF PubMed Scopus (777) Google Scholar, 2Newburger J.W. Takahashi M. Burns J.C. Beiser A.S. Chung K.J. Duffy C.E. et al.The treatment of Kawasaki syndrome with intravenous gamma globulin.N Engl J Med. 1986; 315: 341-347Crossref PubMed Scopus (1168) Google Scholar, 3Newburger J.W. Takahashi M. Beiser A.S. Burns J.C. Bastian J. Chung K.J. et al.A single intravenous infusion of gamma globulin as compared with four infusions in the treatment of acute Kawasaki syndrome.N Engl J Med. 1991; 324: 1633-1639Crossref PubMed Scopus (1004) Google Scholar Therefore, early and successful detection is critical for treating KD. So far, few cytokine-based biomarker platforms have been developed.4Ko T.M. Kuo H.C. Chang J.S. Chen S.P. Liu Y.M. Chen H.W. et al.CXCL10/IP-10 is a biomarker and mediator for Kawasaki disease.Circulation Res. 2015; 116: 876-883Crossref PubMed Scopus (64) Google Scholar Therefore, the detection of KD still mainly relies on the judgment of clinical physicians based on patients' symptoms. However, infectious diseases by pathogens may also result in symptoms similar to those of KD, making it difficult to accurately diagnose KD and leading to a delay in optimal and timely treatment of KD. Previous studies have shown that blood miRNAs can be used as disease biomarkers.5Leidinger P. Backes C. Deutscher S. Schmitt K. Mueller S.C. Frese K. et al.A blood based 12-miRNA signature of Alzheimer disease patients.Genome Biol. 2013; 14: R78Crossref PubMed Scopus (355) Google Scholar, 6Rowley A.H. Backes C. Deutscher S. Schmitt K. Mueller S.C. Frese K. et al.A study of cardiovascular miRNA biomarkers for Kawasaki disease.Pediatr Infect Dis J. 2014; 33: 1296-1299Crossref PubMed Scopus (20) Google Scholar, 7Shimizu C. Kim J. Stepanowsky P. Trinh C. Lau H.D. Akers J.C. et al.Differential expression of miR-145 in children with Kawasaki disease.PLoS One. 2013; 8: e58159Crossref PubMed Scopus (54) Google Scholar So, we enrolled 70 fever control (FC, denoting patients without KD with fever) and 50 subjects with KD (see this article's Methods section in the Online Repository at www.jacionline.org). Among them, 37 FC and 31 KD subjects comprised the training set, used for developing the KD biomarker panel with next-generation sequencing (NGS) and/or quantitative PCR (qPCR) assays. The remaining 33 FC and 19 KD subjects comprised the blind test set, used for examining the performance of the developed KD biomarker panel. Table I shows that the most prevalent diagnosis of FC subjects was upper or lower respiratory tract infection (28 and 27 patients, respectively). In patients with KD, bilateral conjunctivitis, oral mucosa changes, and rash were the most prevalent clinical features.Table IDemographic data of subjects in this studyCharacteristicFC (N = 70)KD (N = 50)P valueSex: male, n (%)33 (47.1)25 (50).853Clinical featuresDiagnosis:Upper respiratory tract infection (28)Lower respiratory tract infection (27)Gastroenteritis (3)Urticaria (3)Septic arthritis (2)Dengue fever (1)Scarlet fever (1)Systemic lupus erythematosus (1)Henoch-Schönlein purpura (1)Atopic dermatitis (1)Acute hepatitis (1)Urinary tract infection (1)Bilateral conjunctivitis: 46 (92%)Oral mucosal change: 43 (86%)Lymphadenopathy: 23 (46%)Swelling/peeling of hands and feet: 36 (72%)Rash: 43 (86%)CALs: 15 (30%)IVIG resistance: 2 (4%)We collected the FC set subjects when the patients without KD had fever. In addition, all subjects, including FC and KD, did not suffer from other diseases except for the ones mentioned in this table. CALs were defined if any of the following findings were seen on 2-dimensional echocardiography: coronary artery internal diameter of greater than 3 mm in children younger than 5 years, or greater than 4 mm in children older than 5 years, or if the internal diameter of a segment was more than 1.5 times greater than that of an adjacent segment. Of the 50 patients with KD included in this study, 15 patients (30%) had positive CAL findings on 2-dimensional echocardiography and 2 patients (4%) were IVIG resistant.CAL, Coronary artery lesion; IVIG, intravenous immunoglobulin. Open table in a new tab We collected the FC set subjects when the patients without KD had fever. In addition, all subjects, including FC and KD, did not suffer from other diseases except for the ones mentioned in this table. CALs were defined if any of the following findings were seen on 2-dimensional echocardiography: coronary artery internal diameter of greater than 3 mm in children younger than 5 years, or greater than 4 mm in children older than 5 years, or if the internal diameter of a segment was more than 1.5 times greater than that of an adjacent segment. Of the 50 patients with KD included in this study, 15 patients (30%) had positive CAL findings on 2-dimensional echocardiography and 2 patients (4%) were IVIG resistant. CAL, Coronary artery lesion; IVIG, intravenous immunoglobulin. We extracted RNA samples from white blood cells (see this article's Methods section in the Online Repository). Then, we had 2 pooled FC and 2 pooled KD RNA libraries sequenced with Illumina NGS platform, followed by miRSeq8Pan C.T. Tsai K.W. Hung T.M. Lin W.C. Pan C.Y. Yu H.R. et al.miRSeq: a user-friendly standalone toolkit for sequencing quality evaluation and miRNA profiling.Biomed Res Int. 2014; 2014: 462135Crossref PubMed Scopus (16) Google Scholar analysis for determining micro (mi)-RNA expression profiles (see Table E1 in this article's Online Repository at www.jacionline.org). According to miRSeq result, almost 80% of the initial reads belonged to clean read, the sequence read with 3′ adaptor recognized and trimmed. In addition, most clean reads were 22-nt long (see Fig E1 in this article's Online Repository at www.jacionline.org), reflecting high performance of the sample preparation procedures and implying the dominance of miRNAs, which was proven by Fig E2 in this article's Online Repository at www.jacionline.org. To examine whether the FC and KD samples were distinguishable on the basis of miRNA expression profiles, we conducted clustering analysis. The heat map showed that the duplicated libraries had similar and consistent patterns (Fig 1, A). Moreover, there were more than 20 miRNAs able to distinguish FC from KD samples so that they can serve as biomarkers of KD. For further validation with qPCR in the training set, 37 FC and 31 KD subjects, we selected 10 miRNAs with NGS abundance larger than 1000 transcript per million in at least 1 library and with average fold change larger than 1.25 between FC and KD libraries. As a result, we observed 10 miRNAs whose expression preferences by qPCR were consistent with the ones by NGS (see Table E2 in this article's Online Repository at www.jacionline.org). Fig 1, B, demonstrates that 8 of the 10 examined miRNAs were significantly differentially expressed. Although the remaining 2 miRNAs were not statistically significant, the expression preferences can be observed. Because males have a higher KD incidence rate than do females, we further investigated whether the 10 miRNAs are significantly differentially expressed between male and female subjects, causing bias when served as a disease biomarker. Although the expression of hsa-miR-941 is significant between males and females (P = .0282), the difference between control and disease is much more significant than that between sex (P = 6.7319 × 10−07 vs P = .0282). The expressions of the remaining 9 biomarker miRNAs were not sex specific. Therefore, we used the ΔCt values of the 10 miRNAs to develop a KD biomarker panel, training a Support Vector Machine (SVM) classification model, and diagnosing KD. SVM is a machine learning algorithm and is good in dealing with binary classification problems, treatment versus control or disease versus normal.9Kvon E.Z. Kazmar T. Stampfel G. Yáñez-Cuna J.O. Pagani M. Schernhuber K. et al.Genome-scale functional characterization of Drosophila developmental enhancers in vivo.Nature. 2014; 512: 91-95Crossref PubMed Scopus (268) Google Scholar We used the ΔCt values of the 10 panel miRNAs (Table E2) to train the SVM classification model with 5-fold cross-validation specified to eliminate overfitting and underfitting. As shown in Fig 2, A, the final SVM classification model has a sensitivity of 83.3% and a specificity of 92.5%, leading to an overall accuracy of 87.9% and a 0.9 area under receiver operating characteristic (auROC) value. An independent cohort, composed of 33 FC and 19 KD subjects, was applied to conduct a blind test. We determined the miRNA ΔCt values of the 52 blind test subjects (see Table E3 in this article's Online Repository at www.jacionline.org). After SVM model alignment, 16 of the 19 KD samples were diagnosed as KD and 27 of the 33 FC samples were diagnosed as not KD, demonstrating a sensitivity of 84.20% and a specificity of 81.8%. The SVM performance examined with the blind test set was similar to the one examined with the training set, reflecting the robustness and reliability of the SVM classification model. In addition, it reflected that the overfitting and underfitting problems were slight in our SVM classification model. To facilitate the early detection of KD, we built the Web-based user-friendly interface, Kawasaki disease Detection Platform (KDP, Fig 2, B), accessible via http://formosa3.nchc.org.tw:50190/KDP/index.php. To detect KD, the physicians or researchers may submit the ΔCt values of the 10 panel miRNAs onto the KDP. KDP calls the SVM alignment and then reports the detection result in both graphical (Fig 2, B) and text formats (see Fig E3 in this article's Online Repository at www.jacionline.org). In summary, our biomarker panel provides a useful tool for researchers and physicians, facilitating early detection of KD. We thank the Genomics & Proteomics Core Laboratory, Department of Medical Research, Kaohsiung Chang Gung Memorial Hospital for technical support. We thank Mr Chao-Yu Pan for helping construct the KDP interface. Download .doc (.37 MB) Help with doc files Online Repository text