The genomic human transcriptome methylation data and mRNA expression data were measured from the whole blood samples followed by white blood cell enrichment. The blood samples used for genome-wide methylation level probing were collected from 24 pre-IVIG KD patients (before IVIG treatment), 12 post-IVIG KD patients (at least 3 weeks after IVIG treatment), 12 healthy children, and 12 fever control subjects. And the blood samples used for expression level analysis were gathered from 50 pre-IVIG KD patients (before IVIG treatment), 18 post-IVIG KD patients (at least 3 weeks after IVIG treatment), 18 healthy children, and 18 fever control subjects. Post-intravenous immunoglobulin (IVIG) treatment KD samples were gathered from the same KD patients that were given a high-dose IVIG treatment (2 g/kg) over 12 hours. The diagnosis of KD patients followed the American Heart Association KD criteria. The patient sample collection was approved by the Institutional Review Board of Chang Gung Memorial Hospital (IRB No.:101-4618A3), and the written informed consents from the parents/guardians of the patients were also ensured. Overall the samples can be categorized into four different groups: the normal controls, the fever controls, the KD samples before IVIG treatment, and the KD samples after IVIG treatment. The samples gathered for genome-wide methylation profiling were treated with bisulfite to convert non-methylated cytosine into uracil for methylated DNA extraction. These DNA extracts underwent genomic DNA methylation probing by Illumina HumanMethylation450 (M450K) BeadChip, which contains around 450K human CpG marker probe beads. For the samples collected for expression level measurement, we used the isolation kit (mirVana miRNA Isolation Kit, Catalog no. AM1560, Life Technologies, Carlsbad, CA) to extract the RNAs from the samples. The quality and quantity of the total RNAs were estimated by Bioanalyzer (ABI) and Qubit (Thermo) to ensured the RIN values were larger than 7. And around six RNA samples of the same group were evenly pooled to obtain unbiased expression measurement, resulting 3 healthy pools (N = 6, 6, 6), 3 fever pools (N = 6, 6, 6), 9 pre-IVIG pools (N = 6, 6, 6, 6, 6, 6, 6, 5, 3), and 3 post-IVIG pools (N = 6, 6, 6). These sample pools were hybridized on the GeneChip Human Transcriptome Array 2.0 (HTA 2.0, Affymetrix, Santa Clara). The microarray experiment quality control was subjected to criteria proposed in previous studies. The DNA methylation data and microarray data were deposited in NCBI GEO (GSE109430, GSE109351).

Genomic methylation profiling for the four sample groups was measured by M450K BeadChip, as described above. The methylation level of one CpG probe is computed using the β value (β = M / (M+ U + ɑ), where M is the methylated cytosine intensity, U is the unmethylated cytosine probing intensity, and ɑ is the calibration number). The raw data is processed using Partek. Gene expression profiling for pooled sample groups was measured by the Affymetrix HTA 2.0 GeneChip. The mRNA expression level of different human genes is also analyzed and calculated by the commercial microarray data analysis tool Partek.

We compared the differential methylation/expression between the four sample groups. β values and expression measurements are used to calculate the differential significance between any two sample groups for differential methylation profiling probing and differential expression comparison, respectively. All combinations of any of the two groups from the four experimental samples (healthy subject group, fever patient group, pre-IVIG KD patient group, and the post-IVIG KD patient group) were considered in the data comparison analysis. In KDmarkers, three statistical test results are available for users: the independent t test, the rank-sum test, and the Kolmogorov-Smirnov test. We calculated the p-values against the null hypothesis that the average/median/distribution of Group 1 is smaller/larger than the corresponding statistics of Group 2. Multiple hypotheses bias occurred in the analysis process is corrected using two different ways: the FDR approach and the Bonferroni method.

After calculating the differential methylation/expression of each bead of the BeadChip/GeneChip microarray, we used the Infinium annotation file/HTA annotation file to map the value reads back to the human genome. Notice that multiple values may be mapped to one gene. And multiple genes may be annotated for one bead value read.

In this function, users can find potential differentially methylated/expressed genes between KD patients and normal kids.

a) Users need to specify the two groups and the search conditions to search for differentially methylated/expressed genes between these two groups.
b) The differentially methylated genes between the selected two groups. Details of the bead probes can be found by clicking on the bead name.
c) The heat map of the methylation levels between the two groups.
d) Users can click on the tabs to switch the table listing into differentially methylated genes, differentially expressed genes, and genes that are both differentially methylated and expressed.

In this function, users can find potentially affected patient groups of the given gene.

a) Users can type in the gene of interest and the search conditions to find the potentially affected patient groups.
b) The two patient groups that bear differential methylation/expression levels on the given gene.
c) The differentially methylated genes between the selected two groups. Details of the bead probes can be found by clicking on the bead name. Users can click on the tabs to switch the table listing into differentially methylated genes, differentially expressed genes, and genes that are both differentially methylated and expressed.

In this function, users can input the gene of interest and obtain the methylation and expression levels of this gene between different groups.

a) The listing of the beads that are mapped to this gene.
b) A comparison visualization on the methylation/expression levels of the selected bead.