Sung-Chou Li

Genome-Wide Association Study Identifies Novel Susceptibility Genes Associated with Coronary Artery Aneurysm Formation in Kawasaki Disease

Kawasaki disease (KD) or Kawasaki syndrome is known as a vasculitis of small to medium-sized vessels, and coronary arteries are predominantly involved in childhood. Generally, 20–25% of untreated with IVIG and 3–5% of treated KD patients have been developed coronary artery lesions (CALs), such as dilatation and aneurysm. Understanding how coronary artery aneurysms (CAAs) are established and maintained in KD patients is therefore of great importance. Upon our previous genotyping data of 157 valid KD subjects, a genome-wide association study (GWAS) has been conducted among 11 (7%) CAA-developed KD patients to reveal five significant genetic variants passed pre-defined thresholds and resulted in two novel susceptibility protein-coding genes, which are NEBL (rs16921209 (P = 7.44 × 10−9; OR = 32.22) and rs7922552 (P = 8.43 × 10−9; OR = 32.0)) and TUBA3C (rs17076896 (P = 8.04 × 10−9; OR = 21.03)). Their known functions have been reported to associate with cardiac muscle and tubulin, respectively. As a result, this might imply their putative roles of establishing CAAs during KD progression. Additionally, various model analyses have been utilized to determine dominant and recessive inheritance patterns of identified susceptibility mutations. Finally, all susceptibility genes hit by significant genetic variants were further investigated and the top three representative gene-ontology (GO) clusters were regulation of cell projection organization, neuron recognition, and peptidyl-threonine phosphorylation. Our results help to depict the potential routes of the pathogenesis of CAAs in KD patients and will facilitate researchers to improve the diagnosis and prognosis of KD in personalized medicine.

Microarray Study of Pathway Analysis Expression Profile Associated with MicroRNA-29a with Regard to Murine Cholestatic Liver Injuries

Accumulating evidence demonstrates that microRNA-29 (miR-29) expression is prominently decreased in patients with hepatic fibrosis, which consequently stimulates hepatic stellate cells’ (HSCs) activation. We used a cDNA microarray study to gain a more comprehensive understanding of genome-wide gene expressions by adjusting miR-29a expression in a bile duct-ligation (BDL) animal model. Methods: Using miR-29a transgenic mice and wild-type littermates and applying the BDL mouse model, we characterized the function of miR-29a with regard to cholestatic liver fibrosis. Pathway enrichment analysis and/or specific validation were performed for differentially expressed genes found within the comparisons. Results: Analysis of the microarray data identified a number of differentially expressed genes due to the miR-29a transgene, BDL, or both. Additional pathway enrichment analysis revealed that TGF-β signaling had a significantly differential activated pathway depending on the occurrence of miR-29a overexpression or the lack thereof. Furthermore, overexpression was found to elicit changes in Wnt/β-catenin after BDL. Conclusion: This study verified that an elevated miR-29a level could alleviate liver fibrosis caused by cholestasis. Furthermore, the protective effects of miR-29a correlate with the downregulation of TGF-β and associated with Wnt/β-catenin signal pathway following BDL.

MicroRNA-29a Alleviates Bile Duct Ligation Exacerbation of Hepatic Fibrosis in Mice through Epigenetic Control of Methyltransferases

MicroRNA-29 (miR-29) is found to modulate hepatic stellate cells’ (HSCs) activation and, thereby, reduces liver fibrosis pathogenesis. Histone methyltransferase regulation of epigenetic reactions reportedly participates in hepatic fibrosis. This study is undertaken to investigate the miR-29a regulation of the methyltransferase signaling and epigenetic program in hepatic fibrosis progression. miR-29a transgenic mice (miR-29aTg mice) and wild-type littermates were subjected to bile duct-ligation (BDL) to develop cholestatic liver fibrosis. Primary HSCs were transfected with a miR-29a mimic and antisense inhibitor. Profibrogenic gene expression, histone methyltransferases and global genetic methylation were probed with real-time quantitative RT-PCR, immunohistochemical stain, Western blot and ELISA. Hepatic tissue in miR-29aTg mice displayed weak fibrotic matrix as evidenced by Sirius Red staining concomitant with low fibrotic matrix collagen 1α1 expression within affected tissues compared to the wild-type mice. miR-29a overexpression reduced the BDL exaggeration of methyltransferases, DNMT1, DNMT3b and SET domain containing 1A (SET1A) expression. It also elevated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling within liver tissue. In vitro, miR-29a mimic transfection lowered collagen 1α1, DNMT1, DNMT3b and SET1A expression in HSCs. Gain of miR-29a signaling resulted in DNA hypomethylation and high PTEN expression. This study shines a new light on miR-29a inhibition of methyltransferase, a protective effect to maintain the DNA hypomethylation state that decreases fibrogenic activities in HSC. These robust analyses also highlight the miR-29a regulation of epigenetic actions to ameliorate excessive fibrosis during cholestatic liver fibrosis development.

Anti-inflammatory effect of resveratrol in human coronary arterial endothelial cells via induction of autophagy: implication for the treatment of Kawasaki disease

BackgroundKawasaki disease (KD) is an acute febrile vasculitis in childhood, which is the leading cause of acquired heart disease in children. If untreated, KD can result in coronary aneurysms in 25% of patients, and even under intravenous immunoglobulin (IVIG) treatment, 10–20% of children will have IVIG resistance and increased risk of developing coronary arteritis complication. Additional therapies should be explored to decrease the incidence of coronary artery lesions and improve the prognosis in KD. Autophagy has been reported to play a critical role in a variety of heart diseases. Resveratrol (RSV) confers cardio protection during ischemia and reperfusion in rats via activation of autophagy. Serum TNF-alpha levels are elevated in KD, which might activate the endothelial cells to express intercellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1(VCAM-1), inducible nitric oxide synthase (iNOS) and IL-1β.MethodsHuman coronary arterial endothelial cells (HCAECs) were either untreated or treated by TNF-α 10xa0ng/ml for 2xa0h in the presence or absence of RSV or autophagy-related protein 16-like 1 (Atg16L1) siRNA. Total RNA was analyzed by real-time quantitative PCR for ICAM-1, VCAM-1, iNOS and IL-1β mRNA expressions. The involvement of autophagy proteins was investigated by Western blot.ResultsPretreatment with resveratrol significantly inhibited TNF-α-induced ICAM-1, iNOS and IL-1β mRNA expression in HCAECs. Western blot revealed the enhanced autophagy proteins LC3B and Atg16L1 expression by RSV. The suppressive effects of RSV were obviously counteracted by Atg16L1 siRNA.ConclusionsWe demonstrated RSV had anti-inflammatory effects on HCAECs via induction of autophagy. Our results suggest that resveratrol may modulate the inflammatory response of coronary artery in KD and explore the role of autophagy in the pathogenesis and alternative therapy of coronary arterial lesions in KD.

Comparison of the Functional microRNA Expression in Immune Cell Subsets of Neonates and Adults

Diversity of biological molecules in newborn and adult immune cells contributes to differences in cell function and atopic properties. Micro RNAs (miRNAs) are reported to involve in the regulation of immune system. Therefore, determining the miRNA expression profile of leukocyte subpopulations is important for understanding immune system regulation. In order to explore the unique miRNA profiling that contribute to altered immune in neonates, we comprehensively analyzed the functional miRNA signatures of eight leukocyte subsets (polymorphonuclear cells, monocytes, CD4+ T cells, CD8+ T cells, natural killer cells, B cells, plasmacytoid dendritic cells, and myeloid dendritic cells) from both neonatal and adult umbilical cord and peripheral blood samples, respectively. We observed distinct miRNA profiles between adult and neonatal blood leukocyte subsets, including unique miRNA signatures for each cell lineage. Leukocyte miRNA signatures were altered after stimulation. Adult peripheral leukocytes had higher let-7b-5p expression levels compared to neonatal cord leukocytes across multiple subsets, irrespective of stimulation. Transfecting neonatal monocytes with a let-7b-5p mimic resulted in a reduction of LPS-induced interleukin (IL)-6 and TNF-α production, while transfection of a let-7b-5p inhibitor into adult monocytes enhanced IL-6 and TNF-α production. With this functional approach, we provide intact differential miRNA expression profiling of specific immune cell subsets between neonates and adults. These studies serve as a basis to further understand the altered immune response observed in neonates and advance the development of therapeutic strategies.

Hepcidin-Induced Iron Deficiency Is Related to Transient Anemia and Hypoferremia in Kawasaki Disease Patients

Kawasaki disease (KD) is a type of systemic vasculitis that primarily affects children under the age of five years old. For sufferers of KD, intravenous immunoglobulin (IVIG) has been found to successfully diminish the occurrence of coronary artery lesions. Anemia is commonly found in KD patients, and we have shown that in appropriately elevated hepcidin levels are related to decreased hemoglobin levels in these patients. In this study, we investigated the time period of anemia and iron metabolism during different stages of KD. A total of 100 patients with KD and 20 control subjects were enrolled in this study for red blood cell and hemoglobin analysis. Furthermore, plasma, urine hepcidin, and plasma IL-6 levels were evaluated using enzyme-linked immunosorbent assay in 20 KD patients and controls. Changes in hemoglobin, plasma iron levels, and total iron binding capacity (TIBC) were also measured in patients with KD. Hemoglobin, iron levels, and TIBC were lower (p < 0.001, p = 0.009, and p < 0.001, respectively) while plasma IL-6 and hepcidin levels (both p < 0.001) were higher in patients with KD than in the controls prior to IVIG administration. Moreover, plasma hepcidin levels were positively and significantly correlated with urine hepcidin levels (p < 0.001) prior to IVIG administration. After IVIG treatment, plasma hepcidin and hemoglobin levels significantly decreased (both p < 0.001). Of particular note was a subsequent gradual increase in hemoglobin levels during the three weeks after IVIG treatment; nevertheless, the hemoglobin levels stayed lower in KD patients than in the controls (p = 0.045). These findings provide a longitudinal study of hemoglobin changes and among the first evidence that hepcidin induces transient anemia and hypoferremia during KD’s acute inflammatory phase.

Major methylation alterations on the CpG markers of inflammatory immune associated genes after IVIG treatment in Kawasaki disease

BackgroundKawasaki 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.MethodsIn 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.ResultsWe 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.ConclusionsIn summary, our result provides researchers with another way into the regulation mechanism through which IVIG represses excessive inflammatory responses.

HAMP promoter hypomethylation and increased hepcidin levels as biomarkers for Kawasaki disease

Kawasaki disease (KD) is the most common coronary vasculitis to appear in children with anemia and has been associated with elevated plasma hepcidin levels. We recruited a total of 241 cases, including 18 KD patients, who were tested both prior to receiving intravenous immunoglobulin (IVIG) and at least 3u202fweeks after IVIG treatment, and 18 febrile controls, who were observed in the Illumina HumanMethylation450 BeadChip study for their CpG markers. The remaining cases consisted of another 92 KD patients and 113 controls that were used for validation by pyrosequencing. We performed a genetic functional study using Luciferase assays. A support vector machine (SVM) classification model was adopted to identify KD patients and control subjects. In this study, KD patients clearly demonstrated a significantly epigenetic hypomethylation of HAMP promoter compared to controls. After receiving IVIG treatment, the hypomethylation status in KD patients was restored, and we observed a significant opposite tendency between the DNA methylation of target CpG sites (cg23677000 and cg04085447) and the hepcidin level. Furthermore, reporter gene assays were used to detect target CpG sites, the methylation of which displayed decreased levels of HAMP gene expression. Of particular note, we developed a SVM classification model with a 90.9% sensitivity, a 91.9% specificity, and 0.94 auROC in the training set. An independent blind cohort also had good performance (96.1% sensitivity and 89.7% specificity). In this study, we demonstrate HAMP promoter hypomethylation, which upregulates hepcidin expression in KD patients. Furthermore, the reliability and robustness of our SVM classification model can accurately serve as KD biomarkers.

Blood-Bourne MicroRNA Biomarker Evaluation in Attention-Deficit/Hyperactivity Disorder of Han Chinese Individuals: An Exploratory Study

Background: Attention-deficit/hyperactivity disorder (ADHD) is a highly genetic neurodevelopmental disorder, and its dysregulation of gene expression involves microRNAs (miRNAs). The purpose of this study was to identify potential miRNAs biomarkers and then use these biomarkers to establish a diagnostic panel for ADHD. Design and methods: RNA samples from white blood cells (WBCs) of five ADHD patients and five healthy controls were combined to create one pooled patient library and one control library. We identified 20 candidate miRNAs with the next-generation sequencing (NGS) technique (Illumina). Blood samples were then collected from a Training Set (68 patients and 54 controls) and a Testing Set (20 patients and 20 controls) to identify the expression profiles of these miRNAs with real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). We used receiver operating characteristic (ROC) curves and the area under the curve (AUC) to evaluate both the specificity and sensitivity of the probability score yielded by the support vector machine (SVM) model. Results: We identified 13 miRNAs as potential ADHD biomarkers. The ΔCt values of these miRNAs in the Training Set were integrated to create a biomarker model using the SVM algorithm, which demonstrated good validity in differentiating ADHD patients from control subjects (sensitivity: 86.8%, specificity: 88.9%, AUC: 0.94, p < 0.001). The results of the blind testing showed that 85% of the subjects in the Testing Set were correctly classified using the SVM model alignment (AUC: 0.91, p < 0.001). The discriminative validity is not influenced by patients age or gender, indicating both the robustness and the reliability of the SVM classification model. Conclusion: As measured in peripheral blood, miRNA-based biomarkers can aid in the differentiation of ADHD in clinical settings. Additional studies are needed in the future to clarify the ADHD-associated gene functions and biological mechanisms modulated by miRNAs.

Roles of microRNA in the immature immune system of neonates

Neonates have an immature immune system; therefore, their immune activities are different from the activities of adult immune systems. Such differences between neonates and adults are reflected by cell population constitutions, immune responses, cytokine production, and the expression of cellular/humoral molecules, which contribute to the specific neonatal microbial susceptibility and atopic properties. MicroRNAs (miRNAs) have been discovered to modulate many aspects of immune responses. Herein, we summarize the distinct manifestations of the neonatal immune system, including cellular and non-cellular components. We also review the current findings on the modulatory effects of miRNAs on the neonatal immune system. These findings suggest that miRNAs have the potential to be useful therapeutic targets for certain infection or inflammatory conditions by modulating the neonatal immune system. In the future, we need a more comprehensive understanding in regard to miRNAs and how they modulate specific immune cells in neonates.