Molecular Phenotyping of Kawasaki Disease


Project Summary

The focus of this project was on Kawasaki disease (KD)—the most common cause of acquired heart disease in children.  Although the cause of KD is unknown, researchers suspect an infectious agent that triggers an immunologic reaction in genetically susceptible hosts.  In this project, we aimed to create microRNA (miR) profiles of patients and relate these profiles to patient demographic and clinical data in order to understand how molecular phenotype relates to clinical phenotype and how they may help predict susceptibility, response to treatment, and risk for cardiovascular sequelae.  After Year 1, we expanded our efforts to include whole genome sequencing (WGS) analysis upon receiving a donation from Illumina for WGS of a family of six individuals including two affected siblings.

 

Overall Results

From this project, we identified differentially expressed miRs associated with acute KD and with adenovirus infection and found new SNPs that are significantly associated with susceptibility.  We also developed new tools to facilitate miR analysis and streamline the process of WGS analysis.  This DBP is now involved with a new privacy-preserving international distributed network as part of a new DBP that started July 2013 (Privacy Preserving Analytics for Kawasaki Disease in African Americans).

 

Results and Outcomes of Specific Aims

Results and outcomes of the project’s specific aims are summarized below. 
A detailed report can be downloaded here.
 

Specific Aim 1: To identify microRNA (miR) profiles specific to Kawasaki disease (KD) and to relate these patterns to clinical outcome.

Results:        

We successfully sequenced small, non-coding RNA species, generated a custom miR analysis tool, called Microrna analysis in GPU Infrastructure (MAGI) , and identified differentially expressed miRs associated with acute KD and with adenovirus infection. miR-145 was differentially expressed during acute KD, and it modulates expression of many genes involved in cardiovascular health including genes in the TGFβ pathway. These findings strengthen our previous observations about the importance of the TGFβ signaling pathway in KD pathogenesis and further support our clinical trial of atorvastatin to block the downstream effects of signaling through this pathway.

Output:

  • This work was published in PlosOne 2013;8(3):e58159.
     

Specific Aim 2: To selectively sequence regions of genes in the TGFβ and calcium signaling pathways to identify rare genetic variants that may play a functional role in disease susceptibility and outcome.

Results:

We proposed to re-sequence targeted regions of 3 genes in the TGFβ pathway (TGFB2, SMAD3, TGFBR2) as well as regions in two additional genes: PLCG2 from the calcium signaling pathway and ATP-binding cassette, subfamily C, member 4 (ABCC4), which were identified from our previous genetic studies.  We completed targeted re-sequencing of about 65Kb located in “hot spots” containing clusters of significantly associated SNPs from 5 genes using 96 KD and 96 controls.  We found 13 new SNPs significantly associated with susceptibility from 3 genes (p<0.05).  Three of these SNPs were rare alleles with minor allele frequencies less than 5% that would not have been identified using association studies or GWAS.  The analysis of genetic influences on aneurysm formation is in progress.  These findings will be combined with previous results and included in a manuscript that focuses on the calcium signaling pathway.

Output:

  • A manuscript for this work is under preparation.
     

Specific Aim 3: To create a KD data warehouse and web-based data analysis system aimed at facilitating discoveries using clinical and molecular data.

Results:

The data from the KD Research Center was migrated into REDCap and custom case report forms were generated for individual patient files. This database now allows sharing data with other sites in a secure and privacy-preserving manner.  REDCap now contains files on 1,043 individual KD patients with linked clinical, demographic, laboratory, and molecular phenotyping data, including gene expression and SNP genotyping data.  Portals are being created to host other KD research centers (Emory University, Atlanta, GA and Kitasato University, Kitasato, Japan), which will allow sharing of data and combined data analysis to support various research projects.

Output:

  • A database of KD files was created to enable sharing of data with other sites in a secure and privacy-preserving manner.


WGS: To analyze WGS performed by Illumina on six members of an African-American KD family in which two children were affected by KD, as well as one unrelated African-American KD patient with aneurysms.

Results:

Analysis of large-scale structural variants and SNP genotypes and small insertions/deletions by iDASH investigators is in progress for these seven sequences. New tools are being created to streamline the process of WGS analysis and manuscripts are being prepared to describe these analytic approaches.  Cloud computing is being used to store the interim analyses and ultimately to share the WGS of these seven individuals with other investigators.

Output:

  • A manuscript describing the analytic approaches for this work is under preparation.

 

Publications & presentations from this project

Papers

  1. Shimizu, C., Kim, J., Stepanowsky, P., Trinh, C., Lau, H.D., Akers, J.C., Chen, C., Kanegaye, J.T., Tremoulet, A., Ohno-Machado, L., and Burns, J.C. “Differential expression of miR-145 in children with Kawasaki disease.” PLoS One. 2013;8(3):e58159. PMID: 23483985. PMCID: PMC3590129.
  2. Shimizu, C., Oharaseki, T., Takahashi, K., Kottek, A., Franco, A., and Burns, J.C. “The Role of TGF-β and Myofibroblasts in the Arteritis of Kawasaki Disease.” Human Pathology. February 2013. PMID: 22955109; PMCID: PMC3518690
  3. Burns, J.C. and Newburger, J.W. “Genetics insights into the pathogenesis of Kawasaki disease.” Circ Cardiovasc Genet. 2012;5(3):277-8. PMID: 22715279. NIHMSID # 388556.
  4. Shimizu, C., Jain, S., Davila, S., Hibberd, M.L., Lin, K.O., Molkara, D., Frazer, J.R., Sun, S., Baker, A.L., and Newburger, J.W. “Transforming growth factor-beta signaling pathway in patients with Kawasaki disease.” Circ Cardiovasc Genet. 2011;4(1):16-25. PMID: 21127203. PMCID: PMC3073847.

Posters

  1. Burns, J. and Shimizu, C. “Whole Blood microRNA Profiles in Adenovirus Infected Children”. Pediatric Academic Societies. Washington, D.C. May 4-7, 2013.
  2. Burns, J. and Shimizu, C. “Lysyl Oxidase Expression in Patients with Kawasaki Disease”. Pediatric Academic Societies. Washington, D.C. May 4-7, 2013.

Presentations

  1. Shimizu, C, Kim J, Stepanowsky P, Trinh C, Lau HD, Arker J, Chen C, Kanegaye JT, Tremoulet AH, Ohno-Machado L, Burns J.C. "Differential Expression of microRNAs in Children with Kawasaki Disease” presented at Pediatric Academic Societies meeting, Washington, D.C., May 4, 2013.
  2. Burns, J. “Whole Blood MicroRNA Profiles in Acute and Convalescent Kawasaki Disease” presented at the 10th international Kawasaki disease symposium, Kyoto, Japan, February 7-10, 2012.
  3. Burns, J. "Kawasaki Disease mirRNA Update” presented at the International KD Genetics Consortium Meeting, Melbourne, Australia, November 21, 2011.