Methylation Microarray Studies Highlight PDGFA Expression as a Factor in Biliary Atresia.

Zenobia C Cofer,John W Tobias,Randolph P Matthews,Kathleen M Loomes,Shuang Cui,Cecilia Kim,Steven F Eauclaire,Hakon Hakonarson,Diego Calvisi

doi:10.1371/journal.pone.0151521

Zenobia C Cofer, John W Tobias + Show 7 more

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https://doi.org/10.1371/journal.pone.0151521

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Abstract

Biliary atresia (BA) is a progressive fibro-inflammatory disorder that is the leading indication for liver transplantation in children. Although there is evidence implicating genetic, infectious, environmental, and inflammatory causes, the etiology of BA remains unknown. We have recently reported that cholangiocytes from BA patients showed decreased DNA methylation relative to disease- and non-disease controls, supporting a potential role for DNA hypomethylation in BA etiopathogenesis. In the current study, we examined the methylation status of specific genes in human BA livers using methylation microarray technology. We found global DNA hypomethylation in BA samples as compared to disease- and non-disease controls at specific genetic loci. Hedgehog pathway members, SHH and GLI2, known to be upregulated in BA, were both hypomethylated, validating this approach as an investigative tool. Another region near the PDGFA locus was the most significantly hypomethylated in BA, suggesting potential aberrant expression. Validation assays confirmed increased transcriptional and protein expression of PDGFA in BA livers. We also show that PDGF-A protein is specifically localized to cholangiocytes in human liver samples. Injection of PDGF-AA protein dimer into zebrafish larvae caused biliary developmental and functional defects. In addition, activation of the Hedgehog pathway caused increased expression of PDGF-A in zebrafish larvae, providing a previously unrecognized link between PDGF and the Hedgehog pathway. Our findings implicate DNA hypomethylation as a specific factor in mediating overexpression of genes associated with BA and identify PDGF as a new candidate in BA pathogenesis.

Highlights

Biliary atresia (BA) is a progressive fibro-inflammatory disorder that is the leading indication for liver transplantation in children
To determine whether DNA methylation of specific genes is decreased in BA livers, we examined global liver DNA methylation from samples taken at transplant
We have shown above that HH pathway genes and PDGFA are hypomethylated in BA liver samples, that activation of both pathways leads to biliary defects in an animal model, and that inhibition of the HH pathway rescues defects caused by inhibition of DNA methylation

Summary

Introduction

Biliary atresia (BA) is a progressive fibro-inflammatory disorder that is the leading indication for liver transplantation in children. Several groups have examined gene expression changes in livers from BA patients [5, 6], noting an increase in imprinted genes [6], supporting a possible role for altered DNA methylation in patients with BA. Recent studies have examined differential methylation in obese adolescents [10] and in patients with ulcerative colitis [11], demonstrating that there are methylation changes in specific genes in a variety of diseases. While these changes may not reflect the complete pathogenic picture, it seems likely that differential gene methylation does play a role in disease pathogenesis for many conditions

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