Society For Pediatric Urology

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Expression Analysis of Prune Belly Syndrome Bladders
Nida Iqbal, PhD1, Adam Kern, MD2, Thomas Jascur, Phd1, Daniel Wong, MS31, Linda Baker, MD3.
1UT Southwestern Medical Center, Dallas, TX, USA, 2Anne Arundel Medical Center, Annapolis, MD, USA, 3Children's Medical Center; UT Southwestern Medical Center, Dallas, TX, USA.

BACKGROUND: Prune belly syndrome (PBS) is a devastating congenital genitourinary disorder for which the cause is unknown for most human cases. Because genetic studies have revealed significant heterogeneity, we chose to identify common molecular mechanisms that drive PBS pathogenesis by coupling global transcriptome networks with histology and ultrastructural information on PBS affected bladder tissue compared to healthy controls. We hypothesize that the myopathic changes in PBS bladders observed by histology will be reflected in gene expression signatures.
METHODS: Bladder tissue was collected from a subset of PBS patients as well as age and sex matched surgical control biopsy specimens from individuals prospectively enrolled in our Pediatric Genitourinary Tissue Repository. Purified RNA from PBS bladder (n=4) vs control bladder (n=3) was subject to the Affymetrix HTA 2.0 RNA expression array and differential mRNA profiles were examined using the Affymetrix Transcriptome Analysis Console (TAC). Differentially regulated genes were validated by quantitative PCR (qPCR). FFPE tissue isolated from independent individuals, PBS bladder (n=3) vs control bladder (n=3), was subject to standard immunohistochemistry analysis on a Dako Autostainer Link 48 system. Light and electron microscopy (EM) was further used to characterize gross tissue morphology and ultrastructural analysis of PBS bladders tissues compared to controls.
RESULTS: Transcriptome analysis from PBS vs control bladders identified 264 differentially expressed genes (135 up-regulated and 129 down-regulated). PANTHER Overrepresentation Pathway analysis of the top 75 upregulated genes (fold change >5) identified the ‘Integrin signaling pathway’ as the top misregulated signaling module (FDR = 8.07E-05) in PBS bladders. Histology and EM confirmed abnormal smooth muscle organization, bundling and fiber size in PBS bladders compared to controls with significant intervening collagen in PBS bladders. Increased collagen observed by EM and light microscopy was corroborated by microarray data identifying changes in collagen family gene expression in PBS bladders. Top (up and downregulated) candidates were confirmed by qPCR.
CONCLUSIONS: PBS is a rare disease for which tissue availability is sparse. We, for the first time, have interrogated alterations in bladder global transcriptional networks in response to PBS pathology in conjunction with histological and ultrastructural analysis of affected tissues. We identified the Integrin signaling pathway as significantly misregulated in PBS bladders. Integrins are a critical physical linkage between cytoskeletal structures and the extracellular matrix (ECM) and participate in various signal transduction processes to modulate diverse cellular functions. The misregulation of this important signaling pathway suggests that the actin cytoskeleton plays a role in PBS pathogenesis. Indeed our work and others in identifying causal PBS genes has pointed to smooth muscle cytoskeletal components (e.g. ACTA2, ACTG2, FLNA) as novel candidates. Our gene expression data further supports the hypothesis that PBS is a smooth muscle myopathy driven by mutation in critical cytoskeletal genes. Further analysis of differentially expressed genes may yield novel targets for PBS pharmacological intervention, therapeutic discovery and identify novel causal candidate genes.

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