Prune Belly Syndrome Bladder Transcriptional Dysregulation characterized by Bulk RNA Sequencing
Nixon Raj, PhD1, Nathalia G. Amado, PhD1, Jeremy A. Mathews, MS2, Alexandria N. Fusco, BS2, Thomas J. Egeland, MS2, Gervaise H. Henry, MS2, Nida S. Iqbal, PhD2, Brandi L. Cantarel, PhD2, Douglas W. Strand, PhD2, Linda A. Baker, MD1.
1Nationwide Children's Hospital, Abigail Wexner Research Institute, Columbus, OH, USA, 2UT Southwestern Medical Center, Dallas, TX, USA.
Background: Prune belly syndrome is characterized by bladder wall thickening, dysmyogenesis, fibrosis, and dysfunction leading to severe urinary tract dilation with poor bladder contraction and elevated postvoid residuals. At present, our understanding of the molecular mechanisms causing and regulating this human bladder phenotype are still limited. Transcriptomics by bulk RNA-sequencing (RNA-Seq) can provide extensive coverage and robust quantification for a broad range of expression levels of genes. We used bulk RNA-Seq to gain deeper insight into the molecular mechanisms that are characteristic to the human PBS bladder.
METHODS: With IRB-approval, 4 unrelated PBS males (mean 5.8years and mean RUBACE Score 15.75) and 2 non-PBS control (CO) males (mean 6.5years) were recruited. At surgery or death, 1 cc of full-thickness bladder was harvested. The bladder RNA was extracted, the Illumina cDNA library was prepared, and bulk RNA was sequenced. The raw reads were trimmed (TrimGalore) and mapped to the GRCh38 human genome (STAR 2-pass method). Gene quantification (featureCount), differential expression gene (DEG) analysis (DESeq2) and functional enrichment analysis (clusterProfiler) were performed. Quantitative PCR on these bladder RNAs (qPCR) for the top 2 DEGs was performed for secondary validation by the delta-delta Ct method.
RESULTS: When compared to controls, human bladder bulk RNA-Seq analysis identified 81 DEGs in PBS bladders with 53 upregulated and 28 downregulated genes (Fig1A); the top 2 up or downregulated genes were validated by qPCR (Fig1B). The highest upregulated DEG was the EGF family member, Delta Like Non-Canonical Notch Ligand 1 (DLK1). This gene encodes a transmembrane protein that is a mesenchymal cell-derived pro-myogenesis factor. It also plays an important role in fibroblast activation and collagen release. Interestingly, DLK1 is in a chromosome region frequently imprinted showing uniparental disomy, expressed only from the paternal allele. DLK1 is also upregulated in Duchenne and Becker Muscular Dystrophy, where the expression reflects skeletal myotube immaturity. Collagen 3A1 (COL3A1) is the second highest upregulated DEG and is increased in bladder hypertrophy and fibrosis. The top two downregulated genes, SHISA3 and GPX3, are Wnt inhibitors. Decreased expression of these genes can be associated with dysregulated mesoderm development. Gene Ontology analysis of the PBS DEGs revealed significantly enriched terms including: “Collagen containing extracellular matrix” and “fibrillar collagen”.
CONCLUSIONS: PBS is a rare disease for which tissue availability is scarce. We, for the first time, have interrogated alterations in PBS bladder global transcriptional networks. This PBS bladder transcriptomic data is a step toward for identifying the molecular basis of PBS and potential markers for diagnosis, including collagens, DLK1 and Wnt/TGF-β signaling pathways. Additionally, we can explore the heterogeneity of PBS bladder gene expression by identifying DEGs, functional enrichment for each individual PBS bladder sample, opening the door for future personalized therapeutic interventions.
NIHDK100483, DK127589 PI: Baker and AUA/UCF Research Scholar Award 2022-2024: Amado
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