No TAP63 promoter mutation is detected in bladder exstrophy-epispadias complex patients.

BACKGROUND/PURPOSE: Bladder exstrophy-epispadias complex (BEEC) is thought to have a genetic component in its pathogenesis. Previously we found that p63(-/-) mice show increased ventral apoptosis and develop a BEEC phenotype. Down-regulation of the anti-apoptotic ΔNP63 and an up-regulation of pro-apoptotic TAP63 isoforms have been demonstrated in BEEC patient bladder tissues. We have previously shown that insertion/deletion polymorphisms of the ΔNp63 promoter are associated with an increased risk of BEEC. In this study, we specifically examined the TAP63 promoter to see if any sequence changes might lead to up-regulation of TAP63 and exaggerated apoptosis in BEEC patients. METHODS: i) Bioinformatic analysis of the TAP63 promoter was performed to identify putative regulatory regions. ii) High-resolution Melt and Sanger sequencing was used to screen targeted regions in 112 BEEC patient DNA samples for potential sequence variants. iii) Sequence variation was analysed for significance against normal population frequency data. RESULTS: i) We identified multiple epigenetic markers of transcriptional regulation within highly conserved areas of the TAP63 promoter sequence. ii) Of the 112 buccal swab DNA samples, adequate and successful screening ranged between 48 and 67 for each region. iii) No novel sequence variation or mutation was uncovered. iv) Two known SNPs were identified. However, allele frequency analysis was not statistically significant. CONCLUSION: Our data do not associate genetic variation within the TAP63 promoter region with an increased risk of BEEC. Our data so far suggests that only ΔNP63 promoter aberration is involved in BEEC pathogenesis.

Identification of Streptococcus parasanguinis DNA contamination in human buccal DNA samples.

BACKGROUND: The use of buccal swabs in clinical and scientific studies is a very popular method of collecting DNA, due to its non-invasive nature of collection. However, contamination of the DNA sample may interfere with analysis. FINDINGS: Here we report the finding of Streptococcus parasanguinis bacterial DNA contamination in human buccal DNA samples, which led to preferential amplification of bacterial sequence with PCR primers designed against human sequence. CONCLUSION: Contamination of buccal-derived DNA with bacterial DNA can be significant, and may influence downstream genetic analysis. One needs to be aware of possible bacterial contamination when interpreting abnormal findings following PCR amplification of buccal swab DNA samples.

New insights into the pathogenesis of bladder exstrophy-epispadias complex.

Bladder exstrophy-epispadias complex (BEEC) is a complex and debilitating congenital disease. Familial and twin studies suggest a possible genetic component in BEEC pathogenesis. Bladder mesenchyme (detrusor) development requires induction by a signal from bladder urothelium, and we and others have shown the Shh-Gli-Bmp4 signalling pathway is likely to be involved. P63 is a master regulator in epithelial stratification and is expressed in urothelium. We have shown that p63 knock-out mice undergo excessive urothelial apoptosis. Failure of mesenchymal induction by epithelium leads to BEEC. We further demonstrated that insertion/deletion (in/del) polymorphisms (1 base pair (bp) ins and 4 bp ins., and 12 bp del) in the ΔNP63 promoter reduce transcriptional efficiency, and are associated with a statistically significant increase in the risk of BEEC in humans. Furthermore, a Genome-Wide Expression Profiling (GWEP) study suggests possible involvement of PERP in human BEEC. Intriguingly, PERP is a direct target of p63 during development, and is also involved in epithelial stratification. PERP co-localizes with desmosome, and both PERP and desmosome are essential for maintaining tissue integrity by cellular adhesion and epithelial stratification. A recent study showed that PERP and desmosome expression levels are abnormal in human BEEC patients. This review describes the role of the P63 > PERP > desmosome pathway in the development of human bladder during embryogenesis. We hypothesize that disruption of this pathway may increase the risk of BEEC.

Insertion/deletion polymorphisms in the ΔNp63 promoter are a risk factor for bladder exstrophy epispadias complex.

Bladder exstrophy epispadias complex (BEEC) is a severe congenital anomaly; however, the genetic and molecular mechanisms underlying the formation of BEEC remain unclear. TP63, a member of TP53 tumor suppressor gene family, is expressed in bladder urothelium and skin over the external genitalia during mammalian development. It plays a role in bladder development. We have previously shown that p63(-/-) mouse embryos developed a bladder exstrophy phenotype identical to human BEEC. We hypothesised that TP63 is involved in human BEEC pathogenesis. RNA was extracted from BEEC foreskin specimens and, as in mice, ΔNp63 was the predominant p63 isoform. ΔNp63 expression in the foreskin and bladder epithelium of BEEC patients was reduced. DNA was sequenced from 163 BEEC patients and 285 ethnicity-matched controls. No exon mutations were detected. Sequencing of the ΔNp63 promoter showed 7 single nucleotide polymorphisms and 4 insertion/deletion (indel) polymorphisms. Indel polymorphisms were associated with an increased risk of BEEC. Significantly the sites of indel polymorphisms differed between Caucasian and non-Caucasian populations. A 12-base-pair deletion was associated with an increased risk with only Caucasian patients (p = 0.0052 Odds Ratio (OR) = 18.33), whereas a 4-base-pair insertion was only associated with non-Caucasian patients (p = 0.0259 OR = 4.583). We found a consistent and statistically significant reduction in transcriptional efficiencies of the promoter sequences containing indel polymorphisms in luciferase assays. These findings suggest that indel polymorphisms of the ΔNp63 promoter lead to a reduction in p63 expression, which could lead to BEEC.