Urothelial Differences in the Exstrophy-Epispadias Complex: Potential Implications for Management.

PURPOSE: The authors examined the urothelium of exstrophy-epispadias complex spectrum patients for histological differences and expression of terminal markers of urothelial differentiation. MATERIALS AND METHODS: Between 2012 and 2017 bladder biopsies were obtained from 69 pediatric exstrophy-epispadias complex patients. These specimens were compared to bladder specimens from normal controls. All bladder specimens underwent histological assessment followed by immunohistochemical staining for uroplakin-II and p63. Expression levels of uroplakin-II and p63 were then assessed by a blinded pathologist. RESULTS: Forty-three classic bladder exstrophy biopsies were obtained (10 newborn closures, 22 delayed closures, and 11 repeat closures). Additional biopsies from 18 cloacal exstrophy patients and 8 epispadias patients were also evaluated. These specimens were compared to 8 normal control bladder specimens. Overall, uroplakin-II expression was lower in exstrophy-epispadias complex patients compared to controls (p <0.0001). Among classic bladder exstrophy patients, there was reduced expression of uroplakin-II in the delayed and repeat closures in comparison to newborn closures (p=0.045). Expression of p63 was lower in patients with exstrophy-epispadias complex compared to controls (p <0.0001). Expression of p63 was similar among classic bladder exstrophy patients closed as newborns when compared to delayed or repeat closures. Classic bladder exstrophy patients had a higher rate of squamous metaplasia when compared to controls (p=0.044). Additionally, there was a higher rate of squamous metaplasia in the patients undergoing delayed closure in comparison to those closed in the newborn period (p <0.001). CONCLUSIONS: The urothelium in the exstrophy-epispadias complex bladder is strikingly different than that of healthy controls. Uroplakin-II expression is greatly reduced in exstrophy-epispadias complex bladders and is influenced by the timing of bladder closure. Reduced uroplakin-II expression and increased rates of squamous metaplasia in exstrophy-epispadias complex patients undergoing delayed closure suggests that exposure of the urothelium may induce these changes. These findings shed light on the molecular changes in exstrophy-epispadias complex bladders and may have implications on the appropriate timing of primary bladder closure, as those closed in the newborn period appear to have a greater potential for growth and differentiation.

Extrofia vesical y vejiga rectal / No disponible

No disponible

Murine expression and mutation analyses of the prostate androgen-regulated mucin-like protein 1 (Parm1) gene, a candidate for human epispadias.

BACKGROUND: Epispadias is the mildest phenotype of the human bladder exstrophy-epispadias complex (BEEC), and presents with varying degrees of severity. This urogenital birth defect results from a disturbance in the septation process, during which separate urogenital and anorectal components are formed through division of the cloaca. This process is reported to be influenced by androgen signaling. The human PARM1 gene encodes the prostate androgen-regulated mucin-like protein 1, which is expressed in heart, kidney, and placenta. METHODS: We performed whole mount in situ hybridization analysis of Parm1 expression in mouse embryos between gestational days (GD) 9.5 and 12.5, which are equivalent to human gestational weeks 4-6. Since the spatio-temporal localization of Parm1 corresponded to tissues which are affected in human epispadias, we sequenced PARM1 in 24 affected patients. RESULTS: We found Parm1 specifically expressed in the region of the developing cloaca, the umbilical cord, bladder anlage, and the urethral component of the genital tubercle. Additionally, Parm1 expression was detected in the muscle progenitor cells of the somites and head mesenchyme. PARM1 gene analysis revealed no alterations in the coding region of any of the investigated patients. CONCLUSIONS: These findings suggest that PARM1 does not play a major role in the development of human epispadias. However, we cannot rule out the possibility that a larger sample size would enable detection of rare mutations in this gene.

Genome-wide expression profiling of urinary bladder implicates desmosomal and cytoskeletal dysregulation in the bladder exstrophy-epispadias complex.

The bladder exstrophy-epispadias complex (BEEC) represents a spectrum of urological abnormalities where part, or all, of the distal urinary tract fails to close during development, becoming exposed on the outer abdominal wall. While the etiology of BEEC remains unknown, strong evidence exists that genetic factors are implicated. To understand the pathways regulating embryonic bladder development and to identify high-probability BEEC candidate genes, we conducted a genome-wide expression profiling (GWEP) study using normal and exstrophic human urinary bladders and human and mouse embryologic bladder-precursor tissues. We identified 162 genes differentially expressed in both embryonic and postnatal human samples. Pathway analysis of these genes revealed 11 biological networks with top functions related to skeletal and muscular system development, cellular assembly and development, organ morphology, or connective tissue disorders. The two most down-regulated genes desmin (DES, fold-change, -74.7) and desmuslin (DMN, fold-change, -53.0) are involved in desmosome mediated cell-cell adhesion and cytoskeletal architecture. Intriguingly, the sixth most overexpressed gene was desmoplakin (DSP, fold-change, +48.8), the most abundant desmosomal protein. We found 30% of the candidate genes to be directly associated with desmosome structure/function or cytoskeletal assembly, pointing to desmosomal and/or cytoskeletal deregulation as an etiologic factor for BEEC. Further findings indicate that p63, PERP, SYNPO2 and the Wnt pathway may also contribute to BEEC etiology. This study provides the first expression profile of urogenital genes during bladder development and points to the high-probability candidate genes for BEEC.

p63 (TP73L) a key player in embryonic urogenital development with significant dysregulation in human bladder exstrophy tissue.

Human bladder exstrophy-epispadias complex (BEEC) comprises a spectrum of urogenital anomalies in which part or all of the distal urinary tract fails to close. Several lines of evidence implicate genetic factors in the formation of BEEC. Among them a murine p63+/+ knockout model showed the full picture of classic exstrophy of the bladder and other urogenital defects within the BEEC spectrum. This led us to study in depth the role of p63 in urogenital development in mice and investigate the implication of p63 in human BEEC. Whole mount in situ analysis in mice was carried out to investigate the ventro-caudal expression of the p63 transcript at gestational days (GD) 9.5-12.5, the equivalent of human gestational weeks 4-6 (postulated time of BEEC organogenesis in humans). In addition, p63 expression analysis was performed in human blood and bladder derived samples of 15 BEEC newborns accompanied by sequencing analysis of their genomic DNA. We also conducted sequencing analysis of genomic DNA in additional 22 BEEC patients. In mouse embryos, p63 expression was detected at days 9.5-12.5 in the cloacal membrane and urethral epithelium, supporting its role in the morphogenesis of the external genitalia and the bladder. Tissue-specific expression of a novel and already-known mRNA isoforms were established and a reproducible dysregulation of variable p63 isoforms was observed in 11 of 15 patients indicating altered gene expression. However, no obvious p63 gene mutations were identified in any of the patients. Our findings strongly suggest that p63 is not only involved in embryonic formation of the urogenital and ventrocaudal anatomy but is also highly dysregulated in human BEEC bladder tissue. Since p63 has been shown to self-regulate its expression through a balance of its isoforms, the dysregulation observed may contribute to the formation of BEEC.

Embryonic expression of the cysteine rich protein 61 (CYR61) gene: A candidate for the development of human epispadias.

BACKGROUND: Isolated epispadias is the mildest phenotype of the exstrophy-epispadias complex, a urogenital birth defect of variable severity. The androgen receptor antagonist flutamide (FLU) is known to cause malformations in the rat genital and reproductive tract, and single-dose prenatal FLU exposure can induce epispadias in rat offspring. The Cyr61 gene exhibited the highest response to FLU in rat fetal testis, and we suggested it a promising candidate gene for epispadias in humans, because its protein product promotes proliferation, migration, and adhesion of endothelial cells and fibroblasts. METHODS: We used whole mount in situ analysis in mice to investigate ventrocaudal expression of the Cyr61 transcript at gestational days 9.5 to 11.5, which is the equivalent of human gestational weeks 4 to 6 (postulated time of epispadias organogenesis in humans). We also performed mutational analysis of the CYR61 gene in 11 patients with isolated epispadias and in additional eight patients with the related classic bladder exstrophy phenotype. RESULTS: Expression of Cyr61 was detected in endothelial cells of vessels surrounding the cloaca and the umbilical cord on gestational days 10 and 11.5. The mutation screening, however, revealed no alterations in the coding region of human CYR61. CONCLUSIONS: The spatiotemporal expression pattern observed suggests a role for Cyr61 in the development of the external genitalia. Our mutation screening study, however, could not confirm that mutations affecting the CYR61 gene are a frequent cause of epispadias or classic bladder exstrophy, although rare mutations might be detectable in larger patient samples.