The utility of gene sequencing in identifying an underlying genetic disorder in prenatally suspected lower urinary tract obstruction.

OBJECTIVE: Fetal megacystis generally presents as suspected lower urinary tract obstruction (LUTO), which is associated with severe perinatal morbidity. Genetic etiologies underlying LUTO or a LUTO-like initial presentation are poorly understood. Our objectives are to describe single gene etiologies in fetuses initially ascertained to have suspected LUTO and to elucidate genotype-phenotype correlations. METHODS: A retrospective case series of suspected fetal LUTO positive for a molecular diagnosis was collected from five centers in the Fetal Sequencing Consortium. Demographics, sonograms, genetic testing including variant classification, and delivery outcomes were abstracted. RESULTS: Seven cases of initially prenatally suspected LUTO-positive for a molecular diagnosis were identified. In no case was the final diagnosis established as urethral obstruction that is, LUTO. All variants were classified as likely pathogenic or pathogenic. Smooth muscle deficiencies involving the bladder wall and interfering with bladder emptying were identified in five cases: MYOCD (2), ACTG2 (2), and MYH11 (1). Other genitourinary and/or non-genitourinary malformations were seen in two cases involving KMT2D (1) and BBS10 (1). CONCLUSION: Our series illustrates the value of molecular diagnostics in the workup of fetuses who present with prenatally suspected LUTO but who may have a non-LUTO explanation for their prenatal ultrasound findings.

Genome-Wide Survey for Microdeletions or -Duplications in 155 Patients with Lower Urinary Tract Obstructions (LUTO).

Lower urinary tract obstruction (LUTO) is, in most cases, caused by anatomical blockage of the bladder outlet. The most common form are posterior urethral valves (PUVs), a male-limited phenotype. Here, we surveyed the genome of 155 LUTO patients to identify disease-causing CNVs. Raw intensity data were collected for CNVs detected in LUTO patients and 4.392 healthy controls using CNVPartition, QuantiSNP and PennCNV. Overlapping CNVs between patients and controls were discarded. Additional filtering implicated CNV frequency in the database of genomic variants, gene content and final visual inspection detecting 37 ultra-rare CNVs. After, prioritization qPCR analysis confirmed 3 microduplications, all detected in PUV patients. One microduplication (5q23.2) occurred de novo in the two remaining microduplications found on chromosome 1p36.21 and 10q23.31. Parental DNA was not available for segregation analysis. All three duplications comprised 11 coding genes: four human specific lncRNA and one microRNA. Three coding genes (FBLIM1, SLC16A12, SNCAIP) and the microRNA MIR107 have previously been shown to be expressed in the developing urinary tract of mouse embryos. We propose that duplications, rare or de novo, contribute to PUV formation, a male-limited phenotype.

Urinary miRNA profiles discriminate between obstruction-induced bladder dysfunction and healthy controls.

Urgency, frequency and incomplete emptying are the troublesome symptoms often shared between benign prostatic obstruction-induced (BLUTD) and neurogenic (NLUTD) lower urinary tract dysfunction. Previously, using bladder biopsies, we suggested a panel of miRNA biomarkers for different functional phenotypes of the bladder. Urine is a good source of circulating miRNAs, but sex- and age-matched controls are important for urinary metabolite comparison. In two groups of healthy subjects (average age 32 and 57 years old, respectively) the total protein and RNA content was very similar between age groups, but the number of secreted extracellular vesicles (uEVs) and expression of several miRNAs were higher in the young healthy male volunteers. Timing of urine collection was not important for these parameters. We also evaluated the suitability of urinary miRNAs for non-invasive diagnosis of bladder outlet obstruction (BOO). A three urinary miRNA signature (miR-10a-5p, miR-301b-3p and miR-363-3p) could discriminate between controls and patients with LUTD (BLUTD and NLUTD). This panel of representative miRNAs can be further explored to develop a non-invasive diagnostic test for BOO. The age-related discrepancy in the urinary miRNA content observed in this study points to the importance of selecting appropriate, age-matched controls.

Bilateral aniridia and congenital ureteral valve: Role of genetic testing.

BACKGROUND: Congenital aniridia involves total or partial hypoplasia of the iris and is due to a deficiency in PAX6 gene expression. WAGR syndrome is comprised of Wilms tumor, aniridia, genitourinary abnormalities, and intellectual disability. Numerous genitourinary pathologies may be associated with WAGR syndrome, necessitating an evaluation of the genitourinary anatomy. The WT1 is vital for the development of kidneys, ovaries in females, and testes in males. WT1 gene mutations result in a WT1 protein with a decreased ability to bind to DNA, leading to uncontrolled growth, and cell division in the kidney which permits the development of Wilms tumor. A congenital ureteral valve is an exceedingly rare cause of obstructive uropathy. RESULTS: A renal and bladder ultrasound demonstrated a renal cyst. A voiding cystourethrogram revealed grade 3 vesicoureteral reflux, and a MAG3 renal scan showed ureteropelvic junction obstruction and hydronephrosis. A ureteral stent was inserted at 3 months of age after which the renal cyst resolved. The patient was urinary tract infection-free at 27 months of age. Genetic testing confirmed a heterozygous alteration in PAX6 (c.495delG, p.Thr166Leufs*41) and no abnormalities of WT1, excluding WAGR syndrome. CONCLUSION: The genitourinary risks potentially associated with aniridia necessitate prompt genetic analysis to evaluate for WAGR syndrome.

Preclinical Study of DNA-Recognized Peptide Compound Pyrrole-Imidazole Polyamide Targeting Human TGF-ß1 Promoter for Progressive Renal Diseases in the Common Marmoset.

Pyrrole-imidazole (PI) polyamides are novel gene silencers that strongly bind the promoter region of target genes in a sequence-specific manner to inhibit gene transcription. We created a PI polyamide targeting human TGF-ß1 (hTGF-ß1). To develop this PI polyamide targeting hTGF-ß1 (Polyamide) as a practical medicine for treating progressive renal diseases, we examined the effects of Polyamide in two common marmoset models of nephropathy. We performed lead optimization of PI polyamides that targeted hTGF-ß1 by inhibiting in a dose-dependent manner the expression of TGF-ß1 mRNA stimulated by PMA in marmoset fibroblasts. Marmosets were housed and fed with a 0.05% NaCl and magnesium diet and treated with cyclosporine A (CsA; 37.5 mg/kg/day, eight weeks) to establish chronic nephropathy. We treated the marmosets with nephropathy with Polyamide (1 mg/kg/week, four weeks). We also established a unilateral urethral obstruction (UUO) model to examine the effects of Polyamide (1 mg/kg/week, four times) in marmosets. Histologically, the renal medulla from CsA-treated marmosets showed cast formation and interstitial fibrosis in the renal medulla. Immunohistochemistry showed strong staining of Polyamide in the renal medulla from CsA-treated marmosets. Polyamide treatment (1 mg/kg/week, four times) reduced hTGF-ß1 staining and urinary protein excretion in CsA-treated marmosets. In UUO kidneys from marmosets, Polyamide reduced the glomerular injury score and tubulointerstitial injury score. Polyamide significantly suppressed hTGF-ß1 and snail mRNA expression in UUO kidneys from the marmosets. Polyamide effectively improved CsA- and UUO-associated nephropathy, indicating its potential application in the prevention of renal fibrosis in progressive renal diseases.

Double knockout of Bax and Bak from kidney proximal tubules reduces unilateral urethral obstruction associated apoptosis and renal interstitial fibrosis.

Interstitial fibrosis, a common pathological feature of chronic kidney diseases, is often associated with apoptosis in renal tissues. To determine the associated apoptotic pathway and its role in renal interstitial fibrosis, we established a mouse model in which Bax and Bak, two critical genes in the intrinsic pathway of apoptosis, were deleted specifically from kidney proximal tubules and used this model to examine renal apoptosis and interstitial fibrosis following unilateral urethral obstruction (UUO). It was shown that double knockout of Bax and Bak from proximal tubules attenuated renal tubular cell apoptosis and suppressed renal interstitial fibrosis in UUO. The results indicate that the intrinsic pathway of apoptosis contributes significantly to the tubular apoptosis and renal interstitial fibrosis in kidney diseases.

p53 induces miR199a-3p to suppress SOCS7 for STAT3 activation and renal fibrosis in UUO.

The role of p53 in renal fibrosis has recently been suggested, however, its function remains controversial and the underlying mechanism is unclear. Here, we show that pharmacological and genetic blockade of p53 attenuated renal interstitial fibrosis, apoptosis, and inflammation in mice with unilateral urethral obstruction (UUO). Interestingly, p53 blockade was associated with the suppression of miR-215-5p, miR-199a-5p&3p, and STAT3. In cultured human kidney tubular epithelial cells (HK-2), TGF-ß1 treatment induced fibrotic changes, including collagen I and vimentin expression, being associated with p53 accumulation, p53 Ser15 phosphorylation, and miR-199a-3p expression. Inhibition of p53 by pifithrin-α blocked STAT3 activation and the expression of miR-199a-3p, collagen I, and vimentin during TGF-ß1 treatment. Over-expression of miR-199a-3p increased TGFß1-induced collagen I and vimentin expression and restored SOCS7 expression. Furthermore, SOCS7 was identified as a target gene of miR-199a-3p, and silencing of SOCS7 promoted STAT3 activation. ChIp analyses indicated the binding of p53 to the promoter region of miR-199a-3p. Consistently, kidney biopsies from patients with IgA nephropathy and diabetic nephropathy exhibited substantial activation of p53 and STAT3, decreased expression of SOCS7, and increase in profibrotic proteins and miR-199a-3p. Together, these results demonstrate the novel p53/miR-199a-3p/SOCS7/STAT3 pathway in renal interstitial fibrosis.

Loss of MeCP2 Causes Urological Dysfunction and Contributes to Death by Kidney Failure in Mouse Models of Rett Syndrome.

Rett Syndrome (RTT) is a neurodevelopmental disorder characterized by loss of acquired skills during development, autonomic dysfunction, and an increased risk for premature lethality. Clinical experience identified a subset of individuals with RTT that present with urological dysfunction including individuals with frequent urinary tract infections, kidney stones, and urine retention requiring frequent catheterization for bladder voiding. To determine if urologic dysfunction is a feature of RTT, we queried the Rett Syndrome Natural History Study, a repository of clinical data from over 1000 individuals with RTT and found multiple instances of urological dysfunction. We then evaluated urological function in a mouse model of RTT and found an abnormal pattern of micturition. Both male and female mice possessing Mecp2 mutations show a decrease in urine output per micturition event. Furthermore, we identified signs of kidney failure secondary to urethral obstruction. Although genetic strain background significantly affects both survival and penetrance of the urethral obstruction phenotype, survival and penetrance of urethral obstruction do not directly correlate. We have identified an additional phenotype caused by loss of MeCP2, urological dysfunction. Furthermore, we urge caution in the interpretation of survival data as an endpoint in preclinical studies, especially where causes of mortality are poorly characterized.

Recurrent Johanson-Blizzard syndrome in a triplet pregnancy complicated by urethral obstruction sequence: a clinical, molecular, and immunohistochemical approach.

We report on a triplet pregnancy of consanguineous parents with one fetus being affected by recurrent Johanson-Blizzard syndrome (JBS). At autopsy in the 35th gestational week, the affected triplet presented with an especially severe and lethal manifestation of the disorder as compared to his elder affected brother and to cases in the literature, thus exemplifying great interfamilial and intrafamilial phenotypic variability. Arhinencephaly and cystic renal dysplasia associated with urethral obstruction sequence were features not described previously in the literature. In addition to the lack of exocrine acini as the characteristic feature of JBS, the pancreas revealed a resorptive inflammatory reaction with infiltration by eosinophilic granulocytes that focally dispersed onto islets of Langerhans, thus favoring a progressive destructive rather than primary dysplastic process and possibly explaining the occurrence of diabetes mellitus in later life. JBS maps to chromosome 15q15-q21.1 and is associated with mutations in the UBR1 gene. Testing the fetus and the affected sibling revealed a homozygous truncating mutation in UBR1. The resulting absence of the UBR1 protein was confirmed by Western blot. Immunohistochemical staining using a commercial anti-UBR1 antibody demonstrated staining, presumably artifactual. This finding suggests that, until an appropriately validated antibody has been identified, this modality should not be utilized for diagnosis or confirmation of this disorder.

Genetic and developmental basis for urinary tract obstruction.

Urinary tract obstruction results in obstructive nephropathy and uropathy. It is the most frequent cause of renal failure in infants and children. In the past two decades studies of transgenic models and humans have greatly enhanced our understanding of the genetic factors and developmental processes important in urinary tract obstruction. The emerging picture is that development of the urinary tract requires precise integration of a variety of progenitor cell populations of different embryonic origins. Such integration is controlled by an intricate signaling network that undergoes dynamic changes as the embryo develops. Most congenital forms of urinary tract obstruction result from the disruption of diverse factors and genetic pathways involved in these processes, especially in the morphogenesis of the urinary conduit or the functional aspects of the pyeloureteral peristaltic machinery.

Obstructive nephropathy: insights from genetically engineered animals.

Congenital obstructive nephropathy is the primary cause for end-stage renal disease (ESRD) in children. An increasingly used animal model of obstructive nephropathy is unilateral ureteral obstruction (UUO). This model mimics, in an accelerated manner, the different stages of obstructive nephropathy leading to tubulointerstitial fibrosis: cellular infiltration, tubular proliferation and apoptosis, epithelial-mesenchymal transition (EMT), (myo)fibroblast accumulation, increased extracellular matrix (ECM) deposition, and tubular atrophy. During the last decade genetically modified animals are increasingly used to study the development of obstructive nephropathy. Although the use of these animals (mainly knockouts) has highlighted some pitfalls of this approach (compensation by closely related gene products, absence of temporal knockouts) it has brought important information about the role of specific gene-products in the pathogenesis of obstructive nephropathy. Besides confirming the important pathologic role for angiotensin II (Ang II) and transforming growth factor-beta (TGF-beta) in obstructive nephropathy, these animals have shown the complexity of the development of tubulointerstitial fibrosis involving a large number of closely functionally related molecules. More interestingly, the use of these animals has led to the discovery of unexpected and contradictory roles (both potentially pro- and antifibrotic) for Ang II, for ECM degrading enzymes matrix metalloproteinase 9 (MMP-9) and tissue plasminogen activators (PAs), for plasminogen activator inhibitor 1 (PAI-1), and for the adhesion molecule osteopontin (OPN) in obstructive nephropathy. Further use of these animals, especially in combination with pharmacologic tools, should help to better identify potential antifibrotic strategies in obstructive nephropathy.

Sexual dysfunction and sudden death in epileptic male EL mice: inheritance and prevention with the ketogenic diet.

PURPOSE: The EL mouse is an animal model for multifactorial idiopathic epilepsy. Although EL mice have been studied extensively for >45 years, the etiology of male sudden death and its relation to seizures have not been defined. Here we investigated the cause of EL male sudden death and its relation to epilepsy. METHODS: For histopathologic analysis, the terminally ill EL mice (n = 15) were killed, and the tissues were fixed. Blood chemical composition was compared between the terminally ill EL (n = 9) and the healthy age-matched EL (n = 17) and DDY (n = 11) males. To determine the effect of the ketogenic diet (KD) on sudden male death, young male EL mice (P30) were randomly separated into two groups that were fed ad libitum with either Agway lab chow (control n = 38) or with the KD (treated, n = 39) for 5 months. The genetic predisposition to sudden death was analyzed in the backcross generation (n = 106) of a cross between EL and the nonepileptic ABP strains. RESULTS: Sudden death coincided with the onset of seizures (70-80 days) and affected 94% of male EL mice by age 300 days. Urethral plugs were observed histologically in 13 of 15 longitudinally sectioned penises. Concentrations of blood urea nitrogen, creatinine, phosphorus, and calcium in the terminally ill mice were significantly elevated when compared with those of healthy animals. None of the mice treated with the KD experienced sudden death, whereas 15 (39%) of the untreated control mice died by age 5 months. The sudden death in male EL mice was inherited as an autosomal recessive sex-limited lethal trait. CONCLUSIONS: The cause of sudden death in male EL mice arises from abnormal ejaculation, which produces a urethral plug with consequent urinary retention and acute severe uremia. The coincident onset of seizures and sudden death in EL males suggests that a sexual dysfunction is associated with epilepsy in this model.

Possible maternal inheritance of a common obstructive urinary tract anomaly. Report of a case of a woman with multiple urinary tract infections and two sons with posterior urethral valves.

BACKGROUND: Posterior urethral valves (PUVs) are the most common cause of lower urinary tract obstruction in boys. Several prior reports have described PUV in siblings and twins, often with a variable phenotype. CASE: PUVs were found in two brothers. The first child had developmental delay, while the second had a branchial cleft cyst and an anteriorly rotated right ear. The children's mother had a history of numerous urinary tract infections of unknown etiology. CONCLUSION: Though most cases of PUV appear to be sporadic, there are reports that suggest a partial genetic etiology, as our case suggests.

Genetic homogeneity of the urofacial (Ochoa) syndrome confirmed in a new French family.

The urofacial syndrome (UFS) or Ochoa syndrome has been reported as a rare autosomal recessive disorder comprising a uropathy and facial abnormalities. The gene was mapped on chromosome region 10q23-q24. We report the first European cases of UFS. Haplotype analyses in our French family were compared with those previously described in patients from Columbia and America (literature data). The results are compatible with the same localization of the critical region and favor the hypothesis of genetic homogeneity.

Epidermolysis bullosa, pyloric atresia, and obstructive uropathy: a report of two case reports with molecular correlation and clinical management.

The epidermolysis bullosa-pyloric atresia-obstructive uropathy (EB-PA-OU) association is a rare, but well-described multisystem disease. While the prognosis at this time is still poor, an increasing number of patients are surviving to adolescence with aggressive care. It is important to understand this syndrome in order to anticipate medical complications and offer preventive strategies where possible. Prompt and expectant management of obstructive uropathy is crucial in these patients. Evidence of ureterovesicular obstruction may require bowel diversion, as excision of the obstructed ureterovesicular junction with reimplantation is often associated with a high risk of reobstruction. Many newborns succumb to sepsis or dehydration and electrolyte imbalance. Those infants who survive need close monitoring for the development of obstructive uropathy, failure to thrive, protein-losing enteropathy, respiratory compromise, and increased susceptibility to invasive infections. Once a clinical diagnosis is made, mutational analysis can confirm it and facilitate genetic counseling, as recurrence risks are 25% for this autosomal recessive condition. Mutational analysis enables direct genetic testing and accurate prenatal diagnosis. As more patients are studied, genotype/phenotype correlations may be possible.

Familial segregation of cervical ribs, Sprengel anomaly, preaxial polydactyly, anal atresia, and urethral obstruction: a new syndrome?

In a consanguineous Jewish family originating from Bombay, India, the propositus presented with anal atresia, micropenis, urethral obstruction with secondary prune belly, omphalocele, patent urachus, and cryptorchidism. The kidneys were dysplastic and he had the Potter phenotype with limb deformities. Additional findings included IUGR with microcephaly, congenital heart defects, spinal anomalies, and hypoplastic lungs. The mother and all three sisters had cervical ribs, and she and one sister had 11 pairs of thoracic ribs. The other two sisters had chronic immune thrombopenia. One of those had bilateral Sprengel deformity with homovertebral bones, club feet, and microcephaly and the other sister also had unilateral preaxial hexadactyly. Although familial segregation of cervical ribs and Sprengel deformity has been reported, the association of the findings in this family is unique and may represent a new syndrome. X-linked dominant transmission may explain the severe manifestations in the affected male, but other modes of inheritance may also apply.

Renal dysplasia, megalocystis, and sirenomelia in four siblings.

First degree relatives of infants with bilateral renal agenesis or dysplasia have an increased risk of renal abnormalities including renal agenesis. We report a family in which four successive offspring exhibited a previously undescribed combination of congenital lethal renal disorders: bilateral renal dysplasia, megalocystis secondary to urethral obstruction and, sirenomelia with associated renal agenesis.