Mutations in TBX18 Cause Dominant Urinary Tract Malformations via Transcriptional Dysregulation of Ureter Development.

Congenital anomalies of the kidneys and urinary tract (CAKUT) are the most common cause of chronic kidney disease in the first three decades of life. Identification of single-gene mutations that cause CAKUT permits the first insights into related disease mechanisms. However, for most cases the underlying defect remains elusive. We identified a kindred with an autosomal-dominant form of CAKUT with predominant ureteropelvic junction obstruction. By whole exome sequencing, we identified a heterozygous truncating mutation (c.1010delG) of T-Box transcription factor 18 (TBX18) in seven affected members of the large kindred. A screen of additional families with CAKUT identified three families harboring two heterozygous TBX18 mutations (c.1570C>T and c.487A>G). TBX18 is essential for developmental specification of the ureteric mesenchyme and ureteric smooth muscle cells. We found that all three TBX18 altered proteins still dimerized with the wild-type protein but had prolonged protein half life and exhibited reduced transcriptional repression activity compared to wild-type TBX18. The p.Lys163Glu substitution altered an amino acid residue critical for TBX18-DNA interaction, resulting in impaired TBX18-DNA binding. These data indicate that dominant-negative TBX18 mutations cause human CAKUT by interference with TBX18 transcriptional repression, thus implicating ureter smooth muscle cell development in the pathogenesis of human CAKUT.

Urinary tract effects of HPSE2 mutations.

Urofacial syndrome (UFS) is an autosomal recessive congenital disease featuring grimacing and incomplete bladder emptying. Mutations of HPSE2, encoding heparanase 2, a heparanase 1 inhibitor, occur in UFS, but knowledge about the HPSE2 mutation spectrum is limited. Here, seven UFS kindreds with HPSE2 mutations are presented, including one with deleted asparagine 254, suggesting a role for this amino acid, which is conserved in vertebrate orthologs. HPSE2 mutations were absent in 23 non-neurogenic neurogenic bladder probands and, of 439 families with nonsyndromic vesicoureteric reflux, only one carried a putative pathogenic HPSE2 variant. Homozygous Hpse2 mutant mouse bladders contained urine more often than did wild-type organs, phenocopying human UFS. Pelvic ganglia neural cell bodies contained heparanase 1, heparanase 2, and leucine-rich repeats and immunoglobulin-like domains-2 (LRIG2), which is mutated in certain UFS families. In conclusion, heparanase 2 is an autonomic neural protein implicated in bladder emptying, but HPSE2 variants are uncommon in urinary diseases resembling UFS.

Whole-genome linkage and association scan in primary, nonsyndromic vesicoureteric reflux.

Primary vesicoureteric reflux accounts for approximately 10% of kidney failure requiring dialysis or transplantation, and sibling studies suggest a large genetic component. Here, we report a whole-genome linkage and association scan in primary, nonsyndromic vesicoureteric reflux and reflux nephropathy. We used linkage and family-based association approaches to analyze 320 white families (661 affected individuals, generally from families with two affected siblings) from two populations (United Kingdom and Slovenian). We found modest evidence of linkage but no clear overlap with previous studies. We tested for but did not detect association with six candidate genes (AGTR2, HNF1B, PAX2, RET, ROBO2, and UPK3A). Family-based analysis detected associations with one single-nucleotide polymorphism (SNP) in the UK families, with three SNPs in the Slovenian families, and with three SNPs in the combined families. A case-control analysis detected associations with three additional SNPs. The results of this study, which is the largest to date investigating the genetics of reflux, suggest that major loci may not exist for this common renal tract malformation within European populations.

Primary, nonsyndromic vesicoureteric reflux and nephropathy in sibling pairs: a United Kingdom cohort for a DNA bank.

BACKGROUND AND OBJECTIVES: Primary vesicoureteric reflux (VUR) can coexist with reflux nephropathy (RN) and impaired renal function. VUR appears to be an inherited condition and is reported in approximately one third of siblings of index cases. The objective was to establish a DNA collection and clinical database from U.K. families containing affected sibling pairs for future VUR genetics studies. The cohort's clinical characteristics have been described. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Most patients were identified from tertiary pediatric nephrology centers; each family had an index case with cystography-proven primary, nonsyndromic VUR. Affected siblings had radiologically proven VUR and/or radiographically proven RN. RESULTS: One hundred eighty-nine index cases identified families with an additional 218 affected siblings. More than 90% were <20 years at the study's end. Blood was collected and leukocyte DNA extracted from all 407 patients and from 189 mothers and 183 fathers. Clinical presentation was established in 122; 92 had urinary tract infections and 16 had abnormal antenatal renal scans. RN was radiologically proven in 223 patients. Four patients had been transplanted; none were on dialysis. In 174 others aged >1 year, estimated GFR (eGFR) was calculated. Five had eGFR 15 to 59 and 48 had eGFR 60 to 89 ml/min per 1.73 m(2). Values were lower in bilateral RN patients than in those with either unilateral or absent RN. CONCLUSIONS: The large DNA collection from families with VUR and associated RN constitutes a resource for researchers exploring the most likely complex, genetic components predisposing to VUR and RN.

Mutational analyses of UPIIIA, SHH, EFNB2 and HNF1beta in persistent cloaca and associated kidney malformations.

OBJECTIVES: 'Persistent cloaca' is a severe malformation affecting females in which the urinary, genital and alimentary tracts share a single conduit. Previously, a Uroplakin IIIA (UPIIIA) mutation was reported in one individual with persistent cloaca, and UPIIIA, Sonic Hedgehog (SHH), Ephrin B2 (EFNB2) and Hepatocyte Nuclear Factor 1beta (HNF1beta) are expressed during the normal development of organs that are affected in this condition. HNF1beta mutations have been associated with uterine malformations in humans, and mutations of genes homologous to human SHH or EFNB2 cause persistent cloaca in mice. PATIENTS AND METHODS: We sought mutations of coding regions of UPIIIA, SHH, EFNB2 and HNF1beta genes by direct sequencing in a group of 20 patients with persistent cloaca. Most had associated malformations of the upper renal tract and over half had impaired renal excretory function. The majority of patients had congenital anomalies outside the renal/genital tracts and two had the VACTERL association. RESULTS: Apart from a previously described index case, we failed to find UPIIIA mutations, and no patient had a SHH, EFNB2 or HNF1beta mutation. CONCLUSION: Persistent cloaca is only rarely associated with UPIIIA mutation. Despite the fact that SHH and EFNB2 are appealing candidate genes, based on their expression patterns and mutant mice phenotypes, they were not mutated in these humans with persistent cloaca. Although HNF1beta mutations can perturb paramesonephric duct fusion in humans, HNF1beta was not mutated in persistent cloaca.

Disruption of ROBO2 is associated with urinary tract anomalies and confers risk of vesicoureteral reflux.

Congenital anomalies of the kidney and urinary tract (CAKUT) include vesicoureteral reflux (VUR). VUR is a complex, genetically heterogeneous developmental disorder characterized by the retrograde flow of urine from the bladder into the ureter and is associated with reflux nephropathy, the cause of 15% of end-stage renal disease in children and young adults. We investigated a man with a de novo translocation, 46,X,t(Y;3)(p11;p12)dn, who exhibits multiple congenital abnormalities, including severe bilateral VUR with ureterovesical junction defects. This translocation disrupts ROBO2, which encodes a transmembrane receptor for SLIT ligand, and produces dominant-negative ROBO2 proteins that abrogate SLIT-ROBO signaling in vitro. In addition, we identified two novel ROBO2 intracellular missense variants that segregate with CAKUT and VUR in two unrelated families. Adult heterozygous and mosaic mutant mice with reduced Robo2 gene dosage also exhibit striking CAKUT-VUR phenotypes. Collectively, these results implicate the SLIT-ROBO signaling pathway in the pathogenesis of a subset of human VUR.

Mutation analyses of Uroplakin II in children with renal tract malformations.

BACKGROUND: Uroplakin (UP) proteins cover urothelial apical surfaces. Mice lacking UPIIIa have elevated urothelial permeability and congenital renal tract anomalies, and UPIIIa mutations have been reported in children with kidney and ureter malformations. Mice with null mutation of another UP family member, UPII, are often born with congenital hydronephrosis. We hypothesized that UPII mutations may be present in humans with renal tract malformations. METHODS: Mutations were sought, using direct sequencing of the five UPII exons, in 42 children with diverse renal tract anomalies. RESULTS: No UPII abnormalities were detected in 41 patients, whereas one index case had a heterozygous frameshift change which, if expressed, would generate a truncated protein. This Caucasian child presented with vesicoureteric reflux (VUR), bilateral nephropathy and renal failure. The genetic change was also found in the index case's mother who had normal renal ultrasonography, but it was absent in 150 healthy Caucasian control individuals (96 assessed by direct sequencing and another 54 assessed by restriction digests). UPII was immunolocalized in urothelium of the normal human embryonic renal pelvis in a pattern similar to UPIIIa. CONCLUSION: This study offers no definitive support for UPII mutations causing human renal tract malformations. In rare patients, UPII variants might be implicated in pathogenesis when acting in conjunction with other yet-to-be-defined, genetic or environmental modifying factors.

De novo Uroplakin IIIa heterozygous mutations cause human renal adysplasia leading to severe kidney failure.

Human renal adysplasia usually occurs sporadically, and bilateral disease is the most common cause of childhood end-stage renal failure, a condition that is lethal without intervention using dialysis or transplantation. De novo heterozygous mutations in Uroplakin IIIa (UPIIIa) are reported in four of 17 children with kidney failure caused by renal adysplasia in the absence of an overt urinary tract obstruction. One girl and one boy in unrelated kindreds had a missense mutation at a CpG dinucleotide in the cytoplasmic domain of UPIIIa (Pro273Leu), both of whom had severe vesicoureteric reflux, and the girl had persistent cloaca; two other patients had de novo mutations in the 3' UTR (963 T-->G; 1003 T-->C), and they had renal adysplasia in the absence of any other anomaly. The mutations were absent in all sets of parents and in siblings, none of whom had radiologic evidence of renal adysplasia, and mutations were absent in two panels of 192 ethnically matched control chromosomes. UPIIIa was expressed in nascent urothelia in ureter and renal pelvis of human embryos, and it is suggested that perturbed urothelial differentiation may generate human kidney malformations, perhaps by altering differentiation of adjacent smooth muscle cells such that the metanephros is exposed to a functional obstruction of urine flow. With advances in renal replacement therapy, children with renal failure, who would otherwise have died, are surviving to adulthood. Therefore, although the mechanisms of action of the UPIIIa mutations have yet to be determined, these findings have important implications regarding genetic counseling of affected individuals who reach reproductive age.

Recent insights into kidney diseases associated with glomerular cysts.

Glomerular cysts can exist in the context of several different kidney diseases. Advances in the last few years have begun to unravel the genetic bases and pathogenesis of some of these entities, many of which have an origin in abnormal development. In this review, we highlight recent insights into three types of disease associated with glomerular cysts: (1) mutations of the hepatocyte nuclear factor 1beta (HNF-1beta)in the recently described renal cysts and diabetes syndrome, (2) mutations of OFD1in the oral facial digital syndrome type 1 and (3) the role of fetal urinary tract obstruction.

Oral-facial-digital syndrome VII is oral-facial-digital syndrome I: a clarification.

We report on further clinical findings in the one single family in the literature classified as oral-facial-digital (OFD) type VII in order to demonstrate that the diagnosis in this kindred should, in fact, be OFD type I. The mother and the daughter described in the original report have since developed polycystic kidney disease. In addition, the daughter recently had a daughter of her own with central nervous system, oral and digital anomalies. Linkage studies have shown that all the affected women share the same haplotype across the previously identified region Xp22.2p22.3 to which OFD I maps. Although the pedigree was too small for a significant lod score, the combination of clinical and molecular information clearly shows that the disease in this family is OFD I. We report this family in order to clarify and simplify the classification of the oral-facial-digital syndrome spectrum and to recommend the removal of OFD VII from the classification system of the oral-facial-digital syndromes.

OFD1 is a centrosomal/basal body protein expressed during mesenchymal-epithelial transition in human nephrogenesis.

OFD1 is the gene responsible for the oral-facial-digital syndrome type 1, a cause of inherited cystic renal disease. The protein contains an N-terminal LisH motif, considered important in microtubule dynamics, and several putative coiled-coil domains. This study used a combination of microscopic, biochemical, and overexpression approaches to demonstrate that OFD1 protein is a core component of the human centrosome throughout the cell cycle. Using a series of GFP-OFD1 deletion constructs, it was determined that the N-terminus containing the LisH domain is not required for centrosomal localization; however, coiled-coil domains are critical, with at least two being necessary for centrosomal targeting. Importantly, most reported OFD1 mutations are predicted to cause protein truncation with loss of coiled-coil domains, presumably leading to loss of centrosomal localization. Kidney development constitutes a classic model of mesenchymal-epithelial transformation. By immunoprobing human metanephroi and kidney epithelial lines, it was found that, during acquisition of epithelial polarity, OFD1 became localized to the apical zone of nephron precursor cells and then to basal bodies at the origin of primary cilia in fully differentiated epithelia. These striking patterns of OFD1 localization within cells place the protein at key sites, where it may play roles not only in microtubule organization (centrosomal function) but also in mechanosensation of urine flow (a primary ciliary function).

OFD1, the gene mutated in oral-facial-digital syndrome type 1, is expressed in the metanephros and in human embryonic renal mesenchymal cells.

Oral-facial-digital syndrome type 1 (OFD1) causes polycystic kidney disease (PKD) and malformations of the mouth, face and digits. Recently, a gene on Xp22, OFD1, was reported to be mutated in a limited set of OFD1 patients. This study describes mutation analysis in six further OFD1 families. Additionally, gene expression was sought in human development. In two OFD1 kindreds affected by PKD, a frameshift mutation and a splice-site mutation were detected. In four apparently sporadic cases, three frameshift and a missense mutation were found. Using RT-PCR of RNA from first-trimester normal human embryos, both alternative splice forms of mRNA (OFD1a and OFD1b) were found to be widely expressed in organogenesis. Northern blot detected OFD1 mRNA in metanephros, brain, tongue, and limb, all organs affected in the syndrome. A polyclonal antibody directed to a C-terminal OFD1a epitope detected a 120-kD protein in the metanephros and in human renal mesenchymal cell lines. In normal human embryos, OFD1a immunolocalized to the metanephric mesenchyme, oral mucosa, nasal and cranial cartilage, and brain. Moreover, using normal human renal mesenchymal cell lines, the immunoreactive protein colocalized with gamma-tubulin, suggesting that OFD1 is associated with the centrosome. First, it is concluded that OFD1 mutations would generally be predicted to result in unstable transcripts or nonfunctional proteins. Second, OFD1 is expressed in human organogenesis; on the basis of the metanephric expression pattern, the results suggest that OFD1 plays a role in differentiation of metanephric precursor cells.

Lack of major involvement of human uroplakin genes in vesicoureteral reflux: implications for disease heterogeneity.

BACKGROUND: Primary vesicoureteral reflux (VUR) is a hereditary disorder characterized by the retrograde flow of urine into the ureters and kidneys. It affects about 1% of the young children and is thus one of the most common hereditary diseases. Its associated nephropathy is an important cause of end-stage renal failure in children and adults. Recent studies indicate that genetic ablation of mouse uroplakin (UP) III gene, which encodes a 47 kD urothelial-specific integral membrane protein forming urothelial plaques, causes VUR and hydronephrosis. METHODS: To begin to determine whether mutations in UP genes might play a role in human VUR, we genotyped all four UP genes in 76 patients with radiologically proven primary VUR by polymerase chain reaction (PCR) amplification and sequencing of all their exons plus 50 to 150 bp of flanking intronic sequences. RESULTS: Eighteen single nucleotide polymorphisms (SNPs) were identified, seven of which were missense, with no truncation or frame shift mutations. Since healthy relatives of the VUR probands are not reliable negative controls for VUR, we used a population of 90 race-matched, healthy individuals, unrelated to the VUR patients, as controls to perform an association study. Most of the SNPs were not found to be significantly associated with VUR. However, SNP1 of UP Ia gene affecting a C to T conversion and an Ala7Val change, and SNP7 of UP III affecting a C to G conversion and a Pro154Ala change, were marginally associated with VUR (both P= 0.08). Studies of additional cases yielded a second set of data that, in combination with the first set, confirmed a weak association of UP III SNP7 in VUR (P= 0.036 adjusted for both subsets of cases vs. controls). CONCLUSION: Such a weak association and the lack of families with simple dominant Mendelian inheritance suggest that missense changes of uroplakin genes cannot play a dominant role in causing VUR in humans, although they may be weak risk factors contributing to a complex polygenic disease. The fact that no truncation or frame shift mutations have been found in any of the VUR patients, coupled with our recent finding that some breeding pairs of UP III knockout mice yield litters that show not only VUR, but also severe hydronephrosis and neonatal death, raises the possibility that major uroplakin mutations could be embryonically or postnatally lethal in humans.