Adrian S. Woolf

New criteria for improved diagnosis of Bardet-Biedl syndrome: results of a population survey

Bardet-Biedl syndrome (BBS) is an autosomal recessive condition characterised by rod-cone dystrophy, postaxial polydactyly, central obesity, mental retardation, hypogonadism, and renal dysfunction. BBS expression varies both within and between families and diagnosis is often difficult. We sought to define the condition more clearly by studying 109 BBS patients and their families, the largest population surveyed to date. The average age at diagnosis was 9 years, which is late for such a debilitating condition, but the slow development of the clinical features of BBS probably accounts for this. Postaxial polydactyly had been present in 69% of patients at birth, but obesity had only begun to develop at around 2-3 years, and retinal degeneration had not become apparent until a mean age of 8.5 years. Our study identified some novel clinical features, including neurological, speech, and language deficits, behavioural traits, facial dysmorphism, and dental anomalies. In the light of these features we propose a revision of the diagnostic criteria, which may facilitate earlier diagnosis of this disorder. We present evidence for an overlapping phenotype with the Laurence-Moon syndrome and propose a unifying, descriptive label be adopted (polydactyly-obesity-kidney-eye syndrome).  We report an increased prevalence of renal malformations and renal cell carcinoma in the unaffected relatives of BBS patients and suggest that these may be a consequence of heterozygosity for BBS genes. Our findings have important implications for the care of BBS patients and their unaffected relatives.

Mutations in the Hepatocyte Nuclear Factor-1β Gene Are Associated with Familial Hypoplastic Glomerulocystic Kidney Disease

Familial glomerulocystic kidney disease (GCKD) is a dominantly inherited condition characterized by glomerular cysts and variable renal size and function; the molecular genetic etiology is unknown. Mutations in the gene encoding hepatocyte nuclear factor (HNF)-1beta have been associated with early-onset diabetes and nondiabetic renal disease-particularly renal cystic disease. We investigated a possible role for the HNF-1beta gene in four unrelated GCKD families and identified mutations in two families: a nonsense mutation in exon 1 (E101X) and a frameshift mutation in exon 2 (P159fsdelT). The family members with HNF-1beta gene mutations had hypoplastic GCKD and early-onset diabetes or impaired glucose tolerance. We conclude that there is genetic heterogeneity in familial GCKD and that the hypoplastic subtype is a part of the clinical spectrum of the renal cysts and diabetes syndrome that is associated with HNF-1beta mutations.

Fraser syndrome and mouse blebbed phenotype caused by mutations in FRAS1/Fras1 encoding a putative extracellular matrix protein.

Fraser syndrome (OMIM 219000) is a multisystem malformation usually comprising cryptophthalmos, syndactyly and renal defects. Here we report autozygosity mapping and show that the locus FS1 at chromosome 4q21 is associated with Fraser syndrome, although the condition is genetically heterogeneous. Mutation analysis identified five frameshift mutations in FRAS1, which encodes one member of a family of novel proteins related to an extracellular matrix (ECM) blastocoelar protein found in sea urchin. The FRAS1 protein contains a series of N-terminal cysteine-rich repeat motifs previously implicated in BMP metabolism, suggesting that it has a role in both structure and signal propagation in the ECM. It has been speculated that Fraser syndrome is a human equivalent of the blebbed phenotype in the mouse, which has been associated with mutations in at least five loci including bl. As mapping data were consistent with homology of FRAS1 and bl, we screened DNA from bl/bl mice and identified a premature termination of mouse Fras1. Thus, the bl mouse is a model for Fraser syndrome in humans, a disorder caused by disrupted epithelial integrity in utero.

The PAX2 tanscription factor is expressed in cystic and hyperproliferative dysplastic epithelia in human kidney malformations.

Human dysplastic kidneys are developmental aberrations which are responsible for many of the very young children with chronic renal failure. They contain poorly differentiated metanephric cells in addition to metaplastic elements. We recently demonstrated that apoptosis was prominent in undifferentiated cells around dysplastic tubules (Winyard, P.J.D., J. Nauta, D.S. Lirenman, P. Hardman, V.R. Sams, R.A. Risdon, and A.S. Woolf. 1996. Kidney Int. 49:135-146), perhaps explaining the tendency of some of these organs to regress. In contrast, apoptosis was rare in dysplastic epithelia which are thought to be ureteric bud malformations. On occasion, these tubules form cysts which distend the abdominal cavity (the multicystic dysplastic kidney) and dysplastic kidneys may rarely become malignant. We now demonstrate that dysplastic tubules maintain a high rate of proliferation postnatally and that PAX2, a potentially oncogenic transcription factor, is expressed in these epithelia. In contrast, both cell proliferation and PAX2 are downregulated during normal maturation of human collecting ducts. We demonstrate that BCL2, a protein which prevents apoptosis in renal mesenchymal to epithelia] conversion, is expressed ectopically in dysplastic kidney epithelia. We propose that dysplastic cyst formation may be understood in terms of aberrant temporal and spatial expression of master genes which are tightly regulated in the normal program of human nephrogenesis.

Evolving Concepts in Human Renal Dysplasia

Human renal dysplasia is a collection of disorders in which kidneys begin to form but then fail to differentiate into normal nephrons and collecting ducts. Dysplasia is the principal cause of childhood end-stage renal failure. Two main theories have been considered in its pathogenesis: A primary failure of ureteric bud activity and a disruption produced by fetal urinary flow impairment. Recent studies have documented deregulation of gene expression in human dysplasia, correlating with perturbed cell turnover and maturation. Mutations of nephrogenesis genes have been defined in multiorgan dysmorphic disorders in which renal dysplasia can feature, including Fraser, renal cysts and diabetes, and Kallmann syndromes. Here, it is possible to begin to understand the normal nephrogenic function of the wild-type proteins and understand how mutations might cause aberrant organogenesis.

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.

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).

HNF1B Mutations Associate with Hypomagnesemia and Renal Magnesium Wasting

Mutations in hepatocyte nuclear factor 1B (HNF1B), which is a transcription factor expressed in tissues including renal epithelia, associate with abnormal renal development. While studying renal phenotypes of children with HNF1B mutations, we identified a teenager who presented with tetany and hypomagnesemia. We retrospectively reviewed radiographic and laboratory data for all patients from a single center who had been screened for an HNF1B mutation. We found heterozygous mutations in 21 (23%) of 91 cases of renal malformation. All mutation carriers had abnormal fetal renal ultrasonography. Plasma magnesium levels were available for 66 patients with chronic kidney disease (stages 1 to 3). Striking, 44% (eight of 18) of mutation carriers had hypomagnesemia (<1.58 mg/dl) compared with 2% (one of 48) of those without mutations (P < 0.0001). The median plasma magnesium was significantly lower among mutation carriers than those without mutations (1.68 versus 2.02 mg/dl; P < 0.0001). Because hypermagnesuria and hypocalciuria accompanied the hypomagnesemia, we analyzed genes associated with hypermagnesuria and detected highly conserved HNF1 recognition sites in FXYD2, a gene that can cause autosomal dominant hypomagnesemia and hypocalciuria when mutated. Using a luciferase reporter assay, we demonstrated HNF1B-mediated transactivation of FXYD2. These results extend the phenotype of HNF1B mutations to include hypomagnesemia. HNF1B regulates transcription of FXYD2, which participates in the tubular handling of Mg(2+), thus describing a role for HNF1B not only in nephrogenesis but also in the maintenance of tubular function.

Identification of a new gene mutated in Fraser syndrome and mouse myelencephalic blebs

Fraser syndrome is a recessive, multisystem disorder presenting with cryptophthalmos, syndactyly and renal defects and associated with loss-of-function mutations of the extracellular matrix protein FRAS1. Fras1 mutant mice have a blebbed phenotype characterized by intrauterine epithelial fragility generating serous and, later, hemorrhagic blisters. The myelencephalic blebs (my) strain has a similar phenotype. We mapped my to Frem2, a gene related to Fras1 and Frem1, and showed that a Frem2 gene-trap mutation was allelic to my. Expression of Frem2 in adult kidneys correlated with cyst formation in my homozygotes, indicating that the gene is required for maintaining the differentiated state of renal epithelia. Two individuals with Fraser syndrome were homozygous with respect to the same missense mutation of FREM2, confirming genetic heterogeneity. This is the only missense mutation reported in any blebbing mutant or individual with Fraser syndrome, suggesting that calcium binding in the CALXβ-cadherin motif is important for normal functioning of FREM2.

Primary, nonsyndromic vesicoureteric reflux and its nephropathy is genetically heterogeneous, with a locus on chromosome 1.

Primary vesicoureteric reflux (VUR) affects 1%-2% of whites, and reflux nephropathy (RN) causes up to 15% of end-stage renal failure in children and adults. There is a 30-50-fold increased incidence of VUR in first-degree relatives of probands, compared with the general population. We report the results of the first genomewide search of VUR and RN; we studied seven European families whose members exhibit apparently dominant inheritance. We initially typed 387 polymorphic markers spaced, on average, at 10 cM throughout the genome; we used the GENEHUNTER program to provide parametric and nonparametric linkage analyses of affected individuals. The most positive locus spanned 20 cM on 1p13 between GATA176C01 and D1S1653 and had a nonparametric LOD score (NPL) of 5.76 (P=.0002) and a parametric LOD score of 3.16. Saturation with markers at 1-cM intervals increased the NPL to 5.94 (P=.00009). Hence, VUR maps to a locus on chromosome 1. There was evidence of genetic heterogeneity at the chromosome 1 locus, and 12 additional loci were identified genomewide, with P<.05. No significant linkage was found to 6p, where a renal and ureteric malformation locus has been reported, or to PAX2, mutations of which cause VUR in renal-coloboma syndrome. Our results support the hypothesis that VUR is a genetic disorder.