Helen M. Stuart

LRIG2 Mutations Cause Urofacial Syndrome

Urofacial syndrome (UFS) (or Ochoa syndrome) is an autosomal-recessive disease characterized by congenital urinary bladder dysfunction, associated with a significant risk of kidney failure, and an abnormal facial expression upon smiling, laughing, and crying. We report that a subset of UFS-affected individuals have biallelic mutations in LRIG2, encoding leucine-rich repeats and immunoglobulin-like domains 2, a protein implicated in neural cell signaling and tumorigenesis. Importantly, we have demonstrated that rare variants in LRIG2 might be relevant to nonsyndromic bladder disease. We have previously shown that UFS is also caused by mutations in HPSE2, encoding heparanase-2. LRIG2 and heparanase-2 were immunodetected in nerve fascicles growing between muscle bundles within the human fetal bladder, directly implicating both molecules in neural development in the lower urinary tract.

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.

Urofacial syndrome: a genetic and congenital disease of aberrant urinary bladder innervation

The urofacial, or Ochoa, syndrome is characterised by congenital urinary bladder dysfunction together with an abnormal grimace upon smiling, laughing and crying. It can present as fetal megacystis. Postnatal features include urinary incontinence and incomplete bladder emptying due to simultaneous detrusor muscle and bladder outlet contractions. Vesicoureteric reflux is often present, and the condition can be complicated by urosepsis and end-stage renal disease. The syndrome has long been postulated to have neural basis, and it can be familial when it is inherited in an autosomal recessive manner. Most individuals with urofacial syndrome genetically studied to date carry biallelic, postulated functionally null mutations of HPSE2 or, less commonly, of LRIG2. Little is known about the biology of the respective encoded proteins, heparanase 2 and leucine-rich repeats and immunoglobulin-like domains 2. Nevertheless, the observations that heparanase 2 can bind heparan sulphate proteolgycans and inhibit heparanase 1 enzymatic activity and that LRIG2 can modulate receptor tyrosine kinase growth factor signalling each point to biological roles relevant to tissue differentiation. Moreover, both heparanase 2 and LRIG2 proteins are detected in autonomic nerves growing into fetal bladders. The collective evidence is consistent with the hypothesis that urofacial syndrome genes code for proteins which work in a common pathway to facilitate neural growth into, and/or function within, the bladder. This molecular pathway may also have relevance to our understanding of the pathogenesis of other lower tract diseases, including Hinman–Allen syndrome, or non-neurogenic neurogenic bladder, and of the subset of individuals who have primary vesicoureteric reflux accompanied by bladder dysfunction.

Heparanase 2, mutated in urofacial syndrome, mediates peripheral neural development in Xenopus

Urofacial syndrome (UFS; previously Ochoa syndrome) is an autosomal recessive disease characterized by incomplete bladder emptying during micturition. This is associated with a dyssynergia in which the urethral walls contract at the same time as the detrusor smooth muscle in the body of the bladder. UFS is also characterized by an abnormal facial expression upon smiling, and bilateral weakness in the distribution of the facial nerve has been reported. Biallelic mutations in HPSE2 occur in UFS. This gene encodes heparanase 2, a protein which inhibits the activity of heparanase. Here, we demonstrate, for the first time, an in vivo developmental role for heparanase 2. We identified the Xenopus orthologue of heparanase 2 and showed that the protein is localized to the embryonic ventrolateral neural tube where motor neurons arise. Morpholino-induced loss of heparanase 2 caused embryonic skeletal muscle paralysis, and morphant motor neurons had aberrant morphology including less linear paths and less compactly-bundled axons than normal. Biochemical analyses demonstrated that loss of heparanase 2 led to upregulation of fibroblast growth factor 2/phosphorylated extracellular signal-related kinase signalling and to alterations in levels of transcripts encoding neural- and muscle-associated molecules. Thus, a key role of heparanase 2 is to buffer growth factor signalling in motor neuron development. These results shed light on the pathogenic mechanisms underpinning the clinical features of UFS and support the contention that congenital peripheral neuropathy is a key feature of this disorder.

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.

Genetics of human congenital urinary bladder disease

Lower urinary tract and/or kidney malformations are collectively the most common cause of end-stage renal disease in children, and they are also likely to account for a major subset of young adults requiring renal replacement therapy. Advances have been made regarding the discovery of the genetic causes of human kidney malformations. Indeed, testing for mutations of key nephrogenesis genes is now feasible for patients seen in nephrology clinics. Unfortunately, less is known about defined genetic bases of human lower urinary tract anomalies. The focus of this review is the genetic bases of congenital structural and functional disorders of the urinary bladder. Three are highlighted. First, prune belly syndrome, where mutations of CHRM3, encoding an acetylcholine receptor, HNF1B, encoding a transcription factor, and ACTA2, encoding a cytoskeletal protein, have been reported. Second, the urofacial syndrome, where mutations of LRIG2 and HPSE2, encoding proteins localised in nerves invading the fetal bladder, have been defined. Finally, we review emerging evidence that bladder exstrophy may have genetic bases, including variants in the TP63 promoter. These genetic discoveries provide a new perspective on a group of otherwise poorly understood diseases.

Determining the pathogenicity of patient-derived TSC2 mutations by functional characterization and clinical evidence

Tuberous sclerosis complex (TSC) is a genetic condition characterized by the growth of benign tumours in multiple organs, including the brain and kidneys, alongside intellectual disability and seizures. Identification of a causative mutation in TSC1 or TSC2 is important for accurate genetic counselling in affected families, but it is not always clear from genetic data whether a sequence variant is pathogenic or not. In vitro functional analysis could provide support for determining whether an unclassified TSC1 or TSC2 variant is disease-causing. We have performed a detailed functional analysis of four patient-derived TSC2 mutations, E92V, R505Q, H597R and L1624P. One mutant, E92V, functioned similarly to wild-type TSC2, whereas H597R and L1624P had abnormal function in all assays, consistent with available clinical and segregation information. One TSC2 mutation, R505Q, was identified in a patient with intellectual disability, seizures and autistic spectrum disorder but who did not fulfil the diagnostic criteria for TSC. The R505Q mutation was also found in two relatives, one with mild learning difficulties and one without apparent phenotypic abnormality. R505Q TSC2 exhibited partially disrupted function in our assays. These data highlight the difficulties of assessing pathogenicity of a mutation and suggest that multiple lines of evidence, both genetic and functional, are required to assess the pathogenicity of some mutations.

Genetic testing in steroid-resistant nephrotic syndrome: why, who, when and how?

Steroid-resistant nephrotic syndrome (SRNS) is a common cause of chronic kidney disease in childhood and has a significant risk of rapid progression to end-stage renal disease. The identification of over 50 monogenic causes of SRNS has revealed dysfunction in podocyte-associated proteins in the pathogenesis of proteinuria, highlighting their essential role in glomerular function. Recent technological advances in high-throughput sequencing have enabled indication-driven genetic panel testing for patients with SRNS. The availability of genetic testing, combined with the significant phenotypic variability of monogenic SRNS, poses unique challenges for clinicians when directing genetic testing. This highlights the need for clear clinical guidelines that provide a systematic approach for mutational screening in SRNS. The likelihood of identifying a causative mutation is inversely related to age at disease onset and is increased with a positive family history or the presence of extra-renal manifestations. An unequivocal molecular diagnosis could allow for a personalised treatment approach with weaning of immunosuppressive therapy, avoidance of renal biopsy and provision of accurate, well-informed genetic counselling. Identification of novel causative mutations will continue to unravel the pathogenic mechanisms of glomerular disease and provide new insights into podocyte biology and glomerular function.

ACTB Loss-of-Function Mutations Result in a Pleiotropic Developmental Disorder

ACTB encodes β-actin, an abundant cytoskeletal housekeeping protein. In humans, postulated gain-of-function missense mutations cause Baraitser-Winter syndrome (BRWS), characterized by intellectual disability, cortical malformations, coloboma, sensorineural deafness, and typical facial features. To date, the consequences of loss-of-function ACTB mutations have not been proven conclusively. We describe heterozygous ACTB deletions and nonsense and frameshift mutations in 33 individuals with developmental delay, apparent intellectual disability, increased frequency of internal organ malformations (including those of the heart and the renal tract), growth retardation, and a recognizable facial gestalt (interrupted wavy eyebrows, dense eyelashes, wide nose, wide mouth, and a prominent chin) that is distinct from characteristics of individuals with BRWS. Strikingly, this spectrum overlaps with that of several chromatin-remodeling developmental disorders. In wild-type mouse embryos, β-actin expression was prominent in the kidney, heart, and brain. ACTB mRNA expression levels in lymphoblastic lines and fibroblasts derived from affected individuals were decreased in comparison to those in control cells. Fibroblasts derived from an affected individual and ACTB siRNA knockdown in wild-type fibroblasts showed altered cell shape and migration, consistent with known roles of cytoplasmic β-actin. We also demonstrate that ACTB haploinsufficiency leads to reduced cell proliferation, altered expression of cell-cycle genes, and decreased amounts of nuclear, but not cytoplasmic, β-actin. In conclusion, we show that heterozygous loss-of-function ACTB mutations cause a distinct pleiotropic malformation syndrome with intellectual disability. Our biological studies suggest that a critically reduced amount of this protein alters cell shape, migration, proliferation, and gene expression to the detriment of brain, heart, and kidney development.

Podocytopathy in primary renal failure and de-novo membranous nephropathy post transplantation

Abstract Background The podocyte is a key component of the glomerular filtration barrier, and mutations in podocyte genes account for familiar and sporadic glomerular disease. After renal transplantation, some patients develop autoimmune glomerular disease with antibodies specific to nascent podocyte antigens, a process that can limit graft survival. We aimed to determine the molecular diagnosis in a family with primary renal failure and de-novo membranous nephropathy post transplantation. Methods We studied two siblings with primary glomerular disease and post-transplant membranous nephropathy using autozygosity mapping, whole exome sequencing, and post-transplant antibody screening. Native and transplant kidney biopsy samples were assessed in immunohistochemical and electron microscopy studies. The functional effects of the affected gene were studied in zebrafish and human podocytes. Findings We identified a novel 129 kb genomic deletion adjacent to exon 1 of IQCJ on chromosome 3 which was homozygous in siblings and heterozygous in their parents. After transplantation, both siblings were seropositive for anti-IQCJ antibodies. Native kidney biopsy tissue identified abnormal podocyte structure and cytoplasmic IQCJ. Transient knockdown of the IQCJ gene in zebrafish caused oedema, consistent with a glomerular phenotype. Human podocytes expressed IQCJ in vesicles resembling autophagosomes. Interpretation We describe a new podocytopathy based on a genomic deletion on chromosome 3 close to IQCJ, which leads to primary renal failure. After renal transplantation, possession of the homozygous IQCJ deletion causes de-novo membranous nephropathy in the allograft within 12 months with rapid graft loss within 2 years. The dysregulation of this gene might have a role in primary glomerular disease or as a target for de-novo membranous nephropathy after transplantation. Funding Wellcome Trust.