The many faces of RET dysfunction in kidney

Signaling pathways that are activated upon interaction of glial cell-line derived neurotrophic factor (Gdnf), its coreceptor Gfrα1, and receptor tyrosine kinase Ret are critical for kidney development and ureter maturation. Outside the kidney, this pathway is implicated in a number of congenital diseases including Hirschsprung disease (intestinal aganglionosis, HSCR) and hereditary cancer syndromes (MEN 2). Total lack of Gdnf, Gfrα1 or Ret in mice results in perinatal lethality due to bilateral renal agenesis or aplasia. In humans, RET mutations have been identified in a spectrum of congenital malformations involving the RET axis including isolated HSCR, isolated congenital anomalies of kidney or urinary tract (CAKUT), or CAKUT and HSCR together. The molecular basis for these pleiotropic effects of RET has just begun to be unraveled. In an effort to delineate the pathogenetic mechanisms that underlie these congenital malformations, we and others have characterized Ret’s role in early kidney and urinary system development. Here we present a brief overview of the “many faces” of Ret dysfunction in kidney with particular emphasis on Ret’s signaling specificity and intergenic interactions that confer normal urinary system development.     

The many faces of RET dysfunction in kidney

Signaling pathways that are activated upon interaction of glial cell-line derived neurotrophic factor (Gdnf), its coreceptor Gfra1, and receptor tyrosine kinase Ret are critical for kidney development and ureter maturation. Outside the kidney, this pathway is implicated in a number of congenital diseases including Hirschsprung disease (intestinal aganglionosis, HSCR) and hereditary cancer syndromes (MEN 2). Total lack of Gdnf, Gfra1 or Ret in mice results in perinatal lethality due to bilateral renal agenesis or aplasia. In humans, RET mutations have been identified in a spectrum of congenital malformations involving the RET axis including isolated HSCR, isolated congenital anomalies of kidney or urinary tract (CAKUT), or CAKUT and HSCR together. The molecular basis for these pleiotropic effects of RET has just begun to be unraveled. In an effort to delineate the pathogenetic mechanisms that underlie these congenital malformations, we and others have characterized Ret''s role in early kidney and urinary system development. Here we present a brief overview of the “many faces” of Ret dysfunction in kidney with particular emphasis on Ret''s signaling specificity and intergenic interactions that confer normal urinary system development.Key words: RET, GDNF, kidney, RTK, CAKUT, branching morphogenesis, ureter     

Disturbances of colonic motility in mouse models of Hirschsprung's disease

Mutations in genes encoding members of the GDNF and endothelin-3 (Et-3) signaling pathways can cause Hirschsprung's disease, a congenital condition associated with an absence of enteric neurons in the distal gut. GDNF signals through Ret, a receptor tyrosine kinase, and Et-3 signals through endothelin receptor B (Ednrb). The effects of Gdnf, Ret, and ET-3 haploinsufficiency and a null mutation in ET-3 on spontaneous motility patterns in adult and developing mice were investigated. Video recordings were used to construct spatiotemporal maps of spontaneous contractile patterns in colon from postnatal and adult mice in vitro. In Ret(+/-) and ET-3(+/-) mice, which have normal numbers of enteric neurons, colonic migrating motor complexes (CMMCs) displayed similar properties under control conditions and following inhibition of nitric oxide synthase (NOS) activity to wild-type mice. In the colon of Gdnf(+/-) mice and in the ganglionic region of ET-3(-/-) mice, there was a 50-60% reduction in myenteric neuron number. In Gdnf(+/-) mice, CMMCs were present, but abnormal, and the proportion of myenteric neurons containing NOS was not different from that of wild-type mice. In the ganglionic region of postnatal ET-3(-/-) mice, CMMCs were absent, and the proportion of myenteric neurons containing NOS was over 100% higher than in wild-type mice. Thus impairments in spontaneous motility patterns in the colon of Gdnf(+/-) mice and in the ganglionic region of ET-3(-/-) mice are correlated with a reduction in myenteric neuron density.     

QTL analysis identifies a modifier locus of aganglionosis in the rat model of Hirschsprung disease carrying Ednrb(sl) mutations

Hirschsprung disease (HSCR) exhibits complex genetics with incomplete penetrance and variable severity thought to result as a consequence of multiple gene interactions that modulate the ability of enteric neural crest cells to populate the developing gut. As reported previously, when the same null mutation of the Ednrb gene, Ednrb(sl), was introgressed into the F344 strain, almost 60% of F344-Ednrb(sl/sl) pups did not show any symptoms of aganglionosis, appearing healthy and normally fertile. These findings strongly suggested that the severity of HSCR was affected by strain-specific genetic factor (s). In this study, the genetic basis of such large strain differences in the severity of aganglionosis in the rat model was studied by whole-genome scanning for quantitative trait loci (QTLs) using an intercross of (AGH-Ednrb(sl)×F344-Ednrb(sl)) F(1) with the varying severity of aganglionosis. Genome linkage analysis identified one significant QTL on chromosome 2 for the severity of aganglionosis. Our QTL analyses using rat models of HSCR revealed that multiple genetic factors regulated the severity of aganglionosis. Moreover, a known HSCR susceptibility gene, Gdnf, was found in QTL that suggested a novel non-coding sequence mutation in GDNF that modifies the penetrance and severity of the aganglionosis phenotype in EDNRB-deficient rats. A further identification and analysis of responsible genes located on the identified QTL could lead to the richer understanding of the genetic basis of HSCR development.     

Mutations in the Extracellular Domain Cause RET Loss of Function by a Dominant Negative Mechanism

The RET proto-oncogene encodes a tyrosine kinase receptor expressed in neuroectoderm-derived cells. Mutations in specific regions of the gene are responsible for the tumor syndromes multiple endocrine neoplasia types 2A and 2B (MEN 2A and 2B), while mutations along the entire gene are involved in a developmental disorder of the gastrointestinal tract, Hirschsprung’s disease (HSCR disease). Two mutants in the extracellular domain of RET, one associated with HSCR disease and one carrying a flag epitope, were analyzed to investigate the impact of the mutations on RET function. Both mutants were impeded in their maturation, resulting in the lack of the 170-kDa mature form and the accumulation of the 150-kDa immature form in the endoplasmic reticulum. Although not exposed on the cell surface, the 150-kDa species formed dimers and aggregates; this was more pronounced in a double mutant bearing a MEN 2A mutation. Tyrosine phosphorylation and the transactivation potential were drastically reduced in single and double mutants. Finally, in cotransfection experiments both mutants exerted a dominant negative effect over protoRET and RET2A through the formation of a heteromeric complex that prevents their maturation and function. These results suggest that HSCR mutations in the extracellular region cause RET loss of function through a dominant negative mechanism.     

Molecular genetics of Hirschsprung disease: a model of multigenic neurocristopathy

Hirschsprung's disease (HSCR, aganglionic megacolon) is a frequent congenital malformation regarded as a multigenic neurocristopathy. Three susceptibility genes have been recently identified in HSCR, namely the RET proto-oncogene, the endothelin B receptor (EDNRB) gene, and the endothelin 3 (EDN3) gene. RET gene mutations were found in significant proportions of familial (50%) and sporadic (15-20%) HSCR, while homozygosity for EDNRB or EDN3 mutations accounted for the rare HSCR-Waardenburg syndrome (WS) association. More recently, heterozygous EDNRB an EDN3 missense mutations have been reported in isolated HSCR patients. Some of these results were obtained after the identification of mouse genes whose natural or site-directed mutations resulted in megacolon and coat color spotting. There is also conclusive evidence for the involvement of other independent loci in HSCR. In particular, the recent identification of neurotrophic factors acting as RET ligands (GDNF and Neurturin) provide additional candidate genes for HSCR. The dissection of the genetic etiology of HSCR disease may then provide a unique opportunity to distinguish between a polygenic and a genetically heterogeneous disease, thereby helping to understand other complex disorders and congenital malformations hitherto considered as multifactorial in origin. Finally, the study of the molecular bases of HSCR is also a step towards the understanding of developmental genetics of the enteric nervous system giving support to the role of the tyrosine kinase and endothelin-signaling pathways in the development of neural crest-derived enteric neurons in human.     

Genetic Background of Hirschsprung Disease: A Bridge Between Basic Science and Clinical Application

Hirschsprung's disease (HSCR) is a congenital disorder, defined by partial or complete loss of the neuronal ganglion cells in the intestinal tract, which is caused by the failure of neural crest cells to migrate completely during intestinal development during fetal life. HSCR has a multifactorial etiology, and genetic factors play a key role in its pathogenesis; these include mutations within several gene loci. These have been identified by screening candidate genes, or by conducting genome wide association (GWAS) studies. However, only a small portion of them have been proposed as major genetic risk factors for the HSCR. In this review, we focus on those genes that have been identified as either low penetrant or high penetrant variants that determine the risk of Hirschsprung's disease. J. Cell. Biochem. 119: 28–33, 2018. © 2017 Wiley Periodicals, Inc.     

Copy number variants in candidate genes are genetic modifiers of Hirschsprung disease

Hirschsprung disease (HSCR) is a neurocristopathy characterized by absence of intramural ganglion cells along variable lengths of the gastrointestinal tract. The HSCR phenotype is highly variable with respect to gender, length of aganglionosis, familiality and the presence of additional anomalies. By molecular genetic analysis, a minimum of 11 neuro-developmental genes (RET, GDNF, NRTN, SOX10, EDNRB, EDN3, ECE1, ZFHX1B, PHOX2B, KIAA1279, TCF4) are known to harbor rare, high-penetrance mutations that confer a large risk to the bearer. In addition, two other genes (RET, NRG1) harbor common, low-penetrance polymorphisms that contribute only partially to risk and can act as genetic modifiers. To broaden this search, we examined whether a set of 67 proven and candidate HSCR genes harbored additional modifier alleles. In this pilot study, we utilized a custom-designed array CGH with ～33,000 test probes at an average resolution of ～185 bp to detect gene-sized or smaller copy number variants (CNVs) within these 67 genes in 18 heterogeneous HSCR patients. Using stringent criteria, we identified CNVs at three loci (MAPK10, ZFHX1B, SOX2) that are novel, involve regulatory and coding sequences of neuro-developmental genes, and show association with HSCR in combination with other congenital anomalies. Additional CNVs are observed under relaxed criteria. Our research suggests a role for CNVs in HSCR and, importantly, emphasizes the role of variation in regulatory sequences. A much larger study will be necessary both for replication and for identifying the full spectrum of small CNV effects.     

Genetic background strongly modifies the severity of symptoms of Hirschsprung disease, but not hearing loss in rats carrying Ednrb(sl) mutations

Hirschsprung disease (HSCR) is thought to result as a consequence of multiple gene interactions that modulate the ability of enteric neural crest cells to populate the developing gut. However, it remains unknown whether the single complete deletion of important HSCR-associated genes is sufficient to result in HSCR disease. In this study, we found that the null mutation of the Ednrb gene, thought indispensable for enteric neuron development, is insufficient to result in HSCR disease when bred onto a different genetic background in rats carrying Ednrb(sl) mutations. Moreover, we found that this mutation results in serious congenital sensorineural deafness, and these strains may be used as ideal models of Waardenburg Syndrome Type 4 (WS4). Furthermore, we evaluated how the same changed genetic background modifies three features of WS4 syndrome, aganglionosis, hearing loss, and pigment disorder in these congenic strains. We found that the same genetic background markedly changed the aganglionosis, but resulted in only slight changes to hearing loss and pigment disorder. This provided the important evidence, in support of previous studies, that different lineages of neural crest-derived cells migrating along with various pathways are regulated by different signal molecules. This study will help us to better understand complicated diseases such as HSCR and WS4 syndrome.     

Contribution of rare and common variants determine complex diseases—Hirschsprung disease as a model

Finding genes for complex diseases has been the goal of many genetic studies. Most of these studies have been successful by searching for genes and mutations in rare familial cases, by screening candidate genes and by performing genome wide association studies. However, only a small fraction of the total genetic risk for these complex genetic diseases can be explained by the identified mutations and associated genetic loci. In this review we focus on Hirschsprung disease (HSCR) as an example of a complex genetic disorder. We describe the genes identified in this congenital malformation and postulate that both common ‘low penetrant’ variants in combination with rare or private ‘high penetrant’ variants determine the risk on HSCR, and likely, on other complex diseases. We also discuss how new technological advances can be used to gain further insights in the genetic background of complex diseases. Finally, we outline a few steps to develop functional assays in order to determine the involvement of these variants in disease development.     

Genetic mosaicism of a frameshift mutation in the RET gene in a family with Hirschsprung disease

Mutations and polymorphisms in the RET gene are a major cause of Hirschsprung disease (HSCR). Theoretically, all true heterozygous patients with a new manifestation of a genetically determined disease must have parents with a genetic mosaicism of some extent. However, no genetic mosaicism has been described for the RET gene in HSCR yet. Therefore, we analyzed families with mutations in the RET gene for genetic mosaicism in the parents of the patients. Blood samples were taken from patients with HSCR and their families/parents to sequence the RET coding region. Among 125 families with HSCR, 33 families with RET mutations were analyzed. In one family, we detected a frameshift mutation due to a loss of one in a row of four cytosines in codon 117/118 of the RET gene (c.352delC) leading to a frameshift mutation in the protein (p.Leu118Cysfs*105) that affected two siblings. In the blood sample of the asymptomatic father we found a genetic mosaicism of this mutation which was confirmed in two independent samples of saliva and hair roots. Quantification of peak-heights and comparison with different mixtures of normal and mutated plasmid DNA suggested that the mutation occurred in the early morula stadium of the founder, between the 4- and 8-cell stages. We conclude that the presence of a RET mutation leading to loss of one functional allele in 20 to 25% of the cells is not sufficient to cause HSCR. The possibility of a mosaicism has to be kept in mind during genetic counseling for inherited diseases.     

Nonsense and frameshift mutations in ZFHX1B, encoding Smad-interacting protein 1, cause a complex developmental disorder with a great variety of clinical features

Mutations in ZFHX1B, encoding Smad-interacting protein 1 (SIP1), have been recently reported to cause a form of Hirschsprung disease (HSCR). Patients with ZFHX1B deficiency typically show mental retardation, delayed motor development, epilepsy, microcephaly, distinct facial features, and/or congenital heart disease, in addition to the cardinal form of HSCR. To investigate the breadth of clinical variation, we studied DNA samples from six patients with clinical profiles quite similar to those described elsewhere for ZFHX1B deficiency, except that they did not have HSCR. The results showed the previously reported R695X mutation to be present in three cases, with three novel mutations-a 2-bp insertion (760insCA resulting in 254fs262X), a single-base deletion (270delG resulting in 91fs107X), and a 2-bp deletion (2178delTT resulting in 727fs754X)-newly identified in the other three. All mutations occurred in one allele and were de novo events. These results demonstrate that ZFHX1B deficiency is an autosomal dominant complex developmental disorder and that individuals with functional null mutations present with mental retardation, delayed motor development, epilepsy, and a wide spectrum of clinically heterogeneous features suggestive of neurocristopathies at the cephalic, cardiac, and vagal levels.     

Exome Sequencing Identified NRG3 as a Novel Susceptible Gene of Hirschsprung’s Disease in a Chinese Population

Hirschsprung’s disease (HSCR) is a complex developmental defect characterized by the absence of enteric ganglia in the gastrointestinal tract. Although the genetic defect of enteric nervous system (ENS) was identified to play a critical role in the progress of HSCR, the systemic genetic dissection of HSCR still needs to be clarified. In this study, we firstly performed exome sequencing of two HSCR patients from a Han Chinese family, including the affected mother and son. After the initial quality filtering (coverage?≥?5X and SNP quality score?≥?40) of the raw data, we identified 13,948 and 13,856 single nucleotide variants (SNVs), respectively. We subsequently compared the SNVs against public databases (dbSNP130, HapMap, and 1000 Genome Project) and obtained a total of 15 novel nonsynonymous SNVs in 15 genes, which were shared between these two patients. Follow-up Sanger sequencing and bioinformatics analysis highlighted variant c.853G>A (p.E285K) in NRG3, a gene involved in the development of ENS. In the validation phase, we sequenced all nine exons of NRG3 in 96 additional sporadic HSCR cases and 110 healthy individuals and identified another nonsynonymous variant c.1329G>A (p.M443I) and two synonymous variants c.828G>A (p.T276T) and c.1365T>A (p.P455P) only in the cases. Our results indicated that NRG3 may be a susceptibility gene for HSCR in a Chinese population.     

Comprehensive analysis of NRG1 common and rare variants in Hirschsprung patients

Hirschsprung disease (HSCR, OMIM 142623) is a developmental disorder characterized by the absence of ganglion cells along variable lengths of the distal gastrointestinal tract, which results in tonic contraction of the aganglionic gut segment and functional intestinal obstruction. The RET proto-oncogene is the major gene for HSCR with differential contributions of its rare and common, coding and noncoding mutations to the multifactorial nature of this pathology. Many other genes have been described to be associated with the pathology, as NRG1 gene (8p12), encoding neuregulin 1, which is implicated in the development of the enteric nervous system (ENS), and seems to contribute by both common and rare variants. Here we present the results of a comprehensive analysis of the NRG1 gene in the context of the disease in a series of 207 Spanish HSCR patients, by both mutational screening of its coding sequence and evaluation of 3 common tag SNPs as low penetrance susceptibility factors, finding some potentially damaging variants which we have functionally characterized. All of them were found to be associated with a significant reduction of the normal NRG1 protein levels. The fact that those mutations analyzed alter NRG1 protein would suggest that they would be related with HSCR disease not only in Chinese but also in a Caucasian population, which reinforces the implication of NRG1 gene in this pathology.     

RET and NTRK1 proto-oncogenes in human diseases

RET and NTRK1 are receptor tyrosine kinase (RTK) proteins which play a role in the development and maturation of specific component of the nervous system. Their alterations have been associated to several human diseases, including some forms of cancer and developmental abnormalities. These features have contributed to the concept that one gene can be responsible for more than one disease. Moreover, both genes encoding for the two RTKs show genetic alterations that belong to either "gain of function" or "loss of function" class of mutations. In fact, receptor rearrangements or point mutations convert RET and NTRK1 in dominantly acting transforming genes leading to thyroid tumors, whereas inactivating mutations, associated with Hirschsprung's disease (HSCR) and congenital insensitivity to pain with anhidrosis (CIPA), impair RET and NTRK1 functions, respectively. In this review we have summarized the main features of the two receptors, their physiological and pathological roles. In addition, we attempted to identify the correlations between the different genetic alterations and the related pathogenetic mechanisms.     

COL4A1 mutations cause ocular dysgenesis, neuronal localization defects, and myopathy in mice and Walker-Warburg syndrome in humans

Muscle-eye-brain disease (MEB) and Walker Warburg Syndrome (WWS) belong to a spectrum of autosomal recessive diseases characterized by ocular dysgenesis, neuronal migration defects, and congenital muscular dystrophy. Until now, the pathophysiology of MEB/WWS has been attributed to alteration in dystroglycan post-translational modification. Here, we provide evidence that mutations in a gene coding for a major basement membrane protein, collagen IV alpha 1 (COL4A1), are a novel cause of MEB/WWS. Using a combination of histological, molecular, and biochemical approaches, we show that heterozygous Col4a1 mutant mice have ocular dysgenesis, neuronal localization defects, and myopathy characteristic of MEB/WWS. Importantly, we identified putative heterozygous mutations in COL4A1 in two MEB/WWS patients. Both mutations occur within conserved amino acids of the triple-helix-forming domain of the protein, and at least one mutation interferes with secretion of the mutant proteins, resulting instead in intracellular accumulation. Expression and posttranslational modification of dystroglycan is unaltered in Col4a1 mutant mice indicating that COL4A1 mutations represent a distinct pathogenic mechanism underlying MEB/WWS. These findings implicate a novel gene and a novel mechanism in the etiology of MEB/WWS and expand the clinical spectrum of COL4A1-associated disorders.     

Mutational Spectrum of Semaphorin 3A and Semaphorin 3D Genes in Spanish Hirschsprung patients

Hirschsprung disease (HSCR, OMIM 142623) is a developmental disorder characterized by the absence of ganglion cells along variable lengths of the distal gastrointestinal tract, which results in tonic contraction of the aganglionic colon segment and functional intestinal obstruction. The RET proto-oncogene is the major gene associated to HSCR with differential contributions of its rare and common, coding and noncoding mutations to the multifactorial nature of this pathology. In addition, many other genes have been described to be associated with this pathology, including the semaphorins class III genes SEMA3A (7p12.1) and SEMA3D (7q21.11) through SNP array analyses and by next-generation sequencing technologies. Semaphorins are guidance cues for developing neurons implicated in the axonal projections and in the determination of the migratory pathway for neural-crest derived neural precursors during enteric nervous system development. In addition, it has been described that increased SEMA3A expression may be a risk factor for HSCR through the upregulation of the gene in the aganglionic smooth muscle layer of the colon in HSCR patients. Here we present the results of a comprehensive analysis of SEMA3A and SEMA3D in a series of 200 Spanish HSCR patients by the mutational screening of its coding sequence, which has led to find a number of potentially deleterious variants. RET mutations have been also detected in some of those patients carrying SEMAs variants. We have evaluated the A131T-SEMA3A, S598G-SEMA3A and E198K-SEMA3D mutations using colon tissue sections of these patients by immunohistochemistry. All mutants presented increased protein expression in smooth muscle layer of ganglionic segments. Moreover, A131T-SEMA3A also maintained higher protein levels in the aganglionic muscle layers. These findings strongly suggest that these mutants have a pathogenic effect on the disease. Furthermore, because of their coexistence with RET mutations, our data substantiate the additive genetic model proposed for this rare disorder and further support the association of SEMAs genes with HSCR.     

Biological and clinical review of stromal tumors in the gastrointestinal tract

Submucosal tumors of the gastrointestinal tract (GI tract) mainly consist of gastrointestinal mesenchymal tumors (GIMTs) that are distributed in the GI tract from the esophagus through the rectum. GIMTs include myogenic tumors, neurogenic tumors and gastrointestinal stromal tumors (GISTs). The term "GIST" is now preferentially used for the tumors that express CD34 and KIT. GIMTs are composed of spindle or epithelioid cells, and 20% to 30% show malignant behavior, including peritoneal dissemination and hematogenous metastasis. KIT expression and mutations in the c-kit gene are found only in GISTs, but not in myogenic or neurogenic tumors. Mutation in the c-kit gene is associated with aggressive features and poor prognosis, and malignant GISTs frequently have mutations in the c-kit gene. The clinicopathological features of GISTs with or without c-kit mutations are markedly different. Therefore, GIMTs may be divided into four major categories based on histochemical and genetic data: myogenic tumors; neurogenic tumors; GISTs with c-kit mutation; and GISTs without c-kit mutation. The origin of GISTs is not fully understood. However, phenotypical resemblance to the interstitial cells of Cajal (ICCs) and gain-of-function mutations in the c-kit gene may suggest origin from ICCs and/or multipotential mesenchymal cells that differentiate into ICCs.     

HumanGFRA1: Cloning, Mapping, Genomic Structure, and Evaluation as a Candidate Gene for Hirschsprung Disease Susceptibility

Congenital aganglionic megacolon, commonly known as Hirschsprung disease (HSCR), is the most frequent cause of congenital bowel obstruction. Germline mutations in theRETreceptor tyrosine kinase have been shown to cause HSCR. Knockout mice forRETand for its ligand, glial cell line-derived neurotrophic factor (GDNF), exhibit both complete intestinal aganglionosis and renal defects. Recently, GDNF and GFRA1 (GDNF family receptor, also known as GDNFR-α), its GPI-linked coreceptor, were demonstrated to be components of a functional ligand for RET. Moreover,GDNFhas been implicated in rare cases of HSCR. We have mappedGFRA1to human chromosome 10q25, isolated human and mouse genomic clones, determined the gene's intron–exon boundaries, isolated a highly polymorphic microsatellite marker adjacent to exon 7, and scanned forGFRA1mutations in a large panel of HSCR patients. No evidence of linkage was detected in HSCR kindreds, and no sequence variants were found to be in significant excess in patients. These data suggest thatGFRA1's role in enteric neurogenesis in humans remains to be elucidated and that RET signaling in the gut may take place via alternate pathways, such as the recently described GDNF-related molecule neurturin and its GFRA1-like coreceptor, GFRA2.