De novo mutation of GDNF, ligand for the RET/GDNFR-alpha receptor complex, in Hirschsprung disease

Hirschsprung disease (HSCR) is a common congenital abnormality characterized by absence of the enteric ganglia in the hind gut. In 10- 40% of HSCR cases, mutations of the RET receptor tyrosine kinase have been found. The recent identification of a multimeric RET ligand/receptor complex suggested that mutations of genes encoding other components of this complex might also occur in HSCR. To investigate this role, we examined the gene for glial cell line-derived neurotrophic factor (GDNF), the circulating ligand of the RET receptor complex, for mutations in a panel of sporadic and familial HSCR. We identified GDNF sequence variants in 2/36 HSCR patients. The first of these was a conservative change which did not affect the GDNF protein sequence. The second variant was a de novo missense mutation in a family with no history of HSCR and without mutation of the RET gene. Thus, our data are consistent with a causative role for GDNF mutations in some HSCR cases.      

GDNF family ligand immunoreactivity in the gut of teleostean fish

Glial-derived neurotrophic factor (GDNF), neurturin (NRTN), persephin (PSPN), and artemin (ARTN) are a group of proteins belonging to the GDNF family ligands (GFLs). GDNF, NRTN, and ARTN support the survival of central, peripheral, and autonomic neuron populations, while PSPN supports the survival of only several central neuron populations. A common receptor, RET, modulates the action of this family and a co-receptor, GFRα, determines RET ligand specificity. GDNF and NRTN appear to be essential for enteric nervous system (ENS) development in mammals, zebrafish, and other teleostean species. GFLs are also essential for the maintenance and plasticity of adult mammalian ENS. In this study, the distribution pattern of GFLs in the intestine of five adult fish (bass, gilt-head, scorpionfish, trout, and zebrafish) was evaluated by immunochemical and immunocytochemical analysis. The results demonstrated the presence of GDNF, NRTN, and ARTN in the gut of all species studied. They appeared to be spread in the ENS and/or endocrine cells of the intestine. These findings suggest that the presence of GFLs in fish gut is not only limited to developmental period, but could be also involved in the enteric physiology of adult species.     

A Novel Point Variant in NTRK3, R645C, Suggests a Role of this Gene in the Pathogenesis of Hirschsprung Disease

Hirschsprung disease (HSCR) is a developmental disorder characterized by the absence of ganglion cells in the myenteric and submucosal plexuses due to a defect in the migration process of neural crest neuroblasts. Manifestation of the disease has been linked to the dysfunction of two principal signalling pathways involved in the enteric nervous system (ENS) formation: the RET-GDNF and the EDN3-EDNRB receptor systems. However, the NTF3/NTRK3 signalling pathway plays an essential role in the development of the ENS suggesting a potential role for those genes in the pathogenesis of HSCR. We have sought to evaluate the candidature of the NTRK3 gene, which encodes the TrkC receptor, as a susceptibility gene for Hirschsprung disease. Using dHPLC technology we have screened the NTRK3 coding region in 143 Spanish HSCR patients. A total of four previously described polymorphisms and 12 novel sequence variants were detected. Of note, the novel R645C mutation was detected in 2 affected siblings of a HSCR family also carrying a RET splicing mutation. Using bioinformatics tools we observed that the presence of an additional cysteine residue might implicate structural alterations in the mutated protein. We propose haploinsufficiency as the most probable mechanism for the NTRK3 R645C mutation. NTRK3 and RET mutations in this family only appear together in the HSCR patients, suggesting that they per se are necessary but not sufficient to produce the phenotype. In addition, it is quite probable that the contribution of other still unidentified modifier genes, may be responsible for the different phenotypes (length of aganglionosis) in the two affected members.     

Hirschsprung associated GDNF mutations do not prevent RET activation

Hirschsprung disease (HSCR) is a complex disorder characterised by aganglia of distal gastrointestinal tracts. The highest proportion of both familial and sporadic cases is due to mutations of the RET proto-oncogene. Five germline mutations in the glial cell-line-derived neurotrophic factor (GDNF) gene, one of the RET ligands, have been detected in HSCR patients. Pedigrees analysis and the observed association between these GDNF alterations and RET variants in the same patients raised the question of whether the GDNF gene plays any causative/predisposing role in HSCR pathogenesis. In the present work, we have studied the ability of GDNF proteins, each bearing one of the reported mutations, to activate RET by performing a functional test in cultured neuroblastoma cells. Consistently with the lack of genotype/phenotype correlation in human subjects, our results indicate absence of detectable alterations of mutant GDNF induced RET activation.     

Analysis of RET, ZEB2, EDN3 and GDNF Genomic Rearrangements in 80 Patients with Hirschsprung Disease (Using multiplex ligation-dependent probe amplification)

Hirschsprung disease (HSCR) is transmitted in a complex pattern of inheritance and is mostly associated with variants in the RET proto-oncogene. However, RET mutations are only identified in 15–20% of sporadic HSCR cases and solely in 50% of the familial cases. Since genomic rearrangements in particularly sensitive areas of the RET proto-oncogene and/or associated genes may account for the HSCR phenotype in patients without other detectable RET variants, the aim of the present study was to identify rearrangements in the coding sequence of RET as well as in three HSCR-associated genes ( ZEB2 , EDN3 and GDNF ) in HSCR patients by using Multiplex Ligation-dependent Probe Amplification (MLPA). We have screened 80 HSCR patients for genomic rearrangements in RET, ZEB2, EDN3 and GDNF and did not identify any deletion or amplification in these four genes in all patients. We conclude that genomic rearrangements in RET are rare and were not responsible for the HSCR phenotype in individuals without identifiable germline RET variants in our group of patients, yet this possibility cannot be excluded altogether because the confidence to identify variation in at least two percent of the individuals was only 95%.     

Analysis of RET,ZEB2, EDN3 and GDNF Genomic Rearrangements in Central Congenital Hyperventilation Syndrome Patients by Multiplex Ligation‐dependent Probe Amplification

Central congenital hypoventilation syndrome (CCHS) is an autonomous control disease producing hypoventilation, high PaCO₂, and low PaO₂ during quiet sleep. The main gene variants detected in CCHS are mutations in the PHOX2b gene in up to 97% of isolated cases. However, CCHS is sometimes associated with autonomic diseases such as Hirschsprung disease (HSCR). Since genomic rearrangements in particularly sensitive areas of the RET protooncogene and/or associated genes may account for the CCHS/HSCR phenotype in patients without other detectable RET variants, the aim of the present study was to identify rearrangements in the coding sequence of RET as well as in three HSCR‐associated genes (ZEB2, EDN3 and GDNF) in CCHS/HSCR patients by using Multiplex Ligation‐dependent Probe Amplification (MLPA). We have screened 27 CCHS and 11 CCHS/HSCR patients for genomic rearrangements in RET, ZEB2, EDN3 and GDNF and did not identify any deletion or amplification in these four genes in all patients. We conclude that genomic rearrangements in RET are rare and were not responsible for the CCHS/HSCR phenotype in individuals without identifiable germline RET variants in our group of patients, yet this possibility cannot be excluded altogether given the size of the cohort.     

Transforming growth factor beta cooperates with persephin for dopaminergic phenotype induction

The aim of the present study was to investigate the putative cooperative effects of transforming growth factor beta (TGF-beta) and glial cell line-derived neurotrophic factor (GDNF) family ligands in the differentiation of midbrain progenitors toward a dopaminergic phenotype. Therefore, a mouse midbrain embryonic day (E) 12 neurospheres culture was used as an experimental model. We show that neurturin and persephin (PSPN), but not GDNF, are capable of transient induction of dopaminergic neurons in vitro. This process, however, requires the presence of endogenous TGF-beta. In contrast, after 8 days in vitro GDNF rescued the TGF-beta neutralization-dependent loss of the TH-positive cells. In vivo, at E14.5, no apparent phenotype concerning dopaminergic neurons was observed in Tgf-beta2(-/-)/gdnf(-/-) double mutant mice. In vitro, combined TGF-beta/PSPN treatment achieved a yield of approximately 20% TH-positive cells that were less vulnerable against 1-methyl-4-phenyl pyridinium ion toxicity. The underlying TGF-beta/PSPN differentiation signaling is receptor-mediated, involving p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways. These results indicate that phenotype induction and survival of fully differentiated neurons are accomplished through distinct pathways and individual factor requirement. TGF-beta is required for the induction of dopaminergic neurons, whereas GDNF is required for regulating and/or maintaining a differentiated neuronal phenotype. Moreover, this study suggests that the combination of TGF-beta with PSPN is a potent inductive cocktail for the generation of dopaminergic neurons that should be considered in tissue engineering and cell replacement therapies for Parkinson's disease.     

Multiple Functional Effects of RET Kinase Domain Sequence Variants in Hirschsprung Disease

The REarranged during Transfection (RET) gene encodes a receptor tyrosine kinase required for maturation of the enteric nervous system. RET sequence variants occur in the congenital abnormality Hirschsprung disease (HSCR), characterized by absence of ganglia in the intestinal tract. Although HSCR‐RET variants are predicted to inactivate RET, the molecular mechanisms of these events are not well characterized. Using structure‐based models of RET, we predicted the molecular consequences of 23 HSCR‐associated missense variants and how they lead to receptor dysfunction. We validated our predictions in biochemical and cell‐based assays to explore mutational effects on RET protein functions. We found a minority of HSCR‐RET variants abrogated RET kinase function, while the remaining mutants were phosphorylated and transduced intracellular signals. HSCR‐RET sequence variants also impacted on maturation, stability, and degradation of RET proteins. We showed that each variant conferred a unique combination of effects that together impaired RET protein activity. However, all tested variants impaired RET‐mediated cellular functions, including cell transformation and migration. Our data indicate that the molecular mechanisms of impaired RET function in HSCR are highly variable. Although a subset of variants cause loss of RET kinase activity and downstream signaling, enzymatic inactivation is not the sole mechanism at play in HSCR.     

HRAS-related epidermal nevus syndromes: Expansion of the spectrum with first branchial arch defects

Epidermal nevus syndrome (ENS) comprises a heterogeneous group of neurocutaneous syndromes associated with the presence of epidermal nevi and variable extracutaneous manifestations. Postzygotic activating HRAS pathogenic variants were previously identified in nevus sebaceous (NS), keratinocytic epidermal nevus (KEN), and different ENS, including Schimmelpenning–Feuerstein–Mims and cutaneous-skeletal-hypophosphatasia syndrome (CSHS). Skeletal involvement in HRAS-related ENS ranges from localized bone dysplasia in association with KEN to fractures and limb deformities in CSHS. We describe the first association of HRAS-related ENS and auricular atresia, thereby expanding the disease spectrum with first branchial arch defects if affected by the mosaic variant. In addition, this report illustrates the first concurrent presence of verrucous EN, NS, and nevus comedonicus (NC), indicating the possibility of mosaic HRAS variation as an underlying cause of NC. Overall, this report extends the pleiotropy of conditions associated with mosaic pathogenic variants in HRAS affecting ectodermal and mesodermal progenitor cells.     

Characterisation of the human GFRalpha-3 locus and investigation of the gene in Hirschsprung disease

BACKGROUND—The GDNF family receptor alpha (GFRα) proteins are extracellular cell surface bound molecules that act as adapters in binding of the GDNF family of soluble neurotrophic factors to the RET receptor. These molecules are essential for development of many neural crest derived cell types and the kidney. Mutations in RET and in two members of the GDNF ligand family are associated with Hirschsprung disease (HSCR), a congenital absence of the enteric ganglia. Members of the GFRα family are also candidates for HSCR mutations. One such gene is GFRα-3, which is expressed in the peripheral nervous system and developing nerves.
OBJECTIVE—We have characterised the structure of the human GFRα-3 locus and investigated the gene for sequence variants in a panel of HSCR patients.
METHODS—Long range PCR or subcloning of PAC clones was used to investigate GFRα-3 intron-exon boundaries. A combination of single strand conformation polymorphism (SSCP) analysis and direct sequencing was used to investigate GFRα-3 sequence variants.
RESULTS—GFRα-3 spans eight coding exons and has a gene structure and organisation similar to that of GFRα-1. We identified three polymorphic variants in GFRα-3 in a normal control population, a subset of which also occurred in HSCR patients. We did not detect any sequence variants within the coding sequence of GFRα-3. We found a base substitution in the 5' UTR of GFRα-3, 15 base pairs upstream of the translation start site. A second substitution was identified in intron 4 (IVS4-30G>A) between the splice branch site and the splice acceptor site. The final variant was a 2 base pair insertion within the splice donor consensus sequence of exon 7 (IVS7+4ins GG).
CONCLUSIONS—We did not detect any correlation between variants of GFRα-3 and the HSCR phenotype. Our data suggest that mutations of this gene are not a cause of HSCR.


Keywords: GFRα-3; Hirschsprung disease; RET     

Investigation of germline GFR alpha-1 mutations in Hirschsprung disease

Inactivating mutations of the RET proto-oncogene and of one of its soluble ligand molecules, glial cell line derived neurotrophic factor (GDNF), have been found in a subset of patients with Hirschsprung disease (HSCR). However, the majority of HSCR mutations remain unidentified. As normal RET function requires a multicomponent ligand complex for activation, other members of the RET ligand complex are primary candidates for these mutations. We investigated the presence of mutations in another member of the RET signalling complex, GDNF family receptor alpha-1 (GFR alpha-1), in a panel of 269 independent cases of HSCR. We identified 10 polymorphisms at the GFR alpha-1 locus. Surprisingly, however, we did not identify any sequence variants in our HSCR population that were not also present in a normal control population. Our data suggest that mutations of the GFR alpha-1 gene are not a common aetiological event in HSCR.     

Mutation analysis of the RET receptor tyrosine kinase in Hirschsprung disease

Hirschsprung disease (HSCR), or congenital agangiionic megacolon,is the most common cause of congenital bowel obstruction withan incidence of 1 in 5000 live births. Recently, linkage ofan incompletely penetrant, dominant form of HSCR was reported,foilowed by identification of mutations in the RET receptortyrosine kinase. To determine the frequency of RET mutationsin HSCR and correlate genotype with phenotype, we have screenedfor mutations among 80 HSCR probands representing a wide rangeof phenotypes and family structures. Polymerase chain reaction(PCR) and single-strand conformation polymorphism (SSCP) analysisof RET's 20 exons for mutations among probands revealed eightputative mutations (10%). Sequence changes, which included missense,frameshift and complex mutations, were detected in both familialand isolated cases, among patients with both long-and short-segmentHSCR and in three kindreds with other phenotypes (maternal deafness,talipes and mairotation of the gut, respectively). Two mutations(C609Y and C620R) we identified have previously been associatedwith multiple endocrine neoplasla type 2A (MEN2A), medullarythyroid carcinoma (MTC) and, on rare occasions, HSCR. Thus,while HSCR family members may be at risk for developing neuroendocrinetumors, it follows that identical mutations in RET may be ableto participate in the pathogenesis of distinct phenotypes. Ourdata suggest that: (i) the overall frequency of RET mutationsin HSCR patients is low and therefore, other genetic and/orenvironmental determinants contribute to the majority of HSCRsusceptibility, and (ii) at present, there is no obvious relationshipbetween RET genotype and HSCR phenotype.     

Functional characterization of mutations in the GDNF gene of patients with Hirschsprung disease

Hirschsprung disease (HSCR) is a congenital disorder characterized by the absence of enteric nervous plexuses in hind gut. Ten to forty percent of HSCR patients carry a dominant loss-of-function mutation in the gene encoding the receptor tyrosine kinase RET, a receptor for glial cell line-derived neurotrophic factor (GDNF). Although several mutations have also been found in the GDNF gene of HSCR patients, their impact on GDNF function is unknown. In this study, we have characterized the effect of these mutations on the ability of GDNF to bind and activate its receptors. Although none of the four mutations analyzed appeared to affect the ability of GDNF to activate RET, two of them resulted in a significant reduction in the binding affinity of GDNF for the binding subunit of the receptor complex, GFR(alpha)1. Our results indicate that, although none of the GDNF mutations identified so far in HSCR patients are per se likely to result in HSCR, two of these mutations (i.e. D150N and I211M) may, in conjunction with other genetic lesions, contribute to the pathogenesis of this disease.     

Balancing neural crest cell intrinsic processes with those of the microenvironment in Tcof1 haploinsufficient mice enables complete enteric nervous system formation

The enteric nervous system (ENS) comprises a complex neuronal network that regulates peristalsis of the gut wall and secretions into the lumen. The ENS is formed from a multipotent progenitor cell population called the neural crest, which is derived from the neuroepithelium. Neural crest cells (NCCs) migrate over incredible distances to colonize the entire length of the gut and during their migration they must survive, proliferate and ultimately differentiate. The absence of an ENS from variable lengths of the colon results in Hirschsprung's disease (HSCR) or colonic aganglionosis. Mutations in about 12 different genes have been identified in HSCR patients but the complex pattern of inheritance and variable penetrance suggests that additional genes or modifiers must be involved in the etiology and pathogenesis of this disease. We discovered that Tcof1 haploinsufficiency in mice models many of the early features of HSCR. Neuroepithelial apoptosis diminished the size of the neural stem cell pool resulting in reduced NCC numbers and their delayed migration along the gut from E10.5 to E14.5. Surprisingly however, we observe continued and complete colonization of the entire colon throughout E14.5-E18.5, a period in which the gut is considered to be non- or less-permissive to NCC. Thus, we reveal for the first time that reduced NCC progenitor numbers and delayed migration do not unequivocally equate with a predisposition for the pathogenesis of HSCR. In fact, these deficiencies can be overcome by balancing NCC intrinsic processes of proliferation and differentiation with extrinsic influences of the gut microenvironment.     

Glial cell line-derived neurotrophic factor family receptors are abnormally expressed in aganglionic bowel of a subpopulation of patients with Hirschsprung's disease

Hirschsprung's disease (HSCR), a congenital disease, is characterized by the absence of ganglion cells in the ganglion plexuses of the caudal most gut. In the aganglionic colon, the plexus remnants are replaced by aggregates of glial cells and hypertrophied nerve fibers. Signaling of glial cell line-derived neurotrophic factor (GDNF)-GFRAs-receptor tyrosine kinase (RET) is crucial for the development and maintenance of ganglion cells. Mutations of genes such as GDNF and RET lead to the perturbation of this signaling pathway, which causes HSCR. To understand the role of GFRAs in ganglion cells and the pathogenesis of HSCR, we intended to determine the specific cell lineages in the enteric nervous system that normally express GFRAs but are affected in HSCR. We studied colon biopsy specimens from 13 patients with HSCR (aged 1 day to 38 months) and 6 age-matched patients without HSCR as normal controls. RT-PCR, in situ hybridization, and immunohistochemistry were performed to examine the expression and cellular distributions of GFRAs in resected bowel segments of normal infants and those with HSCR. In normal infants and normoganglionic colon of patients with HSCR, the expression of GFRA1 was restricted to the glial cells and neurones of the ganglion plexuses. GFRAs expression was found to be markedly reduced in the aganglionic colons of 3 infants with HSCR but was unaffected in the aganglionic colons of 10 other infants with HSCR. Residual GFRA expression was restricted to enteric glial cells in the plexus remnants of the aganglionic colons. Hypertrophied nerve fibers were not found to express GFRA1. We provide the first evidence that abnormal expression of GFRAs in the enteric nervous system may be involved in the pathogenesis of HSCR in a subpopulation of patients.     

Correlation Between Genetic Variations in Hox Clusters and Hirschsprung's Disease

Interactions between migrating neural crest cells and the environment of the gut are crucial for the development of the enteric nervous system (ENS). A key signalling mediator is the RET-receptor-tyrosine-kinase which, when defective, causes Hirschprung's disease (HSCR, colon aganglionosis). RET mutations alone cannot account for the variable HSCR phenotype, invoking interactions with as yet unknown, and probably inter-related, loci involved in ENS development. Homeobox ( HOX ) genes have a major role in gut development as depicted by the enteric Hox code. We investigated whether DNA alterations in HOX genes, either alone or in combination with RET , are implicated in HSCR. Genotyping effort was minimized by applying the HapMap data on Han Chinese from Beijing (CHB). 194 HSCR patients and 168 controls were genotyped using Sequenom technology for 72 tag, single nucleotide polymorphisms (SNPs) distributed along the HOX clusters. The HapMap frequencies were compared to those in our population and standard statistics were used for frequency comparisons. The multifactor-dimensionality-reduction method was used for multilocus analysis, in which RET promoter SNP genotypes were included. Genetic interactions were found between two HOX loci (5'- HOXA13 and 3'UTR- HOXB7 ) and the RET loci tested. Minor allele frequencies (MAF) of the SNPs tested in our sample were not significantly different from those reported by HapMap when the sample sizes of the populations compared were considered. This is the first evaluation of the HOX genes in HSCR and the first application of HapMap data in a Chinese population. The interacting HOX loci may affect the penetrance of the RET risk allele. HapMap data for the CHB population correlated well with the general Chinese population.