Up-regulated FHL1 Expression Maybe Involved in the Prognosis of Hirschsprung's Disease

Background: In a subset of patients with Hirschsprung''s disease (HSCR), gastrointestinal motor dysfunction persisted long after surgical correction. Gastrointestinal motility is achieved through the coordinated activity of the enteric nervous system, interstitial cells of Cajal, and smooth muscle (SMC) cells. Inhibition of four-and-a-half LIM protein-1 (Fhl1) expression by siRNA significantly decreases pulmonary artery SMCs migration and proliferation. Furthermore when up-expressing FHL1 in atrial myocytes, K (+) current density markedly increases, therefore changing myocytes'' response to an electrical stimulus. However whether FHL1 in colon SMCs (the final effector organ) influences intestinal motility in HSCR patients has not been clarified. Methods: FHL1 mRNA and protein expressions were analyzed in 32 HSCR colons and 4 normal colons. Results: Smooth muscle layers were thicken and disorganized in HSCR. FHL1 was expressed in the ganglion cells of the myenteric plexus, submucosa, as well as in the longitudinal and circular muscle layer of the ganglionic colon. FHL1 mRNA relative expression level in aganglionic colons was 1.06±0.49 (ganglionic colon relative expression level was 1) (P=0.44). FHL1 protein gray level relative to GAPDH in normal colons was 0.83±0.09. FHL1 expression level in ganglionic colon (1.66±0.30) or aganglionic colon (1.81±0.35) was significantly higher than that in normal colons (P=0.045 and P=0.041, respectively). Meanwhile, we found FHL1 expression in aganglionic colon was slightly stronger than that in ganglionic colon (P=0.036). Conclusion: These data suggested that up-regulated FHL1 in smooth muscle in HSCR might be associated with intestinal wall remodeling in HSCR and might be one of the risk factors for gastrointestinal motor dysfunction.     

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.     

Expression of dishevelled gene in Hirschsprung’s disease

Hirschsprung’s disease (HSCR) is a congenital disorder of the enteric nervous system and is characterized by an absence of enteric ganglion cells in terminal regions of the gut during development. Dishevelled (DVL) protein is a cytoplasmic protein which plays pivotal roles in the embryonic development. In this study, we explore the cause of HSCR by studying the expression of DVL-1 and DVL-3 genes and their proteins in the aganglionic segment and the ganglionic segment of colon in HSCR patients. Materials and Methods: Specimen of aganglionic segment and ganglionic segment of colon in 50 cases of HSCR patients. Expression levels of mRNA and proteins of DVL-1 and DVL-3 were confirmed by quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry staining between the aganglionic segment and the ganglionic segment of colon in HSCR patients. Results: The mRNA expression of DVL-1 and DVL-3 were 2.06 fold and 3.12 fold in the aganglionic segment colon tissues compared to the ganglionic segment, respectively. Similarly, the proteins expression of DVL-1 and DVL-3 were higher (39.71 ± 4.53 vs and 53.90 ± 6.79 vs) in the aganglionic segment colon tissues than in the ganglionic segment (15.01 ± 2.66 and 20.13 ± 3.63) by western blot. Besides, immunohistochemical staining showed that DVL-1 and DVL-3 have a significant increase in mucous and submucous layers from aganglionic colon segments compared with ganglionic segments. Conclusion: The study showed an association of DVL-1 and DVL-3 with HSCR, it may play an important role in the pathogenesis of HSCR.     

Mutation of the RET ligand, neurturin, supports multigenic inheritance in Hirschsprung disease [published erratum appears in Hum Mol Genet 1998 Oct;7(11):1831]

Hirschsprung disease (HSCR) is a frequent neurocristopathy characterized by the absence of submucosal and myenteric plexuses in a variable length of the gastrointestinal tract. Pedigrees and segregation analyses suggested the involvement of one or several dominant genes with low penetrance in HSCR. Considering that RET and glial cell line-derived neurotrophic factor (GDNF) mutations have been reported in the disease, we regarded the other RET ligand, neurturin (NTN), as an attractive candidate gene, especially as it shares large homologies with GDNF. Here, we report on the finding of a heterozygous missense NTN mutation in a large non-consanguineous family including four children affected with a severe aganglionosis phenotype extending up to the small intestine. Interestingly, it appears that the NTN mutation reported here is not sufficient to cause HSCR, and this multiplex family also segregates a RET mutation. This cascade of independent and additive genetic events fits well with the multigenic pattern of inheritance expected in HSCR, and further support the role of RET ligands in development of the enteric nervous system.      

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.      

Differential expressions of BMPR1α, ACTN4α and FABP7 in Hirschsprung disease

Hirschsprung disease (HSCR) is characterized by the absence of intramural ganglion cells in the nerve plexuses of the distal gut. Recent studies have shown that the bone morphogenetic protein receptor-type IA (BMPR1α), actinin-alpha 4 (ACTN4α) and fatty acid binding protein 7 (FABP7) play important roles in the differentiation and development of neurons. The aganglionic (stenotic) and the ganglionic (normal) colon segment tissues of 60 HSCR patients were collected to investigate the expression pattern of BMPR1α, ACTININ-4α and FABP7 using RT-PCR, quantitative real-time RT-PCR (qRT-PCR) and immunohistochemical staining. The mRNA and protein expressions of BMPR1α and ACTN4α were higher in the stenotic colon segment tissue than those in the normal colon segment tissue. However, the mRNA and protein expressions of FABP7 were lower in the stenotic colon segment tissue than those in the normal colon segment tissue. The study in HSCR patients, findings in mRNA and protein alterations to expecting provide more information to in order to find some clue for the pathomechanism of HSCR disease.     

Long segment and short segment familial Hirschsprung''s disease: variable clinical expression at the RET locus.

Hirschsprung's disease (aganglionic megacolon, HSCR) is a frequent condition of unknown origin (1/5000 live births) resulting in intestinal obstruction in neonates and severe constipation in infants and adults. In the majority of cases (80%), the aganglionic tract involves the rectum and the sigmoid colon only (short segment HSCR), while in 20% of cases it extends toward the proximal end of the colon (long segment HSCR). In a previous study, we mapped a gene for long segment familial HSCR to the proximal long arm of chromosome 10 (10q11.2). Further linkage analyses in familial HSCR have suggested tight linkage of the disease gene to the RET protoncogene mapped to chromosome 10q11.2. Recently, nonsense and missense mutations of RET have been identified in HSCR patients. However, the question of whether mutations of the RET gene account for both long segment and short segment familial HSCR remained unanswered. We have performed genetic linkage analyses in 11 long segment HSCR families and eight short segment HSCR families using microsatellite DNA markers of chromosome 10q. In both anatomical forms, tight pairwise linkage with no recombinant events was observed between the RET proto-oncogene locus and the disease locus (Zmax = 2.16 and Zmax = 5.38 for short segment and long segment HSCR respectively at 0 = 0%) Multipoint linkage analyses performed in the two groups showed that the maximum likelihood estimate was at the RET locus. Moreover, we show that point mutations of the RET proto-oncogene occur either in long segment or in short segment HSCR families and we provide evidence for incomplete penetrance of the disease causing mutation. These data suggest that the two anatomical forms of familial HSCR, which have been separated on the basis of clinical and genetic criteria, may be regarded as the variable clinical expression of mutations at the RET locus.     

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.     

Co-segregation of MEN2 and Hirschsprung's disease: the same mutation of RET with both gain and loss-of-function?

Multiple endocrine neoplasia type 2 (MEN2) and Hirschsprung's disease (HSCR) are two dominantly inherited neurocristopathies ascribed to mutations in the RET gene [Chakravarti, 1996; Pasini et al., 1996; Eng and Mulligan, 1997]. MEN2 is a cancer syndrome comprising three related clinical subtypes: (1) MEN type 2A (MEN2A; MIM# 171400) characterized by the association of medullary thyroid carcinoma (MTC), pheochromocytoma (Pheo), and hyperparathyroidism; (2) MEN type 2B (MEN2B; MIM# 162300), which includes MTC, Pheo, mucosal neuromas, ganglioneuromatosis of the digestive tract, and skeletal abnormalities; and (3) familial MTC (FMTC; MIM# 155240), defined by the sole occurrence of MTC. HSCR (MIM# 142623) is a congenital malformation caused by the absence of enteric plexuses in the hindgut, leading to bowel obstruction in neonates. The RET gene (MIM# 164761) codes for a transmembrane tyrosine kinase, a component of a multimeric complex that also comprises one of four members of a novel family of glycosylphosphatidylinositol (GPI)-anchored receptor, GFRalpha((1-4) (e.g., GFRA1, MIM# 601496; references are detailed in Baloh et al. [1998]. Four structurally related soluble factors-glial cell line-derived neurotrophic factor (GDNF), neurturin, persephin, and artemin-are the ligands of these multimolecular receptors in which the nature of the GFRalpha determines the ligand specificity of the complex [see Baloh et al., 1998, for references]. It is well documented that RET/GFRalpha-1/GDNF delivers a signal critical for the survival of the early neural crest-derived precursors that colonize the intestine below the rostral foregut and give rise to the enteric nervous plexuses [Gershon, 1997; Cacalano et al., 1998; Enomoto et al., 1998].     

The single nucleotide polymorphisms in Smad-interacting protein 1 gene contribute to its ectopic expression and susceptibility in Hirschsprung's disease

Hirschsprung's disease (HSCR) is the third most common congenital disorder of the gastrointestinal tract. It is an anomalous enteric nervous system (ENS) characterized by the absence of ganglion cells in the myenteric and submucosal plexuses. It has been reported that the Smad-interacting protein 1(SIP1) is critical in embryonic development of ENS for its regulation on neural crest cells. In the present study, we analyzed 3 polymorphisms of the SIP1 gene rs41292293 (exon5), rs34961586 (exon6) and rs13017697 (exon8) to determine their potential contributions to the susceptibility of HSCR. Allele frequencies and genotype distributions were analyzed by sequence analysis in 107 HSCR patients and 107 normal controls. The SIP1 expression was carried out by using real-time PCR, western blot and immunohistochemistry. Polymorphic analysis indicated that the genotype distributions and allele frequencies in SIP1 gene rs41292293, rs34961586 and rs13017697 were statistically different between HSCR and normal controls. The expression analysis revealed that SIP1 was ectopically expressed in the aganglionic segments; neither the mRNA nor the protein levels demonstrated that the difference compared with those was in the normal segments. In conclusion, the single nucleotide polymorphisms in SIP1 gene rs41292293, rs34961586 and rs13017697 are associated with the ectopic expression of this gene in human HSCR and contribute to the susceptibility of this disease in population.     

Mutational analysis of RET/GDNF/NTN genes in children with total colonic aganglionosis with small bowel involvement

Hirschsprung disease (HSCR) is characterized by the absence of intramural ganglion cells in the distal gut, resulting in bowel obstruction shortly after birth. Aganglionosis usually affects the distal colon, but may also extensively involve the entire colon and, rarely, the more proximal bowel. Recently, germline mutations of RET, GDNF, and NTN genes have been reported in HSCR. Here we describe the results of mutational analysis of these genes in 15 Japanese child patients with total colonic aganglionosis with small bowel involvement. DNA sequences of all the RET/GDNF/NTN coding regions were determined by the direct dyedeoxy terminator cycle method. Eight different RET mutations were identified in exons 1, 7, 10, 12, 15, and 17 in 10 of the 15 patients. Of these eight mutations, five were found in the tyrosine kinase domain. No GDNF or NTN mutation was found. Compared with typical HSCR, this patient group appeared to exhibit a higher percentage of RET mutations and accumulation of mutations in the tyrosine kinase domain. A homozygous (or hemizygous) RET mutation was found in a male baby with total intestinal aganglionosis, while the heterozygosity of the same mutation resulted in a less severe type of aganglionosis. In familial cases, all heterozygous for the same mutation, aganglionosis was more severe in male than in female siblings. These results also urge us to examine if the RET germline mutation may cause critical alteration of the GDNF/NTN-Ret signal transduction more severely in homo(hemi)zygosity and in male fetuses during organogenesis.     

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.     

Familial form of hirschsprung disease: nucleotide sequence studies reveal point mutations in the RET proto-oncogene in two of six families but not in other candidate genes

Hirschsprung disease (HSCR; McKusick 142623) or aganglionic megacolon is a frequent (1 in 5,000 live births) heritable disorder of the enteric nervous system. By haplotyping with a variety of microsatellite markers, by amplifying all 20 exons of the RET proto-oncogene and by applying a direct DNA sequencing protocol, we have analyzed the DNA from HSCR patients in 6 different families. In one family with a joint occurrence of HSCR and FMTC (follicular medullary thyroid carcinoma), we have identified a mutation in codon 609 in one out of 6 cysteine residues encoded in exon 10 of the RET gene. This C609R point mutation has not previously been reported to cause HSCR. In 2 of the HSCR patients described here from different families, we have found a mutation in exon 2 (R77C) and a silent mutation in exon 3 (Y204Y), respectively, in the extracellular part of the RET proto-oncogene. In introns 2 and 17 of the RET proto-oncogene in 2 families, we have detected single nucleotide exchanges that are probably polymorphisms with unknown, if any, relations to HSCR. The DNA sequences of 5 further genes (GDNF, GDNFRalpha, EDN3, EDNRB, and NTN), that may contribute to the development of HSCR, have not shown mutations in the patients analyzed so far. In 2 of the reported families with several affected children and one grandchild, sequence analyses revealed no mutations in the coding regions of any of the candidate genes analyzed.     

Familial form of Hirschsprung disease: Nucleotide sequence studies reveal point mutations in the RET proto‐oncogene in two of six families but not in other candidate genes

Hirschsprung disease (HSCR; McKusick 142623) or aganglionic megacolon is a frequent (1 in 5,000 live births) heritable disorder of the enteric nervous system. By haplotyping with a variety of microsatellite markers, by amplifying all 20 exons of the RET proto‐oncogene and by applying a direct DNA sequencing protocol, we have analyzed the DNA from HSCR patients in 6 different families. In one family with a joint occurrence of HSCR and FMTC (follicular medullary thyroid carcinoma), we have identified a mutation in codon 609 in one out of 6 cysteine residues encoded in exon 10 of the RET gene. This C609R point mutation has not previously been reported to cause HSCR. In 2 of the HSCR patients described here from different families, we have found a mutation in exon 2 (R77C) and a silent mutation in exon 3 (Y204Y), respectively, in the extracellular part of the RET proto‐oncogene. In introns 2 and 17 of the RET proto‐oncogene in 2 families, we have detected single nucleotide exchanges that are probably polymorphisms with unknown, if any, relations to HSCR. The DNA sequences of 5 further genes (GDNF, GDNFRα, EDN3, EDNRB, and NTN), that may contribute to the development of HSCR, have not shown mutations in the patients analyzed so far. In 2 of the reported families with several affected children and one grandchild, sequence analyses revealed no mutations in the coding regions of any of the candidate genes analyzed. Am. J. Med. Genet. 94:19–27, 2000. © 2000 Wiley‐Liss, Inc.     

Two distinct mutations of the RET receptor causing Hirschsprung's disease impair the binding of signalling effectors to a multifunctional docking site.

The RET gene codes for a transmembrane tyrosine kinase which is a subunit of a multimeric complex that acts as a receptor for four structurally related molecules: the glial cell line-derived neurotrophic factor (GDNF), neurturin, artemin and persephin. Germline mutations of RET cause a dominantly inherited dysgenesis of the enteric nervous system known as Hirschsprung's disease (HSCR; aganglionosis megacolon). The majority of HSCR mutations results either in a reduction of dosage of the RET protein or in the loss of RET function. Two novel distinct mutations of RET that led either to the deletion of codon 1059 (denoted Delta1059) or to the substitution of a Pro for Leu1061 have been identified in five HSCR families. In one large pedigree, two children born from asymptomatic consanguineous parents presented a severe form of HSCR and were found to carry the mutation at codon 1061 in the homozygous state. A tyrosine residue at position 1062 is an intracytoplasmic docking site that enables RET to recruit several signalling molecules, including the Shc adaptor protein. We now report that both HSCR mutations impair the fixation of Shc to RET and consequently prevent its phosphorylation. In addition, quantitative analysis in PC12 cells reveals that mutation Delta1059 inactivates the ability of RET to transduce a downstream signal whereas mutation L1061P only partially inhibits the signalling of RET. Finally, we provide evidence that these effects are partly mediated via the disruption of the RET/Shc interaction. Collectively, these results demonstrate that HSCR can be ascribed to mutations of RET which interfere with the binding of transduction effectors, such as Shc, and further provide a biochemical explanation for the phenotype of patients carrying a homozygous mutation at codon 1061. Finally, these data indicate that Y1062 is a multifunctional docking site that confers to RET the capacity to engage downstream signalling pathways which exert a crucial role during enteric neurogenesis.     

Expression analysis of BMP2, BMP5, BMP10 in human colon tissues from Hirschsprung disease patients

Objective: Bone morphogenetic proteins (BMPs) are members of the transforming growth factor β (TGF β) superfamily. BMP2, BMP5 and BMP10 exert their biological functions by interacting with membrane bound receptors belonging to the serine/threonine kinase family. Hirschsprung disease (HSCR) is characterized by the absence of intramural ganglion cells in the nerve plexuses of the distal gut. However, putative Notch function in enteric nervous system (ENS) development and the etiology of HSCR is unknown. Methods: Aganglionic and ganglionic colon segment tissues of 50 HSCR patients were investigated for the expression pattern of BMP2, BMP5 and BMP10 using real-time RT-PCR, Western blot analysis and immunohistochemical staining. Results: The mRNA levels of BMP2, BMP5 and BMP10 in the stenotic colon segment from HSCR patients were significantly higher than those in the normal ones. Similar increased expressions of them in the stenotic colon segments were detected by Western blotting coupled with densitometry analysis. Lastly, immunohistologicl stain showed significant BMP2, 5 and 10 increases in mucous and muscular layers from stenotic colon segments compared to normal segments. Conclusions: BMP2, BMP5 and BMP10 are elevated in the stenotic colon segment of HSCR, and BMPs signaling plays a pivotal role in the development of HSCR.     

Expression of bcl-2 in enteric neurons in normal human bowel and Hirschsprung disease

OBJECTIVE: The bcl-2 protein has the functional role of blocking apoptosis, ie, programmed cell death. This protein is widely expressed in the developing central and peripheral nervous systems. The purpose of this study was to map bcl-2 expression in the human enteric nervous system, as this has not previously been done. METHODS: Rectal specimens were obtained at autopsy of 13 fetuses at 13 to 31 weeks of gestation. Normal colon was also obtained from 5 children and 2 adults, and, in addition, ganglionic and aganglionic bowel resected in 11 patients with Hirschsprung disease was examined. Specimens were fixed in formalin, embedded in paraffin, and analyzed with immunohistochemical methods, using antibodies raised against bcl-2 and neuron-specific enolase (NSE). RESULTS: The bcl-2 protein was expressed in myenteric and submucous ganglion cells in fetuses, children, and adults. Nerve fibers of the enteric plexuses that were bcl-2 immunoreactive were few compared with the number of NSE-immunoreactive nerve fibers. In aganglionic bowel no bcl-2-or NSE-immunoreactive ganglion cells were revealed. Results of NSE immunohistochemistry showed clearly stained hypertrophic nerve bundles, known to be of extrinsic origin, which were only weakly bcl-2 immunoreactive. CONCLUSION: Expression of bcl-2 in enteric ganglion cells of the myenteric and submucous plexuses is displayed in the fetus and during childhood and is also retained in adult bowel. Immunohistochemical analysis of bcl-2 provides a good marker for identification of ganglion cells in Hirschsprung disease and may also be valuable for the diagnosis of disorders characterized by hypoganglionosis or hyperganglionosis.     

Comparative study of Hsp27, GSK3β, Wnt1 and PRDX3 in Hirschsprung's disease

Hirschsprung's disease (HSCR) is a developmental disorder of the enteric nervous system characterized by aganglionosis in distal gut. In this study, we used two‐dimensional gel electrophoresis (2‐DE) technology coupled with matrix assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) analysis to identify differentially expressed proteins in the aganglionic (stenotic) and ganglionic (normal) colon segment tissues from patients with HSCR. We identified 15 proteins with different expression levels between the stenotic and the normal colon segment tissues from patients with HSCR. Nine proteins were upregulated and six proteins downregulated in the stenotic colon segment tissues compared to the normal colon segment tissues. Based on the biological functions, we selected the Hsp27 upregulated proteins and the PRDX3 downregulated proteins to confirm their expression in 20 patients. The protein and mRNA expressions of Hsp27 were statistically higher in the stenotic colon segment tissues than in the normal colon segment tissues, whereas the protein and mRNA expressions of PRDX3 were statistically lower in the stenotic colon segment tissues than in the normal colon segment tissues. These findings of changes in mRNA and protein in tissues from patients with HSCR provide information which may be helpful in understanding the pathomechanism that is implicated in the disease.     

Novel mutations at RET ligand genes preventing receptor activation are associated to Hirschsprung's disease

Hirschsprung disease (HSCR) is a developmental disorder characterized by the absence of ganglion cells along variable lengths of the distal gastrointestinal tract. The major susceptibility gene for the disease is the RET proto-oncogene, which encodes a receptor tyrosine kinase activated by the glial cell-derived neurotrophic factor (GDNF) family ligands. We analyzed the coding sequence of GDNF, NTRN, and, for the first time, ARTN and PSPN in HSCR patients and detected several novel variants potentially involved in the pathogenesis of HSCR. In vitro functional analysis revealed that the variant R91C in PSPN would avoid the correct expression and secretion of the mature protein. Moreover, this study also highlighted the role of both this variant and F127L in NRTN in altering RET activation by a significant reduction in phosphorylation. To support the role of PSPN R91C in HSCR phenotype, enteric nervous system (ENS) progenitors were isolated from human postnatal gut tissues and expression of GFRα4, the main co-receptor for PSPN, was demonstrated. This suggests that not only GDNF and NRTN but also PSPN might promote survival of precursor cells during ENS development. In summary, we report for the first time the association of PSPN gene with HSCR and confirm the involvement of NRTN in the disease, with the identification of novel variants in those genes. Our results suggest that the biological consequence of the mutations NTRN F127L and PSPN R91C would be a reduction in the activation of RET-dependent signaling pathways, leading to a defect in the proliferation, migration, and/or differentiation process of neural crest cells within the developing gut and thus to the typical aganglionosis of the HSCR phenotype.