Cloning and characterization of the human GFRA2 locus and investigation of the gene in Hirschsprung disease

The glial-cell-line-derived neurotrophic factor (GDNF) family receptors alpha (GFRalpha) are cell surface bound glycoproteins that mediate interactions of the GDNF ligand family with the RET receptor. These interactions are crucial to the development of the kidney and some peripheral nerve lineages. In humans, mutations of RET or RET ligands are associated with the congenital abnormality Hirschsprung disease (HSCR) in which nerves and ganglia of the hind gut are absent. As the GFRalpha family are required for normal activation of the RET receptor, they are also candidates for a role in HSCR. The GFRA2 gene, which is required for the development of the myenteric nerve plexus, is an excellent candidate gene for HSCR. In this study, we cloned the human GFRA2 locus, characterized the gene structure, and compared it with other GFRA family members. We further investigated the GFRA2 gene for mutations in a panel of HSCR patients. GFRA2 has nine coding exons that are similar in size and organization to those of other GFRA family genes. We identified six sequence variants of GFRA2, four of which did not affect the amino acid sequence of the GFRalpha-2 protein. Two further changes that resulted in amino acid substitutions were found in exon 9 and were predicted to lie in the amino acid sequence encoding the glycosylphosphatidylinositol-linkage signal of GFRalpha-2. There was no difference in frequency of any of the sequence variants between control and HSCR populations. Our data indicate that members of the GFRA gene family are closely related in intron/exon structure and in sequence. We have not detected any correlation between sequence variants of GFRA2 and the HSCR phenotype.     

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.     

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.     

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.     

Germline mutations of the paired-like homeobox 2B (PHOX2B) gene in neuroblastoma

Neuroblastoma (NB) is a frequent pediatric tumor for which recurrent somatic rearrangements are known. Germline mutations of predisposing gene(s) are suspected on the basis of rare familial cases and the association of NB with other genetically determined congenital malformations of neural crest-derived cells--namely, Hirschsprung disease (HSCR) and/or congenital central hypoventilation syndrome (CCHS). We recently identified the paired-like homeobox 2B (PHOX2B) gene as the major disease-causing gene in isolated and syndromic CCHS, which prompted us to regard it as a candidate gene in NB. Here, we report on germline mutations of PHOX2B in both a familial case of NB and a patient with the HSCR-NB association. PHOX2B, therefore, stands as the first gene for which germline mutations predispose to NB.     

Phenotypic variation in a family with mutations in two Hirschsprung-related genes (RET and endothelin receptor B)

Hirschsprung disease is a congenital malformation affecting 1 in 5000 live births. The absence of parasympathetic neuronal ganglia (Meissner, Auerbach) in the hindgut results in poor coordination of peristaltic movement, and a varying degree of constipation. Four different genes have been implicated in the pathogenesis of Hirschsprung disease: the RET tyrosine kinase receptor gene; one of its ligands, the glial cell line-derived neurotrophic factor (GDNF) gene; the endothelin receptor B (EDNRB) gene; and its ligand, endothelin-3 (EDN3). Recently, combinations of mutations in two of these genes (RET and GDNF) have been reported in Hirschsprung patients. We report a family with missense mutations in both the RET gene (R982C) and the EDNRB gene (G57S). In this family, three out of five members have the two mutations, but only one, a boy, has the Hirschsprung disease phenotype. This illustrates the complexity of the molecular background of Hirschsprung disease. Received: 23 January 1998 / Accepted: 24 March 1998     

Renal aplasia in humans is associated with RET mutations

In animal models, kidney formation is known to be controlled by the proteins RET, GDNF, and GFRA1; however, no human studies to date have shown an association between abnormal kidney development and mutation of these genes. We hypothesized that stillborn fetuses with congenital renal agenesis or severe dysplasia would possess mutations in RET, GDNF, or GFRA1. We assayed for mutations in these genes in 33 stillborn fetuses that had bilateral or unilateral renal agenesis (29 subjects) or severe congenital renal dysplasia (4 subjects). Mutations in RET were found in 7 of 19 fetuses with bilateral renal agenesis (37%) and 2 of 10 fetuses (20%) with unilateral agenesis. In two fetuses, there were two different RET mutations found, and a total of ten different sequence variations were identified. We also investigated whether these mutations affected RET activation; in each case, RET phosphorylation was either absent or constitutively activated. A GNDF mutation was identified in only one fetus with unilateral agenesis; this subject also had two RET mutations. No GFRA1 mutations were seen in any fetuses. These data suggest that in humans, mutations in RET and GDNF may contribute significantly to abnormal kidney development.     

Human glial cell line-derived neurotrophic factor receptor alpha 4 is the receptor for persephin and is predominantly expressed in normal and malignant thyroid medullary cells

Glial cell line-derived neurotrophic factor (GDNF) family ligands signal through receptor complex consisting of a glycosylphosphatidylinositol-linked GDNF family receptor (GFR) alpha subunit and the transmembrane receptor tyrosine kinase RET. The inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN2), associated with different mutations in RET, is characterized by medullary thyroid carcinoma. GDNF signals via GFRalpha1, neurturin via GFRalpha2, artemin via GFRalpha3, whereas the mammalian GFRalpha receptor for persephin (PSPN) is unknown. Here we characterize the human GFRalpha4 as the ligand-binding subunit required together with RET for PSPN signaling. Human and mouse GFRalpha4 lack the first Cys-rich domain characteristic of other GFRalpha receptors. Unlabeled PSPN displaces (125)I-PSPN from GFRA4-transfected cells, which express endogenous Ret. PSPN can be specifically cross-linked to mammalian GFRalpha4 and Ret, and is able to promote autophosphorylation of Ret in GFRA4-transfected cells. PSPN, but not other GDNF family ligands, promotes the survival of cultured sympathetic neurons microinjected with GFRA4. We identified different splice forms of human GFRA4 mRNA encoding for two glycosylphosphatidylinositol-linked and one putative soluble isoform that were predominantly expressed in the thyroid gland. Overlapping expression of RET and GFRA4 but not other GFRA mRNAs in normal and malignant thyroid medullary cells suggests that GFRalpha4 may restrict the MEN2 syndrome to these cells.     

Expression of PROKR1 and PROKR2 in human enteric neural precursor cells and identification of sequence variants suggest a role in HSCR

Background

The enteric nervous system (ENS) is entirely derived from neural crest and its normal development is regulated by specific molecular pathways. Failure in complete ENS formation results in aganglionic gut conditions such as Hirschsprung''s disease (HSCR). Recently, PROKR1 expression has been demonstrated in mouse enteric neural crest derived cells and Prok-1 was shown to work coordinately with GDNF in the development of the ENS.

Principal Findings

In the present report, ENS progenitors were isolated and characterized from the ganglionic gut from children diagnosed with and without HSCR, and the expression of prokineticin receptors was examined. Immunocytochemical analysis of neurosphere-forming cells demonstrated that both PROKR1 and PROKR2 were present in human enteric neural crest cells. In addition, we also performed a mutational analysis of PROKR1, PROKR2, PROK1 and PROK2 genes in a cohort of HSCR patients, evaluating them for the first time as susceptibility genes for the disease. Several missense variants were detected, most of them affecting highly conserved amino acid residues of the protein and located in functional domains of both receptors, which suggests a possible deleterious effect in their biological function.

Conclusions

Our results suggest that not only PROKR1, but also PROKR2 might mediate a complementary signalling to the RET/GFRα1/GDNF pathway supporting proliferation/survival and differentiation of precursor cells during ENS development. These findings, together with the detection of sequence variants in PROKR1, PROK1 and PROKR2 genes associated to HSCR and, in some cases in combination with RET or GDNF mutations, provide the first evidence to consider them as susceptibility genes for HSCR.     

The RET proto-oncogene induces apoptosis: a novel mechanism for Hirschsprung disease

The RET (rearranged during transfection) proto-oncogene encodes a tyrosine kinase receptor involved in both multiple endocrine neoplasia type 2 (MEN 2), an inherited cancer syndrome, and Hirschsprung disease (HSCR), a developmental defect of enteric neurons. We report here that the expression of RET receptor induces apoptosis. This pro-apoptotic effect of RET is inhibited in the presence of its ligand glial cell line-derived neurotrophic factor (GDNF). Furthermore, we present evidence that RET induces apoptosis via its own cleavage by caspases, a phenomenon allowing the liberation/exposure of a pro-apoptotic domain of RET. In addition, we report that Hirschsprung-associated RET mutations impair GDNF control of RET pro-apoptotic activity. These results indicate that HSCR may result from apoptosis of RET-expressing enteric neuroblasts.     

Geldanamycin promotes nuclear localisation and clearance of PHOX2B misfolded proteins containing polyalanine expansions

Polyalanine expansions in the PHOX2B gene have been detected in the vast majority of patients affected with congenital central hypoventilation syndrome, a neurocristopathy characterized by absence of adequate control of breathing, especially during sleep, with decreased sensitivity to hypoxia and hypercapnia. The correlation between length of the alanine expanded tracts and severity of congenital central hypoventilation syndrome respiratory phenotype has been confirmed by length-dependent cytoplasmic PHOX2B retention with formation of aggregates. To deepen into the molecular mechanisms mediating the effects of PHOX2B polyalanine expansions, we have set up experiments aimed at assessing the fate of cells characterized by PHOX2B polyalanine aggregates. In particular, we have observed that activation of the heat shock response by the drug geldanamycin is efficient both in preventing formation and in inducing clearance of PHOX2B pre-formed polyalanine aggregates in COS-7 cells expressing PHOX2B-GFP fused proteins, and ultimately also in rescuing the PHOX2B ability to transactivate the Dopamine-beta-Hydroxilase promoter. In addition, we have demonstrated elimination of PHOX2B mutant proteins by the proteasome and autophagy, two cellular mechanisms already been involved in the clearance of proteins containing expanded polyglutamine and polyalanine tracts. Moreover, our data suggest that geldanamycin effects on PHOX2B aggregates may be also mediated by the proteasome pathway. Finally, analysis of cellular toxicity due to polyalanine aggregates has confirmed the occurrence of cell apoptosis consequent to expression of PHOX2B carrying the longest expanded alanine tract and shown that geldanamycin can delay cell progression toward the most advanced apoptotic stages.     

Molecular heterogeneity of RET loss of function in Hirschsprung's disease.

The RET proto-oncogene encodes a receptor with tyrosine kinase activity (RET) that is involved in several neoplastic and non-neoplastic diseases. Oncogenic activation of RET, achieved by different mechanisms, is detected in a sizeable fraction of human thyroid tumors, as well as in multiple endocrine neoplasia types 2A and 2B (MEN2A and MEN2B) and familial medullary thyroid carcinoma tumoral syndromes. Germline mutations of RET have also been associated with a non-neoplastic disease, the congenital colonic aganglionosis, i.e. Hirschsprung's disease (HSCR). To analyse the impact of HSCR mutations on RET function, we have introduced into wild-type RET and activated RET(MEN2A) and RET(MEN2B) alleles three missense mutations associated with HSCR. Here we show that the three mutations caused a loss of function of RET when assayed in two model cell systems, NIH 3T3 and PC12 cells. The effect of different HSCR mutations was due to different molecular mechanisms. The HSCR972 (Arg972-->Gly) mutation, mapping in the intracytoplasmic region of RET, impaired its tyrosine kinase activity, while two extracellular mutations, HSCR32 (Ser32-->Leu) and HSCR393 (Phe393-->Leu), inhibited the biological activity of RET by impairing the correct maturation of the RET protein and its transport to the cell surface.     

Immuno-gene therapy of melanoma by tumor antigen epitope modified IFN-γ

Cytokine-based vaccines play a major part in tumor immuno-gene therapy. However, down-regulated antigen expression on tumor cells may diminish the immuno-potentiating aspects of cellular vaccines. In this study, we coexpressed a tumor antigen epitope with IFN- in the same gene by replacing the IFN- signal peptide with an antigen epitope-expressing signal peptide. We then investigated the effect of the antigen epitope-incorporated IFN- on the immunotherapy of murine melanoma B16 tumors. Results showed that TRP-2 epitope-expressing IFN- decreased B16 tumorigenicity and enhanced its immunogenicity after gene transfer. Protective immunity against wild type B16 tumors was induced by vaccination with IFN- transiently gene-modified tumor cells. These data suggest that cellular vaccines engineered to express an antigen epitope within an immunostimulatory cytokine could potentiate the immunization effect.     

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     

PHOX2B genotype allows for prediction of tumor risk in congenital central hypoventilation syndrome

The Phox2b gene is necessary for autonomic nervous-system development. Phox2b-/- mice die in utero with absent autonomic nervous system circuits, since autonomic nervous system neurons either fail to form or degenerate. We first identified the Phox2b human ortholog, PHOX2B, as the gene underlying congenital central hypoventilation syndrome (CCHS, or Ondine curse), with an autosomal dominant mode of inheritance and de novo mutation at the first generation. We have subsequently shown that heterozygous mutations of PHOX2B may account for several combined or isolated disorders of autonomic nervous-system development--namely, tumors of the sympathetic nervous system (TSNS), such as neuroblastoma and late-onset central hypoventilation syndrome. Here, we report the clinical and molecular assessments of a cohort of 188 probands with CCHS, either isolated or associated with Hirschsprung disease and/or TSNS. The mutation-detection rate was 92.6% (174/188) in our series, and the most prevalent mutation was an in-frame duplication leading to an expansion of +5 to +13 alanines in the 20-alanine stretch at the carboxy terminal of the protein. Such findings suggest PHOX2B mutation screening as a simple and reliable tool for the diagnosis of CCHS, independent of the clinically variable phenotype. In addition, somatic mosaicism was detected in 4.5% of parents. Most interestingly, analysis of genotype-phenotype interactions strongly supports the contention that patients with CCHS who develop malignant TSNS will harbor either a missense or a frameshift heterozygous mutation of the PHOX2B gene. These data further highlight the link between congenital malformations and tumor predisposition when a master gene in development is mutated.