Novel pathogenic mechanisms of congenital insensitivity to pain with anhidrosis genetic disorder unveiled by functional analysis of neurotrophic tyrosine receptor kinase type 1/nerve growth factor receptor mutations.

Congenital insensitivity to pain with anhidrosis (CIPA) is a rare genetic disease characterized by absence of reaction to noxious stimuli and anhidrosis. The genetic bases of CIPA have remained long unknown. A few years ago, point mutations affecting both coding and noncoding regions of the neurotrophic tyrosine receptor kinase type 1 (NTRK1)/nerve growth factor receptor gene have been detected in CIPA patients, demonstrating the implication of the nerve growth factor/NTRK1 pathway in the pathogenesis of the disease. We have previously shown that two CIPA mutations, the G571R and the R774P, inactivate the NTRK1 receptor by interfering with the autophosphorylation process. We have extended our functional analysis to seven additional NTRK1 mutations associated with CIPA recently reported by others. Through a combination of biochemical and biological assays, we have identified polymorphisms and pathogenic mutations. In addition to the identification of residues important for NTRK1 activity, our analysis suggests the existence of two novel pathogenic mechanisms in CIPA: one based on the NTRK1 receptor processing and the other acting through the reduction of the receptor activity.     

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     

The Gly571Arg mutation, associated with the autonomic and sensory disorder congenital insensitivity to pain with anhidrosis, causes the inactivation of the NTRK1/nerve growth factor receptor

Point mutations affecting the NTRK1/TRKA gene, encoding one of the receptors for the nerve growth factor (NGF), have been detected in congenital insensitivity to pain with anhidrosis (CIPA), a human hereditary sensory neuropathy characterized by absence of reaction to noxious stimuli and anhidrosis. To define the defect of NTRK1 in CIPA patients, we have introduced one of the previously reported mutations (Gly571Arg) into both the NTRK1 and the TRK-T3 oncogene cDNAs. The expression of the mutated constructs into COS1 cells revealed that the introduced mutation, while not affecting its correct membrane localization, rendered the NTRK1 protein unable to undergo activation upon stimulation with NGF. Similarly, the mutation abolished the constitutive activation of the TRK-T3 oncogene. Transfection into NIH3T3 and PC12 cells showed the loss of transforming and differentiating activity by the mutated constructs. Our results demonstrate clearly that the CIPA mutations cause the inactivation of the NTRK1 receptor, thus exerting a loss of function effect, and provide an experimental approach to distinguish functional mutations from genetic polymorphisms.     

Novel nonsense and frameshift NTRK1 gene mutations in Chinese patients with congenital insensitivity to pain with anhidrosis

Congenital insensitivity to pain with anhidrosis (CIPA; MIM 256800) is a rare autosomal recessive disorder characterized by absence of reaction to noxious stimuli, recurrent episodes of fever, anhidrosis, and mental retardation. It is caused by mutations in the gene coding for neurotrophic tyrosine kinase receptor type 1 (NTRK1; MIM# 191315). We screened two Chinese CIPA cases for mutations in the NTRK1 gene and examined their phenotype. Two novel mutations of the NTRK1 gene and two known mutations were identified. Including our two novel mutations, there are now 62 different NTRK1 gene mutations reported in patients with CIPA. We find that a combination of two null alleles usually leads to the severe phenotype, while the mild form of the CIPA disease is associated with at least one mild allele. Thirty-four among the 62 mutations (55%) are located within the tyrosine kinase domain of the NTRK1 protein. We concluded that the tyrosine kinase domain is a hot spot for mutations.     

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.     

Rearrangements of NTRK1 oncogene in papillary thyroid carcinoma

Papillary thyroid carcinoma (PTC) represents an example of tumour with high incidence of oncogenic sequences, such as RET/PTC and Trk. Both of them arise from the fusion of 3' terminal sequences of TK domain of RET or NTRK1 gene, respectively, with 5' terminal sequences of their activating genes. In case of NTRK1 oncogene, several rearrangement types are observed, characteristic for PTC: Trk (TMP3), Trk-T1, Trk-T2, Trk-T3 and Trk-2h, observed in human breast cancer cell line. Studies from different geographical regions, revealed significant population differences in the incidence of Trk rearrangements (0-50%), while within the same population, the frequency of Trk in spontaneous and radiation-associated PTCs is similar. The results of studies, focused on the correlation between tumour genotype and the histopathological type of tumour, involving cases of both RET/PTC and Trk rearrangements in PTC, are not unequivocal. In many studies, no correlation was observed between the presence of RET and/or NTRK1 rearrangement and such parameters, as patient's age at diagnosis, gender, histopathological type of tumour or clinical stage (TNM stage grouping), although the earliest clinical symptoms and the worst disease outcomes were observed for RET/NTRK1 rearrangement positive tumours. Differences in the rearrangement incidence between male and female patients were associated with the latency period of radiation-associated tumours, being significantly lower in women. In general, it is assumed that oncogenic Trk sequences are typical for the spontaneous type of PTC.     

Molecular analysis of congenital central hypoventilation syndrome

Congenital central hypoventilation syndrome (CCHS or Ondines curse; OMIM 209880) is a disorder characterized by an idiopathic failure of the automatic control of breathing. CCHS is frequently complicated with neurocristopathies such as Hirschsprungs disease (HSCR). The genes involved in the RET-GDNF signaling and/or EDN3-EDNRB signaling pathways have been analyzed as candidates for CCHS; however, only a few patients have mutations of the RET, EDN3, and GDNF genes. Recently, mutations of the PHOX2B gene, especially polyalanine expansions, have been detected in two thirds of patients. We studied the RET, GDNF, GFRA1, PHOX2A, PHOX2B, HASH-1, EDN1, EDN3, EDNRB, and BDNF genes in seven patients with isolated CCHS and three patients with HSCR. We detected polyalanine expansions and a novel frameshift mutation of the PHOX2B gene in four patients and one patient, respectively. We also found several mutations of the RET, GFRA1, PHOX2A, and HASH-1 genes in patients with or without mutations of the PHOX2B gene. Our study confirmed the prominent role of mutations in the PHOX2B gene in the pathogenesis of CCHS. Mutations of the RET, GFRA1, PHOX2A, and HASH-1 genes may also be involved in the pathogenesis of CCHS. To make clear the pathogenesis of CCHS, the analysis of more cases and further candidates concerned with the development of the autonomic nervous system is required.     

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.     

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.     

The DNA rearrangement that generates the TRK-T3 oncogene involves a novel gene on chromosome 3 whose product has a potential coiled-coil domain.

Oncogenic rearrangements of the NTRK1 gene (also designated TRKA), encoding one of the receptors for the nerve growth factor, are frequently detected in thyroid carcinomas. Such rearrangements fuse the NTRK1 tyrosine kinase domain to 5'-end sequences belonging to different genes. In previously reported studies we have demonstrated that NTRK1 oncogenic activation involves two genes, TPM3 and TPR, both localized similarly to the receptor tyrosine kinase, on the q arm of chromosome 1. Here we report the characterization of a novel NTRK1-derived thyroid oncogene, named TRK-T3. A cDNA clone, capable of transforming activity, was isolated from a transformant cell line. Sequence analysis revealed that TRK-T3 contains 1,412 nucleotides of NTRK1 preceded by 598 nucleotides belonging to a novel gene that we have named TFG (TRK-fused gene). The TRK-T3 amino acid sequence displays, within the TFG region, a coiled-coil motif that could endow the oncoprotein with the capability to form complexes. The TRK-T3 oncogene encodes a 68-kDa cytoplasmic protein reacting with NTRK1-specific antibodies. By sedimentation gradient experiments the TRK-T3 oncoprotein was shown to form, in vivo, multimeric complexes, most likely trimers or tetramers. The TFG gene is ubiquitously expressed and is located on chromosome 3. The breakpoint producing the TRK-T3 oncogene occurs within exons of both the TFG gene and the NTRK1 gene and produces a chimeric exon that undergoes alternative splicing. Molecular analysis of the NTRK1 rearranged fragments indicated that the chromosomal rearrangement is reciprocal and balanced and involves loss of a few nucleotides of germ line sequences.     

Genetic factors predisposing to the development of papillary thyroid cancer

According to classic theory of neogenesis, cancer arises from well-differentiated cell that in response to variety of factors de-differentiates, becomes able to proliferate without control and/or loses its ability to undergo apoptosis. According to another theory, cancers (at least cancers of some organs) originate from stem cells, which "by definition" are poorly differentiated and able to proliferate indefinitely. Therefore a lower number of abnormal events is necessary for these cells to escape proliferation-controlling mechanisms. With regard to papillary thyroid cancers it is still thought that it arises from well-differentiated thyreocyte. One of the characteristic features of cancer cell is chromosomal instability. Lowest number of such abnormalities is observed in well-differentiated thyroid cancers (including papillary cancer), intermediate - in poorly-differentiated cancers, while highest - in anaplastic cancers. Microarray analysis shows that despite of clinical heterogeneity, gene expression profiles of papillary cancers are very similar. Genetic anomalies predisposing to the development of papillary cancer most commonly regard proteins that possess kinase activity. Kinases phosphorylate other proteins, and play an extremely important role in signal transduction from outside the cell as well as inside the cell. Constitutive activation of some kinases may lead to the excessive and/or permanent activation of some transduction pathways specific for mitogens or growth factors. This results in excessive proliferation. The best known protein of such type which function is altered in papillary thyroid cancers is RET - a membrane-located growth factor-receptor with kinase activity. RET gene undergoes different rearrangements in this type of cancer. There are approximately 10 RET rearrangements known, with RET/PTC3 and RET/PTC1 being most common. In this anomaly kinase domain-encoding 3' end of RET gene is aberrantly bound to 5' end of another gene. Fusion protein synthesized on such hybrid template is not present in the cell membrane but in the cytoplasm, where it permanently activates transduction pathway specific for RET. NTRK1 gene encoding a member of family of neuronal growth factor receptors containing thyrosine kinase domain is also rearranged in papillary cancers. However, genes fused to its kinase domain-encoding sequence are different from the ones fused to RET. MET, a gene encoding another membrane protein with thyrosine kinase activity, which acts as a growth factor-receptor, is overexpressed in 70%-90% of papillary thyroid cancers. BRAF gene encoding another yet kinase transducing signals from RAS and RAF to the cell is mutated at position 1796 (T/A, amino acid substitution V599E) in 38-69% of papillary cancers. The presence of this activatory mutation is associated with higher degree of clinical advancement of the disease. In addition, in majority of papillary cancers tested, mutations of the genes encoding nuclear triiodothyronine receptors were found. Transgenic mice with both TRB allele replaced with dominant-negative TRB mutants develop aggressive thyroid cancers. Progression from papillary to anaplastic cancer is most possibly caused by the occurrence of additional anomalies within P53, RAS, NM23,b-catenin gene and other genes.     

Molecular analysis of structural abnormalities in papillary thyroid carcinoma gene

Rearrangements of the RET proto-oncogene (RET/PTC) and BRAF gene mutations are the major genetic alterations in the etiopathogenesis of papillary thyroid carcinoma (PTC). We have analyzed a series of 118 benign and malignant follicular cell-derived thyroid tumors for RET/PTC rearrangements and BRAF gene mutations. Oncogenic rearrangements of RET proto-oncogene was revealed by semiquantitative RT-PCR of simultaneously generated fragments corresponding to tyrosine kinase (TK) and extracellular RET domains. The clear quantitative shift toward the TK fragment is indicative for the presence of RET rearrangements. The overall frequency of RET/PTC rearrangements in PTC was 14% (12 of 85), including 7 RET/PTC1, 2 RET/PTC3, 1 deltaRFP/RET and 2 apparently uncharacterized rearrangements. The most common T1796A transversion in BRAF gene was detected in 55 of 91 PTC (60%) using mutant-allele-specific PCR. We also identified two additional mutations: the substitution G1753A (E585K) and a case of 12-bp deletion in BRAF exon 15. Moreover, there was no overlap between PTC harboring BRAF and RET/PTC mutations, which altogether were present in 75.8% of cases (69 of 91). Taken together, our observations are consistent with the notion that BRAF mutations appear to be an alternative pathway to oncogenic MAPK activation in PTCs without RET/PTC activation. Neither RET/PTC rearrangements nor BRAF muta-tions were detected in any of 3 follicular thyroid carcinomas, 11 follicular adenomas and 13 nodular goiters. The high prevalence of BRAF mutations and RET/PTC rearrangements in PTCs and the specificity of these alterations to PTC make them potentially important markers for the preoperative tumor diagnosis.     

Molecular modeling of the extracellular domain of the RET receptor tyrosine kinase reveals multiple cadherin-like domains and a calcium-binding site.

Using bioinformatic tools, mutagenesis, and binding studies, we have investigated the structural organization of the extracellular region of the RET receptor tyrosine kinase, a functional receptor for glial cell line-derived neurotrophic factor (GDNF). Multiple sequence alignments of seven vertebrate sequences and one invertebrate RET sequence delineated four distinct N-terminal domains, each of about 110 residues, containing many of the consensus motifs of the cadherin fold. Based on these alignments and the crystal structures of epithelial and neural cadherins, we have generated molecular models of each of the four cadherin-like domains in the extracellular region of human RET. The modeled structures represent realistic models from both energetic and geometrical points of view and are consistent with previous observations gathered from biochemical analyses of the effects of Hirschsprung's disease mutations affecting the folding and stability of the RET molecule, as well as our own site-directed mutagenesis studies of RET cadherin-like domain 1. We have also investigated the role of Ca(2+) in ligand binding by RET and found that Ca(2+) ions are required for RET binding to GDNF but not for GDNF binding to the GFRalpha1 co-receptor. In agreement with these results, RET, but not GFRalpha1, was found to bind Ca(2+) directly. Our results indicate that the overall architecture of the extracellular region of RET is more closely related to cadherins than previously thought. The models of the cadherin-like domains of human RET represent valuable tools with which to guide future site-directed mutagenesis studies aimed at identifying residues involved in ligand binding and receptor activation.     

Detection of a germline mutation at codon 918 of the RET proto-oncogene in French MEN 2B families

Multiple endocrine neoplasia type 2A (MEN2A), type 2B (MEN 2B), and familial medullary thyroid carcinoma (FMTC) are three dominantly inherited disorders linked to the same disease locus on chromosome 10. Two types of germline mutation of the RET proto-oncogene, which codes for a transmembrane tyrosine kinase, are associated with MEN 2. Missense mutations at cysteine residues in the extra-cytoplasmic domain are exclusively associated with MEN 2A and FMTC. In MEN 2B patients, a single point mutation at codon 918 has recently been characterized, leading to the replacement of a methionine by a threonine within the RET tyrosine kinase domain. We now report the identification of a mutation at codon 918 in the germline of 16 patients out of 18 unrelated MEN 2B families analyzed. In these families we have been able to demonstrate that, in five cases, the mutation arose de novo, and that, in one kindred, it was coinherited with the disease. These results indicate that a unique mutation at codon 918 of the RET gene is the most prevalent genetic defect causing MEN 2B, but also that rare MEN 2B cases are associated with different mutations yet to be defined.     

RET tyrosine kinase signaling in development and cancer

The variety of diseases caused by mutations in RET receptor tyrosine kinase provides a classic example of phenotypic heterogeneity. Gain-of-function mutations of RET are associated with human cancer. Gene rearrangements juxtaposing the tyrosine kinase domain to heterologous gene partners have been found in sporadic papillary carcinomas of the thyroid (PTC). These rearrangements generate chimeric RET/PTC oncogenes. In the germline, point mutations of RET are responsible for multiple endocrine neoplasia type 2 (MEN 2A and 2B) and familial medullary thyroid carcinoma (FMTC). Both MEN 2 mutations and PTC gene rearrangements potentiate the intrinsic tyrosine kinase activity of RET and, ultimately, activate the RET downstream targets. Loss-of-function mutations of RET cause Hirschsprung's disease (HSCR) or colonic aganglionosis. A deeper understanding of the molecular signaling of normal versus abnormal RET activity in cancer will enable the development of potential new treatments for patients with sporadic and inherited thyroid cancer or MEN 2 syndrome. We now review the role and mechanisms of RET signaling in development and carcinogenesis.     

Highlights of HRCT imaging in IPF

Background

Aim of this prospective study was to compare clinical and genetic findings in children with idiopathic or heritable pulmonary arterial hypertension (I/HPAH) with children affected with congenital heart defects associated PAH (CHD-APAH).

Methods

Prospectively included were 40 consecutive children with invasively diagnosed I/HPAH or CHD-APAH and 117 relatives. Assessment of family members, pedigree analysis and systematic screening for mutations in TGFß genes were performed.

Results

Five mutations in the bone morphogenetic protein type II receptor (BMPR2) gene, 2 Activin A receptor type II-like kinase-1 (ACVRL1) mutations and one Endoglin (ENG) mutation were found in the 29 I/HPAH children. Two mutations in BMPR2 and one mutation in ACVRL1 and ENG, respectively, are described for the first time. In the 11 children with CHD-APAH one BMPR2 gene mutation and one Endoglin gene mutation were found. Clinical assessment of relatives revealed familial aggregation of the disease in 6 children with PAH (HPAH) and one CHD-APAH patient. Patients with mutations had a significantly lower PVR.

Conclusion

Mutations in different TGFß genes occurred in 8/29 (27.6%) I/HPAH patients and in 2/11 (18.2%) CHD-APAH patients and may influence the clinical status of the disease. Therefore, genetic analysis in children with PAH, especially in those with I/HPAH, may be of clinical relevance and shows the complexity of the genetic background.