Transcriptional regulation of RET by Nkx2-1, Phox2b, Sox10, and Pax3

Background: The rearranged during transfection (RET) gene encodes a single-pass receptor whose proper expression and function are essential for the development of enteric nervous system. Mutations in RET regulatory regions are also associated with Hirschsprung disease (HSCR) (aganglionosis of the colon). We previously showed that 2 polymorphisms in RET promoter are associated with the increased risk of HSCR. These single nucleotide polymorphisms overlap with the NK2 homeobox 1 (Nkx2-1) binding motif interrupting the physical interaction of NKX2-1 with the RET promoter and result in reduced RET transcription. In this study, we further delineated Nkx2-1-mediated RET Transcription. Methods and results: First, we demonstrated that PHOX2B, like SOX10 and NKX2-1, is expressed in the mature enteric ganglions of human gut by immunohistochemistry. Second, subsequent dual-luciferase-reporter studies indicated that Nkx2-1 indeed works coordinately with Phox2b and Sox10, but not Pax3, to mediate RET transcription. In addition, identification of Phox2b responsive region in RET promoter further provides solid evidence of the potential functional interaction between Phox2b and RET. Conclusion: In sum, Phox2b and Sox10 act together with Nkx2.1 to modify RET signaling and this interaction may also contribute to HSCR susceptibility. © 2009 Elsevier Inc. All rights reserved.postprin

Reduced RET expression in gut tissue of individuals carrying risk alleles of Hirschsprung's disease

Receptor tyrosine kinase (RET) single nucleotide polymorphisms (SNPs) are associated with the Hirschsprung's disease (HSCR). We investigated whether the amount of RET expressed in the ganglionic gut of human was dependent on the genotype of three regulatory SNPs (-5G>A rs10900296 and -1A>C rs10900297 in the promoter, and C>T rs2435357 in intron 1). We examined the effects of three regulatory SNPs on the RET gene expression in 67 human ganglionic gut tissues using quantitative real-time PCR. Also, 315 Chinese HSCR patients and 325 ethnically matched controls were genotyped for the three SNPs by polymerase chain reaction (PCR) and direct sequencing. The expression of RET mRNA in human gut tissue did indeed correlate with the genotypes of the individuals. The lowest RET expression was found for those individuals homozygous for the three risk alleles (A-C-T/A-C-T), and the highest for those homozygous for the 'wild-type' counterpart (G-A-C/G-A-C), with expression values ranging from 218.32±125.69 (mean ± SE) in tissues from individuals carrying G-A-C/G-A-C to 31.42±8.42 for individuals carrying A-C-T/A-C-T (P 5 0.018). As expected, alleles -5A, -1C and intron 1 T were associated with HSCR (P 5 5.94 × 10-31, 3.12 3 10-24 and 5.94 × 10-37, respectively) as was the haplotype encompassing the three associated alleles (A-C-T) when compared with the wild-type counterpart G-A-C (χ2 5 155.29, P « 0.0001). To our knowledge, this is the first RET expression genotype-phenotype correlation study conducted on human subjects to indicate common genetic variants in the regulatory region of RET may play a role in mediating susceptibility to HSCR, by conferring a significant reduction of the RET expression. © The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

Role of RET and PHOX2B gene polymorphisms in risk of Hirschsprung's disease in Chinese population [9]

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RET mutational spectrum in Hirschsprungs disease: evaluation of 601 Chinese patients

Poster Session - Session Title: Gene Structure and Gene Product Function: Program no. 778FHirschsprung’s disease (HSCR) is a developmental disorder characterized by the absence of ganglion cells in the lower digestive tract. Aganglionosis is attributed to a disorder of the enteric nervous system (ENS) whereby ganglion cells fail to innervate the lower gastrointestinal tract during embryonic development. There is significant population variation in the incidence of the disease, and it is most often found among Asians (2.8 per 10,000 live births). HSCR most commonly presents sporadically (80% of the cases), with a recurrence risk of 4%, and more males than females affected (4:1). Both rare (<1% in the population) and common germ line variants of the RET gene, acting either alone or in combination, are the main cause of the disease. Yet, while RET common variants are strongly associated with the commonest manifestation of the disease (male, short segment, and sporadic forms), rare coding sequence (CDS) variants are more frequently found in the lesser common and more severe forms of the disease (females, long or total colonic aganglionosis, and familial). Here we present a rare variant (RV) screening of the CDS and intron/exon boundaries of the RET gene in 607 Chinese sporadic HSCR patients, the largest number of patients ever reported. We have found a total of 61 different heterozygous RVs (50 novel) distributed in 100 patients (16.64%). These include 14 silent, 29 missense, 5 nonsense amino-acid changes, 4 frame-shifts, and one in-frame amino-acid deletion in the exonic region and two splice-site deletions, 4 nucleotide substitutions and a 22 bp deletion in intronic or untranslated regions. The exonic variants were mainly clustered in the sequence encoding the extracellular domain of the RET protein. All RVs were predicted to alter the protein function. The highest frequency of rare variants was found among those patients with the most severe form of the disease (24% in long or total vs 15% in short-segment). Phasing of the RVs with the RET risk-haplotype suggested that RET RVs do not underlie the undisputable association of RET common variants with HSCR. None of the variants was found in 250 Chinese controls.link_to_OA_fulltextThe 12th International Congress of Human Genetics (ICHG 2011), Montreal, Canada, 11-15 October 2011

A RET founder mutation in Chinese hirschsprung's patients

768/W/Poster Board: no. 426Hirschsprung’s disease (HSCR) is a congenital disorder associated with the lack of intramural ganglion cells in the myenteric and sub-mucosal plexuses along varying segments of the gastrointestinal tract. Rearranged during transfection (RET) gene is implicated in HSCR and is the major gene of this gastrointestinal disease. To date, over 200 low frequency recurrent RET coding sequence (CDS) mutations have been identified in HSCR patients. However, a highly recurrent RET(R114H) mutation has been identified in 10% of the Chinese HSCR patients which has never been found in Caucasians patients or controls nor in 400 Chinese controls. The high frequency of RET(R114H) in our population together with the fact that it is not a “de novo” mutation in the context of the most HSCR-associated RET-haplotype, suggests that it may be a founder HSCR mutation in the Chinese population. Initial investigation involved applying a Bayesian method to 21 single nucleotide polymorphisms (SNPs; across a 62kb region of RET) genotyped in 421 Chinese HSCR patients (of which 24 individuals had the mutation) to predict the approximate age of RET(R114H). The approach allowed the inference of the mutation age based on the observed linkage disequilibrium (LD) at multiple SNPs which predicted the mutation to be between 12 and 13 generations old. Including SNPs from a recently obtained genome-wide 500K dataset for 181 of the above mentioned patients (which now only included 14 patients who had the RET(R114H) mutation), we applied haplotype estimation methods to determine whether there were any segments shared between patients with the RET(R114H) compared to those without the mutation and controls. Data consisted a total of 92 SNPs spanning a 510kb region over the RET gene. Analysis yielded a 256kb (76 SNP) shared segment over the RET gene (and downstream) in only those patients with the mutation with no similar segments found among other patients or controls. This suggests that RET(R114H) is a possible founder effect for Hirschsprung’s disease in the Chinese population.The 59th Annual Meeting of the American Society of Human Genetics (ASHG), Honolulu, HI., 20-24 October 2009

Novel RET mutation produces a truncated RET receptor lacking the intracellular signaling domain in a 3-generation family with Hirschsprung disease

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Functional analyses of RET mutations in Chinese hirschsprung disease patients

BACKGROUND: Hirschsprung disease (HSCR) is a congenital disease characterized by the absence of ganglion cells in various length of distal digestive tract. The rearranged during transfection gene (RET) is considered the major gene in HSCR. Although an increasing number of HSCR-associated RET coding sequence (CDS) mutations have been identified in recent years, not many have been investigated for functional consequence on the RET protein. METHODS AND RESULTS: We examined the functional implications of the de novo RET-CDS mutations V145G, Y483X, V636fsX1, and F961L that we first identified in sporadic Chinese patients with HSCR. The V145G disrupted RET glycosylation and F961L RET phosphorylation. Presumably, the truncation mutations would affect the translocation or the anchoring of the RET protein onto the cellular membrane. CONCLUSION: The study of RET-CDS mutations that appear de novo is essential not only for understanding the mechanistic of the disease but also for penetrance and recurrence risk estimations, being the ultimate goal for the improvement in disease management and counseling. © 2011 Wiley Periodicals, Inc.link_to_subscribed_fulltex

MNX1 (HLXB9) mutations in Currarino patients

Purpose: The combination of partial absence of the sacrum, anorectal anomalies, and presacral mass constitutes Currarino syndrome (CS), which is associated with mutations in MNX1 motor neuron and pancreas homeobox 1 (previously HLXB9). Here, we report on the MNX1 mutations found in a family segregating CS and in 3 sporadic CS patients, as well as on the clinical characteristics of the affected individuals. Methods: MNX1 mutations were identified by direct sequencing the coding regions, intron/exon boundaries of MNX1 in 5 CS Japanese family members and 3 Chinese sporadic cases and their parents. Results: There were 2 novel (P18PfsX37, R243W) and 2 previously described (W288G and IVS2 + 1G > A) mutations. These mutations were not found in 198 control individuals and are predicted to impair the functioning of the MNX1 protein. Conclusions: The variability of the CS phenotype among related or unrelated patients bearing the same mutation advocates for differences in the genetic background of each individual and invokes the implication of additional CS susceptibility genes. © 2009 Elsevier Inc. All rights reserved.link_to_subscribed_fulltex

Perturbation of Hoxb5 Signaling in Vagal Neural Crests Down-Regulates Ret Leading to Intestinal Hypoganglionosis in Mice

Background & Aims: The enteric nervous system (ENS) controls intestinal peristalsis, and defective development of this system results in hypo/aganglionosis, as seen in Hirschsprung's disease. In the embryo, vagal neural crest cells (NCC) migrate and colonize the intestine rostrocaudally then differentiate into the ganglia of the ENS. Vagal NCC express the homeobox gene Hoxb5, a transcriptional activator, in human and mouse, so we used transgenic mice to investigate the function of Hoxb5 and the receptor tyrosine kinase gene Ret, which is affected in many patients with Hirschsprung's disease, in ENS development. Methods: We perturbed the Hoxb5 pathway by expressing a chimeric protein enb5, in which the transcription activation domain of Hoxb5 was replaced with the repressor domain of the Drosophila engrailed protein (en), in vagal NCC. This enb5 transcriptional repressor competes with wild-type Hoxb5 for binding to target genes, exerting a dominant negative effect. Results: We observed that 30.6% ± 2.3% of NCC expressed enb5 and that these enb5-expressing NCC failed to migrate to the distal intestine. A 34%-37% reduction of ganglia (hypoganglionosis) and slow peristalsis and, occasionally, absence of ganglia and intestinal obstruction were observed in enb5-expressing mice. Ret expression was markedly reduced or absent in NCC and ganglia, and enb5 blocked Hoxb5 induction of Ret in neuroblastoma cells. Conclusions: Our data indicate that Ret is a downstream target of Hoxb5 whose perturbation causes Ret haploinsufficiency, impaired NCC migration, and hypo/aganglionosis, suggesting that Hoxb5 may contribute to the etiology of Hirschsprung's disease. © 2008 AGA Institute.link_to_subscribed_fulltex

TTF-1 and RET promoter SNPs: Regulation of RET transcription in Hirschsprung's disease

Single nucleotide polymorphisms (SNPs) of the coding regions of receptor tyrosine kinase gene (RET) are associated with Hirschsprung's disease (HSCR, aganglionic megacolon). These SNPs, individually or combined, may act as a low penetrance susceptibility locus and/or be in linkage disequilibrium (LD) with another susceptibility locus located in RET regulatory regions. Because two RET promoter SNPs have been found associated with HSCR, in LD with HSCR-associated RET coding region haplotypes, their implication in the transcriptional regulation of RET is of major interest. Analysis of 172 sporadic HSCR patients also revealed the presence of HSCR-associated RET promoter SNPs in LD with the main coding region RET haplotype observed in Chinese patients. By using a weighted logistic regression approach, we determined that of all SNPs tested in our study, the promoter SNPs are the most correlated to the disease. Functional analysis of the RET promoter SNPs in the context of additional 5′ regulatory regions demonstrated that the HSCR-associated alleles decrease RET transcription. These SNPs overlap a TTF-1 binding site and TTF-1-activated RET transcription is also decreased by the HSCR-associated SNPs. Moreover, we identified an HSCR patient with a Gly322Ser TTF-1 mutation that compromises activation of transcription from HSCR-associated RET promoter haplotypes. Interestingly, we show that the pattern of RET and TTF-1 expression is coincident in developing human gut. We also present a detailed profile of the RET gene in our population, which provides an insight into the higher incidence of the disease in China. © Oxford University Press 2005; all rights reserved.link_to_subscribed_fulltex