Transcriptomics of Hirschsprung disease patient-derived enteric neural crest cells reveals a role for oxidative phosphorylation.

Hirschsprung disease is characterized by the absence of enteric neurons caused by the defects of enteric neural crest cells, leading to intestinal obstruction. Here, using induced pluripotent stem cell-based models of Hirschsprung and single-cell transcriptomic analysis, we identify a gene set of 118 genes commonly dysregulated in all patient enteric neural crest cells, and suggest HDAC1 may be a key regulator of these genes. Furthermore, upregulation of RNA splicing mediators and enhanced alternative splicing events are associated with severe form of Hirschsprung. In particular, the higher inclusion rate of exon 9 in PTBP1 and the perturbed expression of a PTBP1-target, PKM, are significantly enriched in these patient cells, and associated with the defective oxidative phosphorylation and impaired neurogenesis. Hedgehog-induced oxidative phosphorylation significantly enhances the survival and differentiation capacity of patient cells. In sum, we define various factors associated with Hirschsprung pathogenesis and demonstrate the implications of oxidative phosphorylation in enteric neural crest development and HSCR pathogenesis.

Whole genome sequencing reveals epistasis effects within RET for Hirschsprung disease.

Common variants in RET and NRG1 have been associated with Hirschsprung disease (HSCR), a congenital disorder characterised by incomplete innervation of distal gut, in East Asian (EA) populations. However, the allelic effects so far identified do not fully explain its heritability, suggesting the presence of epistasis, where effect of one genetic variant differs depending on other (modifier) variants. Few instances of epistasis have been documented in complex diseases due to modelling complexity and data challenges. We proposed four epistasis models to comprehensively capture epistasis for HSCR between and within RET and NRG1 loci using whole genome sequencing (WGS) data in EA samples. 65 variants within the Topologically Associating Domain (TAD) of RET demonstrated significant epistasis with the lead enhancer variant (RET+3; rs2435357). These epistatic variants formed two linkage disequilibrium (LD) clusters represented by rs2506026 and rs2506028 that differed in minor allele frequency and the best-supported epistatic model. Intriguingly, rs2506028 is in high LD with one cis-regulatory variant (rs2506030) highlighted previously, suggesting that detected epistasis might be mediated through synergistic effects on transcription regulation of RET. Our findings demonstrated the advantages of WGS data for detecting epistasis, and support the presence of interactive effects of regulatory variants in RET for HSCR.

Whole-genome analysis of noncoding genetic variations identifies multiscale regulatory element perturbations associated with Hirschsprung disease.

It is widely recognized that noncoding genetic variants play important roles in many human diseases, but there are multiple challenges that hinder the identification of functional disease-associated noncoding variants. The number of noncoding variants can be many times that of coding variants; many of them are not functional but in linkage disequilibrium with the functional ones; different variants can have epistatic effects; different variants can affect the same genes or pathways in different individuals; and some variants are related to each other not by affecting the same gene but by affecting the binding of the same upstream regulator. To overcome these difficulties, we propose a novel analysis framework that considers convergent impacts of different genetic variants on protein binding, which provides multiscale information about disease-associated perturbations of regulatory elements, genes, and pathways. Applying it to our whole-genome sequencing data of 918 short-segment Hirschsprung disease patients and matched controls, we identify various novel genes not detected by standard single-variant and region-based tests, functionally centering on neural crest migration and development. Our framework also identifies upstream regulators whose binding is influenced by the noncoding variants. Using human neural crest cells, we confirm cell stage-specific regulatory roles of three top novel regulatory elements on our list, respectively in the RET, RASGEF1A, and PIK3C2B loci. In the PIK3C2B regulatory element, we further show that a noncoding variant found only in the patients affects the binding of the gliogenesis regulator NFIA, with a corresponding up-regulation of multiple genes in the same topologically associating domain.

Dual Regulatory Functions of SUFU and Targetome of GLI2 in SHH Subgroup Medulloblastoma.

SUFU alterations are common in human Sonic Hedgehog (SHH) subgroup medulloblastoma (MB). However, its tumorigenic mechanisms have remained elusive. Here, we report that loss of Sufu alone is unable to induce MB formation in mice, due to insufficient Gli2 activation. Simultaneous loss of Spop, an E3 ubiquitin ligase targeting Gli2, restores robust Gli2 activation and induces rapid MB formation in Sufu knockout background. We also demonstrated a tumor-promoting role of Sufu in Smo-activated MB (∼60% of human SHH MB) by maintaining robust Gli activity. Having established Gli2 activation as a key driver of SHH MB, we report a comprehensive analysis of its targetome. Furthermore, we identified Atoh1 as a target and molecular accomplice of Gli2 that activates core SHH MB signature genes in a synergistic manner. Overall, our work establishes the dual role of SUFU in SHH MB and provides mechanistic insights into transcriptional regulation underlying Gli2-mediated SHH MB tumorigenesis.

Identification of Genes Associated With Hirschsprung Disease, Based on Whole-Genome Sequence Analysis, and Potential Effects on Enteric Nervous System Development.

BACKGROUND & AIMS: Hirschsprung disease, or congenital aganglionosis, is believed to be oligogenic-that is, caused by multiple genetic factors. We performed whole-genome sequence analyses of patients with Hirschsprung disease to identify genetic factors that contribute to disease development and analyzed the functional effects of these variants. METHODS: We performed whole-genome sequence analyses of 443 patients with short-segment disease, recruited from hospitals in China and Vietnam, and 493 ethnically matched individuals without Hirschsprung disease (controls). We performed genome-wide association analyses and gene-based rare-variant burden tests to identify rare and common disease-associated variants and study their interactions. We obtained induced pluripotent stem cell (iPSC) lines from 4 patients with Hirschsprung disease and 2 control individuals, and we used these to generate enteric neural crest cells for transcriptomic analyses. We assessed the neuronal lineage differentiation capability of iPSC-derived enteric neural crest cells using an in vitro differentiation assay. RESULTS: We identified 4 susceptibility loci, including 1 in the phospholipase D1 gene (PLD1) (P = 7.4 × 10-7). The patients had a significant excess of rare protein-altering variants in genes previously associated with Hirschsprung disease and in the ß-secretase 2 gene (BACE2) (P = 2.9 × 10-6). The epistatic effects of common and rare variants across these loci provided a sensitized background that increased risk for the disease. In studies of the iPSCs, we observed common and distinct pathways associated with variants in RET that affect risk. In functional assays, we found variants in BACE2 to protect enteric neurons from apoptosis. We propose that alterations in BACE1 signaling via amyloid ß precursor protein and BACE2 contribute to pathogenesis of Hirschsprung disease. CONCLUSIONS: In whole-genome sequence analyses of patients with Hirschsprung disease, we identified rare and common variants associated with disease risk. Using iPSC cells, we discovered some functional effects of these variants.

Update on the Role of Stem Cells in the Treatment of Hirschsprung Disease.

Stem cells possess the ability of self-renewal and the potency to differentiate into multiple cell lineages. Somatic stem cells are present in adult tissues, but they usually exhibit limited differentiation capacity and life span. On the other hand, somatic cells from adult tissues can be reprogrammed into induced pluripotent stem cells (iPSCs) that retain a full differentiation capacity with unlimited self-renewal ability. Autologous origin of iPSCs makes them an ideal source of cells for regenerative medicine to replenish the missing or damaged cells in the patients. iPSCs nowadays have also been widely used to build human disease models to study pathological mechanisms of the diseases. Hirschsprung disease (HSCR) is a congenital disorder caused by defects in the development of enteric neural crest stem cells. The failures of the ENCCs to proliferate, differentiate, and/or migrate lead to the absence of enteric neurons in the distal colon, resulting in colonic motility dysfunction. The lack of effective treatment for HSCR urges continuous efforts to develop new therapies for this congenital disorder. In this review, we will discuss the potential applications of somatic stem cells and iPSCs for the cell-based therapy of HSCR. We will also highlight the recent advances in stem cell research for the establishment of human HSCR models for the development of novel therapies.

Uncovering the genetic lesions underlying the most severe form of Hirschsprung disease by whole-genome sequencing.

Hirschsprung disease (HSCR) is a complex birth defect characterized by the lack of ganglion cells along a variable length of the distal intestine. A large proportion of HSCR patients remain genetically unexplained. We applied whole-genome sequencing (WGS) on 9 trios where the probands are sporadically affected with the most severe form of the disorder and harbor no coding sequence variants affecting the function of known HSCR genes. We found de novo protein-altering variants in three intolerant to change genes-CCT2, VASH1, and CYP26A1-for which a plausible link with the enteric nervous system (ENS) exists. De novo single-nucleotide and indel variants were present in introns and non-coding neighboring regions of ENS-related genes, including NRG1 and ERBB4. Joint analysis with those inherited rare variants found under recessive and/or digenic models revealed both patient-unique and shared genetic features where rare variants were found to be enriched in the extracellular matrix-receptor (ECM-receptor) pathway (p = 3.4 × 10-11). Delineation of the genetic profile of each patient might help finding common grounds that could lead to the discovery of shared molecules that could be used as drug targets for the currently ongoing cell therapy effort which aims at providing an alternative to the surgical treatment.

Correction of Hirschsprung-Associated Mutations in Human Induced Pluripotent Stem Cells Via Clustered Regularly Interspaced Short Palindromic Repeats/Cas9, Restores Neural Crest Cell Function.

BACKGROUND & AIMS: Hirschsprung disease is caused by failure of enteric neural crest cells (ENCCs) to fully colonize the bowel, leading to bowel obstruction and megacolon. Heterozygous mutations in the coding region of the RET gene cause a severe form of Hirschsprung disease (total colonic aganglionosis). However, 80% of HSCR patients have short-segment Hirschsprung disease (S-HSCR), which has not been associated with genetic factors. We sought to identify mutations associated with S-HSCR, and used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing system to determine how mutations affect ENCC function. METHODS: We created induced pluripotent stem cell (iPSC) lines from 1 patient with total colonic aganglionosis (with the G731del mutation in RET) and from 2 patients with S-HSCR (without a RET mutation), as well as RET+/- and RET-/- iPSCs. IMR90-iPSC cells were used as the control cell line. Migration and differentiation capacities of iPSC-derived ENCCs were analyzed in differentiation and migration assays. We searched for mutation(s) associated with S-HSCR by combining genetic and transcriptome data from patient blood- and iPSC-derived ENCCs, respectively. Mutations in the iPSCs were corrected using the CRISPR/Cas9 system. RESULTS: ENCCs derived from all iPSC lines, but not control iPSCs, had defects in migration and neuronal lineage differentiation. RET mutations were associated with differentiation and migration defects of ENCCs in vitro. Genetic and transcriptome analyses associated a mutation in the vinculin gene (VCL M209L) with S-HSCR. CRISPR/Cas9 correction of the RET G731del and VCL M209L mutations in iPSCs restored the differentiation and migration capacities of ENCCs. CONCLUSIONS: We identified mutations in VCL associated with S-HSCR. Correction of this mutation in iPSC using CRISPR/Cas9 editing, as well as the RET G731del mutation that causes Hirschsprung disease with total colonic aganglionosis, restored ENCC function. Our study demonstrates how human iPSCs can be used to identify disease-associated mutations and determine how they affect cell functions and contribute to pathogenesis.

Identification of GLI Mutations in Patients With Hirschsprung Disease That Disrupt Enteric Nervous System Development in Mice.

BACKGROUND & AIMS: Hirschsprung disease is characterized by a deficit in enteric neurons, which are derived from neural crest cells (NCCs). Aberrant hedgehog signaling disrupts NCC differentiation and might cause Hirschsprung disease. We performed genetic analyses to determine whether hedgehog signaling is involved in pathogenesis. METHODS: We performed deep-target sequencing of DNA from 20 patients with Hirschsprung disease (16 men, 4 women), and 20 individuals without (controls), and searched for mutation(s) in GLI1, GLI2, GLI3, SUFU, and SOX10. Biological effects of GLI mutations were tested in luciferase reporter assays using HeLa or neuroblastoma cell lines. Development of the enteric nervous system was studied in Sufu(f/f), Gli3(Δ699), Wnt1-Cre, and Sox10(NGFP) mice using immunohistochemical and whole-mount staining procedures to quantify enteric neurons and glia and analyze axon fasciculation, respectively. NCC migration was studied using time-lapse imaging. RESULTS: We identified 3 mutations in GLI in 5 patients with Hirschsprung disease but no controls; all lead to increased transcription of SOX10 in cell lines. SUFU, GLI, and SOX10 form a regulatory loop that controls the neuronal vs glial lineages and migration of NCCs. Sufu mutants mice had high Gli activity, due to loss of Sufu, disrupting the regulatory loop and migration of enteric NCCs, leading to defective axonal fasciculation, delayed gut colonization, or intestinal hypoganglionosis. The ratio of enteric neurons to glia correlated inversely with Gli activity. CONCLUSIONS: We identified mutations that increase GLI activity in patients with Hirschsprung disease. Disruption of the SUFU-GLI-SOX10 regulatory loop disrupts migration of NCCs and development of the enteric nervous system in mice.

Dysregulation of clathrin promotes thyroid cell growth and contributes to multinodular goiter pathogenesis.

A germline mutation (A339V) in thyroid transcription factor-1 (TITF1/NKX2.1) was shown to be associated with multinodular goiter (MNG) and papillary thyroid carcinoma (PTC) pathogenesis. The overexpression of A339V TTF1 significantly promoted hormone-independent growth of the normal thyroid cells, representing a cause of MNG and/or PTC. Nevertheless, the underlying mechanism still remains unclear. In this study, we used liquid chromatography (LC)-tandem mass spectrometry (MS/MS)-based shotgun proteomics comparing the global protein expression profiles of normal thyroid cells (PCCL3) that overexpressed the wild-type or A339V TTF1 to identify key proteins implicated in this process. Proteomic pathway analysis revealed that the aberrant activation of epidermal growth factor (EGF) signaling is significantly associated with the overexpression of A339V TTF1 in PCCL3, and clathrin heavy chain (Chc) is the most significantly up-regulated protein of the pathway. Intriguingly, dysregulated Chc expression facilitated a nuclear accumulation of pStat3, leading to an enhanced cell proliferation of the A339V clones. Down-regulation and abrogation of Chc-mediated cellular trafficking, respectively, by knocking-down Chc and ectopic expression of a dominant-negative (DN) form of Chc could significantly reduce the nuclear pStat3 and rescue the aberrant cell proliferation of the A339V clones. Subsequent expression analysis further revealed that CHC and pSTAT3 are co-overexpressed in 66.7% (10/15) MNG. Taken together, our results suggest that the A339V TTF1 mutant protein up-regulates the cellular expression of Chc, resulting in a constitutive activation of Stat3 pathway, and prompting the aberrant growth of thyroid cells. This extensive growth signal may promote the development of MNG.

Potential use of skin-derived precursors (SKPs) in establishing a cell-based treatment model for Hirschsprung's disease.

BACKGROUND: Hirschsprung's (HSCR) disease is characterized by absence of ganglia in the distant bowel. Skin-derived precursor cells (SKPs) are somatic stem cells located in the bulge of hair follicles with high neural plasticity. In this study, we elucidated the therapeutic potential of SKPs for replenishing absent ganglia in HSCR bowel. METHODS: SKPs were isolated from mouse or human skin and cultured in neural differentiation medium to generate various types of neural cells. Expression of stem cell and neural differentiation markers were monitored by reverse-transcription polymerase chain reaction and immunocytochemistry, respectively. Engraftment and differentiation potentials of SKPs were further assessed using ex vivo gut culture with Ret(k/k) aganglionic gut. RESULTS: Expression studies revealed that SKPs express a panel of neural crest markers and three key stemness factors (Klf4, c-Myc and Sox2), which may account for the multipotency of these cells. Subsequent differentiation assays directly demonstrated that both mouse and human SKPs retain high differentiation capacities to form enteric neurons, and glia. Importantly, with ex vivo gut explants assay, we further showed that SKPs colonize and differentiate in the Ret(k/k) aganglionic hindgut explants. CONCLUSION: Our data suggest that SKPs may represent an alternative source of stem cells for the study of cell-based therapy for HSCR.

RET and NRG1 interplay in Hirschsprung disease.

Hirschsprung disease (HSCR, aganglionic megacolon) is a complex genetic disorder of the enteric nervous system (ENS) characterized by the absence of enteric neurons along a variable length of the intestine. While rare variants (RVs) in the coding sequence (CDS) of several genes involved in ENS development lead to disease, the association of common variants (CVs) with HSCR has only been reported for RET (the major HSCR gene) and NRG1. Importantly, RVs in the CDS of these two genes are also associated with the disorder. To assess independent and joint effects between the different types of RET and NRG1 variants identified in HSCR patients, we used 254 Chinese sporadic HSCR patients and 143 ethnically matched controls for whom the RET and/or NRG1 variants genotypes (rare and common) were available. Four genetic risk factors were defined and interaction effects were modeled using conditional logistic regression analyses and pair-wise Kendall correlations. Our analysis revealed a joint effect of RET CVs with RET RVs, NRG1 CVs or NRG1 RVs. To assess whether the genetic interaction translated into functional interaction, mouse neural crest cells (NCCs; enteric neuron precursors) isolated from embryonic guts were treated with NRG1 (ErbB2 ligand) or/and GDNF (Ret ligand) and monitored during the subsequent neural differentiation process. Nrg1 inhibited the Gdnf-induced neuronal differentiation and Gdnf negatively regulated Nrg1-signaling by down-regulating the expression of its receptor, ErbB2. This preliminary data suggest that the balance neurogenesis/gliogenesis is critical for ENS development.

Caveolin-1 overexpression is associated with hepatocellular carcinoma tumourigenesis and metastasis.

Caveolin-1 (Cav1) has been implicated in diverse human cancers, yet its role in hepatocellular carcinoma (HCC) tumourigenesis and metastasis remains elusive. In the current study, we aim to provide a comprehensive understanding regarding the functional role of Cav1 in HCC tumourigenesis and metastasis. Cav1 expression was examined in a panel of human HCC cell lines using western blotting analysis and quantitative RT-PCR and human tissues by immunohistochemistry. Cav1 was not detected in normal liver cell line and all non-tumourous liver tissues but exclusively expressed in HCC cell lines and tissues. Dramatic expression of Cav1 was found in metastatic HCC cell lines and tumours, indicating a progressive increase of Cav1 expression along disease progression. Cav1 overexpression was significantly correlated with venous invasion (p = 0.036). To investigate the functions of Cav1 in HCC, Cav1 overexpressing and knockdown stable clones were established in HCC cells and their tumourigenicity and metastatic potential were examined. Overexpression of Cav1 promoted HCC cell growth, motility, and invasiveness, as well as tumourigenicity in vivo. Conversely, knockdown of Cav1 in metastatic HCC cells inhibited the motility and invasiveness and markedly suppressed the tumour growth and metastatic potential in vivo. Collectively, our findings have shown the exclusive expression of Cav1 in HCC cell lines and clinical samples and revealed an up-regulation of Cav1 along HCC progression. The definitive role of Cav1 in promoting HCC tumourigenesis was demonstrated, and we have shown for the first time in a mouse model that Cav1 promotes HCC metastasis.

RET mutational spectrum in Hirschsprung disease: evaluation of 601 Chinese patients.

Rare (RVs) and common variants of the RET gene contribute to Hirschsprung disease (HSCR; congenital aganglionosis). While RET common variants are strongly associated with the commonest manifestation of the disease (males; short-segment aganglionosis; sporadic), rare coding sequence (CDS) variants are more frequently found in the lesser common and more severe forms of the disease (females; long/total colonic aganglionosis; familial).Here we present the screening for RVs in the RET CDS and intron/exon boundaries of 601 Chinese HSCR patients, the largest number of patients ever reported. We identified 61 different heterozygous RVs (50 novel) distributed among 100 patients (16.64%). Those include 14 silent, 29 missense, 5 nonsense, 4 frame-shifts, and one in-frame amino-acid deletion in the CDS, two splice-site deletions, 4 nucleotide substitutions and a 22-bp deletion in the intron/exon boundaries and 1 single-nucleotide substitution in the 5' untranslated region. Exonic variants were mainly clustered in RET the extracellular domain. RET RVs were more frequent among patients with the most severe phenotype (24% vs. 15% in short-HSCR). Phasing RVs with the RET HSCR-associated haplotype suggests that RVs do not underlie the undisputable association of RET common variants with HSCR. None of the variants were found in 250 Chinese controls.

Lack of association between nNOS -84G>A polymorphism and risk of infantile hypertrophic pyloric stenosis in a Chinese population.

BACKGROUND: Infantile hypertrophic pyloric stenosis (IHPS) is one of the most common gastrointestinal obstructions in the infancy requiring surgery. Reduced expression of neuronal nitric oxide synthase (nNOS), which plays an important role in the regulation of the human pyloric muscle, is thought to underlie IHPS. The role of nNOS in IHPS has been supported by the genetic association of a functional regulatory nNOS polymorphism (-84G>A) with IHPS in whites. We reasoned that the corroboration of this association in a population of different ethnic origin would prompt follow-up studies and further investigation of the IHPS pathology at molecular level. Thus, we attempted to reproduce the original findings in a Chinese population of comparable size in what would be the first genetic study on IHPS conducted in Chinese. METHODS: nNOS -84G>A genotypes were analyzed in 56 patients and 86 controls by polymerase chain reaction and DNA sequencing. Logistic regression was used to compute odds ratios. RESULTS: Our study could not corroborate the association previously reported. Although the frequency of the IHPS-associated allele (-84A) in controls (0.205) was similar to that reported for white controls, there was a dramatic difference in -84A frequencies between white and Chinese patients (0.198). Similarly, there was no difference in the nNOS -84G>A genotype distribution between patients and controls, even when the GA and AA genotypes were combined to compare GG genotype (odds ratio, 1.01; 95% confidence interval, 0.47-2.19). CONCLUSIONS: Failure to replicate the initial finding does not detract from its validity, because genetic effects may differ across populations. Differences across populations in linkage disequilibrium and/or allele frequencies may contribute to this lack of replication. The role nNOS in IHPS awaits further investigation.

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.

Genome-wide association study identifies NRG1 as a susceptibility locus for Hirschsprung's disease.

Hirschsprung's disease (HSCR), or aganglionic megacolon, is a congenital disorder characterized by the absence of enteric ganglia in variable portions of the distal intestine. RET is a well-established susceptibility locus, although existing evidence strongly suggests additional loci contributing to sporadic HSCR. To identify these additional genetic loci, we carried out a genome-wide association study using the Affymetrix 500K marker set. We successfully genotyped 293,836 SNPs in 181 Chinese subjects with sporadic HSCR and 346 ethnically matched control subjects. The SNPs most associated with HSCR were genotyped in an independent set of 190 HSCR and 510 control subjects. Aside from SNPs in RET, the strongest overall associations in plausible candidate genes were found for 2 SNPs located in intron 1 of the neuregulin1 gene (NRG1) on 8p12, with rs16879552 and rs7835688 yielding odds ratios of 1.68 [CI(95%):(1.40, 2.00), P = 1.80 x 10(-8)] and 1.98 [CI(95%):(1.59, 2.47), P = 1.12 x 10(-9)], respectively, for the heterozygous risk genotypes under an additive model. There was also a significant interaction between RET and NRG1 (P = 0.0095), increasing the odds ratio 2.3-fold to 19.53 for the RET rs2435357 risk genotype (TT) in the presence of the NRG1 rs7835688 heterozygote, indicating that NRG1 is a modifier of HSRC penetrance. Our highly significant association findings are backed-up by the important role of NRG1 as regulator of the development of the enteric ganglia precursors. The identification of NRG1 as an additional HSCR susceptibility locus not only opens unique fields of investigation into the mechanisms underlying the HSCR pathology, but also the mechanisms by which a discrete number of loci interact with each other to cause disease.

Identification of a HOXD13 mutation in a VACTERL patient.

VACTERL acronym is assigned to a non-random association of malformations in humans with poorly known etiology. It is comprised of vertebral defects (V), anal atresia (A), cardiac anomaly (C), tracheoesophageal fistula with esophageal atresia (TE), renal dysplasia (R) and limb lesions (L). Here, we report on, for the first time, a female patient with VACTERL association with a 21 base-pair deletion in the exon 1 triplet repeats of HOXD13, a sonic hedgehog (SHH) downstream target. Our data provide the first piece of clinical evidence of the implication of the SHH pathway in VACTERL. Moreover, HOXD13 may not only be implicated in limb malformations but also in the development of gut and genitourinary structures, as predicted from the mouse models.

Evaluation of the thyroid transcription factor-1 gene (TITF1) as a Hirschsprung's disease locus.

Hirschsprung's disease (HSCR, colonic aganglionosis) is an oligogenic entity that usually requires mutations in RET and other interacting loci. Decreased levels of RET expression may lead to the manifestation of HSCR. We previously showed that RET transcription was decreased due to alteration of the TITF1 binding site by two HSCR-associated RET promoter single nucleotide polymorphisms (SNPs). This prompted us to investigate whether DNA alterations in TITF1 could play a role in HSCR by affecting the RET-regulatory properties of the TITF1 protein. Our initial study on 86 Chinese HSCR patients revealed a Gly322Ser amino acid substitution in the TITF1protein. In this study we have examined an additional 102 Chinese and 70 Caucasian patients, and 194 Chinese and 60 Caucasian unselected, unrelated, subjects as controls. The relevance of the DNA changes detected in TITF1 by direct sequencing were evaluated using bioinformatics, reporter and binding-assays, mouse neurosphere culture, immunohistochemistry and immunofluorescence techniques. Met3Leu and Pro48Pro were identified in 2 Caucasian patients and 1 Chinese patient, respectively. In vitro analysis showed that Met3Leu reduced the activity of the RET promoter by 100% in the presence of the wild-type or HSCR-associated RET promoter SNP alleles. The apparent binding affinity of the TITF1 mutated protein was not decreased. The Met3Leu mutation may affect the interaction of TITF1 with its protein partners. The absence of Titf1 expression in mouse gut but not in human gut suggests that the role of TITF1 in gut development differs between the two species. TITF1 mutations could contribute to HSCR by affecting RET expression through defective interactions with other transcription factors.