Cancer gene mutations in congenital pulmonary airway malformation patients.

BACKGROUND: Newborns affected with congenital pulmonary airway malformations (CPAMs) may present with severe respiratory distress or remain asymptomatic. While surgical resection is the definitive treatment for symptomatic CPAMs, prophylactic elective surgery may be recommended for asymptomatic CPAMs owing to the risk of tumour development. However, the implementation of prophylactic surgery is quite controversial on the grounds that more evidence linking CPAMs and cancer is needed. The large gap in knowledge of CPAM pathogenesis results in uncertainties and controversies in disease management. As developmental genes control postnatal cell growth and contribute to cancer development, we hypothesised that CPAMs may be underlain by germline mutations in genes governing airways development. METHODS: Sequencing of the exome of 19 patients and their unaffected parents. RESULTS: A more than expected number of mutations in cancer genes (false discovery rate q-value <5.01×10-5) was observed. The co-occurrence, in the same patient, of damaging variants in genes encoding interacting proteins is intriguing, the most striking being thyroglobulin (TG) and its receptor, megalin (LRP2). Both genes are highly relevant in lung development and cancer. CONCLUSIONS: The overall excess of mutations in cancer genes may account for the reported association of CPAMs with carcinomas and provide some evidence to argue for prophylactic surgery by some surgeons.

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.

Genetic study of congenital bile-duct dilatation identifies de novo and inherited variants in functionally related genes.

BACKGROUND: Congenital dilatation of the bile-duct (CDD) is a rare, mostly sporadic, disorder that results in bile retention with severe associated complications. CDD affects mainly Asians. To our knowledge, no genetic study has ever been conducted. METHODS: We aim to identify genetic risk factors by a "trio-based" exome-sequencing approach, whereby 31 CDD probands and their unaffected parents were exome-sequenced. Seven-hundred controls from the local population were used to detect gene-sets significantly enriched with rare variants in CDD patients. RESULTS: Twenty-one predicted damaging de novo variants (DNVs; 4 protein truncating and 17 missense) were identified in several evolutionarily constrained genes (p < 0.01). Six genes carrying DNVs were associated with human developmental disorders involving epithelial, connective or bone morphologies (PXDN, RTEL1, ANKRD11, MAP2K1, CYLD, ACAN) and four linked with cholangio- and hepatocellular carcinomas (PIK3CA, TLN1 CYLD, MAP2K1). Importantly, CDD patients have an excess of DNVs in cancer-related genes (p < 0.025). Thirteen genes were recurrently mutated at different sites, forming compound heterozygotes or functionally related complexes within patients. CONCLUSIONS: Our data supports a strong genetic basis for CDD and show that CDD is not only genetically heterogeneous but also non-monogenic, requiring mutations in more than one genes for the disease to develop. The data is consistent with the rarity and sporadic presentation of CDD.

Trans-ethnic meta-analysis of genome-wide association studies for Hirschsprung disease.

Hirschsprung disease (HSCR) is the most common cause of neonatal intestinal obstruction. It is characterized by the absence of ganglia in the nerve plexuses of the lower gastrointestinal tract. So far, three common disease-susceptibility variants at the RET, SEMA3 and NRG1 loci have been detected through genome-wide association studies (GWAS) in Europeans and Asians to understand its genetic etiologies. Here we present a trans-ethnic meta-analysis of 507 HSCR cases and 1191 controls, combining all published GWAS results on HSCR to fine-map these loci and narrow down the putatively causal variants to 99% credible sets. We also demonstrate that the effects of RET and NRG1 are universal across European and Asian ancestries. In contrast, we detected a European-specific association of a low-frequency variant, rs80227144, in SEMA3 [odds ratio (OR) = 5.2, P = 4.7 × 10-10]. Conditional analyses on the lead SNPs revealed a secondary association signal, corresponding to an Asian-specific, low-frequency missense variant encoding RET p.Asp489Asn (rs9282834, conditional OR = 20.3, conditional P = 4.1 × 10-14). When in trans with the RET intron 1 enhancer risk allele, rs9282834 increases the risk of HSCR from 1.1 to 26.7. Overall, our study provides further insights into the genetic architecture of HSCR and has profound implications for future study designs.

Chinese family with diffuse oesophageal leiomyomatosis: a new COL4A5/COL4A6 deletion and a case of gonosomal mosaicism.

BACKGROUND: Diffuse oesophageal leiomyomatosis (DOL) is a rare disorder characterized by tumorous overgrowth of the muscular wall of the oesophagus. DOL is present in 5 % of Alport syndrome (AS) patients. AS is a rare hereditary disease that involves varying degrees of hearing impairment, ocular changes and progressive glomerulonephritis leading to renal failure. In DOL-AS patients, the genetic defect consists of a deletion involving the COL4A5 and COL4A6 genes on the X chromosome. CASE PRESENTATION: We report a two-generation family (4 individuals; parents and two children, one male and one female) with two members (mother and son) affected with oesophageal leiomyomatosis. Signs of potential renal failure, which characterizes AS, were only apparent in the index patient (son) 2 years and three months after the initial diagnosis of DOL. Blood DNA from the four family members were submitted to exome sequencing and array genotyping to perform a genome wide screening for disease causal single nucleotide (SN) and copy number (CN) variations. Analyses revealed a new 40kb deletion encompassing from intron 2 of COL4A5 to intron 1 of COL4A6 at Xq22.3. The breakpoints were also identified. Possible confounding pathogenic exonic variants in genes known to be involved in other extracellular matrices disorders were also shared by the two affected individuals. Meticulous analysis of the maternal DNA revealed a case of gonosomal mosaicism. CONCLUSIONS: This is the first report of gonadosomal mosaicism associated to DOL-AS.

Depletion of the IKBKAP ortholog in zebrafish leads to hirschsprung disease-like phenotype.

AIM: To investigate the role of IKBKAP (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein) in the development of enteric nervous system (ENS) and Hirschsprung disease (HSCR). METHODS: In this study, we injected a morpholino that blocked the translation of ikbkap protein to 1-cell stage zebrafish embryos. The phenotype in the ENS was analysed by antibody staining of the pan-neuronal marker HuC/D followed by enteric neuron counting. The mean numbers of enteric neurons were compared between the morphant and the control. We also studied the expressions of ret and phox2bb, which are involved in ENS development, in the ikbkap morpholino injected embryos by quantitative reverse transcriptase polymerase chain reaction and compared them with the control. RESULTS: We observed aganglionosis (χ2, P<0.01) and a reduced number of enteric neurons (38.8±9.9 vs 50.2±17.3, P<0.05) in the zebrafish embryos injected with ikbkap translation-blocking morpholino (morphant) when compared with the control embryos. Specificity of the morpholino was confirmed by similar results obtained using a second non-overlapping morpholino that blocked the translation of ikbkap. We further studied the morphant by analysing the expression levels of genes involved in ENS development such as ret, phox2bb and sox10, and found that phox2bb, the ortholog of human PHOX2B, was significantly down-regulated (0.51±0.15 vs 1.00±0, P<0.05). Although we also observed a reduction in the expression of ret, the difference was not significant. CONCLUSION: Loss of IKBKAP contributed to HSCR as demonstrated by functional analysis in zebrafish embryos.

Gene network analysis of candidate loci for human anorectal malformations.

Anorectal malformations (ARMs) are birth defects that require surgery and carry significant chronic morbidity. Our earlier genome-wide copy number variation (CNV) study had provided a wealth of candidate loci. To find out whether these candidate loci are related to important developmental pathways, we have performed an extensive literature search coupled with the currently available bioinformatics tools. This has allowed us to assign both genic and non-genic CNVs to interrelated pathways known to govern the development of the anorectal region. We have linked 11 candidate genes to the WNT signalling pathway and 17 genes to the cytoskeletal network. Interestingly, candidate genes with similar functions are disrupted by the same type of CNV. The gene network we discovered provides evidence that rare mutations in different interrelated genes may lead to similar phenotypes, accounting for genetic heterogeneity in ARMs. Classification of patients according to the affected pathway and lesion type should eventually improve the diagnosis and the identification of common genes/molecules as therapeutic targets.

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.

Allele-specific expression at the RET locus in blood and gut tissue of individuals carrying risk alleles for Hirschsprung disease.

RET common variants are associated with Hirschsprung disease (HSCR; colon aganglionosis), a congenital defect of the enteric nervous system. We analyzed a well-known HSCR-associated RET haplotype that encompasses linked alleles in coding and noncoding/regulatory sequences. This risk haplotype correlates with reduced level of RET expression when compared with the wild-type counterpart. As allele-specific expression (ASE) contributes to phenotypic variability in health and disease, we investigated whether RET ASE could contribute to the overall reduction of RET mRNA detected in carriers. We tested heterozygous neuroblastoma cell lines, ganglionic gut tissues (18 HSCR and 14 non-HSCR individuals) and peripheral blood mononuclear cells (PBMCs; 16 HSCR and 14 non-HSCR individuals). Analysis of the data generated by SNaPshot and Pyrosequencing revealed that the RET risk haplotype is significantly more expressed in gut than in PBMCs (P = 0.0045). No ASE difference was detected between patients and controls, irrespective of the sample type. Comparison of total RET expression levels between gut samples with and without ASE, correlated reduced RET expression with preferential transcription from the RET risk haplotype. Nonrandom RET ASE occurs in ganglionic gut regardless of the disease status. RET ASE should not be excluded as a disease mechanism acting during development.

Screening of the RET gene of Vietnamese Hirschsprung patients identifies 2 novel missense mutations.

BACKGROUND/PURPOSE: Hirschsprung disease (HSCR; megacolon, congenital aganglionosis) is a congenital disorder characterized by the absence of ganglion cells along variable segments of the gut. Both rare (RV) and common variants of the RET gene are associated with HSCR. The aim of this study is to assess, for the first time, the variation in the RET gene of Vietnamese HSCR patients. METHODS: We used Sanger sequencing to screen the coding sequence of the RET gene of 97 Vietnamese HSCR patients of Southern Chinese ancestry. The healthy population consisted of 250 Southern Chinese individuals with no diagnosis of HSCR. RESULTS: We detected 8 heterozygous RVs distributed among 13 patients (13.40%) and that were not present in healthy individuals. Among those variants, there were 2 novel and deleterious (R133C [c.397 C>T]; R144C [c.430 C>T]) missense amino acid substitutions, 2 novel silent variants (P667P [c.2001 A>T]; Y809Y [c.2427 C>T]), and 4 previously described missense substitutions (R114H [c.341 G>A]; V292M [c.874 G>A]; G533S [c.1597 G>A]; R982C [c.2944 C>T]). As expected, the common RET coding sequence variants rs1800858 (A45A [c.135 G>A]) and rs1800861 (L769L [c.2307 T>G]) were highly associated with the disease. CONCLUSIONS: The identification of novel deleterious variants together with the fact RET RVs are virtually unique to HSCR patients indicates that the RET gene is a target for mutations among Hirschsprung patients of any population.

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.

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.

Haplotype analysis reveals a possible founder effect of RET mutation R114H for Hirschsprung's disease in the Chinese population.

BACKGROUND: Hirschsprung'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. The RET gene is the major gene implicated in this gastrointestinal disease. A highly recurrent mutation in RET (RET(R114H)) has recently been identified in approximately 6-7% of the Chinese HSCR patients which, to date, has not been found in Caucasian patients or controls nor in Chinese controls. Due to the high frequency of RET(R114H) in this population, we sought to investigate whether this mutation may be a founder HSCR mutation in the Chinese population. METHODOLOGY AND PRINCIPAL FINDINGS: To test whether all RET(R114) were originated from a single mutational event, we predicted the approximate age of RET(R114H) by applying a Bayesian method to RET SNPs genotyped in 430 Chinese HSCR patients (of whom 25 individuals had the mutation) to be between 4-23 generations old depending on growth rate. We reasoned that if RET(R114H) was a founder mutation then those with the mutation would share a haplotype on which the mutation resides. Including SNPs spanning 509.31 kb across RET from a recently obtained 500 K genome-wide dataset for a subset of 181 patients (14 RET(R114H) patients), we applied haplotype estimation methods to determine whether there were any segments shared between patients with RET(R114H) that are not present in those without the mutation or controls. Analysis yielded a 250.2 kb (51 SNP) shared segment over the RET gene (and downstream) in only those patients with the mutation with no similar segments found among other patients. CONCLUSIONS: This suggests that RET(R114H) is a founder mutation for HSCR in the Chinese population.

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.

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.

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.

A germline mutation (A339V) in thyroid transcription factor-1 (TITF-1/NKX2.1) in patients with multinodular goiter and papillary thyroid carcinoma.

BACKGROUND: The genetic factors that determine the risk of papillary thyroid carcinoma (PTC) among patients with multinodular goiter (MNG) remain undefined. Because thyroid transcription factor-1 (TTF-1) is important to thyroid development, we evaluated whether the gene that encodes it, TITF-1/NKX2.1, is a genetic determinant of MNG/PTC predisposition. METHODS: Twenty unrelated PTC patients with a history of MNG (MNG/PTC), 284 PTC patients without a history of MNG (PTC), and 349 healthy control subjects were screened for germline mutation(s) in TITF-1/NKX2.1 by sequencing of amplified DNA from blood. The effects of the mutation on the growth and differentiation of thyroid cells were demonstrated by ectopic expression of wild-type (WT) and mutant proteins in PCCL3 normal rat thyroid cells, followed by tests of cell proliferation, activation of cell growth pathways, and transcription of TTF-1 target genes. All statistical tests were two-sided. RESULTS: A missense mutation (1016C>T) was identified in TITF-1/NKX2.1 that led to a mutant TTF-1 protein (A339V) in four of the 20 MNG/PTC patients (20%). These patients developed substantially more advanced tumors than MNG/PTC or PTC patients without the mutation (P = .022, Fisher exact test). Notably, this germline mutation was dominantly inherited in two families, with some members bearing the mutation affected with MNG, associated with either PTC or colon cancer. The mutation encoding the A339V substitution was not found among the 349 healthy control subjects nor among the 284 PTC patients who had no history of MNG. Overexpression of A339V TTF-1 in PCCL3 cells, as compared with overexpression of WT TTF-1, was associated with increased cell proliferation including thyrotropin-independent growth (average A339V proliferation rate = 134.27%, WT rate = 104.43%, difference = 34.3%, 95% confidence interval = 12.0% to 47.7%, P = .010), enhanced STAT3 activation, and impaired transcription of the thyroid-specific genes Tg, TSH-R, and Pax-8. CONCLUSION: This is the first germline mutation identified in MNG/PTC patients. It could contribute to predisposition for MNG and/or PTC and to the pathogenesis of PTC.

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.

Mutational analysis of SHH and GLI3 in anorectal malformations.

BACKGROUND: Anorectal malformations (congenital absence of the anal opening) are among the most common pediatric surgical problems and carry a significant chronic morbidity. METHODS: Direct sequencing was used to screen 88 anorectal malformations patients for mutations and polymorphisms in SHH and GLI3. These genes were chosen according to the phenotype presented by mutant mice and their expression patterns. RESULTS: We report on 10 GLI3 variants (IVS3+141C>G, T183A, IVS4+124T>C, IVS7+17G>A, IVS8+1 G>C, N503N, P941P, P998L, A1005A, A1039A) and four SHH mutation/variants (IVS1-49C>T, IVS2+111A>C, L214L, G290D). CONCLUSIONS: These variants are not over-represented in the healthy population and most are predicted to be benign. This study conveys the problematic assessment of the pathogenic role in disease of rare point mutations and variants.