Thomas Yuk-Yu Leon

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

Hirschsprungs 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 [CI95%:(1.40, 2.00), P = 1.80 × 10−8] and 1.98 [CI95%:(1.59, 2.47), P = 1.12 × 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.

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.

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 Hirschsprungs 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 Hirschsprungs 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 Hirschsprungs disease.

Fine Mapping of the NRG1 Hirschsprung's Disease Locus

The primary pathology of Hirschsprungs disease (HSCR, colon aganglionosis) is the absence of ganglia in variable lengths of the hindgut, resulting in functional obstruction. HSCR is attributed to a failure of migration of the enteric ganglion precursors along the developing gut. RET is a key regulator of the development of the enteric nervous system (ENS) and the major HSCR-causing gene. Yet the reduced penetrance of RET DNA HSCR-associated variants together with the phenotypic variability suggest the involvement of additional genes in the disease. Through a genome-wide association study, we uncovered a ∼350 kb HSCR-associated region encompassing part of the neuregulin-1 gene (NRG1). To identify the causal NRG1 variants contributing to HSCR, we genotyped 243 SNPs variants on 343 ethnic Chinese HSCR patients and 359 controls. Genotype analysis coupled with imputation narrowed down the HSCR-associated region to 21 kb, with four of the most associated SNPs (rs10088313, rs10094655, rs4624987, and rs3884552) mapping to the NRG1 promoter. We investigated whether there was correlation between the genotype at the rs10088313 locus and the amount of NRG1 expressed in human gut tissues (40 patients and 21 controls) and found differences in expression as a function of genotype. We also found significant differences in NRG1 expression levels between diseased and control individuals bearing the same rs10088313 risk genotype. This indicates that the effects of NRG1 common variants are likely to depend on other alleles or epigenetic factors present in the patients and would account for the variability in the genetic predisposition to HSCR.

Correlation between genetic variations in Hox clusters and Hirschsprung's disease.

Interactions between migrating neural crest cells and the environment of the gut are crucial for the development of the enteric nervous system (ENS). A key signalling mediator is the RET‐receptor‐tyrosine‐kinase which, when defective, causes Hirschprungs disease (HSCR, colon aganglionosis). RET mutations alone cannot account for the variable HSCR phenotype, invoking interactions with as yet unknown, and probably inter‐related, loci involved in ENS development. Homeobox (HOX) genes have a major role in gut development as depicted by the enteric Hox code. We investigated whether DNA alterations in HOX genes, either alone or in combination with RET, are implicated in HSCR. Genotyping effort was minimized by applying the HapMap data on Han Chinese from Beijing (CHB). 194 HSCR patients and 168 controls were genotyped using Sequenom technology for 72 tag, single nucleotide polymorphisms (SNPs) distributed along the HOX clusters. The HapMap frequencies were compared to those in our population and standard statistics were used for frequency comparisons. The multifactor‐dimensionality‐reduction method was used for multilocus analysis, in which RET promoter SNP genotypes were included. Genetic interactions were found between two HOX loci (5′‐HOXA13 and 3′UTR‐HOXB7) and the RET loci tested. Minor allele frequencies (MAF) of the SNPs tested in our sample were not significantly different from those reported by HapMap when the sample sizes of the populations compared were considered. This is the first evaluation of the HOX genes in HSCR and the first application of HapMap data in a Chinese population. The interacting HOX loci may affect the penetrance of the RET risk allele. HapMap data for the CHB population correlated well with the general Chinese population.

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.

Role of RET and PHOX2B gene polymorphisms in risk of Hirschsprung's disease in Chinese population.

Hirschsprung’s disease (OMIM 142623) is a complex congenital disorder characterised by the absence of ganglion cells of the plexus myentericus and plexus submucosus in the variable lengths of the digestive tract.1,2 Aganglionosis is attributed to a defect of the enteric nervous system, in which ganglion cells fail to innervate the lower gastrointestinal tract during embryonic development, resulting in failure to pass meconium, chronic severe constipation and colonic distention in the neonatal period.3 The receptor tyrosine kinase gene RET , which is expressed in neural crest cells during enteric neurogenesis and is required for normal development of the enteric nervous system, is the major susceptibility gene for Hirschsprung’s disease.4 There is growing evidence indicating that functional single nucleotide polymorphisms (SNPs) of RET could act as low susceptibility factors for Hirschsprung’s disease.5,6 In addition, PHOX2B encodes a transcription …

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

Background Hirschsprungs 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 (RETR114H) has recently been identified in ∼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 RETR114H 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 RETR114 were originated from a single mutational event, we predicted the approximate age of RETR114H 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 RETR114H 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 RETR114H patients), we applied haplotype estimation methods to determine whether there were any segments shared between patients with RETR114H 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 RETR114H is a founder mutation for HSCR in the Chinese population.

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.