High incidence of EDNRB gene mutation in seven southern Chinese familial cases with Hirschsprung's disease.

PURPOSE: Hirschsprung's disease (HSCR) is the leading cause of neonatal functional intestinal obstruction, which has been identified in many familial cases. HSCR, a multifactorial disorder of enteric nervous system (ENS) development, is associated with at least 24 genes and seven chromosomal loci, with RET and EDNRB as its major genes. We present a genetic investigation of familial HSCR to clarify the genotype-phenotype relationship. METHODS: We performed whole exome sequencing (WES) on Illumina HiSeq X Ten platform to investigate genetic backgrounds of core family members, and identified the possibly harmful mutation genes. Mutation carriers and pedigree relatives were validated by Sanger sequencing for evaluating the gene penetrance. RESULTS: Four familial cases showed potential disease-relative variants in EDNRB and RET gene, accounting for all detection rate of 57.1%. Three familial cases exhibited strong pathogenic variants as frameshift or missense mutations in EDNRB gene. A novel c.367delinsTT mutation of EDNRB was identified in one family member. The other two EDNRB mutations, c.553G>A in family 2 and c.877delinsTT in family 5, have been reported in previous literatures. The penetrance of EDNRB variants was 33-50% according mutation carries. In family 6, the RET c.1858T>C (C620R) point mutation has previously been reported to cause HSCR, with 28.5% penetrance. CONCLUSION: We identified a novel EDNRB (deleted C and inserted TT) mutation in this study using WES. Heterozygote variations in EDNRB gene were significantly enriched in three families and RET mutations were identified in one family. EDNRB variants showed an overall higher incidence and penetrance than RET in southern Chinese families cases.

Microcytic hypochromic Anemia is a risk factor for postoperative HAEC: A retrospective study.

Background: Hirschsprung-associated enterocolitis (HAEC) is a common and life-threatening complication of Hirschsprung's disease (HSCR), which can occur before and after surgery. The aim of this study was to identify the risk factors associated with the development of HAEC. Methods: We retrospectively reviewed the medical records of HSCR patients admitted to the Children's Hospital of Shanxi Province, China, between January 2011 and August 2021. Diagnosis of HAEC was made using a scoring system with cutoff values ≥4 and included the patient's history, physical examination, and radiological and laboratory findings. The results are shown as frequency (%). The chi-square test was used to analyze a single factor with a significance level of P < 0.05. Logistic regression analysis was used to analyze multiple factors. Results: A total of 324 patients were included in this study, with 266 males and 58 females. In total, 34.3% (111/324) of patients had HAEC, including 85 males and 26 females; 18.9% (61/324) of patients had preoperative HAEC; and 15.4% (50/324) of patients had postoperative HAEC within one year after surgery. Gender, age at definitive therapy, and feeding methods were not found to be associated with preoperative HAEC in univariate analysis. Respiratory infection was associated with preoperative HAEC (P = 0.00003). No association was found between gender and age at definitive therapy and postoperative HAEC. Postoperative HAEC was associated with microcytic hypochromic anemia (P = 0.00058), preoperative history of HAEC (P = 0.00120), the creation of a preoperative stoma (P = 0.00097), long segment or total colon HSCR (P = 0.00057), and hypoalbuminemia (P = 0.03225). Regression analysis showed that microcytic hypochromic anemia (OR=2.716, 95% CI = 1.418-5.203, P = 0.003), preoperative history of HAEC (OR=2.814, 95% CI = 1.429-5.542, P = 0.003), the creation of a preoperative stoma (OR=2.332, 95% CI = 1.003-5.420, P = 0.049), and long segment or total colon HSCR (OR=2.167, 95% CI = 1.054-4.456, P = 0.035) were associated with postoperative HAEC. Conclusion: This study revealed that the incidence of preoperative HAEC at our hospital was associated with respiratory infections. In addition, microcytic hypochromic anemia, preoperative history of HAEC, the creation of a preoperative stoma, and long segment or total colon HSCR were risk factors of postoperative HAEC. The most important finding of this study was that microcytic hypochromic anemia was a risk factor for postoperative HAEC, which has been rarely reported. Further studies with larger sample sizes are necessary to confirm these findings.

The m6A methyltransferase METTL3 affects cell proliferation and migration by regulating YAP expression in Hirschsprung disease.

BACKGROUND: METTL3, an mRNA m6A methyltransferase, has been implicated in various steps of mRNA metabolism, such as stabilization, splicing, nuclear transportation, translation, and degradation. However, whether METTL3 dysregulation is involved in Hirschsprung disease (HSCR) development remains unclear. In this study, we preliminarily elucidated the role of METTL3 in HSCR and sought to identify the associated molecular mechanism. METHODS: The gene expression levels of YAP and several methyltransferases, demethylases, and effectors were evaluated by RT-qPCR. Protein levels were evaluated by western blot and immunohistochemistry. Cell proliferation and migration were detected by CCK-8 and Transwell assays, respectively. The overall levels of m6A modification were determined by colorimetry. RESULTS: We found that m6A levels were reduced in the stenotic intestinal tissue of patients with HSCR. When METTL3 was knocked down in SH-SY5Y and HEK-293T cells, the proliferative and migratory abilities of the cells were inhibited, m6A modification levels were reduced, and YAP expression was increased. Importantly, YAP and METTL3 expression displayed a negative correlation in both cell lines as well as in HSCR tissue. CONCLUSIONS: Our results provide evidence for an interaction between METTL3 and YAP in HSCR, and further suggest that METTL3 is involved in the pathogenesis of HSCR by regulating neural crest cell proliferation and migration upstream of YAP.

Hirschsprung Disease for the Practicing Surgical Pathologist.

OBJECTIVES: Hirschsprung disease (HD) is a congenital condition defined by the absence of ganglion cells in the distal-most portion of the gastrointestinal tract. Biopsies and resections for HD can be adrenaline inducing for the general surgical pathologist because specimens are infrequent; HD is 1 of only a few neuroanatomic diseases that general surgical pathologists diagnose; numerous preanalytic factors (eg, biopsy adequacy, surgeon sampling protocol, processing artifacts) can affect histologic interpretation; and most importantly, the diagnosis has high stakes. METHODS: We provide a comprehensive overview of the background, relevant clinical procedures, and pathologic assessment of HD. Grossing and frozen section protocols, an algorithmic approach to diagnosis, and histologic pearls and pitfalls are also discussed. RESULTS: Evaluation and recognition of the features of HD have evolved significantly in the past 2 decades with the discovery of the value of calretinin immunohistochemistry in the late 2000s and the recent development of straightforward and reproducible histologic criteria for identification of the HD transition zone. CONCLUSIONS: These advancements have substantially improved the pathologist's ability to reliably evaluate for HD. Nonetheless, as with any high-stakes surgical pathology specimen, clear communication with the clinical team is essential.

Association between IKBKAP polymorphisms and Hirschsprung's disease susceptibility in Chinese children.

Background: Hirschsprung's disease (HSCR) is a rare congenital disease in which enteric nervous system (ENS) in the distal intestine is absent. HSCR is a disease involving genetic factors and environmental factors. Despite a series of genes have been revealed to contribute to HSCR, many HSCR associated genes were yet not identified. Previous studies had identified that a potential susceptibility gene of HSCR was an inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein (IKBKAP). The study aimed to explore the association of genetic variants in IKBKAP and HSCR susceptibility in southern Chinese children. Methods: Single nucleotide polymorphism (SNPs) were genotyped by the Mass ARRAY iPLEX Gold system (Sequenom, San Diego, CA, USA) on all samples, which included 1,470 HSCR children (cases) and 1,473 healthy children (controls). The associations between SNPs and HSCR or clinical subtypes were assessed by comparing their allele frequencies in corresponding case and control samples. Different genetic models, including additive, recessive, and dominant models, were tested using PLINK 1.9 software. Results: Further subgroup analysis revealed rs2275630 as a total colonic aganglionosis (TCA)-specific susceptibility locus. The present study is the first to indicate that IKBKAP rs2275630 were associated with HSCR susceptibility, especially in TCA patients. Conclusions: We conclude that IKBKAP rs2275630 is a susceptibility gene of HSCR.

Aberrant expression of LINC00346 regulates cell migration and proliferation via competitively binding to miRNA-148a-3p/Dnmt1 in Hirschsprung's disease.

BACKGROUND: Hirschsprung's disease (HSCR) is a common birth defect caused by dysplasia of neural crest cells in the gut. Long noncoding RNAs (lncRNAs) play an important role in cellular processes, including development and disease. Despite the known engagement of LINC00346 in several human diseases, its biological function in HSCR remains unknown. METHODS: The relative expression levels of LINC00346, miR-148a-3p and Dnmt1 in HSCR colon tissues were detected by quantitative real-time PCR. Western blot assays were conducted to investigate the Dnmt1 protein expression level. Knockdown of LINC00346 and overexpression of miR-148a-3p in SH-SY5Y and SK-N-BE(2) cell lines was conducted. Cell proliferation and migration were detected by cell counting Kit-8 assays, 5-ethynyl-2'-deoxyuridine assays and transwell assays. Cell apoptosis was verified by flow cytometric analysis. Furthermore, the competing endogenous RNA (ceRNA) activity of LINC00346 on miR-148a-5p was investigated via bioinformatics analysis and luciferase reporter assays. RESULT: Downregulation of LINC00346 and Dnmt1 was detected in HSCR tissues. Knockdown of LINC00346 and overexpression of miR-148a-3p in SK-N-BE(2) and SH-SY5Y cells inhibited cell migration and proliferation and promoted apoptosis. Moreover, the miR-148a-3p inhibitor rescued the downregulation of Dnmt1 in LINC00346 knockdown cell lines, which was evidence of the ceRNA regulatory mechanism of Dnmt1 by LINC00346. CONCLUSIONS: LINC00346 was downregulated in HSCR colon tissues and acted as a ceRNA to regulate the expression of Dnmt1 in vitro. Together, these findings indicate that LINC00346 could affect the occurrence of HSCR by participating in the development of enteric neural crest cells.

Disparate phenotypes in two unfavorable pregnancies due to maternal mosaicism of a novel RET gene mutation.

Mutations in RET have been found in multiple diseases including isolated and associated congenital anomalies. Here, we report a case presented with disparate phenotypes in each pregnancy but caused by the same novel mutation. Whole-exome sequencing (WES) was performed on the proband/abortion product-parental trio and a novel missense variant in RET (chr10:43615610C > G; c.2689C > G; p.Arg897Gly) was identified. The mother was a low-level somatic carrier of this new mutation, with 17.3% in blood, 19.1% in oralmucous membrane, and 15.7% in urine by droplet digital polymerase chain reaction (dd PCR). Our finding not only broadens the mutation spectrum of RET but also gives supportive genetic counseling and timely guidance on fertility choices.

Long-term growth outcomes in children with Hirschsprung disease after definitive surgery: A cross-sectional study.

BACKGROUND: The growth of children with Hirschsprung disease (HSCR) can be affected by many factors, including the environment, nutrient intake, and surgery. Our study compared the long-term (i.e., at least 3 years of follow-up) growth outcomes in HSCR children after transabdominal Soave and Duhamel and transanal endorectal pull-through (TEPT) surgeries. METHODS: A cross-sectional study was conducted in children <18 years of age diagnosed histopathologically with HSCR who underwent pull-through between January 1, 2012-December 31, 2015 in our institution. The postoperative anthropometric data were obtained prospectively through interviews during the outpatient clinic appointment or by telephone. RESULTS: We recruited 21 patients (Soave: 7 vs. Duhamel: 4 vs. TEPT: 10; p = 0.06). There were no significant differences between the three surgical methods in terms of preoperative and postoperative nutritional status categories (p = 0.52). Concerning the changes in nutritional status, after Soave surgery, it was improved, steady, and worsened in 28.6%, 57.1%, and 14.3% of the children, respectively. The nutritional status of the Duhamel group was worsened and steady in 25% and 75% of the children, respectively, while in the TEPT group, it was improved and steady in 40% and 60% of the children, respectively. However, these differences were not statistically significant (p = 0.42). CONCLUSIONS: While some HSCR children show an improvement in their nutritional status after Soave and TEPT procedures, the overall nutritional status is similar among different procedures. Further multicenter studies with a larger sample size are important to clarify our findings.

Quality of life of patients with Hirschsprung disease after Duhamel and Soave pull-through procedures: A mixed-methods sequential explanatory cohort study.

BACKGROUND: Quality of life (QoL) is one of the important outcomes for patients with Hirschsprung disease (HSCR) after pull-through that provides qualitative data concerning the long-term outcomes, however, it has not been well-studied. The HSCR/anorectal malformation quality of life questionnaire (HAQL) is considered valid and reliable to evaluate the QoL of HSCR patients. MATERIAL AND METHODS: A mixed-method sequential explanatory cohort study was conducted to compare the QoL of HSCR patients after Duhamel and Soave pull-through at Dr. Sardjito Hospital between 2013 and 2018 using an Indonesian adaptation of the HAQL, followed by a qualitative study. RESULTS: We ascertained eleven HSCR patients (Duhamel: five HAQL parents and one HAQL adolescent vs. Soave: four HAQL parents and one HAQL adult). For the quantitative study, the mean HAQL score was 2.50 and 2.79 for the Duhamel and Soave groups, respectively. For the qualitative study, interviewed patients' parents expressed how their child's life had improved after surgery. However, frequent bloating was a major complaint following Soave surgery, whereas hardened stools were a major problem after Duhamel procedure. CONCLUSION: Here, for the first time using a mixed-method sequential explanatory cohort design, we show that patients with HSCR after Soave tended to have a higher overall QoL score compared to the Duhamel group. Further multicenter study with a larger sample size is mandatory to give better understanding about QoL of HSCR patients following pull-through.

ERNICA guidelines for the management of rectosigmoid Hirschsprung's disease.

BACKGROUND: Hirschsprung's disease (HSCR) is a serious congenital bowel disorder with a prevalence of 1/5000. Currently, there is a lack of systematically developed guidelines to assist clinical decision-making regarding diagnostics and management. AIMS: This guideline aims to cover the diagnostics and management of rectosigmoid HSCR up to adulthood. It aims to describe the preferred approach of ERNICA, the European Reference Network for rare inherited and congenital digestive disorders. METHODS: Recommendations within key topics covering the care pathway for rectosigmoid HSCR were developed by an international workgroup of experts from 8 European countries within ERNICA European Reference Network from the disciplines of surgery, medicine, histopathology, microbiology, genetics, and patient organization representatives. Recommendation statements were based on a comprehensive review of the available literature and expert consensus. AGREE II and GRADE approaches were used during development. Evidence levels and levels of agreement are noted. RESULTS: Thirty-three statements within 9 key areas were generated. Most recommendations were based on expert opinion. CONCLUSION: In rare or low-prevalence diseases such as HSCR, there remains limited availability of high-quality clinical evidence. Consensus-based guidelines for care are presented.

Sequence characterization of RET in 117 Chinese Hirschsprung disease families identifies a large burden of de novo and parental mosaic mutations.

BACKGROUND: Hirschsprung disease (HSCR) is an inherited congenital disorder characterized by the absence of enteric ganglia in the distal part of the gut. RET is the major causative gene and contains > 80% of all known disease-causing mutations. RESULTS: To determine the incidence of RET pathogenic variants, be they Mendelian inherited, mosaic in parents or true de novo variants (DNVs) in 117 Chinese families, we used high-coverage NGS and droplet digital polymerase chain reaction (ddPCR) to identify 15 (12.8%) unique RET coding variants (7 are novel); one was inherited from a heterozygous unaffected mother, 11 were DNVs (73.3%), and 3 full heterozygotes were inherited from parental mosaicism (2 paternal, 1 maternal): two clinically unaffected parents were identified by NGS and confirmed by ddPCR, with mutant allele frequency (13-27%) that was the highest in hair, lowest in urine and similar in blood and saliva. An extremely low-level paternal mosaicism (0.03%) was detected by ddPCR in blood. Six positive-controls were examined to compare the mosaicism detection limit and sensitivity of NGS, amplicon-based deep sequencing and ddPCR. CONCLUSION: Our findings expand the clinical and molecular spectrum of RET variants in HSCR and reveal a high frequency of RET DNVs in the Chinese population.

Factors influencing the incidence of Hirschsprung associated enterocolitis (HAEC).

PURPOSE: This study aims to characterize risk factors for Hirschsprung-associated enterocolitis (HAEC). We hypothesize that earlier pull-through surgery is associated with lower risks of developing postoperative HAEC. METHODS: A comparative study of 171 Hirschsprung patients treated from 1990 to 2017 was performed. Patients without HAEC were compared to patients with preoperative and/or postoperative HAEC. Results are presented as median [IQR] or frequency (%). Pearson's χ2 test and Wilcoxon rank sum test were performed with a significance level at p < 0.05. Multivariable logistic regression analysis was used to adjust for potential confounders. A subanalysis was done to evaluate laparoscopic, laparotomy, and transanal surgeries. RESULTS: Risk of developing preoperative HAEC was significantly associated with congenital malformations (OR 2.63 [1.11, 6.24]; p = 0.02). Birth weight was lower in patients with preoperative HAEC (OR 0.48 [95% CI 0.25, 0.93]; p = 0.03). On regression analysis, intestinal obstruction after surgery was significantly associated with postoperative HAEC (OR 8.2 [3.18, 21.13]; p < 0.0001). Patients with earlier pull-through surgery did not have a lower risk of developing postoperative HAEC. CONCLUSIONS: Timing of surgery does not seem to be associated with a higher risk of developing pre- and postoperative HAEC. Predisposing factors for preoperative HAEC included associated malformations and lower birth weight, whereas intestinal obstruction was found to be associated with postoperative HAEC. TYPE OF STUDY: Treatment study. LEVEL OF EVIDENCE: Level III.

Association analysis of NOX5 polymorphisms with Hirschsprung disease.

BACKGROUND/PURPOSE: Hirschsprung disease (HSCR) is a developmental disease characterized by the absence of ganglion cells in the intestinal region. NADPH oxidase5 (NOX5) has been identified as one of the possible candidate genes for risk of Hirschsprung disease in our recent genome wide association study (GWAS). In this study, we performed a replication study to analyze the association of NOX5 polymorphisms with HSCR risk and conducted an extended analysis to investigate further associations for sub-groups and haplotypes. METHODS: A total of 23 NOX5 single nucleotide polymorphisms (SNPs) were genotyped in 187 HSCR patients and 283 unaffected controls. Statistical analysis was performed to examine the effects of genotype on risk of HSCR and HSCR subtype. RESULTS: Logistic regression analyses revealed that six SNPs (rs59355559, rs62010828, rs34990910, rs11856030, rs311905, and rs8024894) were associated with risk of HSCR (minimum p = 0.007 at rs62010828). Moreover, three SNPs (rs59355559, rs62010828, and rs8024894) were significantly associated with risk of long-segment HSCR (L-HSCR) subtype and 5 SNPs (rs59355559, rs62010828, rs34990910, rs11856030, and rs8024894) were found to be associated with risk of TCA subtype. CONCLUSION: Our results demonstrate that genetic variants in NOX5 have genetic effects on risk of HSCR, which may serve as useful preliminary information for further study. LEVELS OF EVIDENCE: Level III of prognosis study.

Reduced expression of the NLRP6 inflammasome in the colon of patients with Hirschsprung's disease.

PURPOSE: Hirschsprung's associated enterocolitis (HAEC) is the most common cause of morbidity and mortality in Hirschsprung's Disease (HSCR). The pathogenesis of HAEC remains unsatisfactorily understood. Mounting evidence of an altered microbiome in patients with HSCR adds a new angle to the pathogenesis of HAEC. NLRP6 is a member of the nucleotide-binding domain, leucine-rich-repeat-containing (NLR) innate immune receptor family, a multiprotein complex that functions as a sensor of damage-associated molecular patterns. Known as inflammasomes they have been implicated in the regulation of colonic microbial ecology and by alteration, regulators of inflammation. We designed this study to test the hypothesis that NLRP6 expression is altered in the colon of patients with HSCR. METHODS: We investigated NLPR6 protein expression in both the aganglionic and ganglionic regions of HSCR patients (n = 10) versus healthy control colon (n = 10). Protein distribution was assessed by using immunofluorescence and confocal microscopy. Gene and protein expressions were quantified using quantitative real-time polymerase chain reaction (qPCR), Western blot analysis, and densitometry. MAIN RESULTS: qPCR and Western blot analysis revealed that NLRP6 is expressed in the colon of patients with HSCR. NLRP6 expression was significantly decreased (p < 0.003) in the ganglionic and aganglionic bowel in HSCR compared to controls. Confocal microscopy revealed that NLRP6 expression in colonic epithelium was markedly decreased in HSCR specimens compared to controls. CONCLUSION: We demonstrate for the first time the expression and distribution of NLRP6 in patients with HSCR. The observed decreased expression of NLRP6 may contribute to an altered colonic microbiome in patients with HSCR and increases the susceptibility to develop HAEC.

GT-repeat extension in the IL11 promoter is associated with Hirschsprung's disease (HSCR).

Hirschsprung's disease (HSCR) is a congenital disease characterized by intestinal obstruction due to a defective intestinal neural system. Frequently, the disease is associated with an intestinal inflammation. The most common known underlying genetic alterations are in the RET gene but HSCR can also be caused by mutations in other genes that are responsible for the maturation and migration of intestinal neural cells. Recently, a study in an Asian population reported a significant association of several single nucleotide polymorphisms (SNPs) in the IL11 (interleukin 11) gene with HSCR. We further explored the possible association of genetic alterations in the IL11 gene with HSCR and HSCR subtypes in an unrelated Caucasian population. We used a targeted sequencing approach to identify a total of 32 SNPs covering the coding region of the IL11 gene including the proximal part of the promoter and relevant SNPs described in the previous study. Genotype frequencies were compared using additive genetic models in 103 HSCR patients and 128 healthy controls. We failed to observe any significant association of SNPs with HSCR. However, there was a suggestive evidence for an association of the length of a dinucleotide repeat in the IL11 promoter region with HSCR with an over-representation of >7 GT repeat subtypes (OR = 4.982 (1.448-17.040), p-value = 0.0111) in HSCR patients. A similar trend was further observed in a subgroup of patients with long-segment HSCR (L-HSCR). These findings need to be replicated in a well-powered study. Changes in IL11 expression may be a link to the intestinal inflammation frequently observed in patients with HSCR.

Identification of Variants in RET and IHH Pathway Members in a Large Family With History of Hirschsprung Disease.

BACKGROUND & AIMS: Hirschsprung disease (HSCR) is an inherited congenital disorder characterized by absence of enteric ganglia in the distal part of the gut. Variants in ret proto-oncogene (RET) have been associated with up to 50% of familial and 35% of sporadic cases. We searched for variants that affect disease risk in a large, multigenerational family with history of HSCR in a linkage region previously associated with the disease (4q31.3-q32.3) and exome wide. METHODS: We performed exome sequencing analyses of a family in the Netherlands with 5 members diagnosed with HSCR and 2 members diagnosed with functional constipation. We initially focused on variants in genes located in 4q31.3-q32.3; however, we also performed an exome-wide analysis in which known HSCR or HSCR-associated gene variants predicted to be deleterious were prioritized for further analysis. Candidate genes were expressed in HEK293, COS-7, and Neuro-2a cells and analyzed by luciferase and immunoblot assays. Morpholinos were designed to target exons of candidate genes and injected into 1-cell stage zebrafish embryos. Embryos were allowed to develop and stained for enteric neurons. RESULTS: Within the linkage region, we identified 1 putative splice variant in the lipopolysaccharide responsive beige-like anchor protein gene (LRBA). Functional assays could not confirm its predicted effect on messenger RNA splicing or on expression of the mab-21 like 2 gene (MAB21L2), which is embedded in LRBA. Zebrafish that developed following injection of the lrba morpholino had a shortened body axis and subtle gut morphological defects, but no significant reduction in number of enteric neurons compared with controls. Outside the linkage region, members of 1 branch of the family carried a previously unidentified RET variant or an in-frame deletion in the glial cell line derived neurotrophic factor gene (GDNF), which encodes a ligand of RET. This deletion was located 6 base pairs before the last codon. We also found variants in the Indian hedgehog gene (IHH) and its mediator, the transcription factor GLI family zinc finger 3 (GLI3). When expressed in cells, the RET-P399L variant disrupted protein glycosylation and had altered phosphorylation following activation by GDNF. The deletion in GDNF prevented secretion of its gene product, reducing RET activation, and the IHH-Q51K variant reduced expression of the transcription factor GLI1. Injection of morpholinos that target ihh reduced the number of enteric neurons to 13% ± 1.4% of control zebrafish. CONCLUSIONS: In a study of a large family with history of HSCR, we identified variants in LRBA, RET, the gene encoding the RET ligand (GDNF), IHH, and a gene encoding a mediator of IHH signaling (GLI3). These variants altered functions of the gene products when expressed in cells and knockout of ihh reduced the number of enteric neurons in the zebrafish gut.

Long non-coding RNA LOC100507600 functions as a competitive endogenous RNA to regulate BMI1 expression by sponging miR128-1-3p in Hirschsprung's disease.

Recently studies reported that long non-coding RNAs (lncRNAs) may take part in a lot of congenital diseases, meanwhile, Hirschsprung's disease (HSCR) is a major congenital digestive tract malformation. Nevertheless whether lncRNAs participate in the occurrence of HSCR and how it contributes to this disease are still unknown. LOC100507600 was selected from our gene expression microarray data obtained from bowel tissues from HSCR patients and negative controls. Subsequently, we used qRT-PCR to prove the result in 64 pairs of HSCR disease bowel stenosis tissues and negative controls. Transwell assay, CCK-8 assay and flow cytometry were employed to explore whether cellular functions change after knocking down the LOC100507600 in SH-SY5Y cell and human 293T cell. Dual-luciferase reporter assay was used to confirm the competitive relationship between BMI1 and LOC100507600 through their association with hsa-miR128-1-3p. Protein extraction and Western blotting were used to further confirm the relationship between LOC100507600 and BMI1. We found that LOC100507600 was obvious reduced in tissues from HSCR patients with noteworthy correlation with BMI1. Furthermore, Downregulation of LOC100507600 repressed cell migration and proliferation and didn't affect cell apoptosis or cycle. Dual-luciferase reporter assay, qRT-PCR and Western blotting assay verified that LOC100507600 serves as a competitive endogenous RNA of miR128-1-3p and down-regulates BMI1 expression by sponging miR128-1-3p in HSCR. In sum, our study researches the potential diagnostic value of LOC100507600 in HSCR and deduces that LOC100507600 can contributes to HSCR as a competitive endogenous RNA to regulate BMI1 expression by sponging miR128-1-3p.

Genetic variation in the GDNF promoter affects its expression and modifies the severity of Hirschsprung's disease (HSCR) in rats carrying Ednrb(sl) mutations.

Glial cell line-derived neurotrophic factor (GDNF) is necessary for the migration of neural crest stem cells in the gut. However, mutations in GDNF per se are deemed neither necessary nor sufficient to cause Hirschsprung's disease (HSCR). In a previous study, a modifier locus on chromosome 2 in rats carrying Ednrb(sl) mutations was identified, and several mutations in the putative regulatory region of the Gdnf gene in AGH-Ednrb(sl) rats were detected. Specifically, the mutation -232C>T has been shown to be strongly associated with the severity of HSCR. In the present study, the influence of genetic variations on the transcription of the Gdnf gene was tested using dual-luciferase assay. Results showed that the mutation -613C>T, located near the mutation -232C>T in AGH-Ednrb(sl) rats, decreased Gdnf transcription in an in vitro dual-luciferase expression assay. These data suggested an important role of -613C in Gdnf transcription. Expression levels of the Gdnf gene may modify the severity of HSCR in rats carrying Ednrb(sl) mutations.

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.

Nidogen-1 is a common target of microRNAs MiR-192/215 in the pathogenesis of Hirschsprung's disease.

Recent studies have emphasized the important role of microRNA (miRNA) clusters and common target genes in disease progression. Despite the known involvement of the miR-192/215 family in many human diseases, its biological role in Hirschsprung disease (HSCR) remains undefined. In this study, we explored the role of the miR-192/215 family in the pathogenesis of HSCR. Quantitative real-time PCR and western blotting measured relative expression levels of miRNAs, mRNAs, and proteins in 80 HSCR patients and 77 normal colon tissues. Targets were evaluated by dual-luciferase reporter assays, and the functional effects of miR-192/215 on human 293T and SH-SY5Y cells were detected by the Transwell assay, CCK8 assay and flow cytometry. MiR-192/215 was significantly down-regulated in HSCR tissue samples, and their knockdown inhibited cell migration and proliferation in the human 293T and SH-SY5Y cell lines. Nidogen 1 (NID1) was confirmed as a common target gene of miR-192/215 by dual-luciferase reporter gene assay and its expression was inversely correlated with that of miR-192/215 in tissue samples and cell lines. Silencing of NID1 could rescue the extent of the suppressing effects by miR-192/215 inhibitor. The down-regulation of miR-192/215 may contribute to HSCR development by targeting NID1. We proposed the following cascade for the proposed mechanism of miR-192/215 in the pathogenesis of Hirschsprung disease (HSCR) by targeting Nidogen 1 (NID1). Aberrant expression of miR-192/215 inhibits cell migration and cell proliferation via NID1. We think the miR-192/miR-215/NID1 signaling pathway may play an important role in the pathogenesis of HSCR.