A complementary study approach unravels novel players in the pathoetiology of Hirschsprung disease.

Hirschsprung disease (HSCR, OMIM 142623) involves congenital intestinal obstruction caused by dysfunction of neural crest cells and their progeny during enteric nervous system (ENS) development. HSCR is a multifactorial disorder; pathogenetic variants accounting for disease phenotype are identified only in a minority of cases, and the identification of novel disease-relevant genes remains challenging. In order to identify and to validate a potential disease-causing relevance of novel HSCR candidate genes, we established a complementary study approach, combining whole exome sequencing (WES) with transcriptome analysis of murine embryonic ENS-related tissues, literature and database searches, in silico network analyses, and functional readouts using candidate gene-specific genome-edited cell clones. WES datasets of two patients with HSCR and their non-affected parents were analysed, and four novel HSCR candidate genes could be identified: ATP7A, SREBF1, ABCD1 and PIAS2. Further rare variants in these genes were identified in additional HSCR patients, suggesting disease relevance. Transcriptomics revealed that these genes are expressed in embryonic and fetal gastrointestinal tissues. Knockout of these genes in neuronal cells demonstrated impaired cell differentiation, proliferation and/or survival. Our approach identified and validated candidate HSCR genes and provided further insight into the underlying pathomechanisms of HSCR.

Molecular Genetic Anatomy and Risk Profile of Hirschsprung's Disease.

BACKGROUND: Hirschsprung's disease, or congenital aganglionosis, is a developmental disorder of the enteric nervous system and is the most common cause of intestinal obstruction in neonates and infants. The disease has more than 80% heritability, including significant associations with rare and common sequence variants in genes related to the enteric nervous system, as well as with monogenic and chromosomal syndromes. METHODS: We genotyped and exome-sequenced samples from 190 patients with Hirschsprung's disease to quantify the genetic burden in patients with this condition. DNA sequence variants, large copy-number variants, and karyotype variants in probands were considered to be pathogenic when they were significantly associated with Hirschsprung's disease or another neurodevelopmental disorder. Novel genes were confirmed by functional studies in the mouse and human embryonic gut and in zebrafish embryos. RESULTS: The presence of five or more variants in four noncoding elements defined a widespread risk of Hirschsprung's disease (48.4% of patients and 17.1% of controls; odds ratio, 4.54; 95% confidence interval [CI], 3.19 to 6.46). Rare coding variants in 24 genes that play roles in enteric neural-crest cell fate, 7 of which were novel, were also common (34.7% of patients and 5.0% of controls) and conferred a much greater risk than noncoding variants (odds ratio, 10.02; 95% CI, 6.45 to 15.58). Large copy-number variants, which were present in fewer patients (11.4%, as compared with 0.2% of controls), conferred the highest risk (odds ratio, 63.07; 95% CI, 36.75 to 108.25). At least one identifiable genetic risk factor was found in 72.1% of the patients, and at least 48.4% of patients had a structural or regulatory deficiency in the gene encoding receptor tyrosine kinase (RET). For individual patients, the estimated risk of Hirschsprung's disease ranged from 5.33 cases per 100,000 live births (approximately 1 per 18,800) to 8.38 per 1000 live births (approximately 1 per 120). CONCLUSIONS: Among the patients in our study, Hirschsprung's disease arose from common noncoding variants, rare coding variants, and copy-number variants affecting genes involved in enteric neural-crest cell fate that exacerbate the widespread genetic susceptibility associated with RET. For individual patients, the genotype-specific odds ratios varied by a factor of approximately 67, which provides a basis for risk stratification and genetic counseling. (Funded by the National Institutes of Health.).

A PIGN mutation responsible for multiple congenital anomalies-hypotonia-seizures syndrome 1 (MCAHS1) in an Israeli-Arab family.

Mutations in the PIGN gene involved in the glycosylphoshatidylinositol (GPI) anchor biosynthesis pathway cause Multiple Congenital Anomalies-Hypotonia-Seizures syndrome 1 (MCAHS1). The syndrome manifests developmental delay, hypotonia, and epilepsy, combined with multiple congenital anomalies. We report on the identification of a homozygous novel c.755A>T (p.D252V) deleterious mutation in a patient with Israeli-Arab origin with MCAHS1. The mutated PIGN caused a significant decrease of the overall GPI-anchored proteins and CD24 expression. Our results, strongly support previously published data, that partial depletion of GPI-anchored proteins is sufficient to cause severe phenotypic expression.

Organophosphate pesticides induce morphological abnormalities and decrease locomotor activity and heart rate in Danio rerio and Xenopus laevis.

Organophosphate pesticides (OPs), a class of acetylcholinesterase inhibitors, are used widely in agriculture to reduce insect populations. Because of the conservation of acetylcholinesterase between invertebrates and vertebrates, OPs also can adversely affect nontarget species, such as aquatic and terrestrial animals. This study used uniform conditions to analyze the morphological and physiological effects caused by developmental exposure to 3 commonly used OPs-chlorpyrifos, dichlorvos, and diazinon-on 2 aquatic vertebrate species, Danio rerio (zebrafish) and Xenopus laevis. Survival, locomotor activity, heart rate, and gross anatomical abnormalities, including kyphosis and edema, were observed over a 5-d period in response to OP concentrations ranging from 0 µM to 1000 µM. Both zebrafish and Xenopus showed decreased survival for all 3 OPs at higher concentrations. However, Xenopus showed higher mortality than zebrafish at lower chlorpyrifos and dichlorvos concentrations. Both models showed a dose-dependent decrease in heart rate and free-swimming larval activity in response to chlorpyrifos and dichlorvos. In addition, kyphosis and decreased spine length were prominent in Xenopus in response to 10 µM of chlorpyrifos and 0.1 µM dichlorvos. Although diazinon induced no effects on skeletal and cardiac motor activity in either species, it did induce cardiac edemas in zebrafish. Differences in the biological actions of OPs and their differential effects in these 2 vertebrate models demonstrate the importance of using common protocols and multiple models to evaluate the ecotoxicology of OPs.