Identification and characterization of novel candidate genes for Hirschsprung's disease - a developmental disorder of the enteric nervous system

The neurodevelopmental disorder Hirschsprung’s disease (HSCR) represents the most common cause for congenital obstruction and is characterized by a lack of enteric neurons (aganglionosis) in distinct segments of the colon. This aganglionosis is caused by dysfunctions in the neural crest cell (NCC) population which is responsible for enteric nervous system (ENS) generation during embryogenesis. Specifically, either proliferation, migration, differentiation or cell survival of NCC-derived progenitor cells is impaired. The main symptom of this disorder is represented by a megacolon formation. Patients are routinely treated by surgical resections of the aganglionic segment, but gastrointestinal impairments may persist in a fraction of patients even life-long. HSCR is classified as rare and multifactorial disorder. Up to now more than 20 genes are classified as validated disease-causing loci, including the major susceptibility locus RET, but many more genetic factors have been implicated in the pathoaetiology. However, in the majority of patients the genetic disease causes are still unknown. Next-generation sequencing technologies provide the possibility to rapidly uncover the individual’s genetic architecture. Nevertheless, dissecting the genetic findings of importance and correlating them with the pathomechanisms is a major challenge especially in complex diseases as HSCR. This study aimed to establish a complementary research approach for identification and characterization of novel HSCR candidate genes. By taking genetic, bioinformatics, molecular and functional data into account, better insights into the molecular pathogenesis of HSCR should be gained. In this project, two sporadic long-segment HSCR cases were analysed by whole exome sequencing in a trio-based setup and by genotyping of non-coding risk single nucleotide polymorphisms. In both patients, bioinformatic analyses of exome-wide sequencing data led to the identification of rare structural (copy number variations (CNVs)) and single nucleotide variants (SNVs). To narrow down the list of HSCR candidate genes, rare SNVs were further filtered. Finally, four candidate genes (ATP7A, SREBF1, ABCD1 and PIAS2) which were so far not reported in the context of HSCR, were selected for detailed investigations. Extensive mRNA and protein expression analyses confirmed the expression of these candidates in relevant murine gastrointestinal tissues of different developmental stages and thereby validated their putative relevance for the HSCR aetiology. Moreover, additional HSCR patients carrying rare variants in SREBF1 and PIAS2 were identified. To further assess functionally the neuronal specific role of the candidates, the CRISPR/Cas9 technology was applied in a human neuroblastoma cell line. Gene-specific knockout (KO) cell clones were generated for three candidate genes and the major HSCR susceptibility locus RET, while genome editing was not successful for PIAS2. KO clones were investigated on morphological and functional level by a comparison to a mock control clone. Comparative analyses revealed variable differences for the individual gene-specific KO clones in the differentiation behaviour, proliferation and migration capacity as well as in cell survival during neuronal differentiation. To evaluate all findings of this complementary project, a HSCR risk scoring system was applied. According to the gained risk scores, all four selected HSCR candidates could be classified as relevant for the development of HSCR. Like this, the suitability of the presented research approach for identification and characterization of novel HSCR candidates was validated. It is envisioned to apply the established study pipeline to primary ENS-like model systems to confirm the findings of this project. Moreover, these analyses could help to dissect the candidate gene’s relevance for HSCR in detail and gain insights into affected molecular pathways

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

Author summaryHirschsprung disease (HSCR) is a rare developmental disorder. It leads to the absence of enteric nerve cells (aganglionosis) in the large intestine and is caused by functional defects of neuronal precursor cells during embryonic development of the gut nervous system. The aganglionosis manifests as a variety of symptoms including impaired peristalsis and the formation of a pathogenic dilatation of the intestine (megacolon). The etiology of HSCR is considered to be multifactorial. Variants in more than 20 genes have been reported to be overrepresented in HSCR and replicated in independent cohorts. However, variants in those risk genes account for only 30% of all cases, suggesting that many more genes have to be implicated in the development of HSCR. As the identification and the subsequent validation of novel gene variants to be disease-causing or not, still remains a major challenge, we established and applied a complementary study pipeline. This enabled us to identify four novel candidate genes in two HSCR patients and to validate their potential disease relevance. Our approach represents a suitable way to dissect the complex genetic architecture underlying HSCR.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.</p

miR-16 and miR-103 impact 5-HT receptor signalling and correlate with symptom profile in irritable bowel syndrome

Irritable bowel syndrome (IBS) is a gut-brain disorder involving alterations in intestinal sensitivity and motility. Serotonin 5-HT4 receptors are promising candidates in IBS pathophysiology since they regulate gut motor function and stool consistency, and targeted 5-HT4R selective drug intervention has been proven beneficial in subgroups of patients. We identified a single nucleotide polymorphism (SNP) (rs201253747) c.∗61 T > C within the 5-HT4 receptor gene HTR4 to be predominantly present in diarrhoea-IBS patients (IBS-D). It affects a binding site for the miR-16 family and miR-103/miR-107 within the isoforms HTR4b/i and putatively impairs HTR4 expression. Subsequent miRNA-profiling revealed downregulation of miR-16 and miR-103 in the jejunum of IBS-D patients correlating with symptoms. In vitro assays confirmed expression regulation via three 3′UTR binding sites. The novel isoform HTR4b-2 lacking two of the three miRNA binding sites escapes miR-16/103/107 regulation in SNP carriers. We provide the first evidence that HTR4 expression is fine-tuned by miRNAs, and that this regulation is impaired either by the SNP c.∗61 T > C or by diminished levels of miR-16 and miR-103 suggesting that HTR4 might be involved in the development of IBS-D