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