부산대학교 도서관

Hirschsprung disease (HSCR) is a complex genetic disease characterized by absence of ganglia in the intestine. HSCR etiology can be explained by a unique combination of genetic alterations: rare coding variants, predisposing haplotypes and Copy Number Variation (CNV). Approximately 18% of patients have additional anatomical malformations or neurological symptoms (HSCR-AAM). Pinpointing the responsible culprits within a CNV is challenging as often many genes are affected. Therefore, we selected candidate genes based on gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics. Next, we used a zebrafish model to investigate whether loss of these genes affects enteric neuron development in vivo. This study included three groups of patients, two groups without coding variants in disease associated genes: HSCR-AAM and HSCR patients without associated anomalies (HSCR-isolated). The third group consisted of all HSCR patients in which a confirmed pathogenic rare coding variant was identified. We compared these patient groups to unaffected controls. Predisposing haplotypes were determined, confirming that every HSCR subgroup had increased contributions of predisposing haplotypes, but their contribution was highest in isolated HSCR patients without RET coding variants. CNV profiling proved that specifically HSCR-AAM patients had larger Copy Number (CN) losses. Gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics were used to determine plausible candidate genes located within CN losses. Validation in zebrafish using CRISPR/Cas9 targeting confirmed the contribution of UFD1L, TBX2, SLC8A1, and MAPK8 to ENS development. In addition, we revealed epistasis between reduced Ret and Gnl1 expression and between reduced Ret and Tubb5 expression in vivo. Rare large CN losses—often de novo—contribute to HSCR in HSCR-AAM patients. We proved the involvement of six genes in enteric nervous system development and Hirschsprung disease. Author summary: Hirschsprung disease is a congenital disorder characterized by the absence of intestinal neurons in the distal part of the intestine. It is a complex genetic disorder in which multiple variations in our genome combined, result in disease. One of these variations are Copy Number Variations (CNVs): large segments of our genome that are duplicated or deleted. Patients often have Hirschsprung disease without other symptoms. However, a proportion of patients has additional associated anatomical malformations and neurological symptoms. We found that CNVs, present in patients with associated anomalies, are more often larger compared to unaffected controls or Hirschsprung patients without other symptoms. Furthermore, Copy Number (CN) losses are enriched for constrained coding regions (CCR; genes usually not impacted by genomic alterations in unaffected controls) of which the expression is higher in the developing intestinal neurons compared to the intestine. We modelled loss of these candidate genes in zebrafish by disrupting the zebrafish orthologues by genome editing. For several genes this resulted in changes in intestinal neuron development, reminiscent of HSCR observed in patients. The results presented here highlight the importance of Copy Number profiling, zebrafish validation and evaluating all CCR expressed in developing intestinal neurons during diagnostic evaluation. [ABSTRACT FROM AUTHOR]