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.

Efficient isolation of neurons and glia from the human enteric nervous system (ENS) is challenging because of their rare and fragile nature. Here, we describe a staining panel to enrich ENS cells from the human intestine by fluorescence‐activated cell sorting (FACS). We find that CD56/CD90/CD24 co‐expression labels ENS cells with higher specificity and resolution than previous methods. Surprisingly, neuronal (CD24, TUBB3) and glial (SOX10) selective markers appear co‐expressed by all ENS cells. We demonstrate that this contradictory staining pattern is mainly driven by neuronal fragments, either free or attached to glial cells, which are the most abundant cell types. Live neurons can be enriched by the highest CD24 and CD90 levels. By applying our protocol to isolate ENS cells for single‐cell RNA sequencing, we show that these cells can be obtained with high quality, enabling interrogation of the human ENS transcriptome. Taken together, we present a selective FACS protocol that allows enrichment and discrimination of human ENS cells, opening up new avenues to study this complex system in health and disease.

Filamin A (FLNA) is a cytoplasmic actin binding protein, recently shown to be expressed as a long and short isoform. Mutations in FLNA are associated with a wide spectrum of disorders, including an X-linked form of chronic intestinal pseudo-obstruction (CIPO). However, the role of FLNA in intestinal development and function is largely unknown. In this study, we show that FLNA is expressed in the muscle layer of the small intestine from early human fetal stages. Expression of FLNA variants associated with CIPO, blocked expression of the long flna isoform and led to an overall reduction of RNA and protein levels. As a consequence, contractility of human intestinal smooth muscle cells was affected. Lastly, our transgenic zebrafish line showed that the flna long isoform is required for intestinal elongation and peristalsis. Histological analysis revealed structural and architectural changes in the intestinal smooth muscle of homozygous fish, likely triggered by the abnormal expression of intestinal smooth muscle markers. No defect in the localization or numbers of enteric neurons was observed. Taken together, our study demonstrates that the long FLNA isoform contributes to intestinal development and function. Since loss of the long FLNA isoform does not seem to affect the enteric nervous system, it likely results in a myopathic form of CIPO, bringing new insights to disease pathogenesis.

Isolation of neurons and glia from the enteric nervous system (ENS) enables ex vivo studies, including the analysis of genomic and transcriptomic profiles. While we previously reported a fluorescence-activated cell sorting (FACS)-based isolation protocol for human ENS cells, no equivalent exists for mice. As directly applying the human protocol to mouse tissue resulted in low recovery of live ENS cells, we optimized tissue dissociation using mouse colons. A 30 min Liberase-based digestion showed optimal recovery of viable ENS cells, with CD56 and CD24 emerging as the most reliable markers to select and subdivide these cells. ENS’ identity was further validated by FACS, using neuronal (TUBB3) and glial (SOX10) markers and reverse transcriptase quantitative PCR on sorted fractions. Overall, the mouse ENS expression profile significantly overlapped with the human one, showing that current dissociation protocols yield a mixed population of enteric neurons and glia. Nonetheless, using the imaging flow cytometer BD S8 FACS Discover and ELAVL4 as a neuronal soma-associated marker, we observed enrichment of neurons in a CD56/CD24^TIP population. In conclusion, we present here a protocol for high-purity FACS-based isolation of viable mouse ENS cells, suitable for downstream applications.

Ileocolic intussusception is the invagination of ileum into the colon. In a subset of patients, the disease is caused by mesenteric lymphadenopathy in response to (viral) infection. We present a case of an ileocolic intussusception necessitating surgery in a 7-month-old immunocompetent infant with concurrent primary wild-type varicella-zoster virus (VZV) infection, in whom chickenpox rash developed 2 days after surgery. Detailed in situ analyses of resected intestine for specific cell type markers and VZV RNA demonstrated VZV-infected lymphocytes and neurons in the gut wall and in ganglion cells of the myenteric plexus.

Background: Hirschsprung disease (HSCR) is characterized by absence of ganglia in the intestine. Approximately 18% of patients have additional anatomical malformations or neurological symptoms (HSCR-AAM). HSCR is a complex genetic disease in which the loss of enteric ganglia stems from a combination of genetic alterations: rare coding variants, predisposing haplotypes and Copy Number Variation (CNV). Pinpointing the responsible culprits within a large CNV is challenging as often many genes are affected. We investigated if we could find deleterious CNVs and if we could identify the genes responsible for the aganglionosis. Results: Deleterious CNVs were detected in three groups of patients: HSCR-AAM, HSCR patients with a confirmed causal genetic variant and HSCR-isolated patients without a known causal variant and 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 HSCR-AAM patients had larger copy number losses. Gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics were used to determine plausible candidate genes in Copy Number 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 in vivo . Conclusion: Rare large Copy Number losses - often de novo - contribute to the disease in HSCR-AAM patients specifically. We proved the involvement of five genes in enteric nervous system development and Hirschsprung disease.