The long Filamin-A isoform is required for intestinal development and motility: implications for chronic intestinal pseudo-obstruction.

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.

A combinatorial panel for flow cytometry-based isolation of enteric nervous system cells from human intestine.

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.

Size matters: Large copy number losses in Hirschsprung disease patients reveal genes involved in enteric nervous system development.

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.

Multi-disciplinary Insights from the First European Forum on Visceral Myopathy 2022 Meeting.

Visceral myopathy is a rare, life-threatening disease linked to identified genetic mutations in 60% of cases. Mostly due to the dearth of knowledge regarding its pathogenesis, effective treatments are lacking. The disease is most commonly diagnosed in children with recurrent or persistent disabling episodes of functional intestinal obstruction, which can be life threatening, often requiring long-term parenteral or specialized enteral nutritional support. Although these interventions are undisputedly life-saving as they allow affected individuals to avoid malnutrition and related complications, they also seriously compromise their quality of life and can carry the risk of sepsis and thrombosis. Animal models for visceral myopathy, which could be crucial for advancing the scientific knowledge of this condition, are scarce. Clearly, a collaborative network is needed to develop research plans to clarify genotype-phenotype correlations and unravel molecular mechanisms to provide targeted therapeutic strategies. This paper represents a summary report of the first 'European Forum on Visceral Myopathy'. This forum was attended by an international interdisciplinary working group that met to better understand visceral myopathy and foster interaction among scientists actively involved in the field and clinicians who specialize in care of people with visceral myopathy.

The Somatic Mutation Paradigm in Congenital Malformations: Hirschsprung Disease as a Model.

Patients with Hirschsprung disease (HSCR) do not always receive a genetic diagnosis after routine screening in clinical practice. One of the reasons for this could be that the causal mutation is not present in the cell types that are usually tested-whole blood, dermal fibroblasts or saliva-but is only in the affected tissue. Such mutations are called somatic, and can occur in a given cell at any stage of development after conception. They will then be present in all subsequent daughter cells. Here, we investigated the presence of somatic mutations in HSCR patients. For this, whole-exome sequencing and copy number analysis were performed in DNA isolated from purified enteric neural crest cells (ENCCs) and blood or fibroblasts of the same patient. Variants identified were subsequently validated by Sanger sequencing. Several somatic variants were identified in all patients, but causative mutations for HSCR were not specifically identified in the ENCCs of these patients. Larger copy number variants were also not found to be specific to ENCCs. Therefore, we believe that somatic mutations are unlikely to be identified, if causative for HSCR. Here, we postulate various modes of development following the occurrence of a somatic mutation, to describe the challenges in detecting such mutations, and hypothesize how somatic mutations may contribute to 'missing heritability' in developmental defects.

Goldberg-Shprintzen syndrome is determined by the absence, or reduced expression levels, of KIFBP.

Goldberg-Shprintzen syndrome (GOSHS) is caused by loss of function variants in the kinesin binding protein gene (KIFBP). However, the phenotypic range of this syndrome is wide, indicating that other factors may play a role. To date, 37 patients with GOSHS have been reported. Here, we document nine new patients with variants in KIFBP: seven with nonsense variants and two with missense variants. To our knowledge, this is the first time that missense variants have been reported in GOSHS. We functionally investigated the effect of the variants identified, in an attempt to find a genotype-phenotype correlation. We also determined whether common Hirschsprung disease (HSCR)-associated single nucleotide polymorphisms (SNPs), could explain the presence of HSCR in GOSHS. Our results showed that the missense variants led to reduced expression of KIFBP, while the truncating variants resulted in lack of protein. However, no correlation was found between the severity of GOSHS and the location of the variants. We were also unable to find a correlation between common HSCR-associated SNPs, and HSCR development in GOSHS. In conclusion, we show that reduced, as well as lack of KIFBP expression can lead to GOSHS, and our results suggest that a threshold expression of KIFBP may modulate phenotypic variability of the disease.

Loss-of-Function Variants in MYLK Cause Recessive Megacystis Microcolon Intestinal Hypoperistalsis Syndrome.

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital disorder characterized by loss of smooth muscle contraction in the bladder and intestine. To date, three genes are known to be involved in MMIHS pathogenesis: ACTG2, MYH11, and LMOD1. However, for approximately 10% of affected individuals, the genetic cause of the disease is unknown, suggesting that other loci are most likely involved. Here, we report on three MMIHS-affected subjects from two consanguineous families with no variants in the known MMIHS-associated genes. By performing homozygosity mapping and whole-exome sequencing, we found homozygous variants in myosin light chain kinase (MYLK) in both families. We identified a 7 bp duplication (c.3838_3844dupGAAAGCG [p.Glu1282_Glyfs∗51]) in one family and a putative splice-site variant (c.3985+5C>A) in the other. Expression studies and splicing assays indicated that both variants affect normal MYLK expression. Because MYLK encodes an important kinase required for myosin activation and subsequent interaction with actin filaments, it is likely that in its absence, contraction of smooth muscle cells is impaired. The existence of a conditional-Mylk-knockout mouse model with severe gut dysmotility and abnormal function of the bladder supports the involvement of this gene in MMIHS pathogenesis. In aggregate, our findings implicate MYLK as a gene involved in the recessive form of MMIHS, confirming that this disease of the visceral organs is heterogeneous with a myopathic origin.

Neuronal Development and Onset of Electrical Activity in the Human Enteric Nervous System.

BACKGROUND & AIMS: The enteric nervous system (ENS) is the largest branch of the peripheral nervous system, comprising complex networks of neurons and glia, which are present throughout the gastrointestinal tract. Although development of a fully functional ENS is required for gastrointestinal motility, little is known about the ontogeny of ENS function in humans. We studied the development of neuronal subtypes and the emergence of evoked electrical activity in the developing human ENS. METHODS: Human fetal gut samples (obtained via the MRC-Wellcome Trust Human Developmental Biology Resource-UK) were characterized by immunohistochemistry, calcium imaging, RNA sequencing, and quantitative real-time polymerase chain reaction analyses. RESULTS: Human fetal colon samples have dense neuronal networks at the level of the myenteric plexus by embryonic week (EW) 12, with expression of excitatory neurotransmitter and synaptic markers. By contrast, markers of inhibitory neurotransmitters were not observed until EW14. Electrical train stimulation of internodal strands did not evoke activity in the ENS of EW12 or EW14 tissues. However, compound calcium activation was observed at EW16, which was blocked by the addition of 1 µmol/L tetrodotoxin. Expression analyses showed that this activity was coincident with increases in expression of genes encoding proteins involved in neurotransmission and action potential generation. CONCLUSIONS: In analyses of human fetal intestinal samples, we followed development of neuronal diversity, electrical excitability, and network formation in the ENS. These processes are required to establish the functional enteric circuitry. Further studies could increase our understanding of the pathogenesis of a range of congenital enteric neuropathies.

Loss of LMOD1 impairs smooth muscle cytocontractility and causes megacystis microcolon intestinal hypoperistalsis syndrome in humans and mice.

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital visceral myopathy characterized by severe dilation of the urinary bladder and defective intestinal motility. The genetic basis of MMIHS has been ascribed to spontaneous and autosomal dominant mutations in actin gamma 2 (ACTG2), a smooth muscle contractile gene. However, evidence suggesting a recessive origin of the disease also exists. Using combined homozygosity mapping and whole exome sequencing, a genetically isolated family was found to carry a premature termination codon in Leiomodin1 (LMOD1), a gene preferentially expressed in vascular and visceral smooth muscle cells. Parents heterozygous for the mutation exhibited no abnormalities, but a child homozygous for the premature termination codon displayed symptoms consistent with MMIHS. We used CRISPR-Cas9 (CRISPR-associated protein) genome editing of Lmod1 to generate a similar premature termination codon. Mice homozygous for the mutation showed loss of LMOD1 protein and pathology consistent with MMIHS, including late gestation expansion of the bladder, hydronephrosis, and rapid demise after parturition. Loss of LMOD1 resulted in a reduction of filamentous actin, elongated cytoskeletal dense bodies, and impaired intestinal smooth muscle contractility. These results define LMOD1 as a disease gene for MMIHS and suggest its role in establishing normal smooth muscle cytoskeletal-contractile coupling.

Identification of Variants in RET and IHH Pathway Members in a Large Family With History of Hirschsprung Disease.

BACKGROUND & AIMS: Hirschsprung disease (HSCR) is an inherited congenital disorder characterized by absence of enteric ganglia in the distal part of the gut. Variants in ret proto-oncogene (RET) have been associated with up to 50% of familial and 35% of sporadic cases. We searched for variants that affect disease risk in a large, multigenerational family with history of HSCR in a linkage region previously associated with the disease (4q31.3-q32.3) and exome wide. METHODS: We performed exome sequencing analyses of a family in the Netherlands with 5 members diagnosed with HSCR and 2 members diagnosed with functional constipation. We initially focused on variants in genes located in 4q31.3-q32.3; however, we also performed an exome-wide analysis in which known HSCR or HSCR-associated gene variants predicted to be deleterious were prioritized for further analysis. Candidate genes were expressed in HEK293, COS-7, and Neuro-2a cells and analyzed by luciferase and immunoblot assays. Morpholinos were designed to target exons of candidate genes and injected into 1-cell stage zebrafish embryos. Embryos were allowed to develop and stained for enteric neurons. RESULTS: Within the linkage region, we identified 1 putative splice variant in the lipopolysaccharide responsive beige-like anchor protein gene (LRBA). Functional assays could not confirm its predicted effect on messenger RNA splicing or on expression of the mab-21 like 2 gene (MAB21L2), which is embedded in LRBA. Zebrafish that developed following injection of the lrba morpholino had a shortened body axis and subtle gut morphological defects, but no significant reduction in number of enteric neurons compared with controls. Outside the linkage region, members of 1 branch of the family carried a previously unidentified RET variant or an in-frame deletion in the glial cell line derived neurotrophic factor gene (GDNF), which encodes a ligand of RET. This deletion was located 6 base pairs before the last codon. We also found variants in the Indian hedgehog gene (IHH) and its mediator, the transcription factor GLI family zinc finger 3 (GLI3). When expressed in cells, the RET-P399L variant disrupted protein glycosylation and had altered phosphorylation following activation by GDNF. The deletion in GDNF prevented secretion of its gene product, reducing RET activation, and the IHH-Q51K variant reduced expression of the transcription factor GLI1. Injection of morpholinos that target ihh reduced the number of enteric neurons to 13% ± 1.4% of control zebrafish. CONCLUSIONS: In a study of a large family with history of HSCR, we identified variants in LRBA, RET, the gene encoding the RET ligand (GDNF), IHH, and a gene encoding a mediator of IHH signaling (GLI3). These variants altered functions of the gene products when expressed in cells and knockout of ihh reduced the number of enteric neurons in the zebrafish gut.

ACTG2 variants impair actin polymerization in sporadic Megacystis Microcolon Intestinal Hypoperistalsis Syndrome.

Megacystis Microcolon Intestinal Hypoperistalsis Syndrome (MMIHS) is a rare congenital disorder, in which heterozygous missense variants in the Enteric Smooth Muscle actin γ-2 (ACTG2) gene have been recently identified. To investigate the mechanism by which ACTG2 variants lead to MMIHS, we screened a cohort of eleven MMIHS patients, eight sporadic and three familial cases, and performed immunohistochemistry, molecular modeling and molecular dynamics (MD) simulations, and in vitro assays. In all sporadic cases, a heterozygous missense variant in ACTG2 was identified. ACTG2 expression was detected in all intestinal layers where smooth muscle cells are present in different stages of human development. No histopathological abnormalities were found in the patients. Using molecular modeling and MD simulations, we predicted that ACTG2 variants lead to significant changes to the protein function. This was confirmed by in vitro studies, which showed that the identified variants not only impair ACTG2 polymerization, but also contribute to reduced cell contractility. Taken together, our results confirm the involvement of ACTG2 in sporadic MMIHS, and bring new insights to MMIHS pathogenesis.

Genetics of enteric neuropathies.

Abnormal development or disturbed functioning of the enteric nervous system (ENS), the intrinsic innervation of the gastrointestinal tract, is associated with the development of neuropathic gastrointestinal motility disorders. Here, we review the underlying molecular basis of these disorders and hypothesize that many of them have a common defective biological mechanism. Genetic burden and environmental components affecting this common mechanism are ultimately responsible for disease severity and symptom heterogeneity. We believe that they act together as the fulcrum in a seesaw balanced with harmful and protective factors, and are responsible for a continuum of symptoms ranging from neuronal hyperplasia to absence of neurons.

Congenital Short Bowel Syndrome: from clinical and genetic diagnosis to the molecular mechanisms involved in intestinal elongation.

Congenital Short Bowel Syndrome (CSBS) is a rare gastrointestinal disorder in which the mean length of the small intestine is substantially reduced when compared to its normal counterpart. Families with several affected members have been described and CSBS has been suggested to have a genetic basis. Recently, our group found mutations in CLMP as the cause of the recessive form of CSBS, and mutations in FLNA as the cause of the X-linked form of the disease. These findings have improved the quality of genetic counselling for CSBS patients and made prenatal diagnostics possible. Moreover, they provided a reliable starting point to further investigate the pathogenesis of CSBS, and to better understand the development of the small intestine. In this review, we present our current knowledge on CSBS and discuss hypotheses on how the recent genetic findings can help understand the cause of CSBS.

Contribution of rare and common variants determine complex diseases-Hirschsprung disease as a model.

Finding genes for complex diseases has been the goal of many genetic studies. Most of these studies have been successful by searching for genes and mutations in rare familial cases, by screening candidate genes and by performing genome wide association studies. However, only a small fraction of the total genetic risk for these complex genetic diseases can be explained by the identified mutations and associated genetic loci. In this review we focus on Hirschsprung disease (HSCR) as an example of a complex genetic disorder. We describe the genes identified in this congenital malformation and postulate that both common 'low penetrant' variants in combination with rare or private 'high penetrant' variants determine the risk on HSCR, and likely, on other complex diseases. We also discuss how new technological advances can be used to gain further insights in the genetic background of complex diseases. Finally, we outline a few steps to develop functional assays in order to determine the involvement of these variants in disease development.

Promoter hypermethylation of neural-related genes is compatible with stemness in solid cancers.

BACKGROUND: DNA hypermethylation is an epigenetic feature that modulates gene expression, and its deregulation is observed in cancer. Previously, we identified a neural-related DNA hypermethylation fingerprint in colon cancer, where most of the top hypermethylated and downregulated genes have known functions in the nervous system. To evaluate the presence of this signature and its relevance to carcinogenesis in general, we considered 16 solid cancer types available in The Cancer Genome Atlas (TCGA). RESULTS: All tested cancers showed significant enrichment for neural-related genes amongst hypermethylated genes. This signature was already present in two premalignant tissue types and could not be explained by potential confounders such as bivalency status or tumor purity. Further characterization of the neural-related DNA hypermethylation signature in colon cancer showed particular enrichment for genes that are overexpressed during neural differentiation. Lastly, an analysis of upstream regulators identified RE1-Silencing Transcription factor (REST) as a potential mediator of this DNA methylation signature. CONCLUSION: Our study confirms the presence of a neural-related DNA hypermethylation fingerprint in various cancers, of genes linked to neural differentiation, and points to REST as a possible regulator of this mechanism. We propose that this fingerprint indicates an involvement of DNA hypermethylation in the preservation of neural stemness in cancer cells.

Evaluating the molecular and genetic mechanisms underlying gut motility disorders.

INTRODUCTION: Gastrointestinal (GI) motility disorders comprise a wide range of different diseases affecting the structural or functional integrity of the GI neuromusculature. Their clinical presentation and burden of disease depends on the predominant location and extent of gut involvement as well as the component of the gut neuromusculature affected. AREAS COVERED: A comprehensive literature review was conducted using the PubMed and Medline databases to identify articles related to GI motility and functional disorders, published between 2016 and 2023. In this article, we highlight the current knowledge of molecular and genetic mechanisms underlying GI dysmotility, including disorders of gut-brain interaction, which involve both GI motor and sensory disturbance. EXPERT OPINION: Although the pathophysiology and molecular mechanisms underlying many such disorders remain unclear, recent advances in the assessment of intestinal tissue samples, genetic testing with the application of 'omics' technologies and the use of animal models will provide better insights into disease pathogenesis as well as opportunities to improve therapy.

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing.

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric neuromuscular disorders, highlighting the importance to understand the molecular mechanisms underlying GI development and dysfunction. In this study, we present a method for gut isolation from zebrafish larvae at 5 days post fertilization to obtain live, viable cells which can be used for single-cell RNA sequencing (scRNA-seq) analysis. This protocol is based on the manual dissection of the zebrafish intestine, followed by enzymatic dissociation with papain. Subsequently, cells are submitted to fluorescence-activated cell sorting, and viable cells are collected for scRNA-seq. With this method, we were able to successfully identify different intestinal cell types, including epithelial, stromal, blood, muscle, and immune cells, as well as enteric neurons and glia. Therefore, we consider it to be a valuable resource for studying the composition of the GI tract in health and disease, using the zebrafish.

Unbiased characterization of the larval zebrafish enteric nervous system at a single cell transcriptomic level.

The enteric nervous system (ENS) regulates many gastrointestinal functions including peristalsis, immune regulation and uptake of nutrients. Defects in the ENS can lead to severe enteric neuropathies such as Hirschsprung disease (HSCR). Zebrafish have proven to be fruitful in the identification of genes involved in ENS development and HSCR pathogenesis. However, composition and specification of enteric neurons and glial subtypes at larval stages, remains mainly unexplored. Here, we performed single cell RNA sequencing of zebrafish ENS at 5 days post-fertilization. We identified vagal neural crest progenitors, Schwann cell precursors, and four clusters of differentiated neurons. In addition, a previously unrecognized elavl3+/phox2bb-population of neurons and cx43+/phox2bb-enteric glia was found. Pseudotime analysis supported binary neurogenic branching of ENS differentiation, driven by a notch-responsive state. Taken together, we provide new insights on ENS development and specification, proving that the zebrafish is a valuable model for the study of congenital enteric neuropathies.