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.

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.

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.

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.

NPHP4 variants are associated with pleiotropic heart malformations.

RATIONALE: Congenital heart malformations are a major cause of morbidity and mortality, especially in young children. Failure to establish normal left-right (L-R) asymmetry often results in cardiovascular malformations and other laterality defects of visceral organs. OBJECTIVE: To identify genetic mutations causing cardiac laterality defects. METHODS AND RESULTS: We performed a genome-wide linkage analysis in patients with cardiac laterality defects from a consanguineous family. The patients had combinations of defects that included dextrocardia, transposition of great arteries, double-outlet right ventricle, atrioventricular septal defects, and caval vein abnormalities. Sequencing of positional candidate genes identified mutations in NPHP4. We performed mutation analysis of NPHP4 in 146 unrelated patients with similar cardiac laterality defects. Forty-one percent of these patients also had laterality defects of the abdominal organs. We identified 8 additional missense variants that were absent or very rare in control subjects. To study the role of nphp4 in establishing L-R asymmetry, we used antisense morpholinos to knockdown nphp4 expression in zebrafish. Depletion of nphp4 disrupted L-R patterning as well as cardiac and gut laterality. Cardiac laterality defects were partially rescued by human NPHP4 mRNA, whereas mutant NPHP4 containing genetic variants found in patients failed to rescue. We show that nphp4 is involved in the formation of motile cilia in Kupffer's vesicle, which generate asymmetrical fluid flow necessary for normal L-R asymmetry. CONCLUSIONS: NPHP4 mutations are associated with cardiac laterality defects and heterotaxy. In zebrafish, nphp4 is essential for the development and function of Kupffer's vesicle cilia and is required for global L-R patterning.

Phenotypic spectrum of the SMAD3-related aneurysms-osteoarthritis syndrome.

BACKGROUND: Aneurysms-osteoarthritis syndrome (AOS) is a new autosomal dominant syndromic form of thoracic aortic aneurysms and dissections characterised by the presence of arterial aneurysms and tortuosity, mild craniofacial, skeletal and cutaneous anomalies, and early-onset osteoarthritis. AOS is caused by mutations in the SMAD3 gene. METHODS: A cohort of 393 patients with aneurysms without mutation in FBN1, TGFBR1 and TGFBR2 was screened for mutations in SMAD3. The patients originated from The Netherlands, Belgium, Switzerland and USA. The clinical phenotype in a total of 45 patients from eight different AOS families with eight different SMAD3 mutations is described. In all patients with a SMAD3 mutation, clinical records were reviewed and extensive genetic, cardiovascular and orthopaedic examinations were performed. RESULTS: Five novel SMAD3 mutations (one nonsense, two missense and two frame-shift mutations) were identified in five new AOS families. A follow-up description of the three families with a SMAD3 mutation previously described by the authors was included. In the majority of patients, early-onset joint abnormalities, including osteoarthritis and osteochondritis dissecans, were the initial symptom for which medical advice was sought. Cardiovascular abnormalities were present in almost 90% of patients, and involved mainly aortic aneurysms and dissections. Aneurysms and tortuosity were found in the aorta and other arteries throughout the body, including intracranial arteries. Of the patients who first presented with joint abnormalities, 20% died suddenly from aortic dissection. The presence of mild craniofacial abnormalities including hypertelorism and abnormal uvula may aid the recognition of this syndrome. CONCLUSION: The authors provide further insight into the phenotype of AOS with SMAD3 mutations, and present recommendations for a clinical work-up.

TFAP2B Haploinsufficiency Impacts Gastrointestinal Function and Leads to Pediatric Intestinal Pseudo-obstruction.

Background: Pediatric Intestinal Pseudo-obstruction (PIPO) is a congenital enteric disorder characterized by severe gastrointestinal (GI) dysmotility, without mechanical obstruction. Although several genes have been described to cause this disease, most patients do not receive a genetic diagnosis. Here, we aim to identify the genetic cause of PIPO in a patient diagnosed with severe intestinal dysmotility shortly after birth. Methods: Whole exome sequencing (WES) was performed in the patient and unaffected parents, in a diagnostic setting. After identification of the potential disease-causing variant, its functional consequences were determined in vitro and in vivo. For this, expression constructs with and without the causing variant, were overexpressed in HEK293 cells. To investigate the role of the candidate gene in GI development and function, a zebrafish model was generated where its expression was disrupted using CRISPR/Cas9 editing. Results: WES analysis identified a de novo heterozygous deletion in TFAP2B (NM_003221.4:c.602-5_606delTCTAGTTCCA), classified as a variant of unknown significance. In vitro studies showed that this deletion affects RNA splicing and results in loss of exon 4, leading to the appearance of a premature stop codon and absence of TFAP2B protein. Disruption of tfap2b in zebrafish led to decreased enteric neuronal numbers and delayed transit time. However, no defects in neuronal differentiation were detected. tfap2b crispants also showed decreased levels of ednrbb mRNA, a downstream target of tfap2b. Conclusion: We showed that TFAP2B haploinsufficiency leads to reduced neuronal numbers and GI dysmotility, suggesting for the first time, that this gene is involved in PIPO pathogenesis.

Intrinsic Cellular Susceptibility to Barrett's Esophagus in Adults Born with Esophageal Atresia.

The prevalence of Barrett's esophagus (BE) in adults born with esophageal atresia (EA) is four times higher than in the general population and presents at a younger age (34 vs. 60 years). This is (partly) a consequence of chronic gastroesophageal reflux. Given the overlap between genes and pathways involved in foregut and BE development, we hypothesized that EA patients have an intrinsic predisposition to develop BE. Transcriptomes of Esophageal biopsies of EA patients with BE (n = 19, EA/BE); EA patients without BE (n = 44, EA-only) and BE patients without EA (n = 10, BE-only) were compared by RNA expression profiling. Subsequently, we simulated a reflux episode by exposing fibroblasts of 3 EA patients and 3 controls to acidic conditions. Transcriptome responses were compared to the differential expressed transcripts in the biopsies. Predisposing single nucleotide polymorphisms, associated with BE, were slightly increased in EA/BE versus BE-only patients. RNA expression profiling and pathway enrichment analysis revealed differences in retinoic acid metabolism and downstream signaling pathways and inflammatory, stress response and oncological processes. There was a similar effect on retinoic acid signaling and immune response in EA patients upon acid exposure. These results indicate that epithelial tissue homeostasis in EA patients is more prone to acidic disturbances.