Mutations in RAD21 Disrupt Regulation of APOB in Patients With Chronic Intestinal Pseudo-Obstruction
| Autor: | Dustin Dowless, Mauro D'Amato, Ludmila Francescatto, Greger Lindberg, Lina Cordeddu, Kivanc Cefle, Tommaso Pippucci, Giovanni Romeo, Zeynel Mungan, Sukru Ozturk, Vincenzo Stanghellini, Claudio Graziano, Roberto De Giorgio, Sukru Palanduz, Asuman Gedikbasi, Michael J. Bamshad, Nicholas Katsanis, Giovanni Barbara, Francesca Bianco, Umberto Volta, Rosanna Cogliandro, Elena Bonora, Tayfun Ozcelik, Giacomo Caio, Alessandra Gori, Marco Seri |
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| Přispěvatelé: | Bonora, E, Bianco, F, Cordeddu, L, Bamshad, M, Francescatto, L, Dowless, D, Stanghellini, V, Cogliandro, Rf, Lindberg, G, Mungan, Z, Cefle, K, Ozcelik, T, Palanduz, S, Ozturk, S, Gedikbasi, A, Gori, A, Pippucci, T, Graziano, C, Volta, U, Caio, G, Barbara, G, D'Amato, M, Seri, M, Katsanis, N, Romeo, G, De Giorgio, R. |
| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
Male
Apolipoprotein B Morpholino Genomic DNA Cell Cycle Proteins Gene mutation Enteric Nervous System Chromosome 11 Intestinal Motility Gene expression Haplotype Child Nuclear Protein Mutation Messenger RNA Gastroenterology Complementary DNA Immunohistochemistry Reverse transcription polymerase chain reaction Zebrafish Protein Human Genotype Sporadic and Familial Chronic Intestinal Pseudoobstruction Article RAD21 protein Humans RNA Messenger Segregation analysis Aged Zebra fish Animal Intestinal Pseudo-Obstruction Gene frequency Sporadic and Familial Chronic Intestinal Pseudo-obstruction Human cell Case-Control Studies Gene Knockdown Technique Epistasis Protein expression Animal Model Gastrointestinal Motility Unclassified drug Sporadic and Familial Chronic Intestinal Pseudoobstruction Intestinal Motility Animal Model Genetic Analysis Transcription factor RUNX1 medicine.disease_cause Homozygosity Western blotting HEK293 Cell Cell Cycle Protein Exome Intestine pseudoobstruction Regulatory mechanism Middle aged Zebrafish Genetic transfection Priority journal Allele Lamina propria biology Promoter region Nuclear Proteins Genetic Analysis Middle Aged Nerve cell DNA-Binding Proteins Phenotype Real-time polymerase chain reaction Embryo Gene Knockdown Techniques HEK293 cell line Phosphoprotein Apolipoprotein B-100 Core Binding Factor Alpha 2 Subunit Female Case-Control Studie Adult Down regulation NO Young Adult Lymphoblastoid cell Next generation sequencing medicine Animals Immunoprecipitation Chromosome 8 Hepatology Protein Gene Expression Profiling In vitro study Sequence Analysis DNA Zebrafish Proteins Phosphoproteins Nonhuman biology.organism_classification Molecular biology Single nucleotide polymorphism HEK293 Cells Binding affinity Young adult Preschool child Chronic Disease Cancer research biology.protein Genetic Analysi Genetic variability Controlled study |
| Zdroj: | Gastroenterology |
| Popis: | BACKGROUND & AIMS: Chronic intestinal pseudo-obstruction (CIPO) is characterized by severe intestinal dysmotility that mimics a mechanical subocclusion with no evidence of gut obstruction. We searched for genetic variants associated with CIPO to increase our understanding of its pathogenesis and to identify potential biomarkers. METHODS: We performed whole-exome sequencing of genomic DNA from patients with familial CIPO syndrome. Blood and lymphoblastoid cells were collected from patients and controls (individuals without CIPO); levels of messenger RNA (mRNA) and proteins were analyzed by quantitative reverse-transcription polymerase chain reaction, immunoblot, and mobility shift assays. Complementary DNAs were transfected into HEK293 cells. Expression of rad21 was suppressed in zebrafish embryos using a splice-blocking morpholino (rad21a). Gut tissues were collected and analyzed. RESULTS: We identified a homozygous mutation (p.622, encodes Ala>Thr) in RAD21 in patients from a consanguineous family with CIPO. Expression of RUNX1, a target of RAD21, was reduced in cells from patients with CIPO compared with controls. In zebrafish, suppression of rad21a reduced expression of runx1; this phenotype was corrected by injection of human RAD21 mRNA, but not with the mRNA from the mutated p.622 allele. rad21a Morpholino zebrafish had delayed intestinal transit and greatly reduced numbers of enteric neurons, similar to patients with CIPO. This defect was greater in zebrafish with suppressed expression of ret and rad21, indicating their interaction in the regulation of gut neurogenesis. The promoter region of APOB bound RAD21 but not RAD21 p.622 Ala>Thr; expression of wild-type RAD21 in HEK293 cells repressed expression of APOB, compared with control vector. The gut-specific isoform of APOB (APOB48) is overexpressed in sera from patients with CIPO who carry the RAD21 mutation. APOB48 also is overexpressed in sporadic CIPO in sera and gut biopsy specimens. CONCLUSIONS: Some patients with CIPO carry mutations in RAD21 that disrupt the ability of its product to regulate genes such as RUNX1 and APOB. Reduced expression of rad21 in zebrafish, and dysregulation of these target genes, disrupts intestinal transit and the development of enteric neurons. Background Aims Chronic intestinal pseudo-obstruction (CIPO) is characterized by severe intestinal dysmotility that mimics a mechanical subocclusion with no evidence of gut obstruction. We searched for genetic variants associated with CIPO to increase our understanding of its pathogenesis and to identify potential biomarkers. Methods We performed whole-exome sequencing of genomic DNA from patients with familial CIPO syndrome. Blood and lymphoblastoid cells were collected from patients and controls (individuals without CIPO); levels of messenger RNA (mRNA) and proteins were analyzed by quantitative reverse-transcription polymerase chain reaction, immunoblot, and mobility shift assays. Complementary DNAs were transfected into HEK293 cells. Expression of rad21 was suppressed in zebrafish embryos using a splice-blocking morpholino (rad21a). Gut tissues were collected and analyzed. Results We identified a homozygous mutation (p.622, encodes Ala>Thr) in RAD21 in patients from a consanguineous family with CIPO. Expression of RUNX1, a target of RAD21, was reduced in cells from patients with CIPO compared with controls. In zebrafish, suppression of rad21a reduced expression of runx1; this phenotype was corrected by injection of human RAD21 mRNA, but not with the mRNA from the mutated p.622 allele. rad21a Morpholino zebrafish had delayed intestinal transit and greatly reduced numbers of enteric neurons, similar to patients with CIPO. This defect was greater in zebrafish with suppressed expression of ret and rad21, indicating their interaction in the regulation of gut neurogenesis. The promoter region of APOB bound RAD21 but not RAD21 p.622 Ala>Thr; expression of wild-type RAD21 in HEK293 cells repressed expression of APOB, compared with control vector. The gut-specific isoform of APOB (APOB48) is overexpressed in sera from patients with CIPO who carry the RAD21 mutation. APOB48 also is overexpressed in sporadic CIPO in sera and gut biopsy specimens. Conclusions Some patients with CIPO carry mutations in RAD21 that disrupt the ability of its product to regulate genes such as RUNX1 and APOB. Reduced expression of rad21 in zebrafish, and dysregulation of these target genes, disrupts intestinal transit and the development of enteric neurons. |
| Databáze: | OpenAIRE |
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