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      Mutations in RAD21 disrupt regulation of apob in patients with chronic intestinal pseudo-obstruction

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      Author(s)
      Bonora, E.
      Bianco, F.
      Cordeddu, L.
      Bamshad, M.
      Francescatto, L.
      Dowless, D.
      Stanghellini, V.
      Cogliandro, R. F.
      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.
      Date
      2015
      Source Title
      Gastroenterology
      Print ISSN
      0016-5085
      Publisher
      W.B. Saunders
      Volume
      148
      Issue
      4
      Pages
      771 - 782
      Language
      English
      Type
      Article
      Item Usage Stats
      229
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      280
      downloads
      Abstract
      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.
      Keywords
      Animal Model
      Genetic Analysis
      Intestinal Motility
      Sporadic and Familial Chronic Intestinal Pseudo-obstruction
      Apolipoprotein B
      Complementary DNA
      Genomic DNA
      Messenger RNA
      Protein
      RAD21 protein
      Transcription factor RUNX1
      Unclassified drug
      Adult
      Aged
      Allele
      Article
      Binding affinity
      Child
      Chromosome 11
      Chromosome 8
      Controlled study
      Down regulation
      Embryo
      Epistasis
      Exome
      Female
      Gene expression
      Gene frequency
      Gene mutation
      Genetic transfection
      Genetic variability
      Genotype
      Haplotype
      HEK293 cell line
      Homozygosity
      Human
      Human cell
      Immunohistochemistry
      Immunoprecipitation
      In vitro study
      Intestine pseudoobstruction
      Lamina propria
      Lymphoblastoid cell
      Male
      Middle aged
      Nerve cell
      Next generation sequencing
      Nonhuman
      Phenotype
      Preschool child
      Priority journal
      Promoter region
      Protein expression
      Regulatory mechanism
      Reverse transcription polymerase chain reaction
      Segregation analysis
      Single nucleotide polymorphism
      Western blotting
      Young adult
      Zebra fish
      Permalink
      http://hdl.handle.net/11693/23636
      Published Version (Please cite this version)
      http://dx.doi.org/10.1053/j.gastro.2014.12.034
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