Mutations in RAD21 disrupt regulation of apob in patients with chronic intestinal pseudo-obstruction
Author
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
2015Source Title
Gastroenterology
Print ISSN
0016-5085
Publisher
W.B. Saunders
Volume
148
Issue
4
Pages
771 - 782
Language
English
Type
ArticleItem Usage Stats
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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 ModelGenetic 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