Mutations in RAD21 Disrupt Regulation of APOB in Patients With Chronic Intestinal Pseudo-Obstruction

Abstract

Background & Aims Chronic intestinal pseudo-obstruction (CIPO) is characterized by severe intestinal dysmotility that mimicks a mechanical sub-occlusion with no evidence of gut obstruction. We searched for genetic variants associated with CIPO to increase our understanding of its pathogenesis and indentify 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 mRNA and proteins were analyzed by quantitative reverse transcription PCR, immunoblot, and mobility shift assays. cDNAs were transfected into HEK293 cells. Expression of rad21 was suppressed in zebrafish embryos using a splice-blocking morpholino (rad21a MO). 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 MO 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 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 is also overexpressed in sporadic CIPO in sera and gut biopsies. 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 development of enteric neurons.

A Novel Mutation in Nucleoporin 35 Causes Murine Degenerative Colonic Smooth Muscle Myopathy

“…Moreover, unlike patients with the congenital neuropathy, Hirschsprung disease, 42,43 CIPO patients with degenerative myopathy are variable in age. 3 The colonic myopathy in Nup35-mutant mice was not apparent at birth and so is consistent with the degenerative myopathy seen in a subpopulation of CIPO patients.…”

“…3 Mutations linked to CIPO in humans are often associated with neuropathy rather than myopathy (eg, SOX10 and RAD21), 3,12 and so are unlikely to be related to the Nup35-mutant phenotype. The mechanism by which NUP35 deficiency triggers smooth muscle myopathy thus remains unclear.…”

“…3,7e12 Nonetheless, the genetic cause of a large portion of heritable CIPO cases remains unknown. 3 Mouse models have previously been used to investigate the role of genetic pathways in CIPO pathology. 13 Although data from mouse models have implicated the disruption of a number of genetic pathways in intestinal neuropathy, there is a paucity of models that mimic clinical intestinal smooth muscle myopathy.…”

Diagnosis of Chronic Intestinal Pseudo‐obstruction and Megacystis by Sequencing the ACTG2 Gene

“…A homozygous pathogenic variant in the RAD21 gene was reported in one consanguineous family. 18 4/28 of our cohort and 15/27 in the report by Wangler et al 4 were found to harbor an ACTG2 pathogenic variant. The difference between these two randomly collected cohorts remains unexplained, but may reflect inaccurate clinical diagnoses.…”

Enteric neuropathies: Yesterday, Today and Tomorrow

“…Genome-wide linkage analysis and homozygosity mapping approach identifi ed a maximum multipoint lod score of 5.01 in a critical interval of about 13 Mb between D8S1830 and D8S1799 on the chromosome 8q23-q24 (Deglincerti et al 2007 ). Using whole exome sequencing analysis we have shown a novel mutation in the RAD21 gene, cosegregating with the disease phenotype in this family (Bonora et al 2015 ). Therefore, it is clear that even severe forms of ENs show a high degree of genetic heterogeneity, hindering the identifi cation of the molecular causes and of the deranged molecular pathways shared by the different affected individuals.…”