Bonora Elena, Bianco Francesca, Cordeddu Lina, Bamshad Michael, Francescatto Ludmila, Dowless Dustin, Stanghellini Vincenzo, Cogliandro Rosanna F, Lindberg Greger, Mungan Zeynel, Cefle Kivanc, Ozcelik Tayfun, Palanduz Sukru, Ozturk Sukru, Gedikbasi Asuman, Gori Alessandra, Pippucci Tommaso, Graziano Claudio, Volta Umberto, Caio Giacomo, Barbara Giovanni, D'Amato Mauro, Seri Marco, Katsanis Nicholas, Romeo Giovanni, De Giorgio Roberto
Department of Medical and Surgical Sciences, University of Bologna, and St. Orsola-Malpighi Hospital, Bologna, Italy.
Karolinska Institutet, Stockholm, Sweden.
Gastroenterology. 2015 Apr;148(4):771-782.e11. doi: 10.1053/j.gastro.2014.12.034. Epub 2015 Jan 6.
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.
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.
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.
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.
慢性肠假性梗阻（CIPO）的特征是严重的肠道运动障碍，类似于机械性不完全梗阻，但无肠道梗阻的证据。我们寻找与CIPO相关的基因变异，以增进对其发病机制的理解并识别潜在的生物标志物。
我们对家族性CIPO综合征患者的基因组DNA进行了全外显子组测序。从患者和对照者（无CIPO的个体）采集血液和淋巴母细胞；通过定量逆转录聚合酶链反应、免疫印迹和迁移率变动分析来分析信使RNA（mRNA）和蛋白质水平。将互补DNA转染到HEK293细胞中。使用剪接阻断吗啉代寡核苷酸（rad21a）抑制斑马鱼胚胎中rad21的表达。收集并分析肠道组织。
我们在一个患CIPO的近亲家庭的患者中鉴定出RAD21基因的纯合突变（第622位，编码丙氨酸>苏氨酸）。与对照相比，CIPO患者细胞中RAD21的靶标RUNX1的表达降低。在斑马鱼中，抑制rad21a可降低runx1的表达；通过注射人RAD21 mRNA可纠正该表型，但注射来自第622位突变等位基因的mRNA则不能。rad21a吗啉代寡核苷酸斑马鱼的肠道转运延迟，肠神经元数量大幅减少，类似于CIPO患者。在ret和rad21表达受抑制的斑马鱼中，这种缺陷更严重，表明它们在肠道神经发生的调节中相互作用。APOB的启动子区域与RAD21结合，但不与RAD21第622位丙氨酸>苏氨酸结合；与对照载体相比，在HEK293细胞中野生型RAD21的表达抑制了APOB的表达。携带RAD21突变的CIPO患者血清中APOB的肠道特异性同工型（APOB48）过表达。在散发性CIPO患者的血清和肠道活检标本中，APOB48也过表达。
一些CIPO患者携带RAD21突变，这些突变破坏了其产物调节RUNX1和APOB等基因的能力。斑马鱼中rad21表达降低以及这些靶基因的失调，会破坏肠道转运和肠神经元的发育。
