Department of Pediatric Surgery, Key Laboratory of Chinese Health Ministry for Congenital Malformations, Shengjing Hospital of China Medical University, 36 Sanhao Street, Heping District, Shenyang, 110004, People's Republic of China.
Clin Exp Med. 2018 Aug;18(3):445-451. doi: 10.1007/s10238-018-0496-3. Epub 2018 Mar 29.
Hirschsprung's disease (HSCR) is a common congenital malformation of the enteric nervous system. The pathophysiological basis remains unclear. Recently, the SIP1 gene has been recognized as being involved in the pathogenesis of symptomatic HSCR patients with 2q22 chromosomal rearrangement. In this study, mutations in SIP1 were analyzed to explore the relationship between SIP1 and HSCR. All exons of SIP1 were amplified and then analyzed by PCR-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing. SIP1 expression was determined by immunohistochemistry, Western blot and real-time quantitative PCR. By PCR-RFLP, three different electrophoretic bands of 536, 428 and 257 bp representing different genotypes were demonstrated accordingly. DNA sequencing revealed a heterozygous absence of codon 157 GTG → GTA exchange at exon 7. Simultaneously, exchanges of GCC → ACC at codon 351 and ACC → GCC at codon 395 were also observed in exon 8. All the exchanges caused a missense mutation. By immunohistochemistry, SIP1 was ectopically expressed in the aganglionic segment of HSCR without mutation. For comparison, in HSCR with mutation either in exon 7 or exon 8, SIP1 immunoreactivity disappeared in all structures. The protein and mRNA levels determined by Western blot and real-time quantitative PCR were consistent with that of immunohistochemistry. In summary, mutations of the SIP1 gene were detected in HSCR. These mutations in SIP1 were responsible for the absence of its expression in HSCR and contributed to the pathogenesis of this disease.
先天性巨结肠症（HSCR）是一种常见的肠神经系统先天性畸形。其病理生理基础仍不清楚。最近，SIP1 基因已被认为与 2q22 染色体重排的有症状 HSCR 患者的发病机制有关。在本研究中，分析了 SIP1 的突变，以探讨 SIP1 与 HSCR 之间的关系。扩增 SIP1 的所有外显子，然后通过 PCR-限制性片段长度多态性（PCR-RFLP）和 DNA 测序进行分析。通过免疫组织化学、Western blot 和实时定量 PCR 确定 SIP1 的表达。通过 PCR-RFLP，分别显示代表不同基因型的 536、428 和 257 bp 的三种不同电泳带。DNA 测序显示外显子 7 中存在 GTG→GTA 错义突变。同时，在 8 号外显子中还观察到了 GCC→ACC 和 ACC→GCC 的交换。所有的交换都导致了错义突变。通过免疫组织化学，在没有突变的 HSCR 无神经节段中 SIP1 异位表达。相比之下，在 7 号或 8 号外显子中存在突变的 HSCR 中，SIP1 免疫反应性在所有结构中均消失。Western blot 和实时定量 PCR 确定的蛋白和 mRNA 水平与免疫组织化学结果一致。总之，在 HSCR 中检测到 SIP1 基因的突变。SIP1 中的这些突变导致其在 HSCR 中表达缺失，并有助于该疾病的发病机制。
