Ednrbm1yzcm小鼠巨结肠症修饰基因的QTL定位

A mouse model of recessive Hirschsprung Disease（HSCR） was obtained by ENU-induced mutagenesis.The absence of ganglions in the dilated intestine was confirmed by AChE staining.By mapping and identification of the mutant gene, we revealed a T/C transition mutation at nucleotide position 857 in Ednrb that resulted in a substitution of leucine to a proline at position 286 in the protein. Interestingly, variable severity of symptoms of Hirschsprung disease was found in F2 mice obtained by intercrossing [C57BL/6J(B6)×DBA/2J(D2)]F1 mice, indicating a major contribution from genetic background-modifier genes. The following research are divided into two portions:(1) Evaluation of the effects of L286P mutation: The wild-type and the mutant Ednrb are transfected into HEK293 cells. Intracellular Ca2+ transients and Ip1 evoked by various concentrations of ET-3 are then measured;（2）QTL mapping for modifier loci：Microscopic examination of Ednrbm1yzcm intestines stained by AChE is used to appraise the length of aganglionosis gut, then the extent of aganglionosis is calculated as a ratio of length of the aganglionosis intestine to the length of the entire large intestine used as a quantitative trait in individual animals. The genome-wide scan is performed using microsatellite markers and a further identifiacation of responsible genes located on the identified QTL is carried out.Our study could lead to the richer understanding of the neural crest-derived cells migrating regulated by different signal molecules and the identification of the modifier genes of human Hirschsprung disease.

通过ENU诱变手段，我们获得一例隐性遗传的巨结肠症小鼠，乙酰胆碱酯酶染色显示异常肠管段神经节细胞缺失。通过突变基因定位及鉴定发现内皮素受体B 基因（Ednrb）编码区第857位碱基出现T到C的转换，导致第286位亮氨酸被脯氨酸替代（该小鼠被命名为Ednrbm1yzcm）。对[C57BL/6J (B6)×DBA/2J(D2)]F1互交获得的F2突变纯合子观察发现小鼠巨结肠症严重程度不一，提示存在修饰基因的影响。本研究分为两个部分：（1）评价L286P突变对下游信号的影响：通过将正常及突变Ednrb基因瞬时转染于HEK293细胞中，加入内皮素3后，检测Ca2+及IP1水平；（2）修饰基因的QTL定位：以神经节细胞缺失肠管长度与大肠长度比值作为数量性状，以微卫星为标记进行QTL定位，并对候选基因鉴定。本研究是发现与神经细胞迁徙相关信号通路的基础，并为人类巨结肠症修饰基因鉴定提供参考。

在先天性消化道畸形中巨结肠症发生率约为1：5000，该病主要是由于胚胎期神经细胞迁徙故障导致病变肠段神经节细胞缺失引起，目前已鉴定多个巨结肠症相关基因。研究发现同一家系中相同主导基因突变的不同个体巨结肠症严重程度存在明显的差异，提示该病还受到其他基因共同调控，属于多基因调控疾病。通过乙烷基亚硝基脲诱变手段我们获得了一例巨结肠症小鼠。突变基因定位及鉴定发现内皮素受体B基因（Ednrb）编码区第857位碱基出现T到C的转换，导致第286位亮氨酸被脯氨酸替代（L286P）。观察发现F2突变纯合子小鼠巨结肠症严重程度不一，提示存在影响该疾病的修饰基因。本项目（1）通过将正常及突变Ednrb基因瞬时转染细胞后，加入内皮素3刺激，通过检测Ca2+浓度评价Ednrb L286P突变对下游信号是否产生影响；（2）以神经节细胞缺失肠管长度与大肠长度比值作为数量性状，以微卫星为遗传标记进行QTL定位，并对候选修饰基因进行鉴定。结果显示L286P突变可影响Ednrb功能，提示Ednrb L286P突变是引起本例巨结肠症的主要原因。QTL定位发现小鼠第11号与15号染色体上存在影响本例巨结肠症严重程度的修饰基因，测序发现11号染色体上Efnb3基因为强力候选修饰基因。本研究是发现与神经细胞迁徙相关信号分子的基础，并为人类巨结肠症修饰基因的鉴定提供参考。