巨结肠家系RET基因突变功能研究  

Functional study of a novel RET mutation in a Hirschsprung disease family

作　　者：王大佳[1] 张志波[1] 白玉作[1] Wang Dajia;Zhang Zhibo;Bai Yuzuo(Department of Pediatric Surgery,Shengjing Hospital of China Medical University,Shenyang 110004,China)

机构地区：[1]中国医科大学附属盛京医院小儿外科,沈阳110004

出　　处：《中华小儿外科杂志》2023年第8期676-682,共7页Chinese Journal of Pediatric Surgery

基　　金：辽宁省重点研发计划联合计划(2020JH2/10300131);辽宁省兴辽英才计划(XLYC1908008)。

摘　　要：目的研究巨结肠家系RET基因新发无义突变c.2599G>T是否具有致病功能。方法利用定点突变技术构建RET c.2599G>T突变型过表达腺病毒,感染人胚肾细胞HEK-293。实验分为4组:未感染组(Control组)、阴性对照腺病毒对照组(NC组)、RET野生型过表达腺病毒组(WT组)、RET c.2599G>T突变型过表达腺病毒组(c.2599G>T组)。利用RT-PCR、蛋白质印迹法、免疫荧光定位、Transwell迁移实验、CCK-8增殖实验、流式细胞凋亡检测技术,胶质细胞源性神经营养因子(glial-derived neurotrophic factor,GDNF)处理细胞后检测p-RET和p-ERK1/2,探究突变RET基因的功能。多组均数间比较采用Tukey's T检验,多个研究组均数与对照组均数比较采用Dunnett-T检验。结果c.2599G>T组不能检测到RET全长蛋白。Transwell检测细胞迁移个数c.2599G>T组(100.80±14.72)较WT组(155.20±7.89)迁移能力下降(P<0.001)。CCK-8检测细胞增殖能力c.2599G>T组比WT组分别在24 h(0.68±0.06比0.83±0.10)、48 h(0.90±0.10比1.17±0.13)、72 h(1.07±0.11比1.401±0.19)和96 h(1.38±0.12比1.68±0.15)明显下降(P<0.05)。流式细胞技术检测凋亡,c.2599G>T组3.12%较WT组0.85%增加(P<0.001)。应用GDNF处理细胞后,蛋白质印迹法检测p-RET和p-ERK1/2蛋白表达,c.2599G>T组(0.79±0.02,5.60±0.02)较WT组(72.04±0.58,10.72±0.02)均明显下降(P<0.001)。结论RET基因无义突变c.2599G>T,是具有功能的新发突变,具有致病性,对先天性巨结肠的遗传咨询具有指导意义。Objective To conduct an in vitro study of a novel nonsense mutation in RET gene(c.2599G>T)in a Hirschsprung disease(HSCR)family to determine whether or not this mutation is pathogenic.Methods An adenovirus overexpressing RET c.2599G>T mutant was constructed by site-specific mutagenesis and transfected into human embryonic kidney HEK-293 cells.The cells were assigned into four groups of control(uninfected),NC(infected with a negative control adenovirus),WT(infected with an adenovirus overexpressing wild-type RET);c.2599G>T[infected with an adenovirus overexpressing mutant(c.2599g>T)RET].Real-time polymerase chain reaction,Western blot,immunofluorescent localization,Transwell migration assay,cell counting kit-8(CCK-8)proliferation assay and flow cytometry were utilized for exploring the functions of mutated RET gene.RET phosphorylation and activation via extracellular regulated protein kinase(ERK)were assessed by Western blot.Tukey's t-test was used to compare the mean between multiple study groups,and Dunnett-T test was used to compare the mean between multiple study groups and the control group.Results In group c.2599g>T cells,full-length RET protein were not detected.Transwell assay indicated that migratory capacity was lower for group c.2599g>T cells than that for group WT cells(100.80±14.72 vs 155.20±7.89;P<0.001).CCK-8 assay revealed significantly lower(P<0.05)proliferation for group c.2599g>T cells than for group WT cells(0.68±0.06 vs 0.83±0.10 at 24 h,0.90±0.10 vs 1.17±0.13 at 48 h,1.07±0.11 vs 1.41±0.19 at 72 h,and 1.38±0.12 vs 1.68±0.15 at 96 h).Flow cytometry indicated acclerated apoptotic cell in group c.2599g>T(3.12%)as compared with group WT(0.85%;P<0.001).After glial-derived neurotrophic factor(GDNF)treatment,the levels of P-RET and P-ERK1/2 proteins declined markedly in group c.2599g>T(0.79±0.02,5.60±0.02)as compared with those in group WT(72.04±0.58,10.72±0.02)(P<0.001).Conclusions The nonsense mutation in RET gene,c.2599G>T,is a novel functional mutation with pathogenicity.This findi

关 键 词：先天性巨结肠 功能分析 RET基因 无义突变 

分 类 号：R726.5[医药卫生—儿科]