先天性肛门直肠畸形相关基因筛选、表达及多态性研究
|
摘要
前言
先天性肛门直肠畸形(anorectal malformation,ARMs)是小儿常见的消化道畸形,是世界卫生组织常规监测的先天畸形之一,占消化道畸形的1/4,发病率为1/5000～1/1500。术后有近1/3患儿合并不同程度的排便排尿功能障碍,严重影响患儿的生活质量,该病是主要危害我国儿童健康的先天畸形之一。
先天性肛门直肠畸形的病因仍不清楚。目前认为,肛门直肠畸形的发生是遗传因素和环境因素共同作用的结果。流行病学和动物实验表明遗传因素在其发病过程中发挥重要作用。最近国外学者利用肛门直肠畸形动物实验研究发现,Shh基因、Gli2基因、Gli3基因、Hox基因、Fgf10基因、Bmp4基因、Wnt5a基因、Eph和ephrin基因的B亚类、Sall基因在消化道末端发育中发挥重要作用。当上述基因异常均可导致动物出现肛门闭锁、肛门开口异位等畸形。
由于肛门直肠畸形发病机制和病理改变十分复杂,多伴发其他畸形,遗传方式和外显率尚不清楚,涉及的相关基因较多,且可供研究的家系和标本严重匮乏,因而对其致病基因的研究进展缓慢,短时间内难以有所突破。人类先天性肛门直肠畸形基因水平的研究国内外报道较少,至今尚无先天性肛门直肠畸形相关基因得到定位。因此从分子水平探索人类先天性肛门直肠畸形的发病机制具有十分重要的意义。
基因芯片也称为基因微阵列,是指在有限的载体上加载大量基因信号,可以用有限的标本,在短时间内、条件几乎一致的情况下,检测大量基因的表达情况,从而提供全面可靠的基因表达谱。
应用基因表达谱芯片对正常儿及肛门直肠畸形患儿消化道末端进行基因表达分析,筛选出差异表达基因。对差异表达较显著的基因,扩大样本量,经过RT-PCR验证可靠,并且利用医学生物信息学分析。实验观察所筛选的基因在大鼠胚胎中晚期消化道末端表达的时空性;并且应用免疫组织化学、RT-PCR和蛋白印记研究筛选基因在人类肛门直肠畸形的直肠末端的表达;在此基础上应用限制性片段多态性(RFLP)选择筛选基因的单核苷酸多态性标记(SNP),从遗传学探讨筛选基因多态与肛门直肠畸形的关系,寻找肛门直肠畸形致病基因。这将为解决人类先天性肛门直肠畸形的产前诊断、畸形预防和发现新的治疗手段提供可靠线索。
材料和方法
一、实验材料
1、临床组织标本
收集近一年来在我院治疗的各类无肛畸形病例36例,作为病例组。收集直肠外伤、死于非胃肠道畸形患儿等无畸形患儿病例17例,做为对照组。术中无菌取直肠末端全层肠壁,放入无RNA酶的锡纸中,立即至于液氮中保存。
2、实验动物
体重230-250克的Wistar大白鼠由中国医科大学动物部提供。
3、血样标本
65例ARMs患者,115名无血缘关系的辽宁汉族健康个体,采用EDTA抗凝外周静脉全血0.2ml。
二、实验方法
1、基因表达谱芯片对肛门直肠畸形患儿和正常儿直肠末端组织进行表达谱研究
2例先天性高位肛门闭锁患儿和1例死于非胃肠道疾病患儿,取直肠末端组织。采用Trizol一步法提取组织的总RNA,反转录成cDNA,转录合成cRNA,合成的同时进行生物素标记。生物素标记的cRNA探针经片段化处理后与芯片杂交,扫描仪上扫描芯片。应用GCOS软件进行数据分析。将所获探针号于NetAffy网站(www.afrymetrix.com)中查询出对应的基因和生物学信息。
2、部分差异表达基因表达水平的实验验证
中高位ARMS患儿直肠后壁末端标本36例;对照组由8例死于非胃肠道疾病患儿的直肠末端标本,术中无菌留取直肠末端全层标本。应用RT-PCR的方法对筛选出的7个表达差异基因进行了研究,以验证基因芯片的可靠性和可信度。表达上调的基因:RHOB、HOXA5;表达下调的基因:NKX3-1、SALL1、MMP7、SOX11、EPHB2。
3、EphB2与先天性肛门直肠畸形关系的研究
①用乙烯硫脲(ethylenethiourea,ETU)致畸Wistar孕鼠24只肛门直肠畸形动物模型,根据切片观察后分为给药无畸形组(n=90)和畸形组(n=108),胎龄13d-16d、18d和20d的正常胎鼠(n=111)作为正常对照组。正中矢状面、水平面连续切片,EphB2免疫组化染色。连续动态对比观察EphB2在正常和畸形大鼠胚胎泄殖腔及肛门直肠发育过程中的表达。常规制备胎鼠冰冻切片,解剖显微镜下手工显微切割方法准确切取泄殖腔标本,从中提取总RNA,通过RT—PCR方法研究EphB2 mRNA在正常和畸形大鼠胚胎泄殖腔及肛门直肠的表达。②采用免疫组织化学法、免疫荧光分析方法、RT-PCR方法和Western蛋白印迹方法,检测31例不同类型先天性肛门直肠畸形直肠后壁末端、5例后天瘘及8例正常直肠后壁末端EphB2在蛋白水平和mRNA水平的表达情况,应用单因素方差分析比较正常组和畸形组、不同类型的畸形组之间EphB2表达水平的差异。
4、EphB2基因多态性与先天性肛门直肠畸形相关性研究
采用PCR—RFLP方法,对65例ARMS患者和115名正常人EphB2基因-1395A/G多态位点进行基因型检测,用SHEsis在线统计软件分析等位基因频率、基因型频率及其组间差异。
结果
1、基因表达谱芯片对肛门直肠畸形患儿和正常儿直肠末端组织进行表达谱研究
肛门直肠畸形直肠末端与正常直肠末端组织中表达差异在2倍以上的基因有776条,其中ARMs下调的基因有399条,上调基因377条。差异表达的基因4倍以上的基因259条,其中ARMs下调的基因有150条,上调的基因109条。差异表达基因以发育分化、信号转导传递、细胞生理过程及调节基因为主,同时还涉及核酸转录翻译、凋亡调节、细胞骨架、免疫应激、物质合成代谢及运输等不同层次、不同功能的基因。
2、部分差异表达基因表达水平的实验验证
RT-PCR技术验证的7个差异表达基因中,RHOB、HOXA5基因在高位肛门闭锁患者直肠末端组织中的表达水平高于正常对照,而SOX11、MMP7、SALL1、NKX3-1和EPHB2基因在高位肛门闭锁患者直肠末端组织中的表达水平明显低于正常对照。
3、EphB2与先天性肛门直肠畸形关系的研究
(1)在大鼠中晚期胚胎研究中免疫组化实验中,EphB2在大鼠发育过程中正常及给药无畸形组胚胎泄殖腔中表达,在13d和14d主要表达于尿直肠隔和泄殖腔膜,胚胎15d时在尿直肠隔与泄殖腔膜融合处EphB2表达较强,16d时肛膜破裂,肛门直肠形成,EphB2在直肠粘膜层表达。EphB2在肛门直肠畸形胎鼠泄殖腔和直肠粘膜层亦有表达,测量单位面积内累积光密度值,胎龄13-16d时与正常对照组和给药无畸形组比较值明显减小,有统计学意义(P＜0.05)。应用RT-PCR实验发现在正常和畸形组大鼠胚胎中均能检测出EphB2表达,在胎龄13-16d为表达高峰期,胎龄16d后逐渐降低。畸形组与正常对照组和给药无畸形组相比较,EphB2 mRNA的表达在13-16d明显降低,有统计学意义(P＜0.05)。
(2)在人类先天性肛门直肠畸形直肠末端组织中,免疫组织化学检测到EphB2蛋白在畸形组和对照组均有表达,定位在直肠肠壁粘膜层细胞的胞浆和胞膜中,先天性肛门直肠畸形末端肠壁EphB2基因mRNA相对表达量明显低于正常对照组(P＜0.001)。不同类型的畸形组之间,高位组和中位组之间差异无统计学意义(P=0.33),但二者明显低于低位组(P＜0.001)。后天瘘组与正常对照组比较没有明显差异(P=0.63)。Western蛋白印迹结果显示,先天性肛门直肠畸形高位组、中位组和低位组蛋白表达水平均明显低于后天瘘组和正常对照组(P＜0.001)。不同类型的畸形之间,高位组和中位组之间无明显差异(P=0.11),但二者明显低于低位组(P＜0.01)。后天瘘组与正常对照组比较没有明显差异(P=0.67)。
4、EphB2基因多态性与先天性肛门直肠畸形相关性研究
EphB2受体基因第6外显子的-1395A/G多态A等位基因频率及AA基因型频率在ARMs组(0.854、0.754)与正常对照组(0.170、0.043)差异显著(P＜0.01)。
结论
(1)基因芯片可有效筛查出肛门直肠畸形发生的相关基因,包括发育分化、信号转导传递、细胞生理过程及调节、核酸转录翻译、凋亡调节、物质合成代谢及运输等多种基因,这表明在ARMs的发生发展中有多种类型的基因参与。
(2)初步筛查出差异表达基因2倍以上776条,4倍以上259条。其中与发育相关基因,上调基因RHOB和HOXA5;下调基因SOX11、MMP7、SALL1、NKX3-1和EPHB2,验证的表达趋势与在基因表达谱中的变化一致,提示本研究中基因表达谱的实验结果具有较好的可信度和可靠性,深入分析差异表达基因与先天性肛门直肠畸形之间的关系将可能为阐明先天性肛门直肠畸形的病因及病理生理学提供新的理论依据。
(3) EphB2可能在大鼠胚胎期泄殖腔和直肠的正常发育和畸形发生过程中起重要作用。
(4) EphB2在人类先天性肛门直肠畸形直肠末端低表达,EphB2在人类先天性肛门直肠畸形的发生中可能具有重要作用。
(5) EphB2受体基因第6外显子的-1395A/G多态与先天性肛门直肠畸形存在相关性。
(6) EphB2可能在泄殖腔和直肠的发育中起重要作用,与肛门直肠畸形的发生相关。
Anorectal malformations(ARMs) is one of the most common disorders,affecting 1/5000 to 1/1500 live births,one kind of malformations supervised by World Health Organization.Some patients with ARMs continue to suffer from postoperative anal dysfunctions.ARMs severely influences quality of life,and takes great burdens on families and society.Therefore,ARMs is one of the severest congenital disorders.
The etiopathogenisis of ARMs continues poorly understood and controversial.In current study,it has been only demonstrated that ARMs resulted from hereditary factors and environmental factors in the development of embryogenesis.During the research of epidemiology and animal experiments,it is suggested that the hereditary factors play a key role in the development of ARMs.Recently,it was found that Shh,Gli2、Gli3、Hox、Fgf10、Bmp4、Wnt5a、Eph and ephrinB、Sail play crucial role during the development of the termination of digestive tract.Once the foresaid genes dysfunction, there may be ARMs,such as anal atresia,preternatural anus et al.
Because the spectrum of ARMs is fairly wide,pathogenesis and pathological changes are complex.As well,ARMs is often associated with extra anal anomalies,and its phenotypes are rather variable,so it is very difficult to perform gene investigation. There is a few studies about gene level in human ARMs,until now,the pathogenic genes are still unlocated,even the candidate gene remains unclear.Moreover,it is significant to investigate pathogenesis of ARMs on embryonic and molecular level.
Gene chip(gene microarray),is the chief tool for functional genome research.As adopting the high efficient and parallel DNA hybridization technology,can achieve abundant data from each experiment,so the data analysis of microarrays becomes more challenging and meaningful.
By use of Gene array to detect the normal and ARMs the termination of digestive tract,and screen differential genes.To aim directly at differential genes,extend sample size,by the means of RT-PCR to verify,and then analysis the data with medical bioinformatics.Detect the expression of these genes in the development of the termination of digestive tract in rat embryo,and ensure the virulence gene of ARMs. All of the foresaid results provide reliable basis for prenatal diagnosis,prophylaxis and finding the original therapeutic tool for ARMs.
Materials and methods
Materials
(1) Clinical specimens:the case group:36 ARMs infants operated in our department.Tissues were removed from terminal pouch of rectum and stored immediately in fluid nitrogen.In the control group,tissues were obtained at 1 cm superior to dentate line in acquired anal fistula and rectal traumas infants.
(2) Animals:Wistar rats(230-250g) were provided by Medical Animal Center,the SHENGJING Hospital of China Medical University.
(3) Blood samples:took out 0.2ml vein blood from 65 ARMs patients and 115 samples randomly selected from healthy individuals of Han nationality in LiaoNing Province,anticoagulated with ethylene diamine tetraacetic acid(EDTA).
Methods
(1) Total RNA was isolated from the distal part of rectum tissue of congenital anorectal malformation and normal rectal tissues.RNA was purified with QIAGEN RNeasy Mini Kit,converted to cDNA and hybridized using Affymetrix Human Genome U133 Plus 2.0 Array.
(2) To verify the expressions of the different genes:36 intermediate and high type ARMS infants operated in our department.Tissues were removed from terminal pouch of rectum and stored immediately in fluid nitrogen.In the control group,tissues were obtained at point 1 cm to dentate line in acquired anal fistula and rectal traumas infants.RT-PCR was implied to verify the screened genes,which are acquired the micro array,in order to authenticate the reliability and confidence of the micro array. RHOB,HOXA5 are up-regulated,and NKX3-1,SALL1、MMP7,SOX11、EPHB2 genes are down-regulated,which is consistent with the microarray analysis.
(3) ARMs murine model treated by Ethylenethiourea were employed in this study.Reverse transcription polymerized chain reaction techniques(RT-PCR) and immunohistochemistry analyses were carried out to investigate EphB2 mRNA and protein expression and localizations.①Rat embryos with ARMs were obtained by treating pregnant rats(n=24) with administration of ETU on gestation day(Gd) 10. Normal rat embryos(n=111) and embryos treated by ETU without ARMs(n=90) were taken as control group,and embryos with ARMs(n=108) from Gd13 to Gdl6 were divided by sections.Embryos were serial-sectioned in the sagittal and transversal planes,stained with specific antibody to EphB2.Spatiotemporal study was carried out on EphB2 expression.②Individual frozen sections were used to manual microdissection.The cloaca and anorectal specimens were used for total RNA extraction.EphB2 expression was evaluated by RT-PCR.Specimens were obtained from 20 patients with ARMS,and 8 normal controls.Expression of EphB2 was determined by SP immunohistochemistry and immunofluorescence.Specimens were obtained from 31 patients with ARMS,5 patients with infected rectovestibular fistula and 8 normal controls.Expression of EphB2 gene and protein were determined by RT-PCR and Western-blotting respectively.One-way ANOVA was used to compare the differences of EphB2 expression levels between the normal and abnormal,as well as the differences of EphB2 expression levels among different kinds of ARMs.
(4) The genotype of-1395A/G polymorphism in EphB2 gene were detected by PCR-RFLP in the control group including 115 cases and 65 ARMs patients.The frequencies of alleles and genotypes in the two groups and the difference between the two groups were analyzed by SHEsis software online.
Results
(1) The expression of 776 genes were different between the malformation tissue and normal rectum,of which 399 were down-regulated and 377 up-regulated in congenital anorectal malformation,different clusters of genes associated with embryo development,cell differentiation,metabolism,DNA transcription,signal transduction, cell cycle,etc were concerned.Of these,259 were significantly different,150 of them down-regulated and 109 up-regulated.The expression tendency of screened genes were coincidence with the changes of gene expression spectrum,it is replied that the results of gene expression spectrum were reliable and coefficient.The advanced studies of the relation between the differential expression genes and ARMs may provide original theories for the etiopathogenisis and pathophysiology of ARMs.
(2) RT-PCR was carried out to verify the different expression genes,the expressions of RHOB、HOXA5 in the rectal terminal pouch of the high type ARMs were higher than those of the control group.However,the expressions of SOX11、MMP7、SALL1、NKX3-1 and EPHB2 of the terminal pouch of rectum in the high type ARMS were lower than the control group.
(3) The relationship of EphB2 and ARMs:①On the immunolabeling study, EphB2 expression was confined to cloaca in control group.EphB2 expression mainly located in the urorectal septum(URS) and cloacal membrane on Gdl3 and Gd14.The increased positive expression was observed in the fusion tissue between the URS and the cloacal membrane on Gdl 5.On Gd16,anal membrane broke down and the rectum communicated with the anus,meanwhile the EphB2 expression was confined to mucous membrane of rectum.EphB2 expression was observed in the cloacal and anorectal tissue of the embryos with ARMs.By integrated optical density(IOD) measurement,IOD of EphB2 protein was significantly lower in the ARMs group than that in the control group on Gdl3-Gdl6(P＜0.05),respectively.On RT-PCR,EphB2 expression was detected in three groups.EphB2 mRNA level increased on Gdl 3-Gd 16, but gradually decreased after Gd16.The expression level of EphB2 mRNA in the ARMs embryos was lower on Gdl3-Gdl6 than that in control group(P＜0.05).②In the anorectal tissues of the human normal and ARMs,the expression of EPHB2 was mainly located in the cytoplasm and cytomembrane of the mucous layer by immunohistochemistry.The EphB2 mRNA expression levels were significantly higher in the normal rectum than those in malformations(P＜0.001).There was no significant difference between high and intermediate ARMS(P=0.33).However,the expression levels of EphB2 mRNA in high and intermediate ARMs were significant lower than that in low group(P＜0.001).What's more,there was no significant difference of EphB2 mRNA expression level between the infected rectovestibular fistula and the control group(P=0.63).The expression levels of EphB2 protein in high,intermediate and low ARMs had a marked diminished compared with that in controls(0.21±0.05, 0.24±0.04 and 0.30±0.04 vs.0.52±0.03,P＜0.001).There was no significant difference of EphB2 protein expression level between high and intermediate ARMs by Western-blot(P=0.33).However,the expression levels of EphB2 protein in high and intermediate ARMs were significant lower than that in low group(P＜0.001).There was no significant difference of EphB2 protein expression level between the infected rectovestibular fistula and the control group(P=0.67)
(4) The frequencies of A allele and AA genotype of the-1395A/G polymorphism in the 6th exon region of EphB2 receptor gene in the ARMs patients and control groups were(0.854,0.754) and(0.170,0.043) respectively.
Conclusions
(1) cDNA microarray can be used to study possible genetic influence in the pathogenesis of congenital anorectal malformation.Anorectal malformation has a multifactorial and polygenic background.
(2) The tendencies in the expressive alteration of 7 verified differentially expressed genes were in coincidence with the result of gene expression profiles of human ARMs anorectal tissues,such as RHOB,HOXA5,SOX11,MMP7,SALL1, NKX3-1 and EPHB2.It was suggested that the experiment of cDNA microarray be of reliability and confidence.To analysis the relationship between the differentially expressed genes and ARMs may provide the etiology and pathophysiology of congenital anorectal malformations with new theory gist.
(3) EphB2 expression decreased in the ARMs rat embryos and was confined to URS and cloaca while it was higher in control group.Our data,thus,indicated that EphB2 molecules play an important role in anorectal morphogenesis and the decreased expression of EphB2 might be related to the development of ARMs.
(4) There was decreased expression of EphB2 in the mucous layer of ARMs. There are decreased expression levels of EphB2 in ARMs not only in mRNA level but also in protein level.The expression of EphB2 may be related to the development of ARMs.EphB2 may play an important role in the development of ARMs.
(5) There is an association between the polymorphism of-1395A/G in the 6th exon region of EPHB2 receptor gene and ARMs.
(6) EphB2 may play an important role in the development of cloaca and rectum.EphB2 may be related to the development of ARMs.
先天性肛门直肠畸形(anorectal malformation,ARMs)是小儿常见的消化道畸形,是世界卫生组织常规监测的先天畸形之一,占消化道畸形的1/4,发病率为1/5000～1/1500。术后有近1/3患儿合并不同程度的排便排尿功能障碍,严重影响患儿的生活质量,该病是主要危害我国儿童健康的先天畸形之一。
先天性肛门直肠畸形的病因仍不清楚。目前认为,肛门直肠畸形的发生是遗传因素和环境因素共同作用的结果。流行病学和动物实验表明遗传因素在其发病过程中发挥重要作用。最近国外学者利用肛门直肠畸形动物实验研究发现,Shh基因、Gli2基因、Gli3基因、Hox基因、Fgf10基因、Bmp4基因、Wnt5a基因、Eph和ephrin基因的B亚类、Sall基因在消化道末端发育中发挥重要作用。当上述基因异常均可导致动物出现肛门闭锁、肛门开口异位等畸形。
由于肛门直肠畸形发病机制和病理改变十分复杂,多伴发其他畸形,遗传方式和外显率尚不清楚,涉及的相关基因较多,且可供研究的家系和标本严重匮乏,因而对其致病基因的研究进展缓慢,短时间内难以有所突破。人类先天性肛门直肠畸形基因水平的研究国内外报道较少,至今尚无先天性肛门直肠畸形相关基因得到定位。因此从分子水平探索人类先天性肛门直肠畸形的发病机制具有十分重要的意义。
基因芯片也称为基因微阵列,是指在有限的载体上加载大量基因信号,可以用有限的标本,在短时间内、条件几乎一致的情况下,检测大量基因的表达情况,从而提供全面可靠的基因表达谱。
应用基因表达谱芯片对正常儿及肛门直肠畸形患儿消化道末端进行基因表达分析,筛选出差异表达基因。对差异表达较显著的基因,扩大样本量,经过RT-PCR验证可靠,并且利用医学生物信息学分析。实验观察所筛选的基因在大鼠胚胎中晚期消化道末端表达的时空性;并且应用免疫组织化学、RT-PCR和蛋白印记研究筛选基因在人类肛门直肠畸形的直肠末端的表达;在此基础上应用限制性片段多态性(RFLP)选择筛选基因的单核苷酸多态性标记(SNP),从遗传学探讨筛选基因多态与肛门直肠畸形的关系,寻找肛门直肠畸形致病基因。这将为解决人类先天性肛门直肠畸形的产前诊断、畸形预防和发现新的治疗手段提供可靠线索。
材料和方法
一、实验材料
1、临床组织标本
收集近一年来在我院治疗的各类无肛畸形病例36例,作为病例组。收集直肠外伤、死于非胃肠道畸形患儿等无畸形患儿病例17例,做为对照组。术中无菌取直肠末端全层肠壁,放入无RNA酶的锡纸中,立即至于液氮中保存。
2、实验动物
体重230-250克的Wistar大白鼠由中国医科大学动物部提供。
3、血样标本
65例ARMs患者,115名无血缘关系的辽宁汉族健康个体,采用EDTA抗凝外周静脉全血0.2ml。
二、实验方法
1、基因表达谱芯片对肛门直肠畸形患儿和正常儿直肠末端组织进行表达谱研究
2例先天性高位肛门闭锁患儿和1例死于非胃肠道疾病患儿,取直肠末端组织。采用Trizol一步法提取组织的总RNA,反转录成cDNA,转录合成cRNA,合成的同时进行生物素标记。生物素标记的cRNA探针经片段化处理后与芯片杂交,扫描仪上扫描芯片。应用GCOS软件进行数据分析。将所获探针号于NetAffy网站(www.afrymetrix.com)中查询出对应的基因和生物学信息。
2、部分差异表达基因表达水平的实验验证
中高位ARMS患儿直肠后壁末端标本36例;对照组由8例死于非胃肠道疾病患儿的直肠末端标本,术中无菌留取直肠末端全层标本。应用RT-PCR的方法对筛选出的7个表达差异基因进行了研究,以验证基因芯片的可靠性和可信度。表达上调的基因:RHOB、HOXA5;表达下调的基因:NKX3-1、SALL1、MMP7、SOX11、EPHB2。
3、EphB2与先天性肛门直肠畸形关系的研究
①用乙烯硫脲(ethylenethiourea,ETU)致畸Wistar孕鼠24只肛门直肠畸形动物模型,根据切片观察后分为给药无畸形组(n=90)和畸形组(n=108),胎龄13d-16d、18d和20d的正常胎鼠(n=111)作为正常对照组。正中矢状面、水平面连续切片,EphB2免疫组化染色。连续动态对比观察EphB2在正常和畸形大鼠胚胎泄殖腔及肛门直肠发育过程中的表达。常规制备胎鼠冰冻切片,解剖显微镜下手工显微切割方法准确切取泄殖腔标本,从中提取总RNA,通过RT—PCR方法研究EphB2 mRNA在正常和畸形大鼠胚胎泄殖腔及肛门直肠的表达。②采用免疫组织化学法、免疫荧光分析方法、RT-PCR方法和Western蛋白印迹方法,检测31例不同类型先天性肛门直肠畸形直肠后壁末端、5例后天瘘及8例正常直肠后壁末端EphB2在蛋白水平和mRNA水平的表达情况,应用单因素方差分析比较正常组和畸形组、不同类型的畸形组之间EphB2表达水平的差异。
4、EphB2基因多态性与先天性肛门直肠畸形相关性研究
采用PCR—RFLP方法,对65例ARMS患者和115名正常人EphB2基因-1395A/G多态位点进行基因型检测,用SHEsis在线统计软件分析等位基因频率、基因型频率及其组间差异。
结果
1、基因表达谱芯片对肛门直肠畸形患儿和正常儿直肠末端组织进行表达谱研究
肛门直肠畸形直肠末端与正常直肠末端组织中表达差异在2倍以上的基因有776条,其中ARMs下调的基因有399条,上调基因377条。差异表达的基因4倍以上的基因259条,其中ARMs下调的基因有150条,上调的基因109条。差异表达基因以发育分化、信号转导传递、细胞生理过程及调节基因为主,同时还涉及核酸转录翻译、凋亡调节、细胞骨架、免疫应激、物质合成代谢及运输等不同层次、不同功能的基因。
2、部分差异表达基因表达水平的实验验证
RT-PCR技术验证的7个差异表达基因中,RHOB、HOXA5基因在高位肛门闭锁患者直肠末端组织中的表达水平高于正常对照,而SOX11、MMP7、SALL1、NKX3-1和EPHB2基因在高位肛门闭锁患者直肠末端组织中的表达水平明显低于正常对照。
3、EphB2与先天性肛门直肠畸形关系的研究
(1)在大鼠中晚期胚胎研究中免疫组化实验中,EphB2在大鼠发育过程中正常及给药无畸形组胚胎泄殖腔中表达,在13d和14d主要表达于尿直肠隔和泄殖腔膜,胚胎15d时在尿直肠隔与泄殖腔膜融合处EphB2表达较强,16d时肛膜破裂,肛门直肠形成,EphB2在直肠粘膜层表达。EphB2在肛门直肠畸形胎鼠泄殖腔和直肠粘膜层亦有表达,测量单位面积内累积光密度值,胎龄13-16d时与正常对照组和给药无畸形组比较值明显减小,有统计学意义(P＜0.05)。应用RT-PCR实验发现在正常和畸形组大鼠胚胎中均能检测出EphB2表达,在胎龄13-16d为表达高峰期,胎龄16d后逐渐降低。畸形组与正常对照组和给药无畸形组相比较,EphB2 mRNA的表达在13-16d明显降低,有统计学意义(P＜0.05)。
(2)在人类先天性肛门直肠畸形直肠末端组织中,免疫组织化学检测到EphB2蛋白在畸形组和对照组均有表达,定位在直肠肠壁粘膜层细胞的胞浆和胞膜中,先天性肛门直肠畸形末端肠壁EphB2基因mRNA相对表达量明显低于正常对照组(P＜0.001)。不同类型的畸形组之间,高位组和中位组之间差异无统计学意义(P=0.33),但二者明显低于低位组(P＜0.001)。后天瘘组与正常对照组比较没有明显差异(P=0.63)。Western蛋白印迹结果显示,先天性肛门直肠畸形高位组、中位组和低位组蛋白表达水平均明显低于后天瘘组和正常对照组(P＜0.001)。不同类型的畸形之间,高位组和中位组之间无明显差异(P=0.11),但二者明显低于低位组(P＜0.01)。后天瘘组与正常对照组比较没有明显差异(P=0.67)。
4、EphB2基因多态性与先天性肛门直肠畸形相关性研究
EphB2受体基因第6外显子的-1395A/G多态A等位基因频率及AA基因型频率在ARMs组(0.854、0.754)与正常对照组(0.170、0.043)差异显著(P＜0.01)。
结论
(1)基因芯片可有效筛查出肛门直肠畸形发生的相关基因,包括发育分化、信号转导传递、细胞生理过程及调节、核酸转录翻译、凋亡调节、物质合成代谢及运输等多种基因,这表明在ARMs的发生发展中有多种类型的基因参与。
(2)初步筛查出差异表达基因2倍以上776条,4倍以上259条。其中与发育相关基因,上调基因RHOB和HOXA5;下调基因SOX11、MMP7、SALL1、NKX3-1和EPHB2,验证的表达趋势与在基因表达谱中的变化一致,提示本研究中基因表达谱的实验结果具有较好的可信度和可靠性,深入分析差异表达基因与先天性肛门直肠畸形之间的关系将可能为阐明先天性肛门直肠畸形的病因及病理生理学提供新的理论依据。
(3) EphB2可能在大鼠胚胎期泄殖腔和直肠的正常发育和畸形发生过程中起重要作用。
(4) EphB2在人类先天性肛门直肠畸形直肠末端低表达,EphB2在人类先天性肛门直肠畸形的发生中可能具有重要作用。
(5) EphB2受体基因第6外显子的-1395A/G多态与先天性肛门直肠畸形存在相关性。
(6) EphB2可能在泄殖腔和直肠的发育中起重要作用,与肛门直肠畸形的发生相关。
Anorectal malformations(ARMs) is one of the most common disorders,affecting 1/5000 to 1/1500 live births,one kind of malformations supervised by World Health Organization.Some patients with ARMs continue to suffer from postoperative anal dysfunctions.ARMs severely influences quality of life,and takes great burdens on families and society.Therefore,ARMs is one of the severest congenital disorders.
The etiopathogenisis of ARMs continues poorly understood and controversial.In current study,it has been only demonstrated that ARMs resulted from hereditary factors and environmental factors in the development of embryogenesis.During the research of epidemiology and animal experiments,it is suggested that the hereditary factors play a key role in the development of ARMs.Recently,it was found that Shh,Gli2、Gli3、Hox、Fgf10、Bmp4、Wnt5a、Eph and ephrinB、Sail play crucial role during the development of the termination of digestive tract.Once the foresaid genes dysfunction, there may be ARMs,such as anal atresia,preternatural anus et al.
Because the spectrum of ARMs is fairly wide,pathogenesis and pathological changes are complex.As well,ARMs is often associated with extra anal anomalies,and its phenotypes are rather variable,so it is very difficult to perform gene investigation. There is a few studies about gene level in human ARMs,until now,the pathogenic genes are still unlocated,even the candidate gene remains unclear.Moreover,it is significant to investigate pathogenesis of ARMs on embryonic and molecular level.
Gene chip(gene microarray),is the chief tool for functional genome research.As adopting the high efficient and parallel DNA hybridization technology,can achieve abundant data from each experiment,so the data analysis of microarrays becomes more challenging and meaningful.
By use of Gene array to detect the normal and ARMs the termination of digestive tract,and screen differential genes.To aim directly at differential genes,extend sample size,by the means of RT-PCR to verify,and then analysis the data with medical bioinformatics.Detect the expression of these genes in the development of the termination of digestive tract in rat embryo,and ensure the virulence gene of ARMs. All of the foresaid results provide reliable basis for prenatal diagnosis,prophylaxis and finding the original therapeutic tool for ARMs.
Materials and methods
Materials
(1) Clinical specimens:the case group:36 ARMs infants operated in our department.Tissues were removed from terminal pouch of rectum and stored immediately in fluid nitrogen.In the control group,tissues were obtained at 1 cm superior to dentate line in acquired anal fistula and rectal traumas infants.
(2) Animals:Wistar rats(230-250g) were provided by Medical Animal Center,the SHENGJING Hospital of China Medical University.
(3) Blood samples:took out 0.2ml vein blood from 65 ARMs patients and 115 samples randomly selected from healthy individuals of Han nationality in LiaoNing Province,anticoagulated with ethylene diamine tetraacetic acid(EDTA).
Methods
(1) Total RNA was isolated from the distal part of rectum tissue of congenital anorectal malformation and normal rectal tissues.RNA was purified with QIAGEN RNeasy Mini Kit,converted to cDNA and hybridized using Affymetrix Human Genome U133 Plus 2.0 Array.
(2) To verify the expressions of the different genes:36 intermediate and high type ARMS infants operated in our department.Tissues were removed from terminal pouch of rectum and stored immediately in fluid nitrogen.In the control group,tissues were obtained at point 1 cm to dentate line in acquired anal fistula and rectal traumas infants.RT-PCR was implied to verify the screened genes,which are acquired the micro array,in order to authenticate the reliability and confidence of the micro array. RHOB,HOXA5 are up-regulated,and NKX3-1,SALL1、MMP7,SOX11、EPHB2 genes are down-regulated,which is consistent with the microarray analysis.
(3) ARMs murine model treated by Ethylenethiourea were employed in this study.Reverse transcription polymerized chain reaction techniques(RT-PCR) and immunohistochemistry analyses were carried out to investigate EphB2 mRNA and protein expression and localizations.①Rat embryos with ARMs were obtained by treating pregnant rats(n=24) with administration of ETU on gestation day(Gd) 10. Normal rat embryos(n=111) and embryos treated by ETU without ARMs(n=90) were taken as control group,and embryos with ARMs(n=108) from Gd13 to Gdl6 were divided by sections.Embryos were serial-sectioned in the sagittal and transversal planes,stained with specific antibody to EphB2.Spatiotemporal study was carried out on EphB2 expression.②Individual frozen sections were used to manual microdissection.The cloaca and anorectal specimens were used for total RNA extraction.EphB2 expression was evaluated by RT-PCR.Specimens were obtained from 20 patients with ARMS,and 8 normal controls.Expression of EphB2 was determined by SP immunohistochemistry and immunofluorescence.Specimens were obtained from 31 patients with ARMS,5 patients with infected rectovestibular fistula and 8 normal controls.Expression of EphB2 gene and protein were determined by RT-PCR and Western-blotting respectively.One-way ANOVA was used to compare the differences of EphB2 expression levels between the normal and abnormal,as well as the differences of EphB2 expression levels among different kinds of ARMs.
(4) The genotype of-1395A/G polymorphism in EphB2 gene were detected by PCR-RFLP in the control group including 115 cases and 65 ARMs patients.The frequencies of alleles and genotypes in the two groups and the difference between the two groups were analyzed by SHEsis software online.
Results
(1) The expression of 776 genes were different between the malformation tissue and normal rectum,of which 399 were down-regulated and 377 up-regulated in congenital anorectal malformation,different clusters of genes associated with embryo development,cell differentiation,metabolism,DNA transcription,signal transduction, cell cycle,etc were concerned.Of these,259 were significantly different,150 of them down-regulated and 109 up-regulated.The expression tendency of screened genes were coincidence with the changes of gene expression spectrum,it is replied that the results of gene expression spectrum were reliable and coefficient.The advanced studies of the relation between the differential expression genes and ARMs may provide original theories for the etiopathogenisis and pathophysiology of ARMs.
(2) RT-PCR was carried out to verify the different expression genes,the expressions of RHOB、HOXA5 in the rectal terminal pouch of the high type ARMs were higher than those of the control group.However,the expressions of SOX11、MMP7、SALL1、NKX3-1 and EPHB2 of the terminal pouch of rectum in the high type ARMS were lower than the control group.
(3) The relationship of EphB2 and ARMs:①On the immunolabeling study, EphB2 expression was confined to cloaca in control group.EphB2 expression mainly located in the urorectal septum(URS) and cloacal membrane on Gdl3 and Gd14.The increased positive expression was observed in the fusion tissue between the URS and the cloacal membrane on Gdl 5.On Gd16,anal membrane broke down and the rectum communicated with the anus,meanwhile the EphB2 expression was confined to mucous membrane of rectum.EphB2 expression was observed in the cloacal and anorectal tissue of the embryos with ARMs.By integrated optical density(IOD) measurement,IOD of EphB2 protein was significantly lower in the ARMs group than that in the control group on Gdl3-Gdl6(P＜0.05),respectively.On RT-PCR,EphB2 expression was detected in three groups.EphB2 mRNA level increased on Gdl 3-Gd 16, but gradually decreased after Gd16.The expression level of EphB2 mRNA in the ARMs embryos was lower on Gdl3-Gdl6 than that in control group(P＜0.05).②In the anorectal tissues of the human normal and ARMs,the expression of EPHB2 was mainly located in the cytoplasm and cytomembrane of the mucous layer by immunohistochemistry.The EphB2 mRNA expression levels were significantly higher in the normal rectum than those in malformations(P＜0.001).There was no significant difference between high and intermediate ARMS(P=0.33).However,the expression levels of EphB2 mRNA in high and intermediate ARMs were significant lower than that in low group(P＜0.001).What's more,there was no significant difference of EphB2 mRNA expression level between the infected rectovestibular fistula and the control group(P=0.63).The expression levels of EphB2 protein in high,intermediate and low ARMs had a marked diminished compared with that in controls(0.21±0.05, 0.24±0.04 and 0.30±0.04 vs.0.52±0.03,P＜0.001).There was no significant difference of EphB2 protein expression level between high and intermediate ARMs by Western-blot(P=0.33).However,the expression levels of EphB2 protein in high and intermediate ARMs were significant lower than that in low group(P＜0.001).There was no significant difference of EphB2 protein expression level between the infected rectovestibular fistula and the control group(P=0.67)
(4) The frequencies of A allele and AA genotype of the-1395A/G polymorphism in the 6th exon region of EphB2 receptor gene in the ARMs patients and control groups were(0.854,0.754) and(0.170,0.043) respectively.
Conclusions
(1) cDNA microarray can be used to study possible genetic influence in the pathogenesis of congenital anorectal malformation.Anorectal malformation has a multifactorial and polygenic background.
(2) The tendencies in the expressive alteration of 7 verified differentially expressed genes were in coincidence with the result of gene expression profiles of human ARMs anorectal tissues,such as RHOB,HOXA5,SOX11,MMP7,SALL1, NKX3-1 and EPHB2.It was suggested that the experiment of cDNA microarray be of reliability and confidence.To analysis the relationship between the differentially expressed genes and ARMs may provide the etiology and pathophysiology of congenital anorectal malformations with new theory gist.
(3) EphB2 expression decreased in the ARMs rat embryos and was confined to URS and cloaca while it was higher in control group.Our data,thus,indicated that EphB2 molecules play an important role in anorectal morphogenesis and the decreased expression of EphB2 might be related to the development of ARMs.
(4) There was decreased expression of EphB2 in the mucous layer of ARMs. There are decreased expression levels of EphB2 in ARMs not only in mRNA level but also in protein level.The expression of EphB2 may be related to the development of ARMs.EphB2 may play an important role in the development of ARMs.
(5) There is an association between the polymorphism of-1395A/G in the 6th exon region of EPHB2 receptor gene and ARMs.
(6) EphB2 may play an important role in the development of cloaca and rectum.EphB2 may be related to the development of ARMs.
引文
1 Lonnroth C, Andersson M, Arvidsson A, et al. Preoperative treatment with a non-steroidal anti-inflammatory drug (NSAID) increases tumor tissue infiltration of seemingly activated immune cells in colorectal cancer.Cancer Immun.2008; 29: 5.
2 Raponi M, Harousseau JL, Lancet JE, et al. Identification of molecular predictors of response in a study of tipifarnib treatment in relapsed and refractory acute myelogenous leukemia.Clin Cancer Res. 2007; 13:2254-2260.
3 Heanue TA, Pachnis V. Expression profiling the developing mammalian enteric nervous system identifies marker and candidate Hirschsprung disease genes.Proc Natl Acad Sci U S A. 2006; 103:6919-6924.
4 Cuschieri A. Descriptive epidemiology of isolated anal anomalies : a survey of 4. 6 million births in Europe. Am J Med Genet. 2001; 103: 207-215.
5 Stoll C, Alembik Y, Roth MP, et al. Risk factors in congenital anal atresias. Ann Genet. 1997; 40: 197-204.
6 Hori T, Giuffra E, Andersson L, et al. Mapping loci causing susceptibility to anal atresia in pigs, using a resource pedigree. J Pediatr Surg. 2001; 36: 1370-1374.
7 白玉作,王维林,黄英,等．人类肛门直肠畸形基因表达谱的研究。中国医科大学学报．2003：32：493-495．
8 Mo R, Kim JH, Zhang J, et al. Anorectal malformations caused by defects in sonic hedgehog signaling. Am J Pathol. 2001; 159: 765-74.
9 Kimmel SG, Mo R, Hui CC, et al. New mouse models of congenital anorectal malformations. J Pediatr Surg. 2000; 35: 227-30.
10 Mandhan P, Beasley S, Hale T, et al. Sonic hedgehog expression in the development of hindgut in ETU-exposed fetal rats. Pediatr Surg Int. 2006; 22: 31-36.
11 Sasaki Y, Iwai N, Tsuda T, et al. Sonic hedgehog and bone morphogenetic protein 4 expressions in the hindgut region of murine embryos with anorectal malformations. J Pediatr Surg. 2004; 39: 170-173.
12 Mandhan P, Quan QB, Beasley S, et al. Sonic hedgehog, BMP4, and Hox genes in the development of anorectal malformations in Ethylenethiourea-exposed fetal rats. J Pediatr Surg. 2006; 41: 2041-2045.
13 Fairbanks TJ, De Langhe S, Sala FG, et al. Fibroblast growth factor 10 (Fgf10) invalidation results in anorectal malformation in mice. J Pediatr Surg. 2004; 39: 360-365.
14 Kondo T, Dolle P, Zakany J, et al. Function of posterior HoxD genes in the morphogenisis of the anal sphincter. Development. 1996; 122: 2651-2659.
15 Warot X, Fromental Ramain C, Fraulob V, et al. Gene dosage-dependent effects of the Hoxa-13 and Hoxd-13 mutations on morphogenesis of the terminal parts of the digestive and urogenital tracts.Development.1997;124:4781-4791.
16 Sukegawa A,Narita T,Kameda T,et al.The concentric structure of the developing gut is regulated by Sonic hedgehog derived from endodermal epithelium.Development.2000;127:1971-80.
17 Arsic D,ARMSeron V,Ellmers L,et al.Adriamycin Disruption of the Shh-Gli Pathway is Associated With Abnormalities of Foregut Development.Journal of Pediatric Surgery.2004;39:1747-1753.
18 王大佳,陈秋,吉士俊.先天性马蹄内翻足的病因与Hox基因研究进展.中华骨科杂志.2002;22:440-442.
19 Yeo W,Gautier J.Early neural cell death:dying to become neurons.Dev Biol.2004;274:233-244.
20 Yamaguchi TP,Bradley A,McMahon AP,et al.A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo.Development.1999;126:1211-1223.
21 Unified nomenclature for Eph family receptors and their ligands,the ephrins.Eph Nomenclature Committee.Cell.1997;90:403-404.
22 Kiyokawa E,Takai S,Tanaka M,et al.Overexpression of ERK,an EPH family receptor protein tyrosine kinase,in various human tumors.Cancer Res.1994;54:3645-3650.
23 Dravis C,Yokoyama N,Chumley MJ,et al.Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development.Dev Biol.2004;271:272-290.
24 Yucel S,Dravis C,Garc N,et al.Hypospadias and anorectal malformations mediated by defective Eph/ephrin signaling.J Pediatr Urol.2007;3:354-363.
25 Pyati UJ,Cooper MS,Davidson AJ,et al.Sustained Bmp signaling is essential for cloacadevelopment in zebrafish.Development.2006;133:2275-2284.
26 Qi BQ,Beasley SW,Williams AK,et al.Apoptosis during regression of the tailgut and septation of the cloaca.J Pediatr Surg.2000;11:1556-1561.
27 Kubota Y,Shimotake T,Yanagihara J,et al.Development of anorectal malformations using etretinate.J Pediatr Surg.1998;1:127-129.
28 Netzer C,Rieger L,Brero A,et al.SALL1,the gene mutated in Townes-Brocks syndrome,encodes a transcriptional repressor which interacts with TRF1/P1N2 and localizes to pericentromeric heterochromatin.Hum Mol Genet.2001;10:3017-3024.
29 张志波,高红,郭俊斌,等.先天性肛门直肠畸形合并Townes-Brocks综合征SALL1基因突变的研究.中华小儿外科杂志.2005;26:354-356.
30 Bohm J,Munk-Schulenburg S,Felscher S,et al.SALL1 mutations in sporadic Townes-Brocks syndrome are of predominantly paternal origin without obvious paternal age effect.Am J Med Genet A.2006;140:1904-1908.
31 Sakaki-Yumoto M,Kobayashi C,Sato A,et al.The murine homolog of SALL4,a causative gene in Okihiro syndrome, is essential for embryonic stem cell proliferation, and cooperates with Sall1 in anorectal, heart, brain and kidney development. Development. 2006; 133: 3005-3013.
32 Kamasani U, Prendergast GC. Genetic response to DNA damage: proapoptotic targets of RhoB include modules for p53 response and susceptibility to Alzheimer's disease.Cancer Biol Ther. 2005; 4: 282-288.
33 Wang DA, Sebti SM. Palmitoylated cysteine 192 is required for RhoB tumor-suppressive and apoptotic activities. J Biol Chem. 2005; 280: 19243-19249.
34 Jiang K, Sun J, Cheng J, et al. Akt mediates Ras downregulation of RhoB, a suppressor of transformation, invasion, and metastasis. Mol Cell Biol. 2004; 24: 5565-5576.
35 Adini I, Rabinovitz I, Sun JF, et al. RhoB controls Akt trafficking and stage-specific survival of endothelial cells during vascular development. Genes Dev. 2003; 17: 2721-2732.
36 Bowen C, Stuart A, Ju JH, et al. NKX3.1 homeodomain protein binds to topoisomerase I and enhances its activity. Cancer Res. 2007; 67: 455-464.
37 Gelmann EP, Bowen C, Bubendorf L. Expression of NKX3.1 in normal and malignant tissues.Prostate. 2003; 55: 111-117.
38 Sciavolino PJ, Abrams EW, Yang L, et al. Tissue-specific expression of murine Nkx3.1 in the male urogenital system. Dev Dyn. 1997; 209:127-138.
39 Aumailley M, Gayraud B. Structure and biological activity of the extracellular matrix. J Mol Med. 1998; 76: 253-265.
40 Malemud CJ. Matrix metalloproteinases (MMPs) in health and disease: an overview. Front Biosci. 2006; 11: 1696-1701.
41 Pesta M, Holubec L Jr, Topolcan O, et al. Quantitative estimation of matrix metalloproteinases 2 and 7 (MMP-2, MMP-7) and tissue inhibitors of matrix metalloproteinases 1 and 2 (TIMP-1, TIMP-2) in colorectal carcinoma tissue samples. Anticancer Res. 2005; 25: 3387-3391.
42 Chakraborti S, Mandal M, Das S, et al. Regulation of matrix metalloproteinases: an overview. Mol Cell Biochem. 2003; 253: 269-285.
43 Jay P , Goze C ,Marsouier C , et al. The human Soxl1 gene : Cloning , chromosomal assignment and tissue expression. Genomics. 1995; 29: 541-545.
44 Wiebe MS, Nowling TK, Rizzino A. Identification of novel domains within Sox-2 and Sox-11 involved in autoinhibition of DNA binding and partnership specificity. J Biol Chem. 2003; 278:17901-17911.
45 de Martino S, Yan YL, Jowett T, et al. Expression of soxll gene duplicates in zebrafish suggests the reciprocal loss of ancestral gene expression patterns in development. Dev Dyn. 2000; 217: 279-292.
46 Sock E, Rettig SD, Enderich J, et al. Gene targeting reveals a widespread role for the high-mobility-group transcription factor Soxl1 in tissue remodeling. Mol Cell Biol. 2004; 24: 6635-6644.
47 Campbell JS,Prichard L,Schaper F,et al.Expression of suppressors of cytokine signaling during liver regeneration.J Clin Invest.2001;107:1285-1292.
48 Takahashi Y,Dominici M,Swift J,et al.Trophoblast stem cells rescue placental defect in SOCS3-deficient mice.J Biol Chem.2006;281:11444-11445.
49 Wu Z,Wang S,Sorenson CM,et al.Attenuation of retinal vascular development and neovascularization in transgenic mice over-expressing thrombospondin-1 in the lens.Dev Dyn.2006;235:1908-1920.
50 Daniel C,Schaub K,Amann K,et al.Thrombospondin-1 is an endogenous activator of TGF-beta in experimental diabetic nephropathy in vivo.Diabetes.2007;56:2982-2989..
51 Jiang K,Sun J,Cheng J,et al.Akt mediates Ras downregulation of RhoB,a suppressor of transformation,invasion,and metastasis.Mol Cell Biol.2004;24:5565-5576.
52 Kong JY,Rabkin SW.The association between RhoB and caspase-2:changes with lovastatin-induced apoptosis.Biochem Cell Biol.2005;83:608-619.
53 Anselme I,Laclef C,Lanaud M,et al.Defects in brain patterning and head morphogenesis in the mouse mutant Fused toes.Dev Biol.2007;304:208-220.
54 Braun MM,Etheridge A,Bernard A,et al.Writ signaling is required at distinct stages of development for the induction of the posterior forebrain.Development.2003;130:5579-5587.
55 Robinson MD,Speed TP.A comparison of Affymetrix gene expression arrays.BMC Bioinformatics.2007;15:449.
56 Chen JJ,Hsueh HM,Delongcharnp RR,et al.Reproducibility of microarray data:a further analysis of microarray quality control(MAQC) data.BMC Bioinformatics.2007;25:412.
57 Bains W,Smith GC.A novel method for nucleic acid sequence determination.J Theor Biol.1988;135:303-307.
58 Ding C,Cantor CR.Quantitative analysis of nucleic acids—the last few years of progress.J Biochem Mol Biol.2004;37:1-10.
59 Shimkets RA.Gene expression quantitation technology summary.Methods Mol Biol.2004;258:7-12.
60 Wang Y,Barbacioru C,Hyland F,et al.Large scale real-time PCR validation on gene expression measurements from two commercial long-oligonucleotide microarrays.BMC Genomics.2006;21:59.
61 Qi BQ,Beasley SW,Williams AK,et al.Clarification of the process that lead to anorectal malformations in the ETU-induced rat model of imperforate anus.J Pediatr Surg.2002;37:1305-1312.
62 白玉作,王维林,王伟,等.大鼠肛门直肠畸形的胚胎发育研究.中华小儿外科杂志.2003;24:154-157.
63 Bovolenta P. Morphogen signaling at the vertebrate growth cone: a few cases or a general strategy?J Neurobiol. 2005; 15: 405-416.
64 Chong LD, Park EK, Latimer E, et al. Fibroblast growth factor receptor-mediated rescue of x-ephrin B1-induced cell dissociation in Xenopus embryos. Mol Cell Biol. 2000; 20: 724-734.
65 Bruckner K, Pasquale EB, Klein R. Tyrosine phosphorylation of transmembrane ligands for Eph receptors. Science. 1997; 275: 1640-1643.
66 Compagni A, Logan M, Klein R, et al. Control of skeletal patterning by ephrinB1-EphB interactions. Dev Cell. 2003; 5: 217-230.
67 Katakowski M, Zhang Z, deCarvalho AC, et al. EphB2 induces proliferation and promotes a neuronal fate in adult subventricular neural precursor cells. Neurosci Lett. 2005; 385: 204-209.
68 Holmberg J, Genander M, Halford MM, et al. EphB receptors coordinate migration and proliferation in the intestinal stem cell niche. Cell. 2006; 125: 1151-1163.
69 Barrios A, Poole RJ, Durbin L, et al. Eph/Ephrin signaling regulates the mesenchymal-to-epithelial transition of the paraxial mesoderm during somite morphogenesis. Curr Biol. 2003; 13: 1571-1582.
70 Hafner C, Schmitz G, Meyer S, et al. Differential Gene Expression of Eph Receptors and Ephrins in Benign Human Tissues and Cancers. Clinical Chemistry. 2004; 50: 490-499.
71 Wang HU, Chen ZF, Anderson DJ. Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell. 1998; 93: 741-753.
72 Mellitzer G, Xu Q , Wilkinson DG. Eph receptor and ephrins restrict cell intermingling and communication. Nature. 1999; 400: 77-81.
73 Batlle E, Henderson JT, Beghtel H, et al. β -Catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB. Cell. 2002; 111: 251-263.
74 Hafner C, Schmitz G, Meyer S, et al. Differential Gene Expression of Eph Receptors and Ephrins in Benign Human Tissues and Cancers. Clinical Chemistry. 2004; 50: 490-499.
75 Gu J, Gu X. Natural history and functional divergence of protein tyrosine kinases. Gene. 2003; 23:49-57.
76 Himanen JP, Rajashankar KR, Lackmann M, et al. Crystal structure of an Eph receptor-ephrin complex. Nature. 2001; 414: 933-938.
77 Chrencik JE, Brooun A, Recht MI, et al. Three dimensional structure of the EPHB2 receptor in complex with an antagonistic peptide reveals a novel mode of inhibition. J Biol Chem. 2007; 282:36505-36513.
78 Wilkinson DG. Multiple roles of EPH receptors and ephrins in neural development. Nat Rev Neurosci. 2001; 2: 155-164.
79 Mandhan P, Quan QB, Beasley S, et al. Sonic hedgehog, BMP4, and Hox genes in the development of anorectal malformations in Ethylenethiourea-exposed fetal rats. J Pediatr Surg. 2006; 41: 2041-2045.
80 Bovolenta P. Morphogen signaling at the vertebrate growth cone: a few cases or a general strategy?J Neurobiol. 2005; 15: 405-416.
81 Schilling TF, Concordet JP, Ingham PW. Regulation of left-right asymmetries in the zebrafish by Shh and BMP4. Dev Biol. 1999; 15: 277-287.
82 Salsi V, Zappavigna V. Hoxd13 and Hoxa13 directly control the expression of the EphA7 Ephrin tyrosine kinase receptor in developing limbs. J Biol Chem. 2006; 281: 1992-1999.
83 Chong LD, Park EK, Latimer E, et al. Fibroblast growth factor receptor-mediated rescue of x-ephrin B1-induced cell dissociation in Xenopus embryos. Mol Cell Biol. 2000; 20: 724-734.
84 Picco V, Hudson C, Yasuo H. Ephrin-Eph signalling drives the asymmetric division of notochord/neural precursors in Ciona embryos. Development. 2007; 134: 1491-1497.
85 Holmberg J, Genander M, Halford MM, et al. EphB receptors coordinate migration and proliferation in the intestinal stem cell niche. Cell. 2006; 165: 1151-1163.
86 Clevers H, Batlle E. EphB/EphrinB receptors and Wnt signaling in colorectal cancer. Cancer Res. 2006; 66: 2-5.
87 Shi YY, He L. SHEsis, a powerful software platform for analyses of linkage disequilibrium, haplotype construction, and genetic association at polymorphism loci. Cell Res. 2005; 15: 97-98.
88 Nielsen R. Population genetic analysis of ascertained SNP data. Hum Genomics. 2004; 1: 218-224.
89 Taylor JG, Choi EH, Foster CB, et al. Using genetic variation to study human disease. Trends Mol Med. 2001; 7: 507-512.
90 Chasman D, Adams RM. Predicting the functional consequences of nonsynonymous single nucleotide polymorphism: structure based assessment of amino acid variation. J Mol Biol. 2001; 302:683-706.
91 CargillM, Altshuler D, Ireland J, et al. Characterization of single nucleotide polymorphisms in coding regions of human genes. Nat Genet. 1999; 22: 231-238.
1 Cuschieri A.Descriptive epidemiology of isolated anal anomalies:a survey of 4.6 million births in Europe.Am J Med Genet.2001;103:207-215.
2 Stoll C,Alembik Y,Roth MP,et al.Risk factors in congenital anal atresias.Arm Genet.1997;40:197-204.
3 Hori T,Giuffra E,Andersson L,et al.Mapping loci causing susceptibility to anal atresia in pigs,using a resource pedigree.J Pediatr Surg.2001;36:1370-1374.
4 Schwoebel MG,Hirsig J,Schinzel A,et al.Familial incidence of congenital anorectal anomalies.J Pediatr Surg.1984;19:179-182.
5 Martinez-Frias ML,Frias JL,Opitz JM.Errors of morphogenesis and developmental field theory.Am J Med Genet.1998;76:291-296.
6 Kohlhase J,Taschner PE,Burfeind P,et al.Molecular analysis of SALL1 mutations in Townes-Brocks syndrome.Am J Hum Genet.1999;64:435-445.
7 Salerno A,Kohlhase J,Kaplan BS,et al.Yownes-Brocks syndrome and renal dysplasia:a novel mutation in the SALL1 gene.Pediatr Nephrol.2000;14:25-28.
8 Surka WS,Kohlhase J,Neunert CE,et al.Unique family with Townes- Brocks syndrome,SALL1 mutation,and cardiac defects.Am J Med Genet.2001;102:250-257.
9 Keegan CE,Mulliken JB,Wu BL,et al.Townes-Brocks syndrome versus expanded spectrum hemifacial microsomia:review of eight patients and further evidence of a "hot spot" for mutation in the SALL1 gene.Genet Med.2001;3:310-313.
10 Netzer C,Rieger L,Brero A,et al.SALL1,the gene mutated in Townes-Brocks syndrome,encodes a transcriptional repressor which interacts with TRF1/PIN2 and localizes to pericentromeric heterochromatin.Hum Mol Genet.2001;10:3017-3024.
11 张志波,高红,郭俊斌,等.先天性肛门直肠畸形合并Townes-Brocks综合征SALL1基因突变的研究.中华小儿外科杂志.2005;26:354-356.
12 Bohm J,Munk-Schulenburg S,Felscher S,et al.SALL1 mutations in sporadic Townes-Brocks syndrome are of predominantly paternal origin without obvious paternal age effect.Am J Med Genet A.2006;140:1904-1908.
13 Lynch SA,Bond PM,Copp AJ,et al.A gene for autosomal dominant sacral agenesis maps to the holoprosencephaly region at 7q36.Nat Genet.1995;11:93-95.
14 Ross AJ,Ruiz-Perez V,Wang Y,et al.A homeobox gene,HLXB9,is the major locus for dominantly inherited sacral agenesis.Nat Genet.1998;20:358-361.
15 Wang RY,Jones JR,Chen S,et al.A previously unreported mutation in a Currarino syndrome kindred.Am J Med Genet A.2006;140:1923-1930.
16 Cretolle C,Zerah M,Jaubert F,et al.New clinical and therapeutic perspectives in Currarino syndrome(study of 29 cases).J Pediatr Surg.2006;41:126-131.
17 Quaderi NA, Schweiger S, Gaudenz K, et al. Opitz G/BBB syndrome, a defect of midline development, is due to mutations in a new RING finger gene on Xp22. Nat Genet. 1997; 17: 285-291.
18 So J, Suckow V, Kijas Z, et al. Mild phenotypes in a series of patients with Opitz GBBB syndrome with MID1 mutations. Am J Med Genet A. 2005; 132: 1-7.
19 Perry J, Short KM, Romer JT, et al. FXY2/MID2, a gene related to the X-linked Opitz syndrome gene FXY/MID1, maps to Xq22 and encodes a FNIII domain-containing protein that associates with microtubules. Genomics. 1999; 62: 385-394.
20 Mears AJ, Duncan AM, Budarf ML, et al. Molecular characterization of the marker chromosome associated with cat eye syndrome. Am J Hum Genet. 1994; 55: 134-142.
21 Riazi MA, Brinkman-Mills P, Nguyen T, et al. The human homolog of insect-derived growth factor, CECR1, is a candidate gene for features of cat eye syndrome. Genomics. 2000; 64: 277-285.
22 Kang S, Allen J, Graham JM Jr, et al. Linkage mapping and phenotypic analysis of autosomal dominant Pallister-Hall syndrome. J Med Genet. 1997; 34: 441-446.
23 Killoran CE, Abbott M, McKusick VA, et al. Overlap of PIV syndrome, VACTERL and Pallister-Hall syndrome: clinical and molecular analysis. Clin Genet. 2000; 58: 28-30.
24 Mortlock DP, Innis JW. Mutation of HOXA13 in hand-foot-genital syndrome. Nat Genet. 1997; 15: 179-180.
25 Goodman FR, Bacchelli C, Brady AF, et al. Novel HOXA13 mutations and the phenotypic spectrum of hand-foot-genital syndrome. Am J Hum Genet. 2000; 67: 197-202.
26 Graham JM Jr, Tackels D, Dibbern K, et al. FG syndrome: report of three new families with linkage to Xq12-q22.1. Am J Med Genet. 1998; 80: 145-156.
27 Gil da Silva Lopes VL, Guion-Almeida ML, de Oliveira Rodini ES. Blepharocheilodontic (BCD) syndrome: expanding the phenotype? Am J Med Genet A. 2003; 121: 266-270.
28 Mendoza-Londono R, Lammer E, Watson R, et al. Characterization of a new syndrome that associates craniosynostosis, delayed fontanel closure, parietal foramina, imperforate anus, and skin eruption: CDAGS. Am J Hum Genet. 2005; 77: 161-168.
29 Kleist-Retzow JC, Cormier-Daire V, Viot G. Antenatal manifestations of mitochondrial respiratory chain deficiency. J Pediatr. 2003; 143: 208-212.
30 Takano T, Yamanouchi Y, Mori Y, et al. Interstitial deletion of chromosome 1q [del(1)(q24q25.3)] identified by fluorescence in situ hybridization and gene dosage analysis of apolipoprotein A-II, coagulation factor V, and antithrombin III. Am J Med Genet. 1997; 68: 207-210.
31 Mansouri MR, Carlsson B, Davey E, et al. Molecular genetic analysis of a de novo balanced translocation t(6;17)(p21.31;q11.2) associated with hypospadias and anorectal malformation. Hum Genet.2006;119:162-168.
32 Titomanlio L,Giurgea I,Baumann C,et al.A locus for sacral/anorectal malformations maps to 6q25.3 in a 0.3 Mb interval region.Eur J Hum Genet.2006;14:971-974.
33 Park JP,McDermet MK,Moeschler JB,et al.A case of de novo translocation 7;10 and the duplication 7p,deletion 10p phenotype.Ann Genet.1993;36:217-220.
34 Steinbach P,Rehder H.Tetrasomy for the short ARMs of chromosome 12 with accessory isochromosome(+i(12p]and a marked LDH-B gene dosage effect.Clin Genet.1987;32:1-4.
35 Van Buggenhout G,Trommelen J,Hamel B,et al.13q deletion syndrome in an adult mentally retarded patient.Genet Couns.1999;10:177-181.
36 Kuhnle U,Bartsch O,Werner W,et al.Penoscrotal inversion,hypospadias,imperforate anus,facial anomalies,and developmental delay:definition of a new clinical syndrome.Pediatr Surg Int.2000;16:396-399.
37 Chung JL,Choi JR,Park MS,et al.A case of del(13)(q22) with multiple major congenital anomalies,imperforate anus and penoscrotal transposition.Yonsei Med J.2001;42:558-562.
38 Van Hove JL,McConkie-Rosell A,Chen YT,et al.Unbalanced translocation 46,XY,-15,+der(22) t(15;22)(q13;q11) pat:case report and review of the literature.Am J Med Genet.1992;44:24-30.
39 Quan F,Zonana J,Gunter K,et al.An atypical case of fragile X syndrome caused by a deletion that includes the FMR1 gene.Am J Hum Genet.1995;56:1042-1051.
40 Merello E,De Marco P,Mascelli S,et al.HLXB9 homeobox gene and caudal regression syndrome.Birth Defects Res A Clin Mol Teratol.2006;76:205-209.
41 De Marco P,Merello E,Mascelli S,et al.Mutational screening of the CYP26A1 gene in patients with caudal regression syndrome.Birth Defects Res A Clin Mol Teratol.2006;76:86-95.
42 Bird LM,Newbury RO,Ruiz-Velasco R,et al.Recurrence of diaphragmatic agenesis associated with multiple midline defects:evidence for an autosomal gene regulating the midline.Am J Med Genet.1994;53:33-38.
43 De Smet L,Fryns JP.Anal atresia and abdominal wall defect as unusual symptoms in EEC syndrome.Genet Couns.1995;6:127-130.
44 张志波,王练英,李正,等.应用TDT检验确定先天性肛门直肠畸形与HoxA13基因的关系.中华小儿外科杂志.2001;22:240-241.
45 张志波,王练英,李正,等.先天性无肛畸形HoxA-13基因的改变.中华小儿外科杂志.2001;23:269-270.
46 Yu Zuo Bai,Hong Gao,Wei Lin Wang.Expression of Hoxd-13 gene in children with congenital anorectal malformation.Chin J Contemp Pdiatr.2003;5:201-204.
47 邢茂青,王俊文,张金哲.先天性肛门直肠畸形Hoxc-8基因表达的研究及其意义。中华 小儿外科杂志.2004;25:258-260.
48 白玉作,王维林,黄英,等.人类肛门直肠畸形基因表达谱的研究。中国医科大学学报.2003;32:493-495.
49 Kimmel SG,Mo R,Hui CC,et al.New mouse models of congenital anorectal malformations.J Pediatr Surg.2000;35:227-230.
50 Mo R,Kim JH,Zhang J,et al.Anorectal malformations caused by defects in sonic hedgehog signaling.Am J Pathol.2001;159:765-774.
51 Fukuda K,Yasugi S.Versatile roles for sonic hedgehog in gut development.J Gastroenterol.2002;37:239-246.
52 Arsic D,ARMSeron V,Ellmers L,et al.Adriamycin Disruption of the Shh-Gli Pathway is Associated With Abnormalities of Foregut Development.Journal of Pediatric Surgery.2004;39:1747-1753.
53 Roberts DJ,Johnson RL,Burke AC,et al.Sonic hedgehog is an endodermal signal inducing Bmp4 and Hox genes of chick hindgut.Development.1995;121:3163-3174.
54 Sasaki Y,Iwai N,Tsuda T,et al.Sonic hedgehog and bone morphogenetic protein 4expressions in the hindgut region of murine embryos with anorectal malformations.J Pediatr Surg.2004;39:170-173.
55 Mandhan P,Beasley S,Hale T,et al.Sonic hedgehog expression in the development of hindgut in ETU-exposed fetal rats.Pediatr Surg Int.2006;22:31-36.
56 Mortell A,O'Donnell AM,Giles S,et al.Adriamycin induces notochord hypertrophy with conservation of sonic hedgehog expression in abnormal ectopic notochord in the adriamycin rat model.J Pediatr Surg.2004;39:859-863.
57 Bose J,Grotewold L,Ruther U,et al.Pallister-Hall syndrome phenotype in mice mutant for Gli3.Hum Mol Genet.2002;11:1129-1135.
58 靳曙光,赵玉元,高明太,等.乙烯硫脲致畸胎鼠直肠末端Gli2基因的表达.实用儿科临床杂志.2005;20:180-182.
59 Cassini P,Montironi A,Botti S,et al.Genetic analysis of anal atresia in pigs:evidence for segregation at two main loci.Mamm Genome.2005;16:164-170.
60 王大佳,陈秋,吉士俊.先天性马蹄内翻足的病因与Hox基因研究进展.中华骨科杂志.2002;22:440-442.
61 Kondo T,Dolle P,Zakany J,et al.Function of posterior HoxD genes in the morphogenisis of the anal sphincter.Development.1996;122:2651-2659.
62 Warot X,Fromental-Ramain C,Fraulob V,et al.Gene dosage- dependent effects of the Hoxa-13 and Hoxd-13 mutations on morphogenesis of the terminal parts of the digestive and urogenital tracts.Development.1997;124:4781-4791.
63 Pyati UJ,Cooper MS,Davidson AJ,et al.Sustained Bmp signaling is essential for cloaca development in zebrafish. Development. 2006; 133: 2275-2284.
64 Freeman M. Feedback control of intercellular signalling in development. Nature. 2000; 408: 313-319.
65 Itoh N. FGFs as multifunctional signaling molecules: diversity of structure and function. Seikagaku. 2001; 73: 525-535.
66 Fairbanks TJ, De Langhe S, Sala FG, et al. Fibroblast growth factor 10 (Fgf10) invalidation results in anorectal malformation in mice. J Pediatr Surg. 2004; 39: 360-365.
67 Conover JC, Doetsch F, Garcia-Verdugo JM, et al. Disruption of Eph /ephrin signaling affects migration and proliferation in the subventricular zone. Nat Neurosci. 2000; 3: 1091-1097.
68 Mellitzer G, Xu Q, Wilkinson DG, et al. Eph receptors and ephrins restrict cell intermingling and communication. Nature. 1999; 400: 77-81.
69 Dravis C, Yokoyama N, Chumley MJ, et al. Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development. Dev Biol. 2004; 271: 272-290.
70 Salsi V, Zappavigna V. Hoxd13 and Hoxa13 directly control the expression of the EphA7 Ephrin tyrosine kinase receptor in developing limbs. J Biol Chem. 2006; 281: 1992-1999.
71 Yamaguchi TP, Bradley A, McMahon AP, et al. A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo. Development. 1999; 126: 1211-1223.
72 Lee H, Kimelman D. A dominant-negative form of p63 is required for epidermal proliferation in zebrafish. Dev Cell. 2002; 2: 607-616.
73 Ince TA, Cviko AP, Quade BJ, et al. p63 Coordinates Anogenital Modeling and Epithelial Cell Differentiation in the Developing Female Urogenital Tract. American Journal of Pathology. 2002; 161: 1111-1117.
74 郭俊斌,张志波,王练英,等．维甲酸诱导大鼠肛门直肠畸形胚胎中RARMsRNA表达的研究．中华小儿外科杂志．2005；26：256-259．
75 Sakaki-Yumoto M, Kobayashi C, Sato A, et al. The murine homolog of SALL4, a causative gene in Okihiro syndrome, is essential for embryonic stem cell proliferation, and cooperates with Sall1 in anorectal, heart, brain and kidney development. Development. 2006; 133: 3005-3013.
76 Wiedemann S, Fries R, Thaller G. Genomewide scan for anal atresia in swine identifies linkage and association with a chromosome region on Sus scrofa chromosome 1. Genetics. 2005; 171: 1207-1217.
77 Papapetrou C, Drummond F, Reardon W, et al. A genetic study of the human T gene and its exclusion as a major candidate gene for sacral agenesis with anorectal atresia. J Med Genet. 1999; 36:208-213.
78 Seri M, Martucciello G, Paleari L, et al. Exclusion of the Sonic Hedgehog gene as responsible for Currarino syndrome and anorectal malformations with sacral hypodevelopment. Hum Genet. 1999; 104:108-110.
79 Stoll C, De Saint Martin A, Donato L, et al. Pallister-Hall syndrome with stenosis of the cricoid cartilage and microphallus without hypopituitarism. Genet Couns. 2001; 12: 231-235.
80 Bao Quan Qi, Beasley SW, Williams AK, et al. Apoptosis during regression of the tailgut and septation of the cloaca. J. Pediatr Surg. 2000; 35: 1556-1561.
81 Sasaki C, Yamaguchi K, Akita K. Spatiotemporal distribution of apoptosis during normal cloacal development in mice. Anatomical Record A. 2004; 279: 761-767.
2 Raponi M, Harousseau JL, Lancet JE, et al. Identification of molecular predictors of response in a study of tipifarnib treatment in relapsed and refractory acute myelogenous leukemia.Clin Cancer Res. 2007; 13:2254-2260.
3 Heanue TA, Pachnis V. Expression profiling the developing mammalian enteric nervous system identifies marker and candidate Hirschsprung disease genes.Proc Natl Acad Sci U S A. 2006; 103:6919-6924.
4 Cuschieri A. Descriptive epidemiology of isolated anal anomalies : a survey of 4. 6 million births in Europe. Am J Med Genet. 2001; 103: 207-215.
5 Stoll C, Alembik Y, Roth MP, et al. Risk factors in congenital anal atresias. Ann Genet. 1997; 40: 197-204.
6 Hori T, Giuffra E, Andersson L, et al. Mapping loci causing susceptibility to anal atresia in pigs, using a resource pedigree. J Pediatr Surg. 2001; 36: 1370-1374.
7 白玉作,王维林,黄英,等．人类肛门直肠畸形基因表达谱的研究。中国医科大学学报．2003：32：493-495．
8 Mo R, Kim JH, Zhang J, et al. Anorectal malformations caused by defects in sonic hedgehog signaling. Am J Pathol. 2001; 159: 765-74.
9 Kimmel SG, Mo R, Hui CC, et al. New mouse models of congenital anorectal malformations. J Pediatr Surg. 2000; 35: 227-30.
10 Mandhan P, Beasley S, Hale T, et al. Sonic hedgehog expression in the development of hindgut in ETU-exposed fetal rats. Pediatr Surg Int. 2006; 22: 31-36.
11 Sasaki Y, Iwai N, Tsuda T, et al. Sonic hedgehog and bone morphogenetic protein 4 expressions in the hindgut region of murine embryos with anorectal malformations. J Pediatr Surg. 2004; 39: 170-173.
12 Mandhan P, Quan QB, Beasley S, et al. Sonic hedgehog, BMP4, and Hox genes in the development of anorectal malformations in Ethylenethiourea-exposed fetal rats. J Pediatr Surg. 2006; 41: 2041-2045.
13 Fairbanks TJ, De Langhe S, Sala FG, et al. Fibroblast growth factor 10 (Fgf10) invalidation results in anorectal malformation in mice. J Pediatr Surg. 2004; 39: 360-365.
14 Kondo T, Dolle P, Zakany J, et al. Function of posterior HoxD genes in the morphogenisis of the anal sphincter. Development. 1996; 122: 2651-2659.
15 Warot X, Fromental Ramain C, Fraulob V, et al. Gene dosage-dependent effects of the Hoxa-13 and Hoxd-13 mutations on morphogenesis of the terminal parts of the digestive and urogenital tracts.Development.1997;124:4781-4791.
16 Sukegawa A,Narita T,Kameda T,et al.The concentric structure of the developing gut is regulated by Sonic hedgehog derived from endodermal epithelium.Development.2000;127:1971-80.
17 Arsic D,ARMSeron V,Ellmers L,et al.Adriamycin Disruption of the Shh-Gli Pathway is Associated With Abnormalities of Foregut Development.Journal of Pediatric Surgery.2004;39:1747-1753.
18 王大佳,陈秋,吉士俊.先天性马蹄内翻足的病因与Hox基因研究进展.中华骨科杂志.2002;22:440-442.
19 Yeo W,Gautier J.Early neural cell death:dying to become neurons.Dev Biol.2004;274:233-244.
20 Yamaguchi TP,Bradley A,McMahon AP,et al.A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo.Development.1999;126:1211-1223.
21 Unified nomenclature for Eph family receptors and their ligands,the ephrins.Eph Nomenclature Committee.Cell.1997;90:403-404.
22 Kiyokawa E,Takai S,Tanaka M,et al.Overexpression of ERK,an EPH family receptor protein tyrosine kinase,in various human tumors.Cancer Res.1994;54:3645-3650.
23 Dravis C,Yokoyama N,Chumley MJ,et al.Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development.Dev Biol.2004;271:272-290.
24 Yucel S,Dravis C,Garc N,et al.Hypospadias and anorectal malformations mediated by defective Eph/ephrin signaling.J Pediatr Urol.2007;3:354-363.
25 Pyati UJ,Cooper MS,Davidson AJ,et al.Sustained Bmp signaling is essential for cloacadevelopment in zebrafish.Development.2006;133:2275-2284.
26 Qi BQ,Beasley SW,Williams AK,et al.Apoptosis during regression of the tailgut and septation of the cloaca.J Pediatr Surg.2000;11:1556-1561.
27 Kubota Y,Shimotake T,Yanagihara J,et al.Development of anorectal malformations using etretinate.J Pediatr Surg.1998;1:127-129.
28 Netzer C,Rieger L,Brero A,et al.SALL1,the gene mutated in Townes-Brocks syndrome,encodes a transcriptional repressor which interacts with TRF1/P1N2 and localizes to pericentromeric heterochromatin.Hum Mol Genet.2001;10:3017-3024.
29 张志波,高红,郭俊斌,等.先天性肛门直肠畸形合并Townes-Brocks综合征SALL1基因突变的研究.中华小儿外科杂志.2005;26:354-356.
30 Bohm J,Munk-Schulenburg S,Felscher S,et al.SALL1 mutations in sporadic Townes-Brocks syndrome are of predominantly paternal origin without obvious paternal age effect.Am J Med Genet A.2006;140:1904-1908.
31 Sakaki-Yumoto M,Kobayashi C,Sato A,et al.The murine homolog of SALL4,a causative gene in Okihiro syndrome, is essential for embryonic stem cell proliferation, and cooperates with Sall1 in anorectal, heart, brain and kidney development. Development. 2006; 133: 3005-3013.
32 Kamasani U, Prendergast GC. Genetic response to DNA damage: proapoptotic targets of RhoB include modules for p53 response and susceptibility to Alzheimer's disease.Cancer Biol Ther. 2005; 4: 282-288.
33 Wang DA, Sebti SM. Palmitoylated cysteine 192 is required for RhoB tumor-suppressive and apoptotic activities. J Biol Chem. 2005; 280: 19243-19249.
34 Jiang K, Sun J, Cheng J, et al. Akt mediates Ras downregulation of RhoB, a suppressor of transformation, invasion, and metastasis. Mol Cell Biol. 2004; 24: 5565-5576.
35 Adini I, Rabinovitz I, Sun JF, et al. RhoB controls Akt trafficking and stage-specific survival of endothelial cells during vascular development. Genes Dev. 2003; 17: 2721-2732.
36 Bowen C, Stuart A, Ju JH, et al. NKX3.1 homeodomain protein binds to topoisomerase I and enhances its activity. Cancer Res. 2007; 67: 455-464.
37 Gelmann EP, Bowen C, Bubendorf L. Expression of NKX3.1 in normal and malignant tissues.Prostate. 2003; 55: 111-117.
38 Sciavolino PJ, Abrams EW, Yang L, et al. Tissue-specific expression of murine Nkx3.1 in the male urogenital system. Dev Dyn. 1997; 209:127-138.
39 Aumailley M, Gayraud B. Structure and biological activity of the extracellular matrix. J Mol Med. 1998; 76: 253-265.
40 Malemud CJ. Matrix metalloproteinases (MMPs) in health and disease: an overview. Front Biosci. 2006; 11: 1696-1701.
41 Pesta M, Holubec L Jr, Topolcan O, et al. Quantitative estimation of matrix metalloproteinases 2 and 7 (MMP-2, MMP-7) and tissue inhibitors of matrix metalloproteinases 1 and 2 (TIMP-1, TIMP-2) in colorectal carcinoma tissue samples. Anticancer Res. 2005; 25: 3387-3391.
42 Chakraborti S, Mandal M, Das S, et al. Regulation of matrix metalloproteinases: an overview. Mol Cell Biochem. 2003; 253: 269-285.
43 Jay P , Goze C ,Marsouier C , et al. The human Soxl1 gene : Cloning , chromosomal assignment and tissue expression. Genomics. 1995; 29: 541-545.
44 Wiebe MS, Nowling TK, Rizzino A. Identification of novel domains within Sox-2 and Sox-11 involved in autoinhibition of DNA binding and partnership specificity. J Biol Chem. 2003; 278:17901-17911.
45 de Martino S, Yan YL, Jowett T, et al. Expression of soxll gene duplicates in zebrafish suggests the reciprocal loss of ancestral gene expression patterns in development. Dev Dyn. 2000; 217: 279-292.
46 Sock E, Rettig SD, Enderich J, et al. Gene targeting reveals a widespread role for the high-mobility-group transcription factor Soxl1 in tissue remodeling. Mol Cell Biol. 2004; 24: 6635-6644.
47 Campbell JS,Prichard L,Schaper F,et al.Expression of suppressors of cytokine signaling during liver regeneration.J Clin Invest.2001;107:1285-1292.
48 Takahashi Y,Dominici M,Swift J,et al.Trophoblast stem cells rescue placental defect in SOCS3-deficient mice.J Biol Chem.2006;281:11444-11445.
49 Wu Z,Wang S,Sorenson CM,et al.Attenuation of retinal vascular development and neovascularization in transgenic mice over-expressing thrombospondin-1 in the lens.Dev Dyn.2006;235:1908-1920.
50 Daniel C,Schaub K,Amann K,et al.Thrombospondin-1 is an endogenous activator of TGF-beta in experimental diabetic nephropathy in vivo.Diabetes.2007;56:2982-2989..
51 Jiang K,Sun J,Cheng J,et al.Akt mediates Ras downregulation of RhoB,a suppressor of transformation,invasion,and metastasis.Mol Cell Biol.2004;24:5565-5576.
52 Kong JY,Rabkin SW.The association between RhoB and caspase-2:changes with lovastatin-induced apoptosis.Biochem Cell Biol.2005;83:608-619.
53 Anselme I,Laclef C,Lanaud M,et al.Defects in brain patterning and head morphogenesis in the mouse mutant Fused toes.Dev Biol.2007;304:208-220.
54 Braun MM,Etheridge A,Bernard A,et al.Writ signaling is required at distinct stages of development for the induction of the posterior forebrain.Development.2003;130:5579-5587.
55 Robinson MD,Speed TP.A comparison of Affymetrix gene expression arrays.BMC Bioinformatics.2007;15:449.
56 Chen JJ,Hsueh HM,Delongcharnp RR,et al.Reproducibility of microarray data:a further analysis of microarray quality control(MAQC) data.BMC Bioinformatics.2007;25:412.
57 Bains W,Smith GC.A novel method for nucleic acid sequence determination.J Theor Biol.1988;135:303-307.
58 Ding C,Cantor CR.Quantitative analysis of nucleic acids—the last few years of progress.J Biochem Mol Biol.2004;37:1-10.
59 Shimkets RA.Gene expression quantitation technology summary.Methods Mol Biol.2004;258:7-12.
60 Wang Y,Barbacioru C,Hyland F,et al.Large scale real-time PCR validation on gene expression measurements from two commercial long-oligonucleotide microarrays.BMC Genomics.2006;21:59.
61 Qi BQ,Beasley SW,Williams AK,et al.Clarification of the process that lead to anorectal malformations in the ETU-induced rat model of imperforate anus.J Pediatr Surg.2002;37:1305-1312.
62 白玉作,王维林,王伟,等.大鼠肛门直肠畸形的胚胎发育研究.中华小儿外科杂志.2003;24:154-157.
63 Bovolenta P. Morphogen signaling at the vertebrate growth cone: a few cases or a general strategy?J Neurobiol. 2005; 15: 405-416.
64 Chong LD, Park EK, Latimer E, et al. Fibroblast growth factor receptor-mediated rescue of x-ephrin B1-induced cell dissociation in Xenopus embryos. Mol Cell Biol. 2000; 20: 724-734.
65 Bruckner K, Pasquale EB, Klein R. Tyrosine phosphorylation of transmembrane ligands for Eph receptors. Science. 1997; 275: 1640-1643.
66 Compagni A, Logan M, Klein R, et al. Control of skeletal patterning by ephrinB1-EphB interactions. Dev Cell. 2003; 5: 217-230.
67 Katakowski M, Zhang Z, deCarvalho AC, et al. EphB2 induces proliferation and promotes a neuronal fate in adult subventricular neural precursor cells. Neurosci Lett. 2005; 385: 204-209.
68 Holmberg J, Genander M, Halford MM, et al. EphB receptors coordinate migration and proliferation in the intestinal stem cell niche. Cell. 2006; 125: 1151-1163.
69 Barrios A, Poole RJ, Durbin L, et al. Eph/Ephrin signaling regulates the mesenchymal-to-epithelial transition of the paraxial mesoderm during somite morphogenesis. Curr Biol. 2003; 13: 1571-1582.
70 Hafner C, Schmitz G, Meyer S, et al. Differential Gene Expression of Eph Receptors and Ephrins in Benign Human Tissues and Cancers. Clinical Chemistry. 2004; 50: 490-499.
71 Wang HU, Chen ZF, Anderson DJ. Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell. 1998; 93: 741-753.
72 Mellitzer G, Xu Q , Wilkinson DG. Eph receptor and ephrins restrict cell intermingling and communication. Nature. 1999; 400: 77-81.
73 Batlle E, Henderson JT, Beghtel H, et al. β -Catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB. Cell. 2002; 111: 251-263.
74 Hafner C, Schmitz G, Meyer S, et al. Differential Gene Expression of Eph Receptors and Ephrins in Benign Human Tissues and Cancers. Clinical Chemistry. 2004; 50: 490-499.
75 Gu J, Gu X. Natural history and functional divergence of protein tyrosine kinases. Gene. 2003; 23:49-57.
76 Himanen JP, Rajashankar KR, Lackmann M, et al. Crystal structure of an Eph receptor-ephrin complex. Nature. 2001; 414: 933-938.
77 Chrencik JE, Brooun A, Recht MI, et al. Three dimensional structure of the EPHB2 receptor in complex with an antagonistic peptide reveals a novel mode of inhibition. J Biol Chem. 2007; 282:36505-36513.
78 Wilkinson DG. Multiple roles of EPH receptors and ephrins in neural development. Nat Rev Neurosci. 2001; 2: 155-164.
79 Mandhan P, Quan QB, Beasley S, et al. Sonic hedgehog, BMP4, and Hox genes in the development of anorectal malformations in Ethylenethiourea-exposed fetal rats. J Pediatr Surg. 2006; 41: 2041-2045.
80 Bovolenta P. Morphogen signaling at the vertebrate growth cone: a few cases or a general strategy?J Neurobiol. 2005; 15: 405-416.
81 Schilling TF, Concordet JP, Ingham PW. Regulation of left-right asymmetries in the zebrafish by Shh and BMP4. Dev Biol. 1999; 15: 277-287.
82 Salsi V, Zappavigna V. Hoxd13 and Hoxa13 directly control the expression of the EphA7 Ephrin tyrosine kinase receptor in developing limbs. J Biol Chem. 2006; 281: 1992-1999.
83 Chong LD, Park EK, Latimer E, et al. Fibroblast growth factor receptor-mediated rescue of x-ephrin B1-induced cell dissociation in Xenopus embryos. Mol Cell Biol. 2000; 20: 724-734.
84 Picco V, Hudson C, Yasuo H. Ephrin-Eph signalling drives the asymmetric division of notochord/neural precursors in Ciona embryos. Development. 2007; 134: 1491-1497.
85 Holmberg J, Genander M, Halford MM, et al. EphB receptors coordinate migration and proliferation in the intestinal stem cell niche. Cell. 2006; 165: 1151-1163.
86 Clevers H, Batlle E. EphB/EphrinB receptors and Wnt signaling in colorectal cancer. Cancer Res. 2006; 66: 2-5.
87 Shi YY, He L. SHEsis, a powerful software platform for analyses of linkage disequilibrium, haplotype construction, and genetic association at polymorphism loci. Cell Res. 2005; 15: 97-98.
88 Nielsen R. Population genetic analysis of ascertained SNP data. Hum Genomics. 2004; 1: 218-224.
89 Taylor JG, Choi EH, Foster CB, et al. Using genetic variation to study human disease. Trends Mol Med. 2001; 7: 507-512.
90 Chasman D, Adams RM. Predicting the functional consequences of nonsynonymous single nucleotide polymorphism: structure based assessment of amino acid variation. J Mol Biol. 2001; 302:683-706.
91 CargillM, Altshuler D, Ireland J, et al. Characterization of single nucleotide polymorphisms in coding regions of human genes. Nat Genet. 1999; 22: 231-238.
1 Cuschieri A.Descriptive epidemiology of isolated anal anomalies:a survey of 4.6 million births in Europe.Am J Med Genet.2001;103:207-215.
2 Stoll C,Alembik Y,Roth MP,et al.Risk factors in congenital anal atresias.Arm Genet.1997;40:197-204.
3 Hori T,Giuffra E,Andersson L,et al.Mapping loci causing susceptibility to anal atresia in pigs,using a resource pedigree.J Pediatr Surg.2001;36:1370-1374.
4 Schwoebel MG,Hirsig J,Schinzel A,et al.Familial incidence of congenital anorectal anomalies.J Pediatr Surg.1984;19:179-182.
5 Martinez-Frias ML,Frias JL,Opitz JM.Errors of morphogenesis and developmental field theory.Am J Med Genet.1998;76:291-296.
6 Kohlhase J,Taschner PE,Burfeind P,et al.Molecular analysis of SALL1 mutations in Townes-Brocks syndrome.Am J Hum Genet.1999;64:435-445.
7 Salerno A,Kohlhase J,Kaplan BS,et al.Yownes-Brocks syndrome and renal dysplasia:a novel mutation in the SALL1 gene.Pediatr Nephrol.2000;14:25-28.
8 Surka WS,Kohlhase J,Neunert CE,et al.Unique family with Townes- Brocks syndrome,SALL1 mutation,and cardiac defects.Am J Med Genet.2001;102:250-257.
9 Keegan CE,Mulliken JB,Wu BL,et al.Townes-Brocks syndrome versus expanded spectrum hemifacial microsomia:review of eight patients and further evidence of a "hot spot" for mutation in the SALL1 gene.Genet Med.2001;3:310-313.
10 Netzer C,Rieger L,Brero A,et al.SALL1,the gene mutated in Townes-Brocks syndrome,encodes a transcriptional repressor which interacts with TRF1/PIN2 and localizes to pericentromeric heterochromatin.Hum Mol Genet.2001;10:3017-3024.
11 张志波,高红,郭俊斌,等.先天性肛门直肠畸形合并Townes-Brocks综合征SALL1基因突变的研究.中华小儿外科杂志.2005;26:354-356.
12 Bohm J,Munk-Schulenburg S,Felscher S,et al.SALL1 mutations in sporadic Townes-Brocks syndrome are of predominantly paternal origin without obvious paternal age effect.Am J Med Genet A.2006;140:1904-1908.
13 Lynch SA,Bond PM,Copp AJ,et al.A gene for autosomal dominant sacral agenesis maps to the holoprosencephaly region at 7q36.Nat Genet.1995;11:93-95.
14 Ross AJ,Ruiz-Perez V,Wang Y,et al.A homeobox gene,HLXB9,is the major locus for dominantly inherited sacral agenesis.Nat Genet.1998;20:358-361.
15 Wang RY,Jones JR,Chen S,et al.A previously unreported mutation in a Currarino syndrome kindred.Am J Med Genet A.2006;140:1923-1930.
16 Cretolle C,Zerah M,Jaubert F,et al.New clinical and therapeutic perspectives in Currarino syndrome(study of 29 cases).J Pediatr Surg.2006;41:126-131.
17 Quaderi NA, Schweiger S, Gaudenz K, et al. Opitz G/BBB syndrome, a defect of midline development, is due to mutations in a new RING finger gene on Xp22. Nat Genet. 1997; 17: 285-291.
18 So J, Suckow V, Kijas Z, et al. Mild phenotypes in a series of patients with Opitz GBBB syndrome with MID1 mutations. Am J Med Genet A. 2005; 132: 1-7.
19 Perry J, Short KM, Romer JT, et al. FXY2/MID2, a gene related to the X-linked Opitz syndrome gene FXY/MID1, maps to Xq22 and encodes a FNIII domain-containing protein that associates with microtubules. Genomics. 1999; 62: 385-394.
20 Mears AJ, Duncan AM, Budarf ML, et al. Molecular characterization of the marker chromosome associated with cat eye syndrome. Am J Hum Genet. 1994; 55: 134-142.
21 Riazi MA, Brinkman-Mills P, Nguyen T, et al. The human homolog of insect-derived growth factor, CECR1, is a candidate gene for features of cat eye syndrome. Genomics. 2000; 64: 277-285.
22 Kang S, Allen J, Graham JM Jr, et al. Linkage mapping and phenotypic analysis of autosomal dominant Pallister-Hall syndrome. J Med Genet. 1997; 34: 441-446.
23 Killoran CE, Abbott M, McKusick VA, et al. Overlap of PIV syndrome, VACTERL and Pallister-Hall syndrome: clinical and molecular analysis. Clin Genet. 2000; 58: 28-30.
24 Mortlock DP, Innis JW. Mutation of HOXA13 in hand-foot-genital syndrome. Nat Genet. 1997; 15: 179-180.
25 Goodman FR, Bacchelli C, Brady AF, et al. Novel HOXA13 mutations and the phenotypic spectrum of hand-foot-genital syndrome. Am J Hum Genet. 2000; 67: 197-202.
26 Graham JM Jr, Tackels D, Dibbern K, et al. FG syndrome: report of three new families with linkage to Xq12-q22.1. Am J Med Genet. 1998; 80: 145-156.
27 Gil da Silva Lopes VL, Guion-Almeida ML, de Oliveira Rodini ES. Blepharocheilodontic (BCD) syndrome: expanding the phenotype? Am J Med Genet A. 2003; 121: 266-270.
28 Mendoza-Londono R, Lammer E, Watson R, et al. Characterization of a new syndrome that associates craniosynostosis, delayed fontanel closure, parietal foramina, imperforate anus, and skin eruption: CDAGS. Am J Hum Genet. 2005; 77: 161-168.
29 Kleist-Retzow JC, Cormier-Daire V, Viot G. Antenatal manifestations of mitochondrial respiratory chain deficiency. J Pediatr. 2003; 143: 208-212.
30 Takano T, Yamanouchi Y, Mori Y, et al. Interstitial deletion of chromosome 1q [del(1)(q24q25.3)] identified by fluorescence in situ hybridization and gene dosage analysis of apolipoprotein A-II, coagulation factor V, and antithrombin III. Am J Med Genet. 1997; 68: 207-210.
31 Mansouri MR, Carlsson B, Davey E, et al. Molecular genetic analysis of a de novo balanced translocation t(6;17)(p21.31;q11.2) associated with hypospadias and anorectal malformation. Hum Genet.2006;119:162-168.
32 Titomanlio L,Giurgea I,Baumann C,et al.A locus for sacral/anorectal malformations maps to 6q25.3 in a 0.3 Mb interval region.Eur J Hum Genet.2006;14:971-974.
33 Park JP,McDermet MK,Moeschler JB,et al.A case of de novo translocation 7;10 and the duplication 7p,deletion 10p phenotype.Ann Genet.1993;36:217-220.
34 Steinbach P,Rehder H.Tetrasomy for the short ARMs of chromosome 12 with accessory isochromosome(+i(12p]and a marked LDH-B gene dosage effect.Clin Genet.1987;32:1-4.
35 Van Buggenhout G,Trommelen J,Hamel B,et al.13q deletion syndrome in an adult mentally retarded patient.Genet Couns.1999;10:177-181.
36 Kuhnle U,Bartsch O,Werner W,et al.Penoscrotal inversion,hypospadias,imperforate anus,facial anomalies,and developmental delay:definition of a new clinical syndrome.Pediatr Surg Int.2000;16:396-399.
37 Chung JL,Choi JR,Park MS,et al.A case of del(13)(q22) with multiple major congenital anomalies,imperforate anus and penoscrotal transposition.Yonsei Med J.2001;42:558-562.
38 Van Hove JL,McConkie-Rosell A,Chen YT,et al.Unbalanced translocation 46,XY,-15,+der(22) t(15;22)(q13;q11) pat:case report and review of the literature.Am J Med Genet.1992;44:24-30.
39 Quan F,Zonana J,Gunter K,et al.An atypical case of fragile X syndrome caused by a deletion that includes the FMR1 gene.Am J Hum Genet.1995;56:1042-1051.
40 Merello E,De Marco P,Mascelli S,et al.HLXB9 homeobox gene and caudal regression syndrome.Birth Defects Res A Clin Mol Teratol.2006;76:205-209.
41 De Marco P,Merello E,Mascelli S,et al.Mutational screening of the CYP26A1 gene in patients with caudal regression syndrome.Birth Defects Res A Clin Mol Teratol.2006;76:86-95.
42 Bird LM,Newbury RO,Ruiz-Velasco R,et al.Recurrence of diaphragmatic agenesis associated with multiple midline defects:evidence for an autosomal gene regulating the midline.Am J Med Genet.1994;53:33-38.
43 De Smet L,Fryns JP.Anal atresia and abdominal wall defect as unusual symptoms in EEC syndrome.Genet Couns.1995;6:127-130.
44 张志波,王练英,李正,等.应用TDT检验确定先天性肛门直肠畸形与HoxA13基因的关系.中华小儿外科杂志.2001;22:240-241.
45 张志波,王练英,李正,等.先天性无肛畸形HoxA-13基因的改变.中华小儿外科杂志.2001;23:269-270.
46 Yu Zuo Bai,Hong Gao,Wei Lin Wang.Expression of Hoxd-13 gene in children with congenital anorectal malformation.Chin J Contemp Pdiatr.2003;5:201-204.
47 邢茂青,王俊文,张金哲.先天性肛门直肠畸形Hoxc-8基因表达的研究及其意义。中华 小儿外科杂志.2004;25:258-260.
48 白玉作,王维林,黄英,等.人类肛门直肠畸形基因表达谱的研究。中国医科大学学报.2003;32:493-495.
49 Kimmel SG,Mo R,Hui CC,et al.New mouse models of congenital anorectal malformations.J Pediatr Surg.2000;35:227-230.
50 Mo R,Kim JH,Zhang J,et al.Anorectal malformations caused by defects in sonic hedgehog signaling.Am J Pathol.2001;159:765-774.
51 Fukuda K,Yasugi S.Versatile roles for sonic hedgehog in gut development.J Gastroenterol.2002;37:239-246.
52 Arsic D,ARMSeron V,Ellmers L,et al.Adriamycin Disruption of the Shh-Gli Pathway is Associated With Abnormalities of Foregut Development.Journal of Pediatric Surgery.2004;39:1747-1753.
53 Roberts DJ,Johnson RL,Burke AC,et al.Sonic hedgehog is an endodermal signal inducing Bmp4 and Hox genes of chick hindgut.Development.1995;121:3163-3174.
54 Sasaki Y,Iwai N,Tsuda T,et al.Sonic hedgehog and bone morphogenetic protein 4expressions in the hindgut region of murine embryos with anorectal malformations.J Pediatr Surg.2004;39:170-173.
55 Mandhan P,Beasley S,Hale T,et al.Sonic hedgehog expression in the development of hindgut in ETU-exposed fetal rats.Pediatr Surg Int.2006;22:31-36.
56 Mortell A,O'Donnell AM,Giles S,et al.Adriamycin induces notochord hypertrophy with conservation of sonic hedgehog expression in abnormal ectopic notochord in the adriamycin rat model.J Pediatr Surg.2004;39:859-863.
57 Bose J,Grotewold L,Ruther U,et al.Pallister-Hall syndrome phenotype in mice mutant for Gli3.Hum Mol Genet.2002;11:1129-1135.
58 靳曙光,赵玉元,高明太,等.乙烯硫脲致畸胎鼠直肠末端Gli2基因的表达.实用儿科临床杂志.2005;20:180-182.
59 Cassini P,Montironi A,Botti S,et al.Genetic analysis of anal atresia in pigs:evidence for segregation at two main loci.Mamm Genome.2005;16:164-170.
60 王大佳,陈秋,吉士俊.先天性马蹄内翻足的病因与Hox基因研究进展.中华骨科杂志.2002;22:440-442.
61 Kondo T,Dolle P,Zakany J,et al.Function of posterior HoxD genes in the morphogenisis of the anal sphincter.Development.1996;122:2651-2659.
62 Warot X,Fromental-Ramain C,Fraulob V,et al.Gene dosage- dependent effects of the Hoxa-13 and Hoxd-13 mutations on morphogenesis of the terminal parts of the digestive and urogenital tracts.Development.1997;124:4781-4791.
63 Pyati UJ,Cooper MS,Davidson AJ,et al.Sustained Bmp signaling is essential for cloaca development in zebrafish. Development. 2006; 133: 2275-2284.
64 Freeman M. Feedback control of intercellular signalling in development. Nature. 2000; 408: 313-319.
65 Itoh N. FGFs as multifunctional signaling molecules: diversity of structure and function. Seikagaku. 2001; 73: 525-535.
66 Fairbanks TJ, De Langhe S, Sala FG, et al. Fibroblast growth factor 10 (Fgf10) invalidation results in anorectal malformation in mice. J Pediatr Surg. 2004; 39: 360-365.
67 Conover JC, Doetsch F, Garcia-Verdugo JM, et al. Disruption of Eph /ephrin signaling affects migration and proliferation in the subventricular zone. Nat Neurosci. 2000; 3: 1091-1097.
68 Mellitzer G, Xu Q, Wilkinson DG, et al. Eph receptors and ephrins restrict cell intermingling and communication. Nature. 1999; 400: 77-81.
69 Dravis C, Yokoyama N, Chumley MJ, et al. Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development. Dev Biol. 2004; 271: 272-290.
70 Salsi V, Zappavigna V. Hoxd13 and Hoxa13 directly control the expression of the EphA7 Ephrin tyrosine kinase receptor in developing limbs. J Biol Chem. 2006; 281: 1992-1999.
71 Yamaguchi TP, Bradley A, McMahon AP, et al. A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo. Development. 1999; 126: 1211-1223.
72 Lee H, Kimelman D. A dominant-negative form of p63 is required for epidermal proliferation in zebrafish. Dev Cell. 2002; 2: 607-616.
73 Ince TA, Cviko AP, Quade BJ, et al. p63 Coordinates Anogenital Modeling and Epithelial Cell Differentiation in the Developing Female Urogenital Tract. American Journal of Pathology. 2002; 161: 1111-1117.
74 郭俊斌,张志波,王练英,等．维甲酸诱导大鼠肛门直肠畸形胚胎中RARMsRNA表达的研究．中华小儿外科杂志．2005；26：256-259．
75 Sakaki-Yumoto M, Kobayashi C, Sato A, et al. The murine homolog of SALL4, a causative gene in Okihiro syndrome, is essential for embryonic stem cell proliferation, and cooperates with Sall1 in anorectal, heart, brain and kidney development. Development. 2006; 133: 3005-3013.
76 Wiedemann S, Fries R, Thaller G. Genomewide scan for anal atresia in swine identifies linkage and association with a chromosome region on Sus scrofa chromosome 1. Genetics. 2005; 171: 1207-1217.
77 Papapetrou C, Drummond F, Reardon W, et al. A genetic study of the human T gene and its exclusion as a major candidate gene for sacral agenesis with anorectal atresia. J Med Genet. 1999; 36:208-213.
78 Seri M, Martucciello G, Paleari L, et al. Exclusion of the Sonic Hedgehog gene as responsible for Currarino syndrome and anorectal malformations with sacral hypodevelopment. Hum Genet. 1999; 104:108-110.
79 Stoll C, De Saint Martin A, Donato L, et al. Pallister-Hall syndrome with stenosis of the cricoid cartilage and microphallus without hypopituitarism. Genet Couns. 2001; 12: 231-235.
80 Bao Quan Qi, Beasley SW, Williams AK, et al. Apoptosis during regression of the tailgut and septation of the cloaca. J. Pediatr Surg. 2000; 35: 1556-1561.
81 Sasaki C, Yamaguchi K, Akita K. Spatiotemporal distribution of apoptosis during normal cloacal development in mice. Anatomical Record A. 2004; 279: 761-767.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |