Screening, Expression And Polymorphism Of The Candidate Gene Of Congenital Anorectal Malformation

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 methodsMaterials(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.