【作者】 俎树禄；

【导师】 赵升田； Agneta Nordenskjold；

【作者基本信息】 山东大学 ， 外科学， 2009， 博士

【摘要】 膀胱输尿管反流(Vesicoureteral reflux,VUR)是指由于输尿管膀胱连接部异常造成的先天性泌尿系统畸形。这一畸形导致尿液由膀胱逆流到输尿管甚至肾内,在排尿期尤为明显,膀胱输尿管反流在儿童中的发病率约为1%。膀胱输尿管反流与反复性尿路感染、肾畸形及肾疤痕相关,反流性肾病是导致儿童及青少年终末性肾病的常见原因之一。目前膀胱输尿管反流患者根据反流严重程度不同可以分为Ⅰ-Ⅴ度,其中部分Ⅳ和Ⅴ度患者需要手术治疗。膀胱输尿管反流的诊断金标准是排尿性膀胱尿道造影。很多膀胱输尿管患者无临床症状,利用排尿性膀胱尿道造影对每个儿童进行筛查是不可行的。目前,膀胱输尿管反流被公认具有遗传性和家族史,因此寻找膀胱输尿管反流的致病基因,对于早期诊断,了解膀胱输尿管反流的发病机制具有重大意义。膀胱输尿管反流具有家族聚集性说明遗传因素是其发病机制之一,其中同卵双生的双胞胎同时患膀胱输尿管反流的几率为80-100%,而异卵双生的双胞胎同时患膀胱输尿管反流的几率为35-50%。如果父母双方有一方是膀胱输尿管反流患者,则子女的患病机率为55-66%。目前研究认为,膀胱输尿管反流的遗传方式为多因子疾病或常染色体显性遗传但不完全外显。在膀胱输尿管反流家系中进行的全基因组连锁研究中,发现1p13、2q37、3q22.2-23等位点与膀胱输尿管反流密切相关。在胚胎的发育过程中,后肾间质诱导附近的午非氏(Wolffian)管长出输尿管芽,输尿管芽近端与后肾间质相互诱导形成肾脏,输尿管芽远端则形成输尿管、输尿管膀胱连接部及膀胱三角。输尿管芽的出芽位置能影响输尿管在膀胱开口的位置和膀胱输尿管连接部的正常结构,形成膀胱输尿管反流。在后肾间质诱导附近的午非氏管长出输尿管芽的过程中,很多基因参与了这一诱导过程,例如:GDNF(Glial cell line-derived neurotrophic factor),PAX-2(Paired box gene 2),RET(Rearranged during transfection protooncogene)等。在小鼠胚胎肾发育过程中,gdnf沿着午非氏(Wolffian)管表达,slit2和它的受体robo2能影响gdnf的表达,进而影响输尿管芽的生长,基因敲除robo2或slit2的小鼠,会形成多个肾及输尿管。美国科学家进而发现一例原发性膀胱输尿管反流患者的Y染色体和3号染色体发生了易位,断裂点在ROBO2基因的第一个外显子和第二个外显子之间。后来,他们在124个具有膀胱输尿管反流或者是肾脏等其它泌尿系先天性畸形的家系中,对ROBO2基因进行突变扫描,在两个不相关的家系里新发现了两个错义突变。意大利科学家在95个膀胱输尿管反流或合并肾脏等其它泌尿系先天性畸形的病例中,对ROBO2基因进行测序扫描发现了4个错义突变,具有较高的突变率(5.1%)。RET基因在肾脏不同发育阶段(中肾管,输尿管芽,膀胱)广泛表达。RET基因敲除的小鼠的输尿管发育异常,输尿管远端不是连接到膀胱,而是与性腺管相连。另外加拿大科学家研究发现,小鼠过度表达Ret会造成膀胱输尿管反流。在加拿大魁北克居住的法国移民后代中,RET基因错义突变Gly691Ser与原发性膀胱输尿管反流密切相关。综上所述,ROBO2、SLIT2及RET都有可能是家族性膀胱输尿管反流的致病基因。我们收集了54个膀胱输尿管反流家系的标本,每个家系中至少有两名膀胱输尿管反流患者。本论文进一步研究上述基因在家族性膀胱输尿管反流发病过程中的作用。共分两部分:第一部分从每个家系中挑选一名膀胱输尿管反流患者,分离外周血DNA,利用基因测序方法对ROBO2和SLIT2这一对基因进行突变扫描,对发现的基因突变进行连锁分析和功能预测研究。第二部分是利用Taqman基因分型技术,在54个家系的先证者及亲属、163个健康对照者中,对RET基因单核苷酸多态性rs1799939:G＞A进行基因分型,分析其是否与膀胱输尿管反流这一疾病相关。第一部分ROBO2和SLIT2基因在家族性膀胱输尿管反流中的作用机制研究目的:研究ROBO2和SLIT2这一对基因在家族性膀胱输尿管反流中的作用机制,同时进一步明确泌尿系统的正常发育机制。材料:我们从瑞典哥德堡地区收集了54个家系,每个家系均有2个或者2个以上无症状膀胱输尿管反流患者,从每个家系中挑选出1名患者用于测序。我们从卡罗林斯卡大学附属医院收集了96个健康人的DNA作为对照,用来辨别新发现的基因突变是否是基因多态性。54个家系中,有17个家系存在兄弟姐妹共同患病,16个家系中是从父母遗传,其中14个从母亲遗传。膀胱输尿管反流的严重程度分级:Ⅰ度:1例,Ⅱ度:5例,Ⅲ度12例,Ⅳ度17例,Ⅴ度8例,其余11例情况未明。7例先证者有重复肾输尿管,2例有肾盂输尿管狭窄,1例有双侧重复肾输尿管和输尿管末端囊肿。方法:(1)收集54例患者的外周血,分离DNA。(2)利用Primer3软件针对ROBO2和SLIT2两个基因的每一个外显子设计引物,利用PCR扩增出相应片段。(3)纯化PCR产物,去除多余的引物,分别利用正向和反向引物对相应片段进行测序反应,然后在ABI3730测序仪上进行扫描,结果用Seqscape2.5软件进行分析。(5)在线软件Homologene检测人ROBO2和SLIT2蛋白的同源氨基酸序列,利用在线软件BGDP Splice Site Prediction、NetGene2、GeneSplicer、HumanSplicing Finder(Version2.4)检测基因突变对剪切位点的影响。利用在线软件NNPREDICT检测基因突变对蛋白质二级结构的影响,利用在线软件PolyPhen检测基因突变对蛋白质三级结构和功能的影响。结果:ROBO2所有的26个外显子均未发现突变。在靠近外显子的内含子区域发现6个突变,其中2个为新发现突变:ⅣS13+43C＞T,ⅣS21-40T＞C。在一个患者中发现杂合突ⅣS13+43C＞T,且未在95个对照健康人DNA中发现。一个患者携带ⅣS21-40T＞C杂合突变,另外一名患者携带ⅣS21-40T＞C纯合突变,在95个健康对照着发现一个杂合突变。ⅣS3-3C＞T离编码区最近,4个患者携带这一突变,但并没有在家系中共分离,而在对95个正常人中也发现2个人携带这一杂合突变,因此我们认为它是单核苷酸多态性。另外还发现3个单核苷酸多态性:rs585033、rs17525412、rs3821735,这三个单核苷酸多态性在家系里不与膀胱输尿管反流共分离。以上这些突变没有改变基因的剪切位点。SLIT2所有的37个外显子发现4个突变:c.4253C＞T/p.Ala1418Val,c.1209A＞T,c.2211C＞T(rs7690492),c.2550A＞C(rs17542486),3’UTR*51A＞G(rs1379659)。其中c.1209A＞T,c.2211C＞T(rs7690492),c.2550A＞C(rs17542486)均为同义突变,不改变氨基酸,而c.4253C＞T/p.Ala1418Val是唯一的错义突变,分别在一个男性(53个家系)和女性患者(50个家系)中发现。男性患者的突变来自父亲,而母亲正常。男性患者的姐姐亦患有膀胱输尿管反流,也携带相同的杂合突变,而他患病的弟弟和姑姑则不携带这一突变。在第50个家系中,只有先证者的姑姑具有这一突变,先证者及其母亲则没有。这一突变将丙氨酸转换成缬氨酸,能改变蛋白质的二级结构,但对蛋白质的三级结构和功能影响不大,而这一突变未在95个健康人中发现。此外在SLIT2基因的靠近外显子内含子区域还发现以下突变:rs61790829,rs16869654,rs2290752,ⅣS7+81C＞G,rs4443280,rs543593,rs519813,rs55972345,rs10428394,rs55798019,rs2290750,rs12506323,rs2290751,rs3733510,rs35891001。ⅣS7+81C＞G为新发现的突变,其中8名患者呈杂合状态,3名患者呈纯合状态,而在95个健康对照者中,也发现18人患者有杂合突变,3名患者有纯合突变。稀有等位基因频率为0.123,我们推测它是一单核苷酸多态性。以上这些突变没有改变基因的剪切位点和蛋白质结构。结论:ROBO2和SLIT2基因突变与部分膀胱输尿管反流患者的发病有关系,我们第一次在膀胱输尿管反流患者中发现SLIT2基因错义突变,但其功能及是否也存在在其他人群中需要进一步研究。此外,样本数量较小也降低了发现致病突变的可能性。但是我们尽量采用了家族性患者作为扫描对象,以提高发现膀胱输尿管反流的致病基因。ROBO2和SLIT2基因突变在膀胱输尿管反流患者中少见也进一步证明膀胱输尿管反流是一多基因疾病。第二部分RET基因单核苷酸多态性rs1799939:G＞A与家族性膀胱输尿管反流的相关性研究目的:研究RET基因单核苷酸多态性rs1799939:G＞A是否与瑞典家族性膀胱输尿管反流患者的发病相关。材料及方法:我们从瑞典哥德堡地区收集了54个家系,每个家系均有2个以上膀胱输尿管反流患者,收集先证者及亲属共计212人的外周血,并分离DNA,另外分离163名健康人的DNA作为对照。利用Taqman基因分型技术对RET基因单核苷酸多态性rs1799939:G＞A进行基因分型,然后利用统计学分析检验这一多态性是否与膀胱输尿管反流这一疾病关联。结果:54名先证者中,35名患者具有GG基因型,15名患者具有GA基因型,2名患者具有AA基因型,另外2名不清楚。163例对照者,108人具有GG基因型,47名具有GA基因型,8人具有AA基因型。利用X~2检验,稀有等位基因A的频率在膀胱输尿管患者及健康人群中没有显著差异。分析每个家系,发现RET基因单核苷酸多态性rs1799939:G＞A在5个家系里与膀胱输尿管反流这一疾病共分离。结论:rs1799939:G＞A(Gly691Ser突变)可能只在一部分VUR患者中导致输尿管发育缺陷。膀胱输尿管反流的致病基因可能因不同人群而异。此外,我们样本数较少也是研究的局限性之一。我们的结果进一步表明膀胱输尿管反流为一多基因疾病。更多还原

【Abstract】 Vesicoureteral reflux（VUR） is a congenital urinary tract defect caused by abnormal insertion of the ureter within the bladder wall.This malformation leads to a defective ureterovesical junction in which urine flows retrogradely from the bladder to the ureters and kidneys especially during micturiation.VUR affects approximately 1%of the population and is associated with recurrent urinary tract infections,renal malformations,and renal scarring defined as reflux nephrology（RN）.Reflux nephrology is the most common cause of the end-stage renal disease in children and adolensents.VUR can be classified intoⅠ-Ⅴgrades.VUR patients who belong toⅣandⅤgrade need surgical theraphy.The voiding cystourethrography（VUCG） is the gold standard diagnostic methods and must include the voiding phase.It is not feasible to screen the whole population for reflux.The hereditary and familial nature of vesicoureteral reflux is now well recognized.To screen the candidate genes for vesicoureteral reflux is helpful for early diagnosis and the pathogenesis of VUR will be better understood by clarifying the molecular background.Familial clustering of VUR implies that genetic factors may have an important role in the pathogenesis of reflux.The concordance is higher in monozygotic twins than in dizygotic ones（80-100%and 35-50%,respectively）.The risk for children if one parent had been diagnosed with VUR is reported to be 50-66%.The model of inheritance for VUR is multifactorial or autosomal dominant with reduced penetrance. Three genome-wide linkage studies reported to date with linkage to chromosome 1p13,chromosome 2q37 and chromosome 3q22.2-23,respectively. During the development of the embryo,the metanephric mesenchyme induces the nearby Wolffian duct to evaginate an epithelial tube,the ureteric bud.This invades the metanephric mesenchyme,which in response condenses,proliferates,and undergoes an epithelial transformation.The proximal end of the ureteric bud remains attached to the mesonephric duct and elongates,becoming the distal ureter,ureter-vesico junction and the bladder trigone.The ureteric bud position on the Wolffian duct to the nephrogenic blastema can affect the orifice in the bladder and urethra and the normal structure of the vesico-ureteric junction.Many genes involved in ureteric bud induction.Such as:Glial cell line-derived neurotrophic factor（GDNF）,Paired box gene2（PAX-2）,Rearranged during transfection protooncogene（RET）.Recent studies in mice have shown that gdnf is expressed along the Wolffian duct during embryological development and that the amount of gdnf is determined by slit2 and its receptor robo2 thereby affecting the outgrowth of the ureteric bud. Homozygous deletion of either robo2 and slit2 in the mouse results in supemumberary ureteric buds that form multiple kidneys and ureters.Interestingly one patient with isolated VUR was described with a translocation between the Y-chromosome and chromosome 3 with a break point between exon 1 and 2 of the ROBO2 gene.Two novel ROBO2 missense variants were found in two unrelated families by screening on in 124 families with VUR or congenital abnormalities of kidney/urinary tract（CAKUT）.Another study on 95 unrelated cases has identified a relatively high frequency of ROBO2 variants（5.1%） in familial cases of VUR or VUR/CAKUT.RET is expressed by the mesonephric duct,the ureteric bud,and the future bladder. The RET-/- mice have ureteral abnormalities such that the distal ureters are not connected to the bladder but instead remain attached to sex ducts.Overexpression of RET leads to vesicoureteric reflux in mice.RET Gly691Ser mutation is associated with primary vesicoureteral reflux in the French-Canadian population from Quebec. In summary,ROBO2,SLIT2 and RET are all candidate genes for familial vesico-ureteric reflux.We collected 54 families with reflux from Gothenburg, Sweeden.Every family has at least 2 person with vesico-ureteral reflux.Then overall aim of this thesis is to study these candidate genes in familial vesico-ureteric reflux. This thesis includes two parts:the first part is to further study the ROBO2 and SLIT2 genes on vesico-ureteric reflux by sequencing on 54 index patients.And do assiociation studies and function prediction.The second part is to study the RET polymorphism rs1799939:G>A and familial vesico-ureteral reflux by genotyping on 54 index patients and their relatives and 163 controls.Part one:Study the roles of ROBO2 and SLIT2 genes in familial vesieo-ureteral refluxObjective:This study the mechanism of ROBO2 and SLIT2 genes in familial vesico-ureteral reflux by mutation screening on 54 unrelated patients with primary VUR from Sweden.Material:Fifty-four families with more than one affected relative with non-syndromic vesico-ureteral reflux were identified through medical records in Sweden.One affected from each family was selected for mutation screening.As control group for selected polymorphisms,we used DNA from an anonymous sample constituted by 96 healthy voluntary blood donors at Karolinska University Hospital, Stockholm.In families with 2 affected,altogether in 17 families sibs were affected.In 16 families VUR was inherited from a parent and in our study in 14 cases of 16 it was maternally inherited.The grade of VUR in the probands was gradeⅠ（n=1）,Ⅱ（n=5）,Ⅲ（n=12）,Ⅳ（n=17）,andⅤ（n=8） degree vesico-ureteral reflux.In the remaining 11 cases,the severity of vesico-ureteral reflux was not available.7 of the probands have duplex collecting systerm,2 have pyeloureteral junction stegnosis,1 has duplex collecting systerm and ureterocele bilateral.Methods:（1） DNA was extracted from EDTA preserved blood,and isolated according to standard procedures.（2） Exon-flanking primers（exon 1-26 in ROBO2 gene,exon 1-37 in SLIT2 gene） were designed using Primer 3 program.PCR reactions were performed with AmpliTaq Gold and DyNAzyme^TM EXT DNA Polymerase in a final volume of 25 ul.（3） After ExoSap-IT enzyme treatment,the PCR fragments were sequenced on both direction using BigDye（?） Terminator v3.1 kit and analyzed in ABI Prism 3730 Sequencer.Sequence analysis was preformed with the program SeqScape v2.5.（4） HomoloGene detected putative homologs of human SLIT2 sequences.Splicing prediction was performed with four computer programs:BGDP Splice Site Prediction,NetGene2,GeneSplicer,Human Splicing Finder.Secondary structures were predicted with NNPREDICT.The program Polyphen was used to predict the possible impact of an amino acid substitution on the structure and function of ROBO2 and SLIT2 Proteins.Results.Exons 1-26 of the ROBO2 gene were normal in all of the 54 cases.Six sequence variants were observed in the exon-intron boundary area.Two of them: IVS13+43C>T,IVS21-40T>C have not been described before.The sequence variant IVS13+43C>T was found in heterozygous in one patient and not found in 95 control DNA samples.It may represent a private mutation.The sequence variant IVS21-40T>C was found in two patients respectively in heterozygous and homozygous form.One of 95 control DNA samples presented this variant in heterozygous form.Four patients presented the heterozygous sequence variant IVS3-3 （allele frequency 3.6%） which also found by Bertoli-Avella.This sequence variant did not co-segregate with VUR in four families.Two of the 95 control DNA samples presented this variant in heterozygous form and it may thus represent a single-nucleotide polymorphism（minor allele frequency 1%）.Three SNPs:rs5850333, rs17525412,rs3821735 were also found in the patients.All of them didn’t co-segregate with VUR.None of the above sequence variants was predicted to alter gene splicing.Several single-nucleotide variations were found in the SLIT2 gene.The variants c.4253C>T/p.Ala1418Val,c.1209A>T,c.2211C>T（rs7690492）,c.2550A>C （rs17542486） are all within the exons,and 3’UTR*51A>G（rs1379659） in the 3’UTR. The variant c.4253C>T（p.Ala1418Val） is the only non-synonymous sequence variant found.It converts the amino acid 1418 from alanine to valine,both being hydrophobic amino acids.The variant was found in heterozygous state in one male and one female （from family 50 and 53）.The boy’s variant was inherited from the father,whereas his mother had a normal sequence.His sister with VUR had the same sequence variant, however his affected brother and aunt did not.In family 50,only the affected aunt had the sequence alteration,the proband and his nephew didn’t have.This missence variant changed the secondary structure of the SLIT2 protein,but may have little effect on the three-dimension structure and function.The sequence changes c.1209A>Y,c.2211C>T（rs7690492） and c.2550A>C （rs17542486） are all synonymous.The site 1209bp was described as a SNP （rs34224151,A>G） before but we found a heterozygous variant A>T on this site in one patient.None of the 95 control DNA samples presented this variant and it may thus represent a private mutation.Other gene variants:rs61790829,rs16869654, rs2290752,IVS7+81C>G（not described before）,rs4443280,rs543593,rs519813, rs55972345,rs10428394,rs55798019,rs2290750,rs12506323,rs2290751,rs3733510, rs35891001 were found in the exon-intron boundary area of the SLIT2 gene.The new gene variant IVS7+81C>G were found in heterozygous in 8 cases and in homozygous in 3 cases.It didn’t segregate with VUR in families.18 of 95 control DNA samples presented this variant in heterozygous form.3 presented in homozygous form.The minor allele frequency is 0.123.It may thus represent a single-nucleotide polymorphism.None of these sequence variants was predicted to cause any splicing alteration or protein structure modifications by the previously mentioned methods.Conclusion:In summary,gene variants in ROBO2 and SLIT2 are rare causes for VUR. To our knowledge this study demonstrates for the first time the SLIT2 gene missense variant in familial vesico-ureteral reflux patients,however,functional studies and validation in other cohorts are warranted.The reduced sample size probably limited the power of our study for finding novel pathogenic variants.Neverthe-less,our study populations consisted of familial cases,and this design was aimed at increasing the chance of finding genetic causes of VUR.In summary,gene variants in ROBO2 and SLIT2 genes are rare causes of VUR in humans,providing further evidence for the genetic hetero- geneity of this disorder. Part two:Study if the RET polymorphism rs1799939.G>A associate with familial Vesicoureteral refluxObjective:To investigate the association between the RET polymorphism rs1799939: G>A and familial vesico-ureteric reflux in swedish population.Material and methods:Fifty-four families with more than one affected relative with non-syndromic vesico-ureteral reflux were identified through medical records in Sweden.We collected 54 index patients and their relatives’ DNA and 163 control’s DNA.We use Teqman genotyping method to genotype the RET polymorphism rs1799939:G>A.Results:35 of the index vesico-ureteral reflux patients have the GG genotype,15 have the GA genotype.2 have the AA genotype.107 of the control DNA have the GG genotype.48 have the GA genotype.8 have the AA genotype.By X² test,there was no significant difference between the cases and controls on A allele frequency.The RET polymorphism rs 1799939:G>A co-segeregated with VUR in 5 families.Conclusion:The RET polymorphism rs 1799939:G>A（Gly691 Ser） could generate a spectrum of deficiencies of ureteral development in some VUR patients.The cause of VUR can vary in different populations.The reduced sample size is probably limiting the power of our study in finding association between the RET polymorphism rs 1799939 and VUR.In summary,our results giving further evidence for the genetic heterogeneity of this disorder.更多还原