【作者】 田甜；

【导师】 任爱国；

【作者基本信息】 北京大学医学部 ， 流行病与卫生统计学， 2021， 博士

【摘要】 [背景]神经管缺陷(Neural Tube Defects,NTDs)是由于胚胎发育早期神经管闭合不全导致的一组严重先天畸形。NTDs的病因十分复杂,其发病机制仍不完全清楚。已有研究提示,遗传因素在NTDs发生中起重要作用。平面细胞极化(PCP)信号通路是调控神经管闭合的重要通路之一,多种动物模型和人群测序研究报道了 PCP通路与NTDs的关系。然而,既往研究仅限于基于种系遗传的单核苷酸位点变异,且绝大多数检测到的人群变异使用的是胎儿血液或唾液DNA。而其他突变类型,如基于大片段变异的拷贝数变异(CNVs)和仅发生于特定部位的体细胞突变未有报道。越来越多的证据表明,基因拷贝数变异和体细胞突变在神经系统疾病的发生发展中起重要作用。[目的]研究目的包括:(1)在NTDs病例中筛选PCP通路关键基因的基因拷贝数变异位点,尤其是罕见的de novo基因拷贝数变异,探讨其与NTDs的关系;(2)检验中枢神经组织DNA的PCP通路基因的体细胞突变是否与NTDs发生有关。[方法]本课题包括两部分研究内容,采用人群测序、细胞实验和动物模型研究相结合的研究设计方法。第一部分:PCP通路基因基因拷贝数变异与NTDs(1)基因拷贝数变异筛选和验证本次研究选取PCP通路关键基因,包括:VANGL1、VANGL2、CELSR1、SCRIB、DVL2、DVL3和PTK7。采用CNVplex?高通量DNA拷贝数检测技术,对175例NTDs病例脐带组织进行基因拷贝数检测,初步筛选NTDs相关的CNVs。采用实时荧光定量PCR(qPCR)验证筛选到的CNVs。(2)罕见de novo基因拷贝数变异位点分析对于患儿的生物学父母,采用qPCR进行靶向CNVs检测。CNVs仅发生于胎儿DNA,而不发生于父母DNA时,定义该突变为新生突变,即de novo突变。将在本NTDs人群中筛选到的de novo CNVs位点频率与其在公共对照数据库和本研究纳入的本地对照人群中的频率进行比较。采用Pearson χ2检验分析病例组和对照组间CNVs频率差异。当病例中该拷贝数变异的频率显著大于以上对照人群时,提示其为NTDs发生的危险因素。第二部分:PCP通路基因体细胞突变与NTDs(1)人群研究本研究以PCP通路基因为目标基因,包括两部分:1)PCP通路已有人群种系突变报道的关键基因,包括CELSR1、DACT1、DVLs、FZD6、PRICKLE2、SCRIB、和VANGLs;2)在小鼠动物模型中已经报道该基因突变可出现NTDs表型,但在NTDs人群中尚无报道的基因,包括:CDX1、CDX2、CTHRC1、FZD1、FZD2、MED12、SFRP1、SFRP2、SFRP5、SMURF1、SMURF2。收集48例NTDs患儿的神经系统病变处和脐带组织 DNA,采用 Ion Torrent Personal Genome Machine(PGM)测序,筛选 PCP通路NTDs相关体细胞突变位点,将病变组织检测到而非病变组织未检测到的突变定义为体细胞突变;采用Sanger测序验证筛选到的位点,并进一步纳入同一个患儿的由外胚层发育来的表皮、由中胚层发育来的心脏和肌肉组织、由内胚层发育而成的胸腺和肺组织进行测序,探讨体细胞突变在不同胚层的器官中的分布;此外,为进一步确认突变是发生于胎儿体内而非从双亲遗传得到,纳入携带体细胞突变的患儿父母血液标本进行Sanger测序。(2)细胞功能实验针对经过Sanger验证和生物信息分析后纳入的目标突变,构建野生型和突变质粒。采用免疫荧光染色法检测突变位点对蛋白亚细胞定位的影响;采用蛋白质印迹法检测目标蛋白的表达;采用荧光素酶报告系统检测突变位点对于经典/非经典WNT信号通路的影响;采用活细胞成像技术检测突变位点对于细胞迁移的影响。(3)小鼠基因敲除模型采用6周龄雌性C57BL/6小鼠构建CRISPR/Cas9小鼠模型。将6周龄雌性小鼠与雄鼠合笼受孕后第二天将受精卵取出,在37℃,5%CO2的培养箱中培养2小时。将Cas9 蛋白(20ng/μL)、Med12基因的 sgRNAs(10ng/μL)和Med12单核苷酸链(10ng/μL)混合液采用显微注射技术注入受精卵,将注射后的受精卵转移回雌性小鼠体内。在胚胎发育第12.5天解剖小鼠,检查胚胎畸形并记录神经管缺陷表型等相关表型。[结果]第一部分:PCP通路基因基因拷贝数变异与NTDs(1)CNVs筛选和验证:在175例患儿中,通过CNVplex检测,筛选到了位于5个基因上的16个CNVs位点,其发生于11例NTDs患儿中。该16个CNVs中,DVL2第1-15外显子发生了重复突变,其他15个则为缺失突变。采用qPCR技术,11个CNVs经过了验证。其中,DVL2第1-15外显子重复突变在无脑患儿中得到验证;VANGL1第1-7外显子缺失突变分别在两个脊柱裂患儿中得到验证;VANGL1第8外显子的缺失突变分别在8个NTDs患儿中得到验证。(2)罕见de novo基因拷贝数变异的分析:对携带已验证CNVs的患儿父母血液测序,发现VANGL1第1-7外显子缺失突变和DVL2第1-15外显子重复突变在父母血液中缺失,仅发生于胎儿,提示为de novo突变。在公共CNVs数据库中检索以上两个位点发现其在对照数据库中频率小于0.01%,提示其为罕见突变。与对照数据库比较,在本NTDs人群中,VANGL1外显子1-7缺失突变的发生率为1.14%,DVL2第1-15外显子重复突变发生率为0.57%,均显著高于其在正常人中的频率(P<0.05),表明VANGL1和DVL2的罕见de novo基因拷贝数变异在NTDs中具有聚集性。第二部分:PCP通路基因体细胞突变与NTDs(1)人群研究PGM测序技术在48例NTD患儿的神经系统病变处组织共筛选到位于15个PCP通路基因上的27个体细胞突变位点,其中17个属于有害突变;10个PGM测序突变等位基因检测频率高于40%;16个在正常人群中暂无报道,为新发突变,其余11个在正常人群中为罕见突变。进一步Sanger测序,有10个位点经过了验证,进一步探究该10个突变位点在不同组织器官中的分布,结果表明,目标突变在来自不同胚层的不同组织中表现分布情况各异,其中CELSR1 c.6375G>c、DACT c.1192 C>T、DVL2 c.1195A>G、FZD6 c.1991A>C、FZD6 c.262C>G、VANGL1 c.1121G>A 和 MED12 c.5344C>T突变仅发生在NTDs患儿的神经系统病变组织中,其他组织器官均未检出。对于以上体细胞突变位点进行生物学分析后,发现FZD6 c.262C>G(p.Gln88Glu),VANGLl c.1121 G>A(p.Arg374His),MED12c.5344C>T(p.Arg 1782Cys)可能影响蛋白功能,因此进行后续细胞功能实验加以验证。(2)细胞功能研究FZD6 p.Gln88Glu突变影响FZD6蛋白定位,并且影响FZD6与CELSR1蛋白相互作用,阻碍其共定位到细胞-细胞接触点。此外,FZD6 p.Gln88Glu可影响FZD6对于非经典WNT信号通路的调控,导致非经典WNT信号通路表达上调。VANGL1 p.Arg374His突变影响VANGL1蛋白和CELSR1蛋白相互定位到细胞-细胞接触点,并可以上调VANGL1蛋白水平,导致细胞迁移速度和距离增加。MED12 p.Arg1782Cys突变导致MED12蛋白水平下降,升高经典WNT信号通路表达,从而影响细胞功能。(3)小鼠基因敲除模型人群突变MED12p.Arg1782Cys位点在小鼠中同源突变位点为Med12 p.Arg1784Cys,采用CRISPR/Cas9技术构建Med12p.Arg1784Cys小鼠突变模型。将Med12靶向sgRNA,Med12单核苷酸链和Cas9蛋白复合物,注入受精卵中。进行两次注射后,将192个胚胎植入6个受体小鼠中(每个受体携带32个胚胎),在胚胎发育12.5天处死母鼠,共收集到12和活胎和1个死胚。活胎中,10个为雄性,2个为雌性。两个雄性胎鼠基因型为Med12 p.Arg1784Cys/Y,其余均为野生型。2个Med12 p.Arg1784Cys/Y小鼠均表现出严重的露脑畸形、尾部脊柱裂、卷尾等神经管缺陷相关表型,表明Med12 p.Arg1784Cys突变可导致小鼠NTDs表型,为人群发现提供了有力证据。[结论]1.PCP通路关键基因VANGL1和DVL2基因的罕见de novo基因拷贝数变异与人类NTDs的发生有关。2.中枢神经系统组织PCP通路相关基因VANGL1、FZD6、MED12等的体细胞突变是NTDs发生的遗传机制之一。更多还原

【Abstract】 BackgroundNeural tube defects(NTDs)are congenital malformations of the central nervous system resulting from a failure of the neural tube to close during the third-or fourth-weeks postfertilization.Although NTDs are considered as multifactorial disorders arising from complex interactions of genetic and environmental factors,genetic components are considered dominant factors contributing to the susceptibility of failed neural tube closure.Planar cell polarity(PCP)signaling pathway plays an essential role in the polarization and coordinated movement of cells during embryonic morphogenesis.Mutations of PCP genes could lead to NTD-related phenotypes in mice,and the digenic,trigenic,and oligogenic combinations of PCP variants may also cause mouse NTDs.At present,almost all of the reported human NTDrelated mutations are from studies that used DNA extracted from blood or saliva samples,and the NTD-related mutations identified in these studies were all considered as single-nucleotide germline mutations.At present,the importance of somatic mutations and copy number variants(CNVs)are well documented in neuro-developmental diseases and have been recognized recently.Considering that NTDs are a group of severe disabilities,and the parents of the NTDs cases are often normal,we aimed to identify the role of CNVs and somatic mutations of PCP pathway genes in the occurrence of NTDs.Objectives(1)To explore if rare de novo CNVs in PCP genes are related to NTDs occurrence.(2)To examine if somatic mutations in PCP pathway genes in central nervous tissues contribute to the occurrence of NTDs.MethodsThis study consisted of two parts:Part 1 focused on CNVs of PCP pathway genes,and part 2 focused on somatic mutations of PCP pathway genes.PART 1.CNVs of PCP pathway genes and NTDs(1)Copy number variation assayWe performed a quantitative analysis of copy numbers of all exon regions in the VANGL1,VANGL2,CELSR1,SCRIB,DVL2,DVL3,and PTK7 in DNA samples of umbilical cord tissues CNVplex assay,which is a high-throughput,multiplex CNV analysis method.Quantitative real-time PCR(qPCR)was carried out to confirm the results of the CNV analysis.(2)Bioinformatic analysisPearson’s chi-square test was used to examine the associations between the risk for NTDs and selected CNVs.A two-tailed P value of<0.05 was considered to be statistically significant.Statistical analysis was performed using the Statistical Package for the Social Sciences,version 18.0(SPSS Inc.,Chicago,IL,USA).PART 2.Somatic mutations of PCP pathway genes and NTDs(1)Human gene sequencing48 pairs of lesion sites and umbilical cord tissue DNA were collected from NTDs cases.Ion Torrent Personal Genome Machine(PGM)sequencing was performed to identify the somatic mutations.The targeted gene panel included 19 genes:CELSR1,DACT1,DVLs,FZD6,PRICKLE2,SCRIB,VANGLs,GRHL3,CDX1,CDX2,CTHRC1,FZD1,FZD2,MED12,SFRP1,SFRP2,SFRP5,SMURF1,SMURF2.The inclusion criteria of somatic mutation during this stage were:1)the mutation has a sequencing depth≥100x and alternate-allele read frequency in PGM>5%;2)p-value ≤5x 10-5;3)the frequency of the mutation in the general population was lower than 5%,and 4)the mutation was missense or loss of function.Sanger sequencing was used to validate mutations in the target genes.The source and distribution were further explored for the validated mutants when DNA samples from tissues of skin,heart,muscle,thymus,and lung of fetuses or blood of the parents were available.(2)Cellular functional studiesImmunostaining assays were carried out to examine the influence of target mutations on the subcellular localization of proteins.The western blotting assay was performed to examine the influence of variants on protein levels.Luciferase reporter assays were performed to examine the influence of variants on the expression of canonical and non-canonical WNT pathway signaling.Live-cell imaging for cell migration analysis was performed to measure the influence of variants on the cell migration process.All of the biological experiments were repeated at least twice.(3)Mouse CRISPR/Cas9 editingSix-week-old female C57BL/6 mice were used to provide the embryos on which to perform CRISPR/Cas9 assay.The six-week-old female mice mated with males,and then the zygotes were obtained the next day and cultured.Zygotes were injected with a mixture of Cas9 protein,Med 12 sgRNA,and SSODN to the targeting site.The injected zygotes were then transferred to pseudopregnant mice(32 zygotes per mouse for a total of 6 mice)to be carried to parturition.Mice were dissected at embryonic day 12.5 to screen the embryos for thepresence of any malformations.NTDs and embryo lethality was recorded.The Sanger sequencing method was performed to confirm the genetic variants in the mice,while wholeexome sequencing was used to make sure that the NTD phenotypes of knock-in mice were caused by Med12 variants rather than other NTD-causing genes.ResultsPART 1.CNVs of PCP pathway genes and NTDs(1)CNV analysis:A total of 16 CNVs were identified among 11 NTD probands.These CNVs were found at 5 loci involving DVL2(exons 1-15),VANGL1(exons 1-7,exon 8),and VANGL2(exons 5-8,exon 7-8).One CNV(DVL2 exons 1-15)was a duplication,and the remaining 15 were deletions.Eleven of the 16 CNVs were confirmed by qPCR.Relative copy number gains were found on exons 1-15 of DVL2 in one AN case;relative copy number losses were found on exons 1-7 of VANGL1 in two SB cases,and on exon 8 of VANGL1 in 8 NTD cases with different phenotypes.(2)Association analysis of rare de novo CNVs in VANGL1 and DVL2 with the risk of NTDs:Maternal and paternal inheritance was examined.Two CNVs were found to be de novo.The two de novo CNVs included a loss of exons 1-7 of VANGL1 in one SB case and a gain of exons 1-15 of D VL2 in one AN case.CNVs of VANGL1 and DVL2 public databases were used as controls.Both CNVs showed frequencies of 0.01%in the control population,indicating rare mutations in unaffected individuals.Compared with controls,the frequency of VANGL1 exon 1-7 deletion(1.14%)and DVL2 exon 1-15 duplication(0.57%)showed significant(P<0.05)enrichments in our NTDs cohort.PART 2.Somatic mutations of PCP pathway genes and NTDs(1)Human gene sequencingA total of 27 protein-altering somatic single nucleotide variants located in 15 PCP genes were identified;17 of them were predicted to be harmful to protein functions,and 10 had alternate-allele read frequencies greater than 40%.16 were novel,and 11 mutations were rare in control populations with minor allele frequency(MAF)<1%.Sanger sequencing showed 10 mutations were validated.The mutations CELSR1 c.6375G>C,DACT c.1192C>T,DVL2 c.l195A>G,FZD6 c.1991A>C and c.262C>G,VANGL1 c.1121G>A and MED12 c.5344C>T only occurred in neural tube lesion tissues,but not in other control tissues.Based on the bioinformatics data,we selected FZD6 c.262C>G(p.Gln88Glu),VANGL1 c.1121G>A(p.Arg374His),MED 12 c.5344C>T(p.Arg1782Cys)on which to perform further functional studies.(2)Cellular functional studiesFZD6 p.Gln88Glu caused mislocalization of its protein from the cytoplasm to the nucleus and disrupted the colocalization of CELSR1 and FZD6.This mutation also affected the expression of non-canonical WNT signaling.VANGL1 p.Arg374His impaired co-localization of CELSR1 and VANGL1,increased the protein levels of VANGL1,and influenced cell migration.MED12 p.Arg1782Cys decreased MED12 protein level and affected the regulation of MED12 on the canonical WNT signaling pathway.(3)Mouse CRISPR/Cas9 editingCRISPR/Cas9 mutagenesis editing was employed to generate Med12 p.Arg1784Cys(homologous position of p.Arg1782Cys in human)knock-in mice.12 viable embryos and 1 dead embryo were observed.10 embryos were male,and 2 were female.Of the 10 male embryos,2 were Med12 p.Arg1784Cys knock-in hemizygotes with NTD phenotypes.The others were wildtype and no obvious structural malformations.The two female embryos were wildtype.The two hemizygous Med12 p.Arg1784Cys/Y mouse embryos exhibited 100%penetrant NTD phenotypes,including exencephaly and spina bifida,and curly tails.The result indicates that Med12 p.Arg1784Cys found in a human NTD patient can also cause NTDs in mice.Conclusions(1)Rare de novo CNVs in the PCP genes VANGL1 and DVL2 are associated with human NTDs.(2)Somatic mutations of PCP pathway genes(e.g.,VANGL1,FZD6,and MED12)in neural tissues are one of the genetic mechanisms underlying NTD occurrence.更多还原