不同肢端畸形中基因突变鉴定及致病机制研究
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摘要
前言
先天性肢端畸形是人类最常见的出生缺陷之一,不仅影响肢体的外观形态和功能运动,甚至使患者丧失劳动和生活能力。近年来,随着人类基因组计划的完成,人类单基因遗传病致病基因鉴定不断取得进展,各种四肢先天畸形的致病基因也逐渐被发现,人类先天性肢端畸形研究的数据不仅将补充以往动物和细胞实验的不足,还可以加深对胚胎发育过程的理解,而且可以为人类组织工程学研究和基因治疗药物的研制提供强有力的理论依据,因此对肢端畸形的致病基因突变鉴定及致病机制研究至关重要。
引起先天性肢端畸形的原因有环境因素和遗传因素两大类。遗传因素主要指基因突变或染色体畸变,其导致的肢端畸形既可单独发生又可是某种综合征的一部分。多种基因突变和染色体畸变都会直接引起肢端形态发生(morphogenesis)的异常,表现不同类型的肢端畸形,如短指(趾)(brachydactyly,BD)、缺指(趾)(ectrodactyly,ED)、多指(趾)(polydactyly,PD)和并指(趾)(syndactyly,SD)畸形等。
近端指(趾)间关节粘连(proximal symphalangism,SYM1,MIM 185800)以肢端骨骼融合和短指(趾)为主要表型,常被认为是BDC(Brachydactyly type C,MIM 113100)的一部分。SYM1为常染色体显性遗传,致病基因是GDF5(growth/differentiation factor 5,MIM 601146)或NOG(MIM 602991)。
缺指(趾)/手足裂畸形(ectrodactyly/split-hand/split-foot malformation,SHFM)是四肢端骨(atttopod)中部指轴发育不全而剩余指(趾)呈不同模式融合导致严重影响患者精细活动的先天性肢端畸形,曲型表现为龙虾爪(中部指轴缺陷)或独指(桡侧指轴缺陷而无裂隙,第5指(趾)总不受累)。目前已定位的人类SHFM遗传位点包括:SHFM1(7q21,MIM 183600)、SHFM2(Xq26,MIM 313350)、SHFM3(10q24,MIM 600095)、SHFM4(3q27,MIM 605289)和SHFM5(2q31,MIM 606708)。SHFM3和SHFM4位点的致病突变已经明确,分别是染色体10q24区域内约0.5Mb的DNA串联重复和染色体3q27区域内TP63(MIM 603273)基因的点突变。
手足生殖器综合征(Hand-foot-genital syndrome,HFGS,MIM 140000)是累及手足和泌尿生殖系统的畸形,肢端畸形包括短指(趾)、延迟骨化、融合及腕骨和跗骨短小等。泌尿系统的异常包括输尿管口异位、膀胱输尿管反流和肾盂输尿管接合处梗阻导致慢性肾盂肾炎、肾功能不全和肾移植。生殖系统畸形包括男性的尿道下裂和女性的苗勒(氏)管融合导致阴道纵隔和双子宫。HFGS为常染色体显性遗传,致病基因为染色体7p15的同源盒基因HOXA13(Homeobox A13,MIM142959)。
本文以上述肢端畸形的三个中国人家系(SYM1、SHFM和HFGS)为研究对象,首先利用单体型分析进行了致病基因染色体定位,然后对候选基因DNA测序或定量PCR进行突变鉴定,进一步通过western blot、双萤光素酶报告基因检测系统和染色质免疫沉淀等技术对突变基因功能进行初步研究。
材料与方法
1、家系资料
对家系中可能追溯到的家庭成员进行详细调查,绘制家系系谱图:签署“知情同意书”;对患者进行体格检查和手足骨骼X光检查;采集外周静脉血。
2、单体型分析定位候选致病基因
利用UCSC基因组生物信息学网站在5个SHFM位点选择20对微卫星多态标记,PCR扩增后经变性聚丙烯酰胺凝胶电泳和银染,读出每个个体标记的基因型。再根据个体基因型和系谱中的亲缘关系,按照孟德尔遗传定律推导出同一条染色体上不同标记构成的单体型。
3、候选基因编码区DNA测序
根据单体型分析确定候选基因后,PCR扩增患者候选基因外显子,PCR产物纯化后测序。
4、定量PCR检测基因组拷贝数变异
利用实时荧光定量PCR扩增患者候选基因,与正常个体比较其基因组拷贝数是否发生改变。
5、长距离PCR结合DNA测序确定断裂点
定量PCR将两侧断裂点范围缩小到2-4kb后,以患者基因组DNA为模板,利用端粒侧重复的正向引物R2-2F及中心粒侧重复的反向引物L10-5R(outfacingprimers)行PCR扩增,PCR产物纯化后测序。
6、构建野生型和突变型真核表达载体
PCR扩增目的基因GDF5和HOXA13编码序列,分别与真核表达载体pLXSN和p3xFLAG-CMV7连接后转化感受态细胞,阳性克隆测序验证;以野生型载体为模板,定点诱变产生突变型载体。
7、Western blot检测GDF5~(L373R)二聚体形成和分泌
G418筛选稳定整合野生型和突变型GDF5的COS7细胞系,收集细胞培养上清和细胞总蛋白,经非还原SDS-PAGE电泳后检测GDF5~(L373R)二聚体形成和分泌情况。
8、双萤光素酶报告基因检测系统检测HOXA13~(V375F)对EphA7基因的转录激活能力
将野生型和突变型HOXA13真核表达载体分别与报告基因pGL3-EphA7及内参基因pRL-CMV共转染C3H10 T1/2细胞,计算萤光素酶比活性。
9、染色质免疫共沉淀检测HOXA13~(V375F)对下游靶基因启动子区的结合能力
将野生型和突变型HOXA13真核表达载体分别转染C3H10 T1/2细胞,利用anti-Flag单克隆抗体进行染色质免疫沉淀,然后利用定量PCR检测免疫沉淀的DNA量是否有变化。
实验结果
1、家系资料
根据患者的肢端畸形特征及临床表现和X光片等将收集的3个家系分别诊断为SYM1、SHFM和HFGS。
2、单体型分析
染色体20q11区域的微卫星标记D20S787、D20S601、D20S909和D20S914与SYM1家系畸形共分离;染色体10q24区域的微卫星标记LBXlT1、D10S1239、DactylinTAAT、Dactylin2和Dactylin3与SHFM家系畸形共分离。
3、候选基因编码区DNA测序
SYM1家系患者检测到GDF5基因杂合错义突变c.1118T＞G(p.L373R);HFGS家系患者检测到HOXA13基因杂合错义突变c.1123G＞T(p.V375F)。突变直接导致限制性片段长度多态性(restriction fragment length polymorphism,RFLP),经限制性内切酶酶切验证,家系的患者都携带突变,而家系的正常个体和50名正常无关对照个体都不携带此突变。
4、定量PCR检测基因组拷贝数变异
定量PCR结果表明SHFM家系患者在染色体10q24区域存在包括LBX1、BTRC、POLL、DPCD和FBXW4等五个基因在内的重复突变,DNA增加一个拷贝。
5、长距离PCR结合DNA测序确定重排断裂点
利用正向引物R2-2F和反向引物L10-5R行PCR扩增,SHFM家系患者的基因组都可以扩出约2000bp的特异性片段,而家系的正常个体扩增不出此片段,测序结果表明端粒侧和中心粒侧的断裂点分别在103453895bp处和102965035bp处,重复范围为488859bp,重复方式为串联重复。根据断裂点碱基信息,推测其重复机制可能为非同源末端连接(nonhomologous end joining,NHEJ)或复制叉停滞和模板转换(replication fork stalling and template switching,FoSTeS)。
6、构建野生型和突变型真核表达载体
测序结果表明野生型和突变型GDF5真核表达载体(pLmycGDF5~(WT)SN和pLmycGDF5~(L373R)SN)和HOXA13真核表达载体(p3xFLAG-HOXA13及p3xFLAG-HOXA13~(V375F))构建成功,无突变,阅读框正确。
7、Western blot检测GDF5~(L373R)二聚体形成和分泌
Western blot检测结果表明GDF5~(L373R)突变体可以二聚化并分泌至细胞外。
8、双萤光素酶报告基因检测系统检测HOXA13~(V375F)对EphA7基因的转录激活能力
双萤光素酶报告基因检测结果表明突变型HOXA13~(V375F)转录激活EphA7基因的能力下降至野生型的66.72%。
9、染色质免疫共沉淀检测HOXA13~(V375F)对下游靶基因启动子区的结合能力
定量PCR检测染色质免疫沉淀下游靶基因DNA量,结果表明与HOXA13~(V375F)突变体结合的EphA7、EphA6和Bmp2启动子及Sostdcl 3'UTR的量分别降低至野生型的65.98%、72.95%、42.89%和68.03%;与HOXA13~(V375F)突变体结合的Bmp7和Sostdcl启动子的量与野生型比较无明显差异。
结论
(1)SYM1家系致病基因突变为GDF5基因杂合错义突变c.1118T＞G(p.L373R)。GDF5~(L373R)突变体可以二聚化并分泌至细胞外。
(2)SHFM家系致病基因突变为染色体10q24区域包括LBX1、BTRC、POLL、DPCD和FBXW4等五个基因在内的488859bp串联重复突变。
(3)HFGS家系致病基因突变为HOXA13基因杂合错义突变c.1123G＞T(p.V375F)。HOXA13~(V375F)突变体对EphA7转录激活能力下降,对EphA7、EphA6和Bmp2启动子区及Sostdcl 3'UTR结合能力下降。
Introduction
Congenital limb malformation(LM) is one of the most common birth defects in general population,it may affect the appearance and functional movement,even makes the individuals loss of the ability to work and selfcare.For the past few years,along with the completion of the human genome project(HGP),The identification of causatibe gene mutation about the human single gene inheritance diseases is continuously developed, and various pathopoiesis genes of LM had been identified gradually,the study data of human congenital LM not only could replenish the deficiency of animal and cell experiments,but also help to understand the process of embryonic development, Moreover,it could provide solid therotical basis for the study of human tissue engineering and medicine of gene therapy.So it is important to study the mutation identification and pathogenic mechanism in limb malformations
The etiology can be subdivided into two general categories including environmental and genetic factors.The genetic factors refer to gene mutations and chromosomal aberrations.These LMs may present as an isolated trait or as part of a genetic syndrome.Many gene mutations and chromosomal aberrations may lead to abnormal morphogenesis thereby causing various developmental defects,such as brachydactyly(BD),ectrodactyly(ED),polydactyly(PD) and syndactyly(SD).
Proximal symphalangism(SYM1,MIM 185800),which is characterized by brachydactyly and fusions of autopod skeletal elements and sometimes regarded as part of BDC(Brachydactyly type C,MIM 113100).SYM1 would be transmitted in antosomal dominant pattern and be caused by mutation of GDF5(growth/ differentiation factor 5,MIM 601146) or NOG(MIM 602991 ).
Ectrodactyly,also known as split-hand/split-foot malformation(SHFM),is a congenital autopod malformation characterized by cleft of the hand and/or foot due to the absence of the central rays.It showed variable limb phenotype ranging from typical lobster-claw malformations to monodactyly.Up to now,five loci of SHFM have been identified,including SHFM1(MIM 183600),SHFM2(MIM 313350),SHFM3(MIM 600095),SHFM4(MIM 605289) and SHFM5(MIM 606708),at human chromosome regions 7q21 Xq26,10q24,.3q27 and 2q31,respectively.An about 0.5Mb large-scale DNA duplication at the SHFM3 locus and point mutations in TP63(MIM 603273) at the SHFM4 locus have been identified.
Hand-foot-genital syndrome(HFGS,MIM 140000) is a malformation that affected the distal limbs and genitourinary.In the limbs,the abnormality is brachydactyly,delayed ossification,fusion,and shortening of the carpals and tarsals. Urinary abnormalities include ectopic ureteric orifices,vesicoureteric reflux and pelviureteric junction obstruction and can lead to chronic pyelonephritis,renal insufficiency,and renal transplant.Genital abnormalities include hypospadias in males and Mullerian duct fusion defects in females,the latter defects range from isolated longitudinal vaginal septum to double uterus with double cervix.HFGS would be transmitted in antosomal dominant pattern and be caused by mutation of Hox gene HOXA13(Homeobox A13,MIM 142959) at chromosome 7p15.
In the present study,we recruited three Chinese families with different LMs (SYM1,SHFM and HFGS) for research.First of all,Haplotyping was performed to locate the chromosome position of pathopoiesis genes,then,we undertook DNA sequencing or quantitative PCR to identify the mutations,further,western blot, dual-luciferase reporter assay and chromatin immunoprecipitation assay were undertaken to analyze the preliminary functional characterization of the mutant genes
Materials and methods
1、Subject
Family investigations were performed in the three LM Chinese families, including family medical history and pedigree,then medical examinations or X-ray examinations were performed and peripheral venous blood samples from all available family members were collected after informed consent.
2、Haplotype analysis was performed to locate the causative genes
Using the UCSC Genome Browser,20 perfect microsatellite markers covering five SHFM loci were selected for haplotype analysis.The PCR products of the microsatellite markers were separated by electrophoresis on denaturing polyacrylamide gel and allele fragments were detected with routine silver staining,then on the basis of individual's genotype and kinship,the haplotypes were deduced according to the Mendel's law of inheritance.
3、DNA sequencing of the candidate gene coding sequence
After determining the candidate gene by haplotyping,PCR was performed to amplify the exons of the gene,and then the amplicons were sequenced.
4、Quantitative PCR was performed to detect genomic copy number variation
Real time fluorescence quantitative PCR was performed to compare the affected individual's genomic copy number to that of control.
5、Long range PCR coupled with DNA sequencing was performed to determine the break point of the duplication
After minimizing the extent of the breakpoint to about 2-4kb by quantitative PCR, the proximal and distal breakpoint positions were alnplified by long range PCR by telomeric forward primer R2-2F and centromeric reverse primer L10-5R,then the amplicons were sequenced.
6、Clone of the wild and mutant type eukaryotic expression vector
The wild-type GDF5 or HOXA13 coding sequences were amplified by PCR and then were ligated with pLXSN vector or p3xFLAG-CMV vector,respectively,and transformed the competent cell.then the positive clones were verified by sequencing. The mutant expression vectors were cloned by site-directed mutagenesis using the wild-type vectors as templates.
7、Western blot was performed to detect the dimerization and secretion of GDF5~(1.373R)
COS7 cells that stable expression wild type and mutant GDF5 were obtained by G418 selection,nonreducing SDS-PAGE and immunodetection was applied to detect dimerization and secretion of GDF5(1.373R) in the transgene cell lysate and medium supernate.
8、Using dual luciferase reporter assay to investigate the consequences of the HOXA13~(V375F) in transactivating the promoter of EphA7
We transfected the wild-type and mutant expression vector,luciferase reporter driven by EphA7 promoter and renilla luciferase reporter into mouse C3H10T1/2 cell, relative luciferase activity was tested by luminometer.
9、ChIp was undertook to test the binding ability of HOXA13~(V375F) to downstream target gene promoter
Chromatin immunoprecipitation(ChIP) assay was performed using anti-Flag monoclonal antibody after transfection of the expression vectors of wild type or mutant HOXA13 into nlouse C3H10 T1/2 cell.Then quantitative PCR were perfomed to test the enrichment of downstream target gene promoter region containing HOXA13 binding site.
Results
1、Subject
We performed clinical classification for the limb malformations families that had been recruited,and regarded them as SYM1.SHFM and HFGS,respectively.
2、Haplotype analysis
Microsatellite markers,D20S787,D20S601,D20S909 and D20S914 at chromosome 20q11 were cosegregation with malformations of SYM1 individuals;The potential haplotype shared by all affected individuals of SHFM was detected in the microsatellite markers of LBX1T1,D10S1239,DactylinTAAT,Dactylin2 and Dactylin3 at chromosome 10q24.
3、DNA sequence of the candidate gene coding sequence
One heterozygosis missence mutation c.1118T＞G(p.L373R) in GDF5 in SYM1 family,and another heterozygosis missence mutation c.1123G＞T(p.V375F) in HOXA13 in HFGS family were identified,both mutations caused restriction fragment length polymorphism(RFLP),and were confirmed by restriction analysis to cosegregate with the LM phenotypes in all affected individuals,but not detected in all unaffected individuals of the families or 50 unrelated control individuals.
4、Quantitative PCR was performed to detect genomic copy number variation
Results of quantitative PCR suggested that all affected individuals had an extra copy in the rearrangement region including LBX1,BTRC,POLL,DPCD and FBXW4 at chromosome 10q24 locus.
5、Long range PCR coupled with DNA sequence was performed to determine the break point of the duplication
The proximal and distal breakpoint positions were refined by quantitative PCR and were amplified by long range PCR using forward primer R2-2F and reverse primer L10-5R with a 2000bp production in 3 affected individuals,but no production in the normal family members.Sequence and blat analysis of the chimeric sequence localized the telomeric and the centromeric breakpoint to position 103453895bp and 102965035bp,respectively.The duplication were tandem head-to-tail in orientation region,spanning 488,859bp in length,and encompassed the entire LBXI,BTRC,POLL, DPCD and FBXW4 genes,We concluded that the rearrangement may be NHEJ (nonhomologous end joining) or a simplified FoSTeS event(replication fork stalling and template switching,FoSTeS).
6、Clone of the wild-type and mutant eukaryotic expression vector
The wild-type and the mutant GDF5 or HOXA13 coding sequences were cloned into the pLXSN vector or p3xFLAG-CMV vector to drive expression of the fusion proteins tagged with myc or flag,respectively.The sequence and reading frame of clone vectors were verified by sequencing.
7、Western blot was performed to detect the dimerization and secretion of GDF5~(L373R)
Specific band was detectable in the cell lysate and medium supernate of cells transfected with the mutant GDF5 as well as the wild-type GDF5 by western blot, indicating that the mutant proteins could be dimerizated and secreted out of cytoplasm as the wild-type one.
8、Using dual luciferase reporter assay to investigate the consequences of the HOXA13~(V3755F) in transactivating the promoter of EphA7
The dual luciferase reporter assay results showed that the mutant HOXA13~(V375F) impaired HOXA13's capacity to transactivate EphA 7 promoter,remaining 66.72%of the reporter activity compared to the wild-type counterpart.
9、ChIP was undertook to test the binding ability of HOXA13~(V375F) to downstream target gene promoter
Chromatin immunoprecipitation(ChIP) assay results indicated that mutant HOXA13~(V375F) impaired the DNA binding ability of HOXA13 at EphA7,EphA6 and Bmp2 promoters or Sostdc13'UTR,remaining 65.98%.72.95%,42.89%and 68.03%of the binding ability compared to the wild-type counterpart,respectively.No significant change was observed in theBmp 7 and Sostdcl promoters.
Conclusion
(1) The pathogenic mutation for SYM1 patients was a novel missence mutation in GDF5,c.1118T＞G(p.L373R).The mutant protein GDF5~(L373R) could be dimerizated and secreted out of cytoplasm as the wild-type one.
(2) The pathogenic mutation for SHFM patients was a tandem duplication spanning 488859 bp in length,and encompassed the entire LBX1,BTRC,POLL,DPCD and FBXW4 genes.
(3) The pathogenic mutation for HFGS patients was a novel missence mutation in HOXA13,c.1123G＞T(p.V375F).The mutant protein HOXA13~(V375F) impaired HOXA13's capacity to transactivate EphA7 promoter,and damaged the DNA binding ability of HOXA13 at EphA 7,EphA6 and Bmp2 promoters or Sostdc13'UTR.
先天性肢端畸形是人类最常见的出生缺陷之一,不仅影响肢体的外观形态和功能运动,甚至使患者丧失劳动和生活能力。近年来,随着人类基因组计划的完成,人类单基因遗传病致病基因鉴定不断取得进展,各种四肢先天畸形的致病基因也逐渐被发现,人类先天性肢端畸形研究的数据不仅将补充以往动物和细胞实验的不足,还可以加深对胚胎发育过程的理解,而且可以为人类组织工程学研究和基因治疗药物的研制提供强有力的理论依据,因此对肢端畸形的致病基因突变鉴定及致病机制研究至关重要。
引起先天性肢端畸形的原因有环境因素和遗传因素两大类。遗传因素主要指基因突变或染色体畸变,其导致的肢端畸形既可单独发生又可是某种综合征的一部分。多种基因突变和染色体畸变都会直接引起肢端形态发生(morphogenesis)的异常,表现不同类型的肢端畸形,如短指(趾)(brachydactyly,BD)、缺指(趾)(ectrodactyly,ED)、多指(趾)(polydactyly,PD)和并指(趾)(syndactyly,SD)畸形等。
近端指(趾)间关节粘连(proximal symphalangism,SYM1,MIM 185800)以肢端骨骼融合和短指(趾)为主要表型,常被认为是BDC(Brachydactyly type C,MIM 113100)的一部分。SYM1为常染色体显性遗传,致病基因是GDF5(growth/differentiation factor 5,MIM 601146)或NOG(MIM 602991)。
缺指(趾)/手足裂畸形(ectrodactyly/split-hand/split-foot malformation,SHFM)是四肢端骨(atttopod)中部指轴发育不全而剩余指(趾)呈不同模式融合导致严重影响患者精细活动的先天性肢端畸形,曲型表现为龙虾爪(中部指轴缺陷)或独指(桡侧指轴缺陷而无裂隙,第5指(趾)总不受累)。目前已定位的人类SHFM遗传位点包括:SHFM1(7q21,MIM 183600)、SHFM2(Xq26,MIM 313350)、SHFM3(10q24,MIM 600095)、SHFM4(3q27,MIM 605289)和SHFM5(2q31,MIM 606708)。SHFM3和SHFM4位点的致病突变已经明确,分别是染色体10q24区域内约0.5Mb的DNA串联重复和染色体3q27区域内TP63(MIM 603273)基因的点突变。
手足生殖器综合征(Hand-foot-genital syndrome,HFGS,MIM 140000)是累及手足和泌尿生殖系统的畸形,肢端畸形包括短指(趾)、延迟骨化、融合及腕骨和跗骨短小等。泌尿系统的异常包括输尿管口异位、膀胱输尿管反流和肾盂输尿管接合处梗阻导致慢性肾盂肾炎、肾功能不全和肾移植。生殖系统畸形包括男性的尿道下裂和女性的苗勒(氏)管融合导致阴道纵隔和双子宫。HFGS为常染色体显性遗传,致病基因为染色体7p15的同源盒基因HOXA13(Homeobox A13,MIM142959)。
本文以上述肢端畸形的三个中国人家系(SYM1、SHFM和HFGS)为研究对象,首先利用单体型分析进行了致病基因染色体定位,然后对候选基因DNA测序或定量PCR进行突变鉴定,进一步通过western blot、双萤光素酶报告基因检测系统和染色质免疫沉淀等技术对突变基因功能进行初步研究。
材料与方法
1、家系资料
对家系中可能追溯到的家庭成员进行详细调查,绘制家系系谱图:签署“知情同意书”;对患者进行体格检查和手足骨骼X光检查;采集外周静脉血。
2、单体型分析定位候选致病基因
利用UCSC基因组生物信息学网站在5个SHFM位点选择20对微卫星多态标记,PCR扩增后经变性聚丙烯酰胺凝胶电泳和银染,读出每个个体标记的基因型。再根据个体基因型和系谱中的亲缘关系,按照孟德尔遗传定律推导出同一条染色体上不同标记构成的单体型。
3、候选基因编码区DNA测序
根据单体型分析确定候选基因后,PCR扩增患者候选基因外显子,PCR产物纯化后测序。
4、定量PCR检测基因组拷贝数变异
利用实时荧光定量PCR扩增患者候选基因,与正常个体比较其基因组拷贝数是否发生改变。
5、长距离PCR结合DNA测序确定断裂点
定量PCR将两侧断裂点范围缩小到2-4kb后,以患者基因组DNA为模板,利用端粒侧重复的正向引物R2-2F及中心粒侧重复的反向引物L10-5R(outfacingprimers)行PCR扩增,PCR产物纯化后测序。
6、构建野生型和突变型真核表达载体
PCR扩增目的基因GDF5和HOXA13编码序列,分别与真核表达载体pLXSN和p3xFLAG-CMV7连接后转化感受态细胞,阳性克隆测序验证;以野生型载体为模板,定点诱变产生突变型载体。
7、Western blot检测GDF5~(L373R)二聚体形成和分泌
G418筛选稳定整合野生型和突变型GDF5的COS7细胞系,收集细胞培养上清和细胞总蛋白,经非还原SDS-PAGE电泳后检测GDF5~(L373R)二聚体形成和分泌情况。
8、双萤光素酶报告基因检测系统检测HOXA13~(V375F)对EphA7基因的转录激活能力
将野生型和突变型HOXA13真核表达载体分别与报告基因pGL3-EphA7及内参基因pRL-CMV共转染C3H10 T1/2细胞,计算萤光素酶比活性。
9、染色质免疫共沉淀检测HOXA13~(V375F)对下游靶基因启动子区的结合能力
将野生型和突变型HOXA13真核表达载体分别转染C3H10 T1/2细胞,利用anti-Flag单克隆抗体进行染色质免疫沉淀,然后利用定量PCR检测免疫沉淀的DNA量是否有变化。
实验结果
1、家系资料
根据患者的肢端畸形特征及临床表现和X光片等将收集的3个家系分别诊断为SYM1、SHFM和HFGS。
2、单体型分析
染色体20q11区域的微卫星标记D20S787、D20S601、D20S909和D20S914与SYM1家系畸形共分离;染色体10q24区域的微卫星标记LBXlT1、D10S1239、DactylinTAAT、Dactylin2和Dactylin3与SHFM家系畸形共分离。
3、候选基因编码区DNA测序
SYM1家系患者检测到GDF5基因杂合错义突变c.1118T＞G(p.L373R);HFGS家系患者检测到HOXA13基因杂合错义突变c.1123G＞T(p.V375F)。突变直接导致限制性片段长度多态性(restriction fragment length polymorphism,RFLP),经限制性内切酶酶切验证,家系的患者都携带突变,而家系的正常个体和50名正常无关对照个体都不携带此突变。
4、定量PCR检测基因组拷贝数变异
定量PCR结果表明SHFM家系患者在染色体10q24区域存在包括LBX1、BTRC、POLL、DPCD和FBXW4等五个基因在内的重复突变,DNA增加一个拷贝。
5、长距离PCR结合DNA测序确定重排断裂点
利用正向引物R2-2F和反向引物L10-5R行PCR扩增,SHFM家系患者的基因组都可以扩出约2000bp的特异性片段,而家系的正常个体扩增不出此片段,测序结果表明端粒侧和中心粒侧的断裂点分别在103453895bp处和102965035bp处,重复范围为488859bp,重复方式为串联重复。根据断裂点碱基信息,推测其重复机制可能为非同源末端连接(nonhomologous end joining,NHEJ)或复制叉停滞和模板转换(replication fork stalling and template switching,FoSTeS)。
6、构建野生型和突变型真核表达载体
测序结果表明野生型和突变型GDF5真核表达载体(pLmycGDF5~(WT)SN和pLmycGDF5~(L373R)SN)和HOXA13真核表达载体(p3xFLAG-HOXA13及p3xFLAG-HOXA13~(V375F))构建成功,无突变,阅读框正确。
7、Western blot检测GDF5~(L373R)二聚体形成和分泌
Western blot检测结果表明GDF5~(L373R)突变体可以二聚化并分泌至细胞外。
8、双萤光素酶报告基因检测系统检测HOXA13~(V375F)对EphA7基因的转录激活能力
双萤光素酶报告基因检测结果表明突变型HOXA13~(V375F)转录激活EphA7基因的能力下降至野生型的66.72%。
9、染色质免疫共沉淀检测HOXA13~(V375F)对下游靶基因启动子区的结合能力
定量PCR检测染色质免疫沉淀下游靶基因DNA量,结果表明与HOXA13~(V375F)突变体结合的EphA7、EphA6和Bmp2启动子及Sostdcl 3'UTR的量分别降低至野生型的65.98%、72.95%、42.89%和68.03%;与HOXA13~(V375F)突变体结合的Bmp7和Sostdcl启动子的量与野生型比较无明显差异。
结论
(1)SYM1家系致病基因突变为GDF5基因杂合错义突变c.1118T＞G(p.L373R)。GDF5~(L373R)突变体可以二聚化并分泌至细胞外。
(2)SHFM家系致病基因突变为染色体10q24区域包括LBX1、BTRC、POLL、DPCD和FBXW4等五个基因在内的488859bp串联重复突变。
(3)HFGS家系致病基因突变为HOXA13基因杂合错义突变c.1123G＞T(p.V375F)。HOXA13~(V375F)突变体对EphA7转录激活能力下降,对EphA7、EphA6和Bmp2启动子区及Sostdcl 3'UTR结合能力下降。
Introduction
Congenital limb malformation(LM) is one of the most common birth defects in general population,it may affect the appearance and functional movement,even makes the individuals loss of the ability to work and selfcare.For the past few years,along with the completion of the human genome project(HGP),The identification of causatibe gene mutation about the human single gene inheritance diseases is continuously developed, and various pathopoiesis genes of LM had been identified gradually,the study data of human congenital LM not only could replenish the deficiency of animal and cell experiments,but also help to understand the process of embryonic development, Moreover,it could provide solid therotical basis for the study of human tissue engineering and medicine of gene therapy.So it is important to study the mutation identification and pathogenic mechanism in limb malformations
The etiology can be subdivided into two general categories including environmental and genetic factors.The genetic factors refer to gene mutations and chromosomal aberrations.These LMs may present as an isolated trait or as part of a genetic syndrome.Many gene mutations and chromosomal aberrations may lead to abnormal morphogenesis thereby causing various developmental defects,such as brachydactyly(BD),ectrodactyly(ED),polydactyly(PD) and syndactyly(SD).
Proximal symphalangism(SYM1,MIM 185800),which is characterized by brachydactyly and fusions of autopod skeletal elements and sometimes regarded as part of BDC(Brachydactyly type C,MIM 113100).SYM1 would be transmitted in antosomal dominant pattern and be caused by mutation of GDF5(growth/ differentiation factor 5,MIM 601146) or NOG(MIM 602991 ).
Ectrodactyly,also known as split-hand/split-foot malformation(SHFM),is a congenital autopod malformation characterized by cleft of the hand and/or foot due to the absence of the central rays.It showed variable limb phenotype ranging from typical lobster-claw malformations to monodactyly.Up to now,five loci of SHFM have been identified,including SHFM1(MIM 183600),SHFM2(MIM 313350),SHFM3(MIM 600095),SHFM4(MIM 605289) and SHFM5(MIM 606708),at human chromosome regions 7q21 Xq26,10q24,.3q27 and 2q31,respectively.An about 0.5Mb large-scale DNA duplication at the SHFM3 locus and point mutations in TP63(MIM 603273) at the SHFM4 locus have been identified.
Hand-foot-genital syndrome(HFGS,MIM 140000) is a malformation that affected the distal limbs and genitourinary.In the limbs,the abnormality is brachydactyly,delayed ossification,fusion,and shortening of the carpals and tarsals. Urinary abnormalities include ectopic ureteric orifices,vesicoureteric reflux and pelviureteric junction obstruction and can lead to chronic pyelonephritis,renal insufficiency,and renal transplant.Genital abnormalities include hypospadias in males and Mullerian duct fusion defects in females,the latter defects range from isolated longitudinal vaginal septum to double uterus with double cervix.HFGS would be transmitted in antosomal dominant pattern and be caused by mutation of Hox gene HOXA13(Homeobox A13,MIM 142959) at chromosome 7p15.
In the present study,we recruited three Chinese families with different LMs (SYM1,SHFM and HFGS) for research.First of all,Haplotyping was performed to locate the chromosome position of pathopoiesis genes,then,we undertook DNA sequencing or quantitative PCR to identify the mutations,further,western blot, dual-luciferase reporter assay and chromatin immunoprecipitation assay were undertaken to analyze the preliminary functional characterization of the mutant genes
Materials and methods
1、Subject
Family investigations were performed in the three LM Chinese families, including family medical history and pedigree,then medical examinations or X-ray examinations were performed and peripheral venous blood samples from all available family members were collected after informed consent.
2、Haplotype analysis was performed to locate the causative genes
Using the UCSC Genome Browser,20 perfect microsatellite markers covering five SHFM loci were selected for haplotype analysis.The PCR products of the microsatellite markers were separated by electrophoresis on denaturing polyacrylamide gel and allele fragments were detected with routine silver staining,then on the basis of individual's genotype and kinship,the haplotypes were deduced according to the Mendel's law of inheritance.
3、DNA sequencing of the candidate gene coding sequence
After determining the candidate gene by haplotyping,PCR was performed to amplify the exons of the gene,and then the amplicons were sequenced.
4、Quantitative PCR was performed to detect genomic copy number variation
Real time fluorescence quantitative PCR was performed to compare the affected individual's genomic copy number to that of control.
5、Long range PCR coupled with DNA sequencing was performed to determine the break point of the duplication
After minimizing the extent of the breakpoint to about 2-4kb by quantitative PCR, the proximal and distal breakpoint positions were alnplified by long range PCR by telomeric forward primer R2-2F and centromeric reverse primer L10-5R,then the amplicons were sequenced.
6、Clone of the wild and mutant type eukaryotic expression vector
The wild-type GDF5 or HOXA13 coding sequences were amplified by PCR and then were ligated with pLXSN vector or p3xFLAG-CMV vector,respectively,and transformed the competent cell.then the positive clones were verified by sequencing. The mutant expression vectors were cloned by site-directed mutagenesis using the wild-type vectors as templates.
7、Western blot was performed to detect the dimerization and secretion of GDF5~(1.373R)
COS7 cells that stable expression wild type and mutant GDF5 were obtained by G418 selection,nonreducing SDS-PAGE and immunodetection was applied to detect dimerization and secretion of GDF5(1.373R) in the transgene cell lysate and medium supernate.
8、Using dual luciferase reporter assay to investigate the consequences of the HOXA13~(V375F) in transactivating the promoter of EphA7
We transfected the wild-type and mutant expression vector,luciferase reporter driven by EphA7 promoter and renilla luciferase reporter into mouse C3H10T1/2 cell, relative luciferase activity was tested by luminometer.
9、ChIp was undertook to test the binding ability of HOXA13~(V375F) to downstream target gene promoter
Chromatin immunoprecipitation(ChIP) assay was performed using anti-Flag monoclonal antibody after transfection of the expression vectors of wild type or mutant HOXA13 into nlouse C3H10 T1/2 cell.Then quantitative PCR were perfomed to test the enrichment of downstream target gene promoter region containing HOXA13 binding site.
Results
1、Subject
We performed clinical classification for the limb malformations families that had been recruited,and regarded them as SYM1.SHFM and HFGS,respectively.
2、Haplotype analysis
Microsatellite markers,D20S787,D20S601,D20S909 and D20S914 at chromosome 20q11 were cosegregation with malformations of SYM1 individuals;The potential haplotype shared by all affected individuals of SHFM was detected in the microsatellite markers of LBX1T1,D10S1239,DactylinTAAT,Dactylin2 and Dactylin3 at chromosome 10q24.
3、DNA sequence of the candidate gene coding sequence
One heterozygosis missence mutation c.1118T＞G(p.L373R) in GDF5 in SYM1 family,and another heterozygosis missence mutation c.1123G＞T(p.V375F) in HOXA13 in HFGS family were identified,both mutations caused restriction fragment length polymorphism(RFLP),and were confirmed by restriction analysis to cosegregate with the LM phenotypes in all affected individuals,but not detected in all unaffected individuals of the families or 50 unrelated control individuals.
4、Quantitative PCR was performed to detect genomic copy number variation
Results of quantitative PCR suggested that all affected individuals had an extra copy in the rearrangement region including LBX1,BTRC,POLL,DPCD and FBXW4 at chromosome 10q24 locus.
5、Long range PCR coupled with DNA sequence was performed to determine the break point of the duplication
The proximal and distal breakpoint positions were refined by quantitative PCR and were amplified by long range PCR using forward primer R2-2F and reverse primer L10-5R with a 2000bp production in 3 affected individuals,but no production in the normal family members.Sequence and blat analysis of the chimeric sequence localized the telomeric and the centromeric breakpoint to position 103453895bp and 102965035bp,respectively.The duplication were tandem head-to-tail in orientation region,spanning 488,859bp in length,and encompassed the entire LBXI,BTRC,POLL, DPCD and FBXW4 genes,We concluded that the rearrangement may be NHEJ (nonhomologous end joining) or a simplified FoSTeS event(replication fork stalling and template switching,FoSTeS).
6、Clone of the wild-type and mutant eukaryotic expression vector
The wild-type and the mutant GDF5 or HOXA13 coding sequences were cloned into the pLXSN vector or p3xFLAG-CMV vector to drive expression of the fusion proteins tagged with myc or flag,respectively.The sequence and reading frame of clone vectors were verified by sequencing.
7、Western blot was performed to detect the dimerization and secretion of GDF5~(L373R)
Specific band was detectable in the cell lysate and medium supernate of cells transfected with the mutant GDF5 as well as the wild-type GDF5 by western blot, indicating that the mutant proteins could be dimerizated and secreted out of cytoplasm as the wild-type one.
8、Using dual luciferase reporter assay to investigate the consequences of the HOXA13~(V3755F) in transactivating the promoter of EphA7
The dual luciferase reporter assay results showed that the mutant HOXA13~(V375F) impaired HOXA13's capacity to transactivate EphA 7 promoter,remaining 66.72%of the reporter activity compared to the wild-type counterpart.
9、ChIP was undertook to test the binding ability of HOXA13~(V375F) to downstream target gene promoter
Chromatin immunoprecipitation(ChIP) assay results indicated that mutant HOXA13~(V375F) impaired the DNA binding ability of HOXA13 at EphA7,EphA6 and Bmp2 promoters or Sostdc13'UTR,remaining 65.98%.72.95%,42.89%and 68.03%of the binding ability compared to the wild-type counterpart,respectively.No significant change was observed in theBmp 7 and Sostdcl promoters.
Conclusion
(1) The pathogenic mutation for SYM1 patients was a novel missence mutation in GDF5,c.1118T＞G(p.L373R).The mutant protein GDF5~(L373R) could be dimerizated and secreted out of cytoplasm as the wild-type one.
(2) The pathogenic mutation for SHFM patients was a tandem duplication spanning 488859 bp in length,and encompassed the entire LBX1,BTRC,POLL,DPCD and FBXW4 genes.
(3) The pathogenic mutation for HFGS patients was a novel missence mutation in HOXA13,c.1123G＞T(p.V375F).The mutant protein HOXA13~(V375F) impaired HOXA13's capacity to transactivate EphA7 promoter,and damaged the DNA binding ability of HOXA13 at EphA 7,EphA6 and Bmp2 promoters or Sostdc13'UTR.
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