【作者】 吕丹；

【导师】 张学；

【作者基本信息】 中国协和医科大学 ， 遗传学， 2008， 博士

【摘要】 三种先天性肢端畸形的分子遗传学研究先天性肢端畸形在活产儿中的发生率约为0.5‰-1‰,主要包括指(趾)的数目、长度以及解剖形态的异常,是由于遗传进化过程中的变异或发育过程中的不良因素(如异常子宫内环境)所致。研究先天性肢端畸形,寻找并鉴定致病突变对于指导遗传咨询,了解肢端发育调控机制具有十分重要的意义和价值。本文分别对短/并指(趾)复合畸形、并多指(趾)畸形和缺指(趾)畸形三种先天性肢端畸形的四个家系进行了致病基因的突变筛查,并对其中一个表型罕见的短/并指(趾)复合畸形家系进行了致病突变的初步功能研究。第一部分一种新型短/并指(趾)复合畸形家系致病突变的研究短指(趾)畸形(Brachydactyly,BD)是由指(趾)骨、掌(跖)骨发育异常而导致的指(趾)的缩短畸形,可以作为独立性状单独出现,亦可成为某些综合征的表现之一。非综合征性BD可以分为A-E五种类型,其中B型短指(趾)(Brachydactylytype B,BDB)又可分为BDB1(MIM 113000)和BDB2(MIM 611377)两个亚型。BDB1以远节指(趾)骨短小、指(趾)甲发育不良、中节指(趾)骨发育不全、不同程度的指(趾)关节粘连以及伴有拇指(趾)宽大畸形为主要特征,是由位于染色体9q22的ROR2基因突变导致,其突变类型主要为杂合性无义突变和移码突变,集中分布于ROR2基因酪氨酸激酶结构域(Tyrosine kinase domain,TK)5’近端、TK区与跨膜区之间以及TK结构域3’远端、TK区与S/T1之间的两个区域内。并指(趾)畸形(Syndactyly,SD)是相邻指(趾)间皮肤软组织融合形成的手足畸形,伴有或不伴有指(趾)间的骨性融合,可分为I-V五种类型,其中Ⅰ型并指(趾)(SD1,MIM185900)是最常见的并指(趾)类型,主要表现为3、4指和2、3趾并指(趾),可发生于单侧,表现为不对称性。国外已将SD1定位于染色体2q34-36区域内,迄今尚未发现致病基因。本文首先对一种新型短/并指(趾)复合畸形家系进行了致病突变的筛查。以一个BDB1合并SD1的复合表型家系为研究对象,通过连锁分析,在染色体9q22附近的两个标记(D9S1815和D9S1841),获得最高LOD值2.71(θ=0),高度提示疾病表型与这两个位点之间可能存在连锁关系:然后通过PCR扩增ROR2基因外显子并直接测序的方法,在ROR2基因第九外显子内发现一个单碱基缺失突变c.2243delC,使突变蛋白失去C-末端两个丝/苏氨酸富集区(S/T1、S/T2)和脯氨酸富集区三个结构域,并产生一小段由24个氨基酸残基组成的新肽链;最后我们在家系所有成员中对该突变进行了酶切验证,进一步确认此突变为该复合肢端畸形家系所有患者共有的特点。在对ROR2基因突变导致BDB1合并SD1的初步功能研究中,我们首先分别构建了野生型(ROR2~WT)和突变型(ROR2~W749fsX24)GFP-ROR2融合表达载体,转染HeLa细胞系,观察融合蛋白的亚细胞定位情况,结果发现ROR2蛋白定位有所改变,即野生型ROR2蛋白主要分布于细胞质膜,而突变型ROR2则趋向分散于细胞质内。国内外迄今为止,未见关于突变ROR2蛋白亚细胞定位情况的报道。在对U2OS细胞的转染过程中还发现:ROR2~W749fsX24组细胞伪足形成与ROR2~WT组相近,明显多于ROR2~W749X组。接下来,我们根据国外关于ROR2能够与14-3-3β相互作用的先期报道,分别将野生型与突变型ROR2蛋白胞质内结构域克隆至相应的表达载体上,再通过酵母双杂交系统和哺乳动物双杂交系统检测野生型与突变型ROR2蛋白与14-3-3β的相互作用情况;同时,也利用野生型与突变型ROR2蛋白全长,联合14-3-3β,在体外进行免疫共沉淀的检测。经由以上方法均未发现野生型或突变型ROR2与14-3-3β相互作用的直接证据。总之,我们报道了一种新型BDB1合并SD1的复合表型家系,通过两点连锁分析将致病基因定位至染色体9q22,并对候选基因ROR2进行突变筛查,发现了一个新的ROR2单碱基缺失突变c.2243delC,同时观察到两个突变组细胞的伪足数目差异较大,即ROR2~W749fsX24组细胞伪足形成与ROR2~WT组相近,明显多于ROR2~W749X组;且发现ROR2~W749fsX24突变型蛋白亚细胞定位有所改变,这是首次关于突变ROR2蛋白亚细胞定位情况的报道。第二部分两个并多指(趾)畸形家系HOXD13基因致病突变的鉴定HOX基因在进化上高度保守,参与机体生长、分化等重要的生理过程。人类具有39个HOX基因,形成4个基因簇(HOXA-HOXD),分布于染色体7p15、17p21、12q13和2q31上。HOXD13基因位于HOXD基因簇5’最末端,包含两个外显子,编码335个氨基酸构成的转录因子。其第一外显子内含有一个45bp的不完全三核苷酸(GCN,N为A、C、G或T之一)重复序列,编码蛋白质N端15个丙氨酸残基。当丙氨酸延长至22-29个时,即可导致并多指(趾)(Synpolydactyly,SPD;MIM 186000)畸形。SPD属于非综合征性并指(趾)Ⅱ型,呈常染色体显性遗传,主要表现为第3、4指和第4、5趾的膜性并指(趾),并可在指(趾)蹼中发现部分或完全复制的额外的指(趾)。所有SPD患者均以并指(趾)为共有的主要表型,伴有或不伴有多指(趾)表现。本研究的研究对象为两个SPD家系。患者表型多样,且表现度不一,除具有典型SPD表现外,家系1的一名患者还具有双手轴前多指,双足轴后多趾的罕见表型,此为国际首例SPD合并轴前多指的病例报道;家系2患者还同时具有D型和E型短指(趾)的特征,亦为比较罕见的SPD类型。我们通过PCR扩增测序以及T-A克隆测序的方法,对整个HOXD13基因进行了突变筛查,在上述两个家系中均检测出HOXD13基因的多聚丙氨酸链延展突变,增加的丙氨酸残基数目分别为7个和9个,与国外先期报道相一致。同时,通过T-A克隆测序,明确了两个插入突变的具体插入位置以及增加的丙氨酸残基的碱基组成,为更好地理解SPD发病机制提供理论依据。上述结果也为丰富SPD的表现度变异提供了宝贵的资料。第三部分缺指(趾)畸形致病突变的研究缺指(趾)(Ectrodactyly,ECD)又称手足裂畸形(Split-hand/split-foot malformation,SHFM),是一种严重影响患者精细活动的先天性肢端畸形,以手足正中裂隙、并指(趾)以及指(趾)骨和掌(跖)骨发育不全为主要特征,可呈现出龙虾爪或独指(趾)的典型表现。SHFM可以单独发生,亦可伴随有其它四肢骨骼的畸形或其它器官的发育异常。SHFM具有高度的遗传异质性,截至目前,共发现六个遗传位点与之密切相关,分别为:SHFM1(MIM 183600),定位于7q21;SHFM2(MIM 313350),定位于Xq26;SHFM3(MIM 600095),定位于10q24.3;SHFM4(MIM 605289),定位于3q27;SHFM5(MIM 606708),定位于2q31以及一个新近报道的遗传位点8q21.11-q22.3。除了SHFM3位点致病突变为染色体10q24区域内约0.5Mb的DNA串联重复;SHFM4位点的致病突变为TP63基因点突变外,其他类型的SHFM均未找到相关致病基因或致病突变。本研究以一个本课题组前期收集的SHFM家系为研究对象,对其进行致病突变的筛查。首先补充完成了高分辨率染色体核型分析,未见异常。之后根据前期微卫星标记的连锁分析结果,将染色体7q21.3位点上SHFM1的最小关键区域内DLX5、DLX6基因及其附近染色体区域作为重点筛查范围。通过PCR扩增测序,将DLX6基因上游至DLX5基因5’末端约40kb的区间范围全部测通,共发现20个碱基改变,其中14个为已知的SNP,其余6个为未知的碱基改变,并未发现明显的致病突变。同时,我们还在SHFM1关键区端粒侧约100kb范围内,选择物种间高度保守的序列元件对患者基因组DNA进行实时荧光定量PCR检测,亦未发现明显的DNA拷贝数目的异常。结合PCR扩增测序和实时荧光定量PCR发现的SNP分布情况,初步推测在chr7:96,469,328-96,474,996范围内有产生缺失突变的可能。更多还原

【Abstract】 Molecular Genetic Studies of Three Congenital Limb MalformationsCongenital limb malformations occur in 1 in 500 to 1 in 1000 human live births, including the alterations of the number,length and anatomic morphology of the digits.The major causes are the abnormal genetic programming and the infaust factors during the development,such as the poor uterine environment.The identification of the responsible gene mutations is important for genetic counseling and the understanding of the mechanisms controlling limb development.In this paper,we performed the molecular genetic studies in three pedigrees with different kinds of congenital limb malformations including brachydactyly type B1（BDB1） combinds syndactyly typel（SD1）,synpolydactyly （SPD） and split-hand/split-foot malformation（SHFM）.PartⅠ:Detection and Functional Analysis of a Pathogenic MutationAssociated with a Novel complex malformation with BDB1 and SD1Brachydactyly（BD） refers to shortening of the fingers or toes due to hypoplasia or aplasia of metacarpals（metatarsals） or phalanges,which can occur as an isolated trait or in association with other malformations.Isolated BD has been categorized to five types, among which the brachydactyly type B（BDB） can be further divided into two subtypes, which are BDB1（MIM 113000） and BDB2（MIM 611377）.The prominent features of BDB1 are hypoplasia or absence of the distal phalanges and nails,hypoplastic middle phalanges and symphalangism.Nonsense and frameshift mutations,either proximal or distal with respect to the tyrosine kinase domain in ROR2,have been identified to be the main cause of this disease.Syndactyly refers to the fusion of soft tissues of fingers and/or toes with or without the fusion of bones.It can be divided into five types,and the most common one is syndactyly typeⅠ（SD1,MIM 185900）.SD1 has been linked to chromosome 2q34-36,manifested as complete or partial webbing between the 3^rd and 4^th fingers associated with the 2^nd and 3^rd toes.It can occur on both sides of the limb,and can also appear asymmetry.Till now,no genes have been reported to be associated with it. We identified a three-generation Chinese Han family with complex phenotypes of BDB1 and SD1.Two-point linkage analysis was performed and a maximal LOD score of 2.71 was obtained for the markers D9S1815 and D9S1841,showing the great possibility of linkage between the positions and this disease.We then directly sequenced the 8^th and 9^th exons of ROR2 gene and found a 1-bp deletion,c.2243delC,in exon 9,which leads to a frame shift mutation at Trp749 and predicts a truncated protein with 24 novel amino acids before the first stop codon.This mutation was further confirmed by restriction analysis among all the family members.During the functional analysis of this novel mutation,we constructed the ROR2^WT/ ROR2^W749fsX24-GFP-fusion expression vector to observe the subcellular location of these two proteins.With the use of HeLa cells as targeting transfected cells,we noticed that ROR2^WT was located in the cytoplasma membrane while ROR2^W749fsX24 was scattered throughout the cytoplasm.When we use U2OS cells,we found that the filopodia of ROR2 ^W749fsX24 were much more than those of ROR2^W749X.To investigate the interaction between ROR2^WT/ROR2^W749fsX24and 14-3-3β,we performed both yeast two-hybrid assay and mammalian two-hybrid assay by using the cytoplasmic part of ROR2^WT/ ROR2 ^W749fsX24and the full length of 14-3-3β.Immunoprecipitation was also used as a test in vitro.No obvious clues of interactions between the two proteins were found.In conclusion,we report a new limb malformation of BDB1 associated with SDL The disease gene in this family was mapped to 9q22 by two-point linkage analysis and a novel mutation,c.2243delC,was identified by directly sequencing of ROR2 gene,which is the first report of this mutation internationally.Further functional analysis reveals that there’s obvious differences in the subcellular location between ROR2^WT and ROR2^W749fsX24,and filopodia formation between ROR2^W749X and ROR2^W749fsX24,which give us the inspiration that ROR2^WT,ROR2^W749fsX and ROR2^W749fsX24may play different roles in cells or even during the limb development.Further study should be done to confirm this. PartⅡ:Mutation Identifications of HOXD13 gene in Two ChineseFamilies with SynpolydactylyThe HOX genes encode a highly conservative family of transcriptional factors,which play a fundamental role in embryonic morphogenesis.In human,as in most vertebrates, there are 39 HOX genes organized into four clusters named HOXA through HOXD,which are believed to have arisen from a single ancestral cluster by duplication and divergence. The four clusters are distributed on different chromosomes including 7p15,17p21,12q13 and 2q31.HOXD 13 gene is located at the 5’ end of HOXD cluster,containing two coding exons and encoding 335 amino acids.There is an imperfect GCN（N=A or C or G of T） triplet repeats in exon 1,encoding a 15-residue polyalanine tract.The expansion of this repeat,resulting in an additional 7-14 alanine residues,would lead to synpolydactyly（SPD, MIM 186000）.SPD is a rare,dominantly- inherited limb malformation,which belongs to Syndactyly typeⅡ.Typically,patients have 3^th/4^th fingers and 4^th/5^th toes syndactyly,with partial or complete digit duplications of the digits in the syndactylous web.All patients, with or without polydactyly,will have syndactyly.The subject of this study is two SPD families with incomplete penetrance and variable expressivity as the common traits.One affected individual in family 1 has the rare phenotype of pre-axial polydactyly in hands and post-axial polydactyly in feet.This is the first report of SPD associated with pre-axial polydactyly in the world so far.The affected individuals in family 2 also have the phenotype of BDD and BDE.This is also a rare case of SPD.We found a 7-alanin expansion in the patients in family 1 and a 9-alanin expansion in family 2 using T-A cloning and sequencing of HOXD13 gene.Meanwhile,the exact insertion site and the specific constitution of the bases in additional alanine residues were revealed,which may provide strong theoretical knowledge to the understanding of the mechanism of SPD. PartⅢ:Analysis of Genettic Loci and Pathogenic MutationsAssociated with EctrodactylyEctrodactyly,also known as split-hand/split-foot malformation（SHFM）,is a congenital autopod malformation characterized by cleft of the hands and/or feet due to the absence of the central rays.Typical cases may be the lobster-claw variety（absence of central rays） or monodactyly type（deficiency of radial rays with no cleft）.It may occur as an isolated entity or as a part of a syndrome.SHFM is a disease of great clinical heterogeneity.Till now,six genetic loci have been identified,including SHFM1（MIM 183600）,SHFM2（MIM 313350）,SHFM3（MIM 600095）,SHFM4（MIM 605289）, SHFM5（MIM 606708） and a new locus reported in 2006,which are on human chromosome regions of 7q21,Xq26,10q24,3q27 2q31 and 8q21.11-q22.3,respectively. Among them,only two pathogenic mutations have been identified,which are large-scale DNA duplications in SHFM3 locus and point mutations in TP63 gene in SHFM4 locus.According to the prior study,we chose DLX5,DLX6 and the adjacent region of chromosome 7q21 as the major screening region.The range of the sequencing started from the 5’ end of DLX5 to about 20kb upstream of DLX6.hi total,20 variations have been identified,14 of which are known SNPs and the others are changes unreported in introns. We also selected some highly conserved elements in this region to perform Real-time PCR considering the possibility of copy number variations in this region.No obvious pathogenic mutations have been found.Associated with the distribution of SNPs identified in sequencing and Real-time PCR,we deduce that if deletions are the main reason for this disease,they would be present in chr7:96,469,328-96,474,996,where we have not detected any SNPs.Array CGH or TAR（transformation-associated recombination） cloning should be performed to confirm this conclusion in the future.更多还原