【作者】 袁林天；

【导师】 文玲英；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2004， 博士

【摘要】 先天缺牙是比较常见的牙齿发育异常疾病。除了智齿以外，其他牙齿的先天缺失率从1.6％到9.6％不等。先天缺牙又称牙齿数目不足，是在牙胚形成过程中或牙胚发育的早期，即牙蕾形成期的异常。先天缺牙可分为个别牙先天缺失(hypodontia)、多数牙先天缺失(oligodontia)和先天无牙症(anodontia)三种类型。多数牙先天缺失是指缺牙数目在6个和6个以上的牙齿(不包括第三磨牙)，通常有综合征并发型和非综合征并发型等两种类型，可以表现为常染色体的显性或隐性遗传、X-连锁遗传特性，有的是散发的，有的则是具有家族遗传史的。 人体任何组织、器官的发育都受到基因的调控，而任何组织、器官的发育异常都与基因突变有关。在牙齿的发育形成过程中有多达200多个基因参与调控，它们决定各个牙齿的位置、数目、大小和形状等。在牙胚发育的各个时期，上皮细胞和间充质细胞之间形成复杂的分子网络，这些网络中的信号分子单独或协同作用，负责牙齿各个性状的发育。但这些信号分子和转录因子与疾病的关系的研究却进展缓慢，例如多数牙先天缺失的分子机制究竟是什么?至今还不甚明确。 研究表明多数牙先天缺失与PAX9密切相关，而少数牙先天缺失主要与MSX1基因突变或缺失有关。PAX9为Pax家族新发现成第四军医大学博士学位论文员，存在所有脊椎动物中，是牙齿发育过程中起关键调控作用的转录因子。同源异型盒基因MSxl在牙齿发育各个时期的许多组织中表达，是FGFS、BMpS信号传导网络中的重要调控分子。 尽管多数学者认为PAXg是引起多数牙先天缺失的主要致病基因，但对其突变的方式和位点仍有争议，不尽相同，这主要与缺牙的数目、位置及患者的遗传背景(种族、国籍等)有关。为此，本课题目的是对多数牙先天缺失相关致病基因进行研究，通过对4例不同类型的多数牙先天缺失病例和1例少数牙先天缺失并有唇愕裂患者的临床资料分析，且在此基础上，采用基因组DNA提取、PCR扩增、纯化、测序、结果分析以及TaqMan一MGB探针进行SNP分析等方法分别进行以下几部分研究: 多数牙先天缺失的临床研究 从常规口腔检查、全口曲面断层X线片、实验室检查和系谱分析等四个方面，对临床就诊的4例不同类型的多数牙先天缺失和1例少数牙先天缺失并有唇愕裂的病例进行临床研究。其中，1例为少汗型外胚叶发育不全综合征并发的多数牙先天缺失，无家族遗传史，且其孪生姐姐全口牙列完好;2例为非综合征性的多数牙先天缺失，但有家族遗传史;1例也为非综合征性的多数牙先天缺失，却是散发的，无家族遗传史。 结果表明:(l)多数牙先天缺失患者缺牙的数目、位置等临床表现不同，遗传方式也多种多样，但临床实验室检查均未见明显异常。(2)处在环境完全相同的二卵双生胚胎，一个表现出外胚叶发育不全的特征，另一个却毫无类似异常，说明本病例的遗传物质起着相当重要的作用。(3)病例2中只有父亲和儿子为多数牙先天缺失患者，家系中的女性无此现象，说明多数牙先天缺失可能为Y连锁遗传病。(4)病例3母女二人临床表现的不尽相同，说明在第四军医大学博士学位论文遗传背景相同条件下环境因素可能是疾病表型多样性的决定因素。(5)病例5母亲妊娠期间吸食毒品可能影响牙胚发育而造成多数牙先天缺失，说明该环境因素也许是引起先天缺牙的因素之一。总之，遗传物质和环境因素是导致牙齿先天缺失疾病表型多样性的决定因素。2.多数牙先天缺失患者PAXg基因突变的检测 为了探讨PAXg基因是否是引起多数牙先天缺失的致病基因及其突变的位点和方式。本部分从上述5个病例及其父母和1例牙列完整儿童共14人的静脉血中提取DNA，在PAxg基因内设计4对引物，采用PCR方法扩增PAXg基因的外显子2、3、4的编码区，而后通过对分段PCR纯化产物的测序，并结合系谱进行序列比对分析，结果发现:(l) PAXg基因外显子3的第88、89位核昔酸是2个不相关联的SNPs，它们出现了杂合或纯合突变。其中，88位点是C一T的同义突变，密码子由CAC一CAT，但仍编码组胺酸。89位点是G~C的错义突变，对应的密码子由GCG一CCG，氨基酸则由丙氨酸(Ala)变为脯氨酸(Pro)。(2)多数牙先天缺失可能与PAXg基因外显子3的第89位核昔酸上出现的由G~C的错义突变有关，而与PAxg基因外显子3的第88位核普酸出现的同义突变无关。(3)多数牙先天缺失可能是由PAXg基因突变引起的不完全外显遗传病，且男性杂合可发病，女性杂合不发病，不管男女性纯合均发病。有的病例符合半显性遗传的特征。3.多数牙先天缺失患者MSxl基因突变的检测 为了对多数牙先天缺失的致病相关基因MSxl及其突变的位点和方式进行探讨，本部分又从上述14人的静脉血中提取DNA，第四军医大学博七学位论文而后在MSxl基因内设计引物，采用PCR方法扩增MSXI基因外显子1、2的编码区，通过对外显子l、2的PCR纯化产物测序，与野生型基因序列比对后，结果发现:(l)3位多数牙先天缺失患者在MSXI基因的内含子2个位点上和外显子1的3个位点上同时出现了杂合突变，它们是5个可能的单核昔酸多态性位点。(2) MSxl外显子1的311位点由G变A更多还原

【Abstract】 Tooth agenesis is a common tooth dysplasia. Except for wisdom tooth, the congenital deficiency rate is about 1.6%-9.6%. Tooth agenesis also named teeth number deficiency. It happens in tooth forming process or earlier period of tooth germ development. Congenital missing tooth can be divided into hypodontia, oligodontia and anodontia. Oligodontia is defined as absence of six or more teeth. It has two types: syndromic oligodontia and non-syndromic oligodontia. The condition can be inherited as an autosome-dominant , autosome-recessive, or X-linked trait, some of which happens individually, some has familial heredity background.Any tissue or organ’s development is controlled by genes, and any tissue or organ’s displasia is related togene’ s mutation as well. There are more than 200 genes controlling and adjusting teeth development. They decide tooth position, number, size and shape, etc. In the period of tooth germ development, complicated molecule networks are established between epithelial cells and mesenchymal cells. The signal molecules of the networks are responsible for the tooth development alone or coordinated with other molecules. However, the research about the relationship between these signal molecules and transcription factors with diseases got few achievement. For example, what is the molecule mechanism of oligodontia? No scientificanswerers are got up to now.The research found that oligodontia is intimately related to PAX9, and hypodontia is mainly related to MSX1. PAX9 is a newly discovered member of Pax family, existing in all vertebrates, which is a transcription factor that takes a key responsibility in tooth development. MSX1 expressed in many tissues of teeth in each period of tooth growth, and is an important controlling molecule in FGFs> BMPs signal networks.Most of researchers suggested that PAX9 is a chief pathogenic gene that causes oligodontia, but the ways and positions of mutation are disputable , that is related to the number, position of missing tooth and patient’s genetic background(races, nationalities, et al). To study the pathogenic genes of oligodontia further, this article select PAX9 and MSX1 as candidate pathogenic genes of oligodontia for further study by PCR, sequencing and SNPdetection using TaqMan-MGB probe, based on analyzing the clinical data of 4 different cases of oligodontia and 1 case of hypodontia accompanied with lip cleft palate. As follows:1 The clinical study on oligodontiaWe studyed the clinical character of 4 different types of cases on. oligodontia and one case on hypodontia accompanied with lip cleft palate by common oral examination, pantomoagram radiographs of dentition, laboratory examination and pedigree analysis. The results were as following: (1) The number and positions of missing teeth are different, and oligodontia patients have varieties heredity styles, but clinical laboratory examinations have no obvious abnormorlity. (2)In the same environmental condition, One of twins manifests Ectodermal dysplasia syndrome, another don’t have the similar abnormality, so genetic materials are important factors in case 1. (3)In case 2 the proband and his father have oligodontia, and the female of the family are normal, it suggest that oligodontia may be Y-linked hereditary disease. (4)In case 3 the patient and his mother have different clinical situations, and it suggest that environmental factors may determine the phenotype diversity of oligodontia. (5) In case 5 drug taking of the patient’s mother in gestational period resulted in children’s oligodontia, suggest that drug taking may be a factor causing oligodontia. All of above results suggest that Environmental factors and geneticmaterials are determinative factors, which result in phenotype diversity of congenital missing tooth.2 The detection of PAX9 gene mutation in patients of oligodontiaTo study whether PAX9 is the pathogenic gene causing oligodontia, as well as mutation sites and styles, we got out DNA from the venous blood of 14 persons composed of all 5 patients above, as well更多还原