单纯性先天缺牙患者BMP2/BMP4基因位点分析

目的对BMP2/BMP4基因在单纯性先天缺牙患者中的基因表达进行观察,探讨其在先天缺牙疾病中可能的发病机制。方法提取单纯性先天缺牙患者40例及其家系成员外周静脉血基因组DNA,另选择100例非先天缺牙患者作为对照,应用聚合酶链式反应（PCR）扩增BMP2/BMP4基因编码外显子,纯化、测序,应用DNASTAR软件对测序结果进行对比分析。采用SPSS13.0软件包对数据进行统计学处理。结果40例先天缺牙患者中,共检测到BMP2/BMP4基因 5个突变位点,其中BMP2基因4个突变点：3个错义突变c.109T>G, c.166C>G,c.570A>T,检出率分别为7.5%、2.5%和95%;1个同义突变（c.261A>G）,检出率为100%。3个为dbSNP 数据库中已报道过的多态位点：BMP2SNPs c.109T>G p.Ser37Ala; c.261A>G p.Ser87Ser;c.570A>T p.Arg190Ser;家系先证者BMP2 c.166C>G突变在正常对照组未检出,dbSNP 数据库未报道,也未被收录于致病基因数据库,为新突变位点。BMP4基因检测出错义突变c.455T>C,检出率为55%,为dbSNP 数据库中已报道过的多态位点。与对照组等位基因及基因型比较,无显著差异。结论单纯性先天缺牙可能与BMP2/BMP4基因检出SNPs相关。BMP2c.166C>G杂合突变是新发现的突变,可能是单纯性先天缺牙家系的致病突变。     

重度先天缺牙患者WNT10A基因新突变位点的检测及其种植修复

目的 探讨WNT10A基因突变重度先天缺牙（恒牙先天缺失数目≥6）患者的临床表型及其相关种植修复治疗预后情况。方法 对在临床收集到的先天缺牙患者进行口内检查、遗传病史采集和全外显子测序,筛查并纳入WNT10A基因出现突变的患者。对先证者及其家系成员进行Sanger测序后与正常人群的WNT10A基因序列进行比对。利用基因突变功能预测、基因保守性分析和蛋白结构预测分析来评估患者WNT10A基因突变的致病性,并对缺牙患者进行相关种植修复治疗。结果 在6例无血缘关系的重度先天缺牙患者中共检测到5个WNT10A基因突变,其中c.26G>A(p.Trp9X)和c.1036delT(p.Cys346fs)为新检出突变。突变功能预测的结果显示这些突变表现出一定程度的致病性。患者平均缺牙数目为（15.33±8.64）颗,其中上颌尖牙缺失率最高（100%）,下颌第一磨牙缺失率最低（25%）。对患者采取种植修复,种植体获得了良好的骨结合,患者口腔功能得到了恢复。结论 本研究丰富了重度先天缺牙患者的WNT10A基因突变谱,为遗传诊断和产前咨询提供了新的证据。同时,种植修复可有效恢复这类患者缺牙及口颌功能...     

PAX9基因上新近发现的一个单核苷酸多态性位点的SNP分析

目的:研究位于PAX9基因外显子3的第89位点上的SNP与多数牙先天缺失的相关性.方法:分别从11名多数牙先天缺失病人,5名少数牙先天缺失病人和38名正常对照者的静脉血样品中提取DNA.采用TaqMan-MGB探针技术对该SNP位点进行单核苷酸多态性研究,利用软件对实验结果进行SNP分型,并行统计分析.结果:突变为纯合子C/C的绝大多数表现为先天缺牙(多数牙先天缺失5例,少数牙先天缺失2例,正常1例).但31例杂合子中只有7例表现为先天缺牙(多数牙先天缺失6例,少数牙先天缺失1例).13例野生型纯合子只有2例表现为少数牙先天缺失,11例完全正常.统计结果表明该位点与多数牙先天缺失相关(P<0.001).结论:多数牙先天缺失可能与PAX9基因外显子3的第89位点上的SNP有关.     

AXIN2基因的3个SNP位点与新疆维吾尔族先天缺牙的相关性

目的：探讨维吾尔族单纯型先天缺牙患者标本中AXIN2基因的突变位点。方法：收集维吾尔族非单纯型先天缺牙家系3个,采集家系患者颊黏膜拭子提取DNA,采用聚合酶链反应技术,对分段纯化的PCR产物进行DNA双向测序检测患者的DNA。结果：3个家系的单纯型先天缺牙临床表型符合常染色体显性遗传规律,患者出现不同数量的缺失牙或伴发锥形牙。测序后检测出AXIN2的3个可能的SNP位点。结论：AXIN2基因片段中某些编码基因的改变可能与维吾尔族单纯型先天缺牙有关。     

PITX2突变导致非综合征型先天缺牙的遗传研究

目的:研究1例非综合征型先天缺牙(non-syndromic tooth agenesis, NSTA)患者家系的遗传学病因。方法:采集先证者及家庭成员的外周血,利用全外显子组测序技术和生物信息学工具进行变异检测及分析。锁定先证者候选基因后,通过Sanger测序技术验证变异位点。运用Protean、I-TASSER软件分别分析突变蛋白的二级结构和三维(3D)模型,并与野生型进行比较分析。结果:先证者及母亲均携带同一PITX2新发杂合移码突变(c.176delT,p.I59fsX149)。该突变改变同源结构域和OAR结构域,影响DNA结合活性及对下游基因的激活诱导能力。二级结构预测显示突变蛋白α螺旋数量减少,3D模型空间构象变化显著。结论:PITX2移码突变(c.176delT)可能是先证者患有NSTA的病因之一。本研究扩展了PITX2基因突变谱,为进一步探讨PITX2与NSTA之间的相关性提供临床与遗传学证据。     

单纯性多数牙缺失家系的AXIN2基因突变分析

目的：通过对AXIN2基因多态性与单纯性多数牙先天缺失关系的探讨，寻找该病发病的可能基因因素。方法对单纯性多数牙先天缺失家系中患者进行临床和实验室相关检查；而后提取基因组DNA ，通过RT?PCR方法对AXIN2基因的外显子进行扩增、测序，同时分析基因突变类型。结果 DNA测序显示，在AXIN2基因的3号外显子及附近的内含子区2例患者出现3个相同类型的基因突变，分别是：c.1365A>G（p.Pro455=），c.956+16A>G（Ⅱ?1：纯合型；Ⅲ?1：杂合型），c.1200+71A>G（纯合型），同时先证者的母亲（Ⅱ?2）出现c.1365A>G和c.1200+71A>G的杂合突变类型。经生物信息软件分析认为均是多态性位点。结论 AXIN2基因的c.956+16A>G，c.1365A>G和c.1200+71A>G突变可能与该家系的单纯性多数牙齿缺失密切相关，但AXIN2在多数牙缺失发生发展各阶段的确切作用还需要进一步的研究。     

PAX9基因在2个新疆维吾尔族非综合征型先天缺牙家系中的表达

目的　通过对新疆维吾尔族非综合征型先天缺牙患者PAX9基因突变的检测,为维吾尔族该病发病的分子机制提供依据。方法　采集2个新疆维吾尔族非综合征型先天缺牙家系颊黏膜拭子,提取DNA,采用聚合酶链反应技术结合DNA双向测序技术对患者DNA进行检测。结果　PAX9基因外显子3的85、86位点检测出两个单核苷酸多态性（single nucleotide polymorphisms,SNPs）位点。结论　PAX9基因外显子3的85、86位点的改变可能与新疆维吾尔族非综合征型先天缺牙的发生有关。     

EDA基因在4个少汗型外胚层发育不全家系中的检测及分析

目的 对4个少汗型外胚层发育不全家系的EDA基因进行测序分析,研究突变的位置、类型,为临床诊断提供遗传学依据.方法 提取先证者及其亲属的基因组DNA,其中患者5人,无症状者12人,另外抽取100名无先天缺牙家族史的正常成人外周血,提取基因组DNA作为对照,设计EDA基因8个外显子的引物,通过聚合酶链反应和DNA测序的方法与正常序列比对.结果 4个家系的患者均存在EDA基因不同位点的突变,分别为c.466C>T、c.663-697缺失、c.587-615缺失、c.878T>G,携带者存在杂合突变,正常对照不存在以上突变.结论 EDA基因的c.466C>T、c.663-697缺失、c.587-615缺失、c.878T>G突变是导致家系先证者及患者出现少汗型外胚层发育不全的病因.其中,EDA基因的c.663-697缺失、c.587-615缺失、c.878T>G是未报道的新突变.     

WNT10A基因杂合突变致多数牙先天缺失伴前牙反1例

本文报道1例以多数牙先天缺失伴前牙反为临床表现的患者, 其父母临床表型未见异常, 采用二代测序技术对先证者及其父母进行致病基因鉴定, 测序结果显示先证者携带WNT10A基因的杂合突变c.791G>T;p.Cys264Phe, 家系鉴定证实该错义突变遗传自母亲。对该家系的临床和遗传资料进行分析, 讨论先证者的临床治疗并深入理解WNT10A突变在先天缺牙致病机制中的作用。     

Van der Woude综合征家系先天缺牙患者MSX1基因突变的检测

目的探讨MSX1基因与Van der Woude综合征(VWS)家系中缺牙的关系。方法从VWS家系9中伴发缺牙患者2人及家系正常成员2人、60个牙列完整的健康者共64人的静脉血中提取DNA,设计MSX1基因引物,采用PCR方法扩增MSX1基因外显子1、2的编码区,而后对外显子1、2的PCR纯化产物测序,进行序列比对分析。结果 VWS家系9两个缺牙患者MSX1基因中有ivs2+68 C>T多态;伴IRF6基因突变的VWS患者缺牙较多。结论 VWS家系9中先天缺牙患者的牙先天缺失与MSX1基因的ivs2+68 C>T多态可能相关。     

Functional analysis of a novel missense mutation in AXIN2 associated with non‐syndromic tooth agenesis

Tooth agenesis is a congenital anomaly frequently seen in humans. Several genes have been associated with non‐syndromic tooth agenesis, including msh homeobox 1 (MSX1), paired box 9 (PAX9), axis inhibition protein 2 (AXIN2), ectodysplasin A (EDA), and wingless‐type MMTV integration site family member 10A (WNT10A). In this study, we investigated a Chinese family with non‐syndromic tooth agenesis. A novel missense mutation (c.C1978T) in AXIN2 was identified in affected members. The mutation results in a His660Tyr substitution located between the Axin beta‐catenin binding domain and the DIX domain of the axis inhibition protein 2 (AXIN2). We analysed this novel AXIN2 mutant, together with two reported AXIN2 mutants [c.1966C>T (p.Arg656Stop) and c.1994delG (p.Leu688Stop)] that cause colorectal cancer with and without oligodontia, to study the effect of the mutant p.His660Tyr on the Wnt/β‐catenin signaling pathway and to compare the molecular pathogenesis of different AXIN2 mutants in tooth agenesis and carcinogenesis. Further in vitro experiments indicated that the mutant p.His660Tyr caused inhibition of the Wnt/β‐catenin pathway, and the mutants p.Arg656Stop and p.Leu688Stop resulted in over‐activation of the Wnt/β‐catenin pathway. In line with previous AXIN2 mutation studies, we suggest that AXIN2 mutations with different levels of severity may have distinct effects on the Wnt pathway and the phenotype of disease. Our study provides functional evidence supporting the notion that both inhibition and over‐activation of the Wnt pathway may lead to tooth agenesis.     

Novel mutation in the paired box sequence of PAX9 gene in a sporadic form of oligodontia

Tooth development is regulated through a series of reciprocal interactions between the dental epithelium and mesenchyme and requires protein products of a number of genes. It has been reported that selective tooth agenesis is associated with mutations in human MSX and PAX9 genes. Mutational analysis of the two genes was performed in 25 individuals with familial or sporadic form of permanent tooth agenesis. Single-stranded conformational polymorphism analysis revealed no mutations in the entire coding sequence of the MSX1 gene. In PAX9, a novel, heterozygous G151A transition in the sequence encoding the paired domain of the PAX9 protein was detected in a patient with agenesis of third molars, second premolars and incisors, but not in her parents, the remaining patients or 162 individuals with normal dentition. This is the first de novo mutation described in PAX9. Our results support the view that mutations in PAX9 could constitute a causative factor of oligodontia. We hypothesize that the G151A transition in PAX9 might be responsible for the sporadic form of tooth agenesis in this patient.     

Genetic study of non‐syndromic tooth agenesis through the screening of paired box 9, msh homeobox 1, axin 2, and Wnt family member 10A genes: a case‐series

Non‐syndromic tooth agenesis (NSTA) is the most common developmental anomaly in humans. Several studies have been conducted on dental agenesis and numerous genes have been identified. However, the pathogenic mechanisms responsible for NSTA are not clearly understood. We studied a group of 28 patients with sporadic NSTA and nine patients with a family history of tooth agenesis. We focused on four genes – paired box 9 (PAX9), Wnt family member 10A (WNT10A), msh homeobox 1 (MSX1), and axin 2 (AXIN2) – using direct Sanger sequencing of the exons and intron–exon boundaries. The most prevalent variants identified in PAX9 and AXIN2 genes were analyzed using the chi‐square test. The sequencing results revealed a number of variants in the AXIN2 gene, including one novel missense mutation in one patient with agenesis of a single second premolar. We also identified one variant in the AXIN2 gene as being a putative risk factor for tooth agenesis. Only one missense mutation was identified in the WNT10A gene and this mutation was found in two patients. Interestingly, WNT10A is reported as the most prevalent gene mutated in the European population with NSTA.     

Novel nonsense mutation in MSX1 in familial nonsyndromic oligodontia: subcellular localization and role of homeodomain/MH4

Nonsyndromic tooth agenesis is one of the most common anomalies in human development. Part of the malformation is inherited and is associated with paired box 9 (PAX9), msh homeobox 1 (MSX1), and axin 2 (AXIN2) mutations. To obtain a comprehensive understanding of the genetic and molecular mechanisms that underlie this genetic disease, we investigated six familial and seven sporadic Japanese cases of nonsyndromic tooth agenesis. Searches for mutations in these candidate genes detected a novel nonsense mutation (c.416G>A) in exon 1 of MSX1 from a family with oligodontia. This mutation co‐segregated in the affected family members. Moreover, this mutation produced a termination codon in the first exon and therefore the gene product (W139X) was truncated at the C terminus, hence, the entire homeodomain/MH4, which has many functions, such as DNA binding, protein‐protein interaction, and nuclear localization, was absent. We characterized the properties of this truncated MSX1 by investigating the subcellular localization of the mutant gene product in transfected cells. The wild‐type MSX1 localized exclusively at the nuclear periphery of transfected cells, whereas the mutant MSX1 was stable but localized diffusely throughout the whole cell. These results indicate that W139X MSX1 is responsible for tooth agenesis.     

一个范德伍德综合征家系的IRF6基因突变检测

目的:对收集的1个湖北Van der Woude综合征(VWS)家系进行临床和遗传特点分析,并进行IRF6基因的突变检测。方法:通过先证者及现场家系调查、临床检查和系谱分析收集VWS家系。在IRF6基因的外显子-内含子接头及9个外显子编码区分别设计引物,经聚合酶链式反应扩增并纯化后直接测序。结果:收集的VWS家系符合常染色体显性遗传特征,家系受累患者共3名(1名男性和2名女性),患者表现为典型的下唇瘘管或凹陷,且合并有唇腭裂和先天缺牙。患者表型在同一家系内有明显差异,且呈逐代加重趋势。在所有患者IRF6基因第412位密码子发现与表型一致的CGA>TGA(c.1234C>T)改变,经查证为一个已知的无义突变。结论:该VWS家系疾病表现度极不一致,是由IRF6基因的1个已知无义突变导致,IRF6是参与颌面部发育的重要基因。     

一个掌跖角化-牙周破坏综合征患者家系组织蛋白酶C基因突变分析

目的 对一个掌跖角化-牙周破坏综合征（PLS）患者及其家系组织蛋白酶C基因（CTSC）突变位点进行分析,探讨PLS的分子致病机制。方法 提取1例PLS先证者及其直系血亲（父母、弟弟）的基因组DNA,应用聚合酶链反应和DNA直接测序技术分析先证者及其直系血亲CTSC基因的突变情况。结果 PLS先证者CTSC基因存在复合型杂合突变。先证者位于外显子6的第800位碱基发生了一个杂合错义突变,该碱基对中的碱基T被C取代（c.800T>C）,其编码的氨基酸由亮氨酸改变为脯氨酸（p.L267P）;位于外显子7的第1015位碱基发生了一个杂合错义突变,该碱基对中的碱基C被T取代（c.1015C>T）,其编码的氨基酸由精氨酸改变为半胱氨酸（p.R339C）。其中,c.800T>C来自母亲,c.1015C>T来自父亲,弟弟的CTSC基因未见突变。结论 PLS的临床表征与CTSC基因突变有关。