一个单纯性先天缺牙家系的临床及基因突变分析

目的探讨多数牙先天缺失患者的人类成对盒基因（PAX9）和肌节同源盒基因（MSX1）突变位点,为该疾病的病因学研究提供依据。方法对于该例患者与部分正常家庭成员进行口内检查及家系调查,取研究模型测量其牙冠宽度,并与正常值比较。拍摄曲面断层片和头颅侧位片进行头影测量分析,对比颅面形态和错畸形类型及特点。从静脉血中提取DNA,根据PAX9和MSX1的全序列设计引物,采用聚合酶链式反应（PCR）扩增PAX9基因的外显子1、2、3、4及MSX1基因外显子1、2,而后通过对分段PCR纯化产物的测序,并结合系谱进行突变分析。结果患者伴有牙齿形态畸形,与中国人正常值相比较,牙冠宽度较小。头影测量分析结果提示先证者在骨面型及颌骨形态等方面无明显遗传倾向。基因筛查结果显示先证者及其母亲的PAX9外显子3第718位点上由G变为C,属错义突变,导致与之对应的第240位氨基酸由丙氨酸变为脯氨酸;MSX1未见突变。结论多数牙先天缺失可能与PAX9基因外显子3的第718位点上的错义突变有关。     

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。     

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。     

多数牙先天缺失可能与MSX1上的3个SNPs相关

目的:探讨多数牙先天缺失患者的MSXl基因突变位点.方法:从4个多数牙先天缺失患者与家庭部分成员、1个唇腭裂并发少数牙先天缺失的患者、1个牙列完整的对照儿童共14人的静脉血中提取DNA,在MSXl基因内设计引物,采用PCR方法扩增MSXl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,结合系游进行序列比对分析.结果:发现3个可能的单核苷酸多态性位点(single nucleotide pol-ymorphisms,SNPs).这3个SNPs均位于外显子1中,且来自不同家系的3个患者在这3个位点上同时出现杂合突变.其中,311位点由G变A,对应的密码子由编码甘氨酸的GGC变为编码天门冬氨酸的GAC,发生了错义突变;402位点由C变A,对应的密码子由CCC变为CCA,但仍编码脯氨酸,属同义突变;458位点由C变T,对应的密码子由编码丙氨酸的GCC变为编码缬氨酸的GTC,发生了错义突变.结论:多数牙先天缺失可能与MSX1基因上该3个单核苷酸多态性位点有关.     

中国一少牙畸形家系MSX1基因新突变

目的 研究一个中国少牙畸形家系MSX1基因的突变情况.方法 收集先证者和部分家系成员的外周血标本,采用聚合酶链式反应(PCR)结合DNA直接双向测序的方法,检测了该家系中7例患者及7名表型正常者和100名无亲缘关系健康个体的MSX1基因突变.结果 所有患者的MSX1基因上均存在剪切突变(IVS1-2A＞G),该突变在家系正常个体及100名健康对照个体中均未发现.结论 在MSX1基因上发现的IVS1-2A＞G为一个新的剪切突变,它可能是造成该家系先天性缺牙的致病突变.     

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。     

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。     

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.     

选择性先天缺牙家系的表型与基因型分析：附2例报告

目的: 探讨选择性先天缺牙患者的遗传学病因。方法: 对2例非综合征型先天缺牙患者进行临床检查、家系调查、影像学检查以及外周血采集,通过全外显子测序后与正常人类基因组比对,并进行Sanger测序验证,确定致病基因及突变位点后,进行蛋白结构预测和多物种保守性分析。结果: 2例选择性先天缺牙家系中家系1为散发型,家系2为家族型。全外显子测序结果显示,先证者1和先证者2分别存在LRP6无义突变（II:1, c.C1573T,p.R525X）以及移码突变（II-1, c.4611delT ,p.C1537fs）。蛋白结构分析表明,p.R525X使LRP6蛋白截短,为失功能突变。多物种保守性分析揭示位点在进化过程中高度保守,提示突变具备有害性。结论: 2例选择性先天缺牙家系可能由于LRP6突变导致,为遗传咨询和产前诊断提供了参考。     

非综合征型先天缺牙致病基因和分子机制的研究进展

先天缺牙是牙齿发育过程中常见的牙数目发育异常,对患者的颌面部发育及美观和咀嚼功能产生严重的影响。根据有无伴发全身症状,先天缺牙可分为综合征型先天缺牙与非综合征型先天缺牙。近几年发现新的相关基因和新的突变位点及分子机制已成为目前非综合征型先天缺牙基因研究的主要方向。本文通过对近年来文献的回顾,对与非综合征型先天缺牙主要相关的Wnt/β-catenin信号通路、TGF-β/BMP信号通路、PAX9基因和MSX1基因、EDA/EDAR/NF-κb信号通路的分子机制以及相互调节的紧密联系进行综述,为未来先天缺牙的防治提供了新的理论基础。非综合征型先天缺牙致病基因的分子机制的研究目前甚少,对于其机制的精准探索将成为先天缺牙未来主要的研究方向之一。     

非综合征性唇腭裂与同源异型盒基因1多态性的相关性研究

目的采用聚合酶链反应- 单链构象多态性（PCR- SSCP）方法研究同源异型盒基因（MSX）1外显子1的编码区，探讨非综合征性唇腭裂（NSCL/P）患者MSX1基因外显子1的编码区内是否存在基因突变。方法采用聚合酶链反应（PCR）和单链构象多态性（SSCP）方法，以45名健康人为对照组，45名NSCL/P患者作为研究对象，分析MSX1基因多态性。结果SSCP分析显示NSCL/P患者（45名）与对照组（45名）样本的电泳速率相同，提示无多态性存在。结论MSX1基因外显子1未发现多态性的存在，其与NSCL/P患者之间无明显相关性。     

Two missense mutations in the IRF6 gene in two Japanese families with Van der Woude syndrome

Van der Woude syndrome (VWS) is a common autosomal dominant disorder with cleft lip and/or palate and lower lip pits. Its prevalence is estimated to be 1/33,600 in the Finnish Population, and 1/47,813 in the Japanese. We performed mutation analysis of the IRF6 gene by direct sequencing in 2 unrelated Japanese families that consist of a total of 3 affected members with cleft lip and palate associated with lower lip pits. Consequently, we found novel base substitutions, 25C>T, in IRF6-exon 3 in a boy, his mother, and his phenotypically normal maternal grandmother in one of the families. A known mutation, 250C>T, was identified in exon 4 of a girl and her unaffected father in the other family. The same mutations were never observed among 190 healthy Japanese. The results indicate incomplete penetrance and variable expressivity in the families. Because 25C>T and 250C>T predict to lead to R9W and R84C substitutions, respectively, at the most conserved DNA binding domain of IRF6, and because arginine at positions 9 and 84 is highly conserved among IRFs, the 2 mutations may lead to abolish the DNA binding activity in the developing craniofacial region. To our knowledge, this is the first report of IRF6 mutations observed in Japanese VWS patients.     

干扰素调节因子6基因致病的Van der Woude综合征的家系调查和遗传特点分析

目的 探讨中国人Van der Woude综合征（VWS）的临床表型及遗传学特点。方法 先证者法收集14个VWS家系并进行口腔专科检查、家系调查及基因突变分析,分析不同VWS家系个体或同一家系不同个体的临床表型,绘制家系图谱,明确遗传方式及致病基因,计算表型分布频率和表型基因频率。结果 VWS家系基本符合常染色体显性遗传特征,患者多数表现为典型的VWS,致病基因为干扰素调节因子6（IRF6）。VWS表型分布频率为：唇瘘91.9%,唇腭裂73.0%,牙畸形8.1%。不同家系个体和同一家系的不同个体临床表型存在明显差异。结论 收集的家系均为常染色体显性遗传,表现度变异大。中国人群VWS致病基因为IRF6,为Ⅰ型VWS。     

福建范德伍兹综合征1例家系IRF6基因突变的检测

目的检测福建省一范德伍兹综合征(VWS)家系IRF6基因的突变。方法在IRF6(interferonregulatoryfactor6)基因内设计引物,经分段聚合酶链反应(PCR)和DNA测序进行突变检测,运用PIX-ProteinIdentification软件对检测结果进行蛋白二级结构分析。结果在所有患者IRF6基因的第379密码子发现TGG>TGA(r.1400g→a)的碱基变化,该突变引入终止码,引起IRF6蛋白转录提前终止。结论范德伍兹综合征由IRF6基因突变引起,IRF6基因与唇腭、牙齿发育密切相关。     

Van der Woude syndrome: dentofacial features and implications for clinical practice

Background:  Van der Woude syndrome (VWS) is the most common clefting syndrome in humans. It is characterized by the association of congenital lower lip fistulae with cleft lip and/or cleft palate. VWS individuals have a high prevalence of hypodontia. Although caused by a single gene mutation, VWS has variable phenotypic expression. This study aimed to describe the range of clinical presentations in 22 individuals with VWS to facilitate its diagnosis.
Methods:  A retrospective study of 22 patients with a diagnosis of VWS was undertaken at the Australian Craniofacial Unit (ACFU) in Adelaide. Three extended families with affected members were included in the study cohort.
Results:  The overall prevalence of lip pits in this study cohort was 86%. Cleft phenotypes included bilateral cleft lip and palate (32%); unilateral cleft lip and palate (32%); submucous cleft palate (23%); and isolated cleft hard and soft palate (9%). Missing permanent teeth were reported in 86% of affected individuals.
Conclusions:  Submucous cleft palate in VWS may go undiagnosed if the lower lip pits are not detected. Associated hypodontia and resultant malocclusions will also require management by a dental team.     

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 IRF6 mutations in Chinese patients with Van der Woude syndrome

Van der Woude syndrome (VWS) (OMIM 119300) is a dominantly inherited, developmental disorder that is characterized by pits and/or sinuses of the lower lip and a cleft lip and/or cleft palate. Mutations in the interferon regulatory factor 6 gene (IRF6) have been recently identified in patients with VWS, with more than 60 mutations reported. However, the VWS phenotype, IRF6 mutation genotypes, and their interrelationships in Chinese VWS patients have not been studied. Here, we report 11 Chinese families with variable clinical phenotypes of VWS and identified mutations in all patients. Of the 11 mutations, 8 appeared to be novel: CC5.6GT, T342A, 566delA, C748T, C756A, C989A, C1209G, and 1316delT. Seven mutations caused a change or loss of the IRF6 domain. The marked phenotypic variation may be caused by the action of certain modifier genes on IRF6 function.     

Clinical and genetic studies of Van der Woude syndrome in Sweden.

Van der Woude syndrome (VWS) is an autosomal dominant craniofacial disorder characterized by pits of the lower lip, hypodontia and cleft lip and/or cleft palate. It has been reported as the most common form of syndromic orofacial clefting with very high penetrance and varied expressivity. The disease locus for VWS has been mapped to chomosome 1q32, but the gene is yet to be cloned. Here we report a total of 11 Swedish VWS patients: 9 familial cases from two families and two isolated cases. Clinical examination of these patients showed phenotypic variability, even between patients from the same family. Genetic studies were performed using four microsatellite markers from chromosome 1q32. Constitutional deletion in this region was not demonstrated in any of the familial or isolated cases. However, in the two VWS families, linkage analysis using these markers showed positive LOD (logarithm of the odds) scores ranging from 2.56 to 2.88 to all individual markers. The highest LOD score of 3.75 was obtained with the combined haplotypes of D1S491 and D1S205, thus confirming linkage of VWS in these two families to 1q32. We conclude that there is varied expressivity but no evidence of genetic heterogeneity in VWS.