非综合征型先天缺牙致病基因的筛查及分子发病机制的研究
摘要
先天缺牙是一种较为常见的口腔遗传性疾病,临床上仅仅表现为牙齿数目的缺失。正常人中约20%的个体有智齿缺失,其余牙缺失约在3%-10%之间,以下颌第二前磨牙、上颌侧切牙多见,第一和第二恒磨牙缺失少见。研究发现,在牙发育期环境因素,如创伤、感染、大剂量放疗、化疗等可引起缺牙,同时也有相当数量的缺牙与遗传有关。其遗传模式以常染色体显性为主,而常染色体隐性和X-连锁遗传方式及散发病例均有报道。缺牙按缺失数目可分为少数牙缺失(少于6颗)和多数牙缺失(6颗和6颗以上),极端缺牙表现为全口无牙。按临床表型可分为非综合征型缺牙和伴其他体征的综合征型缺牙,其中非综合征型缺牙较常见,且有明显遗传异质性。迄今为止,有证据表明家族遗传或部分散发的非综合型先天缺牙与MSX1、PAX9、AXIN2和EDA等基因的突变有关。
我国是多民族人口大国,和其它国家相比有不可比拟的遗传资源优势,但相对国外而言,国内对中国人非综合征型先天缺牙的致病基因的定位较少,且分子遗传学病因研究报道并也多见,而国外学者对中国人群中非综合征型先天缺牙的研究报道屡见不鲜。而且随着我国人口政策的深入实行,具有研究意义的大型先天缺牙病家系越来越少,因此在现阶段开展先天缺牙家系的收集和保护工作,对先天缺牙疾病机制的研究以及人类牙齿发育研究等课题意义重大。
因此本研究立足于中国人群先天缺牙家系的收集和保护。对参与调查的家系,调查前每位成员均做到知情同意,并签署知情同意书,未满14岁的儿童由其监护人代理。整个研究已通过武汉大学伦理委员会审查。对各家系成员进行全身健康状况及口腔专科检查,了解相应病史及疾病在各家系中的遗传特征,对受累成员年龄、性别、临床表现、X线特点进行详细记录和分析。绘制完整的家系图谱,明确可能的遗传方式。对家系中的病人及家系中直系亲属,每人采集外周静脉血两份,一份为EDTA抗凝,后用改良盐析法提取外周血基因组DNA,-20℃保存；一份为肝素钠抗凝,用于淋巴细胞建系,建系成功后液氮冻存,并由专人保管。
本研究共收集到8个多代家系,均为非综合征型先天缺牙,其中一个家系部分患者还伴发有单侧唇裂。对这8个家系的遗传特点和表型分析后,先对4个候选基因进行突变位点的筛查,并在其中三个家系中分别发现了三处新的突变：两个错义突变L27P和I29T,均位于PAX9的成对结构域；一个无义突变Q189X,位于MSX1的同源异形盒结构域。这三个突变在正常人群对照中均无出现。对PAX蛋白的同源性分析和结构预测提示突变可能影响蛋白功能。通过体外定点诱变技术获得突变质粒；将野生型与突变质粒瞬时转染细胞系；对于PAX9突变的功能研究：通过细胞免疫荧光和蛋白印迹对突变蛋白进行亚细胞定位,免疫荧光和蛋白印迹结果显示,突变未改变PAX9在细胞中的定位,凝胶迁移实验和萤光素酶分析表明突变的PAX9蛋白失去了与特定DNA序列结合的能力,同时也丧失了激活下游BMP4的能力。对于MSX1突变的功能研究：通过实时定量PCR表明突变MSX1在mRNA表达水平上显著降低。我们的发现扩大了遗传性先天缺牙PAX9和MSX1基因的突变谱,通过功能分析我们认为PAX9和MSX1的这三个突变分别是造成相应家系中缺牙的原因。
第一部分先天缺牙家系调查和样本收集
目的：主要收集非综合征型先天缺牙家系,对家系成员作详细的调查和遗传特点分析,观察是否存在新的疾病表型。保存人类遗传资源,绘制完整的家系图谱,明确可能的遗传方式,为开展进一步的致病基因突变分析及功能研究等工作打下基础。
材料和方法：通过先证者法我们现已收集8个多代无血缘关系的先天缺牙家系。在知情同意原则下,对上述家系成员作详细的家系调查、口腔专科检查和样本采集。
结果：8个家系均为中国人汉族家系,其中家系3除单纯缺牙的表型外,部分患者还伴有先天性唇裂。其它7个家系均为非综合征先天缺牙,主要累及恒牙列,无拔牙或其他导致牙齿脱落的病史,排除患者母亲妊娠期患病服药史,检查指(趾)甲,毛发,汗腺及身体其他系统均无异常。先天缺牙的表现度在不同家系及同一家系的不同个体间存在较大差异,患者的缺牙牙位和缺牙数目不同。
结论：8个家系中,除家系6不排除X连锁遗传外,其它家系均可确定为常染色体显性遗传。
第二部分先天缺牙患者候选基因突变的检测
目的：以收集到的8个家系为研究对象,对家系患者进行候选基因MSX1、PAX9、 AXIN2和EDA的筛查,分析存在的突变类型。
材料和方法：用改良盐析法提取外周血基因组DNA,并将相应淋巴细胞建立细胞系永久保存；应用Primer-blast设计针对MSX1、PAX9、AXIN2和EDA四个基因的引物,对家系患者进行候选基因PCR后直接测序,寻找突变位点,分析存在的突变类型,相应氨基酸变化、疾病基因型与表型的相互关系,确定是否存在突变热区。
结果：分别在三个无血缘关系的家系中发现了三处新的突变：两个错义突变L27P和I29T,均位于PAX9的成对结构域；一个无义突变Q189X,位于MSX1的同源异形盒结构域。这三个突变在正常人群对照中均无出现。其余5个家系的候选基因筛查均未发现可疑突变位点。
结论：这三个突变扩大了PAX9和MSX1的突变谱,且突变均位于相应基因的重要结构域,为下一步进行突变与疾病的关联分析奠定了基础。
第三部分PAX9和MSX1突变的功能分析
目的：对以上三个突变进行功能研究,分析其可能的致病机制
材料和方法：通过体外定点诱变技术获得突变质粒pCMV6-L27PPAX9-MycDdk、 pCMV6-I29TPAX9-MycDdk和pCMV6-Q189X-MSX1;将野生型与突变质粒瞬时转染细胞系；对于PAX9突变的功能研究：通过细胞免疫荧光和蛋白印迹对突变蛋白进行亚细胞定位；通过凝胶迁移实验检测突变蛋白与特定DNA的结合能力；通过双萤光素酶分析检测突变蛋白调控下游BMP4的能力。对于MSX1突变的功能研究：通过实时定量PCR直接检测突变MSX1在mRNA表达水平的变化。
结果：对PAX蛋白的同源性分析和结构预测提示突变可能影响蛋白功能。免疫荧光和蛋白印迹结果显示,突变未改变PAX9在细胞中的定位,凝胶迁移实验和萤光素酶分析表明突变的PAX9蛋白失去了与特定DNA序列结合的能力,同时也丧失了激活下游BMP4的能力。MSX1突变导致了终止密码产生,通过实时定量PCR表明突变MSX1在mRNA表达水平上显著降低。
结论：PAX9和MSX1的这三个突变分别是造成相应家系中缺牙的原因
Non-syndromic tooth agenesis is one of the most common oral heditary diseases, which manifested as missing tooth. Non-syndromic tooth agenesis shows a wide phenotypic heterogeneity and is classified as a sporadic or familial form, inherited in an autosomal-dominant (AD), autosomal-recessive (AR) or X-linked mode. Population studies have revealed that the most commonly missing teeth are third molars followed by deficiency of lower second premolars or upper lateral incisors. Agenesis of the first and second molars is very rare. When more than six teeth, excluding third molars, are missing, the condition is referred to as oligodontia. To date, non-syndromic familial and sporadic tooth agenesis has associated with mutations in MSX1, PAX9, AXIN2and EDA. The purpose of the present study is to investigate the mutational characteristics of MSX1,PAX9, AXIN2and EDA genes in Chinese patients with tooth agenesis and further to perform functional analysis on the molecular pathogenesis of non-syndromic tooth agenesis.
PART1Pedigree collection
Objectives:Collect non-syndromic tooth agenesis families to lay a basis for candidate gene detection and functional analysis.
Materials and Methods:We collected eight tooth agenesis families. Pedigree construction was conducted on the basis of the results of clinical examination and interview of the available family members. Panoramic radiographs were taken to verify tooth agenesis. Blood samples were collected from available members of the family.
Results:These families are all Chinese Han families, in which seven families manifested as non-syndromic tooth agenesis except one family had congenital cleft lip as well. The patients denied tooth extraction or medical history that led to tooth falling out. No special medicine was taken as pregnancy. The expressivity varied among patients even in the same family.
Conclusions:All families conform to AD mode, while in one family X-linked mode can't be excluded.
PART2Mutation detection
Objectives:Detect candidate genes in the eight families to look for mutations.
Materials and Methods:Genomic DNA was isolated from the peripheral blood lymphocytes of all the available members and controls, according to a modified technique using sodium dodecyl sulfate, proteinase K, and chloroform. Pairs of primers were designed for amplifying MSX1, PAX9, AXIN2and EDA genes for all families. Mutation analysis was performed by amplifying the coding regions and splicing junctions of these genes, sequencing the products.
Results:We identified two novel missense mutations, L27P and I29T, in the paired-domain of PAX9and a novel nonsense mutation, Q189X, in the homeodomain of MSX1. But there is no mutation detected in the other five families.
Conclusion:Two novel missense mutations in PAX9and a novel nonsense mutation in MSX1enlarged the mutation spectrum.
PART3Fuctional analysis of PAX9and MSX1
Objectives:Perform functional analysis of the three mutations causing tooth agenesis.
Materials and Methods:We conducted site-directed mutagenesis to generate the mutated vectors. The wild-type and mutated PAX9vectors were then transfected separately to NIH3T3cells. Immunolocalization, electrophoretic mobility shift assay (EMSA) and luciferase reporter assay were performed to analyze the effects of mutations on protein function. As to MSX1, real-time PCR was performed to test the mRNA level of mutated MSX1in transfected COS7cell lines.
Results:Analysis of homology PAX proteins indicated that these two substitutions may affect the function of PAX9protein. Results of immunofluorescence and westemblot showed that the mutations did not alter nuclear localization of PAX9. Gel shift assays and luciferase reporter assays indicated that both mutant proteins can't bind DNA or transactivate BMP4promoter. Real-time PCR showed that the expression level of the mutated MSX1was dramatically reduced compared with that of the wild type.
Conclusion:Two novel missense mutations in PAX9and a novel nonsense mutation in MSX1have been indentified in Chinese families causing oligodontia.
我国是多民族人口大国,和其它国家相比有不可比拟的遗传资源优势,但相对国外而言,国内对中国人非综合征型先天缺牙的致病基因的定位较少,且分子遗传学病因研究报道并也多见,而国外学者对中国人群中非综合征型先天缺牙的研究报道屡见不鲜。而且随着我国人口政策的深入实行,具有研究意义的大型先天缺牙病家系越来越少,因此在现阶段开展先天缺牙家系的收集和保护工作,对先天缺牙疾病机制的研究以及人类牙齿发育研究等课题意义重大。
因此本研究立足于中国人群先天缺牙家系的收集和保护。对参与调查的家系,调查前每位成员均做到知情同意,并签署知情同意书,未满14岁的儿童由其监护人代理。整个研究已通过武汉大学伦理委员会审查。对各家系成员进行全身健康状况及口腔专科检查,了解相应病史及疾病在各家系中的遗传特征,对受累成员年龄、性别、临床表现、X线特点进行详细记录和分析。绘制完整的家系图谱,明确可能的遗传方式。对家系中的病人及家系中直系亲属,每人采集外周静脉血两份,一份为EDTA抗凝,后用改良盐析法提取外周血基因组DNA,-20℃保存；一份为肝素钠抗凝,用于淋巴细胞建系,建系成功后液氮冻存,并由专人保管。
本研究共收集到8个多代家系,均为非综合征型先天缺牙,其中一个家系部分患者还伴发有单侧唇裂。对这8个家系的遗传特点和表型分析后,先对4个候选基因进行突变位点的筛查,并在其中三个家系中分别发现了三处新的突变：两个错义突变L27P和I29T,均位于PAX9的成对结构域；一个无义突变Q189X,位于MSX1的同源异形盒结构域。这三个突变在正常人群对照中均无出现。对PAX蛋白的同源性分析和结构预测提示突变可能影响蛋白功能。通过体外定点诱变技术获得突变质粒；将野生型与突变质粒瞬时转染细胞系；对于PAX9突变的功能研究：通过细胞免疫荧光和蛋白印迹对突变蛋白进行亚细胞定位,免疫荧光和蛋白印迹结果显示,突变未改变PAX9在细胞中的定位,凝胶迁移实验和萤光素酶分析表明突变的PAX9蛋白失去了与特定DNA序列结合的能力,同时也丧失了激活下游BMP4的能力。对于MSX1突变的功能研究：通过实时定量PCR表明突变MSX1在mRNA表达水平上显著降低。我们的发现扩大了遗传性先天缺牙PAX9和MSX1基因的突变谱,通过功能分析我们认为PAX9和MSX1的这三个突变分别是造成相应家系中缺牙的原因。
第一部分先天缺牙家系调查和样本收集
目的：主要收集非综合征型先天缺牙家系,对家系成员作详细的调查和遗传特点分析,观察是否存在新的疾病表型。保存人类遗传资源,绘制完整的家系图谱,明确可能的遗传方式,为开展进一步的致病基因突变分析及功能研究等工作打下基础。
材料和方法：通过先证者法我们现已收集8个多代无血缘关系的先天缺牙家系。在知情同意原则下,对上述家系成员作详细的家系调查、口腔专科检查和样本采集。
结果：8个家系均为中国人汉族家系,其中家系3除单纯缺牙的表型外,部分患者还伴有先天性唇裂。其它7个家系均为非综合征先天缺牙,主要累及恒牙列,无拔牙或其他导致牙齿脱落的病史,排除患者母亲妊娠期患病服药史,检查指(趾)甲,毛发,汗腺及身体其他系统均无异常。先天缺牙的表现度在不同家系及同一家系的不同个体间存在较大差异,患者的缺牙牙位和缺牙数目不同。
结论：8个家系中,除家系6不排除X连锁遗传外,其它家系均可确定为常染色体显性遗传。
第二部分先天缺牙患者候选基因突变的检测
目的：以收集到的8个家系为研究对象,对家系患者进行候选基因MSX1、PAX9、 AXIN2和EDA的筛查,分析存在的突变类型。
材料和方法：用改良盐析法提取外周血基因组DNA,并将相应淋巴细胞建立细胞系永久保存；应用Primer-blast设计针对MSX1、PAX9、AXIN2和EDA四个基因的引物,对家系患者进行候选基因PCR后直接测序,寻找突变位点,分析存在的突变类型,相应氨基酸变化、疾病基因型与表型的相互关系,确定是否存在突变热区。
结果：分别在三个无血缘关系的家系中发现了三处新的突变：两个错义突变L27P和I29T,均位于PAX9的成对结构域；一个无义突变Q189X,位于MSX1的同源异形盒结构域。这三个突变在正常人群对照中均无出现。其余5个家系的候选基因筛查均未发现可疑突变位点。
结论：这三个突变扩大了PAX9和MSX1的突变谱,且突变均位于相应基因的重要结构域,为下一步进行突变与疾病的关联分析奠定了基础。
第三部分PAX9和MSX1突变的功能分析
目的：对以上三个突变进行功能研究,分析其可能的致病机制
材料和方法：通过体外定点诱变技术获得突变质粒pCMV6-L27PPAX9-MycDdk、 pCMV6-I29TPAX9-MycDdk和pCMV6-Q189X-MSX1;将野生型与突变质粒瞬时转染细胞系；对于PAX9突变的功能研究：通过细胞免疫荧光和蛋白印迹对突变蛋白进行亚细胞定位；通过凝胶迁移实验检测突变蛋白与特定DNA的结合能力；通过双萤光素酶分析检测突变蛋白调控下游BMP4的能力。对于MSX1突变的功能研究：通过实时定量PCR直接检测突变MSX1在mRNA表达水平的变化。
结果：对PAX蛋白的同源性分析和结构预测提示突变可能影响蛋白功能。免疫荧光和蛋白印迹结果显示,突变未改变PAX9在细胞中的定位,凝胶迁移实验和萤光素酶分析表明突变的PAX9蛋白失去了与特定DNA序列结合的能力,同时也丧失了激活下游BMP4的能力。MSX1突变导致了终止密码产生,通过实时定量PCR表明突变MSX1在mRNA表达水平上显著降低。
结论：PAX9和MSX1的这三个突变分别是造成相应家系中缺牙的原因
Non-syndromic tooth agenesis is one of the most common oral heditary diseases, which manifested as missing tooth. Non-syndromic tooth agenesis shows a wide phenotypic heterogeneity and is classified as a sporadic or familial form, inherited in an autosomal-dominant (AD), autosomal-recessive (AR) or X-linked mode. Population studies have revealed that the most commonly missing teeth are third molars followed by deficiency of lower second premolars or upper lateral incisors. Agenesis of the first and second molars is very rare. When more than six teeth, excluding third molars, are missing, the condition is referred to as oligodontia. To date, non-syndromic familial and sporadic tooth agenesis has associated with mutations in MSX1, PAX9, AXIN2and EDA. The purpose of the present study is to investigate the mutational characteristics of MSX1,PAX9, AXIN2and EDA genes in Chinese patients with tooth agenesis and further to perform functional analysis on the molecular pathogenesis of non-syndromic tooth agenesis.
PART1Pedigree collection
Objectives:Collect non-syndromic tooth agenesis families to lay a basis for candidate gene detection and functional analysis.
Materials and Methods:We collected eight tooth agenesis families. Pedigree construction was conducted on the basis of the results of clinical examination and interview of the available family members. Panoramic radiographs were taken to verify tooth agenesis. Blood samples were collected from available members of the family.
Results:These families are all Chinese Han families, in which seven families manifested as non-syndromic tooth agenesis except one family had congenital cleft lip as well. The patients denied tooth extraction or medical history that led to tooth falling out. No special medicine was taken as pregnancy. The expressivity varied among patients even in the same family.
Conclusions:All families conform to AD mode, while in one family X-linked mode can't be excluded.
PART2Mutation detection
Objectives:Detect candidate genes in the eight families to look for mutations.
Materials and Methods:Genomic DNA was isolated from the peripheral blood lymphocytes of all the available members and controls, according to a modified technique using sodium dodecyl sulfate, proteinase K, and chloroform. Pairs of primers were designed for amplifying MSX1, PAX9, AXIN2and EDA genes for all families. Mutation analysis was performed by amplifying the coding regions and splicing junctions of these genes, sequencing the products.
Results:We identified two novel missense mutations, L27P and I29T, in the paired-domain of PAX9and a novel nonsense mutation, Q189X, in the homeodomain of MSX1. But there is no mutation detected in the other five families.
Conclusion:Two novel missense mutations in PAX9and a novel nonsense mutation in MSX1enlarged the mutation spectrum.
PART3Fuctional analysis of PAX9and MSX1
Objectives:Perform functional analysis of the three mutations causing tooth agenesis.
Materials and Methods:We conducted site-directed mutagenesis to generate the mutated vectors. The wild-type and mutated PAX9vectors were then transfected separately to NIH3T3cells. Immunolocalization, electrophoretic mobility shift assay (EMSA) and luciferase reporter assay were performed to analyze the effects of mutations on protein function. As to MSX1, real-time PCR was performed to test the mRNA level of mutated MSX1in transfected COS7cell lines.
Results:Analysis of homology PAX proteins indicated that these two substitutions may affect the function of PAX9protein. Results of immunofluorescence and westemblot showed that the mutations did not alter nuclear localization of PAX9. Gel shift assays and luciferase reporter assays indicated that both mutant proteins can't bind DNA or transactivate BMP4promoter. Real-time PCR showed that the expression level of the mutated MSX1was dramatically reduced compared with that of the wild type.
Conclusion:Two novel missense mutations in PAX9and a novel nonsense mutation in MSX1have been indentified in Chinese families causing oligodontia.
引文
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3. Gullikson, J.S., Tooth morphology in rubella syndrome children. ASDC J Dent Child,1975.42(6):p. 479-82.
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6. Axrup K, D.A.M., Hellgren K, Children with thalidomide embryopathy:odontological observations and aspects. Acta Odontol Scand,1966(24):p.3-21.
7. Kjaer, I., et al., Aetiological aspects of mandibular tooth agenesis--focusing on the role of nerve, oral mucosa, and supporting tissues. Eur J Orthod,1994.16(5):p.371-5.
8. Andersen, E., et al., The influence of jaw innervation on the dental maturation pattern in the mandible. Orthod Craniofac Res,2004.7(4):p.211-5.
9. Zhang, Y.D., et al., Making a tooth:growth factors, transcription factors, and stem cells. Cell Res, 2005.15(5):p.301-16.
10. De Coster, P.J., et al., Dental agenesis:genetic and clinical perspectives. J Oral Pathol Med,2009. 38(1):p.1-17.
11. Vastardis, H., The genetics of human tooth agenesis:new discoveries for understanding dental anomalies. Am J Orthod Dentofacial Orthop,2000.117(6):p.650-6.
12. Peters, H. and R. Balling, Teeth. Where and how to make them. Trends Genet,1999.15(2):p.59-65.
13. Ranta R. A review of tooth formation in children with cleft lip/palate. Am J Orthod Dentofacial Orthop 1986; 90:11-18.
14. Shapira Y, Lubit E, Kuftinec MM. Congenitally missing second premolars in cleft lip and cleft palate children. Am J Orthod Dentofacial Orthop 1999; 115:396-400.
15. Garn SM,Lewis AB.The gradient and the pattern of crown-size reduction in simple hypodontia. Angle Orthod 1970; 40:51-8.
16. Brook AH. A unifying aetiological explanation for anomalies of human tooth number and size. Arch Oral Biol 1984; 29:373-8.
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19. Schalk-van der Weide Y,Bosman F.Tooth size in relatives of individuals with oligodontia. Arch Oral Biol 1996; 41:469-72.
20. McKeown HF,Robinson DL,Elcock C,al-Sharood M,Brook AH.Tooth dimensions in hypodontia patients, their unaffected relatives and a control group measured by a new image analysis system. Eur J Orthod 2002; 24:131-41.
21. Albashaireh ZS,Khader YS.The prevalence and pattern of hypodontia of the permanent teeth and crown size and shape deformity affecting upper lateral incisors in a sample of Jordanian dental patients. Community Dent Health 2006; 23:239-3.
22. Lind V.Short root anomaly. Scand J Dent Res 1972; 80:85-93.
23. Apajalahti S,Arte S,Pirinen S.Short root anomaly in families and its association with other dental anomalies. Eur J Oral Sci 1999; 107:97-101.
24. Brin I,Becker A,Shalhav M. Position of the maxillary permanent canine in relation to anomalous or missing lateral incisors:a population study. Eur J Orthod 1986; 8:12-6.
25. Schalk-van der Weide Y,Steen WH,Bosman F.Taurodontism and length of teeth in patients with oligodontia J Oral Rehabil 1993; 20:401-12.
26. Mossey PA,Campbell HM,Luffingham JK.The palatal canine and the adjacent lateral incisor:a study of a westof Scotland population. Br J Orthod 1994; 21:169-74.
27. Peck S,Peck L,Kataja M. Prevalence of tooth agenesis and peg-shaped maxillary lateral incisor associated with palatally displaced canine(PDC)anomaly. Am J Orthod Dentofacial Orthop 1996; 110:441-3.
28. Uslenghi S,Liversidge HM,Wong FS.A radiographic study of tooth development in hypodontia. Arch Oral Biol 2006; 51:129-33.
29. Ezoddini AF,Sheikhha MH,Ahmadi H.Prevalence of dental developmental anomalies:a radiographic study. Community Dent Health 2007; 24:140-4.
30. Vastardis A. The genetics of human tooth agenesis:new discoveries for understanding dental anomalies. Am J Orthod Dentofacial Orthop 2000; 117:650-6.
31. Arte S, Nieminen P, Apajalahti S,Haavikko K,Thesleff I, Pirinen S.Characteristics of incisor-premolar hypodontia in families. J Dent Res 2001; 80:1445-50.
32. Fekonja A. Hypodontia in orthodontically treated children. Eur J Orthod 2005; 27:457-60.
33. Zilberman Y,Cohen B,Becker A.Familial trends in palatal canines,a nomalous lateral incisors and related phenomena. Eur J Orthod 1990; 12:135-9.
34. Svinhufvud E,Myllarniemi S,Norio R.Dominant inheritance of tooth malpositions and their association to hypodontia. Clin Genet 1988; 34:373-81.
35. Pirinen S,Arte S,Apajalahti S.Palatal displacement of canine is genetic and related to congenital absence of teeth.J Dent Res 1996; 75:1742-6.
36. Peck L,Peck S,Attia Y.Maxillary canine-first premolar transposition, associated dental anomalies and genetic basis. Angle Orthod 1993; 63:99-109.
37. Camilleri S.Maxillary canine anomlies and tooth agenesis. Eur J Orthod 2005; 27:450-6.
38. Seow WK,Lai PY.Association of taurodontism with hypodontia:a controlled study. Pediatr Dent 1989; 11:214-9.
39. Calvano Kuchler E,De Andrade Risso P,De Castro Costa M,et al.Assessing the proposed association between tooth agenesis and taurodontism in 975 paediatric subjects. Int J Paediatr Dent 2008; 18:231-4.
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