摘要
第一部分遗传性牙釉质发育不全相关基因Fam83h突变的鉴定与分析
[目的]对五个患有遗传性牙釉质发育不全的家族进行Fam83h测序分析，以探讨其发病是否与Fam83h突变有关。
[方法]对五个家族所有成员取外周血2ml，用QIAGEN公司的QIAamp~(?) DNABlood Maxi Kit提取基因组DNA。根据人Fam83h基因组DNA全长序列设计7对引物，首先PCR扩增出五个家族先证者的编码区Fam83h DNA，然后对所有PCR产物进行测序，对比Genebank的人Fam83h源序列，寻找Fam83h突变位点。若先证者的某Fam83h位点存在突变，再扩增该家族其他所有招募成员的Fam83h编码序列并测序，若所有患者在此位点均有相同变异，而未受影响成员在此位点无变异，则可确认该位点变异为致病突变。
[结果]检测的五个AI家族中，有两个家族鉴定出两个新的致病性的Fam83h突变。家族1为西班牙人，按照标准命名法，Fam83h变异位点为：Fam83h c．1330C＞T，p．Q444X，位于外显子5，突变类型为过早引入终止密码子，C末端氨基酸缺失735个；家族2为高加索人，Fam83h突变位于Fam83h c．1192C＞T，p．Q398X，位于外显子5，突变类型也为过早引入终止密码子，C末端氨基酸缺失781个。结合遗传方式及表型分析，此两个家族均为常染色体显性遗传钙化不全型AI(ADHCAI)。另外三个招募的AI家族未筛选到Fam83h变异。
[结论]Fam83h cDNA1330位点和1192位点C＞T的突变会导致ADHCAI。
第二部分Fam83h亚细胞定位的研究
实验一Fam83h真核表达载体的构建
[目的]构建融合绿色荧光的Fam83h真核表达载体。
[方法]从日本Yokohama公司购买含有小鼠Fam83h cDNA的质粒pBluescriptⅡKS+-Fam83h cDNA，设计Fam83h引物，引入SalⅠ和BglⅡ酶切位点，PCR扩增出小鼠Fam83h cDNA全长编码区后，连入PCR2．1-TOPO T载体，转化大肠杆菌TOP10，铺板，扩增，提取PCR2．1-TOPO-Fam83h质粒，SalⅠ和BglⅡ双酶切初步鉴定正确克隆，将酶切正确的克隆测序。SalⅠ和BglⅡ双酶切测序正确的克隆从而释放出Fam83h，连入BamHⅠ和SalⅠ双酶切过的phrGFP-C绿色荧光表达载体，转化大肠杆菌TOP10，铺板，扩增，提取phrGFP-C-Fam83h质粒，SalⅠ和NdeⅠ双酶切初步鉴定正确克隆，将酶切正确的克隆再次测序确认。
[结果]成功扩增出3651bp的小鼠Fam83h编码区序列，PCR2．1-TOPO-Fam83h质粒测序结果显示扩增出的Fam83h序列和genebank收录的Fam83h源序列完全一致。phrGFP-C-Fam83h质粒测序结果显示Fam83h序列被连入到phrGFP-C正确区域。
[结论]成功构建phrGFP-C-Fam83h荧光真核表达载体，为实验二和实验三打下基础。
实验二检测Fam83h是否为细胞内蛋白
[目的]体外检测Fam83h是否为胞内蛋白或为分泌性蛋白。
[方法]复苏HEK293细胞，待细胞生长状态良好时，接种入腔室载玻片进行培养，接种密度为2-6×10~5个细胞/cm~2。24小时后，将实验一构建的phrGFP-C-Fam83h质粒，与脂质体Lipofectamine 2000按1μg：1μl的比例转染入HEK293细胞，放入培养箱继续培养，6小时后更换培养液。24-72小时后倒置显微镜下观察细胞形态及存活率，4％多聚甲醛固定细胞，分别用DiI和DAPI进行胞膜和胞核染色，封片，在荧光显微镜下观察来自Fam83h的绿色荧光是否位于细胞内并拍照。
[结果]在HEK293细胞中，来自Fam83h的绿色荧光总是位于染成红色的胞膜之内，这充分表明Fam83h是一个胞内蛋白，而不是被分泌性到胞外。而且，Fam83h的绿色荧光信号很少和胞核重叠，绝大多数是位于胞核周围，特别是高尔基体常在的胞核前沿内陷处，排除拍照的角度问题，我们推测Fam83h很可能位于胞核周围的细胞器，特别是高尔基体。
[结论] Fam83h是一个非分泌性蛋白，它位于细胞内，但不在胞核内。Fam83h很可能位于胞核周围的细胞器，特别是与高尔基体密切相关。
实验三检测Fam83h的亚细胞定位
[目的]体外探讨Fam83h亚细胞定位与高尔基体的关系。
[方法]复苏HEK293细胞，待细胞生长状态良好时，接种入腔室载玻片进行培养，接种密度为2-6×10~5个细胞/cm~2。24小时后，将实验一构建的phrGFP-C-Fam83h质粒，与脂质体Lipofectamine 2000按1μg：1μl的比例转染入HEK293细胞，放入培养箱继续培养，6小时后更换培养液。24-72小时后倒置显微镜下观察细胞形态及存活率，4％多聚甲醛固定细胞，并用DAPI进行胞核染色及BODIPY进行高尔基体染色，封片，在荧光显微镜下观察Fam83h绿色荧光信号与高尔基体的关系并拍照。
[结果]在HEK293细胞中，来自Fam83h的绿色荧光总是位于高尔基体的红色荧光之内，且面积总是小于红色荧光，这充分表明Fam83h是位于高尔基体之内。并且，Fam83h绿色荧光所在位置的高尔基体红色荧光明显变淡，甚至无红色荧光，表明Fam83h蛋白可能定位于高尔基体膜表面。
[结论] Fam83h蛋白在细胞内定位与高尔基体密切相关，可能位于高尔基体膜表面。
PartⅠThe identification of Amelogenesis ImperfectacausingFam83h mutation
[Objective] To identify whether the AI phenotypes are caused by Fam83h mutationsin five AI families by DNA sequencing.
[Methods] Genomic DNA was isolated from 2 mL of peripheral whole blood of allrecruited members with QIAamp~(?) DNA Blood Maxi Kit. 7 pairs of primers weredesigned based on human Fam83h cDNA. Full length of Fam83h encoding regions offive probands were first amplified by PCR. All the PCR products were sequenced andthen compared with the original data of Genebank to detect whether there wereFam83h mutations. If there was some Fam83h mutation in the proband, then wewould amplify Fam83h encoding regions of all the recruited members of this familyand send for sequence. If all the affected people had the same Fam83h mutation andall the unaffected people were normal in this site, we could determine that it was thisFam83h mutation that caused AI.
[Results] Two new AI-causing Fam83h mutations were found in two families amongfive recruited families. The proband of family 1 was Hispanic. DNA sequencingchromatograms revealed a C to T transition in one of the two FAM83H alleles,changing codon 444 from a glutamine (Q) to a stop (X) codon (g.2452C＞T;c.1330C＞T; p.Q444X), the following 735 amino acids were lost. The proband of family 2 was Caucasian. DNA sequencing chromatograms showed an A to Gtransition in one of the two FAM83H alleles, changing codon 398 from a glutamine(Q) to a stop (X) codon (g.2314C＞T; c.1192C＞T; p.Q398X), the following 781 aminoacids were lost. No Fam83h mutation was found in the other three families
[Conclusion] Mutations C＞T at Fam83h cDNA 1330 and 1192 sites lead to AI.
PartⅡSubcellular localization of Fam83h
SectionⅠConstruction of Fam83h eukaryotic expression vector
[Objective] To construct a Fam83h eukaryotic expression vector fused to greenfluorescence.
[Methods] Full-length mouse Fam83h cDNA encoding region was amplified by PCRfrom pBluescriptⅡKS+-Fam83h obtained from K.K. DNAFORM (Yokohama, Jpn).SalⅠand BglⅡdigestion sites were introduced into the forward and reverse primersrespectively. The PCR product was then inserted into PCR2.1-TOPO vector andtransformed into TOP10. We plated, shaked bacterias and then extracted the PCR2.1-TOPO-Fam83h plasminds. The correct plasmids were first checked by SalⅠ/ BglⅡdouble digestion and then sended for sequencing. Fam83h was released from correctPCR2.1-TOPO-Fam83h by BglⅡ/SalⅠdouble digestion and then linked into phrGFPCvector which was digested by BamHⅠ/SalⅠ. We Transformed Phr-GFP-C-Fam83hinto TOP10 and plated, shaked bacterias and then extracted the phrGFP-C-Fam83hplasminds. The correct plasmids were first checked by SalⅠ/NdeⅠdouble digestionand then sended for sequencing again.
[Results] 3651bp mouse Fam83h encoding region was amplified successfully.Sequence results of PCR2.1-TOPO-Fam83h were coincidence with the genebank data.Sequence results of phrGFP-C-Fam83h showed that Fam83h was inserted into correctregion of phrGFP-C.
[Conclusion] We constructed phrGFP-C-Fam83h plasmid successfully, which laidthe first stone for SectionⅡand SectionⅢ
SectionⅡDetecting if Fam83h is an intracellular protein
[Objective] To determine if Fam83h is an intracellular or secreted protein in vitro.
[Methods] HEK293 cells were refreshed and then seeded into chamber slides whenthey were in good condition, with seeding density 2-6×10~5 cells/cm~2. 24 hrs later, thecorrect phrGFP-Fam83h plasmid was transfected into HEK293 cell lines by use ofLipofectamine 2000. The ratio of phrGFP-Fam83h: Lipofectamine 2000 was 1μg: 1μl.Cell culture media was changed for normal media after 6 hrs. 24-72 hrs later aftertransfection cells were fixed by 4% PFA, the cell membrane was stained into red byDiI and nucleus were stained into blue by DAPI. The green fluorescence fromFam83h was detected in fluorescence microscope and then taken pictures.
[Results] We detected green fluorescence of Fam83h was always within the redmembrane in HEK293 cell lines, which suggested definitely that Fam83h should bean intracellular protein instead of being secreted extracellularly. Further more, wefound that green fluorescence of Fam83h seldom overlapped with nucleus, most ofthem located in perinuclear region, especially the front edge of membraneinvagination, where usually contains Golgi apparatus. Regard the inaccuracy causedby angle of taking pictures, we propose that Fam83h probably locates in cell organsaround nucleus, especially Golgi apparatus
[Conclusion] Fam83h is an intracellular protein, but not in nucleus. The subcellularlocalization of Fam83h is probably in cell organs around nucleus, it is speculatedclosely related with Golgi complex.
SectionⅢStudy of the subcellular localization of Fam83h
[Objective] To determine the relation between the subcellular localization of Fam83hand Golgi complex.
[Methods] HEK293 cells were refreshed and then seeded into chamber slides whenthey were in good condition, with seeding density 2-6×10~5 cells/cm~2. 24 hrs later, thecorrect phrGFP-Fam83h plasmid was transfected into HEK293 cell lines by use ofLipofectamine 2000. The ratio of phrGFP-Fam83h: Lipofectamine 2000 was 1μg: 1μl.Cell culture media was changed for normal media after 6 hrs. 24-72 hrs later aftertransfection cells were fixed by 4% PFA, the nucleus were stained into blue by DAPIand Golgi complex was stained into red by BODIPY. The relation between greenfluorescence from Fam83h and red fluorescence from Golgi complex was detected influorescence microscope and then taken pictures.
[Results] In HEK293 cells, green fluorescence signal from Fam83h was always inand smaller than the red fluorescence from Golgi complex, which suggested stronglythat Fam83h shoul be in Golgi complex. Typically the Golgi stain was weaker ornegative where the GFP localized, it seemed that Fam83h located in the membrane ofGolgi complex.
[Conclusion] The subcellular localization of Fam83h is closely related with Golgicomplex, Fam83h may locate in the membrane of Golgi complex.
引文
1. Hu JC, Chun YH, Al Hazzazzi T, Simmer JP. Enamel formation and amelogenesis imperfecta. Cells Tissues Organs. 2007; 186(1):78-85.
2. Simmer JP. Evolution and genetics of teeth. Cells Tissues Organs. 2007; 186(1):4-6.
3. Hart PS, Hart TC, Simmer JP, Wright JT. A nomenclature for X-linked amelogenesis imperfecta. Arch Oral Biol. 2002 Apr; 47(4):255-260.
4. Witkop CJ, Sauk JJ. Heritable defects of enamel. St Louis: Stewart RE eds, 1976, 7: 187.
5. Kim JW, Simmer JP, Hu YY, Lin BP, Boyd C, Wright JT, Yamada CJ, Rayes SK, Feigal RJ, Hu JC. Amelogenin p.M1T and p.W4S mutations underlying hypoplastic X-linked amelogenesis imperfecta. J Dent Res. 2004 May; 83(5):378-383.
6. Rajpar MH, Harley K, Laing C, Davies RM, Dixon MJ. Mutation of the gene encoding the enamel-specific protein, enamelin, causes autosomal-dominant amelogenesis imperfecta. Hum Mol Genet. 2001 Aug 1; 10(16):1673-1677.
7. Papagerakis P, Lin HK, Lee KY, Hu Y, Simmer JP, Bartlett JD, Hu JC. Premature stop codon in MMP20 causing amelogenesis imperfecta. J Dent Res. 2008 Jan; 87(1):56-59.
8. Hart PS, Hart TC, Michalec MD, Ryu OH, Simmons D, Hong S, Wright JT. Mutation in kallikrein 4 causes autosomal recessive hypomaturation amelogenesis imperfecta. J Med Genet. 2004 Jul;41(7):545-9.
9. Wright JT, Hart TC, Hart PS, Simmons D, Suggs C, Daley B, Simmer J, Hu J, Bartlett JD, Li Y, Yuan ZA, Seow WK, Gibson CW. Human and mouse enamel phenotypes resulting from mutation or altered expression of AMEL, ENAM,MMP20 and KLK4. Cells Tissues Organs. 2009; 189(1-4):224-229.
10. Kim JW, Simmer JP, Lin BP, Seymen F, Bartlett JD, Hu JC. Mutational analysis of candidate genes in 24 amelogenesis imperfecta families. Eur J Oral Sci. 2006 May; 114 Suppl 1:3-12.
11. Kim JW, Simmer JP, Hart TC, Hart PS, Ramaswami MD, Bartlett JD, Hu JC. MMP-20 mutation in autosomal recessive pigmented hypomaturation amelogenesis imperfecta. J Med Genet. 2005 Mar; 42(3):271-275.
12. Stephanopoulos G, Garefalaki ME, Lyroudia K. Genes and related proteins involved in amelogenesis imperfecta. J Dent Res. 2005 Dec; 84(12): 1117-1126.
13. Mendoza G, Pemberton TJ, Lee K, Scarel-Caminaga R, Mehrian-Shai R, Gonzalez-Quevedo C, Ninis V, Hartiala J, Allayee H, Snead ML, Leal SM, Line SR, Patel PI. A new locus for autosomal dominant amelogenesis imperfecta on chromosome 8q24.3. Hum Genet. 2007 Jan; 120(5):653-662.
14. Lee SK, Hu JC, Bartlett JD, Lee KE, Lin BP, Simmer JP, Kim JW. Mutational spectrum of FAM83H: the C-terminal portion is required for tooth enamel calcification. Hum Mutat. 2008 May 16; 29(8):E95-E99.
15. Kim JW, Lee SK, Lee ZH, Park JC, Lee KE, Lee MH, Park JT, Seo BM, Hu JC, Simmer JP. Fam83h mutations in families with Autosomal-dominant hypocalcified amelogenesis imperfecta. Am J Hum Genet, 2008 Feb; 82(2): 489-494.
1. Milbury CA, Li J, Makrigiorgos GM. PCR-based methods for the enrichment of minority alleles and mutations. Clin Chem. 2009 Apr; 55 (4):632-640.
2. Korf BR. Overview of molecular genetic diagnosis. Curr Protoc Hum Genet. 2006 Feb; Chapter 9: Unit 9.1.
3. Simmer JP, Fincham AG. Molecular mechanisms of dental enamel formation. Crit Rev Oral Biol Med. 1995; 6(2):84-108.
4. Lu Y, Papagerakis P, Yamakoshi Y, Hu JC, Bartlett JD, Simmer JP. Functions of KLK4 and MMP-20 in dental enamel formation. Biol Chem. 2008 Jun;389(6):695-700.
5. Gopinath VK, Yoong TP, Yean CY, Ravichandran M. Identifying polymorphism in enamelin gene in amelogenesis imperfecta (AI). Arch Oral Biol. 2008 Oct; 53(10):937-940.
6. Tanimoto K, Le T, Zhu L, Witkowska HE, Robinson S, Hall S, Hwang P, Denbesten P, Li W. Reduced amelogenin-MMP20 interactions in amelogenesis imperfecta. J Dent Res. 2008 May; 87(5):451-455.
7. Richard B, Delgado S, Gorry P, Sire JY. A study of polymorphism in human AMELX. Arch Oral Biol. 2007 Nov; 52(11):1026-1031.
8. Hu JC, Chun YH, Al Hazzazzi T, Simmer JP. Enamel formation and amelogenesis imperfecta. Cells Tissues Organs. 2007; 186(1):78-85.
9. Kang HY, Seymen F, Lee SK, Yildirim M, Tuna EB, Patir A, Lee KEM, Kim JW. Candidate gene strategy reveals ENAM mutations. J Dent Res. 2009 Mar;88(3):266-269.
10. Seedorf H, Klaften M, Eke F, Fuchs H, Seedorf U, Hrabe de Angelis M. A mutation in the enamelin gene in a mouse model. J Dent Res. 2007 Aug; 86(8):764-768.
11. Kim JW, Seymen F, Lin BP, Kiziltan B, Gencay K, Simmer JP, Hu JC. ENAM mutations in autosomal-dominant amelogenesis imperfecta. J Dent Res. 2005 Mar; 84(3):278-82.
12. Hart TC, Hart PS, Gorry MC, Michalec MD, Ryu OH, Uygur C, Ozdemir D, Firatli S, Aren G, Firatli E. Novel ENAM mutation responsible for autosomal recessive amelogenesis imperfecta and localised enamel defects. J Med Genet.2003 Dec; 40(12):900-906.
13. Mardh CK, Backman B, Holmgren G, Hu JC, Simmer JP, Forsman-Semb K. A nonsense mutation in the enamelin gene causes local hypoplastic Autosomal dominant amelogenesis imperfecta (AIH2). Hum Mol Gen. 2002; 11:1069-1074.
14. Kim JW, Lee SK, Lee ZH, Park JC, Lee KE, Lee MH, Park JT, Seo BM, Hu JC, Simmer JP. Fam83h mutations in families with Autosomal-dominant hypocalcified amelogenesis imperfecta. Am J Hum Genet. 2008 Feb; 82(2):489-494.
15. Lee SK, Hu JC, Bartlett JD, Lee KE, Lin BP, Simmer JP, Kim JW. Mutational spectrum of FAM83H: the C-terminal portion is required for tooth enamel calcification. Hum Mutat. 2008 May 16; 29(8): E95-E99. [Epub ahead of print].
16. Hart PS, Becerik S, Cogulu D, Emingil G, Ozdemir-Ozenen D, Han ST, Sulima PP, Firatli E, Hart TC. Novel FAM83H mutations in Turkish families with autosomal dominant hypocalcified amelogenesis imperfecta. Clin Genet. 2009 Feb 11. [Epub ahead of print]
1. Mendoza G, Pemberton TJ, Lee K, Scarel-Caminaga R, Mehrian-Shai R, Gonzalez-Quevedo C, Ninis V, Hartiala J, Allayee H, Snead ML, Leal SM, Line SR, Patel PI. A new locus for autosomal dominant amelogenesis imperfecta on chromosome 8q24.3. Hum Genet. 2007 Jan; 120(5):653-662.
2. Okazaki Y, Furuno M, Kasukawa T, Adachi J, Bono H, Kondo S, Nikaido I, Osato N, Saito R, Suzuki H, et al. Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs. Nature. 2002 Dec 5;420(6915):563-573.
3. Marchler-Bauer A, Anderson JB, Derbyshire MK, DeWeese-Scott C, Gonzales NR, Gwadz M, Hao L, He S, Hurwitz DI, Jackson JD, Ke Z, Krylov D, Lanczycki CJ, Liebert CA, Liu C, Lu F, Lu S, Marchler GH, Mullokandov M, Song JS, Thanki N, Yamashita RA, Yin JJ, Zhang D, Bryant SH. CDD: a conserved domain database for interactive domain family analysis. Nucleic Acids Res. 2007 Jan; 35(Database issue):D237-240.
4. Izzotti A. DNA damage and alterations of gene expression in chronic-degenerative diseases. Acta Biochim Pol. 2003; 50(1): 145-154.
5. Kim JW, Lee SK, Lee ZH, Park JC, Lee KE, Lee MH, Park JT, Seo BM, Hu JC,Simmer JP. Fam83h mutations in families with Autosomal-dominant hypocalcified amelogenesis imperfecta. Am J Hum Genet. 2008 Feb; 82(2):489-494.
6. Lee SK, Hu JC, Bartlett JD, Lee KE, Lin BP, Simmer JP, Kim JW. Mutational spectrum of FAM83H: the C-terminal portion is required for tooth enamel calcification. Hum Mutat. 2008 May 16; 29(8): E95-E99. [Epub ahead of print].
7. Hart PS, Becerik S, Cogulu D, Emingil G, Ozdemir-Ozenen D, Han ST, Sulima PP, Firatli E, Hart TC. Novel FAM83H mutations in Turkish families with autosomal dominant hypocalcified amelogenesis imperfecta. Clin Genet. 2009 Feb 11. [Epub ahead of print]
8. Takagi T, Suzuki M, Baba T, Minegishi K, Sasaki S. Complete amino acid sequence of amelogenin in developing bovine enamel. Biochem Biophys Res Commun. 1984 Jun 15; 121(2):592-597.
9. Hu CC, Zhang C, Qian Q, Ryu OH, Moradian-Oldak J, Fincham AG, Simmer JP. Cloning, DNA sequence, and alternative splicing of opossum amelogenin mRNAs. J Dent Res. 1996 Oct; 75(10):1728-1734.
10. Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P, Mann M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell. 2006 Nov 3; 127(3):635-648.
11. Villen J, Beausoleil SA, Gerber SA, Gygi SP. Large-scale phosphorylation analysis of mouse liver. Proc Natl Acad Sci U S A. 2007 Jan 30; 104(5): 1488-1493.
12. Sun Y, Chen J. mTOR signaling: PLD takes center stage. Cell Cycle. 2008 Oct; 7(20):3118-3123.
13. Roth MG. Molecular mechanisms of PLD function in membrane traffic. Traffic. 2008 Aug;9(8):1233-1239.
14. Singh IN, Hall ED. Multifaceted roles of sphingosine-1-phosphate: how does this bioactive sphingolipid fit with acute neurological injury? Neurosci Res. 2008 May 15; 86(7): 1419-1433.
15. Huang P, Frohman MA. The potential for phospholipase D as a new therapeutic target. Expert Opin Ther Targets. 2007 May; 11(5):707-716.
16. Oude Weernink PA, L(?)pez de Jes(?)s M, Schmidt M. Phospholipase D signaling: orchestration by PIP2 and small GTPases. Naunyn Schmiedebergs Arch Pharmacol. 2007 Feb; 374(5-6):399-411.
1. Hu JC, Chun YH, Al Hazzazzi T, Simmer JP. Enamel formation and amelogenesis imperfecta. Cells Tissues Organs. 2007; 186(1):78-85.
2. Simmer JP. Evolution and genetics of teeth. Cells Tissues Organs. 2007; 186(1):4-6.
3. Stephanopoulos G, Garefalaki ME, Lyroudia K. Genes and related proteins involved in amelogenesis imperfecta. J Dent Res. 2005 Dec; 84(12): 1117-1126.
4. Richard B, Delgado S, Gorry P, Sire JY. A study of polymorphism in human AMELX. Arch Oral Biol. 2007 Nov; 52(11):1026-1031.
5. Simmer JP, Fincham AG. Molecular mechanisms of dental enamel formation. Crit Rev Oral Biol Med. 1995; 6(2):84-108.
6. Rajpar MH, Harley K, Laing C, Davies RM, Dixon MJ. Mutation of the gene encoding the enamel-specific protein, enamelin, causes autosomal-dominant amelogenesis imperfecta. Hum Mol Genet. 2001 Aug 1; 10(16):1673-1677.
7. Gopinath VK, Yoong TP, Yean CY, Ravichandran M. Identifying polymorphism in enamelin gene in amelogenesis imperfecta (AI). Arch Oral Biol. 2008 Oct; 53(10):937-940.
8. Kobayashi K, Yamakoshi Y, Hu JC, Gomi K, Arai T, Fukae M, Krebsbach PH, Simmer JP.Splicing determines the glycosylation state of ameloblastin.J Dent Res. 2007 Oct;86(10):962-967.
9. Nakamura Y, Slaby I, Spahr A, Pezeshki G, Matsumoto K, Lyngstadaas SP.Ameloblastin fusion protein enhances pulpal healing and dentin formation in porcine teeth.Calcif Tissue Int. 2006 May;78(5):278-284.
10. Fukumoto S, Yamada A, Nonaka K, Yamada Y.Essential roles of ameloblastin in maintaining ameloblast differentiation and enamel formation. Cells Tissues Organs. 2005; 181(3-4):189-195.
11. Tieleman LN, van Wezel GP, Bibb MJ, Kraal B.Growth phase-dependent transcription of the Streptomyces ramocissimus tuf1 gene occurs from two promotersJ Bacteriol. 1997 Jun; 179(11):3619-3624.
12. Lu Y, Papagerakis P, Yamakoshi Y, Hu JC, Bartlett JD, Simmer JP. Functions of KLK4 and MMP-20 in dental enamel formation. Biol Chem. 2008 Jun;389(6):695-700.
13. Wright JT, Hart TC, Hart PS, Simmons D, Suggs C, Daley B, Simmer J, Hu J, Bartlett JD, Li Y, Yuan ZA, Seow WK, Gibson CW. Human and mouse enamel phenotypes resulting from mutation or altered expression of AMEL, ENAM,MMP20 and KLK4. Cells Tissues Organs. 2009; 189(1-4):224-229.
14. Simmer JP, Hu JC.Expression, structure, and function of enamel proteinases.Connect Tissue Res. 2002; 43(2-3):441-449.
15. Sun Z, Fan D, Fan Y, Du C, Moradian-Oldak J.Enamel proteases reduce amelogenin-apatite binding.J Dent Res. 2008 Dec; 87(12):1133-1137.
16. Hu JC, Chun YH, Al Hazzazzi T, Simmer JP. Enamel formation and amelogenesis imperfecta. Cells Tissues Organs. 2007; 186(1):78-85.
17. Witkop CJ, Sauk JJ. Heritable defects of enamel. St Louis: Stewart RE eds, 1976, 7: 187.
18. Kirkham J, Zhang J, Brookes SJ, Shore RC, Wood SR, Smith DA, Wallwork ML, Ryu OH, Robinson C.Evidence for charge domains on developing enamel crystal surfaces.J Dent Res. 2000 Dec;79(12):1943-1947.
19. Robinson C, Brookes SJ, Kirkham J, Bonass WA, Shore RC.Crystal growth in dental enamel: the role of amelogenins and albumin.Adv Dent Res. 1996 Nov;10(2):173-179;discussion 179-180.
20. Wright JT, Hall K, Yamauchi M.The protein composition of normal and developmentally defective enamel.Ciba Found Symp. 1997; 205:85-99; discussion 99-106.
21. Kim JW, Simmer JP, Hu YY, Lin BP, Boyd C, Wright JT, Yamada CJ, Rayes SK, Feigal RJ, Hu JC.Amelogenin p.M1T and p.W4S mutations underlying hypoplastic X-linked amelogenesis imperfecta. J Dent Res. 2004 May; 83(5):378-383.
22. Smith RN, Elcock C, Abdellatif A, B(?)ckman B, Russell JM, Brook AH.Enamel defects in extracted and exfoliated teeth from patients with Amelogenesis Imperfecta, measured using the extended enamel defects index and image analysis.Arch Oral Biol. 2008 Sep 1. [Epub ahead of print]
23. Takagi Y, Fujita H, Katano H, Shimokawa H, Kuroda T.Immunochemical and biochemical characteristics of enamel proteins in hypocalcified amelogenesis imperfecta.Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1998 Apr;85(4):424-430.
24. Weikard R, Pitra C, Kuhn C.Amelogenin cross-amplification in the family Bovidae and its application for sex determination.Mol Reprod Dev. 2006 Oct;73(10):1333-1337.
25. Hart PS, Hart TC, Simmer JP, Wright JT. A nomenclature for X-linked amelogenesis imperfecta. Arch Oral Biol. 2002 Apr; 47(4):255-260.
26. Brookes SJ, Lyngstadaas SP, Robinson C, Shore RC, Kirkham J.Intracellular nanosphere subunit assembly as revealed by amelogenin molecular cross-linking studies.Eur J Oral Sci. 2006 May;114 Suppl 1:280-284; discussion 285-286, 382.
27. Kim JW, Seymen F, Lin BP, Kiziltan B, Gencay K, Simmer JP, Hu JC. ENAM mutations in autosomal-dominant amelogenesis imperfecta. J Dent Res. 2005 Mar; 84(3):278-82.
28. Mendoza G, Pemberton TJ, Lee K, Scarel-Caminaga R, Mehrian-Shai R, Gonzalez-Quevedo C, Ninis V, Hartiala J, Allayee H, Snead ML, Leal SM, Line SR, Patel PI. A new locus for autosomal dominant amelogenesis imperfecta on chromosome 8q24.3. Hum Genet. 2007 Jan; 120(5):653-662.
29. Lee SK, Hu JC, Bartlett JD, Lee KE, Lin BP, Simmer JP, Kim JW. Mutational spectrum of FAM83H: the C-terminal portion is required for tooth enamel calcification. Hum Mutat. 2008 May 16; 29(8):E95-E99.
30. Kim JW, Lee SK, Lee ZH, Park JC, Lee KE, Lee MH, Park JT, Seo BM, Hu JC, Simmer JP. Fam83h mutations in families with Autosomal-dominant hypocalcified amelogenesis imperfecta. Am J Hum Genet, 2008 Feb; 82(2): 489-494.
31. Kim JW, Simmer JP, Hart TC, Hart PS, Ramaswami MD, Bartlett JD, Hu JC. MMP-20 mutation in autosomal recessive pigmented hypomaturation amelogenesis imperfecta. J Med Genet. 2005 Mar; 42(3):271-275.
32. Papagerakis P, Lin HK, Lee KY, Hu Y, Simmer JP, Bartlett JD, Hu JC. Premature stop codon in MMP20 causing amelogenesis imperfecta. J Dent Res.2008 Jan; 87(1):56-59.
33. Tanimoto K, Le T, Zhu L, Witkowska HE, Robinson S, Hall S, Hwang P,Denbesten P, Li W. Reduced amelogenin-MMP20 interactions in amelogenesis imperfecta. J Dent Res. 2008 May; 87(5):451-455.
34. Hart PS, Hart TC, Michalec MD, Ryu OH, Simmons D, Hong S, Wright JT. Mutation in kallikrein 4 causes autosomal recessive hypomaturation amelogenesis imperfecta. J Med Genet. 2004 Jul; 41(7):545-549.
35. Yamakoshi Y, Hu JC, Fukae M, Yamakoshi F, Simmer JP.How do enamelysin and kallikrein 4 process the 32-kDa enamelin?Eur J Oral Sci. 2006 May;114 Suppl 1:45-51; discussion 93-95, 379-380.
36. Wazen RM, Moffatt P, Zalzal SF, Yamada Y, Nanci A.A mouse model expressing a truncated form of ameloblastin exhibits dental and junctional epithelium defects.Matrix Biol. 2009 Apr 15. [Epub ahead of print]
37. Ravindranath RM, Devarajan A, Uchida T.Spatiotemporal expression of ameloblastin isoforms during murine tooth development.J Biol Chem. 2007 Dec 14; 282(50):36370-36376.
38. Leiser Y, Blumenfeld A, Haze A, Dafni L, Taylor AL, Rosenfeld E, Fermon E, Gruenbaum-Cohen Y, Shay B, Deutsch D.Localization, quantification, and characterization of tuftelin in soft tissues.Anat Rec (Hoboken). 2007 May;290(5):449-454.