RESEARCH REPORTS Biological

H. Seedorf1*, M. Klaften2, F. Eke1, H. Fuchs2, U. Seedorf3*, A Mutation in the and M. Hrabe de Angelis2 in a Mouse Model 1Department of Prosthetic Dentistry, University Medical Center, Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany; 2GSF National Research Center for Environment and Health, Institute of Experimental Genetics, Ingolstaedter Landstrasse 1, D-85764 Oberschleissheim, Germany; and 3Center of Internal Medicine, Department of Internal Medicine, Nephrology/Rheumatology with Section Endocrinology, University Medical Center, Hamburg- Eppendorf 52, D-20246, Germany; *corresponding author, [email protected] INTRODUCTION ooth enamel, the exterior tissue of vertebrate teeth, is a biomineral with J Dent Res 86(8):764-768, 2007 Tremarkable hardness and resistance to physical and biochemical attack (Schroeder, 1992). Its unique properties are related to large, highly ABSTRACT organized hydroxyapatite crystals that contribute more than 90% to the total imperfecta is an inherited disorder weight of mature enamel (Brudevold and Störenmark, 1967). The affecting formation. We previously hydroxyapatite crystals' high level of organization is the result of a complex isolated a mouse strain with an amelogenesis biomineralization process, which is controlled by a regulated expression of et al. imperfecta phenotype (ATE1 mice) from a multiple (Paine , 2001). Disorders in this biomineralization dominant ethylnitrosourea screen and mapped the process of enamel can lead to diseases, one of which is amelogenesis disease-causing defect to a 9-cM region of mouse imperfecta. To date, mutations in 5 genes [ (AMELX), enamelin et 5. In the current study, we tested the (ENAM), kallikrein-4 (KLK4), enamelysin (MMP-20), and DLX3 (Dong al. hypothesis that there is a mutation in enamelin , 2005)] have been found to cause . (ENAM) or (AMBN), both of which Ameloblastin, which has been reported to have a modulating effect on et al. are located wihin the linkage region, by calcium phosphate crystal morphology (Moradian-Oldak , 2003), is sequencing these two candidate genes. Analysis of another candidate gene. Ameloblastin-null mice show severe enamel et al. our data shows that the amelogenesis imperfecta hypoplasia and develop odontogenic tumors (Fukumoto , 2004). phenotype is linked to a C > T transition in exon 8 However, the involvement of ameloblastin in amelogenesis imperfecta has et al. of the enamelin gene. The mutation predicts a not yet been fully understood (Stephanopoulos , 2005). We previously C826T transition, which is present in the enamelin introduced a mouse strain with an amelogenesis imperfecta phenotype, transcript and changes the glutamine (Gln) codon which can serve as a new mouse model for hypoplastic autosomal-co- et al., at position 176 into a premature stop codon dominant amelogenesis imperfecta (ATE1 mice) (Seedorf 2004). The (Gln176X). Conversely, no mutation could be phenotype was visible to the naked eye in homozygotic and heterozygotic detected in the ameloblastin gene. These results animals. Microradiographs and toluidine-blue staining of non-decalcified define the ATE1 mice as a model for local sections showed complete loss of enamel in the front and molar teeth of hypoplastic autosomal-dominant amelogenesis homozygotic ATE mutants, cracked enamel of reduced width (approx. 50%) imperfecta (AIH2), which is caused by enamelin in incisor and molar teeth of heterozygotic ATE mutants, and regular truncation mutations in humans. structure of incisor and molar teeth of wild-type mice. Scanning electron micrographs of teeth of homozygotic ATE mutants showed loss of enamel et al. KEY WORDS: amelogenesis imperfecta, and exposed dentinal tubules (Seedorf , 2004). We mapped the defect ethylnitrosourea-induced mutagenesis, mutational to a 9-cM region of mouse chromosome 5, corresponding to human analysis, mouse disease models, dental enamelin chromosome 4q21, the location of the human ameloblastin and enamelin et al. . genes) (Hu , 2000), and hence considered ameloblastin and enamelin to be the strongest candidates within this region to cause the disease in ATE1 mice (Seedorf et al., 2004). Ameloblastin (also called amelin or sheathlin) is part of the organic enamel matrix. It is expressed at high levels by during enamel formation (Fong et al., 1996a), and also by odontoblasts (Nagano et al., 2003), in Hertwig's epithelial root sheath (Fong et al., 1996b) and in odontogenic tumors (Toyosawa et al., 2000). The mature is composed of 447 amino acids. Soon after secretion, the initial cleavages Received July 13, 2006; Last revision February 9, 2007; generate small polypeptides containing the N-terminus and relatively large Accepted April 16, 2007 polypeptides containing the C-terminus. The role of the protein in A supplemental appendix to this article is published mineralization remains obscure. The murine gene consists of 11 exons and electronically only at http://www.dentalresearch.org. 10 introns. The putative start codon is at 82 base pairs in exon 1. The

764 J Dent Res 86(8) 2007 ENAM Mutation in a Mouse Model 765 transcript is alternatively spliced in humans (Mardh et al., 2001) and mice (Simmons et al., 1998). No amelogenesis-imperfecta-causing mutations in ameloblastin have been reported (Stephanopoulos et al., 2005), while several mutations causing an amelogenesis imperfecta phenotype have been described for enamelin Figure 1. Phenotype of the ATE1 mutant mice. Homozygotes and heterozygotes differ from wild-type et al. mice based on the shown tooth abnormalities. Wild-type mice (left) have brown incisors, whereas teeth (Stephanopoulos , 2005). in heterozygous ATE1 mice (middle) have a whitish, chalk-like appearance. Homozygotes (right) show Some of these mutations were complete loss of enamel in the front and molar teeth. induced by N-ethyl-N-nitrosourea (ENU) (Masuya et al., 2005). Enamelin is the largest and least abundant of the 3 enamel matrix proteins (enamelin, was applied. Genotyping of this panel was performed with amelogenin, and ameloblastin), representing about 5% of the MassEXTEND, a MALDI-TOF high-throughput genotyping total proteins found in developing enamel (Uchida et al., 1991). system supplied by Sequenom (San Diego, CA, USA). It is a tooth-specific gene expressed by the enamel organ and, Mutation Screening and Genotyping at a low level, in odontoblasts (Nagano et al., 2003). The Genomic DNA was extracted from mouse tail tips with the mouse gene consists of 10 exons, while the human enamelin DNeasy Tissue Kit (Qiagen, Hilden, Germany), according to the gene contains 9 exons, because the sequence corresponding to supplier's instructions. Each exon including the intron boundary mouse exon 2 is absent in humans, though the intron between regions of the ameloblastin and enamelin genes was individually the first and second exons in humans shows a sequence amplified by PCR with the HotStarTaq Master Mix Kit (Qiagen, homologous to mouse exon 2. This region is flanked by splice Hilden, Germany), as recommended by the manufacturer. The junctions, so that alternative splicing may occur (Hu et al., genomic nucleotide sequences used in the construction of primers 2001). Like all enamel matrix proteins, enamelin goes through were derived from a Mus musculus chromosome 5 genomic contig, proteolytic processing that leads to cleavage products of 155 strain C57BL/6J, containing the complete ameloblastin and kDa, 142 kDa, 89 kDa, 34 kDa, 32 kDa, and 25 kDa (Fukae et enamelin gene sequences (GenBank accession number, NT al., 1996). This processing is believed to be of great importance 039308). All primers consisted of 20 nucleotides and had a G/C for proper enamel biomineralization (Stephanopoulos et al., content of 50% (APPENDIX). The following PCR conditions were 2005). used in all PCR amplifications: 94°C, 15 min; 94°C, 0:40 min In this study, by sequencing the enamelin and ameloblastin followed by 19 cycles at 93°C for 0:40 min, at 68°C for 1:30 min gene, we tested the hypothesis that a mutation in the enamelin (-0.5°C/cycle), at 93°C for 0:40 min, and at 58°C for 1:30 min (+1 or ameloblastin gene is the cause of the disease in ATE1 mice. sec/cycle). PCR products were purified with the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany), and DNA sequencing MATERIALS & METHODS was performed on an ABI 310 capillary sequencer (Applied Animals Biosystems, Darmstadt, Germany) with sequencing reactions The ATE1 mutants were obtained by intraperitoneal injection of prepared with the BigDye Terminator v1.1 Cycle Sequencing Kit ethylnitrosourea in wild-type C3HeB/FeJ animals at the National (Applied Biosystems, Darmstadt, Germany) as described by the Research Center for Environment and Health GmbH, Helmholtz supplier. Association, according to the German law governing the protection Heterozygotes and homozygotes were categorized according of animals. Isolation of the ATE1 strain and breeding conditions to their phenotypes (see Fig. 1 for details), and 3 mice of each were described previously (Seedorf et al., 2004). group were genotyped by DNA sequencing of exon 8. Genome-wide Linkage Analysis For linkage analysis of the chromosomal site of the mutation, RESULTS heterozygous male ATE001 mice on a C3HeB/FeJ background Genome-wide linkage analysis revealed a strong linkage [␹2 = were mated to C57BL/6Jico female mice. F1 hybrid males were 57, -log10 (P) = 13.36] on chromosome 5 with SNP marker backcrossed to C57BL/6Jico female mice. Fifty-seven N2 animals rs31585424 (Mouse Genome Assembly [MGA] 36.1: 80.03 showing amelogenesis imperfecta were selected for linkage Mb). Haplotype analysis of this region showed no recombinant analysis. Spleen subsamples were homogenized and treated allele at marker rs31585424, one recombinant allele at overnight by incubation at 57°C with a lysis buffer consisting of 10 rs13478429, and 6 recombinant alleles at marker rs29635956 mM Tris-HCl (pH 8.0), 1% (w/v) SDS, 50 mM EDTA, and 300 (Fig. 2). The maximum 95% confidence interval of rs31585424 ␮g/mL Proteinase K (Sigma-Aldrich Chemie GmbH, Taufkirchen, and the putative mutated gene is 6.2 cM, either proximal or Germany). Automated DNA extraction from the lysates was distal to that marker. This can be narrowed down by calculation performed with the AGOWA Mag Maxi DNA Isolation Kit of the minimum confidence intervals of the surrounding (AGOWA GmbH, Berlin, Germany). DNA concentration was markers. At a 95% level, these are 5 cM for rs29635956 and determined by absorption measurement at 260 nm. 0.4 cM for rs13478429. Considering the genetic distances of For linkage analysis, a genome-wide mapping panel 10.7 cM between rs29635956 and rs31585424 and 1.8 cM consisting of 157 single-nucleotide polymorphism (SNP) markers between rs31585424 and rs13478429, this means that the target 766 Seedorf et al. J Dent Res 86(8) 2007

Table. Results of the Genome-wide Linkage* Analysis

␹2 ␹2 Chromosome Highest Highest -log10 (P) of

1 5.07 1.61 2 1.42 0.63 3 3.95 1.33 4 2.96 1.07 5 57 13.36 6 2.12 0.84 7 6.33 1.93 8 2.12 0.84 9 0.86 0.45 10 0.86 0.45 Figure 2. Haplotype diagram of 5 SNP markers on chromosome 5. The 11 0.86 0.45 haplotype structure of the analyzed markers between 55 Mb and 112 12 2.96 1.07 Mb shows the strongest linkage with marker rs31585424. 13 0.44 0.29 14 3.5 1.21 15 2.96 1.07 16 2.96 1.07 region must start 5.7 cM proximal and end 1.4 cM distal to 17 0.86 0.45 rs31585424. Roughly translated to physical distances, the 18 2.12 0.84 linkage region spans an area from 73 Mb to 100 Mb on 19 0.44 0.29 chromosome 5. Linkage with markers on other could not be * Linkage could be shown on chromosome 5 with a high probability < < ␹2 established with a probability P 0.01 or -log10 (P) 2 (Table). [ = 57, -log10 (P) = 13.36]. No other chromosome reached the > Two candidate genes, enamelin (Enam, MGA 36.1: 89.58 minimum probability level of -log10 (P) 2. Mb) and ameloblastin (Ambn, MGA 26.1: 89.53 Mb), are located next to each other within the linkage region. Hence, we conducted mutation screening of the enamelin and ameloblastin resulted from a dominant ENU mutagenesis screen (Seedorf et genes, but not of Dmp1, osteopontin, or integrin-binding al., 2004). Our results demonstrated that abnormal tooth sialoprotein. enamel formation is linked to a mutation in exon 8 of the To screen for amelogenesis-imperfecta-causing mutations, enamelin gene. The mutation, a C826T transition which is we sequenced all exons, including flanking intron sequences and predicted to be present in the enamelin transcript, converts the two overlapping DNA fragments of ca. 500 bp, located upstream Gln codon at position 176 into a premature stop codon of the transcription initiation sites, which harbor the putative core (Gln176X). promoters of the two genes. In the case of the ameloblastin gene, ENU is a powerful alkylating mutagene acting on identical sequences were obtained from homozygous spermatogonial stem cells in mice. Although the most amelogenesis imperfecta mice and mice from the C57BL/6 commonly reported ENU-induced mutations are AT-to-TA reference strain (data not shown). However, a sequence deviation transversions or AT-to-GC transitions, our finding of a GC-to- could be detected in the 8th exon of the enamelin gene (Fig. 3a). AT transition is consistent with the ENU mutagenesis The normally occurring cytosine at position 40 of the exon was mechanism, and ENU-induced GC-to-AT transitions have been replaced by thymine in the homozygote. This substitution described previously in several cases (Jansen et al., 1994; corresponds to a C826T transition with respect to the cDNA Masuya et al., 2005). In a study of ENU-induced mutations, sequence (numbering according to GenBank accession No. NM Noveroske et al. (2000) found that 63% of mutations were 017468), which is predicted to change the normally occurring missense mutations, 26% caused abnormal splicing, and 10% glutamine (Gln) residue at position 176 of the precursor protein resulted in nonsense mutations. into a premature termination signal (Gln176X). Detection of a Like all enamel matrix proteins, enamelin goes through a G>A transition mutation in the sequence of the reverse strand complex pathway of proteolytic processing that involves confirmed the presence of the mutation in exon 8 (Fig. 3a). To multiple cleavage products (Fukae et al., 1996). This confirm further that the Gln176X allele is associated with processing is believed to be crucial for proper enamel amelogenesis imperfecta in ATE1 mice, we next genotyped 3 biomineralization (Stephanopoulos et al., 2005). Intact mice with the severe phenotype and 3 mice with the milder enamelin and the cleavage products containing the C-terminus heterozygous form of the phenotype. All 3 severely affected are exclusively present at the superficial layers of the mice lacking enamel were homozygous for the mutated T allele, developing enamel matrix, whereas the downstream cleavage whereas the less severely affected mice were heterozygous for products accumulate predominantly in the deeper layers (Hu et the C826T mutation (Fig. 3b). al., 1997). In porcine enamel, enamelin is secreted by ameloblasts as a 186-kDa precursor, which accumulates along DISCUSSION the secretory face of the Tomes' process. In this manuscript, we present the positional cloning of the Proteolytic processing toward the C-terminus via a 155-kDa disease-causing gene in the ATE1 mutant mouse strain, which intermediate leads to the 142-kDa form of enamelin. J Dent Res 86(8) 2007 ENAM Mutation in a Mouse Model 767

Subsequent cleavage steps result in the 89-kDa fragment consisting of the first 627 amino acids of the enamelin precursor and a 34-kDa polypeptide starting at residue 632. Further processing of the 89-kDa fragment results in polypeptides of 32 kDa (containing amino acids 136–238) and 25 kDa (containing amino acids 477-638) (Fukae et al., 1996). Since the Gln176X mutation leads to a truncated peptide containing only the first 175 amino acids of the 1142-amino-acid enamelin precursor, all functionally important enamelin-processing products would either be absent or truncated significantly in the ATE1 mice. Thus, it seems plausible to us that the Gln176X variant would not be able to mediate proper enamel biomineralization, and that the variant is essentially non- functional. Figure 3a. Identification of a C826T (Gln176X) mutation in exon 8 of the ENAM gene. Shown are The amelogenesis imperfecta sequence tracings (left — plus strand, forward; right — minus strand, reverse) obtained for exon 8 of the ENAM gene from a homozygous ATE1 mouse (top) and a C57BL/6 control (bottom). The amino phenotype has previously been acid sequence derived after translation of the ENAM cDNA is given below the nucleotide sequence. The mapped to a ~ 9-cM region between mutated nucleotide is underlined; ###, translational termination signal. markers D5Mit10 and D5Mit18 at map position 45–54 cM, according to the Mouse Genome Informatics (MGI©) database of the Jackson Laboratory© gene map, version 3.5 (Seedorf et al., 2004). Since ameloblastin and enamelin are located next to each other at map position 46 cM in this map, our previous linkage data are consistent with our current finding of a nonsense mutation in enamelin. In contrast, the most recent version of the mouse Ensemble gene map assigns the two markers to map positions 94.4 and 104.5 Mb, while the ameloblastin-enamelin gene cluster is placed outside the linkage region at map positions 89.53 to 89.58 Mb. At first glance, the findings of the previous study would therefore be in contradiction to the data presented here. However, the assembly and annotation of the physical map of the mouse genome are still incomplete, and contigs may be relocated in the future. Additionally, analysis of the linkage data based on a set of markers with a proven physical location Figure 3b. Comparison of sequence tracings obtained for wild-type and heterozygous or homozygous showed strong linkage with the region ATE1 mice. Mice were categorized according to their phenotypes (see Fig. 1), and 3 mice of each where the enamelin/ameloblastin group were genotyped by DNA sequencing for the C826T allele as described in the METHODS section. cluster is located. The mutated nucleotide is underlined. 768 Seedorf et al. J Dent Res 86(8) 2007

The phenotype of the C826T is consistent with that Hu CC, Fukae M, Uchida T, Qian Q, Zhang CH, Ryu OH, et al. (1997). reported for the EnamRgsc521 by Masuya et al. (2005). Both Cloning and characterization of porcine enamelin mRNAs. J Dent Res mutants have a truncated enamelin-protein, due to a premature 76:1720-1729. Hu CC, Hart TC, Dupont BB, Chen JJ, Sun X, Qian Q, et al. (2000). stop codon, and, for both, homozygotes differed from Cloning human enamelin cDNA, chromosomal localization, and heterozygotes based on their enamel abnormalities. Whereas analysis of expression during tooth development. J Dent Res 79:912- the teeth in heterozygotes had a whitish, chalk-like appearance, 919. and microradiographs and toluidine-blue staining of non- Hu JC, Zhang CH, Yang Y, Karrman-Mardh C, Forsman-Semb K, Simmer decalcified sections showed cracked enamel of reduced width JP (2001). Cloning and characterization of the mouse and human J Dent Res (approx. 50%), homozygote C826T and EnamRgsc521 showed enamelin genes. 80:898-902. complete loss of enamel. Thus, the mutation is inherited as a Jansen JG, Mohn GR, Vrieling H, van Teijlingen CM, Lohman PH, van Zeeland AA (1994). Molecular analysis of hprt gene mutations in skin co-dominant trait. fibroblasts of rats exposed in vivo to N-methyl-N-nitrosourea or N- In summary, our results provide further support for ethyl-N-nitrosourea. Cancer Res 54:2478-2485. enamelin as a prominent factor involved in the pathogenesis of Mardh CK, Backman B, Simmons D, Golovleva I, Gu T, Holmgren G, et al. amelogenesis imperfecta, and define ATE1 mice as a model for (2001). Human ameloblastin gene: genomic organization and mutation local hypoplastic autosomal-dominant amelogenesis imperfecta analysis in amelogenesis imperfecta patients. Eur J Oral Sci 109:8-13. et al. (AIH2), which is caused by enamelin truncation mutations in Masuya H, Shimizu K, Sezutsu H, Sakuraba Y, Nagano J, Shimizu A, (2005). Enamelin (Enam) is essential for amelogenesis: ENU-induced humans as well. mouse mutants as models for different clinical subtypes of human amelogenesis imperfecta (AI). Hum Mol Genet 14:575-583. ACKNOWLEDGMENTS Moradian-Oldak J, Iijima M, Bouropoulos N, Wen HB (2003). Assembly of amelogenin proteolytic products and control of octacalcium phosphate The authors thank Helga Reschke for expert technical crystal morphology. Connect Tissue Res 44:58-64. assistance regarding PCR amplifications and DNA sequencing. Nagano T, Oida S, Ando H, Gomi K, Arai T, Fukae M (2003). Relative Financial support was provided by each of the institutions levels of mRNA encoding enamel proteins in enamel organ epithelia involved in the study. and odontoblasts. J Dent Res 82:982-986. Noveroske JK, Weber JS, Justice MJ (2000). The mutagenic action of N- ethyl-N-nitrosourea in the mouse. Mamm Genome 11:478-483. REFERENCES Paine ML, White SN, Luo W, Fong H, Sarikaya M, Snead ML (2001). Brudevold F, Störenmark R (1967). Chemistry of the mineral phase of Regulated gene expression dictates enamel structure and tooth function. enamel. New York: Academic Press. Matrix Biol 20:273-292. Dong J, Amor D, Aldred M, Gu T, Escamilla M, MacDougall M (2005). Schroeder H (1992). Oral structure biology. New York: Thieme. DLX3 mutation associated with autosomal dominant amelogenesis Seedorf H, Springer IN, Grundner-Culemann E, Albers HK, Reis A, Fuchs imperfecta with taurodontism. Am J Med Genet A 133:138-141. H, et al. (2004). Amelogenesis imperfecta in a new animal model—a Fong CD, Hammarström L, Lundmark C, Wurtz T, Slaby I (1996a). mutation in chromosome 5 (human 4q21). J Dent Res 83:608-612. Expression patterns of RNAs for amelin and amelogenin in developing Simmons D, Gu TT, Krebsbach PH, Yamada Y, MacDougall M (1998). rat molars and incisors. Adv Dent Res 10:195-200. Identification and characterization of a cDNA for mouse ameloblastin. Fong CD, Slaby I, Hammarström L (1996b). Amelin: an enamel-related Connect Tissue Res 39:3-12. protein, transcribed in the cells of epithelial root sheath. J Bone Miner Stephanopoulos G, Garefalaki ME, Lyroudia K (2005). Genes and related Res 11:892-898. proteins involved in amelogenesis imperfecta. J Dent Res 84:1117- Fukae M, Tanabe T, Murakami C, Dohi N, Uchida T, Shimizu M (1996). 1126. Primary structure of the porcine 89-kDa enamelin. Adv Dent Res Toyosawa S, Fujiwara T, Ooshima T, Shintani S, Sato A, Ogawa Y, et al. 10:111-118. (2000). Cloning and characterization of the human ameloblastin gene. Fukumoto S, Kiba T, Hall B, Iehara N, Nakamura T, Longenecker G, et al. Gene 256:1-11. (2004). Ameloblastin is a cell adhesion molecule required for Uchida T, Tanabe T, Fukae M, Shimizu M (1991). Immunocytochemical maintaining the differentiation state of ameloblasts. J Cell Biol and immunochemical detection of a 32 kDa nonamelogenin and related 167:973-983. proteins in porcine tooth germs. Arch Histol Cytol 54:527-538.