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linical Article Amelogenesis Imperfecta Due to a Mutation of the Enamelin Gene: Clinical Case With Genotype-phenotype Correlations Rochelle G. Lindemeyer. DMD' • Carolyn W. Gibson. PhD-' • Timothy]. Wright, DDS,

Abstract: The major protein components of the enamel matrix include the most abundant omelogenin proteins as well as less plentiful proteins such as enamelin and ameloblastin. The enamel defect in amelogenesis imperfecta (AI) generally results In enamel that is too thin (hypoplastic) or too soft (hypocalafication or hypomaturation).

Previous reports indicate that mutations in the human enamelin gene (ENAM) cause hypoplastic Al through

autosomal-

dominant inheritance patterns and patisnts may also exhibit an anterior open bite. Although crown résorption of unerupted teeth occurs more frequently In Al patients, this finding has not been previously associated with known ENAM mutations. The purpose of this article was to report the genotypephenotype correlations for a 9-year, V-month-old boy with a homozygous ENAM mutation (c.1258J259insAG).

(Pediatr Dent ¿0í0:3¿:56-60) Received

September 3,2008 I Last Revision January 29,2009 I Revision Accepted January 30.2009

KEVWORDS: DENTAL DEVELOPMENT. GENETICS, RESTORATIVE DENTISTRY

At the beginning of the formation of dental enamel, ameloblast cells within the dental organ secrete an organic matrix which immediately begins to mineralize. The major enamel protein components of this matrix include the most abundant amelogcnin proteins as well as less plentiful proteins such as enamelin and ameloblastin.' Mutations in the genes encoding these and other proteins lead to the enamel defect ameiogenesis imperfecta (AI).-^ The defect in AI generally results in enamel that is too thin (hypopiastic) or too soft due to abnormal mineralization (hypocalcification or hypomaturation}.^'' AI has been classified according to both the appearance of the enamel and the pattern of genetic inheritance.-'^"^ As sufficient information is learned about the genetic causes of AI, a gene-based classification system will most likely be adopted. In the past, treatment has generally been provided without knowledge of the genetic defect. With most AI cases now diagnosable at the molecular level, however, this will not be the case in the future. The AI trait caused by mutations in the enamelin (ENAM) gene is transmitted predominantly through an autosomaldominant mode of inheritance.''"^' ENAM mutations generally result in hypoplastic AI, in which the hypoplastic teeth can

have proximal spacing due to reduced enamel thickness. Depending on the specific ENAM mutation, the hypoplastic enamel may be localized as pits or horizontal ridges or can be generalized with the entire enamel thickness being markedly reduced. In addition, several of the reported cases of ENAM gene mutations report an anterior open bite malocclusion.'"''''' Crown résorption of unerupted teeth and delayed eruption occurs more frequently in AI patients than non-Al patients.'^ '' This has not, however, been specifically linked to patients with ENAM mutations. These defects have aesthetic implications with frequent psychosocial impact requiring extensive treatment of primary and permanent dentitions as well as counseling therapies.•" A case of autosomal hypoplastic AI presented to the pédiatrie dentistry clinJc at Children's Hospital of Philadelphia (CHOP), Philadelphia, Pa. In addition to the enamel defects usually associated with AI, there appeared to be evidence of byperplastic gingival tissue and several impacted permanent molars. Unlike several previous reports on autosomal hypoplastic Al, the purpose of this report was to describe the phenotypical/ clinical findings for this patient as well as the histological findings and mutational analysis.

Case description The patient and parents provided consent for treatment and 'Dr. Lindemeyer is assistant professai: Pédiatrie Dentistry. School of Dental Medicine, genetic analysis. Saliva collection and mutational analysis was The University of Pennsylvania, and director. Pédiatrie Dental Residency Program Chilapproved by the Institutional Review Board of the University dren's Hospital of Philadelphia, and ^DT. Gibson 1,5 professor. Department of Anatomy of Pennsylvania, Philadelphia, Pa. A 9-year, Il-month-old AI and Cell Biology. School of Dental Medicine. The University of Pennsylvania, both in fíí\ladelphia. Pa; ^Dr. Wright is Bawden distinguished professor and chair department. male was referred to CHOP by a private pédiatrie dentist for Pédiatrie Dentistry, School of Dentistry. The University of North Carolina. Chapel Hill. NC. dental evaluation and treatment. His chief complaint was that Correspond with Dr. Lindemeyer at Undemey^dentai.upenn.edu the permanent posterior molars were not erupting. His prenatal

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and medical hisrory was noncontributory. His mother reported a normal pregnancy and delivery without complications. His father indicated a history significant for AI, as reported to him by his family dentist, who had performed extensive dental rchabilitarion on him over the years (Figure 1). The father also stated that his mother had received dentures at a young age and that another cousin (not included on the pedigree chart)

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and his niece had been told by their dentists that they bad AI. The mother denied any history of AI. Clinical examination revealed a mixed dentition. Ihe patient denied any sensitivity of the teeth to thermal stimuli, eating difficulty, or pain. The teeth appeared smooth and lacking proximal contacts with a generalized yellow color and tbin enamel consistent with the findings of hypoplastic AI. There

Figure I. Pedigree chari. (Males are itidicaicd by squares, ftmalcs .ivf circles, sex unknown arc tiiamuiids, LilFfctcd shaded in, and iion-pc'iicirant iiidicuicd by dark circle» in [he unshaded symbiilx. Extepi for ilie proband (Indicated by arrow), affected kindred were repnried by the probarid's faitier. Figure 2. Clinica] presentation of paiieni (male: 9y/l Im) wiih autosonial hypoplastic AI. Teeth arc smooth and lacking proxim.il ctintaces wiib a genera i iicd yellow color. Figure .Î, Panopraph ot paiieni (male; 9y/l Im) with auioiomal hypopiasiic Al. Note the pcrmatieru molars arc uneruptcd. wiih ihe maxillary first motafs appearing externally resorbed. Figure 4. Extracted maxillary permanent first miilars with crown résorption. Figure 5. Scanning electron micrographs nf fractured teeth. A) Notmal enamel súrtate (!•,] and prismatic Iracrured enamel layei. B) in the prohatid's permanctit maxillary molar, the enamel latkcd any prismatic architecture and was exiremely tliin (t.inc-lO jim) (E = Enamel. D = Dentin; original magnification (1400X).

n

Figure 6. Chromatograms for Enam mutation (c.1 258_Í259insAü); A. DNA sequence of the proband, shtiwing he is homo/ygntis tor the mutation. 1 he AC! insertion is indicated by a bracket, and gettcraies a premature stop at codon 448. B. DNA sequence ot the prohand's father, who is heterozygous for [he insertion, wiih tbe location indicated bv aii arrow.

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was no clinical evidence of pitting, grooves, or otiier irregularities in the enamel (Figure 2). All of the first permanent molars were unerupted, despite having complete root formation. The primary maxillary posterior teeth appeared ankylosed, as they were well below the occlusa! plane. This may have been due to the hyperplastic gingival tissue and uneven résorption of some of the primary roots. The primary mandibular central incisors were retained, and the permanent mandibular central incisors appeared to be congenitally missing. Soft tissues overlying che affected teeth were intact, and no periodontal pockets were detected on any adjacent teeth. There was enlargement of the gingival tissues in the molar region. His occlusion appeared to be a bilateral Class I molar with an overbite of 100% (no anterior open bice) and an overjet of 2mm. Radiographically, the unerupted permanent maxillary first molars appeared malformed wicb résorption of a portion of the clinical crowns (Figure 3). The permanent mandibular right first molar appeared to have a marked area of résorption on its mesial surface. There was no evidence of taurodoncism. After consulting with an oral surgeon and an orthodontist, a treatment plan was formulated to surgically expose the 4 permanent molars and extract all remaining primary molars. Saliva samples were collected from the patient and his parents ior DNA analysis. The patient was taken co the operating room for oral rehabilitation under genera! anesthesia, and surgical exposure ot the permanent firsc molars was performed. The 2 permanent maxillary molars appeared co have deformed dentin, a direct communication with the pulp chambers in both teech, and little or no enamel (Figure 4). They were clinically nontestorable and subsequently extracted. The permanent mandihular molars were exposed, and an area of résorption was evident on the permanent mandibular right first molar's buccal surface. The mandibular tooth was restored, and the extracted teeth were preserved for evaluation by scanning electron microscopy (SEM). Histological evaluation. The ceeth were photographed and then fractured, mounted on aluminum stubs wich silver paint, and coaced with Au-Pd for SEM.-^' The fractured surfaces were evaluated co assess che enamel thickness and structure ac various magnifications using a JEOL jSM 6300 SEM (JEOL USA, Peabody, Mass). The analysis revealed thac the enamel lacked atiy prismatic architecture and was extremely thin (Figure 5). Mutation analysis. Genomic DNA was isolated from saliva using the QIAamp kic (Qiagen, Santa Clara, Calif). The ENAM gene was sequenced as the mosc likely candidate gene in this AI family due co che generalized chin hypoplastic phenotype that has been previously associated with several different ENAM mucacions. The exons and exon/intron boundaries of the ENAM gene were amplified by PCR using forward and reverse primers previously described.'^ Aniplicons were electrophoresed through agarose gels, and the products were extracted using the Qiagen gel extraction kit. Extracted products were sequenced in both directions. The proband was homozygous for ENAM mutation c.l258_1259insAG, while the parents were both heterozygous (Figure 6). This insertion generates a premature scop codon at codon 448.'^

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Discussion This report illustrates the diverse manifestations that can be associated with Al and is chefirscto document abnormal tooch eruption and coronal résorption with an ENAM mutation. Enamelin is a glycosylated protein, considered co be an enamelspecific protein secreted by ameloblasts in relatively low amounts (1-5% of the matrix).-^•^' While the specific role of enamelin in amelogenesis is unknown, it is thought to play a role in crystallite growth regulation and elongation. In enamelin mutations thac result in a secreted but altered protein, a generalized thin hypoplascic phcnotype is generally seen.' There is no known role of enamelin in tooth eruption, and abnormal tooth eruption is not generally reported with ENAM mucacions. The purpose of this paper was co illustrate how the clinical phenotype of generalized hypoplastic AI led us to suspect a mutation in the ENAM gene and the identification of che molecular defect. The ENAM mutation c.l258_1259insAG has been described previously and was associated with a generalized hypoplastic phenotype in individuals homozygous for che niticant alíele.' The child in chis report also was homozygous for the ENAM mutant alíele and had smooth but very thin enamel, Ic was, however, devoid of any pitting or grooving, as has been described with several previous cases.'" The unusual finding of unerupted permanent molars that undergo résorption has been reporced in other cases of liypoplastic AI and is most commonly associated with aucosomal recessive rough hypoplastic AI. The associated genetic mutation, however, has not been previously identified. The etiology of abnormal tooch eruption and coronal résorption remains unclear, but appears to be the result of abnormal function of the enamel epithelium and amelobiascs. Failure of erupción in pacients with this Al cype may be a result of an abnormality in che molecular control of the eruption process chac is known to be at least partially driven by the odontogenic epithelium. Some AI-associated, nonenamel manifestations could result from modifying genes or environmental effects.'**The marked phenotypic diversity observed in amelogenin gene mutations associated with AI illustrates how allelic mutations altering different functional domains of the amelogenin protein are responsible for markedly different enamel phenotypes.-' The proband's mother reported not having Als clinical manifestations. It is possible chac, upon microscopic examination, however, she could have subclinical pitting or hypoplascic enamel defeccs chat were not previously choughc CO be associated with AI, but which have been described in individuals who are heterozygous for the ENAM c. 1258_ 12')9insAG mutation.'" The father was also heterozygous for this mutation and appeared to have the same generalized thin enamel phenotype as seen in the proband. This suggests that: the generalized thin hypoplastic AI traie associaced with che c.]258_1259insAG ENAM mutación is, indeed, transmitted in an autosomal dominant manner; and there is highly variable expression in people who are heterozygous for this alíele. Whether this is correct remains to be validated by additional AI cases that are carefully phenotyped and evaluated at the molecular level.


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7. Aldred MJ, Gtawford PJ. Amelogenesis imperfecta: Toward a new classification. Oral Dis 1995;l:2-5. 8. Aldred MJ, Savarirayan R, Grawford PJ. Amelogenesis imperfecta: A classification and catalogue for the 21" century. Oral Dis 2003;9:19-23. 9. Rajpar MH, Harley K, Laing G, Davies RM, Dixon MJ. Mutation of the gene encoding the enamel-specific protein, enameiin, causes autosomal-dominant amelogenesis imperfecta. Hum Mol Genet 2001; 10:1673-7. 10. Kida M, Ariga T, Shirak.iwa T, Oguchi H, Sakiyama Y. Autosomal-dominant hypopiastic form of amelogenesis imperfecta caused by an enameiin gene mutation at the exon-intron boundary. J Dent Res 2002;81:738-42. 11. Mardh GK, Backman B, Holmgren G, Hu JG, Simmer JP, Forsman-Semb K. A nonsense mutation in the enameiin gene causes local hypopiastic autosomal dominant amelogenesis imperfecta (AIH2). Hum Mol Genet 2002;ll: 1069-74. 12. Hart PS, Michalec MD, Seow WK, Hart TG, Wright JT. Identification of the enameiin {g.8344detG) mutation in a new kindred and presentation of a standardized ENAM nomenclature. Arch Oral Biol 2003:48:589-96. 13. Gutierrez SJ, Ghaves M, I'orres DM, Briceño I. Identification of a novel mutation in the enamalin gene in a family with autosomal-dominant amelogenesis imperfecta. Arch Oral Biol 2007;52:503-6. 14. Pavlic A, Petelin M, Battelino T. Phenotype and enamel ultrastructure characteristics in patients with ENAM gene mutations g. 131 85-1 31 86insAG and 8344delG. Arch Oral Biol 2007;52:209-17. 15. Kim JW, Seymen F, Lin BP, et al. ENAM mutations in autosomal-dominant amelogenesis imperfecta. J Dent Res 2005;84:278-82. 16. Gopinath VK, Yoong TP, Yean GY, Ravichandran M. Acknowledgments Identifying polymorphism in enameiin gene in ameloThis work was supported by National Institute of Health genesis imperfecta (AI). Arch Oral Biol 2008;53:937-40. grants DE016079 and DEOl 1089. The authors wish to thank U. Simmons BA, UNC Pédiatrie Dentistry for assistance with 17. HartTG, Hart PS, Gorry MG, et al. Novel ENAM mutaDNA .sequence determination. tion responsible for autosomal recessive amelogenesis imperfecta and localized enamel defects. J Med Genet 2003; 40:900-6. References 1. Robinson C, Brookes SJ, Shore RC, Kirkham J. The deve18. Gollins MA, Mauriello SM, Tyndall DA, Wright JT. loping enamel matrix: Nature and function. Eur J Oral Dental anomalies associated with amelogenesis imperSei I998;106(suppl 1}:282-91. fecta: A radiographie assessment. Oral Surg Oral Med Oral Pathoi Oral Radiol Endod 1999;88:358-64. 2. Wright JT. The molecular etiologies and associated phenotypes of amelogenesis imperfecta. Am J Med Genet 19. Korbmacher HM, Lemke R, Kahl-Nieke B. Progressive 2006;l40:2547-55. pre-eruptive crown résorption in autosomal recessive generalized hypopiastic amelogenesis imperfecta. Oral 3. Simmer JP, Hu JC. Dental enamel formation and its Surg Oral Med Oral Pathoi Oral Radiol Endod 2007; impact on clinical dentistry. J Dent Educ 2Ü01;65: 104:540-4. 896-905. 20. Goffield KD, Phillips G, Brady M, Roberts MW. Strauss 4. Hu JC, Yamakoshi Y. Esianielin and autosomal-dominant RP, Wright J l . The psychosocial impact of developmental amelogenesis imperfecta. Grit Rev Oral Biol Med 2Ü03; dental defects in people with hereditary amelogenesis im14:387-98. perfecta. J Am Dent Assoc 2005;I36:62n-30. 5. Witkop C] SJ. Heritable defects of enamel. In: RE Stewart RE, G H Prescott, eds. Oral Facial Generics. St. Louis, Mo: 21. Wright JT, Daly B, Simmons D, et al. Human enamel Mosby; 1976:151-226. phenotype associated with amelogenesis imperfecta and a kallikrein-4 (g.2l42G>A) proteinase mutation. Eur J Oral 6. Witkop GJ Jr. Amelogenesis imperfecta, dentinogenesis Sei 2006; 114(suppl 1):7, 13; discussion 39-41, 379. imperfecta, and dentin dysplasia revi.sitcd: Problems in classification. J Oral Pathoi 1988; 17:547-53.

Selecting appropriate treatment approaches for AI patients can be extremely complex, as demonstrated in this case. SHM analysis revealed a lack of prismatic enamel structure, leading one to question whether or not conventional bondingbased restorative therapies are appropriate. Seow and Amararunge performed acid etching on the extracted teeth of patients with hypopiastic and hypomineralized Al. They concluded that the lack of typical etching patterns in these variants may be the result of abnormal prism structure, and the standard etching time and/or acid concentration may be inappropriate for the abnormal enamel."'' Several studies have concluded that there is no relationship between bond strength to acid-etched enamel and etching conditions,"'' "' but these studies were all performed on sound enamel. Acid etch bonding therapies are frequently applied in hypopiastic AI cases and generally appear to have a reasonable level of success, suggesting that even having a thin, nonprismatic enamel layer could be sufficient to adequately retain bonded materials. Selecting appropriate treatment options and materials is ideally predicated on understanding the type of enamei defect that is present. For example, pretreatment with NaOCI may be used in hypomineralized AI types to improve bonding," but may not help in hypopiastic enamel that is wcll-mineraIÍ7xd and does not have increased protein content. It may one day be practical and highly beneficial to determine the specific Al genotype and associated AI phenotype before rendering treatment to optimize preventive and restorative care. By studying the outcomes of various restorative procedures for each genotypc/phenotype condition, practicing dentists will be able to use gene-based diagnoses to choose among various treatment options and thereby restore the dentition in a way that achieves the best results.''

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22. Hu JC, Zhang CH, Yang Y, Karrman-Mardh C, ForsmanSemb K, Simmer JP. Cloning and characterization of the mouse and human enamelin genes. J Dent Res 200Í;80: 898-902. 23. Hu CC, Hart TC, Dupont BR, et al. Cloning human enamelin cDNA. chromosomal localization, and analysis of expression during tooth development. J Dent Res 2000:79:912-9. 24. Wright JT. Hatt PS, Aldred MJ, et al. Relationship of phenotype and genotype in X-linked amelogenesis imperfecta. Connect Tissue Res 2003;44(suppl l):72-8. 25. Seow WK, Amaratunge A. The effects of acid-etching on enamel from different ciinicai variants of amelogenesis imperfecta: An SEM study. Pediatr Dent 1998;20:37-42. 26. Shinchi MJ, Soma K, Nakabayashi N. The effect of phosphoric acid concentration on resin tag length and bond strength of a photo-cured resin to acid-etched enamel. Dent Mater 2000; 16:324-9.

27. Orellana MF, Nelson AE, Carey JP, Heo C, Boychuk DG, Major PW. Surface analysis of etched molar enamel hy gas adsorption. J Dent Res 2008;87:532'6. 28. Legier LR. Retief DH, Bradley EL, Denys FR, Sadowsky PL. Effects of phosphoric acid concentration and etch duration on the shear bond strength of an orthodontic bonding resin to enamel: An in vitro study. Am J Orthod Dentofacial Orthop 1989;96:483-92. 29. Hotta K, Mogi M, Miura F, Nakabayashi N. Effect of 4-MET on bond strength and penetration of monomers into enamel. Dent Mater 1992:8:173-5. 30. Batkmeier WW, Gwinnett AJ, Shaffer SE. Effects of reduced acid concentration and etching time on bond strength and enamel morphology. J Clin Orthod 1987;21:395-8. 31. Saroglu 1, Aras S, Oztas D. Effect of deproteinization on composite bond strength in hypocalcified amelogenesis imperfecta. Oral Dis 2006; 12:305-8.

Absiract; olthe Scientific Literature What do children remember about their operative experience? The aim of this study was to evaluate children's intraoperative awareness, what were the contributing factors of this awareness, and what were the stiort term psychological effects. This study recruited 1830 children aged 5-15 years old, ASA status I- IV. English speaking with scheduled GA procedures of at least 30 minutes in length. Sites involved with the study were University of Michigan, University of Colorado, and Emory University. All IRB's. consents and assents were completed. Data was gathered via in person or phone interviews on postoperative days I 3, and 14 employing a Brice questionnaire simplified for chiidren. Ail primary investigators were trained to review children's answers to the question, "Do you remember anything between falling asleep and waking up?" There were 135 children who answered "yes' when asked this question. A total of 1554 children had no recall or memories after falling asleep. Statistical analysis were applied to the collected data for association between several child and perioperative outcomes. Psychological follow-up was refused by parents of children reporting intraoperative awareness. As reported previously, children may have awareness during their GA experiences. Comments: General anesthesia is routinely used to treat pédiatrie dental patients. From this study, we must be cognizant that sounds and conversation during the procedure may be heard by our young patients. To paraphrase what we often tell the children, "little tongue be careful what you say. in the OR." J6J Address correspondence to Dr. Shobha Malviya. MD E3900 C S. Mott Hospital. SPC 5211. 1500 E. Medical Center Dr.. Ann Arbor. Ml 48109-5ZIV, e-mail smalviya&umich.edu Malviya S, et al. The incidence of intraoperative awareness in children: childhood awareness and recall evaluation. Anesth & Anal 2009: W9:l4Zh7, 20 references

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