Eur J Oral Sci 2006; 114 (Suppl. 1): 3–12 Copyright Ó Eur J Oral Sci 2006 Printed in Singapore. All rights reserved European Journal of Oral Sciences Jung-Wook Kim1,2, James P. Mutational analysis of candidate genes Simmer1, Brent P.-L. Lin3, Figen Seymen4, John D. Bartlett5, Jan C.-C. in 24 amelogenesis imperfecta families Hu1 1University of Michigan School of Dentistry, University of Michigan Dental Research 2 Kim J-W, Simmer JP, Lin BP-L, Seymen F, Bartlett JD, Hu JC-C. Mutational analysis Laboratory, Ann Arbor, MI, USA; Seoul National University, College of Dentistry, of candidate genes in 24 amelogenesis imperfecta families. Eur J Oral Sci 2006; 114 Department of Pediatric Dentistry & Dental (Suppl. 1): 3–12 Ó Eur J Oral Sci, 2006 Research Institute, Seoul, Korea; 3UCSF School of Dentistry, Department of Growth and Amelogenesis imperfecta (AI) is a heterogeneous group of inherited defects in dental Development, San Francisco, CA, USA; 4 enamel formation. The malformed enamel can be unusually thin, soft, rough and University of Istanbul, Faculty of Dentistry, stained. The strict definition of AI includes only those cases where enamel defects Department of Pedodontics, apa, Istanbul, Turkey; 5The Forsyth Institute, Harvard-Forsyth occur in the absence of other symptoms. Currently, there are seven candidate genes for Department of Oral Biology, Boston, MA, USA AI: amelogenin, enamelin, ameloblastin, tuftelin, distal-less homeobox 3, enamelysin, and kallikrein 4. To identify sequence variations in AI candidate genes in patients with isolated enamel defects, and to deduce the likely effect of each sequence variation on Jan C.-C. Hu, BDS, PhD, Department of protein expression and structure, families with isolated enamel defects were recruited. Orthodontics and Pediatric Dentistry, University The coding exons and nearby intron sequences were amplified for each of the AI of Michigan Dental Research Laboratory, 1210 candidate genes by using genomic DNA from the proband as template. The amplifi- Eisenhower Place, Ann Arbor, MI 48108, USA cation products for the proband were sequenced. Then, other family members were Telefax: +1–734–9759329 tested to determine their genotype with respect to each sequence variation. All subjects E-mail: [email protected] received an oral examination, and intraoral photographs and dental radiographs were obtained. Out of 24 families with isolated enamel defects, only six disease-causing Key words: amelogenesis imperfecta; AMELX; mutations were identified in the AI candidate genes. This finding suggests that many ENAM; enamel; MMP20 additional genes potentially contribute to the etiology of AI. Accepted for publication October 2005 Inherited enamel defects that occur in the absence of a twice as many affected females as males, and affected generalized syndrome are collectively designated as males transmit the AI trait to all of their daughters but to amelogenesis imperfecta (AI). For reviews of the clinical none of their sons. Potentially more diagnostic than the features and classification of AI, refer to the classic pattern of inheritance (which may not be obvious in works of Witkop (1,2). Currently, there are five proven small families with few affected members) is the distinc- candidate genes for AI: amelogenin (AMELX), enamelin tive vertical banding pattern on the enamel of affected (ENAM), enamelysin (MMP20), kallikrein 4 (KLK4), females. These vertical bands are believed to be caused and distal-less homeobox 3 (DLX3). There are also two by the presence of alternating bands of ameloblasts unproven candidate genes: ameloblastin (AMBN) and secreting normal and defective amelogenin during tuftelin (TUFT1). In the subsequent text, we review the amelogenesis (1,5). mutations that have been shown, to cause different forms The X and Y chromosomal copies of the human of AI. Then, we present our mutational analyses of the amelogenin genes do not undergo homologous recom- AI candidate genes in 24 families with inherited enamel bination, so, over time, their sequences have diverged (6). defects, and discuss what the low success rate in finding The differences make AMELX and AMELY useful for new mutations means in terms of our understanding of sex determination in forensics. For this purpose, oligo- the molecular participants in normal enamel formation nucleotide primer pairs are used that give different-size and the state of our understanding of the genetic etiol- polymerase chain reaction (PCR) amplification products ogies of AI. for AMELX and AMELY (7). There is a small failure rate for the amelogenin sex test, caused by the rare deletion of AMELY (8), which can reach as high as 3.6% X-linked AI in particular ethnic groups in Malaysia and India (9). X-linked AI accounts for 5% of all AI cases (3), and is Two individuals with AMELY deletions reportedly had caused by defects in the amelogenin gene on the normal teeth (10). X-chromosome (Xp22.3–p22.1). While there is a second As of the time of writing this report, 14 different dis- amelogenin gene on the Y-chromosome (AMELY), this ease-causing mutations have been identified in AMELX, gene is expressed at low levels (4), does not appear to be which are described by a standardized nomenclature necessary for proper dental enamel formation, and does (11). The designations, based upon their predicted effect not contribute to the etiology of AI. X-linked AI has a on translation of the amelogenin protein, are: p.0 (12); distinctive pattern of inheritance: there are likely to be p.W4S and p.M1T (13); p.W4X (14); p.H129fs187 (15); 4 Kim et al. p.I5-A8delinsT (16); p.T51I, p.P158fs187, and p.E191X monly affected tissues: hair, teeth, and bones (48,49). The (17); p.P52fsX53 (18,19); p.P70T (20–22); p.H77L (23); genetic cause of TDO was linked to the DLX3 gene on p.Y141fs187 (24); and p.L181fs187 (23,25). Different chromosome 17q21 (50), and mutational analyses identi- phenotypic patterns appear to correlate with mutations fied a 4-bp deletion in DLX3 that caused the disease affecting three different regions of the amelogenin pro- (51,52). While the principal clinical features of TDO tein (26). include kinky or curly hair in infancy, enamel hypoplasia, taurodontism, and increased thickness and density of cranial bones, the most penetrant feature in some kindreds Autosomal-dominant AI may be the dental phenotype, as a 2-bp deletion in the An autosomal-dominant pattern of inheritance is easily DLX3 gene has now been reported to cause AIHHT (53). recognized by analysis of the pedigree. Men and women are affected equally, and an affected person and an Autosomal-recessive AI unaffected person are likely to have an equal number of affected and unaffected children. The candidate genes for When a condition is inherited in an autosomal-recessive autosomal-dominant forms of AI (ADAI) are the genes pattern, the affected person must have two abnormal encoding enamel matrix proteins: enamelin and amelo- alleles of the disease gene to manifest a phenotype. blastin (4q11–q21) (27,28), and tuftelin (1q21–31) (29). Heterozygotes are phenotypically normal (even though Autosomal-dominant AI was first linked to chromosome they usually express a reduced amount of the given 4q (30), in a region since shown to contain the amelo- protein), but are carriers of the trait. Both parents are blastin (31,32) and enamelin genes (28). Subsequent usually normal phenotypically and one in four of the studies, however, only detected mutations in the children are likely to be affected. When one parent shows enamelin gene (33–39). Other genes besides enamelin are the phenotype and the other parent is a carrier, half of certain to participate in the etiology of ADAI; as linkage their children are likely to be affected. Consanguinity outside the 4q region (40), as well as outside the loci for (inbreeding) may be a factor as related persons are more all of the known AI candidate genes (41), has been likely to share the same mutant allele. The diversity of established. clinical enamel phenotypes observed in autosomal- The enamelin gene (ENAM, 4q13) has 10 exons, eight recessive AI (ARAI) is remarkable and may be indicative of which are coding (42,43). As of the time of writing this of a large number of potential candidate genes (54). report, five different disease-causing mutations have been Two proteinases – enamelysin (55) and kallikrein-4 identified in ENAM, which are described by a stan- (previously designated enamel matrix serine proteinase 1, dardized nomenclature (35). The designations of the or EMSP1) – are normally secreted into the enamel mutations are based upon their predicted effect on extracellular space during amelogenesis (56). Enamelysin translation of the enamelin protein: p.K53X (39); p.M71- (MMP-20) is a matrix metalloproteinase that is primarily Q157del (33) and p.A158-Q178del (36); p.N197fsX277 expressed during the secretory or early stage of amelo- (33,35,38); and p.P422fsX448 (34). Mutations in ENAM genesis (57–59), while KLK4 is a serine protease that is produce a hypoplastic (thin enamel) phenotype. In its primarily secreted during the transition/maturation or mildest form, very minor pits are evident (34). Sometimes later stages (57,60). Mutations in the genes encoding these there are horizontal lines of hypoplastic enamel, especi- Ôenamel proteasesÕ form part of the etiology of ARAI. ally in the cervical third of the crown. In humans, enamelysin is expressed from the No disease-causing mutations have yet been found in MMP20 gene on chromosome 11q22.3–q23, which has the ameloblastin gene (AMBN, 4q13). Ameloblastin (27), 10 exons. All are coding (61–63). The dental phenotype which has also been referred to as amelin (44) and caused by a splice junction mutation (IVS6-2A>T), sheathlin (45), is a proven constituent of the enamel affecting both MMP20 alleles, corresponded to an matrix of developing teeth (46).