PEDIATRIC DENTAL JOURNAL 18(2): 79–85, 2008 79

Analysis of mutations in the and the in severe caries in Japanese pediatric patients

Ken Ouryouji*1#, Yasuhiro Imamura*2#, Yoshihisa Fujigaki*3, Yuriko Oomori*4, Shigeru Yanagisawa*3, Hiroo Miyazawa*1 and Pao-Li Wang*2

*1 Department of Pediatric Dentistry, Matsumoto Dental University, *2 Department of Pharmacology, Matsumoto Dental University, *3 Department of Oral Preventive Dentistry, Matsumoto Dental University, *4 Department of Oral Health Promotion, Graduate School of Oral Medicine, Matsumoto Dental University 1780 Gobara, Hirooka, Shiojiri, Nagano 399-0781, JAPAN (#: These authors contributed equally to this work.)

Abstract Caries and periodontitis are major oral diseases, both widespread Key words and serious. Caries is thought to be caused by multifactorial, lifestyle-related Amelogenin X, factors as well as the genetic background of the patient. However, little is Caries, known about relevant genetic factors. Since the quality and quantity of enamel Enamelin, plays a direct role in the susceptibility to caries, we set out our quest for genetic Single nucleotide polymorphism factors from the two crucial to the formation of dental enamel: amelogenin and enamelin. We isolated genomic DNAs from lingual mucosal cells derived from healthy and caries subjects, and examined the frequency of single nucleotide polymorphisms (SNPs) by the polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) method. We focused 522ם and 287ם on the SNPs of the amelogenin (AMELX) at positions position of the 2452ם in both cases, a C to T substitution), and on position) enamelin gene (ENAM; here, too, a C to T substitution). The subjects were all of Japanese extraction, of which 67 individuals served as healthy controls and 80 patients with severe caries served as test subjects. The occurrence of ,were 12.2 (2452ם)and ENAM (522ם) ,(287ם)the substitutions at AMELX 0.0 and 11.9% in the control group, and 2.5, 0.0 and 5.0% in the caries group, respectively. The P-values for the statistical frequency of SNPs for were 0.142 and 0.143, respectively. Our (2452ם)and ENAM (287ם)AMELX data suggest that there was no significant association between the SNPs of those genes and caries susceptibility in the Japanese pediatric population.

␣ Introduction reported that levels of -defensin 1–3 (an antimicro- bial peptides from saliva) were significantly higher Caries and periodontitis are common dental diseases. in children with no caries than in those with caries, It is thought that dental caries is the results of bacte- in contrast to cathelicidin LL37 and ␤-defensin 33). rial infection and many contributory environmental Thus, dental caries is a multifactorial disease. factors, like poor oral hygiene, insufficient fluoride are extracellular matrix proteins exposure and inadequate diet1,2). Host factors, such which make up 90% of the enamel organic matrix. as salivary flow, salivary buffering capacity and They are expressed specifically in the developing surface characteristics of the teeth, are also likely tooth bud and cleaved in a regulated process during factors in the etiology of caries. Recently, it has been enamel maturation4,5). Amelogenin proteins have been implicated in the control of enamel crystal Received on December 10, 2007 growth, but their precise role has not yet been Accepted on April 3, 2008 defined6). Amelogenin proteins also inhibit apatite

79 80 Ouryouji, K., Imamura, Y., Fujigaki, Y. et al.

Table 1 Diagnostic criteria of the caries subjects and healthy subjects

Age (years) Gender Present teeth Decayed teeth Caries n dmf (%) SD Range Male Female (total number) (total number) incidenceעMean

0.0 0.0 0 1,340 38 29 6–3 1.2עControl 67 4.8 2.6ע12.5 64.4 1,002 1,557 29 51 6–3 0.92עCaries 80 5.4

crystal growth when bound to crystal surfaces7). It We have previously studied the relationship between is likely that amelogenin proteins are hydrolyzed by the frequency of single nucleotide polymorphism proteases in the enamel matrix, like kallikrein-4 and (SNP) in the IL-1A, IL-1B, TLR2 and TLR4 genes MMP-208). The amelogenin X and Y genes (AMELX and susceptibility of periodontitis in the Japanese and AMELY, respectively) map to p22.3-p22.1 of population and in the Japanese Down syndrome X- and p11 of the Y-chromosome9). patients18,19). Analysis of SNP in relation to caries Known mutations of AMELX include deletions of has just started. The identification of genetic risk parts of the gene, single base mutations and prema- factors, which has the potential to lead to the ture stop codons. They appear to be critical in the development of diagnostic tools, is necessary for control of enamel thickness and mineralization10). caries. We have started our quest for genetic factors imperfecta (AI) is a genetic disorder, involved in caries from the two proteins crucial to the prevalence of which varies from 1:700 to the formation of dental enamel: amelogenin and 1:14,000 according to the populations studies. AI enamelin, focusing on a Japanese pediatric patients. involves hypoplasia, hypomineralization (or both) 11,12) of the dental enamel . The phenotype of the Materials and Methods amelogenin null mice revealed that amelogenins are required for the organization of crystal pattern and Subjects regulation of enamel thickness13). Thus, amelogenins All subjects who have no AI were unrelated may play very important roles for dental enamel individuals of Japanese extraction. The 67 healthy formation. controls (male: 29, female: 38) and 80 caries patients Enamelin is another tooth-specific matrix , (The caries might be caused by influence of environ- which is also related to AI. The gene encoding mental factors and genetic background rather than enamelin (ENAM) has been mapped to chromosome AI., male: 51, female: 29) ranged from 3 to 6 years ,years 0.92עand 5.4 1.2עSD 4.8ע4q2114). Subsequently, a mutation of ENAM was of age (mean identified in a family with the autosomal-dominant respectively, Table 1). Numbers of total present hypoplastic form of AI15). Their mutation was a and decayed teeth were also indicated in Table 1, heterozygous, single-G deletion at the exon 9-intron respectively. All patients visited Matsumoto Dental 9 boundary of ENAM, specifically present in AI University Hospital for their caries, and had no patients of the family, but not in other unaffected other disease of the oral hard and soft tissues. None family members or control individuals16). Moreover, used orthodontic appliances and none needed pre- N-ethyl-N-nitrosourea (ENU)-induced dominant medication for dental treatment. No subject was mouse mutants display AI-like phenotypes and taking anti-inflammatory drugs chronically. None present a single-base substitution (putative missense had a history of diabetes, hepatitis virus or HIV mutation) or substitution at the splicing donor site infection, a state of declining immunity (immuno- in intron 4 of ENAM17). These results indicate that suppressive chemotherapy, history of any disease enamelin is essential for amelogenesis. known to severely compromise immune function), Recently, the Project has been current acute necrotizing ulcerative gingivitis. The completed and the sequences of human chromosomal diagnostic classification of the control subjects and DNAs have been determined. Many researchers caries patients was calculated by dmf (decayed, have studied the relationship between gene poly- missing and filled primary teeth). Subjects with and Ն10 were classified in the control and in 0סmorphisms and susceptibility to various diseases. dmf SNPS ANALYSIS OF THE AMELX AND ENAM GENES IN CARIES 81

Table 2 PCR primer pairs used for the detection of SNPs in the amelogenin and the enamelin genes Gene Primer Sequences (position) Fw 5Ј-GGCTGCACCACCAAATCATCCCCGTGCTGTCCCAAATGCA-3Ј(287ם)AMELX hAMELX ם Rv 5Ј-GGAGGTTTGGCTGGTGGTGTTGG-3Ј(287ם)hAMELX (287 )

Fw 5Ј-GCCAACACTCCATGACTCCAATCC-3Ј(522ם)AMELX hAMELX ם Rv 5Ј-GCATGGGGAACATCGGAGGCAGAGGTGGCTGTGGCGTTA-3Ј(522ם)hAMELX (522 )

Fw 5Ј-AGGATTTTTATTACAGTGAATTTTACCCATGGGGCC-3Ј(2452ם)ENAM hENAM ם Rv 5Ј-CTTGTATCCTGTGGTCCCAGGAATTCC-3Ј(2452ם)hENAM (2452 )

the severe caries groups, respectively (The patients extension at 74°C for 7 min. The amplified fragment who have 10 or more decayed teeth are treated under (180 bp) was digested with 3 units of EcoT22 I general anesthesia in Matsumoto Dental University (Toyobo) at 37°C for 2 hr. (522ם)Hospital. Table 1). The survey was conducted under For the PCR amplification of the AMELX the supervision of a pediatric dentist with 6 years of fragment, the reaction solution and PCR cycle con- ,(287ם)clinical experience. ditions were the same as those for AMELX Fw(522ם)except that the specific primers hAMELX Rv were used. The amplified(522ם)Ethics and hAMELX The cells used in this study were obtained from fragment (180 bp) was digested with 0.3 units of subjects with appropriate informed consent from Mse I (New England Biolabs) at 37°C for 2 hr. their parents or legal guardians. The Ethics Committee As a control for restriction digestion, a fragment of Matsumoto Dental University gave approval to of the AMELX gene was amplified from the the study protocol (No. 0061). genomic DNA of a control patient using primers Rv1 (5Ј-GCATGGGGAACATCG(522ם)hAMELX Extraction of genomic DNA GAGGCAGAGGTGGCTGTGGCGTTAAGGGC- Fw. The amplified product(522ם)Lingual mucosal cells were collected with a 3Ј) and hAMELX toothbrush and DNA extraction was performed as (180 bp) was digested with Mse I. (2452ם)described before20,21). The concentration of purified For PCR amplification of the ENAM DNAs was measured by spectrophotometry and fragment, the reaction solution and PCR conditions ,(287ם)electrophoresis. were the same as those for the AMELX except that the specific primers used were Rv. The(2452ם)Fw and hENAM(2452ם)Genotype hENAM PCR amplification of the amelogenin and the amplified fragment (180 bp) was digested with 2 enamelin genes was carried out using Gene Amp® units of Apa I (Takara) at 37°C for 2hr. All of PCR System 9700 (Applied Biosystems). The the digested and undigested PCR products were sequences of the primers used are listed in Table 2. separated by electrophoresis through a 5% poly- acrylamide gel (19:1) and stained with ethidium (287ם)For the PCR amplification of the AMELX .Fw bromide(287ם)fragment, the specific primers hAMELX Rv were used. The reactions(287ם)and hAMELX were carried out in a volume of 50␮l containing Statistical analysis Taq buffer, 1.5 mM The differences in the frequencies of genotypes andן0.5␮g genomic DNA, 1–0.1

MgCl2, 0.2 mM dNTP, 0.2␮M of the primers and 1 alleles for the target genes were analyzed by Fisher’s unit of Taq Polymerase (Biotech International). The exact test. PCR cycle conditions were an initial denaturation at 96°C for 2 min, followed by 45 cycles of denatur- Results ation at 94°C for 1 min, annealing at 58°C for 1.5 min, and extension at 72°C for 1 min, with a final We have investigated the possibility of a relationship 82 Ouryouji, K., Imamura, Y., Fujigaki, Y. et al.

Fig. 1 Determination of polymorphisms of the amelogenin and the enamelin genes on normal and caries populations PCR amplification of the amelogenin and the enamelin genes was carried out using specific primers each and genomic DNA as a of the amelogenin gene was digested with EcoT22 I. (B) For 287ם template. (A) The amplified 180bp fragment from the position of the amelogenin gene, the amplified 180bp fragment was digested with Mse I. Control, the PCR products that 522ם the position of the enamelin gene, the amplified 180bp fragment was 2452ם could be digested with restriction enzyme. (C) For the position digested with Apa I. All samples were electrophoresed on a 5% polyacrylamide gel, followed by staining with ethidium bromide. 100bp ladder as size marker.

between severe caries and the incidence of known fragments of the genomes containing the target polymorphisms in the amelogenin gene (C to T positions. The PCR products were then subjected the SNP database ID of to restriction digestion to distinguish between the) 287ם mutations at position the National Center for Biotechnology Information mutants and the wild types (PCR-RFLP method), (NCBI: http://www.ncbi.nlm.nih.gov/): rs2106416) and the incidence was calculated. (287ם)rs6639060) and in the enamelin gene Digestion of the PCR product of AMELX) 522ם and rs3796703)) with EcoT22 I yields a single 180 bp fragment if the) 2452ם a C to T mutation at position) in a Japanese pediatric populations. The analysis sequence is wild type, whereas two fragments of 140 was carried out in subjects with severe caries as and 40 bp are expected in case the sequence contains Fig. 1A .287ם against a healthy control group. the C to T substitution at position We purified genomic DNAs from the subjects shows a typical pattern of restriction digestions of carried out single nucleotide polymorphism analysis individuals from both the control and caries groups. by PCR using appropriate specific primers to amplify The control group comprised 87.8% wild types and SNPS ANALYSIS OF THE AMELX AND ENAM GENES IN CARIES 83

Table 3 Frequencies of SNPs in the amelogenin and the enamelin genes in a healthy population (control) and caries subjects Control Caries Gene Allele Genotype P-value (80סn) (67סn)

AMELX Wild type (C) C/C61(87.8%) 78 (97.5%) 0.142 ם ( 287) Mutant (T) C/T6(12.2%) 2 (2.5%) T/T0(0.0%) 0 (0.0%) AMELX Wild type (C) C/C67(100.0%) 80 (100.0%) ם ( 522) Mutant (T) C/T0(0.0%) 0 (0.0%) T/T0(0.0%) 0 (0.0%) ENAM Wild type (C) C/C59(88.1%) 76 (95.0%) 0.143 ם ( 2452) Mutant (T) C/T8(11.9%) 4 (5.0%) T/T0(0.0%) 0 (0.0%)

12.2% mutants, whereas the caries group comprised products consistently digested only partially. This occurs 2452ם wild types and 2.5% mutants (Table 3). suggests that the mutation at position 97.5% Interestingly, the specimen from those individuals mostly, if not exclusively, in a heterozygous context. that gave digestible products consistently digested We then determined the statistical significance only partially. This suggests that the mutation at of these results. The P-values of the SNPs-frequency (2452ם)and the ENAM (287ם)occurs mostly, if not exclusively, in a for the AMELX 287ם position heterozygous context (No homozygous mutants or between healthy control and caries patients were wild types were observed). 0.142 and 0.143, respectively (Table 3). The AMELX map to p22.3-p22.1 of X-chromosome. Then, we (522ם)Digestion of the PCR product of AMELX with Mse I yields a 180 bp fragment if the sequence also determined the statistical significance of .for male and female, respectively (287ם)is wild type, whereas two fragments of 141 and AMELX 39 bp are expected in case the sequence contains However, there is no correlation (data not shown). Fig. 1B These seem to suggest that there is no significant .522ם the C to T substitution at position shows the typical pattern of restriction digestion of association between the SNPs of those genes (at individuals from both the control and caries groups. least at the positions tested) and caries susceptibility. None were digested. This means that both groups ם are strictly homozygous wild type at position 522. Discussion This is not due to an artifact in which the restriction enzyme is for whatever reason inactive: The It has been previously reported that there is no sig- “control” lanes in Fig. 1B demonstrate that a PCR nificant association between SNP of the amelogenin product of comparable size and containing Mse I or of the enamelin gene and caries susceptibility for restriction sites yielded the expected fragments. the subjects from mixture of various races, at least not any involving a C to T substitution at position (2452ם)Digestion of the PCR product of ENAM or ,273ם a C to G substitution at position ,236ם with Apa I yields a 180 bp fragment if the sequence as in this) 287ם is wild type, whereas two fragments of 144 and a C to T substitution at position 36 bp are expected in case the sequence contains study) in exon 5 of the amelogenin gene and a G to in exon 9 of the 2569ם Fig. 1C A substitution at position .2452ם the C to T substitution at position shows typical pattern of restriction digestions of enamelin gene. And the greatest severity of disease homozygous wild type subjects (left panel) and was found among the Caucasian children22). It seems heterozygous mutants (right panes). In the control that caries susceptibility is related to the races group, 88.1% resulted wild type, whereas 11.9% (different races might have different susceptibility was mutant. In the caries group, 95.0% were of caries). wild type and 5.0% mutant (Table 3). Again, the Various SNPs of those genes are observed in specimen from those individuals that gave digestible various positions, like as exons and introns. Selection 84 Ouryouji, K., Imamura, Y., Fujigaki, Y. et al. of the SNP-positions for those genes are based on not in relation to SNPs at the positions of the Slayton and amelogenin and the enamelin genes which we :(287ם)the following reasons. AMELX co-workers have recently reported22), but it has not analyzed. This came somewhat as a disappointment been studied about those in the Japanese population and a surprise, since a recent report indicated that the It was described bellow. frequency of a SNP in the intron 2 of the amelogenin :(522ם)so far. AMELX ;the SNP has been registered into gene is associated with higher DMFT (DMFTՆ20 :(2452ם)ENAM in high caries incidence in the (0.0000001סthe database. In particular, its allele frequency in the P Japanese (population diversity) has been identified. Guatemalan-Mayan population26). Nevertheless, we Therefore, in this study, we have first assessed the should think that our data could contribute to the possibility whether the frequency of polymorphisms field of pediatric dentistry not a little. for the amelogenin and the enamelin genes are We must conclude that further analysis will be correlated with caries susceptibility in the Japanese needed for the amelogenin and the enamelin genes, pediatric population. and comparisons with different races. We further Our results indicate that there are no significant envisage the study of other target genes suspected association between the prevalence of caries and the to be involved in caries, and more specifically of position of the amelogenin gene candidate targets. And we will aim at the potential to 287ם SNP at the of the enamelin gene in a purely lead to the development of a caries risk assessment 2452ם and at the and 0.143, tool and a gene diagnosis with the usage of SNPs 0.142סJapanese population (Table 3, P respectively). These results cannot be imputed to analysis, thereby opening up the possibility of more the size of the cohorts, since the incidence of both effective therapy or prevention. wild type and mutant genotypes roughly matched ם the allelic frequencies of AMELX( 287) in East Acknowledgments Asians (ss3025358: 0.95 (C) and 0.05 (T)) and in the Japanese population We are very grateful to the individuals who graciously (2452ם)of ENAM (rs3796703: 0.932 (C) and 0.068 (T)), as reported consented to be the subjects of this investigation. We by the SNPs database of the NCBI. thank Dr. Ivo Galli for proofreading this manuscript. We also found no incidence of mutations at This research was supported by a Grant-in-Aid for .(of the amelogenin gene. 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