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Acidic Proline-rich Protein Db and Caries in Young Children
1 Department of Biochemistry and Molecular Biology, Colleges of Medicine and Correspondence: * corresponding author, martin-levine{at}ouhsc.edu
Polymorphic, acidic proline-rich proteins (PRPs) in saliva influence the attachment of bacteria associated with caries. Our aims were to detect one of three acidic PRP alleles of the PRH1 locus (Db) using polymerase chain-reaction (PCR) on genomic DNA, and to determine its association with caries. DNA was obtained from buccal swabs from Caucasian and African-American children, and their caries experience was recorded. PCR primers designed around exon 3 of the PRH1 locus gave a 416-base product representing Db and a 353-base product representing the other two alleles (Pa or Pif). In Caucasians, Db gene frequency was 14%, similar to Db protein from parotid saliva. In African-Americans, however, it was 37%, 18% lower than Db from parotid saliva (reported previously). Compared with African-Americans, all Caucasians had significantly greater Streptococcus mutans colonization, but only Db-negative Caucasians had significantly more caries. Alleles linked to Db may explain racial differences in caries experience.
Key Words: acidic PRPs • caries • saliva • genetics • polymerase chain-reaction • Streptococcus mutans
Dental caries is caused by acidogenic and aciduric bacteria that colonize tooth-adherent biofilms and grow on dietary carbohydrate (Marsh, 2003). Streptococcus mutans is the best-characterized of these bacteria, but its association with the extent of dental caries is weak (Sullivan et al., 1989). Other bacteria—Lactobacillus spp., Actinomyces spp., Bifidobacterium spp. and non-mutans streptococci—are increased in the biofilms from high-caries individuals, regardless of S. mutans colonization (Beighton, 2005). Caries severity and S. mutans colonization have strong genetic components (Bretz et al., 2005; Corby et al., 2005), and, after adjustment for socio-nutritional factors, different racial groups significantly differ in caries experience (Zoitopoulos et al., 1996). Much of these genetically determined differences in caries experience might be due to polymorphic, acidic proline-rich proteins (PRP) in saliva (Lenander-Lumikari and Loimaranta, 2000) encoded at two loci, PRH1 and PRH2. Three alleles (Db, Pa, and Pif) provide polymorphisms at the PRH1 locus, and 3 (Pr1, Pr1', and Pr2) at the PRH2 locus (Azen, 1993, 1998). The Pr1' allele is present in 16% of African-Americans, who can therefore express up to 36 combinations, whereas Caucasians express only up to 18 combinations (polymorphisms). The acidic PRPs comprise 37% of the salivary proteins that adhere to freshly cleaned teeth (Bennick et al., 1983). They attach to apatite by their acidic N-terminal domain, and this exposes their proline-rich C-terminal domain to bind to oral bacteria and initiate biofilm development (Gibbons et al., 1991; Li et al., 1999). A common S. mutans strain (serotype c) binds to an expressed allele of the PRH2 locus (Pr1) more strongly than to any other tooth-attached salivary protein (Gibbons and Hay, 1989), but different mixtures of the acidic PRPs influence S. mutans binding independently. For example, S. mutans binding to parotid saliva-coated apatite is enhanced along with caries if the Db allele of PRH1 is expressed as the Db protein in saliva, but Actinomyces naeslundii binding is enhanced if the Db protein is absent (Stenudd et al., 2001). Thus, in the absence of Db, increased colonization by A. naeslundii may promote a high antibody response that associates with less caries in adults (Levine et al., 2005). Different acidic PRP mixtures may therefore promote different mutualistic bacterial interactions (Yoshida et al., 2006) that influence the extent of biofilm colonization by acidogenic and aciduric bacteria responsible for caries. The use of genomic DNA to detect PRH1 and PRH2 polymorphisms would overcome investigators’ difficulties in obtaining parotid saliva from children and the need for the different protein alleles expressed by different individuals to be identified. The aims of this study were to detect the Db allele in whole, genomic DNA, to confirm that the allele’s presence corresponds to Db protein expression in parotid saliva, and to determine how Db associates with caries in young children.
Db Gene Determination in Adult Caucasians Prior to the study, a consent document was approved by the University of Oklahoma HSC Institutional Review Board. Thirty-nine local volunteers provided genomic DNA, of whom 10 also provided parotid saliva (5 mL) using a Lashley canula. DNA was collected by the gentle rubbing of sterile nylon bristle brushes on the inside of the cheek. A protocol and reagents for DNA purification from the bristle-adherent buccal cells were supplied with the brushes (Gentra Systems Inc., Minneapolis, MN, USA). Additional samples of DNA from 50 random US adult Caucasians were purchased from the Coriell Cell Repository (Camden, NJ, USA).
In allele Db, a 63-base duplication of part of the nucleotide sequence of exon 3 encodes an additional 21 amino acids not present in Pa and Pif (Azen et al., 1987). Db is therefore 63 base pairs larger than Pa and Pif (Fig.
Polymerase chain-reaction (PCR) was performed on purified DNA with a PTC-100 Programmable Thermal Controller with Peltier Effect Cycling (Levine et al., 2001). A PCR reaction (total volume 50 µL) contained 20 ng genomic DNA, 2.5 mM deoxyribonucleotide triphosphates, 20 units/mL Taq polymerase, 5 µL Taq Reaction Buffer (Taq Mastermix; Continental Lab Products, San Diego, CA, USA), and 10 pmol of each primer. A cycle of heating to 94°C, annealing at 62°C, and extension at 72°C (each 1 min) was repeated 38 times, followed by a final extension for 10 min and cooling to 4°C. Products were visualized with ethidium bromide on a 2.5% agarose gel in trisborate-buffer, pH 8.3 (tris base, 0.1 M; boric acid, 0.1 M; and EDTA, 2 mM). To ensure that the correct nucleotide sequences were obtained, we cut selected PCR products from the gel and purified them using a QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, CA, USA); products were taken up in 50 µL of tris-HCl buffer, pH 8.5. The samples were reacted with BigDye® Terminator v1.1 Sequencing Reagents and sequenced with an AB3731 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). The presence or absence of the Db allele (gene) was determined by PCR and compared with whether the corresponding protein (Db) was present in parotid saliva from local volunteers. The saliva (5 mL) was dialyzed for 3 hrs against 6 changes of a hundred-fold excess volume of 0.1 M ammonium carbonate, freeze-dried overnight, and dissolved in 1.0 mL of 4.0 M urea. Approximately 20 3L was applied to a non-denaturing alkaline Mini-gel (BioRad Labs Inc., Hercules, CA, USA) and stained with Coomassie blue in 20% v/v trichloroacetic acid (Azen and Denniston, 1981).
Eligible children had to be enrolled in the US Head Start Program for low-income (low socio-economic status) families and to reside in Iowa in communities with optimally fluoridated water. Prior to entry, consent for a dental examination and collection of DNA from buccal oral mucosa was obtained from at least one parent, according to University of Iowa Institutional Review Board (IRB) guidelines (Slayton et al., 2005). Participants were 89 children of Caucasian parentage, 96 of African-American parentage, and 23 of mixed parentage (one Caucasian and one African-American). They were examined once for caries by means of a flashlight and mouth mirror. We determined the extent of caries by recording the decayed, missing, and filled surfaces of primary teeth in each child and calculating the dmft and dmfs. The Child-Care Centers were racially mixed, and all children received two meals a day, which controlled for differences in diet that could affect caries development as far as practicable. ’Outside’ dietary habits, i.e., between-meals habits, were not evaluated. We obtained microbiological data by touching the dorsal surface of each child’s tongue with a wooden blade until wet. We inoculated a growth medium selective for either Streptococcus mutans or Lactobacillus sp. (CRT Bacteria Kit, Ivoclar Vivadent, Schaan, Liechtenstein) by placing the blade onto the agar surface. Samples were incubated at 37°C for 48 hrs and then compared with visual standards (Edelstein and Tinanoff, 1989). Although low, medium, or high bacterial growth was noted, the plates were simply recorded as positive or negative for the respective bacteria for this study. Each Caucasian and African-American adult and child participant has 2 PRH1 loci in their genome. Allele Db may be absent (no copies), present at one locus (heterozygous), or present at both loci (homozygous). We therefore determined the gene frequency by totaling all heterozygous individuals, adding each homozygous individual twice, and dividing by twice the total number of individuals in the population. The population was divided into control individuals (dmfs 0) and cases (dmfs > 3), and the frequencies of Db and race were compared by chi-square. Caries experience (dmfs or dmft) was not normally distributed, and persons with dmfs 1, 2, and 3 were excluded. Differences in dmfs and dmft by Db and race were examined by the non-parametric Kruskal-Wallis test, followed by Dunn’s procedure for pair-wise comparisons.
Db Detection, Occurrence, and Relationship to Expressed Protein Db The 416-base segment representing Db and the 353-base segment representing Pa or Pif were detected as strong, ethidium-bromide-staining bands after PCR of genomic DNA (Fig.  , B). Most individuals had only the 353-base lower band (no Db), or both bands (heterozygous; Db present with either Pa or Pif). One Caucasian (Fig., C) and three African-American children showed only the 416-base upper band (homozygous for Db). DNA from all local and Coriell individuals, and 10 randomly selected children from Iowa, was tested at least twice, with identical results. The duplicates included two individuals whose DNA produced only the 416-bp upper band after PCR. In addition, the products, 11 upper bands from six Coriell and five local individuals, and a lower band from one Coriell and one local individual, all had the expected sequence (Fig., A).
The Db gene frequency in Caucasian local adult volunteers and Coriell adults was, respectively, 10.3% and 17%. The overall gene frequency of this combined Caucasian population was 14.0%, similar to that in 89 Caucasian children from Iowa (12.9%). Compared with Caucasians, the mixed-race group had a two-fold and African-Americans a three-fold greater Db gene frequency (Table 1
Saliva was obtained from five local individuals whose DNA gave the 416-bp PCR product and five others whose DNA gave only the 353-bp PCR product. The Db proteins were present in saliva from only those individuals whose DNA gave the 416-bp product on PCR (Fig. , D). The proteins appeared on Coomassie-blue-stained gels of parotid saliva as a double band (Db) that served as a unique identifier (see DISCUSSION).
Db and Dental Caries
The racial difference between cases and control individuals was not significant for Db-positive individuals (X2 = 2.3, n.s.), despite being significant for Db-negative individuals (X2 = 5.6, p < 0.03). The Kruskal-Wallis (KW) test confirmed racial and Db differences (observed KW statistic = 15.0, critical KW = 7.8, degrees of freedom = 2, p < 0.002). The Dunn Multiple Comparisons Test indicated that the median dmfs of African-Americans, regardless of Db, was significantly less than the median dmfs of Db-negative Caucasians, whereas that for Db-positive Caucasians did not differ significantly from that of either Db-negative Caucasians or African-Americans, regardless of Db (Table 3  ). Similar findings were obtained for dmft.
The results of this study indicate that, in young children of low socio-economic status, African-Americans are protected from caries compared with Caucasians; they had more control individuals, fewer cases, and less caries experience. This agrees with The U.S. Surgeon General’s 2000 Report on Oral Health: 41% of African-Americans aged 12 to 17 yrs are caries-free, compared with 32% of Caucasians. Indeed, in London, UK, less caries and a lower frequency of S. mutans isolation were previously reported in three- to four-year-old African-Caribbean children, compared with Caucasian children of similar socio-economic status (Zoitopoulos et al., 1996). In this study, the Db gene frequency in Caucasians was 14%, in agreement with a 15 to 17% expression frequency reported previously (Hay et al., 1994). The correspondence between Db protein expression and PCR-determined Db gene presence has been confirmed by the detection of Db in polyacrylamide gels of parotid saliva (Azen and Yu, 1984). The present study also confirmed a greater frequency of Db in African-Americans, but the gene frequency of 37.5% was 18.5% lower than that reported previously (Azen and Maeda, 1988). One possibility is that the Pr1' protein (Azen, 1998), which is absent from Caucasians, may have been confused with Db in parotid saliva. Antibody detection is impractical, because the additional amino acid sequence that characterizes Db duplicates part of the Pa and Pif sequences (Azen et al., 1987). The findings of this study call into question the reported association of Db gene expression with greater caries experience in African-American adults (Friedman et al., 1980). Compared with African-Americans, the caries experience of Db-positive Caucasians was less in magnitude and not significant. In Caucasian young adults, factors that enhance antibody responses to actinomyces, presumably by increasing the extent of actinomyces colonization, minimize caries development (Levine et al., 2005). This study suggests that acidic PRP alleles in linkage disequilibrium with Db could enhance the mutualistic growth of actinomyces in biofilms, and promote the observed antibodies and reduced caries experience observed in young adult Caucasians. Thus, the complete genotype of an individual at both acidic PRP loci (PRH1 and PRH2) may yet account for genetic differences in caries susceptibility. Results from a study in which 100 African-Americans and 206 Caucasians, aged 5 to 15 yrs, were typed for PRH1 and PRH2 alleles (Yu et al., 1986) suggested a greater increase in caries with age in children whose saliva was homozygous for Pr2 and expressed Pa. Unfortunately, the Db frequency in African-Americans may have been over-estimated, and the Pr1' protein was not examined. The present study suggests that the PRH1 and PRH2 alleles in an individual’s parotid saliva may be determined more accurately from genomic DNA than by the identification of expressed proteins. A powerful modification of the procedure in this paper would be the use of the dye-labeled oligonucleotide ligation (DOL) procedure (Chen et al., 1998).
We sincerely thank Ms. Carole Davis (OUHSC Dept. of Biochemistry & Molecular Biology) for technical advice and assistance, and Prof. Roger W.A. Linden (Division of Physiology, Guy’s, King’s and St Thomas’ School of Biomedical Sciences, King’s College London, Guy’s Campus, London Bridge, London SE1 1UL, UK) for providing Lashley canulae. This study was supported by grants from the J. Dean Robertson Society, College of Dentistry, University of Oklahoma HSC to G.M. Zakhary, and by NIH grants R03 DE014445.03 (R. Slayton, PI) and P60 DE13076 (J.C. Murray, PI). Preliminary reports were presented at the International Association for Dental Research meetings in March, 2005 (http://iadr.confex.com/iadr/2005Balt/techprogram/abstract_61785.htm), and March, 2006 (http://iadr.confex.com/iadr/2006Orld/techprogram/abstract_73973.htm).
a present address, 193 Hallette Dr., Shreveport, LA 71115, USA 
b present address, Harris County Medical Examiner’s Office, 1885 Old Spanish Trail, Houston, TX 77054–2098, USA Received for publication June 5, 2006. Revision received May 8, 2007. Accepted for publication August 28, 2007.
Journal of Dental Research, Vol. 86, No. 12,
1176-1180 (2007)
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