Association of Polymorphisms in the Carbonic Anhydrase 6 Gene with Salivary Buffer Capacity, Dental Plaque Ph, and Caries Index in Children Aged 7–9 Years

Association of polymorphisms in the carbonic anhydrase 6 gene with salivary buffer capacity, dental plaque pH, and caries index in children aged 7–9 years

RCR Peres1, G Camargo2, Carbonic anhydrase VI is a secreted enzyme that catalyzes the hydration of 2 3 carbon hydroxide in saliva and other body fluids. This enzyme has been LS Mofatto , KL Cortellazzi , implicated in taste and gastrointestinal dysfunctions, tooth erosion, and 2 1 MCLG Santos , MN Santos , caries. The purpose of this study was to analyze the allele and genotype CC Bergamaschi4 and SRP Line2 distribution of three polymorphisms in the coding sequences of (CA6) gene and check for possible associations with salivary buffer capacity, number of 1Department of Pediatric Dentistry, Piracicaba decayed, missing, and filled teeth in deciduous and permanent teeth (dmft/ Dental School, University of Campinas-Unicamp, DMFT, Decayed/Missing/Filled Teeth), plaque index (PI), and the plaque pH Sao Paulo, Brazil; 2Department of Morphology, Piracicaba Dental School, University of variation (DpH) in children aged 7–9 years. Two hundred and forty-five Campinas-Unicamp, Sao Paulo, Brazil; children from both genders, residents in area with fluoridated water 3Department of Preventive Dentistry and Public (Piracicaba, Sa˜o Paulo, Brazil) were divided into two groups: caries free and Health, Piracicaba Dental School, University of with caries. The clinical examinations were conducted by a single previously Campinas-Unicamp, Sao Paulo, Brazil and 4Department of Physiological Sciences, calibrated examiner (k ¼ 0.91) in an outdoor setting using a mirror and a Piracicaba Dental School, University of probe, according to WHO criteria index (dmft/DMFT). Approximately 2 h Campinas-Unicamp, Sao Paulo, Brazil after the first daily meal, the buffer capacity (BC) and the plaque pH were analyzed by means of a pH meter and an ion selective electrode. Plaque pH Correspondence: was measured immediately and 5 min after a mouth rinse with a 10% sucrose Dr RCR Peres, Department of Pediatric 2 Dentistry, Piracicaba Dental School, University solution. The data were submitted to w , Student’s, and Mann–Whitney tests of Campinas (UNICAMP), Av. Limeira, 901. CP (a ¼ 0.05). The PI and DpH of the upper and lower teeth were significantly 52, 13414-903 Piracicaba, Sao Paulo, Brazil. higher in the carious group than control (Po0.05). There was no difference E-mail: [email protected] between the groups in relation to BC. There was no association between the alleles and genotypes distributions for polymorphisms in the CA6 gene exons 2 and 3 and caries experience (P40.05). There was a positive association between buffer capacity and the rs2274327 (C/T) polymorphism. The allele T and genotype TT were significantly less frequent in individuals with the highest buffer capacity (P ¼ 0.023 and 0.045, respectively). This finding encourages future studies relating CA6 gene polymorphisms and their association with malfunctions, such as taste and gastrointestinal alterations, or the differential effect of chemical modulators on the protein products originated from the distinct genotypes of the CA6 gene. The Pharmacogenomics Journal (2010) 10, 114–119; doi:10.1038/tpj.2009.37; published online 1 September 2009

Keywords: buffer capacity; carbonic anhydrase 6; caries; saliva

Received 4 March 2009; revised 5 May 2009; accepted 27 July 2009; published online 1 September 2009 Salivary CA6 gene polymorphisms RCR Peres et al 115

Introduction genetic polymorphisms found in the coding sequences that may modulate the activity of this enzyme. Gene poly- Carbonic anhydrases (CAs) are a class of enzymes that have morphisms are a mechanism by which individuals may the function of maintaining the pH homeostasis in body exhibit variations within the range of what is considered tissues and fluids by catalyzing the hydration of carbon biologically normal. Single nucleotide polymorphisms occur À þ hydroxide in the reaction CO2 þ H2O3HCO3 þ H . at a high frequency in human genome and can affect gene Carbonic anhydrase VI (CAVI) is the only secreted form of function. Polymorphisms in the exon sequences of CA6 CA. This enzyme was first described and characterized in have not been associated with enzyme activity or diseases. saliva, in which it is produced by the serous acinar cells of The purpose of this study was to analyze the distribution the parotid and submandibular glands.1,2 CAVI was shown of three polymorphisms in the coding sequences of CA6 to be expressed in the lacrimal glands,3 lowers airways and gene, which alters the amino-acid sequence of this enzyme, lungs of rats,4 in the mouse nasal gland,5 in the lingual and checks for possible association of these polymorphisms serous glands of von Ebner in humans,6 in human and rat with salivary buffer capacity, dmft/DMFT (Decayed/Missing/ mammary gland and present in milk,7 as well as in the Filled Teeth) index, plaque index (PI), and variation in the lining epithelial cells of large intestine, stomach, and dental plaque pH in children aged 7–9 years. esophagus.8 The importance of CAVI is evidenced by its involvement in several malfunctions. Decreased secretion of salivary Results CAVI has been associated with loss of taste (hypogeusia) and smell (hyposmia) and distorted taste (dysgeusia) and smell The characteristics of the sample (age, gender, dmft/DMFT) (dysosmia).9 Salivary bicarbonate secretion is also known to are presented in Table 1. PI and pH and salivary buffer be important in the maintenance of esophageal pH home- capacity are presented in Tables 2 and 3, respectively. The PI ostasis.10,11 Additionally, Parkkila et al. 12 showed that and DpH of the upper and lower teeth were significantly salivary CAVI is present in the gastric mucus, in which it higher in the caries than control group (Po0.05). There was can contribute to maintain the pH gradient on the surface no difference between the groups in relation to buffer epithelial cells, thus protecting from gastric ulcers. capacity. There was no association of alleles and genotypes Salivary factors are considered important for dental with caries experience for polymorphisms in the CAVI gene health, as rampant caries is seen in patients with high exons 2 and 3 (P40.05). The genotype distribution for the salivary hypofunction.13 In this context, the salivary buffer three polymorphisms studied is shown in Table 4. A positive capacity is a factor of primary importance in maintaining association between buffer capacity and the rs2274327 (C/T) oral homeostasis. The bicarbonate system is the main buffer polymorphism was observed when the 245 individuals were that contributes to the total buffer capacity of saliva.14 divided into three groups containing the 1/3 highest (pH Bicarbonate ions can neutralize lactic and acetic acids 8.0–7.45, 82 individuals), 1/3 intermediate (pH 7.24–6.78, 81 produced by plaque bacteria and reduce demineralization. individuals), and the 1/3 lowest (6.77–3.77, 81 individuals) CAVI can adsorbs to the crystals of hydroxyapatite of enamel;15 it is present in the enamel pellicle16 and in dental biofilm.17 Salivary buffer capacity seems to be a determinant Table 2 Mean and s.d. of plaque index and pH in caries and factor on dental erosion, which is becoming a major dental caries-free children problem in both children and adults.18 Low CAVI concen- Variables Caries Caries free P-value trations in the saliva were associated with the prevalence of dental caries, especially in individuals with poor oral Mean s.d. Mean s.d. hygiene.19 Experimental evidences indicate that CAVI in saliva penetrates plaque and facilitates acid neutralization 17 PI 1.19 0.37 1.06 0.35 0.004 by salivary bicarbonate. Although CAVI was originally D1 1.19 0.42 0.93 0.43 o0.0001 20,21 predicted to regulate salivary pH or buffer capacity, D2 0.97 0.39 0.73 0.33 o0.0001 some studies indicate that these variables are not directly associated with CAVI concentration in saliva.22,23 Therefore, PI, plaque index; D1 (plaque pH difference in the upper arch—resting pH and 5 min after 2 min mouthrinse of 10% sucrose); D2 (plaque pH difference in the it is possible that besides concentration, CAVI activity in lower arch—resting pH and 5 min after 2 min mouthrinse of 10% sucrose); saliva can also be influenced by other variables such as by P-values derived from Student’s t-test.

Table 1 Characteristics of the study sample

Total Gender (~/#) Age dmft DMFT

Caries 125 63/62 (7.79±0.79) (3.00±2.26) (0.71±1.16) Caries free 120 56/64 (7.9±0.83) (0±0) (0±0)

(Mean±s.d.), ~ (female), # (male).

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Table 3 Mean (s.d.) salivary buffer capacity in caries and caries-free children

Variables Caries Caries free Significance (P)

Buffer capacity (BC) 6.88 (0.72) 6.9 (0.6) 0.87

P-value derived from Mann–Whitney test.

Table 4 Gene polymorphisms of CA6 gene

SNP Caries-free group Caries group P-value

n % n % rs2274333 AA 61 51.3 66 52.8 exon 3 GG 09 6.7 7 5.6 0.82 Figure 1 PSIPRED prediction of protein secondary structure of CAVI AG 50 42.0 52 41.6 region that includes polymorphism rs2274327 (amino acid 55). rs2274328 AA 52 43.3 60 48.0 (a) Allele T coding for Met (M). (b) Allele C coding for Thr (T). Note exon 2 CC 6 5.0 8 6.4 0.62 that distinct secondary structures are formed by the two alleles. AC 62 51.7 57 45.6 rs2274327 CC 72 60.0 82 65.6 exon 2 TT 15 12.5 12 9.6 0.62 shown). Analysis with the PSIPRED software showed CT 33 27.5 31 24.8 that the two alleles had a local effect on the secondary structure of CAVI, giving rise to distinct protein conforma- SNP, single nucleotide polymorphism; P-values derived from w2. Distribution of genotypes in the caries and caries-free groups. tions (Figure 1). It is worth mentioning that the population analyzed in this study was composed of 213 Caucasians and 32 Black individuals. Statistical analysis excluding specific Table 5 Gene polymorphisms of CA6 gene races did not change significantly from the one results obtained with the total population (that is Caucasians and SNP Genotype Buffer capacity P-value Blacks).

3.77–6.77 6.78–7.24 7.25–8.0 Discussion n % n % n % Gene polymorphisms are a mechanism by which individuals rs2274333 AA 43 53.1 43 52.4 41 50.0 may exhibit variations within the range of what is exon 3 GG 6 7.4 6 7.3 4 4.9 0.915 considered biologically normal. Single nucleotide poly- AG 32 39.5 33 40.3 37 45.1 morphisms occur at a high frequency in human genome rs2274328 AA 2 2.5 6 7.3 6 7.3 and can affect gene function. Polymorphisms in the coding exon 2 CC 42 51.8 33 40.3 37 45.1 0.426 sequences of CA6 gene have neither been studied earlier nor AC 37 45.7 43 52.4 39 47.6 have they been associated with disorders. It is known that salivary CAVI, the only secreted isoenzyme of the CA rs2274327 CC 45 55.6 48 58.5 61 74.4 enzyme family, is one of the major protein constituents in exon 2 TT 12 14.8 12 14.6 3 3.6 0.046 human parotid saliva.24 Polymorphisms in exons 2 and 3 CT 24 29.6 22 26.9 18 22.0 were selected because they produce changes in the amino- Allele acid sequence in the secreted protein (www.ncbi.nih. C 114 70.4 118 72.0 140 85.4 0.002 gov/SNP) and have, therefore, a potential to interfere with T 48 29.6 46 28.0 24 14.6 the function of the enzyme. In fact, our results showed that SNP, single nucleotide polymorphism. Allele frequencies for rs2274327 were also the rs2274327 (C/T) polymorphism is associated with included as significant differences (Po0.05) were observed in genotype salivary buffer capacity. A comparative analysis (http:// distributions. P-values derived from w2. Distribution of genotypes according to the buffer capacity. genome.ucsc.edu/) showed that the C allele, whose codon corresponds to the amino acid, Thr, is the ancestral form, as it is present in Chimp, Macaque, and Orangutan. The T buffer capacity. The allele T and genotype TT were signifi- allele produces the codon Met, and disrupts a highly cantly less frequent in individuals in the group with the conserved short beta sheet between amino acids 50 and highest buffer capacity (P ¼ 0.002 and 0.046, respectively, 52, which is present in the rat, cow, horse, and the three Table 5). No significant association was observed when these primates listed above (not shown). Therefore, it is likely that three groups were analyzed in regard to DpH (P40.05, not this change will interfere with the function of CAVI.

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Interesting, the younger T allele is associated with decreased polymorphisms and malfunctions, such as taste, gastro- buffer capacity. The distribution of CAVI genotypes and alleles intestinal alterations, and periodontitis or the differential in the population studied is similar to the frequencies described effect of chemical modulators on the protein products in other human populations (http://www.hapmap.org/). originated from the distinct genotypes of CA6 gene. The etiology of caries involves a combination of factors, such as environmental aspects, diet, infection, and genetic Materials and methods background that influence the chemical and physical 25,26 properties of teeth and saliva. It is worth mentioning Study population that children had the main meals at school (breakfast, snack, A convenience sample of 245 schoolchildren (aged 7–9 and lunch), which partially reduce the effects of variations years) was selected from a group of the city of Piracicaba, Saõ in diet composition. It is known that bicarbonate in saliva Paulo, Brazil. The inclusion criteria were lack of systemic þ diffuses into dental plaque and combines with H to form diseases, severe fluorosis, hypoplasia, orthodontic braces, carbonic acid. CAVI presence contributes to the neutraliza- use of antimicrobials, and communication or neuromotor tion of plaque acid, mainly in stimulated saliva whose impairments. Schoolchildren were chosen because they had 14 buffering is mainly performed by bicarbonate. Saliva the two principal meals and snacks in the school. The meals controls the pH of dental plaque after exposure to were composed of nutrients and food with cariogenic fermentable carbohydrate, and thus helps to prevent dental agents. The mean age was 7.84 years (s.d. ¼ 0.81) and the caries. When plaque pH was measured before and after a boys/girls ratio in the sample was 51.4%/48.6%. For dmft/ sucrose rinse (DpH), the children with caries had a higher pH DMTF study, children were divided into caries and caries- fall when compared with caries free; however, there was no free groups. The caries-free group (n ¼ 120) presented difference between these two groups regarding salivary absence of frank cavitations. The children for caries group buffer capacity. This indicates that the salivary buffer (n ¼ 125) were screened for presence of frank cavitation and capacity may not be the only factor that controls plaque presence of fillings, representing past caries experience. pH. The size of plaque and the amount of cariogenic Children were requested to withhold toothbrush and other bacterial cells present in plaque are probable factors that oral hygiene measures for 1 day before the examination to also influence the DpH. In fact, PI was significantly higher in encourage plaque accumulation. This study was approved caries group. The effect of buffer capacity on caries by the Ethical Committee of Piracicaba Dental School— development is still a matter of debate. The results of this University of Campinas, Saõ Paulo, Brazil (#110/2005). work were in accordance with the majority of the recent studies reporting that buffering capacity values are not Clinical examination related to dental caries.27–29 Although it seems logical that Clinical examinations were carried out using standardized salivary buffer capacity is a contributing factor on enamel criteria, after calibration of the examination methodology demineralization, its effect may be overshadowed by several (k ¼ 0.91). Caries index, expressed as mean dmft/DMFT was other factors that contribute to caries development such as scored as recommended by the WHO report on oral health oral hygiene, presence of cariogenic bacterial strains, sugar surveys.32 Caries detection was carried out by the same consumption, and exposure of dental surfaces to fluoride. dentist under a natural light source and dental mirror. Oral This rationale also explains the lack of association hygiene level was assessed using the PI described by Silness between CA6 polymorphisms and dental caries observed in and Loë 33 and was scored on the buccal surfaces of primary this study. or permanent teeth 16 (55), 21 (61), 24 (64), 36(75), 44 (84), It is also plausible to assume that besides caries, CAVI may and 41 (81), using a mirror and a WHO CPTIN type E probe. participate in the homeostasis of oral mucosa. In fact, The PI was calculated as the mean score for the examined periodontal disease, which occurs mainly in adults, was surfaces and was used as a measure for the average level of associated with salivary pH.30 The implication of CAVI in plaque accumulation. taste and gastrointestinal dysfunctions has raised the interest in the development of drugs that modulate the Dental plaque pH measurement function of CAVI. Several clinically used compounds, such Plaque pH measurements were made using an iridium oxide as acetazolamide, methazolamide, ethoxzolamide, dichlor- touch electrode 2 mm in length and 0.1 mm in diameter ophenamide, dorzolamide, brinzolamide, topiramate, sul- (Beetrode model NMPH1, WPI, Sarasota, FL, USA) inserted piride, indisulam, and benzolamide showed effective CAVI into an interdental space apical to the contact point in each inhibitory activity.31 These authors proposed that CAVI of the four quadrants in the mouth, between the upper inhibitors might be used as additives in toothpastes for canine and first primary molar, and between the lower first reducing the acidification produced by the CO2 hydrase primary and second primary molars, in the region with activity of CAVI, which leads to the formation of protons higher quantity of plaque.34 Children had been asked not to and bicarbonate and may have a role in cariogenesis. eat or drink for 3 h before the measurements. The measure- Despite the lack of association between CA6 gene poly- ment was only performed if the electrode was inserted into morphisms and dental caries, the finding that buffer the biofilm. The electrode was connected to a battery- capacity is associated with a polymorphism in the CA6 gene powered meter, and the circuit completed by having the opens new possibilities for association studies relating gene subject dipping a finger into 3 mol lÀ1 KCL containing the

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reference electrode. The electrode was standardized in pH CA6 gene exon 3 was also PCR amplified with primers 50- 4.0 and 7.0 standard buffers at the start of each subject’s GGA GGT CAG GTG GAG CAG AG -30 (forward); 50-GCC measurements and the response in pH 7.0 buffer checked CTG TCC ATC GAG GAC GC -30 (reverse). PCR was carried again at the end. The electrode tip was dipped in 70% out in a total volume of 25 ml, containing 250 ng of genomic ethanol and the electrode holder wiped with a tissue soaked DNA; PCR Master Mix 2 Â (50 units/ml of Taq DNA in 70% ethanol between subjects, to assure rigorously strict polymerase supplied with reaction buffer (pH 8.5), 400 mM cross-infection control measures. dATP, 400 mM dGTP, 400 mM dCTP, 400 mM dTTP, 3 mM MgCl2, To determine plaque acidogenicity, each subject rinsed his Promega Corporation, Madison, WI, USA); 1 mM of each mouth with a 10% sucrose solution at room temperature for primer (upstream and downstream); nuclease-free water of 2 min, and pH measurements were taken immediately and 25 ml. For exon 2, the reaction was incubated for 5 min at after 5 min. Each measurement took about 30 s, including 95 1C, followed by 35 cycles of 1 min at 95 1C, 1 min at 59 1C, 10–15 s for a stable reading to be obtained. The electrode was and 1 min at 72 1C, with a final extension of 72 1C for 7 min. inserted into the interdental space of the teeth 53/54, 63/64, For exon 3, the solution was incubated for 5 min at 95 1C, 74/75, and 84/85. Thus, the plaque pH variation (DpH) was followed by 35 cycles of 1 min at 95 1C, 1 min at 63 1C, and determined and averages of measures of upper and lower 1 min at 72 1C, with a final extension of 72 1C for 7 min. For teeth were obtained. the analysis of the rs2274327 (C/T) polymorphism in exon 2 of CA6 gene, a 3 ml aliquot of PCR products was mixed with a Buffer capacity 17 ml solution containing 2 ml10Â NE Buffer (50 mM NaCl,

Wax-stimulated whole saliva was collected at the same time 10 mM Tris–HCl, 10 mM MgCl2,1mM dithiothreitol, pH 7.9), of the day, 3 h after breakfast. Buffer capacity of saliva was 0.1 ml BtsCI (20 000 U mlÀ1) (New England Biolabs Inc., measured immediately after collection using the method Beverly, MA, USA) and 14.9 ml sterile deionized H2O. The described by Frostell.35 Briefly, saliva secretion was stimu- solution was incubated at 50 1C for 16 h. For the analysis of lated by the chewing of parafilm (Sigma, St Louis, MO, USA) the rs2274328 (A/C) polymorphism in exon 2 of CA6,a for 3 min. The saliva produced during the first 30 s was 3 ml aliquot of PCR products was mixed with a 17 ml ignored and the remainder was collected. The buffer solution containing 2 ml10Â NE Buffer, 0.2 ml Hpy188I capacity of saliva was determined by adding 1.5 ml of (10 000 U mlÀ1) (New England Biolabs Inc.), and 14.8 ml

5mM HCl to a tube containing 0.5 ml of stimulated saliva. sterile deionized H2O. The solution was incubated at 37 1C The tube was shaken and opened to release the CO2 for 16 h. For the analysis of the polymorphism in exon 3 of dissolved in the saliva, and pH was determined after 5 min. CA6 gene, a 3 ml aliquot of PCR products was mixed with a 17 ml solution containing 2 ml10Â NE, 0.2 ml HaeIII Analysis of genetic polymorphisms (10 000 U mlÀ1) (New England Biolabs Inc.), and 14.8 ml

The gene for CA6 is located at chromosome 1, region 1p36.2 sterile deionized H2O. The digest was mixed with 5 mlof (gene ID: 765). Individuals were genotyped for polymorph- loading buffer and electrophoresed on a 10% vertical isms rs2274327 (C/T), rs2274328 (A/C), located at exon 2, polyacrylamide gel. The DNA bands were evidenced by and rs2274333 (A/G) located at exon 3 of the gene for CA6 silver staining.37 (www.ncbi.nih.gov/SNP). These polymorphisms result in an exchange of amino acids Met-Thr (rs2274327), Gly-Ser (rs.2274333), and Leu-Met (rs2274328). For sampling of Analysis of protein structure epithelial buccal cells, the volunteers undertook 5 ml of 3% The effect of the genetic polymorphisms on the secondary glucose mouthwash for 2 min and the oral mucosa was structure of the CA6 gene was analyzed using the PSIPRED scraped with a sterile wooden spatula. The tip of the spatula secondary structure prediction software (http://bioinf.cs. was then shacked into the retained mouthwash solution. ucl.ac.uk/psipred/).38 Oral epithelial cells were pelleted by centrifugation at 2000 r.p.m. for 10 min. The supernatant was discarded and the cell pellet was resuspended in 500 ml extraction buffer Statistical analysis (10 mM Tris–HCl, pH 7.8, 5 mM EDTA, 0.5% SDS). The The results were statistically analyzed at a significance level 39 samples were then frozen at À20 1C until used for DNA of 5% by using the SAS procedure Proc NLMIXED software. extraction. After defrost, samples were incubated overnight A two-tailed Student’s test was used to determine the with 100 ng mlÀ1 proteinase K (Sigma Chemical Co ) at 37 1C significance of caries group differences. When the variances with agitation. DNA was then purified by sequential of the used data were nonparametric, Mann—Whitney two- 2 ammonium acetate.36 DNA was dissolved in 70 ml TE buffer tailed test was used. The w test was used to test the (10 mM Tris (pH 7.8), 1 mM EDTA). The concentration was differences in genotype frequencies between the groups with and without caries. estimated by measurements of OD260. The primers were synthesized by the IDT (Integrated DNA Technologies, Coralville, IA, USA). A fragment of 200 bp of the CA6 gene exon 2 was PCR amplified with primers 50-TGT Conflict of interest CTT AGA AGG GGC ACT GG-30 (forward); 50-CCT TCC TCT TAC CTG TGT GG-30 (reverse). A fragment of 202 bp of the The authors declare no conflict of interest.

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