ORIGINAL ARTICLE

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,LS Mofatto2, KL Cortellazzi3,MCLG Santos2, MN Santos1,CC Bergamaschi4 and SRP Line2

1Department of Pediatric Dentistry, PiracicabaDental School, University of Campinas-Unicamp,Sao Paulo, Brazil; 2Department of Morphology,Piracicaba Dental School, University ofCampinas-Unicamp, Sao Paulo, Brazil;3Department of Preventive Dentistry and PublicHealth, Piracicaba Dental School, University ofCampinas-Unicamp, Sao Paulo, Brazil and4Department of Physiological Sciences,Piracicaba Dental School, University ofCampinas-Unicamp, Sao Paulo, Brazil

Correspondence:Dr RCR Peres, Department of PediatricDentistry, Piracicaba Dental School, Universityof Campinas (UNICAMP), Av. Limeira, 901. CP52, 13414-903 Piracicaba, Sao Paulo, Brazil.E-mail: rochaperes@fop.unicamp.br

Received 4 March 2009; revised 5 May 2009;accepted 27 July 2009; published online1 September 2009

Carbonic anhydrase VI is a secreted enzyme that catalyzes the hydration ofcarbon hydroxide in saliva and other body fluids. This enzyme has beenimplicated in taste and gastrointestinal dysfunctions, tooth erosion, andcaries. The purpose of this study was to analyze the allele and genotypedistribution of three polymorphisms in the coding sequences of (CA6) geneand check for possible associations with salivary buffer capacity, number ofdecayed, missing, and filled teeth in deciduous and permanent teeth (dmft/DMFT, Decayed/Missing/Filled Teeth), plaque index (PI), and the plaque pHvariation (DpH) in children aged 7–9 years. Two hundred and forty-fivechildren from both genders, residents in area with fluoridated water(Piracicaba, Sao Paulo, Brazil) were divided into two groups: caries free andwith caries. The clinical examinations were conducted by a single previouslycalibrated examiner (k¼0.91) in an outdoor setting using a mirror and aprobe, according to WHO criteria index (dmft/DMFT). Approximately 2 hafter the first daily meal, the buffer capacity (BC) and the plaque pH wereanalyzed by means of a pH meter and an ion selective electrode. Plaque pHwas measured immediately and 5 min after a mouth rinse with a 10% sucrosesolution. The data were submitted to w2, Student’s, and Mann–Whitney tests(a¼0.05). The PI and DpH of the upper and lower teeth were significantlyhigher in the carious group than control (Po0.05). There was no differencebetween the groups in relation to BC. There was no association between thealleles and genotypes distributions for polymorphisms in the CA6 gene exons2 and 3 and caries experience (P40.05). There was a positive associationbetween buffer capacity and the rs2274327 (C/T) polymorphism. The alleleT and genotype TT were significantly less frequent in individuals with thehighest buffer capacity (P¼0.023 and 0.045, respectively). This findingencourages future studies relating CA6 gene polymorphisms and theirassociation with malfunctions, such as taste and gastrointestinal alterations,or the differential effect of chemical modulators on the protein productsoriginated 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

The Pharmacogenomics Journal (2010) 10, 114–119& 2010 Nature Publishing Group All rights reserved 1470-269X/10 $32.00

www.nature.com/tpj

Introduction

Carbonic anhydrases (CAs) are a class of enzymes that havethe function of maintaining the pH homeostasis in bodytissues and fluids by catalyzing the hydration of carbonhydroxide in the reaction CO2þH2O3HCO3

�þHþ .Carbonic anhydrase VI (CAVI) is the only secreted form ofCA. This enzyme was first described and characterized insaliva, in which it is produced by the serous acinar cells ofthe parotid and submandibular glands.1,2 CAVI was shownto be expressed in the lacrimal glands,3 lowers airways andlungs of rats,4 in the mouse nasal gland,5 in the lingualserous glands of von Ebner in humans,6 in human and ratmammary gland and present in milk,7 as well as in thelining epithelial cells of large intestine, stomach, andesophagus.8

The importance of CAVI is evidenced by its involvementin several malfunctions. Decreased secretion of salivaryCAVI has been associated with loss of taste (hypogeusia) andsmell (hyposmia) and distorted taste (dysgeusia) and smell(dysosmia).9 Salivary bicarbonate secretion is also known tobe important in the maintenance of esophageal pH home-ostasis.10,11 Additionally, Parkkila et al. 12 showed thatsalivary CAVI is present in the gastric mucus, in which itcan contribute to maintain the pH gradient on the surfaceepithelial cells, thus protecting from gastric ulcers.

Salivary factors are considered important for dentalhealth, as rampant caries is seen in patients with highsalivary hypofunction.13 In this context, the salivary buffercapacity is a factor of primary importance in maintainingoral homeostasis. The bicarbonate system is the main bufferthat contributes to the total buffer capacity of saliva.14

Bicarbonate ions can neutralize lactic and acetic acidsproduced by plaque bacteria and reduce demineralization.CAVI can adsorbs to the crystals of hydroxyapatite ofenamel;15 it is present in the enamel pellicle16 and in dentalbiofilm.17 Salivary buffer capacity seems to be a determinantfactor on dental erosion, which is becoming a major dentalproblem in both children and adults.18 Low CAVI concen-trations in the saliva were associated with the prevalence ofdental caries, especially in individuals with poor oralhygiene.19 Experimental evidences indicate that CAVI insaliva penetrates plaque and facilitates acid neutralizationby salivary bicarbonate.17 Although CAVI was originallypredicted to regulate salivary pH or buffer capacity,20,21

some studies indicate that these variables are not directlyassociated with CAVI concentration in saliva.22,23 Therefore,it is possible that besides concentration, CAVI activity insaliva can also be influenced by other variables such as by

genetic polymorphisms found in the coding sequences thatmay modulate the activity of this enzyme. Gene poly-morphisms are a mechanism by which individuals mayexhibit variations within the range of what is consideredbiologically normal. Single nucleotide polymorphisms occurat a high frequency in human genome and can affect genefunction. Polymorphisms in the exon sequences of CA6have not been associated with enzyme activity or diseases.

The purpose of this study was to analyze the distributionof three polymorphisms in the coding sequences of CA6gene, which alters the amino-acid sequence of this enzyme,and checks for possible association of these polymorphismswith salivary buffer capacity, dmft/DMFT (Decayed/Missing/Filled Teeth) index, plaque index (PI), and variation in thedental plaque pH in children aged 7–9 years.

Results

The characteristics of the sample (age, gender, dmft/DMFT)are presented in Table 1. PI and pH and salivary buffercapacity are presented in Tables 2 and 3, respectively. The PIand DpH of the upper and lower teeth were significantlyhigher in the caries than control group (Po0.05). There wasno difference between the groups in relation to buffercapacity. There was no association of alleles and genotypeswith caries experience for polymorphisms in the CAVI geneexons 2 and 3 (P40.05). The genotype distribution for thethree polymorphisms studied is shown in Table 4. A positiveassociation between buffer capacity and the rs2274327 (C/T)polymorphism was observed when the 245 individuals weredivided into three groups containing the 1/3 highest (pH8.0–7.45, 82 individuals), 1/3 intermediate (pH 7.24–6.78, 81individuals), and the 1/3 lowest (6.77–3.77, 81 individuals)

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).

Table 2 Mean and s.d. of plaque index and pH in caries andcaries-free children

Variables Caries Caries free P-value

Mean s.d. Mean s.d.

PI 1.19 0.37 1.06 0.35 0.004D1 1.19 0.42 0.93 0.43 o0.0001D2 0.97 0.39 0.73 0.33 o0.0001

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

lower arch—resting pH and 5 min after 2 min mouthrinse of 10% sucrose);

P-values derived from Student’s t-test.

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buffer capacity. The allele T and genotype TT were signifi-cantly less frequent in individuals in the group with thehighest buffer capacity (P¼ 0.002 and 0.046, respectively,Table 5). No significant association was observed when thesethree groups were analyzed in regard to DpH (P40.05, not

shown). Analysis with the PSIPRED software showedthat the two alleles had a local effect on the secondarystructure of CAVI, giving rise to distinct protein conforma-tions (Figure 1). It is worth mentioning that the populationanalyzed in this study was composed of 213 Caucasians and32 Black individuals. Statistical analysis excluding specificraces did not change significantly from the one resultsobtained with the total population (that is Caucasians andBlacks).

Discussion

Gene polymorphisms are a mechanism by which individualsmay exhibit variations within the range of what isconsidered biologically normal. Single nucleotide poly-morphisms occur at a high frequency in human genomeand can affect gene function. Polymorphisms in the codingsequences of CA6 gene have neither been studied earlier norhave they been associated with disorders. It is known thatsalivary CAVI, the only secreted isoenzyme of the CAenzyme family, is one of the major protein constituents inhuman parotid saliva.24 Polymorphisms in exons 2 and 3were selected because they produce changes in the amino-acid sequence in the secreted protein (www.ncbi.nih.gov/SNP) and have, therefore, a potential to interfere withthe function of the enzyme. In fact, our results showed thatthe rs2274327 (C/T) polymorphism is associated withsalivary buffer capacity. A comparative analysis (http://genome.ucsc.edu/) showed that the C allele, whose codoncorresponds to the amino acid, Thr, is the ancestral form, asit is present in Chimp, Macaque, and Orangutan. The Tallele produces the codon Met, and disrupts a highlyconserved short beta sheet between amino acids 50 and52, which is present in the rat, cow, horse, and the threeprimates listed above (not shown). Therefore, it is likely thatthis change will interfere with the function of CAVI.

Table 3 Mean (s.d.) salivary buffer capacity in caries andcaries-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 52.8exon 3 GG 09 6.7 7 5.6 0.82

AG 50 42.0 52 41.6rs2274328 AA 52 43.3 60 48.0exon 2 CC 6 5.0 8 6.4 0.62

AC 62 51.7 57 45.6rs2274327 CC 72 60.0 82 65.6exon 2 TT 15 12.5 12 9.6 0.62

CT 33 27.5 31 24.8

SNP, single nucleotide polymorphism; P-values derived from w2.

Distribution of genotypes in the caries and caries-free groups.

Table 5 Gene polymorphisms of CA6 gene

SNP Genotype Buffer capacity P-value

3.77–6.77 6.78–7.24 7.25–8.0

n % n % n %

rs2274333 AA 43 53.1 43 52.4 41 50.0exon 3 GG 6 7.4 6 7.3 4 4.9 0.915

AG 32 39.5 33 40.3 37 45.1

rs2274328 AA 2 2.5 6 7.3 6 7.3exon 2 CC 42 51.8 33 40.3 37 45.1 0.426

AC 37 45.7 43 52.4 39 47.6

rs2274327 CC 45 55.6 48 58.5 61 74.4exon 2 TT 12 14.8 12 14.6 3 3.6 0.046

CT 24 29.6 22 26.9 18 22.0AlleleC 114 70.4 118 72.0 140 85.4 0.002T 48 29.6 46 28.0 24 14.6

SNP, single nucleotide polymorphism. Allele frequencies for rs2274327 were also

included as significant differences (Po0.05) were observed in genotype

distributions. P-values derived from w2.

Distribution of genotypes according to the buffer capacity.

Figure 1 PSIPRED prediction of protein secondary structure of CAVI

region that includes polymorphism rs2274327 (amino acid 55).

(a) Allele T coding for Met (M). (b) Allele C coding for Thr (T). Note

that distinct secondary structures are formed by the two alleles.

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Interesting, the younger T allele is associated with decreasedbuffer capacity. The distribution of CAVI genotypes and allelesin the population studied is similar to the frequencies describedin other human populations (http://www.hapmap.org/).

The etiology of caries involves a combination of factors,such as environmental aspects, diet, infection, and geneticbackground that influence the chemical and physicalproperties of teeth and saliva.25,26 It is worth mentioningthat children had the main meals at school (breakfast, snack,and lunch), which partially reduce the effects of variationsin diet composition. It is known that bicarbonate in salivadiffuses into dental plaque and combines with Hþ to formcarbonic acid. CAVI presence contributes to the neutraliza-tion of plaque acid, mainly in stimulated saliva whosebuffering is mainly performed by bicarbonate.14 Salivacontrols the pH of dental plaque after exposure tofermentable carbohydrate, and thus helps to prevent dentalcaries. When plaque pH was measured before and after asucrose rinse (DpH), the children with caries had a higher pHfall when compared with caries free; however, there was nodifference between these two groups regarding salivarybuffer capacity. This indicates that the salivary buffercapacity may not be the only factor that controls plaquepH. The size of plaque and the amount of cariogenicbacterial cells present in plaque are probable factors thatalso influence the DpH. In fact, PI was significantly higher incaries group. The effect of buffer capacity on cariesdevelopment is still a matter of debate. The results of thiswork were in accordance with the majority of the recentstudies reporting that buffering capacity values are notrelated to dental caries.27–29 Although it seems logical thatsalivary buffer capacity is a contributing factor on enameldemineralization, its effect may be overshadowed by severalother factors that contribute to caries development such asoral hygiene, presence of cariogenic bacterial strains, sugarconsumption, and exposure of dental surfaces to fluoride.This rationale also explains the lack of associationbetween CA6 polymorphisms and dental caries observed inthis study.

It is also plausible to assume that besides caries, CAVI mayparticipate in the homeostasis of oral mucosa. In fact,periodontal disease, which occurs mainly in adults, wasassociated with salivary pH.30 The implication of CAVI intaste and gastrointestinal dysfunctions has raised theinterest in the development of drugs that modulate thefunction of CAVI. Several clinically used compounds, suchas acetazolamide, methazolamide, ethoxzolamide, dichlor-ophenamide, dorzolamide, brinzolamide, topiramate, sul-piride, indisulam, and benzolamide showed effective CAVIinhibitory activity.31 These authors proposed that CAVIinhibitors might be used as additives in toothpastes forreducing the acidification produced by the CO2 hydraseactivity of CAVI, which leads to the formation of protonsand bicarbonate and may have a role in cariogenesis.Despite the lack of association between CA6 gene poly-morphisms and dental caries, the finding that buffercapacity is associated with a polymorphism in the CA6 geneopens new possibilities for association studies relating gene

polymorphisms and malfunctions, such as taste, gastro-intestinal alterations, and periodontitis or the differentialeffect of chemical modulators on the protein productsoriginated from the distinct genotypes of CA6 gene.

Materials and methods

Study population

A convenience sample of 245 schoolchildren (aged 7–9years) was selected from a group of the city of Piracicaba, SaoPaulo, Brazil. The inclusion criteria were lack of systemicdiseases, severe fluorosis, hypoplasia, orthodontic braces,use of antimicrobials, and communication or neuromotorimpairments. Schoolchildren were chosen because they hadthe two principal meals and snacks in the school. The mealswere composed of nutrients and food with cariogenicagents. The mean age was 7.84 years (s.d.¼0.81) and theboys/girls ratio in the sample was 51.4%/48.6%. For dmft/DMTF study, children were divided into caries and caries-free groups. The caries-free group (n¼ 120) presentedabsence of frank cavitations. The children for caries group(n¼125) were screened for presence of frank cavitation andpresence of fillings, representing past caries experience.Children were requested to withhold toothbrush and otheroral hygiene measures for 1 day before the examination toencourage plaque accumulation. This study was approvedby the Ethical Committee of Piracicaba Dental School—University of Campinas, Sao Paulo, Brazil (#110/2005).

Clinical examination

Clinical examinations were carried out using standardizedcriteria, after calibration of the examination methodology(k¼0.91). Caries index, expressed as mean dmft/DMFT wasscored as recommended by the WHO report on oral healthsurveys.32 Caries detection was carried out by the samedentist under a natural light source and dental mirror. Oralhygiene level was assessed using the PI described by Silnessand Loe 33 and was scored on the buccal surfaces of primaryor permanent teeth 16 (55), 21 (61), 24 (64), 36(75), 44 (84),and 41 (81), using a mirror and a WHO CPTIN type E probe.The PI was calculated as the mean score for the examinedsurfaces and was used as a measure for the average level ofplaque accumulation.

Dental plaque pH measurement

Plaque pH measurements were made using an iridium oxidetouch electrode 2 mm in length and 0.1 mm in diameter(Beetrode model NMPH1, WPI, Sarasota, FL, USA) insertedinto an interdental space apical to the contact point in eachof the four quadrants in the mouth, between the uppercanine and first primary molar, and between the lower firstprimary and second primary molars, in the region withhigher quantity of plaque.34 Children had been asked not toeat or drink for 3 h before the measurements. The measure-ment was only performed if the electrode was inserted intothe biofilm. The electrode was connected to a battery-powered meter, and the circuit completed by having thesubject dipping a finger into 3 mol l�1 KCL containing the

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reference electrode. The electrode was standardized in pH4.0 and 7.0 standard buffers at the start of each subject’smeasurements and the response in pH 7.0 buffer checkedagain at the end. The electrode tip was dipped in 70%ethanol and the electrode holder wiped with a tissue soakedin 70% ethanol between subjects, to assure rigorously strictcross-infection control measures.

To determine plaque acidogenicity, each subject rinsed hismouth with a 10% sucrose solution at room temperature for2 min, and pH measurements were taken immediately andafter 5 min. Each measurement took about 30 s, including10–15 s for a stable reading to be obtained. The electrode wasinserted into the interdental space of the teeth 53/54, 63/64,74/75, and 84/85. Thus, the plaque pH variation (DpH) wasdetermined and averages of measures of upper and lowerteeth were obtained.

Buffer capacity

Wax-stimulated whole saliva was collected at the same timeof the day, 3 h after breakfast. Buffer capacity of saliva wasmeasured immediately after collection using the methoddescribed by Frostell.35 Briefly, saliva secretion was stimu-lated by the chewing of parafilm (Sigma, St Louis, MO, USA)for 3 min. The saliva produced during the first 30 s wasignored and the remainder was collected. The buffercapacity of saliva was determined by adding 1.5 ml of5 mM HCl to a tube containing 0.5 ml of stimulated saliva.The tube was shaken and opened to release the CO2

dissolved in the saliva, and pH was determined after 5 min.

Analysis of genetic polymorphisms

The gene for CA6 is located at chromosome 1, region 1p36.2(gene ID: 765). Individuals were genotyped for polymorph-isms rs2274327 (C/T), rs2274328 (A/C), located at exon 2,and rs2274333 (A/G) located at exon 3 of the gene for CA6(www.ncbi.nih.gov/SNP). These polymorphisms result in anexchange of amino acids Met-Thr (rs2274327), Gly-Ser(rs.2274333), and Leu-Met (rs2274328). For sampling ofepithelial buccal cells, the volunteers undertook 5 ml of 3%glucose mouthwash for 2 min and the oral mucosa wasscraped with a sterile wooden spatula. The tip of the spatulawas then shacked into the retained mouthwash solution.Oral epithelial cells were pelleted by centrifugation at2000 r.p.m. for 10 min. The supernatant was discarded andthe cell pellet was resuspended in 500 ml extraction buffer(10 mM Tris–HCl, pH 7.8, 5 mM EDTA, 0.5% SDS). Thesamples were then frozen at �20 1C until used for DNAextraction. After defrost, samples were incubated overnightwith 100 ng ml�1 proteinase K (Sigma Chemical Co ) at 37 1Cwith agitation. DNA was then purified by sequentialammonium acetate.36 DNA was dissolved in 70 ml TE buffer(10 mM Tris (pH 7.8), 1 mM EDTA). The concentration wasestimated by measurements of OD260.

The primers were synthesized by the IDT (Integrated DNATechnologies, Coralville, IA, USA). A fragment of 200 bp ofthe CA6 gene exon 2 was PCR amplified with primers 50-TGTCTT AGA AGG GGC ACT GG-30 (forward); 50-CCT TCC TCTTAC CTG TGT GG-30 (reverse). A fragment of 202 bp of the

CA6 gene exon 3 was also PCR amplified with primers 50-GGA GGT CAG GTG GAG CAG AG -30 (forward); 50-GCCCTG TCC ATC GAG GAC GC -30 (reverse). PCR was carriedout in a total volume of 25 ml, containing 250 ng of genomicDNA; PCR Master Mix 2� (50 units/ml of Taq DNApolymerase supplied with reaction buffer (pH 8.5), 400 mM

dATP, 400 mM dGTP, 400 mM dCTP, 400 mM dTTP, 3 mM MgCl2,Promega Corporation, Madison, WI, USA); 1 mM of eachprimer (upstream and downstream); nuclease-free water of25ml. For exon 2, the reaction was incubated for 5 min at95 1C, followed by 35 cycles of 1 min at 95 1C, 1 min at 59 1C,and 1 min at 72 1C, with a final extension of 72 1C for 7 min.For exon 3, the solution was incubated for 5 min at 95 1C,followed by 35 cycles of 1 min at 95 1C, 1 min at 63 1C, and1 min at 72 1C, with a final extension of 72 1C for 7 min. Forthe analysis of the rs2274327 (C/T) polymorphism in exon 2of CA6 gene, a 3 ml aliquot of PCR products was mixed with a17ml solution containing 2 ml 10� NE Buffer (50 mM NaCl,10 mM Tris–HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9),0.1 ml BtsCI (20 000 U ml�1) (New England Biolabs Inc.,Beverly, MA, USA) and 14.9 ml sterile deionized H2O. Thesolution was incubated at 50 1C for 16 h. For the analysis ofthe rs2274328 (A/C) polymorphism in exon 2 of CA6, a3 ml aliquot of PCR products was mixed with a 17 mlsolution containing 2 ml 10� NE Buffer, 0.2ml Hpy188I(10 000 U ml�1) (New England Biolabs Inc.), and 14.8 mlsterile deionized H2O. The solution was incubated at 37 1Cfor 16 h. For the analysis of the polymorphism in exon 3 ofCA6 gene, a 3 ml aliquot of PCR products was mixed with a17ml solution containing 2 ml 10� NE, 0.2 ml HaeIII(10 000 U ml�1) (New England Biolabs Inc.), and 14.8 mlsterile deionized H2O. The digest was mixed with 5ml ofloading buffer and electrophoresed on a 10% verticalpolyacrylamide gel. The DNA bands were evidenced bysilver staining.37

Analysis of protein structure

The effect of the genetic polymorphisms on the secondarystructure of the CA6 gene was analyzed using the PSIPREDsecondary structure prediction software (http://bioinf.cs.ucl.ac.uk/psipred/).38

Statistical analysis

The results were statistically analyzed at a significance levelof 5% by using the SAS procedure Proc NLMIXED software.39

A two-tailed Student’s test was used to determine thesignificance of caries group differences. When the variancesof the used data were nonparametric, Mann—Whitney two-tailed test was used. The w2 test was used to test thedifferences in genotype frequencies between the groups withand without caries.

Conflict of interest

The authors declare no conflict of interest.

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Acknowledgments

This study was supported by CAPES/PRODOC (# 0120/05-7) andFAPESP (# 05/57649-2).

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