paixao-cortes_2011_pax9 and msx1 transcription factor genes in non-syndromic

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PAX9 and MSX1 transcription factor genes in non-syndromic

dental agenesis

Vanessa Rodrigues Paixao-Cortes a,1, Tatiana Braga b,1, Francisco Mauro Salzano a,Karina Mundstock b, Carlos Alberto Mundstock b, Maria Ca tira Bortolini a,* a Departamento de Gene tica, Instituto de Biociencias, Universidade Federal do Rio Grande do Sul,

Caixa Postal 15053, 91501-970 Porto Alegre, RS, BrazilbFaculdade de Odontologia, Universidade Federal do Rio Grande do Sul, Caixa Postal 1118, 90035-003 Porto Alegre, RS, Brazil

1. Introduction

Therole of heterodontyfor themammalianevolutionaryhistory

is well-recognized.1,2 For humans, teeth have also a prominent

relevance to socio-cultural interactions and at an individual

level can represent a bad or good life quality.3,4 Agenesis of one

or more teeth is the most common anomaly observed in the

human craniofacial development.1,3,5–7 Amongst all non-syn-

dromic (familial or sporadic) agenesis conditions detected in

humans, the most common is the absence of third molar(s) – in

average about20% ofthe individuals ina population donot have

at least one third molar. Upper lateral incisors and second

premolar ageneses are also common, being second in frequen-

cies (2.2% and 3.4%, respectively).8–10

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 3 3 7 – 3 4 4

a r t i c l e i n f o

Article history:

Accepted 27 October 2010

Keywords:

PAX9 gene

MSX1 gene

Dental agenesis

Developmental genes

a b s t r a c t

Objective: The molecular variation of paired domain box gene 9 (PAX9) was previously

investigated by ourresearch group and a high degree of evolutionary conservation in coding

and non-coding regions was observed except in exon 3. PAX9 is a transcription factor

importantin tooth development, andwe wanted to verify itsrole in dentalagenesis in detail.

Since dental development is a complex trait we also decided to examine the influence of

another transcription factor, muscle segment homeodomain-homeobox 1 (MSX1) on it.

Design: A total of 360 consecutively ascertained patients seeking orthodontic treatment

were screenedfor tooth agenesis and 33% of them were found to haveit. Thirty-five of those

with agenesis and 15 controls had their DNA studied for PAX9 exons 2, 3, 4 and adjacent

regions (totalof 1476basepairs,bp) aswellas MSX1 exon2 (698 bp).A trio(a proband and her

parents) was also studied.Results: Six polymorphic sites were found, three in PAX9 exon 3 and three in MSX1 exon2.

MSX1 rs1095 derived allele occurred in individuals with agenesis only, and two other

mutations in this gene had been earlier associated with tooth agenesis. Homozygosity

for the PAX9 Ala240Pro mutation was studied in a family (proband and her parents),

suggesting recessive inheritance with variable expressivity for the dental agenesis found.

Conclusion: Common variants located out of the DNA binding domain of the two PAX9 and

MSX1 genes can also be related to tooth agenesis.

# 2010 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: +55 51 3308 9844.E-mail address: [email protected] (M.C. Bortolini).

1 These two authors contributed equally to this work.

a v a i l a b l e a t w w w . s c i e nc e d i r e c t . c o m

journal homepage: http://www.elsevier.com/locate/aob

0003–9969/$ – see front matter # 2010 Elsevier Ltd. All rights reserved.

doi:10.1016/j.archoralbio.2010.10.020

http://dx.doi.org/10.1016/j.archoralbio.2010.10.020

mailto:[email protected]



mailto:[email protected]




Variation in these frequencies between and within conti-

nental human groups has been found. Third molar agenesis

occurrence, for example, increases in a gradient from Sub-

Saharan Africa (2%) to Europe (20%) and Asia (30%).11–21

Polder et al.22, in a meta-analysis, observed that gender

differences can also be found, females being 1.4 times more

susceptible to non-syndromic dental agenesis than males.

Changesin the expressionand/or structure of transcriptionfactors are common genetic causes of absence of one or more

teeth in non-syndromic agenesis. Mutations in the Paired Box

9 (PAX9) andin the muscle segment homeodomain-homeobox

1 (MSX1) transcription factor genes have been linked to failure

in tooth development.23–29 Up to now, 16 and 11 distinct

mutations in the PAX9 and MSX1 genes, respectively, have

been identified in humans (http://www.ncbi.nlm.nih.gov/

omim – OMIM#167416; OMIM#142893), all resulting in dental

agenesis. The majority of these mutations are located in the

PAX9 paired box domain (segment of 381 bp inside exon 2) and

in the MSX1 homeodomain (segment of 180 bp inside exon 2),

both DNA binding regions. The only mutation in the OMIM list

that is not located in a DNA binding domain, considering bothgenes cited here, is a nucleotide substitution in PAX9 exon 4,

which introduces one premature stop codon.25

Pereira et al.30 demonstrated that a common polymor-

phism ( Ala240Pro; rs4904210) in PAX9 exon 3 is probably

functional and could be associated with third molar agenesis

and its different distributions around the world. Their results

are in agreement with a family study that showed that the

derived allele (240Pro) has a significant role in third molar

agenesis.31,32 Pawlowska et al.29 on the other hand, suggested

that two polymorphisms in MSX1 exon 2 untranslated region

(rs8670 and rs12532) were involved with familial and sporadic

agenesis in humans. These results introduced the idea that

regions out of the DNA binding domain of these twotranscription factor genes could also be related to tooth

development.

The present report reviews the influenceof genetic factors in

tooth development and describes our observations of tooth

agenesisinafamilytrioandapilotstudyonasampleofpatients

who received orthodontic treatment at an orthodontic clinic of

the Federal University of Rio Grande do Sul (UFRGS), Porto

Alegre, Brazil. Patients with tooth agenesis were screened for

molecular variation in PAX9 and MSX1 genes.

2. Subjects and methods

2.1. Participants and clinical examination

An initial group of 360 consecutively ascertained patients

who received orthodontic treatment at the UFRGS were

selected. Forty-three of them were Blacks and the remaining

(317s) were Whites. The urban complex formed by Porto

Alegre and neighbouring cities has 3,152,596 inhabitants, 7%

and 88% of whom are classified as Blacks ( pretos, in

Portuguese) and Whites (brancos), respectively (Brazilian

Institute of Geography and Statistics-IBGE, www.ibge.gov.br,

2000 census). InBrazil,skincolour ratherthancloseor remote

ancestry is used to define an equivalent to ‘‘race’’, and in the

present study the word ‘‘Black’’ was employed to refer to

pretos or any person identified or self-identified with another

term that suggests major African ancestry, such as mulato or

pardo. ‘‘White’’ was used to define those who, based on their

physical traits and information, show no admixture with

non-Europeans. One-hundred and fifty eight of them were

males and 202 females. A total of 119 of these 360 patients

presented congenital non-syndromic dental agenesis (ab-

sence ofat least onesecondary tooth,including thirdmolars).Thirty-five of them (all White) accepted to participate in the

genetic investigation. Parents of one proband were also

studied.

Tooth agenesis was characterized by panoramic radio-

graphs and careful examination of their clinical charts.

Medical, birth defects, and family histories were gathered to

identify possible associated anomalies and to differentiate the

non-syndromic from the syndromic agenesis. No other dental

anomalies beyond agenesis were observed. We also randomly

included 15 individuals, without any dental agenesis, as a

control group (all Whites).

2.2. DNA extraction, PAX9/MSX1 PCR amplification andsequencing

Genomic DNA was extracted from saliva using the QIAamp

DNA MiniKit (Qiagen). PAX9 exon 3 (138 bp) and its 5 0and 30

flanking intronic segments (232 bp and 219 bp, respectively)

were amplified using primers and conditions described in

Pereira et al.30 Primers were designed to amplify PAX9 exons 2

(640 bp) and 4 (247 bp) (Table S1, Supplementary Data). With

this approach all PAX9 coding regions were covered (since

exon 1 presents just the initiation code) MSX1 exon 2 (698 bp,

involving both the homeodomainand the untranslatedregion)

was amplified using primers and conditions described in Xuan

et al.33. PCR products were purified using exonuclease I andalkaline phosphatase (Amersham Biosciences). Both DNA

strands were sequenced using ABI Prism BigDye and an ABI

310 Genetic Analyzer.

2.3. Data analysis

Information about the 360 patients was collected and

organized in a database with complete dental description.

The SPSS program (version 16) was used to analyse the data

concerning dental agenesis. Nonparametric tests (distribution

free Kruskal–Wallis and Chi-square) were used to compare

agenesis by gender, age, skin colour (White or Black), tooth

types (third molars, molars, premolars, canine and incisors)and other dental categories (left and right quadrants; upper

and lower arches).

Sequences were aligned and their quality, as well as the

precision of the resulting data, was ascertained using the

PHRED, PHRAP and CONSED program (http://www.genome.-

washington.edu). All chromatograms were visualized and

checked manually to detect possible mutations in the

sequence. Deviations from the reference sequence were

compared with available genome databases (Ensembl –

http://www.ensembl.org/index.html, UCSC Genome Browser

– http://genome.ucsc.edu) and SNP banks (dbSNP http://

www.ncbi.nlm.nih.gov/snp/, Hapmap – http://hapmap.nc-

bi.nlm.nih.gov/).


http://www.ncbi.nlm.nih.gov/omim


http://www.ibge.gov.br/

http://www.genome.washington.edu/


http://www.ensembl.org/index.html

http://genome.ucsc.edu/

http://www.ncbi.nlm.nih.gov/snp/


http://hapmap.ncbi.nlm.nih.gov/






http://genome.ucsc.edu/

http://www.ensembl.org/index.html



http://www.ibge.gov.br/





Allele frequencies were determined by direct counting.

Haplotypes for the PAX9 and MSX1 genes were estimated from

phase-unknown multi-site genotypes using multiple locus

haplotype analysis (MLOCUS).34,35

Observed allele and genotype frequencies of patients andcontrols were compared by Chi-square with a 95% confidence

interval using the SPSS program (version 16).

Genes related to odontogenesis were compiled in the Gene

Ontology website database using the AmiGO browser (http://

amigo.geneontology.org/cgi-bin/amigo/browse.cgi). Associa-

tion between these genes and dental development was tested

using the STRING software (known and predicted protein–

protein interactions – http://string.embl.de/database), assum-

ing the highest confidence value (0.900).

3. Results

3.1. Dental agenesis frequencies

Table 1 shows that a total of 119 (33%) of the 360 patients

presented non-syndromic dental agenesis. Amongst them,

third molar absence was the most common, 66 (55%) of the

individuals showing it in isolation. Twenty-eight (23%)

presented agenesis of other teeth, whilst agenesis of other

dental groups plus third molar was present in 25 (21%). Four

patients had oligodontia (at least six teeth missing).

Fig. 1 illustrates the dental agenesis distribution by tooth

considering the whole dentition. As indicated, third molar is

the most common missing tooth (14%), followed by premolar

(2%) and incisor (1%), whereas the occurrence of canine and

other molar ageneses is much less frequent (0.3% and 0.9%,

respectively). Comparisons between left and right quadrants,or upper and lower arches agenesis showed no significant

statistical differences.

Females presented more tooth agenesis than males when

only upper teeth were considered (9, 10, 11, 12, 13, 14, 15, 16, 8,

7, 6, 5, 4, 3, 2, 1 teeth; Fisher’s exact test p-value = 0.037).

However, removing the upper third molars (18 and 28 teeth) of

the analysis, the significance is lost ( p-value = 0.064). A

significant value between genders was also obtained when

all incisors were compared (9, 10, 8, 7, 25, 26, 24, 23 teeth;

Fisher’s exact test p-value = 0.022) (data not shown).

Whites presentedmore tooth agenesis thanBlackswhen the

upper teeth, left and right quadrant, as well as molar dental

groups were considered separately. However, the significanceof these differences is lost(with exceptionof the rightquadrant)

when third molars are excluded from the analysis (Table S2).

Third molar agenesis frequency differences are expected, since

it is well known that third molarabsence is rare in Sub-Saharan

Africans as compared to Europeans.11–21

3.2. PAX9 and MSX1 gene polymorphisms

Sequences of the DNA binding domain and of other regions of

the MSX1 and PAX9 genes were obtained for 35 patients with

distinct tooth agenesis and respective controls (Table 2). The

following PAX9 and MSX1 nucleotide sequences were submit-

ted to GenBank, IDs: HM213907–HM214140. No mutation wasfound in PAX9 exons 2 and 4. Sequencing revealed, however,

six nucleotide substitutions outside the DNA binding domains

of both genes (PAX9 exon 3: rs12881240, rs4904210; 50 flanking

intronic segment of PAX9 exon 3: rs7143727; untranslated

region of MSX1 exon 2: rs8670, rs1095, rs12532), all recognized

as single-nucleotide polymorphisms in the available data-

bases (Table 2).

Table 1 – Number of individuals with dental agenesis.

Group Number

Total of patients 360

Non-syndromic agenesis patients 119

Dental agenesis class

Hypodontia ( 6 teeth missing) 115

Oligodontia ( 6 teeth missing) 4

Dental agenesis categoriesThird molar 66

Othera 28

Third molar + othera 25

a Agenesis in almost all other teeth were found. Exceptions were

the upper central incisors (numbers 9 and 8) and lower canines (22

and 27; Fig. 1).

Fig. 1 – Dental agenesis distribution by tooth. The universal numbering system, which is adopted by the American Dental

Association, was used to identify each tooth ( http://www.ada.org/ ).

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 3 3 7 – 3 4 4 339

http://amigo.geneontology.org/cgi-bin/amigo/browse.cgi


http://string.embl.de/database

http://string.embl.de/


http://string.embl.de/database





Table 2 – Type of tooth missing and genotypes for 35 patients and 15 controls.

Pacienta Dental Agenesisb PAX9 MSX1

Intronc

rs7143727Exon3rs12881240

Exon3rs4904210

Exon2rs8670

Exon2rs1095

Exon2rs12532

BCA001 1, 16, 17, 20 G/C C/C G/G C/C C/C A/A

BCA002 17, 32 G/G C/T G/G C/C C/C A/A

BCA003 7, 16, 17, 32 G/G C/C C/C C/C C/T A/ABCA004 1, 16, 17, 32 G/G C/T G/G ND ND ND

BCA005 1, 2, 4, 5, 6, 11, 12, 13, 15, 16, 17, 20, 21, 28, 29, 32 G/G C/C G/C C/C C/C A/G

BCA006 1, 2, 4, 5, 14, 15, 16, 17, 18, 19, 20, 21, 26, 28, 29, 30, 31, 32 G/G C/C G/G C/C C/T A/A

BCA007 17 G/G C/C G/C C/C C/T A/A

BCA008 10 G/G T/T G/G C/C C/C A/A

BCA009 5, 12, 17, 20, 29, 32 G/C C/C G/G C/T C/C A/A

BCA010 1, 16, 32 G/G C/T G/C C/T C/C A/A

BCA011 1, 2, 4, 5, 12, 13, 15, 16, 17, 18, 20, 21 G/G C/C G/C C/T C/C A/G

BCA012 1, 7, 10, 16, 17, 23, 24, 31, 32 G/G C/C G/G C/T C/C A/A

BCA013 1, 16, 17, 31, 32 G/G C/T G/G C/T C/C A/G

BCA014 1, 16, 20, 29 G/G T/T G/G C/T C/C A/G


BCA016 20, 29 G/G C/C G/C T/T C/C A/A

BCA017 1, 16 G/G C/T G/C C/C C/C A/A

BCA018 1,16, 17, 32 G/G C/C G/G C/C T/T A/ABCA019 16 G/G C/C G/G C/T C/C A/A

BCA020 7, 10, 32 G/G C/C C/C C/T C/C A/A

BCA021 1, 16 G/G C/T G/C C/C C/C A/G


BCA023 29, 20 G/G C/C G/G C/T C/C A/A

BCA024 29, 20 G/G C/T G/G C/C C/C A/G

BCA025 1, 16, 17, 20 G/G C/T G/G C/T C/C ND

BCA026 14 G/G C/C G/G C/T C/C A/G

BCA027 7, 10 G/G C/C G/C C/C C/C A/G

BCA028 1, 16, 32 ND ND ND ND ND ND

BCA029 32 G/G C/C G/G C/T C/T A/G

BCA030 1,16, 17, 32 G/G C/T G/G C/C C/C A/A

BCA031 1,16, 17, 32 ND ND ND ND ND ND

BCA032 20 G/G C/T G/C C/T C/C A/A

BCA033 5 ND ND ND ND ND NDBCA034 1, 16 G/G C/C G/G C/C C/T A/G

BCA035 7, 10 G/G C/C G/G C/C C/C A/A

Controlsd Dental Agenesisb PAX9 MSX1

Intronc

rs7143727Exon3rs12881240

Exon3rs4904210

Exon2rs8670

Exon2rs1095

Exon2rs12532

BSA001 0 G/G C/T G/C C/C C/C A/G

BSA002 0 G/G C/C G/C C/C C/C G/G

BSA003 0 G/G C/C G/C C/T C/C A/A

BSA004 0 G/G C/T G/C C/T C/C A/G

BSA005 0 G/G C/T G C/C C/C A/A

BSA006 0 ND ND ND C/C C/C A/A

BSA007 0 G/G C/C G C/T C/C A/A

BSA008 0 G/G C/C G C/C C/C A/A

BSA009 0 G/G C/C G C/C C/C G/G

BSA010 0 G/G C/C G C/C C/C A/A


BSA012 0 G/G C/C G ND ND ND

BSA013 0 G/G C/C G C/C C/C G/G

BSA014 0 G/G C/C G C/C C/C A/G


BSA016 0 G/G C/C G C/C C/C A/G

ND: No data. All were classified as White.a BCA: Code for patient identification.b Agenesis was characterized by panoramic radiographs and clinical examination. The universal numbering system, which is adopted by the

American Dental Association, was used to identify each tooth (http://www.ada.org/).c 50 flanking intronic segment of PAX9 exon3.d BSA: Code for control identification.


http://www.ada.org/

http://www.ada.org/



There is no statistical difference between allele and

genotype distributions in patients and controls (Table 3).

Kim et al.27 and Nieminen7 suggested that MSX1 and PAX9

differ in their influence for agenesis of specific teeth. Both

genes affect third molars, but significantly higher frequencies

of agenesis forsecondpremolars andmaxillary first premolars

were found in association with MSX1 mutations as compared

to PAX9 mutations. Whilst, agenesis of the maxillary first and

second molars and mandibular second molars was signifi-

cantly more common in association with PAX9 nucleotide

substitutions. Usually the upper lateral or lower incisors are

also affected. Interestingly, some reports on MSX1 mutations

Table 3 – Genotype and allele frequencies (in %) for the 35 agenesis patients and 15 controls.

Variation PAX9

rs7143727 rs12881240 rs4904210 (Ala240Pro)

Patients Controls p-Valuea Patients Controls p-Valuea Samples Controls p-Valuea

Allele 0.832 0.595 0.139

G 97 100 C 75 75 G 80 73C 3 0 T 25 25 C 20 27

Genotype

GG 94 100 CC 57 90 GG 66 73

GC 6 0 CT 37 10 GC 28 27

CC 0 0 TT 6 0 CC 6 0

Variation MSX1

rs8670 rs1095 rs12532

Patients Controls p-Valuea Patients Controls p-Valuea Patients Controls p-Valuea

Allele 0.582 0.144 0.325

C 76 83 C 89 100 C 89 67

T 24 17 T 11 0 T 11 33

GenotypeCC 55 67 CC 81 100 CC 81 53

CT 42 33 CT 16 0 CT 16 27

TT 3 0 TT 3 0 TT 3 20

a p-Values refer to the genotype comparisons.

Fig. 2 – (a) Panoramic radiograph of II-1 (BCA003). Missing teeth are marked with an T; an impacted tooth is marked with an

arrow; (b) pedigree of the family considered; and (c) chromatogram showing a PAX9 exon 3 reference sequence, the

homozygous mutation for 240Pro (rs4904210) in the patient, the heterozygote mother and the homozygote for 240Pro father

profiles.




describe agenesis of the first permanent molars even in the

presence of second molars. We tested each agenesis dental

category for association with the MSX1 and PAX9 polymorph-

isms, and although the same general tendencies listed above

were found, the values were not significant. These results,

however, should be considered with caution since the sample

sizes used for our case–control comparisons were small.

Multiple locus haplotype analysis showed no linkage disequi-librium between the PAX9 or MSX1 alleles.

Although the case–control results showed no association

with the PAX9 and MSX1 variation, it shouldbe mentioned that

in two individuals, BCA003 (7, 16, 1, 17) and BCA020 (10, 7, 32;

Table S2, Supplementary Data) where the derived allele

(240Pro; PAX9 exon 3) appears in homozygosity, third molar(s),

as well as upper lateral incisor(s), are absent. For BCA003, a

woman with absence of three third molars and one upper

lateral incisor, it was possible to obtain sequences of the PAX9

exon 3 of her parents. Interestingly, her father, who presents

the four third molars missing is also homozygote for the

240Pro allele. The mother, on the other hand, is heterozygote

G/C and does not present missing teeth (Fig. 2). No

homozygotes for the 240Pro allele were found in our control

sample.

4. Discussion

In the present study 33% of the subjects who receivedorthodontic treatment had agenesis of one or more teeth.

Third molar is the tooth with the highest agenesis frequency,

followed by the lower premolars and upper lateral incisors.

Some differences between genders and skin colour groups

were found, but generally they disappear if third molars are

excluded of the analysis.

Sequences of the untranslated MSX1 exon 2 region, and of

the PAX9 exons 2, 3 and 4 were obtained for 35 patients with

distinct dental agenesis. Although no new or previously

described mutations located in the DNA binding domain for

both genes were identified, six substitutions located outside

this domain were found.

Fig. 3 – Fourty-two genes of the dental development network. Image created by STRING (Search Tool for the Retrieval of

Interacting Genes/P Genes/Proteins – http://string.embl.de ) with the required highest confidence score – 0.900. Different

types of lines represent the kind of evidence for the association.






Although the case–control results showed no significant

differences, some findings deserve a comment; for instance,

the MSX1 rs1095 derived allele appeared in agenesis affected

patients only (no mutant allele C was found in controls). This

variant was absent in a sample of Euro-descendents studied

earlier (dbSNP database – http://www.ncbi.nlm.nih.gov/snp/).

The other two MSX1 polymorphisms (rs8670 and rs12532) had

earlier been associated with dental agenesis.29 PAX9 rs7143727derived allele also appeared in agenesis affected individuals

only (no mutant allele T was found in controls). However,

differently from the other substitutions, this variant is located

in a non-coding region (50 flanking intronic segment of PAX9

exon 3).

Anearlierfamilystudyshowedthatthe Ala240Pro (PAX9 exon

3) mutation seemsto produce a recessive pattern of inheritance,

since allhomozygotes forit hadmissingthird molar(s) as wellas

lateral incisor(s).31,32 Additionally, Pereira et al.30 based on

population and protein modelling data, suggested that 240Pro

homozygotes might present a PAX9 protein with a slightly

reduced DNA-binding capacity, which could be specifically

associated to third molar(s) absence. Our data reinforce the roleof the Ala240Pro polymorphism in these situations, but if the

inheritanceisrecessivethereisvariablephenotypeexpressivity,

since the number of missing third molars is different for each

patient. Our results also indicate a possible role of this

polymorphism for lateral incisor development but in this case

other factors may be involved, since one 240Pro homozygote

studied here presents all lateral incisors (the father in Fig. 2).

Finally, it should be stressed that non-syndromic congeni-

tal missing tooth is a complex and heterogeneous trait.7 Fig. 3

shows a network involving 42 teeth development genes,

including the two studied here. Table S3 give details of each

gene of this network, their interconnections, and the wide

range of their functions. In this context, and based in ourresults, MSX1 and PAX9 appear to influence different agenesis

phenotypes, with other known and unknown genes as well as

epigenetic factors having an influence in tooth development.

For instance, nine Ala240Pro G/C heterozygote patients

present third molar agenesis, whilst the trio’s mother and

other four controls with thissame condition showno agenesis

(Table 2 and Fig. 2). These results illustrate the importance of

these other factors in tooth development and agenesis.

5. Conclusion

Our results support an earlier finding that the derived 240Proallele (PAX9 exon 3) is related with third molar agenesis and

that it may have a recessive pattern of inheritance with

variable expressivity. On the other hand, MSX1 rs1095 derived

allele appeared in agenesis affected individuals only. These

results suggest that common variants located out of the DNA

binding domain of these two transcription factor genes can

also be related to tooth agenesis.

Acknowledgement

We would like to thank the patients and controls who made

this study possible.

Funding: Thisresearchwas supportedby Conselho Nacional

de Desenvolvimento Cientıfico e Tecnologico and Fundaca ˜ o de

Amparo a Pesquisa do Estado do Rio Grande do Sul.

Competing interest: None declared.

Ethical approval: Informed consent was obtained from all of

the participants, andthe project wasapprovedby theResearch

and Ethics Committee of the Federal University of Rio Grande

do Sul. In the case of children under 15 years of age, consentwas requested from their parents or from the individual

legally in charge of the child.

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.archoral-

bio.2010.10.020.

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