1

Research Article

WNT10A variants in relation to non-syndromic hypodontia in Eastern Slovak

population

Grejtakova D1, Gabrikova- Dojcakova D1, , Boronova I1, Kyjovska L2,

Hubcejova J1, Fecenkova M1, Zigova M1, Priganc M 1, Bernasovska J1

1Department of Biology, Faculty of Humanities and Natural Sciences, University of

Presov, Slovakia

2Dental Clinic and Orthodontics, Kosice, Slovakia

Correspondence to:

Daniela Grejtakova, PhD., Department of Biology, Faculty of Humanities and Natural

Sciences, University of Presov, Slovakia

E-mail: daniela.grejtakova@gmail.com

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ABSTRACT

Objectives - Non-syndromic hypodontia is a congenital absence of less than six

permanent teeth, with a most common subtype maxillary lateral incisor agenesis

(MLIA). Mutations in many genes have been described in severe tooth agenesis. The

aim of this study is search for the variants in Wingless-type MMTV integration site

family member (WNT10A), Paired box 9 (PAX9) and Axis inhibitor 2 (AXIN2) genes,

thus investigate their potential role in the pathogenesis of non-syndromic hypodontia.

Material and Methods - Clinical examination and panoramic radiograph were

performed in the cohort of 60 unrelated Slovak patients of Caucasian origin with non-

syndromic hypodontia including 37 MLIA cases, and 48 healthy controls. Genomic

DNA was isolated from buccal swabs and Sanger sequencing of WNT10A, PAX9 and

AXIN2 was performed.

Results - We identified altogether 23 single-nucleotide variants, 5 which of were novel.

We have found 3 rare non-synonymous variants in WNT10A (p.Gly165Arg;

p.Gly213Ser; p.Phe228Ile) in 8 patients (13.33%) from 60. Analysis showed potentially

damaging WNT10A variant p.Phe228Ile predominantly occurred only in MLIA patients,

and with dominant form of tooth agenesis (ORdom= 9.841; p=0.045; 95% CI 0.492–

196.701; ORrec= 0.773; p= 1.000; 95% CI 0.015 – 39.877). In addition, WNT10A variant

p.Phe228Ile showed trend to associate with familial non-syndromic hypodontia

(p=0.024; OR=1.20; 95%CI= 0.97–1.48). After Bonferroni correction, these effects

remained with borderline tendencies. Using 3D WNT10A protein model we

demonstrated the variant Phe228Ile changes protein secondary structure. In PAX9 and

AXIN2 common variants were detected.Conclusion - Our findings suggest that

identified WNT10A variant p.Phe228Ile could represent the risk for the inherited non-

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syndromic hypodontia underlying MLIA. However, further study in different

populations is required.

Key words: WNT10A; PAX9; AXIN2; tooth agenesis; MLIA

Introduction

In the modern society, a significant role of teeth is to enhance appearance; facial

appearance is very important in determining an individual’s integration into society, and

teeth also play an essential role in speech and communication. Oral disorders are

associated with considerable pain, anxiety and impaired social functioning (Sheiham,

2001). The congenital absence of one or more permanent teeth is the most common

developmental anomaly in humans with prevalence in the general population from 2.6

% to 11.3 % and is more frequent in women than men (Larmour et al., 2005, Amini et

al., 2012). Tooth agenesis of one to six teeth, excluding the third molars is called

hypodontia. The rare form, oligodontia is the congenital absence of 6 or more

permanent teeth (Figure 1). In case of anodontia all permanent teeth are missing, which

is extremely rare (Schalk-van der Weide et al., 1992). Hypodontia can appear in non-

syndromic form (isolated) when only teeth are affected, or in syndromic form, when it

associates with symptoms (Rieger syndrome, Hypohidrotic Ectodermal Dysplasia,

Witkop syndrome) (Pawlowska et al., 2010, Lee et al., 2012).

Non-syndromic hypodontia can be the result of environmental or hereditary

causes or of their interaction (Swinnen et al., 2008). To date, several studies have

addressed the molecular-genetic basis of non-syndromic hypodontia and showed that

msh homeobox 1 (MSX1), paired homeobox 9 (PAX9), axis inhibitor 2 (AXIN2),

ectodysplasin 1 (EDA), ectodysplasin A receptor (EDAR), EDAR- associated death

domain (EDARADD) and wingless-type MMTV integration site family, member 10A

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(WNT10A) play an important role in early tooth development (Stockton et al., 2000,

Lammi et al., 2004, Mostowska et al., 2006, Kantaputra and Sripathomsawat, 2011,

Arzoo et al., 2014, Bergendal et al., 2011, He et al., 2013, Tao et al., 2006). WNT10A

gene is primarily associated with severe ectodermal dysplasias, such as

odontoonychodermal dysplasia (OODD) and Schöpf-Schulz-Passargesyndrome (SPSS)

(Adaimy et al., 2007, Nawaz et al., 2009, Kantaputra and Sripathomsawat, 2011).

Recent study confirmed that mutations in this gene are present in 56% of patients with

non-syndromic tooth agenesis (van den Boogaard et al., 2012), and are also associated

with maxillary and mandibular molar agenesis as well as maxillary lateral incisor

agenesis (MLIA) (Kantaputra and Sripathomsawat, 2011, Arzoo et al., 2014, Song et

al., 2014, Mostowska et al., 2015). Maxillary lateral incisor agenesis is one of the most

frequent subtypes of tooth agenesis, characterized by the absence of formation of

deciduous or permanent lateral incisors (Alves-Ferreira et al., 2014). Its prevalence

varies across population from 0.8% to 4.5% (Pandey et al., 2013). In spite of progress in

the molecular research, there is a little evidence about susceptibility factors of MLIA

and aetiopathogenesis of non-syndromic hypodontia is still not fully elucidated.

Therefore, we aim in the present study to investigate a cohort of 60 Slovak unrelated

patients with non-syndromic hypodontia, including 37 MLIA cases, for mutations in the

WNT10A, PAX9 and AXIN2 genes and to search for genotype–phenotype association.

Materials and methods

Participants

In this study, buccal swabs from 60 unrelated patients with non-syndromic

hypodontia, including 37 MLIA cases and 48 healthy controls without dental agenesis

were obtained in collaboration with Dental and Orthodontic clinic in Kosice, Slovakia

(Table 1). All patients and controls were Caucasians of Slovak origin. Non-syndromic

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hypodontia was confirmed by panoramic radiographs, personal and family medical

history of the individuals. The selection criteria were agenesis of one to six permanent

teeth, excluding third molars. Extraoral examination was performed to exclude any

other ectodermal abnormalities of the nails, hair, skin and sweat glands as well as

craniofacial malformations, including orofacial clefts. No other defects were observed

in patients.

The study was approved by the Ethics Committee of the Faculty of Humanities

and Natural Sciences at the University of Presov (Slovakia) and was conducted under

the written consent of all participants.

Sequence analysis

Genomic DNA was extracted from buccal swabs using a commercial isolation

kit ReliaPrep™ gDNA Tissue Miniprep System (Promega, Madison, USA), according

to the manufacturer's instructions. Exons, exon/intron junctions and UTRs of WNT10A,

PAX9 and AXIN2 genes were sequenced in genomic DNA. Primers were designed by

Primer 3 software (Table 2). Sanger sequencing was performed from both ends (forward

and reverse reads) by standard conditions using BigDye®Terminator v3.1. Cycle

Sequencing Kit (Applied Biosystems, Foster City, California, USA) and sequencer

3500xL Genetic Analyzer (Life Technologies, Foster City, California, USA).

SeqScape®Software and Sequencing Analysis Software (Life Technologies, Foster

City, California, USA) were used for evaluation of the data. The reference sequences

used for the genes were: NC_000002.11 (WNT10A), NC_000014.9 (PAX9) and

NC_000017.10 (AXIN2), all available in NCBI database.

Statistical analysis

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The SPSS program (v.20) was used to analyse the data. Statistical tests

(Independent samples T-test, Chi-square test, Fisher´s Exact test) were used to compare

agenesis by gender, age, heredity and tooth types in missense variants. For the

WNT10A variants we determined the allele and genotypes frequencies. The data were

analyzed under the recessive and dominant model. Furthermore for association with

tooth agenesis we used Cochran-Armitage trend test and OR (95% CI). P values

<0.0125 were considered to be significant (Bonferroni corrected level).

Bioinformatics analysis

Non-synonymous WNT10A variants were analysed by PolyPhen prediction

program (http://genetics.bwh.harvard.edu/pp) for their potential deleterious effect on

protein function. The 3D model of protein were constructed by I-Tasser v3.0 and

visualized using Swiss-PdbViewer v4.1. Detected amino acid substitutions in protein

model structure were predicted using CFSSP Server (http://www.biogem.org).

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Results

Clinical manifestation

Non-syndromic hypodontia was diagnosed in 60 individuals aged from 8 to 50

years. Clinical manifestations included tooth agenesis of 1 to 6 teeth (average 2 ± 1.01).

Altogether, 115 teeth (76 in maxilla and 60 in mandible) were missing in all patients.

The most common missed tooth was a lateral incisor (65 %). We did not observe

significant differences between genders neither in the number of missing teeth (p=

0.900; Independent samples T-test), nor in the type of teeth (p= 0.533; Chi-Square test).

Sequence analysis

The sequence analysis of WNT10A, PAX9 and AXIN2 genes in 60 patients with

non-syndromic hypodontia revealed 23 single nucleotide polymorphisms (SNPs). We

found 13 SNPs in coding region, of which 8 were synonymous and 5 non-synonymous

(Table 3).

In the WNT10A gene we detected 3 non-synonymous variants. All variants were

localized in exon 3 of the WNT10A gene (Figure 2). Four patients had mutation c.493

G>A (p.Gly165Arg). In the three patients were present MLIA, in one case were

assessed the canine and premolar transposition with lateral incisors agenesis. In one

case we observed bilateral mandibular second premolars agenesis. Further, we detected

variant c.637G>A, that cause the amino acid change p.Gly213Ser in one patient with

bilateral MLIA. Non-synonymous mutation c.682T>A (p.Phe228Ile) was present in

three patients. In all patients with p.Phe228Ile was observed maxillary lateral incisor

agenesis. In one patient we observed microdontia of contralateral maxillary incisor

simultaneously with unilateral maxillary incisor agenesis. None of the patients had a

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combination of two or more of these variants. All detected WNT10A variants are rare

among the world population, with minor allele frequency < 1 %.

In the coding region of PAX9 we detected 3 polymorphisms, 2 were common

non-synonymous variants with minor allele frequency >20% (c.717 C>T, p.His239His;

c.718 G>C, Ala240Pro) and one was novel synonymous variant c.705C>T

(p.Ala235Ala). 8 common SNPs were found in the AXIN2 gene: one non-synonymous

variant (c.149C>T, p.Pro50Ser) and 6 synonymous variants (c.1365A>G, p.Pro455Pro;

c.1387C>T, p.Pro462Pro; c.1531G>A, Thr510Thr; c.1546C>T, p.His515His;

c.2062C>T, p.Leu688Leu; c.2125 G>C, p.Ser708Ser).

The family history of patients with non-syndromic hypodontia showed 18

patients with first or second-degree relatives affected by tooth agenesis, 14 of them had

maxillary lateral incisors agenesis. For the next analyses we excluded the common

variants in the PAX9 and AXIN2 genes. Sequence analysis showed the presence of

WNT10A p.Phe228Ile and p.Gly213Ser variants only in hypodontia patients,

furthermore p.Phe228Ile especially in MLIA cases with no evidence in healthy controls,

whereas the variant p.Gly165Arg was found in hypodontia patients and also in healthy

controls. When genotype frequencies of coding variants of WNT10A in all non-

syndromic patients were analysed, the WNT10A p.Phe228Ile variant was found

associated with familial form of non-syndromic hypodontia (p=0.024; Fisher exact test;

OR=1.20; 95% CI= 0.97 – 1.48), suggesting that the variant c.682T>A with minor allele

frequency 2.5% could be a risk factor for inherited tooth agenesis, predominantly

occured in patients with maxillary lateral incisor agenesis (ptrend= 0.045 ). (p=0.047;Chi-

square; OR 1.27; 95% CI 0.97 – 1.67) (Table 4, Table 5).

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When the allele frequencies of the all WNT10A variants were compared in

patients with non-syndromic hypodontia (n=60) versus healthy controls (n=48), there

were no significant differences observed neither for p.Gly165Arg (pallelic= 0.387; p=

0.260), p.Gly213Ser (pallelic= 1.123; p= 0.369), nor for the variant p.Phe228Ile (pallelic=

0.275; p= 0.116). In MLIA patients the variant p.Phe228Ile were present only in

heterozygote state, significant differences between AT genotype and TT genotype

carriers were observed (p=0.045). For this variant dominant model was stronger

associated with hypodontia of maxillary incisors (ORdominant= 9.841; p= 0.045; 95% CI

0.492 – 196.701), rather than the recessive model (ORrecessive= 0.773; p= 1.000; 95% CI

0.015 – 39.877). Although after Bonferroni correction, these effects were only with

borderline tendencies. For the WNT10A variants p.Gly165Arg and p.Gly213Ser we

didn´t find association with the higher risk of MLIA (ptrend= 0.193; ptrend= 0.251) (Table

5).

Bioinformatics analysis

Analyse by Polyphen2 prediction software revealed two rare sequence

alterations, c.637G>A (p.Gly213Ser), c.682T>A (p.Phe228Ile) in the WNT10A gene

predicted to be damaging or potentially damaging in 4 patients (6.67%), which in all of

them were present maxillary lateral incisor agenesis. Variant c.493 G>A (p.Gly165Arg)

were predicted as benign. By 3D structure modelling, the variant c.682T>A

(p.Phe228Ile) was detected to change the shape of protein (Figure 3). Using CFSSP tool

we observed changes in secondary protein structure in variant c.682T>A (p.Phe228Ile),

helix structure of the WNT10A c.682T allele was changed to the sheet structure in

WNT10A c.682A allele.

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Discussion

Non-syndromic hypodontia is a congenital absence of one to six teeth and could

be the result of environmental and genetic factors (Jumlongras et al., 2004). To date,

more than 300 genes are known to be involved in the process of odontogenesis.

Mutations in the MSX1, PAX9, AXIN and EDA genes have been previously associated

with non-syndromic hypodontia (Stockton et al., 2000, Lammi et al., 2004, Mostowska

et al., 2006, Kantaputra and Sripathomsawat, 2011, Bergendal et al., 2011, van den

Boogaard et al., 2012, Mostowska et al., 2015, Jumlongras et al., 2004, Vastardis et al.,

1996)( However, recent insights suggest the WNT10A gene as a major candidate gene

for non-syndromic hypodontia (He et al., 2013, Abdalla et al., 2014). WNT10A gene is

primarily associated with severe ectodermal dysplasias, but mutations in WNT10A have

been identified in up to 56% of cases with non-syndromic hypodontia and might be

responsible for the permanent tooth agenesis(Adaimy et al., 2007, van den Boogaard et

al., 2012).

Here we report the sequence analysis of the WNT10A, PAX9 and AXIN2 genes

in the cohort of 60 unrelated Caucasian patients from Eastern Slovakia with non-

syndromic hypodontia including 37 MLIA cases and 48 healthy controls.

In coding region of the WNT10A gene we detected three non-synonymous

variants (p.Gly165Arg, p.Gly213Ser; p.Phe228Ile). Among 60 samples in our study, we

found 3 patients with WNT10A p.Phe228Ile variant in heterozygous constitution

resulting in an allele frequency of 2.5% with present of microdontia in one case. Variant

p.Phe228Ile was predicted as potentially damaging and could represent the potential

disorder-causing variants for non-syndromic hypodontia underlying the MLIA, what

was confirmed in the previous studies (Mostowska et al., 2015, Arte et al., 2013). In our

cohort of patients we observed theassociation pattern of p.Phe228Ile variant with

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familial form of non-syndromic hypodontia. Moreover, variant could represent

potentially higher risk for autosomal-dominant maxillary lateral incisor agenesis. We

did not detect this variant among 48 healthy controls. This variant in heterozygous

constitution was already associated in American family with autosomal-dominant tooth

agenesis involving the lateral incisors and premolars, without other manifestations of

ectodermal dysplasia (Kantaputra and Sripathomsawat, 2011). Mostowska et al.

(Mostowska et al., 2015) described variant p.Phe228Ile in heterozygote state in MLIA

patients in Polish population as aetiological mutation underlying maxillary lateral

incisor agenesis with associated dental anomalies including microdontia and

taurodontism. Microdontia of maxillar lateral incisors is part of the MLIA phenotype,

segregating as an autosomal dominant trait with incomplete penetrance(Pinho et al.,

2010). Furthermore, it has been shown that the same frequent p.Phe228Ile is associated

with autosomal dominant and autosomal recessive forms of ectodermal dysplasias and

isolated hypodontia(van den Boogaard et al., 2012, Bohring et al., 2009, Mostowska et

al., 2013),. According Bohring et al. (Bohring et al., 2009) up to 50% of heterozygous

mutations of the WNT10 gene causing ectodermal dysplasia could be manifested in

phenotype partly, in the form of the tooth agenesis.

Another WNT10A probably damaging variant p.Gly213Ser were previously

identified in 6 of 9 unrelated Thai patients, associated with agenesis of the rare

maxillary permanent canines with autosomal-dominant inheritance, also with present of

peg-shaped (microdontia of the) maxillary lateral incisors with dens invaginatus

(Kantaputra et al., 2014). We detected variant p.Gly213Ser in one patient with bilateral

maxillar lateral incisors agenesis with no agenesis of canines. The variant was present in

patient in heterozygous state and was no present in the healthy controls. We did not

confirm significant association with tooth agenesis in the cohort of Slovak patients in

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comparison with healthy controls, but the most recent studies confirmed the variant

p.Gly213Ser has been shown to be disorder causing (Mostowska et al., 2015, Yang et

al., 2015).

We found WNT10A variant p.Gly165Arg in heterozygous state in 4 patients with

non-syndromic hypodontia, which 3 of them had MLIA and also in one healthy control.

The amino acid change destroys an RGD motif, which represents a binding site for

integrins and causes creation of three consecutive arginines, which has been recognized

as a cytoplasmic signal for retaining proteins in the endoplasmic reticulum (Arte et al.,

2013, Ruoslahti, 1996, Marcello et al., 2010)Variant p.Gly165Arg was predicted as

benign with location in not highly conserved region and has probably no contribution to

the hypodontia phenotype (Bohring et al., 2009, Mostowska et al., 2013, Yang et al.,

2015)

The number of defective WNT10A alleles also correlates with severity of the

tooth agenesis (Yang et al., 2015). In a large study of Sweden families with tooth

agenesis showed that biallelic WNT10A variants were strongly associated with a larger

number of missing teeth when compared to monoallelic WNT10 mutations. The same

study confirmed that WNT10A mutations were associated with absence of maxillary

and mandibular molars as well as mandibular central incisors (Bergendal et al., 2011).

In our study were present only monoallelic WNT10A variants with predominantly

missing maxillary lateral incisors. Genetic contribution of variants to the maxillary

lateral incisor remains largely unknown. Study of familiar aggregation showed the 15-

times higher risk of maxillary lateral incisor agenesis in a relative of an individual with

MLIA (Pinho 2004). Pinho et al. (Pinho et al., 2010) didn´t confirm association between

mutations in MSX1, PAX9 and the in Portuguese patients.

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Common variants were identified in other two sequenced genes for non-

syndromic tooth agenesis, PAX9 and AXIN2. PAX9 polymorphisms c.717C>T

(p.His239His) and c.718G>C (p.Ala240Pro) have already been described in families

with hypodontia and oligodontia (Pawlowska et al., 2010, Mu et al., 2013). Earlier

family studies have shown that p.Ala240Pro polymorphism has a recessive model of

inheritance (Kula et al., 2008). An association of polymorphism p.Ala240Pro with a

crown size of incisor was found in the recent study of the Korean population(Lee et al.,

2012). Mostowska et al. (Mostowska et al., 2006) found the AXIN2 polymorphism

c.2062C>T in the Polish population with tooth agenesis and suggested that transition

c.2062C>T may negatively affect splicing efficiency and the cellular concentration of

AXIN2 and lead to abnormalities in tooth development. Mutations in the AXIN2 are

associated with more sever tooth agenesis called oligodontia and may be an indicator of

specific form of endometrioid ovarian adenocarcinoma(Schwartz et al., 2003). More

recent study excluded the associations of tooth agenesis with colon cancer(Lindor et al.,

2014).

There exist large genotype and phenotype variability behind non-syndromic

hypodontia. The discrepancy in the findings of some authors may be result of diversity

in populations and ethnic differences. Also combination of one of more polymorphisms

in one or more genes can cause decrease of gene expression, changes in secondary

structure of protein and changes in protein-protein interactions and finally lead to

specific phenotype.

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CONCLUSIONS

In conclusion, we provide here the first report of mutational analysis of

candidate genes for non-syndromic hypodontia in Slovak population. Our results

suggest that variant p.Phe228Ile in the WNT10A gene could be involved in the

pathogenesis of inherited form of non-syndromic hypodontia underlying maxillary

lateral incisor agenesis. An understanding the exact function of candidate genes can

significantly contribute to the definition of their role in odontogenesis, which improve

the diagnosis, treatment and prognosis of craniofacial malformations and other related

diseases.

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ACKNOWLEDGMENTS

This publication is the result of the implementation of the projects ITMS 26220120041

and ITMS 26110230100 supported by Research & Development Operational

Programme and LPP-0331-09 funded by Slovak Research and Development Agency.

COMPETING INTERESTS

The authors declare that they have no conflict of interest.

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TABLES

Table 1 Summary of patients with non-syndromic hypodontia

Characteristic Hypodontia patients MLIA patients

n (%) n (%)

Total number of patients 60 (100) 37 (61.67)

Gender Male 30 (50) 17 (45.95)

Female 30 (50) 20 (54.05)

Symetry Unilateral 23 (38.33) 14 (37.83)

Bilateral 37 (61.67) 23 (62.16)

Type of tooth agenesis Incisors (only) 35 31 (83.78)

Premolars (only) 16 –

Combined type 9 6 (16.22)

Other dental anomalies Microdontia 1 (1.67) 1 (1.67)

Tooth transposition 1 (1.67) 1 (1.67)

Persistence of

deciduous teeth

1 (1.67) 1 (1.67)

Tooth retention 1 (1.67) 0 (0.00)

Positive family history 18 (30) 14 (37.84)

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Table 2 Sequences of primers for amplification and sequencing reaction of the PAX9, AXIN2 and

WNT10A genes.

PAX9

Exon (5´→3´) Primer sequence Size (bp) Annealing temperature (°C) 1 F cgctaatatggggaaactgaa

674 60

1 R gcggctaaaaggagcagtc 60

2 F accagcctgattttgctgtc 584

60

2 R agaatgtgagcgcctagtgg 60

3 F ggggacagccccagtagtta 627

60

3 R tgtccctgaggctgcagata 60

4 F ggtctaagccctccagctct 385

60

4 R gaaggatctggctcgtagca 60

5a F gagcattgctggcttactca 976

60

5a R gtcaaaacaccagggagagc 60

5b F tgctacaccctctaatcaaatatgg 991

60

5b R actcacatgctcacacacaca 60

AXIN2

Exon (5´→3´) Primer sequence Size (bp) Annealing temperature (°C) e1 F tcagaactcgggctgagaat

421 60

e1 R gggggctttctttgaagc 60

e2a F agctgggttgcttgatttga 669

60

e2a R tcagctgcttggagacaatg 60

e2b F tgttccgaactttcctggag 661

60

e2b R catggccagcagtcctaact 60

e3 F gttggcgttgagcagattgt 341

60

e3 R cagctgaggatgacagacga 60

e4 F agcaccgatggtatctggag 457

60

e4 R tcacatcactgtgctcacca 60

e5 F ctacacccgaacatgggttt 462

60

e5 R atccacacgcatatgcacac 60

e6 F gcgtgtggatatgtgcttgt 798

60

e6 R tgccgccctcttagaaacta 60

e78 F cccgacttgctgaattgtct 949

60

e78 R tcttctcatgggagggtttg 60

e9 F gggttcgtgtctcttcagga 370

60

e9 R ggacatggatggcaacatct 60

e10 F attgcagccctagtgtttgg 477

60

e10 R ctgctgcttcgttattgctg 60

e11a F cctaggatacctcagtcctcca 837

60

e11a R gcaccaatttctgcatgtgt 60

e11b F cagaggaattatgctttgcact 996

60

e11b R tgggcaaggtaggaaatcaa 60

WNT10A

Exon (5´→3´) Primer sequence Size (bp) Annealing temperature (°C) 1a F aacacatatcccccacacaa

623 60

1a R cggagacacccactctcttg 60

1b F ccccttacccttgagagg 500

60

1b R taccccagcaagagcatc 60

2 F agaagcagaggttggaagagg 494

60

2 R gtgtggggatgggaggat 60

3 F tgggcttcagtttctccttg 571

60

3 R agcaacgtggtcctcagaag 60

4a F cctctgtataatgggagtgggttt 697

60

4a R agagccaaggcgatgctg 60

4b F tggtgctgtttcgtggtct 806

60

4b R acagtggctttgtctgaggag 60

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Table 3 Sequence variations found in the PAX9, AXIN2 and WNT10A genes.

Gene Localization Sequence variation Amino acid

exchange

dbSNP ID n

PAX9 Exon 1

g.99-100insC - rs11373281 44

g.272 C>G - rs4904155 42

g.276 C>T - novel variant 1

Exon 4 g.1431 C>T (c.705 C>T) p.Ala235Ala novel variant 1

g.1443 C>T (c.717 C>T) p.His239His rs12881240 19

g.1444 G>C (c.718 G>C) p.Ala240Pro rs4904210 36

Exon 5 g.2012 C>T - rs17104965 4

g.2181 T>C - novel variant 3

g.2307 G>C - rs72679753 12

g.2471 G>A - novel variant 31

g.2570 C>T - rs11847165 15

g.3017 T>C - novel variant 5

AXIN2 Exon 2 g.241 A>C - rs190687283 1

g.1679 C>T (c.149 C>T) p.Pro50Ser rs2240308 44

Exon 6 g.1679 A>G (c.1365 A>G) p.Pro455Pro rs9915936 57

g.1699 C>T (c.1387 C>T) p.Pro462Pro rs1133683 50

g.1844 G>A (c.1531 G>A) Thr510Thr rs63533624 1

g.1859 C>T (c.1546 C>T) p.His515His rs139316692 1

Exon 7 g.2376 C>T (c.2062 C>T) p.Leu688Leu rs35415678 5

g.2437 G>C (c.2125 G>C) p.Ser708Ser rs143243661 1

WNT10A Exon 3 g.1126 G>A (c.493 G>A) p.Gly165Arg rs77583146 4

g.1270 G>A (c.637 G>A) p.Gly213Ser rs147680216 1

g.1315 T>A (c.682 T>A) p.Phe228Ile rs121908120 3

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Table 4 Association between c.682T>A (p.Phe228Ile) of the WNT10A gene and familial tooth agenesis in

hypodontia group (n=60) and MLIA subgroup (n=37)

Variant Number of probands (n)

Probands with

hypodontia in

family n (%)

Statistical

significance *(p<0.005 )

Odds ratio

(95%

Confidence

interval)

Hy

po

do

nti

a

gro

up

c.682T>A

(p.Phe228Ile)

Mutation positive, n=3 3 (100) 0.024 1.20

( 0.97 – 1.48) Mutation negative, n=57 15(26.32)

Total, n=60 18 (30)

ML

IA

gro

up

c.682T>A

(p.Phe228Ile)

Mutation positive, n=3 3 (100) 0.047 1.27

( 0.97– 1.67) Mutation negative, n=34 11 (32.35 )

Total, n=37 14 (37.84)

* p values were calculated by Fischer exact test

20

Table 5 Association between WNT10A variants and non-syndromic hypodontia.

Variant Genotype

Total (n)/ Frequency

Patients

(n=60)

MLIA

(n=37)

Controls

(n=48)

c.493 G>A

(p.Gly165Arg) Homozygote -mut (AA) 0/0.00 0/0.00 0/0.00

Heterozygote (AG) 4 /0.07 3/0.08 1/0.02

Homozygote (GG)wt 56 /0.93 34/0.92 47/0.98

MAF 0.033 0.041 0.010

*pallelic 0387 0.349

*ptrend 0.260 0.193

c.637 G>A

(p.Gly213Ser) Homozygote -mut (AA) 0/0 0/0 0/0.00

Heterozygote (GA) 1 /0.02 1/ 0.03 0/0.00

Homozygote (GG)wt 59 /0.98 36/0.97 48/1.00

MAF 0.008 0.014 0.000

*pallelic 1.230 0.998

*ptrend 0.369 0.252

c.682 T>A

(p.Phe228Ile) Homozygote -mut(AA) 0/0.00 0/0.00 0/0.00

Heterozygote (AT) 3 /0.05 3/0.08 0/0.00

Homozygote (TT) wt 57 /0.95 34/0.92 48/1.00

MAF 0.025 0.045 0/0.000

*pallelic 0.275 0.152

*ptrend 0.116 0.045

* p values were calculated by Chi-square test and Cochran–Armitage test for trend

21

Figures

Figure 1 Panoramic radiograph of patients with non-syndromic missing tooth (missing teeth are marked

with cross): (A) 19 years old male patient with non-syndromic hypodontia; (B) 6 years old female patient

with several form of non-syndormic oligodontia

22

Figure 2 DNA sequencing chromatogram (SeqScape®Software) showing the non-synonymous SNPs in

the WNT10A gene through comparison with healthy controls: c.493G>A (p.Gly165Arg); c.637G>A

(p.Gly213Ser); c.682T>A (p.Phe228Ile).

23

Figure 3 Visualization of the amino acid changes in WNT10A chain in a protein model. Close-up view of

the 3D structure of protein (FASTA accession number: wingless-type MMTV integration site family,

member 10A, NP_079492.02). Comparison of (A) normal and (B) mutated c.682T>A (p.Phe228Ile)

predicted structures.

24

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Received 15 June 2017; revised 24 January 2018; accepted 16 April 2018