A novel PAX9 mutation causing oligodontia

Accepted Manuscript Title: A novel PAX9 mutation causing oligodontia Authors: Eiman Mohammed Daw, Christian Saliba, Godfrey Grech, Simon Camilleri PII: DOI: Reference:

S0003-9969(17)30300-X https://doi.org/10.1016/j.archoralbio.2017.09.018 AOB 3997

To appear in:

Archives of Oral Biology

Received date: Revised date: Accepted date:

28-10-2016 17-8-2017 24-9-2017

Please cite this article as: Daw Eiman Mohammed, Saliba Christian, Grech Godfrey, Camilleri Simon.A novel PAX9 mutation causing oligodontia.Archives of Oral Biology https://doi.org/10.1016/j.archoralbio.2017.09.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A novel PAX9 mutation causing oligodontia

Eiman Mohammed Daw BDS, MSca Christian Saliba PhDa Godfrey Grech PhDa Simon Camilleri PhD MOrth FDSa* *Corresponding author, [email protected] aDepartment

of Physiology and Biochemistry, Medical School, University of Malta, Tal-

Qroqq, Msida, Malta MSD2020

Highlights

An extended family exhibiting both hypodontia and oligodontia is described Mutations were found in MSX1 and PAX9 genes The PAX9 mutation is novel and associated solely with the oligodontia phenotype

Abstract: Introduction. An extended family presenting with several members affected by developmentally missing teeth was investigated by analysis of the MSX1 and PAX9 genes. Materials and methods. Saliva samples were collected and DNA extracted. Primers were designed to span the exons and intron-exon junctions of the MSX1 and PAX9 genes. These primers were optimised using gradient Polymerase Chain Reaction. The amplified fragments were sent for Sanger sequencing Results. a novel heterozygote missense mutation in exon 3 of PAX9 (c.296G>C, p.A99P), was found in two severely affected members of the family as well as a potentially pathogenic heterozygote variant (c.119C>G, p.A40G) in exon 1 of the MSX1 gene. Conclusion. The PAX9 A99P mutation is in the DNA binding domain and is predicted to be pathogenic.

Keywords Family, Oligodontia, Hypodontia, Mutation, PAX9, MSX1

Introduction: A tooth is defined as developmentally missing if it has never erupted in the oral cavity and is not visible in a radiograph (Bailleul-Forestier, Molla, Verloes, & Berdal, 2008). Agenesis of teeth is one of the commonest developmental anomalies seen in human populations. Failure of development of up to five permanent teeth (not including third molars) is termed hypodontia, six or more missing teeth is known as oligodontia, while total agenesis of teeth is called anodontia (Polder et al 2004). Permanent tooth agenesis differs by continent and gender; the prevalence for both sexes is higher in Europe and Australia than for North American Caucasians and a sex bias exists,

with females affected 1.37 times more than males (Polder, Van't Hof, van der Linden, & Kuijpers-Jagtman, 2004). The second mandibular premolar is the most affected tooth, followed by the lateral maxillary incisor and the second maxillary premolar in Caucasians (Grahnen, 1956). Environmental factors such as irradiation, chemotherapeutic agents or dioxins may arrest tooth development (Alaluusua et al., 2004; Nasman, Forsberg, & Dahllof, 1997). However, most cases are caused by genetic factors as evidenced by association with syndromes, familial clustering and a higher concordance in monozygotic than in dizygotic twins. Family studies show that, as an isolated form, both hypodontia and oligodontia may be inherited as an autosomal dominant trait with incomplete penetrance and variable expression (Burzynski & Escobar, 1983). While no genes have yet been identified as causing nonsyndromic hypodontia, a number have been associated with nonsyndromic oligodontia, i.e. MSX1, PAX9, AXIN2, EDA and WNT10A (Vastardis, Karimbux, Guthua, Seidman, & Seidman, 1996; Stockton, Das, Goldenberg, D'Souza, & Patel, 2000; Lammi et al., 2004; van den Boogaard et al., 2012). In this article, we describe a novel mutation of PAX9 in an extended family, exhibiting both hypodontia and oligodontia.

Materials and Methods: Subjects A twelve year old girl was referred to the Child Dental Health and Orthodontics clinic at Mater Dei Hospital, Malta for investigation and treatment of multiple missing teeth. She gave no history of previous extractions of any sort. A Dental Panoramic Tomogram (DPT) (Fig.1) showed agenesis of all molar teeth and of the lower central incisors. A family history revealed missing teeth in relatives, with the father, an aunt (Fig. 2) and a father’s maternal

uncle also affected. Arrangements were made for the extended family (Fig. 3) to be examined. The study was approved by the University of Malta Research Ethics Committee (Ref 09/2012). Informed consent was obtained. The phenotype was determined by visual and oral examination, supplemented by available radiographs. Inquiries as to a family history of skin, hair, nail or gastrointestinal problems were all answered in the negative. Based on the phenotype, the genes PAX9 and MSX1 were selected for analysis. Mutational analysis Two millilitres of saliva was collected under supervision from the affected and unaffected family members (Fig. 3) in Oragene•DNA self-collection kits (OG-500; DNA Genotek, Ontario, CA) and stored at room temperature until analysed. Genomic DNA was extracted and purified from saliva by using the purifier kit supplied by the manufacturer.

Polymerase Chain Reaction Primer Design: Primer

Sequence 5’ to 3’

Size (bp)

Fragment Size (bp)

PAX9 Exon 2

PAX9 Exon 3

PAX9 Exon 5

Forward - GCC CTC TCG CCT CCT CCT CC

20

Reverse - CCT CCC TCC CTC CCT TGC CC

20

Forward - CGG CAC GGC AGG ATT GAG GG

20

Reverse - GCC GAT GCC CAG GAT GTC GG

20

Forward - AGT AGA GTC AGA GCA TTG CTG GCT 24 Reverse - GGC GAC ACT TGG GCT GGG G

MSX1 Exon 1 Forward - CAG TGC TGC GGC AGA AGG GG Reverse - TCT GCA TCC ACG GGG TCC TC

20

200

476

20 20

2a

20

MSX1 Exon Forward - CAG CCT GCA CCC TCC GCA AA

20

2b

20

Reverse - GCC GCC GAG AGG GAA GGA GA

827

20

MSX1 Exon Forward - TGG AGC GCA AGT TCC GCC AG Reverse - GCC GCC GAG AGG GAA GGA GA

1571

218

248

Table 1. PAX9 and MSX1 primer sequences and their respective PCR details

The PAX9 and MSX1 exons with mutations associated with hypodontia were targeted. Primers were designed to cover the exons and intron-exon junctions of exons 2, 3 and 5 of PAX9

and

exons

1

and

2

of

MSX1

with

the

use

(http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi).

of No

Primer

3

plus

analysis

was

carried out for PAX9 exons 1 and 4. An in silico PCR was performed using BiSearch Polymerase Chain Reaction (http://bisearch.enzim.hu) to ensure specificity of the primers. Primers were synthesised by Integrated DNA Technologies (Belgium). PCR: The Polymerase Chain Reaction (PCR) mix consisted of (1X) buffer, 2mM MgCl2, 200µM dNTPs, 0.1µM forward and 0.1µM reverse primer, 0.036U/µl Taq polymerase, 18.07µl DNAase free water and 1µl (2ng/µl) of genomic DNA sample. The final volume of the mixture was 25µl per reaction. A positive and a negative control were also included with each batch of samples.

PCR thermal profile: The PCR tubes were placed in a TProfessional thermocycler (Biometra), using an optimised thermal profile. The initial denaturation step, carried out at a temperature of 95°C for 5 minutes was followed by 35 cycles of a denaturation step at a temperature of 95°C for 30 seconds, an annealing step of 61°C for 30 seconds and an extension step at a temperature of 72°C for 1 minute. Once the cycles were complete, a final extension step of 7 minutes was carried out at a temperature of 72°C. The PCR products were then stored at 4°C. Agarose gel electrophoresis:

A 1.5% agarose gel pre stained with ethidium bromide was used. Ten μl of each of the PCR products were pipetted directly into separate wells in the gel. Three µl of 100bp DNA ladder were loaded into the first well of each row to act as a reference marker used for comparison with the PCR product to ensure that the length of the fragment obtained was the one expected. Electrophoresis was carried out in 1x Tris-acetate EDTA (TAE), pH 7.3 at a voltage of 80 volts and a current of 400 milliamperes for 30 minutes. The gel was then illuminated using ultraviolet light and photographed. PCR product purification: PCR products to be used for DNA sequencing were purified using AccuPrep® PCR Purification Kit (Bioneer Ltd, Canada). DNA Sequencing and sequencing analysis: The purified PCR products were sent for Sanger sequencing with their corresponding forward and reverse primers. All samples were sent to McGill University and Génome Québec Innovation Centre, Canada. The resultant chromatograms were viewed using Genious (Biomatters Ltd, New Zealand) in order to detect variants. Exonic variants were analysed using SIFT (http://sift.jcvi.org) (Kumar,

Heinikoff

&

Ng

2009),

PolyPhen2

(http://genetics.bwh.harvard.edu/pph2/index.shtml) (Adzhubei et al., 2010) and Mutation Taster (http://www.mutationtaster.org) (Schwarz, Cooper, Schuelke, & Seelow, 2014) in order to estimate the pathogenicity of the detected variant. The variants were checked against dbSNP, the 1000 Genomes database and an in-house database of the allele frequencies of 50 Maltese subjects. Results:

The proband, III:1 (Fig. 3) exhibited absence of all molar teeth and lower central incisors. Her father, II:2, had several missing molar teeth (17, 27, 37, 46 and 47) and his upper lateral incisors (12, 22) were diminutive and peg shaped. He denied ever having had any teeth extracted and this was independently corroborated by his mother. The proband’s aunt, II:4, had developmentally missing upper lateral incisors as did his father’s first cousin II:7. The father’s uncle II:3 was partially edentulous and gave a history of retained deciduous teeth, extracted in adulthood, but was not certain which. The proband’s grandfather was deceased, however her grandmother was unaware of any dental problems as he had long been edentulous, as had she. Sequencing results of PAX9 exon 3 illustrated a novel missense heterozygote mutation, with a guanine (G) to cytosine (C) substitution in codon 99 (c.295G>C, p.A99P) (Fig. 4) This results in the amino acid alanine [GCC] being replaced by proline [CCC] in two cases, the father (II:2) and daughter (III:1). SIFT gave a score of 0 and predicted this to be ‘damaging’. PolyPhen2 gave a score of 1.000, ‘probably damaging’ and Mutation Taster analysis showed this to be ‘disease causing’. This variant was not represented in dbSNP, 1000Genomes, in any other family member, or in the alleles of 50 Maltese individuals.

Species Mutation H sapiens P troglodytes M mulatta M musculus G gallus D rerio D melanogaster C elegans X tropicalis

Match

identical identical identical identical identical identical identical identical

Amino acid 99 99 99 99 99 99 99 104 156 99

Alignment RTYKQRDPGIFPWEIRDRLLADG RTYKQRDPGIFAWEIRDRLLADG RTYKQRDPGIFAWEIRDRLLADG RTYKQRDPGIFAWEIRDRLLADG RTYKQRDPGIFAWEIRDRLLADG RTYKQRDPGIFAWEIRDRLLADG RTYKQRDPGIFAWEIRDRLLADG RELKQRDPGIFAWEIRDRLLSEG RSLKRSDPGIFAWEIRDRLISAD RTYKQRDPGIFAWEIRDRLLADG

Table 2. Phylogenetic table showing the PAX9 A99P mutation to be in a highly conserved area of the paired domain (adapted from Mutation Taster).

Sequencing of MSX1 exon 1 indicated a heterozygote alanine [GCA] to glycine [GGA] substitution at codon 40 (c.119C>G) in several members of the family, I:3, II:2, II:4 and II:6. The SIFT score was 0.66 and was predicted to be ‘tolerated’. Polyphen gave a score of 0.016 with a prediction of ‘benign’ and Mutation Taster classified this as a ‘polymorphism’. Discussion: The genes PAX9 and MSX1 were chosen for analysis, based on the phenotypes of the family and the presence of purely dental anomalies. The expectation was that one gene would be responsible for the hypodontia and oligodontia seen. This was clearly not the case, with two genotypes being present in the same family. It is not possible to determine whether the father’s PAX9 A99P mutation is inherited or is a germline de novo mutation, as his own father is deceased. The MSX1 A40G variant seems to be inherited independently on both sides of the pedigree, as it is present in I:3, but not in I:2. The PAX9 exon 3 A99P variant is novel and is in the highly conserved C terminal subdomain of the paired DNA binding domain (Table 2). This variant is predicted to be harmful and nearby mutations, at residues 87 and 91, have been shown to cause oligodontia (Kapadia, Frazier-Bowers, Ogawa, & D'Souza, 2006; Das et al., 2003). Another rare variant, rs759490130, has been described at the same position, a G/T mutation causing an alanine to serine change. This is also predicted to be pathogenic but no phenotype data is publicly available.

Both affected individuals exhibit typical PAX9 mutation phenotypes, with the father exhibiting multiple absence of molars. Interestingly, he also has diminutive and conical upper lateral incisors. The daughter is more severely affected in that she has aplasia of all molars and lower central incisors, though her upper lateral incisors are of proportionate size to the rest of her dentition. Given that the mode of inheritance in all cases of PAX9 mutations is autosomal dominant, the resulting phenotype may be due to haploinsufficiency, a dominant-negative activity, or a novel activity of the mutant protein (Kapadia, Mues & D’Souza 2007), although, given the nature of the mutation here, the most likely action is loss of function. The differing phenotypes of the father and daughter may be the result of interaction with other genes. MSX1 and PAX9 have been found to interact synergistically throughout mouse lower incisor development and affect multiple signaling pathways that influence incisor size and symmetry (Nakatomi et al., 2010). However evidence is controversial. Wang et al suggest that disturbances in the interaction with PAX9 are probably not the basis of the human phenotypes caused by MSX1 mutations (Wang, Kong, Mues, & D'Souza, 2011). Four members of the family exhibited MSX1 A40G variants, I:3, II:2, II:4 and II:8. This known variant (rs36059701) is found in an area of low complexity, where the change is from GCA to GGA, resulting in an alanine to glycine substitution. It is predicted to have poor conservation among species with a mild pathogenic effect. No variants were found in the two members married into the family, I:4 and II:3. The frequency of the MSX1 A40G variant in the Maltese population is 14%; that quoted by the 1000 Genomes database for Europeans is 20%. It should be pointed out that this variant has been described as A34G, prior to revision of the MSX1 exon start site. There was a suggested association of this variant with simultaneous tooth agenesis and orofacial clefting

(Modesto, Moreno, Krahn, King, & Lidral, 2006). However, there is no history of cleft palate in the family described here. The Maltese population has a high prevalence of lateral incisor agenesis (Camilleri & Mulligan, 2007) and it is possible that the phenotypes of I:3, II:2, II:4 and II:7 may be influenced by the MSX1 A40G variant, as it segregates with lateral incisor aplasia in this family. It is most probable that the variant does not explain the incisor agenesis but it may contribute to it. On the other hand, this variant may well be innocuous, the hypodontia being due to another, as yet unknown, gene. Conclusion A novel mutation in the C terminal subdomain of the PAX9 gene is described in a family with oligodontia. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

The authors declare no conflict of interest

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Figure 1. Diagram of PAX9 and MSX1 genes showing the selected mutations, associated with hypodontia, targeted by the primers. The PAX9 variant A99P and the MSX1 variant A40G are underlined. Figure 2. Dental panoramic tomogram of proband at 12 years of age, showing developmental absence of all molar teeth and of both lower central incisors of the proband. Developmentally missing teeth are starred. Figure 3. A, B. Photographs of the father's dentition, showing absence of molars and diminutive/peg shaped upper lateral incisors (A- Upper arch, B- Lower arch). C. Dental panoramic tomogram of paternal aunt with missing upper lateral incisors. The 46 had been extracted. Developmentally missing teeth are starred. Figure 4. Pedigree of family with dentograms showing phenotypes and mutations. Developmentally missing teeth are starred. Teeth known to have been extracted or of uncertain fate are left blank. Third molars are not included in the analysis. Figure 5.Chromatogram for PAX9 exon 3 showing a G to C heterozygous base substitution and an amino acid change from alanine to proline.