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Genetic analysis of non-syndromic familial multiple

supernumerary premolars

Doo Hwan Bae

The Graduate School

Yonsei University

Department of Dentistry

[UCI]I804:11046-000000516423[UCI]I804:11046-000000516423

Genetic analysis of non-syndromic familial multiple

supernumerary premolars

Directed by Professor Hyung Jun Choi

A Dissertation Thesis

Submitted to the Department of Dentistry and the Graduate School of Yonsei University

in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Dental Science

Doo Hwan Bae

June 2018

감사의 글

긴 세월 동안 기도로 조력하며 힘들게 뒷바라지 해주신 어머니와 고생만

하다 하늘 나라에 가신 아버지, 어렸을 적부터 항상 맏이 역할을 하기 위해

노력한 지혜누나, 우리 집의 여러 가지 일을 잘 조율해준 지선누나, 어머니를

항상 잘 챙겨주는 혜선 누나, 멀리 떨어져 있었던 저 대신에 집안의 여러

가지 일을 잘 챙겨주었던 거원 매형, 태룡 매형, 항상 제가 하는 일을

응원해주는 혜원이에게 감사의 마음을 전하고 싶습니다.

증례발표로 과잉치에 대한 논문을 처음쓰기 시작하여 본 논문을

완성하기까지 벌써 5년이란 시간이 흘렀습니다. 논문을 완성하기까지 지도와

격려를 아끼지 않으신 최형준 지도 교수님, 소아치과 의사로서의 자세를

가르쳐 주신 김지훈 교수님, 실험부터 논문 집필까지 같이 진행해주신 이지현

교수님, 논문의 방향을 잡아주신 송제선 교수님께 감사의 말씀을 드리고

싶습니다. 그리고 논문과 관련하여 많은 도움을 주셨던 차윤선 선생님께도

감사의 마음을 전하고 싶습니다.

많은 분들의 도움과 사랑으로 학위를 받게 되었습니다.

2018년 6월,

배두환 드림.

i

Table of Contents

Abstract ······························································································ v

I. Introduction ······················································································ 1

II. Subjects and Methods ········································································· 4

1. Subjects ························································································ 4

2. Candidate Target Gene Selection ·························································· 11

3. Genetic Analysis ············································································· 17

III. Results ·························································································· 18

1. Mutation Identification ······································································ 18

2. Clinical Findings and Family Pedigree ··················································· 21

3. Causative gene confirmation ······························································· 24

IV. Discussion ······················································································ 25

V. Conclusion ······················································································ 28

VI. References ····················································································· 29

Abstract (in Korean) ············································································ 32

ii

List of Figures

Figure 1. Pedigree of the family ·································································· 6

Figure 2. Panoramic and periapical radiographs of subject II-1 showing

4 supernumerary premolars ···························································· 7

Figure 3. Panoramic and periapical radiographs of subject II-2 showing

3 supernumerary premolars ···························································· 8

Figure 4. Panoramic radiograph of subject II-3 ················································ 9

Figure 5. Panoramic radiograph of subject I-3 showing

two supernumerary premolars ······················································· 10

Figure 6. Prioritization tool for disease gene candidates ····································· 14

Figure 7. Mutation analysis ······································································ 20

iii

Figure 8. 2 year-after-panoramic and periapical radiographs of subject II-3 showing

impacted lateral incisors(marked as ‘a’) and

late developing canines(marked as ‘b’) ·············································· 22

Figure 9. Corrected pedigree of the family ····················································· 23

iv

List of Tables

Table 1. 59 genes associated with tooth number abnormality ······························· 12

Table 2. 10 characteristics used to find the genes associated with seed genes ············ 13

Table 3. Candidate target genes involved in tooth development regulation ··············· 15

Table 4. Rare, non-synonymous, exonic variants detected by targeted sequencing in

2 affected family members ···························································· 19

v

Abstract

Genetic analysis of non-syndromic familial multiple

supernumerary premolars

Doo Hwan Bae

Department of Dentistry

The Graduate School, Yonsei University

(Directed by Professor Hyung Jun Choi)

Supernumerary teeth refer to extra teeth that exceed the normal number in dentition. The

incidence of supernumerary teeth is about 3%. Among them, single supernumerary tooth

accounts for 76-86% of cases, double supernumerary teeth comprise 12-23%, and three or

more supernumerary teeth represent less than 1%. In most cases, multiple supernumerary

teeth are associated with other syndromes or developmental disorders. Multiple

supernumerary teeth without any associated syndromes or conditions, are very rare and

named non-syndromic multiple supernumerary teeth.

vi

Most reported cases related to non-syndromic familial multiple supernumerary teeth are

related to a mesiodens, and genetic analysis has never been performed on non-syndromic

premolar patients. Although incidence of supernumerary premolar is as low as

0.075~0.26%, several members in a family showed supernumerary premolars. For this

reason, genetic etiology was expected, and genetic analyses of family members were

planned to identify the causative genes of these multiple supernumerary teeth.

59 genes associated with tooth number abnormality were found by literature review.

Genes having similar characteristics to the seed gene set known to affect abnormal tooth

number were sorted out, in order to find more genes associated with tooth number

abnormality. Using 10 characteristics, 101 candidate genes were selected. Genomic DNA

from saliva was extracted using Oragene DNA kits. For targeted sequencing, DNA

fragments were enriched by solution-based hybridization capture and followed by

sequencing with an Illumina Hiseq2500 platform. Targeted exome sequence data of the

two subjects affected (I-3 and II-1) showed several shared variants. A frequency cutoff

was set considering the incidence of supernumerary premolars. The variants, which

appeared more than the frequency cutoff, were excluded from candidate genes, and

PDGFRB, MSX2, and FGFR2 were considered candidates. As a result of Sanger

sequencing, the PDGFRB mutation was considered as the most likely cause of the

supernumerary teeth.

PDGF ligands and their receptors are expressed throughout the initial stages of tooth

development, while its precise role in tooth development remains unclear. PDGFRB

vii

proteins are mainly expressed in the dental mesenchyme during initial tooth development

and PDGF-BB signaling is important for dental mesenchymal cell proliferation.

Excessive mesenchymal expression by PDGFRB mutation is a possible cause of

supernumerary tooth formation, because PDGF-BB serves as a mitogen for dental

follicles.

This is the first report of the association between supernumerary premolars and

PDGFRB mutations. Further molecular studies are required to clarify the causative genes

of non-syndromic supernumerary premolars, and this study can be the cornerstone to

understand full pathologies on non-syndromic supernumerary teeth.

Keywords: Non-syndromic supernumerary tooth, targeted exome sequencing, PDGFRB

1

Genetic analysis of non-syndromic familial multiple

supernumerary premolars

Doo Hwan Bae

Department of Dentistry

The Graduate School, Yonsei University

(Directed by Professor Hyung Jun Choi)

I. INTRODUCTION

Supernumerary teeth refer to extra teeth that exceed the normal number in dentition

(Dummett and Thikkurissy, 2013). The incidence of supernumerary teeth is about 3%.

Among them, single supernumerary tooth accounts for 76-86% of cases, double

supernumerary teeth comprise 12-23%, and three or more supernumerary teeth represent

less than 1% (Manrique Mora, et al., 2004; Rajab and Hamdan, 2002). In most cases,

multiple supernumerary teeth are associated with other syndromes or developmental

2

disorders such as cleft palate and cleft lip, cleidocranial dysplasia, and familial

adenomatous polyposis (Aggarwal, et al., 2003; Bufalino, et al., 2012; Jugessur, et al.,

2009). Multiple supernumerary teeth, without any associated syndromes or conditions,

are very rare (Kaya, et al., 2011). In other words, in most cases, multiple supernumerary

teeth appear as the result of a genetic trait. This indicates that there is strong evidence for

a genetic basis of supernumerary teeth.

The beginning stage of tooth development is a key step in determining tooth number.

Studies on odontogenesis-regulating genes have revealed that over 300 genes are

associated with tooth development. Transcription factors such as Barx, Dlx, Gli, Lef and

Lhx, and secreted proteins such as Bmp, Fgf, Hgf, and Shh affect gene expression during

tooth development (Galluccio, et al., 2012; Nanci, 2007).

Although the etiology of supernumerary teeth has not been well-documented, it has been

proposed that genetic factors are associated with the process. Genes related to some

syndromes that cause multiple supernumerary teeth have been identified, such as RUNX2,

APC, Tenascin-XB, NHS, EVC, TRPS1, and ROR2, but genes associated with non-

syndromic supernumerary teeth have not yet been identified (Bufalino, et al., 2012; Hong,

et al., 2014; Li, et al., 2015; Maas, et al., 2015; Mazzeu, et al., 2007; O'Connell, et al.,

2010; Ulucan, et al., 2008).

Most reported cases related to non-syndromic familial multiple supernumerary teeth

were related to a mesiodens, and genetic analysis has never been performed on non-

syndromic premolar patients (Khambete and Kumar, 2012). To date, most genetic studies

3

of the teeth have been carried out using mice. However, mice do not develop both canine

and premolar teeth. Therefore, genetic studies on supernumerary premolars in the human

family can reveal information that cannot be determined with in vivo mouse studies.

The aim of this study was to identify genetic mutations correlated with non-syndromic

supernumerary premolars. Genetic analyses were executed through exome sequencing of

a Korean family that presented with supernumerary premolars in two successive

generations.

4

II. Subjects and Methods

1. Subjects

The index patient (II-1) and his younger brothers (II-2, II-3) presented at the Department

of Pediatric Dentistry, Yonsei University, Wonju Severance Christian Hospital for

extraction of supernumerary teeth and non-eruption of permanent teeth, respectively

(Figure 1). Panoramic radiograph of the index patient (II-1) revealed the presence of four

total supernumerary teeth adjacent to the premolars on both sides (Figure 2). His brothers

also showed supernumerary teeth (Figures 3, 4).

There were no syndromic features of the body, or other relevant developmental or

medical history. Panoramic radiographs were collected from other family members. As a

result, a total of four family members were confirmed to have supernumerary teeth

(Figures 1-5). Characteristically, supernumerary teeth were commonly found in the lower

premolar area.

Multiple supernumerary teeth developed in several members in one family and in the

same area in each family member, despite an extremely low incidence of non-syndromic

multiple supernumerary teeth in the general population. For this reason, genetic etiology

was expected, and genetic analyses of family members were planned to identify the

causative genes of these multiple supernumerary teeth.

Written informed consent was obtained from all participants or their legal guardians. All

clinical investigations were conducted according to the principles expressed in the

5

Declaration of Helsinki. This study was approved by the Institutional Review Board at

Yonsei University Wonju College of Medicine (YWDR-14-9-097).

6

Fig. 1. Pedigree of the family. Family members who participated in this study are denoted

by a number under the symbol. The proband is indicated with a black arrow.

7

Fig. 2. Panoramic and periapical radiographs of subject II-1 showing 4 supernumerary

premolars.

8

Fig. 3. Panoramic and periapical radiographs of subject II-2 showing 3 supernumerary

premolars.

9

Fig. 4. Panoramic radiograph of subject II-3. ‘a’s were suspected to be impacted canines

and ‘b’s were suspected to be supernumerary premolars.

10

Fig. 5. Panoramic radiograph of subject I-3 showing two supernumerary premolars. One

impacted tooth was assumed to be the mandibular left 2nd premolar based on the dental

history not to have extracted that tooth.

11

2. Candidate Target Gene Selection

The first step was to find genes, as many as possible, which were mentioned to be

associated with tooth number abnormality. 59 genes were found by literature review

(Table 1). Genes having similar characteristics to the seed gene set known to affect

abnormal tooth number were sorted out, in order to find more genes associated with tooth

number abnormality. If the seed gene set is enriched in particular biological pathway,

other genes belonging to the pathway can be considered to affect abnormal tooth number.

On the same principle, genes showing similar expression patterns with the seed gene set

can affect abnormal tooth number. 10 characteristics used in screening process are shown

in Table 2.

59 seed genes were set as input. As genes had more similar characteristics to the seed

gene set, higher ranks were given to those genes (Figure 6). Final ranks were determined

by compiling the information of the individual characteristics, and candidate genes were

sorted according to those ranks. Finally, 101 candidate genes were selected by adjusting

the significance level using Bonferroni method (Table 3).

12

Table 1. 59 genes associated with tooth number abnormality

Gene name Gene name Gene name Gene name Gene name Gene name ACVR2A DLX2 FST MSX2 SPRY4 TBX10 ACVR2B DLX3 GLI1 PAX6 TGFA TFAP2A AXIN2 EDA GLI2 PAX9 TRAF6 OSR2 BARX1 EDAR GLI3 PITX2 WNT10A PTHR1 BMP2 ENO1P IFT88 RUNX2 EGFR TP73L BMP4 FGF3 IRF6 SHH NR2F1 LHX8 BMP7 FGF4 LEF1 SHOX2 PDGFA PRRX1

BMPR1A FGF8 LHX6 SMAD2 PDGFC CDKN1A DKK1 FGF9 LTBP3 SOSTDC1 PDGFRA PTCH1 DLX1 FGFR1 MSX1 SPRY2 PDGFRB

13

Table 2. 10 characteristics used to find the genes associated with seed genes

1 EST(Expression Sequence Tag)

2 GO(Gene Ontology)

3 domain interpro

4 pathway kegg

5 sequence swissprot, blast 6 cis regulatory module

7 expression 6 database

8 interaction 7 database

9 motif

10 textmining

14

Fig. 6. Prioritization tool for disease gene candidates

15

Table 3. Candidate target genes involved in tooth development regulation

Gene name Ensemble gene ID Gene name Ensemble gene ID

ACVR2A ENSG00000121989 SPRY2 ENSG00000136158 ACVR2B ENSG00000114739 SPRY4 ENSG00000187678 AXIN2 ENSG00000168646 TBX10 ENSG00000167800 BARX1 ENSG00000131668 TFAP2A ENSG00000137203 BMP2 ENSG00000125845 TGFA ENSG00000163235 BMP4 ENSG00000125378 TP73L ENSG00000073282 BMP7 ENSG00000101144 TRAF6 ENSG00000175104 BMPR1A ENSG00000107779 WNT10A ENSG00000135925 CDKN1A ENSG00000124762 DLX5 ENSG00000105880 DKK1 ENSG00000107984 TGFBR2 ENSG00000163513 DLX1 ENSG00000144355 ALX1 ENSG00000180318 DLX2 ENSG00000115844 SMAD6 ENSG00000137834 DLX3 ENSG00000064195 EGF ENSG00000138798 EDA ENSG00000158813 FGFR2 ENSG00000066468 EDAR ENSG00000135960 ERBB3 ENSG00000065361 EDARADD ENSG00000186197 FGF1 ENSG00000113578 EGFR ENSG00000146648 ERBB4 ENSG00000178568 FGF3 ENSG00000186895 ERBB2 ENSG00000141736 FGF4 ENSG00000075388 FGF5 ENSG00000138675 FGF8 ENSG00000107831 ACVR1B ENSG00000135503 FGF9 ENSG00000102678 ACVR1 ENSG00000115170 FGFR1 ENSG00000077782 MET ENSG00000105976 FST ENSG00000134363 NOG ENSG00000183691 GLI1 ENSG00000111087 HOXD10 ENSG00000128710 GLI2 ENSG00000074047 CAV1 ENSG00000105974

16

GLI3 ENSG00000106571 TFAP2C ENSG00000087510 IFT88 ENSG00000032742 FGF7 ENSG00000140285 IRF6 ENSG00000117595 TAB2 ENSG00000055208 LEF1 ENSG00000138795 BMP6 ENSG00000153162 LHX6 ENSG00000106852 CBLB ENSG00000114423 LHX8 ENSG00000162624 FGFR3 ENSG00000068078 LTBP3 ENSG00000168056 BMPR2 ENSG00000204217 MSX1 ENSG00000163132 BMP5 ENSG00000112175 MSX2 ENSG00000120149 EPHB3 ENSG00000182580 NR2F1 ENSG00000175745 IGF1R ENSG00000140443 OSR2 ENSG00000164920 TGFB3 ENSG00000119699 PAX6 ENSG00000007372 NTRK3 ENSG00000140538 PAX9 ENSG00000198807 PAX3 ENSG00000135903 PDGFA ENSG00000197461 FGF2 ENSG00000138685 PDGFC ENSG00000145431 FGF6 ENSG00000111241 PDGFRA ENSG00000134853 RUNX3 ENSG00000020633 PDGFRB ENSG00000113721 IRAK3 ENSG00000090376 PITX2 ENSG00000164093 BMPR1B ENSG00000138696 PRRX1 ENSG00000116132 TP53 ENSG00000141510 PTCH1 ENSG00000185920 LYN ENSG00000147507 PTHR1 ENSG00000160801 TCF7L1 ENSG00000152284 RUNX2 ENSG00000124813 TGFBR1 ENSG00000106799 SHH ENSG00000164690 CDKN1B ENSG00000111276 SHOX2 ENSG00000168779 CHX10 ENSG00000119614 SMAD2 ENSG00000175387 TGFB2 ENSG00000092969 SOSTDC1 ENSG00000171243

17

3. Genetic Analysis

Genomic DNA from saliva was extracted using Oragene DNA kits (OG-500) (DNA

Genotek, Ontario, Canada) according to the manufacturer’s instructions. For targeted

sequencing, DNA fragments were enriched by solution-based hybridization capture and

followed by sequencing with an Illumina Hiseq2500 platform. Analyses of Next Generation

Sequencing (NGS) data were performed using an in-house analysis pipeline. Specifically,

sequencing reads from the HiSeq2500 raw data were sorted by index and barcode

sequences. Sorted fastq files were aligned to the hg19 reference genome using the Burrows-

Wheeler Aligner algorithm (BWA; ver. 0.7.5a) (Li and Durbin, 2010). Output SAM files

were converted into BAM files and sorted using SAMtools (ver. 0.1.18) (Li, et al., 2009).

Duplicate removal was performed with the Picard tools (ver. 1.95) MarkDuplicates.

Realignment around known indel sites and base quality score recalibration (BQSR) were

performed using GATK (ver. 2.6-5) to create final BAM files (McKenna, et al., 2010).

Variants were identified using the GATK v2.6 Unified Genotyper algorithm for loci

with a sequencing depth greater than or equal to 20X. Variants were annotated with

ANNOVAR (ver. 2013-06-21) (Wang, et al., 2010). Functional effect prediction for

single-nucleotide variants (SNVs) was performed by PolyPhen-2 (v2.2.2), SIFT, and

Mutation Taster. Polymorphisms found in the Korean population (N = 405) and public

databases (1000 Genome project SNP (2012 April release, ESP)) from both Asian and

all-population databases were also filtered. All selected variants were validated by Sanger

sequencing.

18

III. Results

1. Mutation Identification

Using a targeted capture approach, 101 candidate genes involved in tooth development

regulation were sequenced. The average depth of coverage for the targeted regions was

871.7X. Greater than 20X coverage was obtained for 98.1% of the bases sequenced.

Targeted exome sequence data of the two subjects affected (I-3 and II-1) showed several

shared variants. A frequency cutoff was set considering the incidence of supernumerary

premolars. The variants, which appeared more than the frequency cutoff, were excluded

from candidate genes, and PDGFRB, MSX2, and FGFR2 were considered candidates

(Table 4).

Sanger sequencing screening was performed on other family members (II-2, II-3),

PDGFRB mutation was detected in one family member (II-2) but not in the other family

member (II-3), and MSX2 and FGFR2 mutations were not detected in either family

member (II-2, II-3) (Figure 7).

19

Table 4. Rare, non-synonymous, exonic variants detected by targeted sequencing in 2

affected family members.

Chr Gene Changes SIFT PolyPhen-2Mutation

Taster

1000G

MAF

ESP

MAF

Korean

MAF

(N=405)

I-3 II-1

chr5 PDGFRB

NM_002609

c.C2053T

p.R685C

Deleterious

(score: 0.95)

Possibly

damaging

(score: 0.894)

Disease

causing NA NA 0.01 Hetero Hetero

chr5 MSX2

NM_002449

c.A703G

p.I235V

Tolerated

(score: 0.91)

Benign

(score: 0.004)Neutral NA NA 0 Hetero Hetero

Chr

10 FGFR2

NM_000141

c.T557C

p.M186T

Tolerated

(score: 0)

Benign

(score: 0) Polymorphism 0.06 0.10 0.06 Hetero Hetero

Abbreviations: NA, not available; MAF, minor allele frequency.

20

Fig. 7. Mutation analysis. Sanger sequencing chromatograms of the family members are

shown. Nucleotide sequences are shown above the chromatograms. Each red arrow

indicates a mutation (A) PDGFRB (c.C2053T, p.R685C), (B) MSX2 (c.A703G, p.I235V)

and (C) FGFR2 (c.T557C, p.M186T).

21

2. Clinical Findings and Family Pedigree

There was no mutation which was completely segregated with supernumerary teeth in

this family, so research materials were reviewed thoroughly. Reviewing patients’

materials, it was found that third son’s supernumerary teeth appear different from other

family members. In the initial radiograph of subject II-3, ‘b’ teeth showed outline but no

clear shape (Figure 4). Based on the familial history of supernumerary teeth, ‘b’ teeth had

been considered as supernumerary teeth. However, ‘b’ teeth were found to form no more

cusp in the radiograph after two years (Figure 8). They looked like canines rather than

premolars. They were different from other family members’ supernumerary teeth which

looked exactly premolars having two cusps. Additionally, ‘a’ teeth looked like canines in

the panoramic radiograph, but they looked rather lateral incisors having 3 mamelons in

the periapical radiographs (Figure 8). Therefore, the previous assumption that subject II-3

had supernumerary teeth was reversed, and subject II-3 considered to have impacted

lateral incisors and canines which were late developing.

As a result, the family members showed that the father (family member I-3) and his two

sons (family members II-1, II-2) had common supernumerary premolars in the lower

premolar region (Figure 9). No evidence of any other systemic anomalies was observed in

the family members. Of nine members from whom DNA was extracted, three were

affected (all males). Therefore, we determined that the supernumerary tooth mutation

seemed to be inherited in an autosomal dominant manner.

22

Fig. 8. 2 year-after-panoramic and periapical radiographs of subject II-3 showing

impacted lateral incisors(marked as ‘a’) and late developing canines(marked as ‘b’).

23

Fig. 9. Corrected pedigree of the family. Pedigree was corrected, because subject II-3

seemed to have no supernumerary tooth on the 2 year-after radiographs.

24

3. Causative gene confirmation

The PDGFRB mutation was the missense mutation which substituted cytidine with

thymidine in the 2053th coding region. If a mutation occurs in this region, disease is

likely to occur, because amino acids in this region are well conserved in several species

including the human. Additionally, in the table 4 which predicted functional effect of

mutations, PDGFRB mutation showed deleterious, possibly damaging, and disease

causing, while other mutations showed tolerated, benign, and etc. Compared to other

mutations, PDGFRB mutation was more likely to cause disease, and completely

segregated with supernumerary teeth in the family. Therefore, other mutations were

excluded from candidates, and the PDGFRB mutation was considered as the most likely

cause of the supernumerary teeth.

25

IV. DISCUSSION

Associations between PDGFRB mutations and supernumerary premolars were proposed

by targeted exome sequencing results. However, the location of the sequence variants in

PDGFRB is not given, so the effect on protein function cannot be exactly inferred. There

is also missing sequence information for I-3 and II-1. A gene editing experiment on

humans could overcome these weaknesses; however such experiments are not ethically

acceptable and, even if allowed, would take a very long time. The phenotype was almost

the same among the affected family members, but the number of supernumerary teeth

varied. This could be attributed to modifier genes that have not been considered in this

study.

It is known that PDGFRB is important in vasculature support cell development.

According to Klinghoffer et al. (Klinghoffer, et al., 2001) and Hellstrom et al.(Hellstrom,

et al., 1999), PDGFRB mutations can cause cardiac hypertrophy, glomerulosclerosis, and

proliferative retinopathy in mice. The role of PDGFRB in organogenesis has been defined

in mice, but not in humans. Considering that the family had no cardiac hypertrophy,

glomerulosclerosis, or proliferative retinopathy, PDGFRB may have a different role in

human organogenesis. While its precise role in tooth development remains unclear even

in mice, some involvement of PDGFRB in tooth development has been confirmed. PDGF

ligands and their receptors are expressed throughout the initial stages of tooth

development, indicating their involvement in that process (Wu, et al., 2010). Particularly,

26

PDGFRB proteins are mainly expressed in the dental mesenchyme during initial tooth

development and PDGF-BB signaling is important for dental mesenchymal cell

proliferation (Wu, et al., 2010). PDGF-BB serves as a mitogen for dental follicles (Bsoul,

et al., 2003). Morphogenesis is controlled by interactions between epithelial cells and

mesenchymal cells. There is a possibility that the mitogenetic ability of PDGF-BB

induces excessive mesenchymal expression, leading to inductive ability of the epithelium.

Excessive mesenchymal expression by PDGFRB mutation is a possible cause of

supernumerary tooth formation.

The majority of reported mutations in PDGFRB have shown an autosomal dominant

pattern, as was found in the family in this study. Therefore, this study assumed Mendelian

inheritance, which is based on the premise that affected subjects have the same mutations.

However, there is the possibility of non-Mendelian inheritance in cases of supernumerary

teeth. To analyze more complex inheritance patterns, more samples are needed to identify

genetic mutations and diagnostic markers. If non-syndromic supernumerary teeth are

inherited with a non-Mendelian inheritance pattern, many additional samples are needed,

which could prove to be difficult because the incidence of non-syndromic supernumerary

teeth is extremely low (Kaya, et al., 2011). Additional study is planned to be performed

when other families with non-syndromic supernumerary teeth are found.

PDGFRB was considered the single potential causative gene in this study, but some

phenotypes are caused by double mutations, such as MSX1/MSX2 or DLX1/DLX2. There

is a possibility that non-syndromic supernumerary teeth are not caused by a PDGFRB

27

single mutation, but a PDGFRB mutation in combination with another mutation of a gene

such as Spry2 and AXIN2, which were excluded based on the frequency cutoff, or Gas1

which was not included in the candidate genes.

28

V. CONCLUSION

The PDGFRB mutation was completely segregated with supernumerary teeth in this

family. This is the first report of the association between supernumerary premolars and

PDGFRB mutations. Further molecular studies are required to clarify the causative genes

of non-syndromic supernumerary premolars, and this study can be the cornerstone to

understand full pathologies on non-syndromic supernumerary teeth.

29

VI. REFERENCE

Aggarwal VR, Sloan P, Horner K, Macfarlane TV, Clancy T, Evans G, et al.: Dento-

osseous changes as diagnostic markers in familial adenomatous polyposis

families. Oral Dis 9(1): 29-33, 2003.

Bsoul S, Terezhalmy G, Abboud H, Woodruff K, Abboud SL: PDGF BB and bFGF

stimulate DNA synthesis and upregulate CSF-1 and MCP-1 gene expression in

dental follicle cells. Arch Oral Biol 48(6): 459-465, 2003.

Bufalino A, Paranaíba LMR, Gouvêa AF, Gueiros LA, Martelli-Júnior H, Junior JJ, et al.:

Cleidocranial dysplasia: oral features and genetic analysis of 11 patients. Oral

Dis 18(2): 184-190, 2012.

Dummett CO, JR, Thikkurissy S: Anomalies of the developing dentition. In: Pediatric

dentistry: infancy through adolescence 5th ed. Casamassimo PS, Fields HW, Jr,

McTigue DJ, Nowak A, eds. Elsevier Health Sciences. 2013.

Galluccio G, Castellano M, La Monaca C: Genetic basis of non-syndromic anomalies of

human tooth number. Arch Oral Biol 57(7): 918-930, 2012.

Hellstrom M, Kalen M, Lindahl P, Abramsson A, Betsholtz C: Role of PDGF-B and

PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes

during embryonic blood vessel formation in the mouse. Development 126(14):

3047-3055, 1999.

Hong N, Chen YH, Xie C, Xu BS, Huang H, Li X, et al.: Identification of a novel

mutation in a Chinese family with Nance-Horan syndrome by whole exome

sequencing. J Zhejiang Univ Sci B 15(8): 727-734, 2014.

Jugessur A, Farlie PG, Kilpatrick N: The genetics of isolated orofacial clefts: from

genotypes to subphenotypes. Oral Dis 15(7): 437-453, 2009.

Kaya GS, Yapici G, Omezli MM, Dayi E: Non-syndromic supernumerary premolars.

Medicina Oral Patología Oral y Cirugia Bucal 16(4): e522-e525, 2011.

Khambete N, Kumar R: Genetics and presence of non-syndromic supernumerary teeth:

A mystery case report and review of literature. Contemp Clin Dent 3(4): 499-

502, 2012.

30

Klinghoffer RA, Mueting-Nelsen PF, Faerman A, Shani M, Soriano P: The two PDGF

receptors maintain conserved signaling in vivo despite divergent

embryological functions. Mol Cell 7(2): 343-354, 2001.

Li FF, Liu Z, Yan P, Shao X, Deng X, Sam C, et al.: Identification of a novel mutation

associated with familial adenomatous polyposis and colorectal cancer. Int J

Mol Med 36(4): 1046-1056, 2015.

Li H, Durbin R: Fast and accurate long-read alignment with Burrows–Wheeler

transform. Bioinformatics 26(5): 589-595, 2010.

Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al.: The Sequence

Alignment/Map format and SAMtools. Bioinformatics 25(16): 2078-2079,

2009.

Maas SM, Shaw AC, Bikker H, Ludecke HJ, van der Tuin K, Badura-Stronka M, et al.:

Phenotype and genotype in 103 patients with tricho-rhino-phalangeal

syndrome. Eur J Med Genet 58(5): 279-292, 2015.

Manrique Mora MC, Bolanos Carmona MV, Briones Lujan MT: Molarization and

development of multiple supernumerary teeth in the premolar region. J Dent

Child (Chic) 71(2): 171-174, 2004.

Mazzeu JF, Pardono E, Vianna-Morgante AM, Richieri-Costa A, Ae Kim C, Brunoni D, et

al.: Clinical characterization of autosomal dominant and recessive variants of

Robinow syndrome. Am J Med Genet A 143(4): 320-325, 2007.

McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, et al.: The

Genome Analysis Toolkit: A MapReduce framework for analyzing next-

generation DNA sequencing data. Genome Res 20(9): 1297-1303, 2010.

Nanci A: Development of the tooth and its supporting tissues. In: Ten Cate's Oral

Histology-Pageburst on VitalSource: Development, Structure, and Function 7th

ed. Nanci A, ed. Elsevier Health Sciences. 2007. p. 79-107.

O'Connell M, Burrows NP, van Vlijmen-Willems MJ, Clark SM, Schalkwijk J: Tenascin-X

deficiency and Ehlers-Danlos syndrome: a case report and review of the

literature. Br J Dermatol 163(6): 1340-1345, 2010.

Rajab LD, Hamdan MA: Supernumerary teeth: review of the literature and a survey of

152 cases. Int J Paediatr Dent 12(4): 244-254, 2002.

31

Ulucan H, Gul D, Sapp JC, Cockerham J, Johnston JJ, Biesecker LG: Extending the

spectrum of Ellis van Creveld syndrome: a large family with a mild mutation in

the EVC gene. BMC Med Genet 9: 92, 2008.

Wang K, Li M, Hakonarson H: ANNOVAR: functional annotation of genetic variants

from high-throughput sequencing data. Nucleic Acids Res 38(16): e164-e164,

2010.

Wu N, Iwamoto T, Sugawara Y, Futaki M, Yoshizaki K, Yamamoto S, et al.: PDGFs

regulate tooth germ proliferation and ameloblast differentiation. Arch Oral Biol

55(6): 426-434, 2010.

32

국문요약

가족에서 발생한 비증후군성 다수 소구치 과잉치의

유전학적 분석

연세대학교 대학원 치의학과

배 두 환

지도교수: 최형준

과잉치는 정상적인 치아 수보다 많은 수의 치아를 이르는 용어이며 발생률

은 약 3% 정도로 그 중에서 76-86%는 단일 과잉치, 12-23%는 두 개의

과잉치, 3개 이상의 과잉치가 나타날 확률은 1%이하로 보고되고 있다. 대부

분의 경우 다수 과잉치는 전신 질환 또는 증후군과 관련되어 나타난다. 전신

적인 요소나 증후군 없이 다수 과잉치가 발생하는 경우는 매우 드물며 비증후

군성 다수 과잉치라는 용어로 명명한다.

현재까지 이루어진 가족에서 발생한 다수 과잉치에 대한 연구는 정중과잉치

에 대한 연구가 대부분이며, 특히 비증후군성 소구치 과잉치에 대한 유전학적

연구는 시행된 적이 없다. 소구치 과잉치의 발생률은 0.075~0.26%로 매우

적음에도 불구하고 한 가족에서 여러 명의 구성원에게 소구치 과잉치가 발생

33

하였기에 과잉치의 원인 유전자와 치아 수의 이상을 야기하는 분자생물학적인

역학을 밝히는데 도움이 될 것이라고 생각하여 가족 구성원들에 대한 유전학

적 분석을 시행하였다.

문헌 검색을 통해 과잉치와 연관이 있다고 언급이 된 59개의 seed 유전자

를 찾았다. 아직 연구를 통해 밝혀지진 않았지만 치아 수 이상과 관련이 있을

수 있는 유전자를 찾기 위해 앞서 찾은 seed 유전자들과 기능, biological

pathway, sequence, 혹은 domain 등을 공유하거나 발현 양상이 유사한 101

개의 후보 유전자를 선별하였다. 오라진 DNA kits를 사용해서 피험자들의 타

액을 채취하고 DNA를 추출하였다. 타액에서 추출한 DNA를 PCR로 증폭한

후 Illumina HiSeq2500 platforms을 이용하여 sequencing을 시행하였다. 먼

저 과잉치가 나타난 2명에서 Targeted exome sequencing을 시행한 결과

10가지 공통된 변이를 보였고 소구치 과잉치의 발생률 0.075~0.26%를 고려

하여 그 이상의 빈도수를 보이는 변이들은 후보 유전자에서 제외하였다. 최종

적으로 PDGFRB, MSX2, FGFR2 3가지 gene을 후보로 고려하여, Sanger

sequencing을 시행하였고 PDGFRB의 이형접합형 돌연변이를 확인하였다.

치아 발생에서 PDGFRB의 명확한 역할은 밝혀지지 않았지만 PDGF ligand

와 receptor가 치아발생의 초기 단계에 발현된다는 사실은 확인되었다.

PDGFRB 단백질은 주로 치아 발생 초기단계의 치아 중배엽에서 발현되고

PDGF-BB signaling은 치아 중배엽 증식에 중요한 역할을 한다. PDGF-BB

34

는 치배에 대해 mitogen으로 작용하기 때문에 PDGFRB 변이에 의한 중배엽

의 과도한 발현이 과잉치를 발생시키는 원인으로 작용할 것이라고 생각할 수

있다.

본 논문은 비증후군성 다수 과잉치를 가진 한국인 가족에 대한 첫 번째 유

전학적 분석으로서 PDGFRB 돌연변이와 소구치 부위 과잉치와의 관련성에

대해 보여주었다. 명확한 원인유전자를 밝히기 위해서는 좀 더 연구가 필요하

겠지만 본 연구는 비증후군성 다수과잉치의 발생을 이해하는 기초가 될 수 있

을 것이다.

핵심되는 말: 비증후군성 다수 과잉치, targeted exome sequencing, PDGFRB