Association Analysis of Single Nucleotide Polymorphisms (SNPs) within or near the ActivinA (INHBA), ActivinB (INHBB) and/or the , Type IIA (ACVR2A) and Naturally Missing Teeth (NMT)

Kyle Kirk • Anna Vu, DMD, MS • Melissa Gilbey • Gabriel Falcão-Alencar, MS • Paul DiFranco, DDS, MS • James K. Hartsfield, Jr., DMD, PhD • Lorri A Morford, PhD Division of Orthodontics, College of Dentistry, University of Kentucky, Lexington, KY 40536, U.S.A .

Interesting Connections Exist Abstract Figure 5: Percentage of Subjects with Between NMT and Cancer Objectives. The purpose of this study was to investigate potential Hypodontia and Controls for each genetic associations between various single nucleotide Of the genes involved in odontogenesis, they also have polymorphisms (SNPs) within or near the ActivinA (INHBA), rs2877098 Genotype – INHBA important roles in other organs and body systems, therefore it is a ActivinβB (INHBB) and/or Activin Type IIA Receptor (ACVR2A) genes plausible assumption that a genetic mutation resulting in and Naturally Missing Teeth (NMT). hypotontia can lead to abnormalities in other organs. Control Hypodontia 60 Methods. Ethics approval was granted by the UK-Internal Review Interesting connections have been made between Board. Twenty-five unrelated Caucasians diagnosed with Agenic/Naturally Missing Teeth and Cancer : 48.0% 48.5% hypodontia and 68 unrelated Caucasian controls were consented 50 45.6% from the faculty and graduate orthodontic and pediatric dentistry • AXIN2 – Mutations involved in embryonic development & clinics. Hypodontia was defined as having 1 to 5 naturally missing 40 overall cellular homeostasis have been associated with (agenic) teeth in the adult dentition, excluding 3rd molars. Peg- 32.0% colorectal cancer (Lammi et al. Am J Hum Genet 2004; 74(5): 1043-50.) shaped teeth were also noted. DNA was isolated from subject 30

saliva and was genotyped on a Roche LightCycler480 for SNPs Subjects ! • PAX9 – Reductions in expression have led to increased within or near the INHBA (rs2877098), INHBB (rs17625845) and 20.0% malignancy of dysplastic and cancerous esophageal epithelium 20 ACVR2A (rs1364658) genes. Chi squared analysis was utilized to (Gerber et al. J Pathol 2002; 197(3): 293-7.) access Hardy-Weinberg Equilibrium (HWE) and to determine 5.9% 10 potential marker association(s) with hypodontia; significance at • Research done within the UK Orthodontic Program found a

p<0.05. statistical relationship between hypodontia and epithelial ovarian or Hypodontia Control % of Total n=4 n=5 n=31 n =12 n=33 n=8 0 cancer (EOC) (Chalathorn et al. JADA 2008; 139(2): 163-9.) Results. Hypodontia occurred more frequently in females than TT CT CC • Hypodontia was found in up to 20% of women with EOC males. The average ages for the hypodontia and control cases Genotype • Implies that women with EOC are 8.1 times more likely to were 16.8+5.8 and 14.5+4.6 years, respectively. Maxillary-lateral- Co-Dominant Mode of Inheritance: p = 0.085 have hypodontia than women without EOC incisors were the most frequently affected by agenesis and/or peg • Agenic teeth in patients with EOC could not be formation, followed by mandibular-2nd-premolars and maxillary-2nd- explained by a common mutation in the known tooth premolars. All Control genotyping maintained HWE. There was no Figure 6: Percentage of Subjects with development genes PAX9, MSX1, AXIN2, EDA, BARX1, significance association identified between the markers tested and BARX2 and WNT10A or the BRCA1 . Hypodontia and Controls for each hypodontia (p-values 0.085 to 0.451). (Pollan White, S., Genetic studies on the association of ovarian cancer and tooth agenesis., Thesis in Biomedical Sciences 2008, Texas A&M University System Health Science Center: Dallas, Texas. pps. 62.; J. BONDS, S. POLLAN, L. XIANG, G. MUES, and R. D'SOUZA, AADR Annual Meeting 2014 Poster #1607)" rs17625845 Genotype– INHBB Conclusion. While the SNPs tested in this project were not associated with hypodontia, additional data gathered in our laboratory has identified a novel association of SNP rs7576183 near 80

the ActivinbB gene and hypodontia , particularly hypodontia of the Control Hypodontia Figure 3: Materials and Methods 70 68.0% upper arch. . 60.3% • Study Population 60

50 Figure 1: Etiology of Agenic/NMT Teeth • 93 unrelated Caucasian subjects treated with orthodontics at Hypodontia the University of Kentucky College of Dentistry. Based on the 40 38.2% interesting connections between NMT and cancer, we are

obtaining a 3 generation family history of cancer for each Subjects 30 24.0% • Definition of Hypodontia– developmental absence of one to five subject. teeth within the adult dentition. In addition to hypodontia, 25 subjects (with Hypodontia) 20 investigators in this study also made not of variations in tooth size 68 controls (without Hypodontia) 8.0% 10 and shape (i.e. including peg-shaped teeth, small teeth) 1.5% • Control or Total % of n=4 n=17 n=26 n=6 n=1 n=2 Hypodontia phenotype determined by: 0 Causes 1 TT CTn=23 CC • Genetics – Hypodontia is often an autosomal dominant • Radiograph assessment Genotype (AD) trait with incomplete penetrance and variable • Tooth eruption history obtained from dental history form Co-Dominant Mode of Inheritance: Yates p = 0.451 expressivity. • Missing one or more teeth, excluding third molars • Environmental – Non-genetic factors that can lead to • Variations in the general dentition (making note of hypodontia include trauma, infection of developing tooth peg-shape and/or small) Figure 7: Percentage of Subjects with bud, radiation, chemotherapy, drug exposure, and systemic diseases (i.e., Rickets, syphilis, and rubella) • Saliva Collection and DNA Isolation Hypodontia and Controls for each

" rs1364658 Genotype – ACVR2A • 2-4 mL of saliva collected using Oragene-DNA Collection Kits (DNA Genotek Inc., Ottawa, 70 " Ontario, Canada) 62.5% Control Hypodontia Dempsey • Genomic DNA isolated from saliva/OrageneDNA stabilized 60 * * mixture by ethanol precipitation and resuspended in 10mM Photos courtesy of Dr. Jerrod Photos courtesy of Dr. Cynthia Beeman Photos courtesy of Dr. Tris-HCL, 1 mM EDTA pH 8.0 50 • DNA concentrations measured on NanoDrop-1000 HYPODONTIA is the agenesis of 5 or less teeth.! Variations in tooth shape and size, " 41.2% 42.6% ie peg laterals." spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) 40 33.3% Gene knock out (KO) studies in mouse models have helped to • SNP Selection Using SNPBrowser4.0 (Applied Biosystems) 30 define 300+ genes involved in tooth development. Based on Tooth development Subjects these studies, it was shown that ActivinβA (INHBA) and the Activin Reviewsrs2877098 (INHBA) 20 16.2% Type IIA Receptor (ACVR2A) play an important role in mouse rs17625845 (INHBB) incisor and/or mandibular molar development. In mouse models, rs1364658 (ACVR2A) 10 ActivinβB (INHBB) KO did not appear to affect mouse tooth 4.2% development. Control or Hypodontia Total % of n=11 n=1 n=28 n=8 n=29 n=15 FIGURE 3. Reciprocal signaling during the early(Matzuk, M.M.,phases et al. (1995) Natureof tooth374:354-6; Matzuk development, M.M., T.R. Kumar, and A. Bradley (1995) Nature 374:356-60; Ferguson, C.A., et al. 0

(2001) Development 128: 4605-4613; Knight, P.G. and C. Glister (2003) Anim Reprod Sci 78:165-83.) TT CT CC Initiation-stage Bud-stage Cap-stage Genotype

Co-Dominant Mode of Inheritance: p = 0.157 Epithelial signaling Mesenchymal signaling Enamel knot signaling Activin βA –/– Lef1 –/– Dental Dlx1/Dlx2 –/– Dental epithelium" Msx1 –/– Dental epithelium" epithelium Msx1/Msx2–/– Pax9 –/– Conclusions Dental mesenchyme • SNP Genotyping was conducted using Taqman® Genotyping ® Dental Mesenchyme" Dental Mesenchyme" Assay Kits (Applied Biosystems) in the Roche LightCycler 480 TIG February 1999, volume 15, No. 2 Instrument (Roche Applied Science, Indianapolis, IN) • The null hypothesis could not be rejected for this Msx2 sample size of patients and the SNPs: Lef1 Since many difference exist between mouse and humanBMP4/2 tooth SHH BMP4 FGF8 developmentp21 (i.e. mouse incisors grow continuously during their • rs2877098 (INHBA) lifetime, mice do not have only 2 incisors perMsx2 arch and lack p21 Bmp2 • rs17625845 (INHBB) canine and premolar-like teeth), we wanted to investigate the • rs1364658 (ACVR2A) role of these three genes in human tooth agenesis.Apoptosis Fgf3 Proliferation Proliferation ActβA Msx1 Pax9 FGF4/9 Our null hypothesis states: SNP variations within or near the INHBA • Analysis of SNP rs2877098 (INHBA), yielded a p-value of Ptc Msx1 Pax9 (rs2877098), INHBB (rs17625845 )and/or ACVR2A (rs1364658) 0.085 under the co-dominant model of inheritance. Gli1 Dlx2genes are not associated with NMT/agenic teeth. Given a larger sample population, this might prove to Bmp4 • Statistical analysis be significant. We are continuing to consent BMP2 Ptc Proliferation individuals in the clinic to expand this study. Figure 2: Lef1The Activin/Inhibin Genes • Departures from Hardy-Weinberg Equilibrium (HWE) were assessed using Pearson’s chi-square (2) test. Encode which Generate • Chi-Square test for trend was utilized to evaluate the potential • In additional observations that our group has made association of each SNP with hypodontia using a co-dominant (not described in this poster), we have identified a Multiple Dimer Complexes mode of inheritance. The upper panel shows three stages of early tooth morphogenesis, and white arrows indicate the direction of inductive events. The lower panel summarizes the cur- novel association of rs7576183 near the INHBB gene (Preacher, K. J. (2001, April). Calculation for the chi-square test: An interactive calculation tool for chi-square tests of goodness of fit and rent knowledge about the molecular pathways at each stage. At the initiation stage, the epithelium starts to induce tooth development. FGFs and BMPs differen- independence [Computer software]. Available from http://quantpsy.org.) and human hypodontia. This SNPs is associated tially act on the expression of Pax9 and Msx1 in the dental mesenchyme. At the bud stage, the mesenchyme dominates tooth development. Presumably a key func- predominately with hypodontia of incisors of the tion of Pax9 and Msx1 at this stage is the maintenance of mesenchymal Bmp4 expression. BMP4 signals back to the epithelium and induces the expression of p21 maxillary arch. and Msx2 which are associated with programmed cell death. Already at the bud stage, cells at the center of the epithelium are withdrawn from the cell cycle (encir- Figure 4: Clinical Correlate cled by the broken line). At the cap stage, the enamel knot is formed and secretes factors which induce apoptosis in the knot itself. Simultaneously, the enamel knot • It was found that females experience hypodontia secretes FGFs which stimulate proliferation in adjacent cell compartments. Pax9, Msx1 and Activin ␤A are essential in the mesenchyme for tooth morphogenesis to more frequently than males, which could lead to proceed from the bud to the cap stage whereas Lef1 is essential in the tooth epithelium. It should be noted that a simplified model is presented and that signaling Funding cascades in vivo are not as static as implied by the scheme. For details, see text. further studies in dental anomalies.

1 1 A A 3 3 Funding: This project was funded by the National Center for C C 1 Most common affected Research Resources and the National Center for Advancing A tooth these patterns, P. Sharpe has suggested the presence of an developing tooth led to concepts about the molecular con- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Translational Sciences, National Institutes of Health, through Grant nd 2 2 most common affected B odontogenic homeobox code that might determine the trol of organogenesis, which, to a considerable extent, tooth UL1TR000117, an American Association of Women Dentists – 35 32 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 Procter and Gamble Research Scholarship Award, the Southern identity of each individual tooth , a model that assumes a parallels those emerging from the analyses of other organ 3 3rd most common affected C prespecification of neural crest cells. So far, gene disruption systems, such as the developing limb2,36. Experimental tooth Association of Orthodontists Graduate Student Research Grant, a experiments have neither supported nor refuted this model: analyses of early tooth development have recently focussed 2 2 grant from the National Institutes of Health NIH/NIDCR B B 18 R03DE021438, a grant from the NIH Center for Oral/Systemic in Msx1–/– mice, no teeth are formed ; in the absence of on signaling molecules and transcription factors and it Biomedical Research Excellence NIH/NCRR P20RR020145, and the Dlx1 and Dlx2, maxillary molars are not formed whereas can be expected that more factors of both groups will be Preston E. Hicks Endowed Chair. all other teeth are normal31. Both genes are expressed in the characterized in the near future. maxillary and mandibular arch mesenchyme, but in the lat- Interaction of FGF and BMP signaling might be a gen- ter, other members of this gene family (Dlx5, -6, -3, -7) eral mechanism for the regulation of early organ develop- could compensate for the loss of Dlx1, -2. Thus, respecifica- ment. In Xenopus, BMP4 has been suggested to modulate tion of tooth identities have not been observed in these the dorsal–ventral extension of mesoderm induced by experiments. Recently, a transformation of tooth identity FGFs (Ref. 37). In the developing limb of the chicken, from incisor to molar has been experimentally induced by FGF4 mediated outgrowth of the mesenchyme is antag- -mediated inhibition of BMP4 signaling in the distal onized by BMP2 in vitro38. Conversely, FGF4 is able to mesenchyme of the mandibular arch57. The transformation inhibit BMP-induced apoptosis and chondrogenesis, is preceded by a distal expansion of Barx1 expression, which depending on the differentiation stage of the limb mesen- is normally restricted to the future molar region. These find- chyme39. Experimental evidence also indicates that antag- ings support the idea that homeobox genes are involved in onistic BMP and FGF signaling commits cells to either a the determination of individual tooth types. However, the thyrotroph or corticotroph progenitor state during the expression pattern of Barx1 in vivo, like that of other early development of the anterior pituitary40. Moreover, at homeobox genes, appears to be under the control of epithe- the cap stage of tooth development, recent experiments lial signals, rather than predetermined in the neural crest. indicate that combinatorial FGF4 and BMP4 signaling of the enamel knot regulates the balance between cell Outlook proliferation and apoptosis21. Given the large number of In the recent years a substantial number of genes that regu- members in each of the protein family we speculate that late epithelial–mesenchymal interactions during early tooth more examples will be identified in which a combinatorial development have been identified (Table 1) and we are FGF/BMP signaling acts during initiation and morpho- beginning to understand how they interact. Studies on the genesis at other sites of organ formation. However, it will

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