113th AAO Annual Session OVERVIEW

Unraveling an Association between Hypodontia and OUTLINE Epithelial Ovarian Cancer

1. Introduction Anna N Vu, DMD, MS 2. Background

3. Purpose Division of Orthodontics 4. Materials and Methods May 2013 5. Results 6. Discussion 7. Conclusion

U N I V E R S I T Y O F K E N T U C K Y C O L L E G E O F D E N T I S T R Y

HYPODONTIA HYPODONTIA REVIEW & CANCER

• Over 300 genes are involved in odontogenesis including MSX1, PAX9, and AXIN2 HYPODONTIA • Genes involved in dental development also have roles in other organs of the body

Defined as the developmental absence of one or more teeth as well as variations in size, • Mutation in several genes governing tooth development have already been associated with shape, rate of dental development and eruption time. cancer

• Mutations in AXIN2 cause familial tooth agenesis and predispose to colorectal cancer7 Hypodontia is the agenesis of 6 or less teeth. • AXIN2 gene is highly expressed in ovarian tissue so may play a role in epithelial ovarian cancer (EOC)8 Oligodontia is the agenesis of 6 or more teeth. Anodontia is the agenesis of all teeth. • Reduced expression of PAX9 can lead to hypodontia and has been correlated with increased malignancy of dysplastic and cancerous esophageal epithelium9 2.6-11.3% reported prevelance worldwide. 78

• RUNX transcription factor family (RUNX1, 2, and 3) are involved in odontogenesis and has been Women are affected more than males at a ratio of 3:2. the most targeted genes in acute myeloid leukemia and acute lymphoblastic leukemia10

Both genetic and environmental explanations for hypodontia have been reported. • RUNX2 is amplified in various cancers including osteosarcoma and may play a role in breast and prostate cancer

WHAT IS WHAT IS OVARIAN CANCER? OVARIAN CANCER?

• Mainly affects women over the age of 40 OVARIAN CANCER • Symptoms include: • Vaginal bleeding Ovarian cancer is a gynecological cancer that begin in the ovaries. • Pain or pressure in pelvic region or abdominal area • Back pain • Bloating 70% • Change in bathroom habits Although ovarian cancer only ranks of women • Increased risk of EOC is correlated with: 8th in most common cancer in diagnosed with • ovarian cancer Family history of ovarian cancer women…. do not survive. • Increasing age • History of breast, uterine, or colorectal cancer • Infertility …it is the 5th leading cause of cancer • Endometriosis

death in women. Inside Knowledge: Ovarian Cancer. Atlanta, GA: Center for Disease Control and Prevention, May 2010.

OVARIAN CANCER OVARIAN CANCER IN THE LITERATURE IN THE LITERATURE

• Supports a possible association between hypodontia and EOC PURPOSE • Suggests hypodontia may potentially become a risk marker for future cancer development • Improved genetic screenings to identify persons at high risk for developing EOC JADA 2008; 139(2): 163-9. • Refer female patients to get earlier screenings for EOC • Compare prevalence rates • FINDINGS of hypodontia among Earlier EOC diagnosis and treatment could save many lives epithelial ovarian cancer (EOC) subjects and control • Prevalence of hypodontia was 20% for EOC subjects (p< .001) subjects • Explore the possible • Reported family hx of hypodontia and ovarian cancer was higher in genetic association EOC subjects between the two phenotypes • EOC subjects were 8.1 times more likely to have hypodontia than women without EOC

GENE SELECTION: PURPOSE Locus 8q24

Susceptibility Loci for Ovarian Cancer PURPOSE

• Previous genome wide association study (GWAS) identified a ovarian cancer susceptiblity loci at 9q22 and found a 20% reduction in risk with each copy of the minor alleles23 To investigate whether SNP rs10088218 is genetically associated with hypodontia.

• Most recent GWAS identified two new susceptibility loci for ovarian cancer

• 2q31 (p= 3.8 x 10-14): rs2072590 • 8q24 (p= 8.0 x 10-15): rs10088218, rs1519682 and rs10098821 NULL • SNP rs10088218 is NOT associated with hypodontia. SNPs at these two loci showed strong support for an association with ovarian cancer with p-values Hypothesis < 0.001. (HO) • At locus 8q24, minor allele (A) of SNP rs10088218 was associated with a decreased risk for ovarian cancer SNP rs10088218 is associated with hypodontia. ALTERNATIVE Hypothesis

(HA)

Goode EL, et al. Genome wide association study identifies susceptibility loci for ovarian cancer at 2q31 and 8q24. Nat Genet 2010; 42(10): 874-81.

MATERIALS MATERIALS AND METHODS AND METHODS

Saliva Collection Subject Population • 2-4 mm of saliva was collected from all study participants • Oragene-DNA Collection Kits (DNA Genotek Inc., Ottawa, Ontario, Canada) • • 110 Caucasian subjects were recruited from the orthodontic clinic at the University of Preservatives in container will preserve DNA for 5+ years

Kentucky College of Dentistry DNA Isolation • 67 females, 43 males • Genomic DNA extracted and resuspended in 10 mM Tris-HCl, 1 mM EDTA pH 8.0, and stored at 20° C

• DNA isolated and purified via ethanol precipitation • Subjects were classified into two research groups: • DNA concentrations measured on the NanoDrop-1000 spectrophotometer (Thermo Fischer Scientific, Wilmington, DE) • Controls: patients without hypodontia Taqman® Gene Assay Kits • Subjects: patients with hypodontia • Analysis of all SNPs were performed on the genomic DNA utilizing Taqman® Genotyping Assay Kits on Roche LightCycler 480®

• Following RT-PCR amplification, each SNP allele was identified using allelic specific probes • VIC or FAM fluorescence

• Analyze SNP rs10088218

MATERIALS RESULTS AND METHODS SNP rs10088218

Statistical Methods Frequency of Missing • Genotyping of SNP rs10088218 was completed in all 30 subjects with Teeth NMT and 80 controls. • Same as previously • 2 The Chi-Square (x ) Analysis • reported No deviation from HWE was observed in either the controls or the • entire cohort. Used to evaluate the potential association of each SNP with hypodontia • Maxillary lateral incisor • Co-dominant mode on inheritance was utilized for potential association was the most commonly • SNP rs10088218 was significantly associated with the presence of • Significance set at an alpha of 5% (p<0.05) missing tooth hypodontia in 25 individuals versus 80 controls (p=0.019) with an

odds ratio (OR) of 11.51 (95% confidence interval (CI) 1.49-88.98) • The next most commonly under an additive mode of inheritance (MOI). missing tooth type included the mandibular • SNP rs10088218 was also significantly associated with the presence second premolars of hypodontia in 30 individuals versus 80 controls (p=0.021; OR=4.37 followed by maxillary second premolars 95%CI: 1.25-15.35) under an additiven= 3 MOI. n= 0

• Females affected more than males at a ratio of 3:1

RESULTS DISCUSSION SNP rs10088218 & CONCLUSIONS

Number of individuals within the group with Conclusions a given genotype OR* Group (% of the total group) (95% Confidence p-Value • Hypodontia patients GG AG AA Interval) (%) (%) (%) are 11.5x more likely • Hypodontic teeth reported in this study are consistent with those previously Hypodontia to be homozygous for reported missing in women diagnosed with EOC and hypodontia 24 1 0 Subjects Only the G allele than (96.0) (4.0) (0) 11.51 n=25 0.019 (95% CI:1.49-88.98 ) controls • The null hypothesis was rejected Controls 54 23 3 n=80 (67.5) (28.75) (3.75) • Odds ratio reduces • SNP rs10088218 is significantly associated with the occurrence of hypodontia

Hypodontia with addition of (p= 0.019) and 27 3 0 oligodontia patients Oligodontia (90.0) (10.0) (0) 4.37 Subjects 0.021 (95% CI: 1.25-15.35) n=30 Controls 54 23 3 n=80 (67.5) (28.75) (3.75)

FUTURE REFERENCES DIRECTION

1. Arte S and S Pirinen. Hypodontia. Orphanet Encyclopedia. 2004. 2. Peck S et al. Site –specificity of tooth agenesis in subjects with maxillary canine malpositions. Angle Orthod 1996; 66: 473- • 476. Continue to expand the sample size in order to increase the power of the study 3. Al-Nimri KS and E Bsoul. Maxillary palatal canine impaction displacement in subjects with congenitally missing maxillary lateral incisors. Am J Orthod Dentofacial Orthop 2011; 140(1): 81-86. • Test for association of SNP rs10088218 with ovarian cancer in EOC patients 4. Jena AK and R Duggal. The pattern of maxillary canine impaction in relation to anomalous lateral incisors. J Clin Pediatr Dent 2010; 35(1): 37-40. • Look at other potential markers 5. DeCoster PJ et al. Dental Agenesis: Genetic and Clinical Perspectives. J Oral Pathol Med (2009). 38: 1-17. 6. Beeman CS. Prevalence of hypodontia in north america: regional and international comparisons. University of Kentucky. • SNP rs2072590 that has been associated with an increased risk for ovarian caner Lexington, KY 2009. Lecture. 7. Lammi L, et al. Mutations in AXIN2 cause familial tooth agenesis and predispose to colorectal cancer. Am J Hum Genet • WNT 10A which has been associated with hypodontia and has a presence in ovarian tissue 2004; 74(5): 1043-50. 8. Dong X, et al. Genomic structure, chromosome mapping and expression analysis of the human AXIN2 gene. Cytogenet Cell • Test these SNPs in ovarian cancer patients to find a common gene involved with both hypodontia Genet 2001; 93 (1-2): 26-8. and ovarian cancer 9. Gerber JK, et al. Progressive loss of PAX9 expression correlates with increasing malignancy of dysplastic and cancerous epithelium of the human esophagus. J Pathol 2002; 197(3): 293-7. 10. Song H et al. A genome-wide association study identifies a new ovarian cancer susceptibility locus on 9p22.2. Nat Genet 2009; 41: 996-1000.

RESEARCH REFERENCES SUPPORT

11. Ohno K and Ohmori I. Anodontia with hypohidrotic ectodermal dysplasia in a young female: a case report. Pediatric Dentistry 2000; 22(1): 49-52. 12. Fleischmannova J et al. Formation of the tooth-bone interface. J Dent Res 2010;89:108-115. 13. Inside Knowledge: Ovarian Cancer. Atlanta, GA: Center for Disease Control and Prevention, May 2010. 14. U.S. Cancer Statistics Working Group. United States Cancer Statistics: 1999–2007 Incidence and Mortality Web-based American Association of Women Dentists/Proctor & Gamble Research Scholarship Award Report. Atlanta (GA): Department of Health and Human Services, Centers for Disease Control and Prevention, and National Cancer Institute; 2010. Southern Association of Orthodontists 15. Hoskins WJ. Prospective on ovarian cancer: why prevent? J Cell Biochem. 1995. S23: 189-99. E. Preston Hicks Endowed Chair (JKH) 16. Chalothorn LA et al. Hypodontia as a risk marker for epithelial ovarian cancer: a case-controlled study. JADA 2008; 139(2): 163-169. 17. White SP. Evaluation of a common genetic etiology in ovarian cancer and tooth agenesis. Master’s Thesis, Texas A&M University System Health Sciences Center. Ann Arbor: ProQuest/UMI, 2008. (Publication No. UMI 1457516) 18. Venkatesh K et al. Regulation of bone mass by Wnt signaling. J Clin Invest 2006; 116(5): 1202-1209. 19. Kimori T et al. Targeted disruption of Cbfa1 results in complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 1997; 89(5): 755-764. 20. Camilleri S et al. Runx2 and dental development. European Journal of Oral Sciences 2006; 114(5): 361-373. 21. Aberg T et al. Phenotypic changes in dentition of Runx2 homozygote-null mutant mice. J Histochem Cytochem 2004; 52(1): 131-139. 22. Goode EL et al. Genome-wide association study identifies susceptibility loci for ovarian cancer at 2q31 and 8q24. Nat Genet 2010; 42(10): 874-81. 23. Meyer KB et al. Allele-specific up-regulation of FGFR2 increases susceptiblity to breast cancer. PLoS Biol 2008; 6(5): 1098- 103.