| Objective: To determine if variations in WNT10A are the cause of autosomal dominant tooth agenesis phenotype observed in a three-generation multiplex family.;Materials and Methods: This study was approved by the UTHSC Committee for the Protection of Human Subjects (HSC-DB-12-0255). A three-generation multiplex family showing autosomal dominant tooth agenesis was recruited at the Orthodontic clinic at the University of Texas Health Science Center School of Dentistry. Saliva samples from the study family were collected using Oragene-DNA Collection Kits. Genomic DNA from saliva was extracted using established protocols (Trevilato and Line, 2000). To amplify exons 1-4 of the WNT10A gene, specific primers were designed using Primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/). PCR was performed and for the samples that appeared to have the correct exon amplified, the gels were purified using NucleoSpin Gel and PCR Clean-Up (MACHEREY-NAGEL, Bethlehem, PA). For the samples with adequate DNA concentration (>15 ng/microliter), direct sequencing of WNT10A exons was performed. Samples were submitted to SeqWright Genomic Services. Samples that failed the direct sequencing method (likely due to poor DNA quality) were submitted to Taqman genotyping (Hui et al., 2008).;Results: Direct sequencing was completed for exon 1 in I.2, II.2, III.1, and III.3. For exon 2, no direct sequencing was completed. For exon 3, direct sequencing was completed in I.1, I.2, II.2,III.1, and III.3. For exon 4a, direct sequencing was completed in all individuals. For exon 4b, direct sequencing was completed in I.1, I.2, and III.3. Exon 4c direct sequencing was completed for I.1, I.2, II.2, III.1, and III.3 (Table 2). Overall, no mutations were identified by direct sequencing (Figure 9). Two WNT10A mutations were identified in the proband using the Taqman method: c. 149 C>T (Pro50Leu, rs199980023) and c. 827 G>T (Cys276Phe, rs201593552). These mutations are located in exons 2 and 4, respectively. For rs199980023 (Pro50Leu), the proband presented a TT genotype whereas the proband's mother and maternal grandmother, who were also affected presented CT and CT genotypes. For rs201593552 (Cys276Phe), the proband and her mother presented with a TT genotype whereas the proband's grandmother presented GT phenotype (Figure 10).;Conclusions: Two mutations in WNT10A [c. 149 C>T (Pro50Leu, rs199980023) and c. 827 G>T (Cys276Phe, rs201593552)] were found in our study proband that may be contributing to the oligodontia phenotype. These mutations segregated with the phenotype in affected individuals. Both mutations are rare and have not yet been reported in individuals with tooth agenesis. Additional studies are currently underway to investigate the presence of these mutations in other families with tooth agenesis and control families. Overall, this study further supports a role for WNT10A mutations in the etiology of familial tooth agenesis. |
