Functional characterization of ATF1, GREM2 AND WNT10B variants associated with tooth agenesis

To determine the functional effects of ATF1, WNT10B and GREM2 gene variants identified in individuals with tooth agenesis (TA).

Stem cells from human exfoliated deciduous teeth (SHED) were used as an in vitro model system to test the effect of TA-associated variants.

Plasmid constructs containing reference and mutant alleles for ATF1 rs11169552, WNT10B rs833843 and GREM2 rs1414655 variants were transfected into SHED for functional characterization of variants. Allele-specific changes in gene transcription activity, protein expression, cell migration and proliferation, and expression of additional tooth development genes (MSX1, PAX9 and AXIN2) were evaluated. Data analyses were performed using Student's t-test. P-values ≤ .05 were considered statistically significant.

Mutant variants resulted in significantly decreased transcriptional activity of respective genes (P < 0.05), although no changes in protein localization were noted. Expression of MSX1 was significantly decreased in ATF1- and GREM2-mutant cells, whereas PAX9 or AXIN2 mRNA expression was not significantly altered. Mutant WNT10B had no significant effect on the expression of additional TA genes. ATF1- and GREM2-mutant cells presented increased cell migration. Cell proliferation was also affected with all three mutant alleles.

Our results demonstrate that ATF1, WNT10B and GREM2 mutant alleles have modulatory effects on gene/protein function that may contribute to TA.