Identification and functional analysis of two novel PAX9 mutations

The paired-domain transcription factor, PAX9, plays a critical role in early tooth morphogenesis. Heterozygous mutations in PAX9 have been shown to be associated with human hypodontia.

Objectives: The aims of this study were to determine if alterations in PAX9 in Chinese patients were responsible for permanent tooth agenesis, and if so, to investigate consequences on the function of the resulting protein.

Methods: DHPLC analysis and sequencing of PAX9 gene were used to identify the mutations. Site-directed mutagenesis was then used to generate missense mutations in a PAX9 expression vector. Immunofluorescence and immunoblotting were used to determine subcellular protein expression. Gel-shift assay was used to analyze the binding ability of the mutant proteins. Transcriptional activity of the mutant proteins was determined with a Bmp4 luciferase reporter assay.

Results: Two novel missense mutations, G16A (G6R) and G128A and C129A (S43K) in the paired domain of PAX9 were identified in patients with varying degrees of hypodontia. The protein products for both mutations were found to be expressed in the nucleus. Gel-shift assay showed that the G6R mutant protein was able to bind DNA with greater affinity than S43K. Our luciferase reporter assay showed the G6R protein was able to transcriptionally activate a Bmp4-promoter with greater activity than S43K.

Conclusions: Our analysis of tooth agenesis in the two affected families revealed differences in phenotypes, with individuals with the S43K mutation more severely affected than the G6R mutation. Our functional analysis of the mutant proteins showed that the severity of tooth agenesis observed in the patients correspond with the level of defects observed for the respective mutant proteins, thereby supporting the notion of genotype-phenotype correlations for PAX9 mutations.

This work was supported by NIH U24 (DE16472), TAMHSC-VPR grant, NIH K08(DE16346) and Beijing Natural science foundation (7063099).