Identification and Functional Analysis of Two Novel <i>PAX9</i> Mutations

Abstract

The paired-domain transcription factor PAX9 plays a critical role in tooth development, as heterozygous mutations in PAX9 have been shown to be associated with human tooth agenesis. In this study, we report 2 novel missense mutations, gly6arg (G6R) and ser43lys (S43K), in the paired domain of PAX9 in Chinese patients with varying degrees of nonsyndromic tooth agenesis. Excluding third molars, the individual with the G6R mutation was missing 2 mandibular incisors and a maxillary premolar, while the phenotype of individuals with the S43K mutation consisted of peg-shaped upper lateral incisors and missing molars, premolars and canines. As these 2 mutations occur at highly conserved amino acids in the PAX gene family and between different species, we further analyzed the effects of the mutations on the function of the resulting proteins. Immunofluorescence and immunoblotting studies showed that the mutations did not alter nuclear localization in mammalian cells. Gel shift and super shift assays indicate that both mutant proteins bound DNA at a lower level than the normal protein, with G6R having a greater affinity for DNA than S43K. Likewise, the G6R protein was able to transcriptionally activate a Bmp4 promoter construct to a greater extent than S43K. Our finding that the severity of tooth agenesis in the patients was correlated to the DNA-binding capacity of the mutated PAX9 9proteins supports the hypothesis that DNA binding is responsible for the genetic defect.

Novel PAX9 and COL1A2 Missense Mutations Causing Tooth Agenesis and OI/DGI without Skeletal Abnormalities

“…No MSX1 mutation could be identified, but a PAX9 missense mutation substituting isoleucine for a highly conserved phenylalanine (p.Phe15Ile) was identified in the paired box domain crucial for DNA binding. Wang et al identified a PAX9 mutation (p.Gly6Arg) causing a mild hypodontia phenotype almost identical to that of our proband’s mother [19]. Interestingly, our p.Phe15Ile substitution is structurally close to the p.Gly6Arg mutation.…”

PAX9 gene mutations and tooth agenesis: A review

“…Another in vitro study using the PAX9 protein comprising a mutated paired domain with amino acid change Arg28Pro revealed dramatically reduced ability to form protein‐DNA complex . Wang et al made functional analysis on Gly6Arg and Ser43Lys substitutions and reported no effect on nuclear localization of PAX9 but found reduced DNA‐binding activity of the mutant protein compared to the wild type. These findings were provided with additional support by observations of Zhao et al who studied Arg47Trp amino acid change.…”

Mutations in WNT10B Are Identified in Individuals with Oligodontia

“…individuals with hypodontia were mandibular second premolars, followed by maxillary lateral incisors, as reported in a recent meta-analysis, 33 it is likely that there is an overlapping phenotype in some of the individuals with WNT10A and WNT10B mutations. The selective pattern in WNT10B mutants is also different from that of mutations in other oligodontia-associated genes, such as MSX1 (missing second premolar) and PAX9 (agenesis of molars), as described previously 7,10,11,34 and currently in the individuals with the p.Ile97Leufs*217 variant ( Figure S1). Regardless, protein-protein-interaction and genetic studies have shown crosstalk between PAX9 and the Wnt pathway (e.g., AXIN2) or MSX1 in dental tissues, 11 which are essential for the establishment of the odontogenic potential of the mesenchyme.…”

“…5 By Sanger sequencing of these oligodontia-associated genes, we have previously identified mutations in most of these genes in Chinese individuals with non-syndromic oligodontia. [6][7][8][9][10] However, no mutations were detected in nearly half of the examined affected individuals, suggesting the existence of unidentified genetic etiologies. Presumably, additional crucial components in several defined canonical pathways (such as BMP, FGF, NF-kB, P53, PAX, and Wnt, which involve tooth development) could be the candidates.…”