An in vitro and computational validation of a novel loss-of-functional mutation in PAX9 associated with non-syndromic tooth agenesis

Congenital tooth agenesis (CTA) is one of the most common craniofacial anomalies. Its frequency varies among different population depending upon the genetic heterogeneity. CTA could be of familial or sporadic and syndromic or non-syndromic. Five major genes are found to be associated with non-syndromic CTA namely, PAX9, MSX1, EDA1, AXIN2 and WNT10A. In this study, an India family with CTA was investigated and a novel c.336C>G variation was identified in the exon 3 of PAX9, leading to substitution of evolutionary conserved Cys with Trp at 112 amino acid position located at the functionally significant DNA binding paired domain region. Functional analysis revealed that p.Cys112Trp mutation did not prevent the nuclear localization although mutant protein had higher cytoplasmic retention. EMSA using e5 probe revealed that mutant protein was unable to bind with the paired-domain binding site. Subsequently, GST pull-down assay revealed lower binding activity of the mutant protein with its known interactor MSX1. Further RNA-sequencing of PAX9 over-expressed HEK293, identified two potential novel targets, WNT4 and WNT7b those are up-regulated by wild-type PAX9 but not by mutant. These in vitro results were consistent with the computational results. The in vitro and computational observations altogether suggest that c.336C>G (p.Cys112Trp) variation leads to loss-of-function of PAX9 leading to CTA in this family.

Pax9’s Interaction With the Ectodysplasin Signaling Pathway During the Patterning of Dentition

In these studies, we explored for the first time the molecular relationship between the paired-domain-containing transcription factor, Pax9, and the ectodysplasin (Eda) signaling pathway during mouse incisor formation. Mice that were deficient in both Pax9 and Eda were generated, and the status of dentition analyzed in all progeny using gross evaluation and histomorphometric means. When compared to wildtype controls, Pax9+/–Eda–/– mice lack mandibular incisors. Interestingly, Fgf and Shh signaling are down-regulated while Bmp4 and Lef1 appear unaffected. These findings suggest that Pax9-dependent signaling involves the Eda pathway and that this genetic relationship is important for mandibular incisor development. Studies of records of humans affected by mutations in PAX9 lead to the congenital absence of posterior dentition but interestingly involve agenesis of mandibular central incisors. The latter phenotype is exhibited by individuals with EDA or EDAR mutations. Thus, it is likely that PAX9, in addition to playing a role in the formation of more complex dentition, is also involved with EDA signaling in the initiation of odontogenesis within the incisal domain.

Pax7 pioneer factor action requires both paired and homeo DNA binding domains

The pioneer transcription factor Pax7 contains two DNA binding domains (DBD), a paired and a homeo domain. Previous work on Pax7 and the related Pax3 had shown that each DBD can bind a cognate DNA sequence, thus defining two targets of binding and possibly modalities of action. Genomic targets of Pax7 pioneer action leading to chromatin opening are enriched for composite DNA target sites containing juxtaposed binding sites for both paired and homeo domains. The present work investigated the implication of both DBDs in pioneer action. We now show that the composite sequence is a higher affinity Pax7 binding site compared to either paired or homeo binding sites and that efficient binding to this site involves both DBDs. We also show that a Pax7 monomer binds composite sites and that methylation of cytosines within the binding site does not affect binding, which is consistent with pioneer action exerted at methylated DNA sites within nucleosomal heterochromatin. Finally, introduction of single amino acid mutations in either the paired or homeo domain that impair binding to cognate DNA sequences showed that both DBDs must be intact for pioneer action. In contrast, only the paired domain is required for low affinity binding of heterochromatin sites. Thus, Pax7 pioneer action on heterochromatin requires unique protein:DNA interactions that are more complex compared to its simpler DNA binding modalities at accessible enhancer target sites.Significance StatementPioneer transcription factors have the unique ability to recognize DNA target sites within closed heterochromatin and to trigger chromatin opening. Only a fraction of the heterochromatin recruitment sites of pioneers are subject to chromatin opening. The molecular basis for this selectivity is unknown and the present work addressed the importance of DNA sequence affinity for selection of sites to open. The pioneering ability of the pioneer factor Pax7 is not strictly determined by affinity or DNA sequence of binding sites, nor by number or methylation status of DNA sites. Mutation analyses showed that recruitment to heterochromatin is primarily dependent on the Pax7 paired domain whereas the ability to open chromatin requires both paired and homeo DNA binding domains.

PAX6 missense variants in two families with isolated foveal hypoplasia and nystagmus: evidence of paternal postzygotic mosaicism

PAX6 is considered the master regulator of eye development, the majority of variants affecting this gene cause the pan-ocular developmental eye disorder aniridia. Although no genotype-phenotype correlations are clearly established, missense variants affecting the DNA-binding paired domain of PAX6 are usually associated with non-aniridia phenotypes like microphthalmia, coloboma or isolated foveal hypoplasia. In this study, we report two missense heterozygous variants in the paired domain of PAX6 resulting in isolated foveal hypoplasia with nystagmus in two independent families: c.112 C > G; p.(Arg38Gly) and c.214 G > C; p.(Gly72Arg) in exons 5 and 6, respectively. Furthermore, we provide evidence that paternal postzygotic mosaicism is the cause of inheritance, with clinically unaffected fathers and reduced affected allele fraction. This work contributes to increase the phenotypic spectrum caused by PAX6 variants, and to our knowledge, is the first report to describe the presence of postzygotic parental mosaicism causing isolated foveal hypoplasia with nystagmus. These results support the growing evidence that suggest an overestimation of sporadic cases with PAX6 variants, which has strong implications for both genetic counselling and family planning.

Congenital Hypothyroidism Due to Truncating PAX8 Mutations: A Case Series and Molecular Function Studies

Context PAX8 is a transcription factor required for thyroid development, and its mutation causes congenital hypothyroidism (CH). More than 20 experimentally verified loss-of-function PAX8 mutations have been described, and all but one were located in the DNA-binding paired domain. Objective We report the identification and functional characterization of 3 novel truncating PAX8 mutations located outside the paired domain. Methods Three CH probands, diagnosed in the frame of newborn screening, had thyroid hypoplasia and were treated with levothyroxine. Next-generation sequencing-based mutation screening was performed. Functionality of the identified mutations were verified with Western blotting, intracellular localization assays, and transactivation assays with use of HeLa cells. Luciferase complementation assays were used to evaluate the effect of mutations on the interaction between PAX8 and its partner, NKX2-1. Results Each proband had novel truncating PAX8 mutations that were I160Sfs*52, Q213Efs*27, and F342Rfs*85. Western blotting showed destabilization of the I160fs-PAX8 protein. Q213fs-PAX8 and F342fs-PAX8 showed normal protein expression levels and normal nuclear localization, but showed loss of transactivation of the luciferase reporter. By luciferase complementation assays, we showed that PAX8-NKX2-1 interaction was defective in Q213fs-PAX8. We also characterized the recombinant PAX8 proteins, and found that the protein sequence corresponding to exon 10 (363-400 aa residues) was essential for the PAX8-NKX2-1 interaction. Conclusions Clinical and molecular findings of 3 novel truncating PAX8 mutations located outside the paired domain were reported. Experiments using cultured cells and recombinant proteins showed that the C-terminal portion (ie, 363-400 aa) of PAX8 is required for the PAX8-NKX2-1 interaction.