Position of the identified PAX9 mutation.

<p>(A) Locations of P20L and previously reported missense variants are shown with arrows above and below the PAX9 diagram, respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186260#pone.0186260.ref012" target="_blank">12</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186260#pone.0186260.ref025" target="_blank">25</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186260#pone.0186260.ref031" target="_blank">31</a>]. Reported pathogenic variants with nonsense mutations or frameshifts are omitted. NSD, N-terminal subdomain of the paired domain; CSD, C-terminal subdomain of the paired domain. (B) Modeled pair-domain structure of the wild-type (blue and red with green backbone) and P20L (yellow) PAX9. P20L mutation results in Van del Waals clashes with the carbonyl oxygen of Leu²¹ (red disc) and the side chain of Pro⁶⁸ (brown disc). This results in a slight displacement of Pro⁶⁸. DNA is shown in pink with a gray surface.</p

Luciferase reporter assay using the <i>BMP4</i> promoter region as a cis element.

<p>Activity of firefly luciferase expressed from the reporter vector was normalized with <i>Renilla</i> luciferase, and shown as arbitrary units (mean ± SEM). Experiments were repeated three times with independent transfections, and for each experiment luciferase activity was measured in triplicate. Protein samples were prepared from aliquots of transfected cells, and ectopic PAX9 expression was verified by immunoblotting. An alleviating effect of P20L on transactivation was supported statistically, while that of A240P was not, as shown above the graph.</p

Electrophoretic mobility shift assay of PAX9^WT and PAX9^P20L.

<p>Nuclear extract was prepared at 48 h after transfection with PAX9-Myc-expressing (WT or P20L) or 1st ATG-deleted plasmids (Null), and analyzed using the CD19-2(A-ins) probe. Ectopic expression of PAX9-Myc and integrity of nuclear extract were confirmed with immunoblotting (bottom). Excessive amount of the non-biotinylated DNA eliminated the mobility shift signals in PAX9^WT, whereas addition of anti-Myc antibody resulted in a super-shift, confirming the specificity of the bipartite interaction. No detectable degree of signal shift was observed in PAX9^P20L. No NE, no nuclear extract added; L probe, biotin-labelled probe; NL probe, non-labelled probe.</p

Pedigree with tooth genesis involving three patients.

<p>(A) Proband (patient ID 9 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186260#pone.0186260.t001" target="_blank">Table 1</a>), her father (ID 7) and brother (ID 10) are affected (indicated with filled symbols), while her mother is unaffected (indicated with an open symbol). The inheritance pattern is consistent with autosomal dominance. The three affected members invariably show C/T heterozygosity at the second position of the Pro²⁰ codon, while the unaffected member shows C/C homozygosity. (B) Radiogram of the proband (patient ID 9 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186260#pone.0186260.t001" target="_blank">Table 1</a>). Missing teeth other than 3rd molars are indicated with asterisks.</p