Functional study of novel PAX9 variants: The paired domain and non‐syndromic oligodontia

Abstract

Objectives To investigate pathogenic variants of the paired box 9 (PAX9) gene in patients with non‐syndromic oligodontia, and the functional impact of these variants. Subjects and Methods Whole exome sequencing and Sanger sequencing were utilized to detect gene variants in a cohort of 80 patients diagnosed with non‐syndromic oligodontia. Bioinformatic and conformational analyses, fluorescence microscopy and luciferase reporter assay were employed to explore the functional impact. Results We identified three novel variants in the PAX9, including two frameshift variants (c.211_212insA; p.I71Nfs*246 and c.236_237insAC; p.T80Lfs*6), and one missense variant (c.229C > G; p.R77G). Familial co‐segregation verified an autosomal‐dominant inheritance pattern. Conformational analyses revealed that the variants resided in the paired domain, and could cause corresponding structural impairment of the PAX9 protein. Fluorescence microscopy showed abnormal subcellular localizations of frameshift variants, and luciferase assay showed impaired downstream transactivation activities of the bone morphogenetic protein 4 (BMP4) gene in all variants. Conclusions Our findings broaden the spectrum of PAX9 variants in patients with non‐syndromic oligodontia and support that paired domain structural impairment and the dominant‐negative effect are likely the underlying mechanisms of PAX9‐related non‐syndromic oligodontia. Our findings will facilitate genetic diagnosis and counselling, and help lay the foundation for precise oral health therapies.

Patterns of molar agenesis associated with p.P20L and p.R77Q variants in PAX9

“…Microdontia or peg-shaped lateral incisors were observed in 6/10 cases (60%) (II:4 was excluded due to uncertain microdontia phenotype). These findings are consistent with previous studies, reviews, and meta-analyses demonstrating that the heterozygous PAX9 variants are associated with certain tooth agenesis phenotypes, including (1) oligodontia, (2) nonsyndromic, (3) molar dominated, and (4) microdontia or peg-shaped lateral incisors [11,15,16,31,32]. Consistently, a review paper by Fournier et al (2018) showed that the absence of upper and lower third molars was observed in 95% and 90% of the patients with PAX9 variants, respectively.…”

“…We identified that the p.Arg77Gln did not compromise the intracellular localization of PAX9, but significantly reduced the transactivation of BMP4, the downstream target of PAX9. Recently, the PAX9 variant c.229C > G (p.Arg77Gly), adjacent to the c.230G > A, was reported in a patient who had 17 missing teeth (four third molars, four second molars, two first molars, three premolars, one canine, and two maxillary lateral incisors) [11]. The p.Arg77Gly reduced BMP4 transactivation and localized in the nucleus, similar to the p.Arg77Gln.…”

“…Similarly, the other missense variants in the PAX9 paired domain also normally localized in the nucleus, but significantly reduced BMP4 expression [15,35]. In contrast, the truncating variants in the paired domain, although demonstrating reduced BMP4 transactivation, were localized in both the nucleus and cytoplasm [11]. Interestingly, the Proband-2 and her father were only missing their molars.…”

“…Tooth agenesis is one of the most common congenital anomalies in humans with a prevalence ranging from 2.5 to 13.4% [1,2]. The congenital absence of 1-6 teeth is termed hypodontia, whereas the absence of more than six teeth is generally Nonsyndromic tooth agenesis can be caused by pathogenic variants in several odontogenic genes, including MSX1 [6], AXIN2 [7], PITX2 [8], EDA [9], WNT10A [10], and PAX9 [11]. The paired box 9 (PAX9) gene is a member of the pairedbox family of transcriptional factors that regulate organogenesis and neural crest cell development.…”

Four Novel PAX9 Variants and the PAX9-Related Non-Syndromic Tooth Agenesis Patterns

“…To date, a number of genes have been found to be associated with non-syndromic tooth agenesis, including axin inhibition protein 2 ( AXIN2 ), muscle segment homeobox 1 ( MSX1 ), paired box 9 ( PAX9 ), ectodysplasin A ( EDA ), wingless-type mouse mammary tumor virus integration site family member 10A ( WNT10A ), wingless-type mouse mammary tumor virus integration site family member 10B, low-density lipoprotein receptor-related protein 6 ( LRP6 ), and opsin 3 ( OPN3 ) [ 2 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ]. As one of the earliest discovered pathogenic genes of tooth agenesis, PAX9 has been a research hotspot [ 2 ].…”