Exploring the role of TWIST1 in malocclusion and craniofacial morphology

Despite increasing evidence that common genetic variation contributes to variation in jaw and cranial base morphology, the biological mechanisms underlying malocclusion remain poorly defined. This study tested the hypothesis that a noncoding variant near TWIST1 alters craniofacial development by disrupting transcriptional regulation, contributing to skeletal phenotypes associated with malocclusion.

In a cohort of 277 non-syndromic individuals with malocclusion, we performed targeted genotyping and deep sequencing of the TWIST1 locus, followed by multivariate genotype-phenotype correlation analyses. To evaluate regulatory function, we performed luciferase reporter assays and chromatin immunoprecipitation in multiple cell lines. Craniofacial consequences of Twist1 loss of function were characterized using 3D morphometrics and craniometric analysis in conditional knockout mice at postnatal days 14 and 21.

The SNP rs2189000, located 4.2 kb upstream of TWIST1, showed a significant association with mandibular and anterior cranial base shape (P = 0.0003). No coding mutations were detected. Functional assays revealed that rs2189000 disrupts a conserved PITX2 binding site, abolishing PITX2-mediated activation of TWIST1 transcription. In mice, mesoderm-specific deletion of Twist1 produced craniofacial changes, such as domed skulls, mandibular shortening, palatal rotation, and facial asymmetry, that paralleled the human phenotypic associations. Additionally, premature closure of the cranial base synchondroses was observed, indicating a mechanistic link to disrupted postnatal growth trajectories.

This study identifies a putative functional noncoding variant that dysregulates TWIST1 via disruption of PITX2 DNA binding and links this effect to postnatal craniofacial phenotypes in both humans and mice. These findings expand the developmental and genetic framework for understanding malocclusion and suggest a broader role for TWIST1 in cranial base growth and midface patterning.