Mutations in TCF12, encoding a basic helix-loop-helix partner of TWIST1, are a frequent cause of coronal craniosynostosis

Abstract

Craniosynostosis, the premature fusion of the cranial sutures, is a heterogeneous disorder with a prevalence of ∼1 in 2,200 (refs. 1,2). A specific genetic etiology can be identified in ∼21% of cases, including mutations of TWIST1, which encodes a class II basic helix-loop-helix (bHLH) transcription factor, and causes Saethre-Chotzen syndrome, typically associated with coronal synostosis. Using exome sequencing, we identified 38 heterozygous TCF12 mutations in 347 samples from unrelated individuals with craniosynostosis. The mutations predominantly occurred in individuals with coronal synostosis and accounted for 32% and 10% of subjects with bilateral and unilateral pathology, respectively. TCF12 encodes one of three class I E proteins that heterodimerize with class II bHLH proteins such as TWIST1. We show that TCF12 and TWIST1 act synergistically in a transactivation assay and that mice doubly heterozygous for loss-of-function mutations in Tcf12 and Twist1 have severe coronal synostosis. Hence, the dosage of TCF12-TWIST1 heterodimers is critical for normal coronal suture development.

Figure 1

Phenotype associated with TCF12/Tcf12 haploinsufficiency in humans and mice. (a,b) Clinical presentation of individuals with TCF12 mutations, showing facial appearance (left) and computed tomography (CT) images of head (right). Written permission was obtained to publish the clinical photographs. (a) Bilateral coronal synostosis in proband #30: note high forehead and brachycephaly at the age of 13 mo and, on CT images at the age of 4 mo, bilateral fusion of the coronal sutures associated with bony ridging (arrows). (b) Right unilateral coronal synostosis in proband #35: note elevation of right orbit and shifting of midface and mandible to left at the age of 10 mo. CT images at the age of 4 mo demonstrate complete fusion of the coronal suture on the right (arrowhead), an open coronal suture on the left (LC) and relative angulation of the metopic (M) and sagittal (S) sutures. (c-f) Severe bilateral coronal synostosis in EIIa-Cre;Tcf12^flox/+;Twist1^+/− combination mutants. Panels show representative images of postnatal day (P)21 skulls stained with Alizarin Red S (top), 3-dimensional μCT reconstructions (middle) and 2-dimensional μCT images of sagittal slices (10 μm) through the coronal suture (bottom; planes of section are indicated by white lines in 3-dimensional images). Note normal coronal sutures in wild-type (c) and EIIa-Cre;Tcf12^flox/+ skull (d), partial unilateral coronal synostosis in Twist1^+/− skull (e, arrow) and complete bilateral coronal synostosis in EIIa-Cre;Tcf12^flox/+;Twist1^+/− skull (f, arrows). C, coronal suture; FB, frontal bone; M, metopic suture; L: lambdoid suture; PB, parietal bone; S, sagittal suture. Scale bars: 1 mm.

Figure 2

Structure of TCF12 and encoded protein, showing location of mutations identified in coronal synostosis. (a) Gene structure. The major transcript comprizes 21 exons; inclusion or exclusion of exon 15 by alternative splicing generates proteins of 706 or 682 amino acids, termed HEBβ and HEBα, respectively. Alternative transcription starting at exon 9A generates a different protein (HEBAlt) of 512 amino acids. (b) Domains identified in the HEBβ protein (portion encoded by exon 15 shaded gray). The highly conserved bHLH region lies near the C-terminus. Functional studies previously defined Activation Domains 1 and 2 (ref. 24) and the Rep domain. Identical mutations identified in independent families are denoted (x2); note that mutations predicted to affect splicing are omitted from the protein cartoon.