Heterozygous mutations of FREM1 are associated with an increased risk of isolated metopic craniosynostosis in humans and mice

Abstract

The premature fusion of the paired frontal bones results in metopic craniosynostosis (MC) and gives rise to the clinical phenotype of trigonocephaly. Deletions of chromosome 9p22.3 are well described as a cause of MC with variably penetrant midface hypoplasia. In order to identify the gene responsible for the trigonocephaly component of the 9p22.3 syndrome, a cohort of 109 patients were assessed by high-resolution arrays and MLPA for copy number variations (CNVs) involving 9p22. Five CNVs involving FREM1, all of which were de novo variants, were identified by array-based analyses. The remaining 104 patients with MC were then subjected to targeted FREM1 gene re-sequencing, which identified 3 further mutant alleles, one of which was de novo. Consistent with a pathogenic role, mouse Frem1 mRNA and protein expression was demonstrated in the metopic suture as well as in the pericranium and dura mater. Micro-computed tomography based analyses of the mouse posterior frontal (PF) suture, the human metopic suture equivalent, revealed advanced fusion in all mice homozygous for either of two different Frem1 mutant alleles, while heterozygotes exhibited variably penetrant PF suture anomalies. Gene dosage-related penetrance of midfacial hypoplasia was also evident in the Frem1 mutants. These data suggest that CNVs and mutations involving FREM1 can be identified in a significant percentage of people with MC with or without midface hypoplasia. Furthermore, we present Frem1 mutant mice as the first bona fide mouse model of human metopic craniosynostosis and a new model for midfacial hypoplasia.

Figure 1. Schematic overview of CNVs and mutations affecting the FREM1 gene.

(a) Deletions in patients 1-4 are represented by solid red bars, whereas the duplicated segment in patient 2 is represented by a solid green bar. The proximal deletion breakpoint of patient 1 as well as the deletion/duplication breakpoint in patient 2 disrupts FREM1. (b) Partial electropherograms of the de novo mutated nucleotide identified in patient 6. (c) Schematic overview of the FREM1 protein showing the domain structure and positions of mutations in the FREM1-trigonocephaly (above) and BNAR (below) syndromes.

Figure 2. Frem1 expression in the developing frontal and nasal sutures in mice.

(a,b) Frem1 transcripts are detected in the developing cranial sutures and in the regions fated to form the posterior-frontal suture, the metopic suture equivalent that is affected in cases of metopic craniosynostosis (white arrowheads) (c) Immunostaining for Frem1 (red) of the frontal bones at postnatal day 0 reveals expression of the protein in the pericranium and dura mater on either side of the frontal bones (dotted line; fb  =  frontal bone, hf  =  hair follicle). Samples have been counterstained with DAPI (blue) and with an antibody to entactin (green) which marks the basement membrane of the epidermis and hair follicles. The boxed area (yellow) is magnified in (d) highlighting fibrillar staining for Frem1 above and below the frontal bone (fb) (e) Frem1 protein was also noted diffusely in the suture mesenchyme at the medial edge of the frontal bones, with d' and e') showing the identical images in black and white to emphasize the fibrillar localization of Frem1. Scale bars  =  50 µm.

Figure 3. Frem1 mCT and morphometric analysis in mice are consistent with the human MC phenotype.

mCT and morphometric analysis of mouse skulls at postnatal day 28 reveal anterofrontal cranial deformation (a) semi-landmark mesh over nasal and frontal bones of control skull; (b) plot of principal component 1 (PC1) and PC2 reveal that heterozygote (green dots) and homozygote (red dots) bat mice each have distinct craniofacial shapes from age, sex and genetic background matched controls (blue dots); (c) average mesh coordinates of control (blue dots) versus Frem1 homozygotes (red dots) show changes in shape over the frontal bones. These differences can also be seen in (d) by comparison of identical cross-sections. These deformations are reminiscent of the trigonocephalic changes seen in patients with FREM1 mutations.

Figure 4. Variable presentation of midface hypoplasia/assymetry in Frem1 mutant mice.

Multiple views from rendered mCT reconstructions of P28 wildtype (far left column) and homozygote male Frem1 mice. Around 45% of homozygotes exhibited some readily apparent dysmorphology involving the midfacial region. Four examples of homozygote skull morphology observed: (i) pronounced leftward deviation of midface, (ii) uniform midface hypoplasia (shortened snout), (iii) leftward deviation extending from posterior frontal suture through to nasal tip, (iv) pronounced rightward deviation of midface. Frontal views of the homozygote skulls reveal medial depression along the internasal suture and/or marked curvature of the nasal bones (see white arrow in column (ii). Most homozygotes display abnormal maxillary-premaxillary suture morphology (compare asterisks in lateral and ventral views of control and homozygote (i).

Figure 5. Frem1^bat and Frem1^Qbrick mice exhibit advanced posterior frontal suture fusion.

Rendered 3D images of postnatal day 28 heads were generated from microcomputed tomographic scan data and each virtually sectioned in the coronal plane at the same position through the posterior frontal suture (i – dorsal view of position of coronal plane; ii – frontal view of rendered image in (i)). The region indicated by the rectangle is shown in (iii) for a control (+/+) skull, homozygote Frem1^bat (bat/bat) and Frem1^Qbrick (qb/qb) skulls, as well as different heterozygote Frem1^bat (bat/+) and Frem1^Qbrick (qb/qb) skulls. The most severely affected heterozygote Frem1^bat (bat/+) posterior frontal suture is also shown. Control skulls all showed sparse points of contact (typically just on the endocranial surface) between the frontal bones, indicative of the early stages of suture fusion. Fusion of the posterior frontal suture is largely completed by ∼postnatal day 45 in controls. In contrast, the Frem1^bat/bat and Frem1^qb/qb skulls exhibited extensive fusion both on the endocranial and ectocranial surfaces (arrowheads) at day 28, indicating advanced fusion of this suture. Heterozygotes also showed variable suture anomalies, from complete fusion to asymmetry of the suture.