C-type natriuretic peptide analog treatment of craniosynostosis in a Crouzon syndrome mouse model

Abstract

Activating mutations of fibroblast growth factor receptors (FGFRs) are a major cause of skeletal dysplasias, and thus they are potential targets for pharmaceutical intervention. BMN 111, a C-type natriuretic peptide analog, inhibits FGFR signaling at the level of the RAF1 kinase through natriuretic peptide receptor 2 (NPR2) and has been shown to lengthen the long bones and improve skull morphology in the Fgfr3Y367C/+ thanatophoric dysplasia mouse model. Here we report the effects of BMN 111 in treating craniosynostosis and aberrant skull morphology in the Fgfr2cC342Y/+ Crouzon syndrome mouse model. We first demonstrated that NPR2 is expressed in the murine coronal suture and spheno-occipital synchondrosis in the newborn period. We then gave Fgfr2cC342Y/+ and Fgfr2c+/+ (WT) mice once-daily injections of either vehicle or reported therapeutic levels of BMN 111 between post-natal days 3 and 31. Changes in skeletal morphology, including suture patency, skull dimensions, and long bone length, were assessed by micro-computed tomography. Although BMN 111 treatment significantly increased long bone growth in both WT and mutant mice, skull dimensions and suture patency generally were not significantly affected. A small but significant increase in the relative length of the anterior cranial base was observed. Our results indicate that the differential effects of BMN 111 in treating various skeletal dysplasias may depend on the process of bone formation targeted (endochondral or intramembranous), the specific FGFR mutated, and/or the specific signaling pathway changes due to a given mutation.

Conflict of interest statement

LZ, RM, LS, and TO are employees of BioMarin Pharmaceutical, Inc., that sponsored this study. GH and EWJ received funding from BioMarin, but do not have any commercial interest in BioMarin Pharmaceutical, Inc. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. The internal publication review committee of BioMarin Pharmaceutical, Inc., approved the manuscript for publication.

Figures

Fig 1. Locations of sutures and linear dimensions analyzed in the skull.

(A) BMN 111 treatment timeline. (B) 2D sagittal slice of μCT data showing the coronal (Cor) and frontonasal (FN) sutures and the intersphenoidal (ISS) and spheno-occipital (SOS) synchondroses. (C) 3D reconstruction of μCT data showing a dorsal view of the skull with skull, nose, and upper jaw lengths and skull width indicated. (D) 2D sagittal slice of μCT data showing the anterior and posterior cranial base lengths and the foramen magnum sagittal diameter.

Fig 2. Immunohistochemical detection of NPR2 expression in the coronal suture at P0.

Near-adjacent frozen sections were incubated with control IgG serum or antibodies to NPR2 or RUNX2. (A) For orientation, dashed lines overlay the osteoid of frontal (f) and parietal bones (p) and the arrows indicate the osteogenic fronts of these bones. Sagittal sections, oriented with anterior to the left. (B) Same sections as in (A) are shown without dashed lines over the osteoid for clearer visualization. In the coronal suture NPR2 expression is present in osteoblasts along the osteoid of the frontal and parietal bones. RUNX2 is expressed predominantly in the osteoblasts. No signal was seen with control IgG serum. Scale bars are 200 μm.

Fig 3. Immunohistochemical detection of NPR2 expression in growth plates at P0.

Near-adjacent frozen sections were incubated with control IgG serum or antibodies to NPR2 or RUNX2. (A) In the spheno-occipital synchondrosis, NPR2 expression was higher in the prehypertrophic (ph) than the hypertrophic (h) zone, with some expression in the resting (r) zone. RUNX2 was expressed less in the prehypertrophic than the hypertrophic zone. Sagittal sections, oriented with anterior to the left. (B) In the proximal femoral growth plate, NPR2 expression was present in the prehypertrophic and hypertrophic zones, similar to RUNX2 expression. Transverse section. No differences were seen between Fgfr2+/+ and Fgfr2cC342Y/+ mice. No signal was seen with control IgG serum. Scale bars are 200 μm.

Fig 4. Patency of craniofacial sutures and effect of BMN 111.

Graphs show patency of the coronal and frontonasal sutures and the spheno-occipital and intersphenoidal synchondroses at P3 (no treatment) and P31 (treatment of wild type [W] or Crouzon mutant mice [C] with either BMN 111 [B] or vehicle alone [V]). No sutures or synchondroses in the “early fusion” category were observed.

Fig 5. Linear dimensions of the calvaria and skull base and effect of BMN 111.

Graphs show the indicated linear dimensions in millimeters (mm) at P3 (no treatment) and P31 (treatment of wild type [W] or Crouzon mutant mice [C] with either BMN 111 [B] or vehicle alone [V]). (A) Skull length. (B) Nose length. (C) Upper jaw length. (D) Skull width. (E) Anterior cranial base length. (F) Posterior cranial base length. (G) Foramen magnum sagittal diameter. **p<0.01, ***p<0.001, ****p<0.0001.

Fig 6. Tibia length and effect of BMN 111.

(A) 2D slice of μCT data showing tibias at P31. (B) Graph shows the tibia length in millimeters (mm) at P31, males and females combined, after treatment of wild type [W] or Crouzon mutant mice [C] with either BMN 111 [B] or vehicle alone [V]. (C) Graph shows body weight at P31 for males and females, after treatment of wild type [W] or Crouzon mutant mice [C] with either BMN 111 [B] or vehicle alone [V]. *p<0.05, **p<0.01.