An inherited FGFR2 mutation increased osteogenesis gene expression and result in Crouzon syndrome

The research was carried out according to the principles of the Declaration of Helsinki; informed consent was obtained and Shanghai Ninth People’s Hospital Ethics Committee approved the study. The patient photos, which are contained in this article, were taken by a hospital-based photographer at Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine. Permission to use these photos in this report has been obtained from all the subjects including the parents of individual V2 and IV-2 (minor’s parent: legal guardian) who participated in this study.

We present 10 Crouzon syndrome patients from one Chinese family with 5 generation. All patients underwent a standard ophthalmologic assessment. Three-dimensional orbital computed tomography (CT) scan examination was used for bony defects. All CT scans were obtained with a spiral CT scanner (GE LightSpeed 16, Milwaukee, WI). The scans included the range from the maxilla to the skull. Continuous high-resolution scanning with a slice detection of 1.25*16 mm, reconstructed thickness of 1.25 mm, and slice increment of 1.25 mm was performed. The reconstructed field of view was 23 cm, and the reconstruction matrix was 512*512. All CT images were loaded in the DICOM format. Mimics v18.0 software (Materialise, Leuven, Belgium) was used for further calculations and analyses. After the DICOM data were imported into Mimics, the coordinate system was redefined to ensure that all measurements were completed in the same environment. The new coordinate system was defined according to the protocols of the “Online Reslice” and “Reslice Project” functions. The axial plane was defined as a plane parallel to the Frankfurt plane, passing through the inferior margin of the left orbit and the upper margin of each of the external auditory meatuses. The sagittal plane was defined as the median plane passing through the midpoint of the nasion, sellaturcica, and foramen magnum. The oblique sagittal plane was defined as the plane passing through the midpoint of the optic canal orifice and the ipsilateral orbital aperture and was perpendicular to the axial plane. The coronal datum plane was defined as the coronal plane passing through the most posterior point of the lateral orbital rim. The orbital width, orbital height, roof length, floor length, medial wall length, lateral wall length, ocular protrusion, bony orbital volume (BOV), midinterorbital distance and lateral orbital wall angle parameters were calculated according to the protocol in the previous study [12].

Genomic DNA was extracted from peripheral blood leukocytes of the patients and 100 randomly selected healthy volunteers using the Automatic Nucleic Acid Isolation System (QuickGene-610 L; Fujifilm Life Science, Tokyo, Japan). PCR for the target sequence of FGFR2 was performed, the products purified and sequenced as previously described [13, 14]. A three dimensional computer model was used to analyse the structural positioning of FGFR2-G338A mutation site. The immunoglobulin (Ig)-like domain 3 of FGFR2 protein was modeled with SWISS-MODEL (template PDB code, 1iil) as we have previously described [15, 16].

Orbital bones obtained from Crouzon syndrome patient and orbital fracture patient on patient’s consent. After impurities removing, bone samples were soaked in lquid nitrogen and grined into fine particles or a powder. Total RNA was extracted using TRI-REAGENT (Invitro- gen, USA) according to the manufacturer’s instructions, and cDNA was synthesized using the PrimeScript RT re-agent kit (Takara, Japan) as we have previously described [17]. The housekeeping gene GAPDH was used as an endogenous control. The PCR cycle parameters for OC and ALP expression were as follows: 33 cycles of denaturation at 95 °C for 30 s, optimal annealing temperature for 30 s, extension at 72°Cfor30sandafinalextensionat72°Cfor5min.

All of the experiments were performed in triplicate, and the data were expressed as the mean ± standard deviation (SD). The comparative threshold cycle method was applied in the quantitative real-time RT-PCR assay according to the ΔΔ threshold cycle method.

We present 10 Crouzon syndrome patients from one Chinese family with 5 generation (Fig. 1a). These patients had shallow orbits and ocular proptosis, accompanied by craniosynostosis, mid-face hypoplasia, a curved, beaklike nose, but clinically normal hands and feet. They had normal vision since early childhood, just displaying a surprised look, but their vision was getting worse and worse as they aged. The visual acuity of individual III-14 (unmarried, 64 years old) was FC /1 m (figure count, OU). The quality of his life is at stake for serious exposure keratitis and obstinate conjunctivitis. And even more severe visual impairment, because of chronic corneal scarring result from hypophasis (Fig. 1b, III21and IV16), the visual acuity of III21 and IV16 was LP (light perception, OU of III21, OS of IV16).

Individual IV-2 (Fig. 1b, panel 1) the probands was born after a normal pregnancy and was referred for craniofacial assessment at 14 years of age because prominent eyes. He had a Crouzon syndrome brother (IV-1) died around 8 months old. The visual acuity of the IV-2 patient was 0.4 (OD) and 0.1 (OS). The refractive error was − 2.25D, with − 1.00 astigmatism (OD) and + 0.75D, with − 3.00 astigmatism (OS). Vertical interpalpebral fissure height (IPFH) OD 11 mm, OS 7 mm; horizontal palpebral fissure length (HPFL) OD 20 mm, OS 19 mm; levator function OD 5 mm, OS 8 mm; The prominence of the IV-2 patient was 20 mm (OD) and 18 mm (OS). In addition aural atresia was detected. No abnormalities were detected in the lenses, retinas, choroids, or optic nerves.

To measurement the orbital volume, we carried out the morphology study. The data showed the orbital volume of Crouzon syndrome patient was much more shallower and rounder than normal population (Table 1). As the data show the shape of the orbital aperture was more round and bigger than the normal orbits, for both the orbital height and width were larger than what we have previously measured from normal male Chinese adults [12] for the bony orbit have almost reached the adult level in the 14-year old patient [18]. The patient orbits were quite shallow as all length parameters from the orbital aperture to the optic nerve canal orifice were smaller than the normal data, especially the medial wall length (OD 39.97 mm, OS 38.00 mm, normal 46.32 ± 2.67 mm) and the floor length (OD 37.38 mm, OS 34.66 mm, normal 47.93 ± 2.68 mm), which was consistent with the dysplasia of the maxilla,finally resulted in a small orbital cavity (OD 17.15 ml, OS 16.31 ml, normal 26.04 ± 2.60 ml).However,the patient ocular volume was 5.76 ml (OD) and 5.96 ml (OS)which was normal along with the shallow orbits finally caused the “uninhabitable habitation”, then exophthalmos was presented.

Sequencing of the complete coding sequence of the FGFR2 gene derived from genomic DNA revealed a heterozygous mutation (p.G338R, c. 1012G > C) in exon 10 of the human FGFR2 (Fig. 2a), causing the replacement of glycine (Gly) by arginine (Arg) at amino acid position 338, a highly conserved segment of the FGFR2 protein (Fig. 2c). This mutation is located in the immunoglobulin (Ig)-like domain 3 (Fig. 2b). Moreover, the missense mutation was not found in 100 normal people, including III-5 and III-7 from the Crouzon syndrome family who were normal subjects (Fig. 2a).

To study the structural changes in the mutant-type FGFR2, three-dimensional structural model was used to analyse the position of missense mutation. Compared with wild-type FGFR2 (Fig. 2d, a-c), obviously changes were detected in the local spatial structure of the mutant-type FGFR2 (Fig. 2d, d-f) carrying the amino acid substitution in the (Ig)-like domain 3. Notably, in the wild-type FGFR2 there was no hydrogen bond between Tyr328 and Arg330 (Fig. 2d, c). However, in the mutant-type FGFR2one hydrogen bond was formed between Tyr328 and Arg330 that was near the Gly338 amino acid residue (Fig. 2d, f), which may reduced the stability of protein.

To explore the factors that may affect the upgrowth of orbit bone in Crouzon syndrome patients,we examined two osteoblast related genes expressions OC and ALP. The results clearly showed that OC and ALP was remarkably increased in the Crouzon syndrome patient compared with normal orbital bone (Fig. 3a and b). These data further highlight the clinical importance of FGFR2 in craniofacial growth.