Introduction of a Phe377del mutation in ANK creates a mouse model for craniometaphyseal dysplasia

Abstract

Craniometaphyseal dysplasia (CMD) is a monogenic human disorder characterized by thickening of craniofacial bones and flaring metaphyses of long bones. Mutations for autosomal dominant CMD have been identified in the progressive ankylosis gene ANKH. Previous studies of Ank loss-of-function models, Ank(null/null) and Ank(ank/ank) mice, suggest that Ank plays a role in the regulation of bone mineralization. However, the mechanism for Ank mutations leading to CMD remains unknown. We generated the first knockin (KI) mouse model for CMD expressing a human mutation (Phe377 deletion) in ANK. Homozygous Ank knockin mice (Ank(KI/KI)) replicate many typical features of human CMD including hyperostosis of craniofacial bones, massive jawbones, decreased diameters of cranial foramina, obliteration of nasal sinuses, fusion of middle ear bones, and club-shaped femurs. In addition, Ank(KI/KI) mice have increased serum alkaline phosphatase and TRACP5b, as reported in CMD patients. Biochemical markers of bone formation and bone resorption, N-terminal propeptide of type I procollagen and type I collagen cross-linked C-terminal telopeptide, are significantly increased in Ank(KI/KI) mice, suggesting increased bone turnover. Interestingly, Ank(KI/KI) bone marrow-derived macrophage cultures show decreased osteoclastogenesis. Despite the hyperostotic phenotype, bone matrix in Ank(KI/KI) mice is hypomineralized and less mature, indicating that biomechanical properties of bones may be compromised by the Ank mutation. We believe this new mouse model will facilitate studies of skeletal abnormalities in CMD at cellular and molecular levels.

Figures

FIG. 1

Generation and genotyping of AnkKI/KI mice. (A) Deletion of phenylalanine 377 in exon 9 introduced into the mouse Ank gene by homologous recombination. The floxed allele contains a PGK-Neo cassette (loxP indicated by solid triangle) and a TTC1130–1132 deletion in exon 9. The knockin allele after cre-mediated recombination contains one loxP site upstream of mutant exon 9. Genotyping primers (a and b) flank the loxP and the deletion site. (B) PCR genotyping assay for Ank+/+, Ank+/KI, and AnkKI/KI animals. Wildtype allele: 659 bp, mutant allele: 757 bp. (C) Total body weight measurement of Ank+/+, Ank+/KI, and AnkKI/KI male mice at 3–10 wk of age (n ≥ 6). (D) Femur length of 10-wk-old Ank+/+ (n = 12), Ank+/KI (n = 11), and AnkKI/KI (n = 9) male mice; ap < 0.05 and bp < 0.01 indicate statistical significance by one-way ANOVA.

FIG. 2

CMD-like phenotype in AnkKI/KI mice. (A) Representative radiographs of skulls, mandibles, and femurs from 6-mo-old Ank+/+, Ank+/KI, and AnkKI/KI male mice. (B) μCT images of frontal plane through skulls and 3D reconstruction of mandibles from 3-mo-old Ank+/+ and AnkKI/KI male mice. White line indicates sagittal plane through furcation of first mandibular molar. (C) Internal, dorsal view of cranial floor and nasal cavities from horizontal plane of superior semicircular duct and the cribriform plate of ethmoid. Histogram shows dimensions of foramen magnum of Ank+/+ and AnkKI/KI littermates. (D) 2D μCT images of tympanic bulla from frontal plane through cochlea showing fusion of malleus (1) and incus (2). (E) Internal view of Ank+/+ and AnkKI/KI femurs. 3D reconstructions of trabeculation in metaphysis and cross-sectional slices of cortical bone in diaphysis. (F) 3D μCT images of total trabecular bone in femurs from Ank+/+ and AnkKI/KI mice at 10 wk of age. (G) Internal view of femurs of 12-mo-old Ank+/+ and Ank+/KI mice. 3D reconstructions of trabeculation in metaphysis and cross-sectional slices of cortical bone in diaphysis.

FIG. 3

Histology of Ank+/+ and AnkKI/KI male mice. (A) Calvariae from 10-wk-old mice (H&E). (B) H&E staining of 10-wk-old mice: femurs (left panel), metaphyses (top right), and diaphyses (bottom right). (C) TRACP staining of femurs from 4-wk-old mice. Arrows indicate TRACP+ cells. GP, growth plate; T.b, trabecular bone. Scale bar = 500 μm.

FIG. 4

In vitro osteoclast assays. (A) Representative images of TRACP staining at day 5 and of resorption of osteologic slides in BMM cultures. Histograms show number of mature osteoclasts per well and percentile of the resorbed area., Analysis of TRACP staining was performed by two-way ANOVA followed by Bonferroni test compared with the Ank+/+ group for each time point. Resorption assay was evaluated by one-way ANOVA with Tukey's multiple-comparison test. ap < 0.05; bp < 0.01. Scale bar = 200 μm. (B) Representative images of resorption pit assay on bone chips. Scale bar = 300 μm.

FIG. 5

FTIR and X-ray diffraction analysis of bones from 10-wk-old Ank+/+ and AnkKI/KI male mice. (A) Mineral:matrix ratio; collagen cross-linking; carbonate:phosphate ratio; crystallinity of central cortical bones from 10-wk-old Ank+/+ and AnkKI/KI femurs. Numerical scales represent the range of intensity ratios applied for each parameter. Histograms for individual images are shown on the left. (B) Analysis of crystal size in bone powder from tibias and scapulae by X-ray diffraction. (C) Crystalline phases in bone powder identified by wide-angle X-ray diffraction. Data are mean ± SD. Statistical significance by Student's t-test (bp < 0.01).

FIG. 6

Skeletal phenotype of 3-mo-old Ankank/ank male mice. (A) 3D μCT images comparing hind feet of Ank+/+, AnkKI/KI, and Ankank/ank animals. (B) μCT images of skulls, cranial and nasal cavities, and mandibles. (C) Internal view of Ank+/+ and Ankank/ank femurs. 3D reconstructions of trabeculation in metaphysis and cross-sectional slices of cortical bone in diaphysis.