Gain-of-function mutations in a member of the Src family kinases cause autoinflammatory bone disease in mice and humans

Abstract

Autoinflammatory syndromes are characterized by dysregulation of the innate immune response with subsequent episodes of acute spontaneous inflammation. Chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory bone disorder that presents with bone pain and localized swelling. Ali18 mice, isolated from a mutagenesis screen, exhibit a spontaneous inflammatory paw phenotype that includes sterile osteomyelitis and systemic reduced bone mineral density. To elucidate the molecular basis of the disease, positional cloning of the causative gene for Ali18 was attempted. Using a candidate gene approach, a missense mutation in the C-terminal region of Fgr, a member of Src family tyrosine kinases (SFKs), was identified. For functional confirmation, additional mutations at the N terminus of Fgr were introduced in Ali18 mice by CRISPR/Cas9-mediated genome editing. N-terminal deleterious mutations of Fgr abolished the inflammatory phenotype in Ali18 mice, but in-frame and missense mutations in the same region continue to exhibit the phenotype. The fact that Fgr null mutant mice are morphologically normal suggests that the inflammation in this model depends on Fgr products. Furthermore, the levels of C-terminal negative regulatory phosphorylation of Fgr ^Ali18 are distinctly reduced compared with that of wild-type Fgr. In addition, whole-exome sequencing of 99 CRMO patients including 88 trios (proband and parents) identified 13 patients with heterozygous coding sequence variants in FGR, including two missense mutant proteins that affect kinase activity. Our results strongly indicate that gain-of-function mutations in Fgr are involved in sterile osteomyelitis, and thus targeting SFKs using specific inhibitors may allow for efficient treatment of the disease.

Keywords: autoinflammation; bone destruction; chronic recurrent multifocal osteomyelitis; mouse model; tyrosine kinase.

Fig. 1.

Positional candidate cloning of the Ali18 mutation. (A) Ali18/+ (Right) and wild-type C3HeB/FeJ (Left) hind paws. Ali18 mice show reddening and swelling in peripheral paws. (B) Genetic map and the critical interval of the Ali18 locus. The complex modifier effects from the C57BL/6J genetic background prevented further narrowing down of the region. (C) By Sanger sequencing of candidate genes in the region, a point mutation (c.1506A > G) in exon 13 of Fgr, a member of the SFKs, was detected. (D) Mbo II restriction enzyme digestion of PCR products spanning exon 13 correlated to the swollen paw phenotype. Genetic background is described as C3H (C3HeB/FeJ), BL6 (C57BL/6J), and CBF1 (F1 from C3H and BL6 crossing). Ali18 mice were originally derived from C3H parents. (E) Schematic diagram of the p.Asp502Gly (D502G) amino acid change induced by c.1506A > G. Y400 and Y511 indicate the autophosphorylation site and the C-terminal regulatory phosphorylation site, respectively. (F) Alignment of Src family tyrosine kinases. Square encompasses the amino acid residues exchanged by the c.1506A > G mutation.

Fig. 2.

Disruption of the Fgr gene by genome editing alters the autoinflammatory phenotype in Ali18 mice. (A) DNA sequence of guide RNA (fgRNA1 and fgRNA2) and PAM around exon 3 of Fgr are indicated. The p.Asp502Gly mutation in exon 13 is also shown. Sanger sequencing of a PCR fragment around exon 3 and genotyping of p.Asp502Gly were done using genomic DNA from F0 and F1 mice. (B) Schematic strategy of genome editing in the Fgr locus of Ali18 mice. pX330-based constructs were microinjected into Ali18/Ali18 oocytes in C3H (C3HeB/FeJ) genetic background from in vitro fertilization. The founder mice (F0) derived from microinjection were bred with wild-type C3H mice to obtain F1 mice. (C and D) Correlation of Fgr genotypes and lower limb morphology of F1 mice. Loss-of-function mutations show no morphological abnormality (red font). In contrast, missense, in-frame deletion, and synonymous mutations exhibit autoinflammatory paws. SA, splice acceptor; ATG, the translational initiation site. See also SI Appendix, Table S2 for detail.

Fig. 3.

Western blot analysis, tyrosine kinase assays, and phosphorylation of Fgr by C-terminal Src kinase (Csk). Cell lysate of spleen (A) and bone marrow cells (B) from wild-type and Ali18/Ali18 mice were used for Western blotting. As control experiments, transformants of wild-type and p.Asp502Gly (D502G) Fgr expression constructs in murine embryo-derived cultured fibroblast, NIH 3T3, cells were used. Empty vector was transfected as negative control. Anti-Fgr (Upper), anti-phosphotyrosine (Middle), and anti-actin (Lower, loading control) were used. No overt changes in Fgr protein levels of Ali18/Ali18 spleen (relative ratio: 0.900 ± 0.256, P = 0.538, t test) and NIH 3T3 cells (relative ratio: 0.982 ± 0.139, P = 0.833, t test) were detected. (C) In vitro translated wild-type and p.Asp502Gly Fgr proteins were used for kinase assay experiments. Recombinant enolase protein was used as SFK-specific substrate (Lower, loading control). No activity changes were detected in different reaction time. (D) The C-terminal phosphorylation of KD Fgr by Csk was measured. Fgr KD with p.Asp502Gly showed five and two times less phosphorylation levels in 5- and 20-min reaction time, respectively. The proteins used for kinase assays were fractionated by SDS/PAGE and stained by Coomassie Brilliant Blue. Experiments were independently triplicated.

Fig. 4.

Missense mutations of FGR in CRMO and phosphorylation assays. (A and B) Radiograph of case 1 (p.Arg118Trp). Osteolytic lesions with sclerosis and periosteal elevation of the right distal femur are shown. (C) MRI of case 1. Increased STIR signal intensity on the iliac and sacral sides of the left sacroiliac joints is shown. (D) Sanger sequencing chromatogram of p.Arg118Trp in the proband (Top), father (Middle), and mother (Bottom). Proband harbors a de novo C > T mutation, which induces a p.Arg118Trp amino acid change in FGR. (E–G) MRI of case 2 (p.Pro525Ser). Abnormalities included increased signal intensity on STIR images in the left distal fibula (E) and the pelvis at the right acetabulum (F). (G) Repeat MRI 9 mo after naproxen therapy showing improvement in the left acetabular lesion. (H) Schematic diagram of amino acid substitution from the FGR mutations found in human CRMO (Top arrows) and the mouse Ali18 mutation (Lower arrow). (I) SDS/PAGE of affinity purified Csk and FGR used in kinase assays. (J) Kinase activity of FGR with CRMO variants. (J, Left) SDS/PAGE of affinity purified substrate, enolase, used in each assay. (J, Right) Phosphorylation intensity of enolase and FGR indicate enzyme activity. (K) Phosphorylation of FGR_KD with CRMO variants by Csk. (Left) SDS/PAGE of affinity purified FGR_KD of human CRMO variants as substrate used in each assay. (Right) FGR_KD proteins were phosphorylated by Csk in equal intensity. Experiments were independently triplicated.