Recessive coding and regulatory mutations in FBLIM1 underlie the pathogenesis of chronic recurrent multifocal osteomyelitis (CRMO)

Abstract

Chronic recurrent multifocal osteomyelitis (CRMO) is a rare, pediatric, autoinflammatory disease characterized by bone pain due to sterile osteomyelitis, and is often accompanied by psoriasis or inflammatory bowel disease. There are two syndromic forms of CRMO, Majeed syndrome and DIRA, for which the genetic cause is known. However, for the majority of cases of CRMO, the genetic basis is unknown. Via whole-exome sequencing, we detected a homozygous mutation in the filamin-binding domain of FBLIM1 in an affected child with consanguineous parents. Microarray analysis of bone marrow macrophages from the CRMO murine model (cmo) determined that the Fblim1 ortholog is the most differentially expressed gene, downregulated over 20-fold in the cmo mouse. We sequenced FBLIM1 in 96 CRMO subjects and found a second proband with a novel frameshift mutation in exon 6 and a rare regulatory variant. In SaOS2 cells, overexpressing the regulatory mutation showed the flanking region acts as an enhancer, and the mutation ablates enhancer activity. Our data implicate FBLIM1 in the pathogenesis of sterile bone inflammation and our findings suggest CRMO is a disorder of chronic inflammation and imbalanced bone remodeling.

Fig 1. X-ray and MRI knee images from the affected proband.

(A) X-ray showing osteomyelitis and a lesion characteristic of CRMO (red arrow) in the left proximal tibia. (B). MRI of the same knee showing inflammation and bone destruction (red arrow). Similar lesions were found in the clavicle, hip, femur, tibia, foot and toes (not shown). (C) MRI of a healthy knee for comparison.

Fig 2. Microarray analysis of bone marrow macrophages from the cmo mouse model.

(A) Gene expression heatmap from microarray analysis of RNA from bone marrow-derived macrophages of cmo, IL-1R^-/- and IL-1R^+/- mice (n = 3 for each strain). Normalized expression values were analyzed for differential expression using Partek Genomics Suite software (version 6.12, Partek Inc., St. Louis, MO, USA.). The top 25 differentially expressed genes are shown. Fblim1 (red arrow) was the most differentially expressed (downregulated) gene in the cmo mouse. (B, C and D) Representative fixed decalcified tail bone sectioned and stained with H&E from cmo, IL-1R^-/- and IL-1R^+/- mice, respectively. The cmo mouse has extensive mixed inflammatory infiltrate with destruction of the vertebral body, the cmo.IL-1R^+/- has similar but less severe inflammation and bone destruction, whereas, the cmo.IL-1R^-/- mouse has normal bone with no inflammation. Representative tail kinks and foot deformities in these mice were depicted and described previously [10].

Fig 3. Coding mutations found in individuals with CRMO.

(A) Chromatogram showing the homozygous mutation in exon 3 of FBLIM1 in a child from a consanguineous union. The mutation is rs146575757, a G to A nucleotide change, causing an Arg38Gln change in the protein. (B) Chromatogram showing a novel 1-bp frameshift insertion in exon 6 of FBLIM1 in a second proband with CRMO. The mutation is one allele of a compound heterozygote and the frameshift occurs at Glu255. (C) Image of conserved domains in FBLP1 from the Protein Data Bank (PDB) (www.rcsb.org) [32]. The red arrow points to the location of the Arg38Gln position, in the filamin-binding domain. (D) Protein alignment of FBLP1 and its orthologues in other mammals. The red arrow points to the amino acid disrupted by rs146575757.

Fig 4. Structural modeling of patient mutation in FBLIM1.

(A) Disorder prediction of the Filamin-1 binding region of FBLP1 (residues 1–70) was performed with DISOPRED version 3. Disorder probability scores greater than 0.5 are considered to be disordered. The R38Q mutation falls in a predicted disordered region. (B) Secondary structure prediction was performed using PSI-PRED. The confidence scores for coils, helices, and strands are shown. (C) The output model of the Filamin-1 binding region of FBLIM1 generated in Phyre2. A total of 86% of the residues are predicted to be disordered, resulting in 76% of the residues having a low confidence score (<90%). Residues 1–24 are predicted to be ordered. (D) Superimposition of the Phyre2 model onto the NMR structure of the Filamin-1-FBLIM1 complex. Modeling of the complex places the R38Q mutation downstream of the interaction site. (E) Electrostatic potential calculations in APBS reveal a loss of positive charge in FBLP1 as a result of the CRMO mutation.

Fig 5. FBLIM1 enhancer variant in 2^nd proband.

(A) Chromatogram from the second proband. Red arrow indicates rs41310367 (C→T) in the third intron of FBLIM1. (B) From JASPAR [26], the human NR4A2 consensus transcription factor binding site (TFBS) motif. The red arrow shows where rs41310367 is located within the binding site and that the TFBS is disrupted. (C) Screen capture from UCSC genome browser (hg19) showing the regulatory region that includes rs41310367 (red arrow) and a STAT3 binding region. H3K27ac and H3K4me1 peaks from ENCODE are shown, including in osteoblasts. The red box indicates the region that was amplified and cloned into the Firefly reporter vector.

Fig 6. Functional validation of rs41310367 by luciferase assay.

(A) Diagram of the firefly luciferase reporter driven by the cfos promoter plus the putative FBLIM1 enhancer region (hg19:chr1:16,091,437–16,092,574). attB1 and attB2 are the recombination sites for the Gateway recombination system. The diagram shows recombination inserted the regulatory region into the construct backwards. The red arrow shows the position of rs41310367. (B) Firefly to Renilla luciferase ratios in transfected SaOS2 cells (72-hours after transfection). The number of replicates for each experiment is indicated in parentheses. The presence of the enhancer (red) increased luciferase activity compared to the empty vector (blue), and the mutation (gray) significantly reduced enhancer activity, both with and without fluoride treatment; however, the effects were greater and more significant in NaFl-treated SaOS2 cells. In all experiments, luciferase activity was normalized to the co-transfected Renilla luciferase. Data from individual experiments were combined prior to statistical analysis and differences in relative luciferase activity between experimental scenarios were determined using a two-tailed t-test.

Fig 7. Proposed model for FBLIM1’s involvement in CRMO.

FBLIM1 expression is regulated by STAT3 and NR4A2 binding. FBLP1 blocks the phosphorylation of ERK1/2; in its absence or when dysfunctional, the increased phosphorylation of ERK1/2 leads to increased RANKL production, subsequent osteoclast activation, and bone resorption, as well as inflammasome activation. Mutations like rs41310367 disrupt regulation and expression of FBLIM1, resulting in decreased osteoblast activity and subsequent bone loss.