Mutations in Fibronectin Cause a Subtype of Spondylometaphyseal Dysplasia with "Corner Fractures"

Abstract

Fibronectin is a master organizer of extracellular matrices (ECMs) and promotes the assembly of collagens, fibrillin-1, and other proteins. It is also known to play roles in skeletal tissues through its secretion by osteoblasts, chondrocytes, and mesenchymal cells. Spondylometaphyseal dysplasias (SMDs) comprise a diverse group of skeletal dysplasias and often manifest as short stature, growth-plate irregularities, and vertebral anomalies, such as scoliosis. By comparing the exomes of individuals with SMD with the radiographic appearance of "corner fractures" at metaphyses, we identified three individuals with fibronectin (FN1) variants affecting highly conserved residues. Furthermore, using matching tools and the SkelDys emailing list, we identified other individuals with de novo FN1 variants and a similar phenotype. The severe scoliosis in most individuals and rare developmental coxa vara distinguish individuals with FN1 mutations from those with classical Sutcliffe-type SMD. To study functional consequences of these FN1 mutations on the protein level, we introduced three disease-associated missense variants (p.Cys87Phe [c.260G>T], p.Tyr240Asp [c.718T>G], and p.Cys260Gly [c.778T>G]) into a recombinant secreted N-terminal 70 kDa fragment (rF70K) and the full-length fibronectin (rFN). The wild-type rF70K and rFN were secreted into the culture medium, whereas all mutant proteins were either not secreted or secreted at significantly lower amounts. Immunofluorescence analysis demonstrated increased intracellular retention of the mutant proteins. In summary, FN1 mutations that cause defective fibronectin secretion are found in SMD, and we thus provide additional evidence for a critical function of fibronectin in cartilage and bone.

Keywords: FN1; cartilage; corner fractures; extracellular matrix; fibronectin; metaphyses; protein secretion; scoliosis; skeletal dysplasia; spondylometaphyseal.

Figure 1

Pedigrees of the Families with FN1 Mutations Co-segregation of the variants with the trait in families 1 and 4 suggests dominant inheritance. Consistently, de novo FN1 mutations in the affected individuals of simplex families 2, 3, 5, 6, and 7 were observed.

Figure 2

Photographs of Some of the Individuals with FN1 Mutations (A) Photographs in a figure reproduced with permission from Sutton et al. (copyright © 2005 Wiley-Liss, Inc.). On the left are the three affected individuals who have an FN1 mutation and the maternal grandmother. In the middle is the older child at age 5 years, and on the right is the younger child at age 3 years. (B) Affected child from family 2 at age 13 years. Note the short trunk, facial asymmetry, dysplastic left ear, and normal hands and feet. (C) Affected child from family 5 at age 2 years and 11 months. Note the scoliosis, genu varum, and normal hands and feet. (D) Affected child from family 6 at age 8 years. Note the short trunk and scoliotic posture.

Figure 3

Radiographs Showing the “Corner Fractures” and Other Radiological Changes (A) Individual from family 7; note the significant scoliosis. (B) Individual from family 3; note the absence of coxa vara, the presence of irregular metaphyses with corner fractures, and the presence of ovoid vertebral bodies. Additional radiographs from all families are available in Figure S1.

Figure 4

Position and Conservation of Amino Acids Affected by Substitutions (A) Location of the SMD-associated fibronectin amino acid substitutions (in black) and those underlying glomerulopathy (in blue). The fibronectin domains I, II, and III are numbered, V stands for variable domain, and EIIIA and EIIIB indicate the extra type III repeat A and B segments. These three domains in yellow are subject to alternative splicing. Domains I-1 to I-5 in red represent the N-terminal assembly domain. Domains III-9 and III-10, also in red, contain the synergy site and the RGD site. Domains III-1 and III-2, in orange, contain self-interaction sites and are involved in conformational changes promoting fibronectin assembly. (B) Amino acid conservation of the mutated residues across vertebrates. Gray shading indicates non-conserved amino acid residues.

Figure 5

Structural Impact of SMD-Causing Mutations (A) Structural models of fibronectin domains I-1 and I-2 (PDB: 1O9A, without the S. dysgalactiae FnBP B3 peptide). On the left is the wild-type protein, and on the right is the model with the variant. The model for the p.Cys87Phe substitution was generated by the ModWeb Server. The disulfide bond formed between Cys76 and Cys87 stabilizes domain I-1. The p.Cys87Phe substitution is predicted to destabilize the structure by breaking the disulfide bond and by displaying a hydrophobic residue at the surface. (B) Structural models of fibronectin domains I-4 and I-5 (PDB: 2RKY, without the S. aureus FnBPA peptide). To generate the model for the p.Tyr240Asp change, we replaced tyrosine with aspartic acid and manually adjusted the rotomer position to minimize the steric clash with the rest of the protein by using Coot. For the wild-type protein, the side chains of Tyr240 and Trp246 interact through π stacking and stabilize the fibronectin domain. This interaction is predicted to be lost in the mutant protein.

Figure 6

Analysis of Protein Secretion of Recombinant Wild-Type and SMD-Causing Mutants of rF70K and rFN (A) Schematic overview of full-length fibronectin (rFN) and the 70 kDa N-terminal fragment of fibronectin (rF70K). To ensure secretion through the secretory pathway, the expression plasmids contain either the sequence coding for the endogenous fibronectin signal peptide (rFN) or a heterologous BM40 signal peptide (rF70K). The indicated SMD variants were engineered into the expression constructs. (B–E) NT refers to the non-transfected HEK293 controls, WT refers to the wild-type rF70K or rFN cell clones, and the mutant cell clones are indicated in the one-letter amino acid code. (B) Western blot analysis of conditioned cell-culture medium (for 2 days) harvested from HEK293 cells transfected with rF70K (left) and rFN (right). Analysis of rF70K was performed with a rabbit anti-fibronectin antibody (top; primary antibody, Sigma, F3648; secondary goat anti-rabbit horseradish-conjugated antibody, Agilent Technologies, K4008) and a horseradish-conjugated anti-V5 monoclonal antibody (bottom, Thermo Fisher Scientific, MA5-15253-HRP), whereas the rFN analysis was performed with only an anti-V5 antibody, given that the anti-fibronectin antibody also reacts with endogenous fibronectin. All samples were analyzed under reducing conditions, the blots for rF70K were developed with the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, 34080), and the blots for rFN were developed with 0.5 mg/mL 4-chloro-1-naphthol (Sigma, C8890) in Tris-buffered saline including 0.02% H₂O₂. (C) Specific RT-PCR analysis of mRNA coding for rF70K (left; 285 bp) or rFN (right; 440 bp) (excluding the endogenous FN1 mRNA). GAPDH analysis (226 bp) was included as a control. RNA was extracted with the RNeasy Plus Mini Kit (QIAGEN, 74134) according to the manufacturer’s protocol. Reverse transcription was performed with the ProtoScript II First Strand cDNA Synthesis Kit (New England Biolabs, E6560S), and PCR was performed for 40 cycles. (D and E) Immunofluorescence analysis of transfected HEK293 cells 3 days after seeding with anti-V5 antibodies (red; primary antibody, Thermo Fisher Scientific, R960-25; secondary antibody, Cy3-conjugated antibody, Thermo Fisher Scientific, A10521) for rF70K and rFN was performed according to a previously established protocol. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100; nuclei were counter-stained with DAPI (blue). The experiments were confirmed with three to four individual recombinant cell clones. The scale bar indicates 50 μm. Images were taken at 400× magnification with Zen software and an AxioImager M2 microscope (Zeiss) equipped with an ORCA-flash 4.0 camera (Hamamatsu, C11440). Note that all analyzed rF70K and rFN mutant proteins were retained intracellularly, but the wild-type proteins were not.