Cortical-Bone Fragility--Insights from sFRP4 Deficiency in Pyle's Disease

Abstract

Background: Cortical-bone fragility is a common feature in osteoporosis that is linked to nonvertebral fractures. Regulation of cortical-bone homeostasis has proved elusive. The study of genetic disorders of the skeleton can yield insights that fuel experimental therapeutic approaches to the treatment of rare disorders and common skeletal ailments.

Methods: We evaluated four patients with Pyle's disease, a genetic disorder that is characterized by cortical-bone thinning, limb deformity, and fractures; two patients were examined by means of exome sequencing, and two were examined by means of Sanger sequencing. After a candidate gene was identified, we generated a knockout mouse model that manifested the phenotype and studied the mechanisms responsible for altered bone architecture.

Results: In all affected patients, we found biallelic truncating mutations in SFRP4, the gene encoding secreted frizzled-related protein 4, a soluble Wnt inhibitor. Mice deficient in Sfrp4, like persons with Pyle's disease, have increased amounts of trabecular bone and unusually thin cortical bone, as a result of differential regulation of Wnt and bone morphogenetic protein (BMP) signaling in these two bone compartments. Treatment of Sfrp4-deficient mice with a soluble Bmp2 receptor (RAP-661) or with antibodies to sclerostin corrected the cortical-bone defect.

Conclusions: Our study showed that Pyle's disease was caused by a deficiency of sFRP4, that cortical-bone and trabecular-bone homeostasis were governed by different mechanisms, and that sFRP4-mediated cross-regulation between Wnt and BMP signaling was critical for achieving proper cortical-bone thickness and stability. (Funded by the Swiss National Foundation and the National Institutes of Health.).

Figure 1. Clinical, Radiographic, and Molecular Findings

Panel A shows Patient 1 at 16 years of age; he had deformation of the lower limbs with marked valgus of both legs. The expanded distal femora could be easily palpated above the knees. Panel B is a radiograph of the lower limbs in Patient 1, showing expanded metaphyses of the distal femora and proximal and distal tibiae with extremely thin cortexes. The appearance of the bone at the tibial midshaft, with thicker cortex, is relatively normal. Panel C is an anteroposterior skull radiograph of Patient 2, showing expansion of the diploe. Panel D is an anteroposterior and lateral projection of the distal right tibia in Patient 3, showing changes similar to those in Panel B. In addition, two fractures run through the abnormal metaphysis, and a third fracture runs through the distal fibula. Panel E is a hand radiograph of Patient 2 at 14 years of age, showing expanded metaphyses and thin cortexes at the distal radius, as well as expansion of the distal metacarpals and (to a lesser degree) of the phalanges. Panels F, G, and H show the nucleotide sequences of SFRP4 in controls, affected patients, and heterozygous family members. Patients 1 and 2 are homozygous for a nucleotide insertion in exon 2 (Panel F); as a result, amino acid residues 167 and 168 are replaced, and a stop codon is generated at position 169, leading to a truncated protein. Both parents and the brother are heterozygous for the insertion. Circles denote female family members, squares male family members, open symbols unaffected family members, and black symbols affected family members. Patient 3 is homozygous for a C-to-T transition that transforms codon 232 from arginine to a stop codon (Panel G). Patient 4 is homozygous for the deletion of 7 nucleotides; as a consequence, there is a stretch of 10 missense amino acids (codons 161–170), followed by a premature termination at codon 171 (Panel H). Panel I is a diagram of the sFRP4 protein, illustrating the two main domains as well as the positions of the three mutations found in the patients.

Figure 2. Functional Consequences of Sfrp4 Deletion and Correction of the Cortical Phenotype in Sfrp4-Null Mice

Panel A shows representative radiographic images of wild-type and Sfrp4-null female littermates at 10 weeks of age. The arrowhead indicates the expanded metaphyses in the Sfrp4-null mice (four mice were analyzed in each genotype). Panel B shows microcomputed tomography (microCT) analysis of cortical thickness in femur midshaft of wild-type (black bars) and Sfrp4-null (open bars) mice at 4, 7, 17, 24, 60, and 68 weeks of age. The bars show mean values; T bars indicate standard errors. P<0.05 for the comparison with wild-type at each time point (five mice were analyzed in each genotype). Panel C shows representative histologic features of von Kossa–stained trabecular bone in proximal tibiae (top row; scale bar, 1 mm) and cortical bone in the tibial midshaft region (bottom two rows; scale bars, 300 μm) of wild-type, heterozygous, and Sfrp4-null mice at 10 weeks of age (five mice were analyzed in each genotype). Sfrp4 deletion leads to increased trabecular bone, decreased cortical thickness, and increased width. Panel D shows the differential effects of Sfrp4 deletion on canonical and noncanonical Wnt signaling in calvarial osteoblasts and bone marrow–derived osteoblasts. The total amount of active β-catenin, phosphorylated Jnk (p-Jnk), and total Jnk was assessed in calvarial osteoblasts and bone marrow–derived osteoblasts isolated from Sfrp4-null and wild-type littermates. Actin was used as a loading control. Fold differences in protein levels were measured by normalizing to actin and to total Jnk and dividing the normalized protein level by the mean normalized level in wild-type cells; values are expressed as means (±SD). An asterisk indicates P<0.05 for the comparison with wild type (three to five independent experiments were performed). Panel E shows the analysis of Sost mRNA expression in wild-type and Sfrp4-null calvarial osteoblasts and bone marrow–derived osteoblasts after 0, 14, and 28 days of osteogenic differentiation. Sfrp4 deletion affects Sost expression only in calvarial osteoblasts. The bars show mean values; T bars indicate standard deviations. An asterisk indicates P<0.05, and a double asterisk P<0.01 for the comparison with wild type at each time point (three independent experiments were performed). Panel F shows the regulation of bone morphogenetic protein (BMP) signaling by sFrp4. Wnt5a stimulates Bmp2 mRNA expression, which was blunted by sFrp4 in MC3T3-E1 cells. The bars show mean values; T bars indicate standard deviations. An asterisk indicates P<0.05 for the comparison with control, and a dagger P<0.05 for the comparison with Wnt5a-treated cells (three independent experiments were performed). Panel G shows the quantification of Bmp2 mRNA expression in wild-type and Sfrp4-null calvarial osteoblasts and bone marrow–derived osteoblasts. Sfrp4 deletion affects BMP signaling only in calvarial osteoblasts. The bars show mean values; T bars indicate standard deviations. An asterisk indicates P<0.05 for the comparison with wild-type calvarial osteoblasts (three independent experiments were performed). In Panels E, F, and G, fold differences in mRNA expression relative to that in wild-type cells were calculated as described by Livak and Schmittgen. Panel H shows the effects of Sfrp4 deletion on Smad1, Smad5, and Smad8 phosphorylation, which occurs only in calvarial osteoblasts. We measured fold differences in protein levels by normalizing to Smad1 and dividing the normalized protein level by the mean normalized level in wild-type cells; values are expressed as means (±SD). An asterisk indicates P<0.05 for the comparison with wild type (three to five independent experiments were performed). Actin was used as a loading control. Panel I shows representative histologic features of von Kossa–stained tibiae of 3-week-old wild-type and heterozygous mice after vehicle (phosphate-buffered saline) or RAP-661 treatment. Blocking BMP signaling through RAP-661 treatment increases cortical thickness in heterozygous mice (five mice were analyzed in each group). Panel J shows representative images of von Kossa–stained tibiae of wild-type and Sfrp4-null male mice treated with a sclerostin-neutralizing antibody (25 mg per kilogram) or vehicle (phosphate-buffered saline) (three to five mice were analyzed in each group). Blocking sclerostin increases cortical thickness in sFrp4-null mice.