Mutations in the gene encoding the RER protein FKBP65 cause autosomal-recessive osteogenesis imperfecta

Abstract

Osteogenesis imperfecta is a clinically and genetically heterogeneous brittle bone disorder that results from defects in the synthesis, structure, or posttranslational modification of type I procollagen. Dominant forms of OI result from mutations in COL1A1 or COL1A2, which encode the chains of the type I procollagen heterotrimer. The mildest form of OI typically results from diminished synthesis of structurally normal type I procollagen, whereas moderately severe to lethal forms of OI usually result from structural defects in one of the type I procollagen chains. Recessively inherited OI, usually phenotypically severe, has recently been shown to result from defects in the prolyl-3-hydroxylase complex that lead to the absence of a single 3-hydroxyproline at residue 986 of the alpha1(I) triple helical domain. We studied a cohort of five consanguineous Turkish families, originating from the Black Sea region of Turkey, with moderately severe recessively inherited OI and identified a novel locus for OI on chromosome 17. In these families, and in a Mexican-American family, homozygosity for mutations in FKBP10, which encodes FKBP65, a chaperone that participates in type I procollagen folding, was identified. Further, we determined that FKBP10 mutations affect type I procollagen secretion. These findings identify a previously unrecognized mechanism in the pathogenesis of OI.

Figure 1

Pedigrees of OI Families Turkish OI families originating from the Black Sea region of Turkey (Families 1–5) and the Mexican-American OI family (Family 6).

Figure 2

Clinical Phenotype (A) EB at the ankle (arrow) in case R06-113A. (B) Upper extremity joint laxity and long fingers in R06-113A. (C and D) Radiographs showing scoliosis (arrows) in R06-113A and R93-188A, respectively. (E and F) Wedge vertebrae (arrows) in R06-113A and R93-188A, respectively. (G and H) Fractures, bent bones, and osteopenia (arrows) in R06-113A.

Figure 3

Bone Histomorphology Bone histology from the iliac crest from control and an affected individual (R06-113A) demonstrating normal (A) and abnormal (B) lamellar bone patterns (arrows).

Figure 4

FKBP10 Mutation Detection (A and B) Sequence analysis of exon 2 in control and a representative affected individual (R06-113A) with the mutation in the Turkish families. (C and D) Exon 5 sequence analysis in control and a representative affected individual (R93-188A) with the mutation in the Mexican-American family (R93-188A). (E) RT-PCR of FKBP10 cDNA from control and R06-113A fibroblasts showing that a FKBP10 cDNA is synthesized. (F) RT-PCR of FKBP10 cDNA from control and R93-188A fibroblasts showing that a FKBP10 cDNA is not synthesized; lower band demonstrates control cDNA synthesis. (G) Cartoon of the FKBP65 molecule with predicted protein consequences for each mutation. PPIase, peptidyl-prolyl cis-trans isomerase; EF/Hand domain; HEEL, putative ER-retention sequence.

Figure 5

Type I Procollagen/Collagen Biochemical Analysis (A) Cells synthesized normal procollagenous proteins. Normal amounts and mobilities were seen for proα1(I) and proα2(I) chains in control and affected fibroblasts in both the cell layer and the media (R06-016A, Family 3; R93-188A and B, Family 6). (B) Cells also synthesized normal collagenous proteins, with normal amounts and mobilities seen for α1(I) and α2(I) chains in control and affected fibroblasts in both the cell layer and the media (R06-016A, Family 3; R93-188A and B, Family 6).

Figure 6

Transmission Electron Microscopy, Pulse-Chase of Collagenous Proteins (A) TEM of cultured fibroblasts from WT and an affected individual (R06-113A) showing dilated ER in the affected cells (labeled). (B) Pulse-chase. The bands in each set represent the collagen trimers that migrated into the gel under nonreducing conditions. In all cell lines, label was seen in the monomeric proα chains. For controls, by 40 min the amount of trimer in the medium exceeded that remaining in the cells. In R06-113 and R93-188 cells, the amount in the medium exceeded that in the cells by 40 min of chase, but not to the extent seen in control cells. In all experiments, the amount of labeling in the cells with FKBP10 mutations was slightly reduced compared to that in the control cells. (C) Graph of band intensities from each pulse-chase experiment (control, R06-113A, and R93-188A) showing a delay in secretion of the type I collagen trimer.

Figure 7

Intracellular Localization of FKBP65 and Type I Procollagen (A–C) Confocal microscopy demonstrating ER localization of both FKBP65 (green) and type I procollagen (red) and colocalization of the two proteins in control fibroblasts and R06-113 (merge). (C and D) Confocal microscopy of fibroblasts from cases R06-113A and R93-188A, respectively, demonstrating a speckled pattern of type I procollagen suggestive of intracellular aggregates containing this protein.