Figure 1.
Features of Siblings with Mutations of LEPRE1.
A: The pedigree of the affected family. The arrow indicates the proband. Patient II-3 was electively terminated. B: Radiographs of Patient II-2 as a neonate. There were multiple rib fractures, healed fractures of both femora and the right humerus, and a subacute fracture of the left humerus. Metaphyseal osteopenia was significant. C: Postmortem radiographs of Patient II-3. Bilateral femoral bowing, a healed fracture of the right femoral shaft, thin ribs, and metaphyseal demineralization were shown. D, E: Histological findings of Patient II-3. Irregular trabeculae of woven bones lined by osteoblasts are observed in the humerus (D) and spine (E). The stroma is cellular and consists of fibroblasts and collagen resembling osteofibrous dysplasia.
Figure 2.
Identification of LEPRE1 mutations.
A: A partial sequence of PCR product of Patient II-3 is shown. Compound heterozygous frame shift mutations (c.484delG, p.A162LfsX22 and c.2155dupC, p.E719RfsX11) are indicated by arrows. The mutations have been confirmed by the subsequent sequencing of subcloned products of normal and mutant alleles. B: Schematic presentation of the positions of the mutation. LEPRE1 cDNA encodes the tetratricopeptide repeat domain (four black regions), the Prolyl/Lysyl/hydroxylase domain (green region), and the KDEL ER- retrieval motif (red region). LEPRE1 with a p.E719RfsX11 change results in the lack of only the KDEL ER-retrieval sequence, whereas other functional domains remain intact.
Figure 3.
Characterization of the LEPRE1 mutations and proband collagen.
A: Patient II-3 LEPRE1 transcripts are about one-half the control level, by real-time RT-PCR. B: Steady-state type I collagen protein from fibroblasts of Patient II-3 and a normal control is shown. In both the cell layer and media, overmodification, detected as back-streaking of collagen alpha chains (α1 (I) and α2 (I)) on gel electrophoresis, is present in Patient II-3. We also detected mild overmodification of type V collagen (α1 (V)). C: Western blots of fibroblast P3H1 in Patient II-3 and control cells confirm absence of intracellular P3H1. D: Immunofluorescent staining of fibroblasts from Patient II-3 and a normal control show colocalization of P3H1 and CRTAP with GRP94 in control cells. Both P3H1 and CRTAP proteins are absent in fibroblasts from Patient II-3.