Prolyl 3-hydroxylase 1 deficiency causes a recessive metabolic bone disorder resembling lethal/severe osteogenesis imperfecta

Abstract

A recessive form of severe osteogenesis imperfecta that is not caused by mutations in type I collagen has long been suspected. Mutations in human CRTAP (cartilage-associated protein) causing recessive bone disease have been reported. CRTAP forms a complex with cyclophilin B and prolyl 3-hydroxylase 1, which is encoded by LEPRE1 and hydroxylates one residue in type I collagen, alpha1(I)Pro986. We present the first five cases of a new recessive bone disorder resulting from null LEPRE1 alleles; its phenotype overlaps with lethal/severe osteogenesis imperfecta but has distinctive features. Furthermore, a mutant allele from West Africa, also found in African Americans, occurs in four of five cases. All proband LEPRE1 mutations led to premature termination codons and minimal mRNA and protein. Proband collagen had minimal 3-hydroxylation of alpha1(I)Pro986 but excess lysyl hydroxylation and glycosylation along the collagen helix. Proband collagen secretion was moderately delayed, but total collagen secretion was increased. Prolyl 3-hydroxylase 1 is therefore crucial for bone development and collagen helix formation.

Figure 1

Clinical and radiological manifestations of severe recessive osteogenesis imperfecta. (a) Fetal anteroposterior and lateral radiographs of proband 2–2 at 19-weeks gestation demonstrate irregular fractures with periosteal reaction and bowing of the femurs. The skull is markedly thin. The metaphyses are irregularly ossified. The vertebrae and ribs are not fractured. (b) Fetal anteroposterior radiograph of proband 4 at 18 weeks gestation. Bones are severely osteopenic with irregular fractures of the long bones. Femurs and tibiae are bowed. Ribs and skull are markedly thin. (c) Proband 5 at 6 years of age. The bones are severely osteopenic. Long bones are thin and gracile with prominent epiphyses and multiple fractures resulting in severe bowing. The ribs are markedly thin. There is significant platyspondyly with thoracolumbar scoliosis. Anteroposterior view of the hand at 9 years of age demonstrates severe osteopenia with disorganized matrix. The phalanges are unusually long and metacarpals are short. Bone age is normal. (d) Proband 3 newborn films (left of dotted line). The skull is thin. Long bones have numerous fractures with irregular callus formation, resulting in ‘accordion’ shaped femurs. There is bowing of the tibia, fibula and radius. Asymmetric platyspondyly is most marked at the lower thoracic vertebrae. Proband radiographs at 16 months of age (right of dotted line) demonstrate healing of fractures with resultant irregular bowing of the long bones, particularly the tibiae. Severe osteopenia has progressed. Vertebra plana has worsened with resultant kyphosis. Anteroposterior view of the hand demonstrates long phalanges with abnormal matrix; bone age is normal.

Figure 2

LEPRE1 mRNA and P3H1 protein expression in probands and parents. (a) Proband fibroblast total RNA was screened for LEPRE1 mRNA transcripts by real-time RT-PCR. Decreased LEPRE1 mRNA was identified in primary fibroblasts from five probands with possible quantitative defects in LEPRE1. Partial rescue of LEPRE1 mRNA expression in fibroblasts incubated with the translational inhibitor emetine (+) suggests that transcripts are degraded by nonsense-mediated decay because of the presence of premature termination codons. (b) Decreased expression of LEPRE1 transcripts was evident only in affected probands by real-time RT-PCR. Parental fibroblasts (F, father; M, mother) showed relatively normal expression of LEPRE1 mRNA compared with normal control fibroblasts. (c) Protein blots of total cell lysates probed with antibody to P3H1. Complete absence of P3H1 protein is seen in fibroblasts of proband 1 (P1), both siblings from family 2 (P2–1, P2–2), proband 3 (P3) and proband 5 (P5). A residual amount of P3H1, about 25% of control, was detected in proband 4 (P4). β-actin was probed as a loading control.

Figure 3

Tandem mass spectrometry of proband and normal control secreted type I collagen. Probands were observed to have absent or decreased 3-hydroxylation at α1(I)Pro986. Left, ions are displayed by their relative abundance and mass/charge (m/z) ratio. Doubly charged ions shown for probands differ from control by 16 a.m.u., because of a lack of an oxygen atom. In the presence of normal 3-hydroxylation, the tryptic peptide ions containing α1(I)Pro986 yield a mass of 782, whereas the peptide ions without 3-hydroxylation have a mass of 774. Right, fragmentation spectra of tryptic peptide ions containing the α1(I)Pro986 residue. N-terminal (b) and C-terminal (y) daughter ions are labeled for control and proband 1 samples. The y6 ion localizes the extra 16 a.m.u. to the C-terminal six amino acid residues containing the α1(I)Pro986 site of 3-hydroxylation.

Figure 4

Collagen chains with normal primary structure are overmodified in the helical region. (a) SDS-urea-PAGE analysis of type I collagens synthesized by proband and available parental fibroblasts. Overmodification, detected as delayed electrophoretic migration of collagen alpha chains, is present in all proband samples, but is absent in all parental samples. (b) Differential scanning calorimetry thermograms of proband type I collagens. The T_m of the proband samples are increased approximately 1 °C compared with normal control collagen, but are comparable to collagen synthesized at 40 °C, consistent with increased modification in the absence of a primary structural defect. A thermogram of overmodified type I collagen containing an α1(I)Gly997Ser substitution (top tracing) shows the decreased T_m characteristic of collagen structural defects.

Figure 5

The effect of P3H1 absence on type I collagen secretion. (a) Pulse-chase studies of collagen synthesis by proband and control cells were conducted as described in Methods. Each scan shows intracellular and secreted collagen from the indicated cell line from t = 0 to 4 h after chase was initiated; sample loading was normalized to cell number. (b) Densitometry of radiographs of pulse-chase gels was used to calculate relative total collagen secretion (ratio of α1(I) band in proband versus control in medium at 4 h), and time course of secretion (proportion of α1(I) band in media versus media + cell at a given time for each cell line).