Severe osteogenesis imperfecta caused by a small in-frame deletion in CRTAP

Abstract

Mutations of proteins involved in posttranslational modification of collagen type I can cause osteogenesis imperfecta (OI) inherited in a recessive pattern. The cartilage-associated protein (CRTAP) is part of a heterotrimeric complex (together with prolyl-3-hydroxylase-1 [P3H1] and cyclophilin B) that 3-hydroxylates the alpha 1 chain of collagen type I at proline residue 986 and plays a collagen chaperon role. CRTAP mutations usually cause severe OI. We report on a patient with OI and a homozygous in-frame deletion in CRTAP and a severe form of OI. The girl was born with markedly deformed long bones. Despite intravenous bisphosphonate treatment, she developed multiple vertebral compression fractures and severe scoliosis and at 4 years of age was able to sit only with support. Although CRTAP transcript levels were normal in the patient's fibroblasts, protein levels of both CRTAP and P3H1 were severely reduced. The degree of 3-hydroxylation at proline residue 986 was also decreased. This report characterizes a patient with a CRTAP small in-frame deletion. We are unaware of prior reports of this finding. We suggest that this deletion affects crucial amino acids that are important for the interaction and/or stabilization of CRTAP and P3H1.

Figure 1

A. Family pedigree. B. Severe thoracolumbar right convex scoliosis in proband at 3 years of age. Severe bowing deformities of the right humerus, ulna, and radius with healing fracture at the apex of the humerus at same age. The hand radiograph shows thin cortices with osteopenia. Severe anterior convex bowing of femora, tibiae and fibulae with a fracture at the proximal shaft of the left femur, and an oblique fracture at the distal shaft of the right femur at the same age. C. Control sequence: Exon 4 in the CRTAP gene (upper panel). In-frame deletion in proband: c.804_809delAGAAGT in exon 4 in the CRTAP gene (lower panel)

Figure 2

A. Real time PCR quantification of CRTAP and P3H1 transcripts normalized against GAPDH. B. Western blot with CRTAP antibody: Lane 1: Human control fibroblasts; lane 2: human control fibroblasts; lane 3: CRTAP hypomorphic mutation; lane 4: new in-frame deletion; lane 5: Crtap^-/- murine osteoblasts used as negative control; lane 6: wild type murine osteoblasts used as positive control. C. CRTAP and P3H1 immunofluorescence on skin fibroblasts from a control, the patient with the in-frame CRTAP deletion presented here and a patient with a hypomorphic CRTAP mutation. D. Representative western blot of CRTAP in 24 h conditioned medium and cell layer showing increased CRTAP secretion in the studied patient compared to a control. E. Mass spectrometry of a tryptic peptide of the collagen type I alpha 1 chain containing Pro986 and showing a significant amount of non 3-hydroxylated proline in the patient fibroblasts compared to a control.