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. 2012 Nov;82(5):453-9.
doi: 10.1111/j.1399-0004.2011.01794.x. Epub 2011 Oct 19.

Deficiency of CRTAP in non-lethal recessive osteogenesis imperfecta reduces collagen deposition into matrix

M Valli  1 , A M Barnes, A Gallanti, W A Cabral, S Viglio, M A Weis, E Makareeva, D Eyre, S Leikin, F Antoniazzi, J C Marini, M Mottes
Affiliations

Deficiency of CRTAP in non-lethal recessive osteogenesis imperfecta reduces collagen deposition into matrix

M Valli et al. Clin Genet. 2012 Nov.

Abstract

Deficiency of any component of the ER-resident collagen prolyl 3-hydroxylation complex causes recessive osteogenesis imperfecta (OI). The complex modifies the α1(I)Pro986 residue and contains cartilage-associated protein (CRTAP), prolyl 3-hydroxylase 1 (P3H1) and cyclophilin B (CyPB). Fibroblasts normally secrete about 10% of CRTAP. Most CRTAP mutations cause a null allele and lethal type VII OI. We identified a 7-year-old Egyptian boy with non-lethal type VII OI and investigated the effects of his null CRTAP mutation on collagen biochemistry, the prolyl 3-hydroxylation complex, and collagen in extracellular matrix. The proband is homozygous for an insertion/deletion in CRTAP (c.118_133del16insTACCC). His dermal fibroblasts synthesize fully overmodified type I collagen, and 3-hydroxylate only 5% of α1(I)Pro986. CRTAP transcripts are 10% of control. CRTAP protein is absent from proband cells, with residual P3H1 and normal CyPB levels. Dermal collagen fibril diameters are significantly increased. By immunofluorescence of long-term cultures, we identified a severe deficiency (10-15% of control) of collagen deposited in extracellular matrix, with disorganization of the minimal fibrillar network. Quantitative pulse-chase experiments corroborate deficiency of matrix deposition, rather than increased matrix turnover. We conclude that defects of extracellular matrix, as well as intracellular defects in collagen modification, contribute to the pathology of type VII OI.

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Conflict of interest statement

Conflict of interest There are no conflicts of interest relevant to this article.

Figures

Fig. 1
Fig. 1
Patient 903 features, mutation identification, and fibril diameters (a) At 5 years, 10 months, the patient has a normal head, white sclerae, small thorax and shortening of the proximal segment of the upper and lower extremities. (b) Detail of short bowed legs. Radiographs of: (c) spine, (d) arm, (e) legs show osteopenia, undertubulation and severe deformities, consistent with a severe deforming form of osteogenesis imperfecta. (f) Sequence tracing of patient 903 gDNA shows a homozygous insertion/deletion in exon 1 of CRTAP. The mutation deletes the 16 nucleotides underlined in the control sequence and inserts the five nucleotides shown in the patient sequence. (g) A MwoI restriction enzyme digest of patient 903 gDNA and parental gDNA confirms the presence of the homozygous mutation in the patient and heterozygosity of the mutation in his father (F) and mother (M), respectively. (h) Patient dermal collagen fibrils from a skin punch biopsy were compared to an age-matched control and examined by transmission electron microscopy. The fibril diameters (n = 200) were increased, and fibrils had slightly irregular borders and increased size variability.
Fig. 2
Fig. 2
Effects of CRTAP mutation on the prolyl 3-hydroxylation complex and on type I collagen. (a) Western blots of cartilage-associated (CRTAP), prolyl 3-hydroxylase 1 (P3H1) and cyclophilin B (CyPB) protein levels in patient 903 fibroblasts compared to actin-loading controls. Due to mutual protection, the absence of CRTAP leads to the loss of P3H1. CyPB levels remain normal. (b) Immunofluorescence staining of fibroblasts of patient 903 and control for the three components of the prolyl 3-hydroxylation complex, CRTAP (top left), P3H1 (bottom left), CyPB (top right), plus type I collagen (bottom right), shown colocalized with the endoplasmic reticulum chaperones GRP94, PDI or HSP47. The immunofluorescence confirms the lack of CRTAP and P3H1 in fibroblasts. (c) Procollagen synthesized during metabolic labeling with tritiated proline by control (C) and patient (903) was partially purified from medium (M) and cell layer (CL), pepsin digested, and analyzed on 6% SDS-Urea PAGE. Alpha chains in medium and cell layer have a delayed migration and appear overmodified. No normal migrating chains are evident. (d) A differential scanning calorimetry thermogram shows that the collagen melting temperature is increased by ∼1°C, consistent with the increased collagen modification.
Fig. 3
Fig. 3
Decreased collagen deposition in ECM after long-term culture. (a) Control and patient fibroblasts were maintained for 21 days after confluence. After medium removal, the cell layers were labeled with a polyclonal anti-type I collagen, LF-67, and nuclei were counterstained with DAPI. The fibrillar network is absent in patient 903. (b) Quantitation of collagen turnover in matrix, with the day 0 time point arbitrarily set to 100, showing similar rates of turnover between control (white) and patient cells (gray). (c) Comparison of α1(I) chain (left) and α2(I) chain (right) deposition and turnover of control and patient matrix, showing much less matrix deposited from patient 903 cells.
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