Clinical Descriptions of C-propeptide Cleavage Site Patients
Patient 1 (P1), a 13.5 yr old girl, was the 2950 g product of an uneventful pregnancy to a 39 yr old G5P3 Swedish woman. She was delivered vaginally at full term and had a normal newborn exam. Her developmental history was normal. At age 3.5 years, she incurred her first fractures (right tibia and fibula) after mild trauma. In subsequent years, the patient suffered 2–5 fractures/yr, including 3 upper extremity long bone, 3 olecranon, 4 tibial, 4 fibular and 4 small bone fractures of hands and feet. She has never been treated with an anti-resorptive drug. Clinical exam at age 12 years revealed white sclerae, normal dentition on inspection and radiographs, normal joint extensibility, straight spine and normal hearing and cardio-pulmonary evaluations. Bruising was slightly increased. Her growth and body habitus were normal, with height age of 161 cm at age 12 years (+1 SD compared to Swedish reference data), head circumference of 54 cm at 12 years (50th percentile for age) and normal proportions (span:height ratio= 1.03; upper segment:lower segment ratio = 1.08).
Multiple evaluations of serum Ca, Mg, Vitamin D, phosphate and PTH were normal. L1-L4 DXA (Lunar Prodigy, GE Medical Systems) spine Z-scores at ages 8, 9.5, 11.5 and 12.5 years were +3.4, +2.8, +3.3, and +3.9; radial pQCT (XCT 2000, Stratec Medizintechnik GmbH, Pforzheim, Germany) at age 11.5 yielded a z-score = +3.1 total vBMD. Family members, including both parents, have lumbar spine DXA z-scores between +0.1 to +1.5, ruling out familial high bone mass. Despite Patient 1’s elevated spine and wrist BMD, radiographic examination did not reveal signs of dense bone (). Long bones, especially fibulae, were not gracile, as would be common in OI. Radiographically, bone mass was diminished, appearing more osteopenic over time. Trabeculation was coarse throughout the skeleton, with persistence of larger trabeculae and loss of smaller trabeculae. There was a loss of modeling in the distal ulna and radius; in the tibia/fibula there was periosteal reactive change. The lumbar and cervical vertebrae had a “bone-in-bone” appearance, despite no prior bisphosphonate therapy. The lumbar vertebrae were not expanded in a way that might artificially increase the BMD z-score. Mineralization of the skull appeared normal, with only a few wormian bones but without basilar invagination.
Figure 2 Patient radiographs show a generalized decrease in bone mass. The skull radiograph of Patient 1 shows few wormian bones, while her vertebral bodies have os en os without compressions. Patient 2 also has os en os vertebral bodies, perhaps a reflection (more ...)
Patient 2 (P2), a 14 year old boy, is the offspring of non-consanguineous Caucasian American parents. He was the 3690 g product, delivered vaginally, of a full term pregnancy in a 32 year old G2P2 mother. Prenatal ultrasounds showed normal fetal development; post-natal development was normal. Starting at age 2 years, he sustained 8 long bone fractures in addition to several small bone fractures. At age 4.5 years, he was referred to a geneticist to rule out OI after osteopenia was noted during assessment of a metatarsal fracture. He has undergone three orthopaedic procedures, the first at age 5 years to stabilize an olecranon fracture with a pin, the second at age 10 for placement of intramedullary rods in the right femur, and the third at age 12 for placement of plate and screws on the distal left ulna.
On clinical exam at age 11.5 years, he had normal facies with light grey scleral hue, normal dentition, and unremarkable audiological and cardiovascular (ECHO/EKG) exams. He exhibited generalized joint hyperextensibility, with a Beighton score of 4/9 for bilateral elbows, the left knee and the ability to place the palms of has hands flat on the ground while bending down at the waist. His spine was straight. There was no bruising. Patient 2 had above average stature, with length that is 50% for a normal 12.5 year old boy, weight is 50% for an 11 year old and head circumference that is 50% for a 16 year old. There were no dysmorphic facial features. Dentinogenesis imperfecta was absent. Mild S-curve scoliosis has recently developed.
Serum Ca, Mg, Vitamin D, phosphate and PTH were normal. Patient 2 had received 6 cyles of bisphosphonate between ages 6–8 yr. His L1-L4 DXA (Hologic QDR 4500) z-score at age 12 years was average (0.00), while L1-L2 volumetric QCT (GE LightSpeed Ultra) z-score is −1.8. Radiographs of his lower extremity showed evidence of mild osteopenia () but no evidence of dense bones. Lower limb long bones had dense metaphyseal transverse lines from cycled bisphosphonate, genu valgum, and tibial bowing. No vertebral compressions were seen, only mild concavity of lumbar vertebrae. Cervical spine CT showed no platybasia or basilar invagination.
Identification of C-propeptide Cleavage Site Mutations
The coding regions of the type I collagen genes, COL1A1 and COL1A2, were completely sequenced. Patient 1 was found to have a heterozygous c.3655G>A substitution, causing an aspartic acid>asparagine change (p.Asp1219Asn) at the C-propeptide cleavage site in COL1A1 ( and ). Her mutation was confirmed by cDNA sequencing. Sequencing of Patient 2 COL1A1 and COL1A2 genes (The Center for Gene Therapy, Tulane University) revealed a heterozygous c.3355G>A substitution causing an alanine>threonine change (p.Ala1119Thr) at the cleavage site of COL1A2. ( and ) We confirmed the mutation, which eliminated a CviKI-I restriction site, by amplification and digestion of patient gDNA. The collagen sequence changes were not found in either set of parents.
Figure 3 Sequence and biochemistry of type I collagen. (A) Sequence tracings of Patient 1 cDNA and gDNA show the heterozygous G>A change in the α1(I) cleavage site. (B) The sequence tracing of Patient 2 cDNA shows the heterozygous G>A mutation (more ...)
Mutations near the N-terminus of LDL-receptor related protein 5 (LRP5
) cause both a benign high bone mass phenotype and high bone mass disease (Van Wesenbeeck, et al., 2003
). Accordingly, we sequenced LRP5
exons 2, 3 and 4, where all high bone mass mutations have been detected, but did not find any defects in our patients.
Collagen biochemistry is minimally altered
The steady-state fibroblast collagen from both children () had minimal electrophoretic abnormalities. Patient 1 had slightly delayed gel migration in the cell and media fractions, while Patient 2 collagen chains had normal mobility. Both patients had slight backstreaking of α1(I) and α2(I) cellular collagen, suggesting only a slight delay in the folding of the chains. Differential scanning calorimetry confirmed that collagen of both patients had normal thermal stability ().
Incorporation of proα1(I) and proα2(I) chains into triple helices was measured (), to determine whether C-propeptide cleavage site mutations delayed chain incorporation, as previously demonstrated for mutations in the C-propeptides (Chessler, et al., 1993
). Patient 1 had normal chain incorporation, compared to control cells, while Patient 2 had a 20–40 minute delay in chain incorporation, suggesting the cleavage site substitution in proα2(I) may alter the structure of the chain recognition or alignment regions.
Procollagen pericellular and in vitro processing is delayed
Processing of the C-propeptide of patient procollagen was examined by pericellular and in vitro assays (). Patients 1 and 2 both had a substantial delay in pericellular procollagen processing compared to controls. () Patient 1 had a severe processing defect, with minimal cleavage of either α chain and accumulation of only a small amount of mature collagen in the media. Although Patient 2 appeared to be able to cleave α1(I) chains, only a minor amount of mature α2(I) chains was detected. In addition, increased amounts of uncleaved procollagen and pC-collagens (procollagen with N- but not C- end processed) were seen in both patients.
Figure 4 Procollagen with a cleavage site mutation has delayed C-propeptide processing. (A) Pericellular processing of type I collagen from Patient 1 shows delayed processing kinetics, increased pro-α1(I) and pCα1(I) and much less mature collagen (more ...)
Pericellular procollagen processing assays reflect the combined activities of the various PCPs and their enhancer proteins, PCPE1 and 2. To examine the effect of the mutation on the activity of BMP1 and PCPE1 separately, in vitro quantitative assays were performed in which the 3H-tryptophan labeled procollagen substrates were incubated with purified BMP1, with or without addition of PCPE1, and the amount of radioactivity in the released C-propeptide trimer was measured. The results of these assays revealed that the activity of BMP1 on Patient 1 and 2 procollagen was about 30% and 46% of control (data not shown), in good agreement with the 25 and 50% cleavage expected if the amino acid substitutions interfered with C-propeptide cleavage. Enhancing activity of PCPE1 was about half of normal toward Patient 1 procollagen, but comparable to control (≈ 4 fold) with Patient 2 procollagen. A marked reduction in C- terminal procollagen processing by BMP1 is also seen on the autoradiogram (, right), with relatively lower amounts of free C1 and C2 in Patients 1 and 2, respectively, suggesting impaired cleavage of the mutant chains. Once again, enhancing activity of PCPE1 toward procollagen from Patient 1 but not from Patient 2, appeared to be reduced. Data obtained by densitometry revealed that processing of the mutant proα chain in both Patient 1 (α1(I)) and Patient 2 (α2(I)) was ~2 fold slower than that of the respective normal chains, seen with and without addition of PCPE1 (). This is also evident from the C1/C2 ratios: ~2:1, 1.5:1 and 4.9:1 for control, Patient 1 and Patient 2 procollagens, respectively (, , right).
Incorporation of pC-collagen alters matrix fibrils
mice, which cannot cleave the type I collagen C-propeptide because the processing enzyme is absent, incorporation of pC-collagen into fibrils results in a "barbed-wire" like appearance (Pappano, et al., 2003
; Suzuki, et al., 1996
). The dermal collagen fibrils of our patients have an irregular border on cross-section, compared to the smooth cross-sectional borders of fibrils from an age-matched control (). This is especially seen in Patient 2 fibrils, many of which have blebs protruding from the surface. Fibril diameters were normal in Patient 1(88.3 ± 7.4), but with significantly increased variance compared to control (p=0.032). Patient 2 (71.2 ± 6.9) fibril diameters were significantly smaller than control (89.0 ± 6.4, p = <0.001) without higher diameter variability. The longitudinal views of patient collagen fibrils did not have an obvious "barbed-wire" appearance.
Figure 5 Fibrils with a cleavage site mutation have irregular cross-sections and normal or decreased diameter. Dermal collagen fibrils from skin punch biopsies were examined by transmission electron microscopy. The fibril diameter was measured (n = 200) and compared (more ...)
Histomorphometry of patient bone samples
Each patient underwent an iliac crest biopsy after declomycin labeling. The analyses of static and kinetic parameters revealed distinct patterns in each child, compared with those found in type I OI (Glorieux, et al., 2000
) and in a reference population of 11–13.9 year old children () (Rauch, et al., 2000
Bone Histomorphometric Parameters
Unlike type I OI patients, Patient 1 has normal ratio of bone volume to bone tissue, with increased trabecular number and reduced trabecular thickness. Osteoid seams, with and without osteoblasts, appeared several fold thicker than normal or type I OI, covering a greater portion of the trabecular surface and giving a picture of osteoidosis. () Osteoclast surface and number and eroded surface were increased. Mineral apposition (MAR) and bone formation (BFR/BS) rates were elevated compared to controls, suggesting that osteoblasts produced a greater amount of matrix which was incompletely mineralized.
Figure 6 . Patients have abnormal osteoid formation. Light microscopic analysis of transiliac bone sections. Goldner’s trichrome staining represents mineralized bone matrix in green and osteoid in purple. (A) Patient 1 has thick osteoid seams in purple (more ...)
For Patient 2, all structural indices were decreased compared to controls. However, bone volume/total volume (BV/TV) and trabecular number (Tb.N.) were increased compared to type I OI, while trabecular thickness (Tb.Th.) was normal. The osteoid thickness was within normal range, and thus markedly thinner than in Patient 1. Interestingly, most of the trabeculae appeared to be covered by thin osteoid seams without osteoblasts (). In occasional areas, active osteoblasts and relatively thick osteoid were found. The osteoblast surface and eroded surface were normal in Patient 2. However, no active osteoclasts were seen in the sections. The portion of surface undergoing mineralization (MS/BS) was much smaller in Patient 2 than in Patient 1 and controls. MAR was elevated resulting in normal range BFR/BS, and indicating less brisk remodelling than in type I OI. Neither child had signs of osteosclerosis.
Increased mineralization of cortical and trabecular bone
To understand the unexpectedly normal or high BMD of our patients, we examined sections of their iliac crest bone by Fourier transform infrared (FT-IR) imaging. The mineral/ matrix ratio was significantly increased in both cortical and trabecular bone of Patient 1 and Patient 2, compared not only to two age-matched samples of control bone but also to bone from patients with classical OI caused by helical mutations in COL1A2 (p.Gly280Val, α2(I)G190V; p.Gly1030Asp, α2(I)G940D)(). The crystal size and perfection (crystallinity) of patient mineral was reduced in cortical bone, comparable to the crystallinity of classical OI cortical bone samples (). The matrix phase of patient trabecular bone had a significant increase in collagen maturity compared to normal or classical OI bone (), which may reflect increased proximity of cross-link sites due to altered folding of incorporated pC-collagen; the increase in maturity was not significant in patient cortical bone. The carbonate/phosphate ratio, a parameter indicative of carbonate substitution in the hydroxyapatite crystal, did not differ significantly among all OI or control samples (). The heterogeneity of the mineral and crystallinity distribution was increased in the two patients for mineral/matrix ratio relative to both classic OI and the controls demonstrating the wide variation in mineral content across the sample in both cortical and trabecular regions. The greatest spread in the data was seen in the mineral/matrix ratio and collagen maturity in Patient 1. This distribution can be seen in the representative images for Patient 1 and classic OI ().
Figure 7 Composition of iliac crest bone from patients with the C-propeptide cleavage site mutation, classical OI, and age-matched controls as determined by Fourier-Transform Infrared Imaging (FT-IR) Spectroscopy. The four parameters listed in the top row were (more ...)
Patient BMDDs show increased matrix mineralization
The BMDDs of the patients were measured using quantitative backscattered electron imaging (qBEI). Both patients exhibited BMDDs with a significant shift towards increased matrix mineralization compared to a young reference cohort (Fratzl-Zelman, et al., 2009
) but their mineralization patterns were different. For Patient 1,
the BMDD peak was remarkably broadened although the peak was shifted to a lesser extent than for Patient 2 (). Patient 1 had matrix areas of both higher (black arrow) and lower mineral content (white arrows) (), while Patient 2 had a more uniformly higher mineral content throughout the bone sample ().
Figure 8 Mineral content and organization of patient bone is altered. (A) Backscattered electron imaging (BEI) of Patient 1 bone samples demonstrates abnormal structure of mineralized bone matrix, both areas of higher (black arrow) and lower (white arrows) mineral (more ...)
The patient BMDDs were compared to two classical OI bone samples and to a previously published cohort of type I OI cases (Fratzl-Zelman, et al., 2010
) ( and ). Patient 1 had increased CaMean
, and CaHigh
, similar to classical OI bone, in which increased mineral content compared to normal pediatric bone was previously demonstrated (Roschger, et al., 2008a
; Weber, et al., 2006
). However, Patient 1 also had a distinctive increase in CaWidth
, reflecting an increase in heterogeneity of mineralization. Patient 2 had a higher CaMean
than all other OI and control samples, demonstrating uniformly high bone mineralization. Furthermore, the bone area undergoing primary mineralization (CaLow
) was approximately doubled in Patient 1 compared to control and OI samples, while the portion of highly mineralized areas (CaHigh
) in Patient 2 was increased 50-fold compared to control and about 3-fold compared to classical OI bone. These findings have not been noted in other OI children before or after bisphosphonate treatment (Weber, et al., 2006