Calcified Tissue International

Paget’s disease of bone (PDB) can adversely affect quality of life, but relatively little is known about the clinical predictors of reduced quality of life in patients with the disease. Here, we studied quality of life and its determinants in a large cohort of PDB patients who had been enrolled into the PRISM study, a randomized comparative trial of intensive versus symptomatic treatment for PDB. Health-related quality of life was assessed using the Short-Form 36 (SF36) questionnaire and other validated assessment instruments in 1,324 subjects with PDB. Clinical predictors of quality of life were identified by multivariate regression analysis. The physical summary (mean ± standard deviation) score of the SF36 was substantially reduced in PDB to 36.3 ± 11.3 compared with the expected population norm of 50 (P < 0.001). The mental summary score was only slightly reduced, to 48.7 ± 11.8, in PDB; but this was statistically significant (P < 0.001). Bone pain due to PDB, previous bisphosphonate therapy, and increasing age were identified as negative predictors of the SF36 physical summary score (P < 0.001); but serum levels of total alkaline phosphatase (ALP) did not predict physical summary score. We conclude that PDB has a substantial negative impact on health-related quality of life, which mainly affects physical functioning. The lack of correlation between ALP and quality of life observed in this study emphasizes the importance of addressing quality-of-life issues when treating PDB and not just focussing on response of ALP levels.

Identification of variants in the calcium-sensing receptor (CASR) gene is an important means of distinguishing between familial hypocalciuric hypercalcaemia (FHH) and primary hyperparathyroidism. However, identification and bioinformatics analysis of genetic variants alone is now considered insufficient as definitive proof; additional functional assessment is required to diagnose FHH with certainty. We identified two novel variants, D433Y and C739Y, and one previously reported variant G509R in the CASR of four kindreds provisionally diagnosed with FHH and aimed to functionally characterise these variants to confirm the diagnosis. Variant receptors were cloned as FLAG-tagged constructs into the mammalian expression vector, pcDNA3.1. Wild type and variant receptor constructs were expressed in HEK293 cells and their expression assessed by Western blot analysis and their functionality analysed using an IP-One assay which measures myo-inositol 1-phosphate accumulation following CaSR activation. Western blot analysis showed that the D433Y receptor had diminished mature glycosylated receptor compared with wild type CaSR whereas the G509R receptor had a complete lack of mature receptor. The C739Y receptor was consistently overexpressed. Functional assessment showed the D433Y receptor to be mildly inactivating at physiological calcium concentrations whereas the G509R receptor was inactive at all calcium concentrations. By contrast, the C739Y variant was activating compared to wild type receptor which is inconsistent with it causing FHH. We conclude that functional assessment of CaSR variants using the IP-One assay was useful in the investigation of suspected FHH probands, confirming the D433Y and G509R variants as likely pathogenic/pathogenic, but dismissing the C739Y variant as causing FHH.

X-linked hypophosphatemic rickets (XLH) and m.3243A>G mitochondrial disease share several clinical findings, including short stature, hearing impairment (HI), nephropathy, and hypertension. Here, we report on a case with the rare coincidence of these two genetic conditions. In early childhood, the patient presented with hypophosphatemia and bone deformities and was clinically diagnosed with XLH. This was genetically verified in adulthood with the identification of a de novo pathogenic deletion in phosphate-regulating endopeptidase homolog X-linked (PHEX). In addition, the patient developed HI and hypertension and when his mother was diagnosed with m.3243A>G, subsequent genetic testing confirmed the patient to carry the same variant. Over the next two decades, the patient developed progressive renal impairment however without nephrocalcinosis known to associate with XLH which could indicate an m.3243A>G-related kidney disease. Parallel with the progression of renal impairment, the patient developed hyperphosphatemia and secondary hyperparathyroidism. In conclusion, this case represents a complex clinical phenotype with the reversal of hypo- to hyperphosphatemia in XLH potentially mediated by the development of an m.3243A>G-associated nephropathy.

The aim of this study was to explore the relationship(s) between histomorphometric measurements of cancellous bone structure and ultrasound. Ultrasonic measurements were made using a CUBA research system consisting of a pair of 1 Mhz unfocused transducers. Speed of sound (SOS) and broadband ultrasonic attenuation (BUA) were determined in 15 human cadaveric heels, with mean precision for all measurements coefficients of variation (CV) = 0.6% and 12%, respectively. The calcaneus was dissected and a 7.5 mm transcortical cylinder was removed from the path of ultrasound (US) transmission. The cortices were removed and the remaining cancellous core was sectioned into approximately 5 mm blocks, numbered from 1 to 6 (medial-lateral). Histomorphometric measurements were performed on decalcified, 5 μm-thick sections from blocks 1–6 using an automatic color image analysis system. There were significant differences between blocks 1 and 3–6 for BS/TV, BV/TV, Tb.N, and Tb.Sp (all P < 0.001), all decreasing in a medial-lateral direction (except Tb.Sp), implying that the medial portion of the calcaneus had more trabeculae with less spacing between them than the lateral portion. Furthermore, Tb.Th and BS/BV variables were uniform across the calcaneus, suggesting that individual trabeculae were of similar dimension. We found no significant correlations between US and histomorphometric parameters either averaged over all blocks or by using each block region separately. In conclusion, this study does not support the notion that US measurements of SOS and BUA through the heel reflect calcaneal cancellous bone structure, however, further studies using larger sample sizes may be warranted.

Clonal osteoblastic cell lines were isolated from neonatal rat calvariae and characterized with regard to a number of features associated with authentic osteoblasts. These included elevated alkaline phosphatase activity (relative to fibroblasts), PTH and PGE2-stimulated increases in cAMP, the predominant synthesis of type 1 collagen, and the production of a mineralized matrixin vitro. By these criteria, five clones with osteoblast-like phenotypes were identified (ROB-C8a, C11, C20, C23, and C26) which varied somewhat in shape, levels of alkaline phosphatase activity, and in responsiveness to PTH and PGE2, C11, C20, and C23 responded to both effector substances, whereas C8a only responded to PTH and C26 only responded strongly to PGE2. Upon further examination, two of the clones (C23 and C26) were also found to exhibit significant muscle myotube formation after reaching confluence, and three of the clones (C8a, C11, and C26) showed marked adipocyte differentiation after treatment with dexamethasone. Overall, these data add further supporting documentation to (1) the suspected ontogenetic relationships of osteoblasts to other connective tissue cells, and (2) the concept that osteoblastic cells associated with neonatal rat calvariae are in various stable stages of differentiation and developmental commitment.

The proteins and peptides of immature enamel were extracted from freshly slaughtered bovine embryos in solutions containing protease inhibitors. No detectable differences were noted in the number of components, their overall amino acid composition, or molecular weights from the proteins and peptides extracted 12–16 h postmortem in solutions which contained no protease inhibitors. These data indicate that the large number of components found in developing bovine enamel is not due to proteolysis occurring during their isolation. Significant amounts of protein components having molecular weights greater than ∼15,000 were not detected. Therefore, if the ameloblasts initially synthesize only a few high molecular weight protein species, the present data imply that in vivo degradation of the high molecular weight enamel proteins occurs very rapidly after their synthesis and precedes the massive loss of protein which accompanies the final stages of enamel mineralization and maturation.

Several studies show that in elderly men bone mineral density (BMD) is not correlated with the insulin-like growth factor (IGF-I) level, but data are scanty in young men. Results of studies correlating insulin-like growth factor binding protein 3 (IGFBP-3) and BMD in men are discordant. As different hypotheses can explain the discordant results, we evaluated the correlation of BMD with serum IGF-I, IGFBP-3, and IGF-I/IGFBP-3 index in a large cohort of 721 men aged 19–85 years taking into account age, body weight, 17β-estradiol, free testosterone, and parathyroid hormone. Serum IGF-I and IGFBP-3 decreased with age (r = −0.44 and r = −0.36, P = 0.0001). After adjustment for confounding variables, IGF-I correlated weakly positively with BMD and with bone mineral apparent density (BMAD) of hip as well as with cortical thickness of femoral neck, both of which are determined mainly by bone resorption, but not with bone size determined by periosteal apposition. IGF-I correlated weakly positively with BMD at the whole body and at the third lumbar vertebra IGFBP-3 and IGF-I/IGFBP-3 index did not correlate with densitometric parameters. In men aged 19–60 years, IGF-I correlated with BMD and BMAD of total hip and with cortical thickness of femoral neck positively and more strongly than in the entire cohort but not with the size of proximal femur. BMD of total hip was 6% higher in men in the highest quartile of IGF-I than in men in the lowest quartile. IGF-I, IGFBP-3, and IGF-I/IGFBP-3 index did not correlate with densitometric parameters of other sites. In the men aged more than 60 years, neither IGF-I nor IGFBP-3 nor IGF-I/IGFBP-3 index correlated with BMD, BMAD, or bone size. In men aged 19–60 years, the most significant hormonal determinants of BMD and BMAD of the hip and of the cortical thickness of femoral neck were 17β-estradiol and IGF-I (P < 0.05–0.0001). In men aged more than 60 years, the most significant determinants of hip BMD were 17β-estradiol and PTH. In conclusion, IGF-I seems to contribute to the inhibition of bone resorption and to maintaining bone mass of the proximal femur during the phase of slow bone loss in men aged less than 60 years. IGFBP-3 and IGF-I/IGFBP-3 index were not correlated with BMD or bone size.