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Sequestosome 1/p62 (p62) mutations are associated with PDB; however, there are limited data regarding functional consequences. We report a novel mutation in exon 7 (K378X) in a patient with polyostotic Paget's disease of bone. p62 mutants increased NF‐κB activation and significantly potentiated osteoclast formation and bone resorption in human primary cell cultures.

Sequestosome 1/p62 (p62) mutations are associated with PDB; however, there are limited data regarding functional consequences. We report a novel mutation in exon 7 (K378X) in a patient with polyostotic Paget's disease of bone. p62 mutants increased NF‐κB activation and significantly potentiated osteoclast formation and bone resorption in human primary cell cultures.Introduction:Sequestosome 1/p62 (p62) mutations are associated with Paget's disease of bone (PDB); however, there are limited data regarding functional consequences. One report has linked the common P392L mutation in the p62 ubiquitin binding associated (UBA) domain with increases in NF‐κB activity, a transcription factor essential for osteoclastogenesis. To further clarify the functional impact of p62 mutations associated with PDB, we assessed the effect of p62 mutation (a novel mutation: K378X, and previously reported mutations: P392L and E396X) on RANK‐induced NF‐κB activation and compared this with the effect of wildtype p62. In addition, we studied the effect of p62 mutation on osteoclast formation and bone resorption.Materials and Methods:We performed co‐transfection experiments with expression plasmids for p62 (wildtype or mutated) and RANK and an NF‐κB luciferase reporter gene. Luciferase activities were recorded after addition of luciferin to cellular lysates. RAW264.7cells stably expressing enhanced green fluorescent protein (EGFP)‐tagged p62 (wildtype, K378X, or P392L) or EGFP alone were assessed for changes in cell proliferation. Additionally, these cells were stimulated with RANKL to produce osteoclast‐like cells (OLCs). Primary human monocytes collected from the K378X‐affected patient and a control subject were stimulated to form OLCs and bone resorption data were obtained.Results:The novel mutation introduces a premature stop codon in place of Lys‐378 and thereby eliminates the entire p62 UBA domain; this and two additional natural mutations (P392L, E396X) increased NF‐κB activation compared with wildtype p62. Wildtype p62 consistently inhibited NF‐κB activation compared with empty vector. UBA mutations (K378X and P392L) significantly increased the number of OLCs formed in response to RANKL and also the number of nuclei of the OLCs. K378X‐affected human monocytes formed more OLCs with more nuclei and increased bone resorption compared with control monocytes.Conclusions:Our data show that mutation of the p62 UBA domain results in increased activation of NF‐κB and osteoclast formation and function compared with wildtype p62. These results may partially explain the mechanism by which p62 mutation contributes to the pathogenesis of PDB.

Bisphosphonate treatment for severe Paget's disease leads to hypocalcemia followed by a secondary hyperparathyroid response to restore normocalcemia. A case is presented of a 60‐year‐old woman with polyostotic Paget's disease and postsurgical hypoparathyroidism. In 1993 her Paget's disease—alkaline phosphatase (ALP), 1260 U/liter (35–135 U/liter), and fasting urinary hydroxyproline excretion, 13.7 μmol/liter GF (0.4–1.9 μmol/liter)—was treated with intravenous pamidronate. Symptomatic hypocalcemia followed the first 60‐mg dose, requiring large doses of calcium supplementation and calcitriol. Pamidronate therapy to a total dose of 360 mg was followed by rapid and prolonged remission with indices of bone turnover in the normal range within 2 months and persisting for at least 19 months after treatment. In 1999 relapse of Paget's disease—ALP, 511 U/liter (35–135 U/liter), and fasting urinary deoxypyridinoline/creatinine 53.1 μmol/mol (5–27 μmol/mol)—was treated with alendronate, 10 mg daily. Symptomatic hypocalcemia occurred again, requiring increased calcium and calcitriol therapy. Indices of bone turnover were within the normal range 9 weeks after the start of therapy. These responses were significantly more rapid and sustained than those observed in euparathyroid subjects. This case suggests that the lack of parathyroid response may modify the response to bisphosphonates by: (a) increasing intrinsic uptake of bisphosphonate into the pagetic skeleton, allowing response to a smaller dose; (b) increasing duration and severity of hypocalcemia after bisphosphonate therapy; and (c) removing the hyperparathyroid drive to reactivation of pagetic osteoclasts, leading to a prolonged remission. These observations have implications for optimizing bisphosphonate therapy both in Paget's disease and in osteoporosis.

Bisphosphonate treatment for severe Paget's disease leads to hypocalcemia followed by a secondary hyperparathyroid response to restore normocalcemia. A case is presented of a 60‐year‐old woman with polyostotic Paget's disease and postsurgical hypoparathyroidism. In 1993 her Paget's disease—alkaline phosphatase (ALP), 1260 U/liter (35–135 U/liter), and fasting urinary hydroxyproline excretion, 13.7 μmol/liter GF (0.4–1.9 μmol/liter)—was treated with intravenous pamidronate. Symptomatic hypocalcemia followed the first 60‐mg dose, requiring large doses of calcium supplementation and calcitriol. Pamidronate therapy to a total dose of 360 mg was followed by rapid and prolonged remission with indices of bone turnover in the normal range within 2 months and persisting for at least 19 months after treatment. In 1999 relapse of Paget's disease—ALP, 511 U/liter (35–135 U/liter), and fasting urinary deoxypyridinoline/creatinine 53.1 μmol/mol (5–27 μmol/mol)—was treated with alendronate, 10 mg daily. Symptomatic hypocalcemia occurred again, requiring increased calcium and calcitriol therapy. Indices of bone turnover were within the normal range 9 weeks after the start of therapy. These responses were significantly more rapid and sustained than those observed in euparathyroid subjects. This case suggests that the lack of parathyroid response may modify the response to bisphosphonates by: (a) increasing intrinsic uptake of bisphosphonate into the pagetic skeleton, allowing response to a smaller dose; (b) increasing duration and severity of hypocalcemia after bisphosphonate therapy; and (c) removing the hyperparathyroid drive to reactivation of pagetic osteoclasts, leading to a prolonged remission. These observations have implications for optimizing bisphosphonate therapy both in Paget's disease and in osteoporosis.

Intravenous disodium 3‐amino‐1‐hydroxypropylidene‐1,1‐bisphosphonate pentahydrate (pamidronate disodium) was used to treat 39 patients (22 males and 17 females, age range 48–85 years) with symptomatic Paget's disease. Patients were stratified into three groups based on the biochemical severity of the disease as assessed by fasting urinary hydroxyproline excretion (HypE, μmol/liter GF, glomerular filtrate): group I (n= 23), HypE< 5.0, treated with 120 mg total dose over 2 or 4 days; group II (n= 6), 5.0 ≤ HypE≤ 10.0, 180 mg over 3 or 6 days; and group III (n= 10), HypE> 10.0, 240 mg over 4 or 8 days. Bone mineral density (BMD) was measured before and 3 and 6 months following treatment in the spine (LI‐4) using dual‐energy x‐ray absorptiometry and in the forearm at an ultradistal and a shaft site using single‐photon absorptiometry. When groups I‐III were combined, nonpagetic and pagetic lumbar spinal BMD had both risen significantly at 3 months compared with the pretreatment values (p< 0.001). In each group, lumbar spinal BMD in pagetic vertebrae rose markedly by 3 months, with no further significant change at 6 months. The percentage rises in the three groups were not different from each other at 3 or 6 months. Nonpagetic lumbar spinal BMD followed a similar and significant trend but with a significantly smaller rise than for pagetic bone. (For the combined groups, nonpagetic BMD rose 5.1 ± 1.1% SEM, above pretreatment at 6 months; pagetic BMD rose 17.8 ± 1.6%: significance of comparison = p< 0.0001). In contrast, forearm BMD in group III had fallen at 6 months by 8.3 ± 2.5% (p< 0.01) and 7.0 ± 1.2% (p< 0.001) in the ultradistal and shaft sites, respectively. There were no significant changes in forearm BMD in groups I and II. When groups I‐III were combined, the maximum observed changes within each individual in ultradistal forearm BMD (seen posttreatment) were correlated inversely with maximum intraindividual changes (seen posttreatment) in intact parathyroid hormone (Spearman's Q= −0.53, p< 0.001). A group of 18 control subjects with untreated Paget's disease were studied for 3–6 months. No changes were seen in nonpagetic or pagetic lumbar spinal BMD or in forearm BMD. Three mechanisms are proposed to explain these findings: (1) in nonpagetic bone, persistence of bone formation following acute reduction in resorption, with a more marked effect in the axial than in the appendicular skeleton; (2) in pagetic bone, magnification of mechanism 1, caused by the presence of abnormally active osteoclasts, leading to a marked divergence between bone resorption and formation, and (3) in cancellous and cortical distal forearm bone, the onset of acute secondary hyperparathyroidism following treatment, leading to resorption. These findings have implications for treatment of Paget's disease using pamidronate or other bisphosphonates in patients with preexisting appendicular osteopenia, particularly if the treatment‐induced appendicular deficit is sustained.

The calcium (Ca) metabolism of established human lactation was studied in 40 adult women (mean age 32.4 years) who had been breast‐feeding for 6 months (Lac) and in 40 age‐matched controls (Con) using fasting urine and blood biochemistry and forearm single‐photon bone mineral densitometry (BMD). Serial studies were performed up to 6 months after weaning in Lac women and repeated once in Con women. During lactationthe significant findings were (1) a selective reduction (7.1%, P< 0.03) in BMD at the ultradistal site containing 60% trabecular bone, but not at two more proximal, chiefly cortical bone sites; (2) increased bone turnover affecting bone resorption [fasting hydroxyproline excretion, Lac 2.22 + 0.12 μmol/liter GF (mean + SEM), Con 1.19 + 0.04, P< 0.001] and affecting bone formation (plasma alkaline phosphatase, Lac 81.9 + 2.5 IU/liter, Con 53.5 + 2.7, P< 0.001, and serum osteocalcin, Lac 14.0 + 0.7 μg/liter, Con 7.3 + 0.4, P< 0.001); and (3) renal conservation in the fasting state of both Ca and inorganic phosphate (Pi) with a resultant moderate increase in plasma Pibut not in plasma Ca (total or ionized). There were no differences between the groups in serum parathyroid hormone (PTH, intact and midmolecule assays), 25‐hydroxy‐ and 1,25‐dihydroxyvitamin D, nephrogenous cyclic AMP production, or plasma creatinine. In 25 of these Lac women restudied at one or more of the times 2, 4, or 6 months after weaning, the findings were (1) an early (2 months) normalization of bone resorption and renal Pihandling with continuing increased bone formation and renal Ca conservation, associated with the onset of increased intact PTH levels; and (2) a recovery, within 4‐6 months in ultradistal BMD associated with normalization of bone formation but with persisting renal Ca conservation and elevated intact PTH levels. We conclude that in established adult human lactation there is increased bone turnover with an accompanying loss of trabecular bone, despite renal conservation of Ca and Pi. After weaning, the deficit in trabecular bone is made up during a period of imbalance between a normal bone resorption rate and an elevated bone formation rate. The moderately elevated PTH after weaning may play a role in the recovery of bone mass by maintaining renal Ca conservation and by an anabolic action on bone.

Spontaneous fractures were reported to be rare (<1%) in 1664 hospital admissions for hip fracture in the 1950s in Sweden. We report 11 fluoride‐treated postmenopausal patients who developed spontaneous fractures of the femoral necks, all subcapital initially. In 7 patients who continued treatment there were later femoral neck or shaft fractures; in 6, these were bilateral (one followed a fall). In all there were 19 spontaneous fractures: 5 were asymptomatic, including 2 with deformity; 12 fractures required surgery. Five were incomplete (stress) fractures. All were treated with supplementary calcium 1 g daily; 10 had vitamin D supplementation. In all patients where the timing was known, the initial and subsequent fractures were preceded by, or associated with increased bone turnover as measured by plasma alkaline phosphatase (pAIP) (i.e., they were all “good responders”). Two had pretreatment hip fractures following falls. We compared these 11 (Group 1) and another identically treated group of 14 patients (Group 2), without spontaneous femoral fractures and not different in mean age, pretreatment vertebral fractures, years since menopause, fluoride dosage, and plasma creatinine. Group 1 had a lower (p< 0.05) index of cortical bone in the femoral neck, as assessed by the ratio “calcar width/femoral neck minimum width.” The 6 biopsied fluorotic patients from Group 1 had a higher (p< 0.05) bone fluoride content than the 4 biopsied fluorotic patients from Group 2. Furthermore, histological cortical features of thinning, increased porosity, and advanced tunneling resorption characterized Group 1 posttreatment biopsies. There were no significant differences in peak pAIP responses in the two groups. Mild asymptomatic vitamin D excess may have been a contributing factor in three Group 1 patients.Two further treatment groups have been studied more recently by forearm single‐photon absorptiometry (SPA) at two sites; a cyclic NaF group (Group 3) and a calcium ± vitamin D group (Group 4). Neither showed significant changes in forearm cortical bone density on treatment for 2 and 1.5 years, respectively, but Group 3 showed a significant increasein density at an ultradistal (60% trabecular) site. The pAIP response in Group 3 was significantly less than in Group 1. Spontaneous femoral neck or shaft fractures did not occur in either Groups 3 or 4. Therefore, we recommend: (1) Avoidance of sodium fluoride (NaF) treatment if pretreatment femoral fracture or thin femoral neck cortices exist. (2) Reduction in NaF dosage if pAIP or osteocalcin increases by over 50% of pretreatment values. (3) Cessation of NaF if femoral neck or shaft fracture occurs during treatment, and also on the attainment of early to moderate spinal fluorosis. (4) Consideration of cyclic NaF treatment, to limit overstimulation of bone turnover and to enhance mineralization.