Michael P. Whyte

Atypical Subtrochanteric and Diaphyseal Femoral Fractures: Report of a Task Force of the American Society for Bone and Mineral Research

Bisphosphonates (BPs) and denosumab reduce the risk of spine and nonspine fractures. Atypical femur fractures (AFFs) located in the subtrochanteric region and diaphysis of the femur have been reported in patients taking BPs and in patients on denosumab, but they also occur in patients with no exposure to these drugs. In this report, we review studies on the epidemiology, pathogenesis, and medical management of AFFs, published since 2010. This newer evidence suggests that AFFs are stress or insufficiency fractures. The original case definition was revised to highlight radiographic features that distinguish AFFs from ordinary osteoporotic femoral diaphyseal fractures and to provide guidance on the importance of their transverse orientation. The requirement that fractures be noncomminuted was relaxed to include minimal comminution. The periosteal stress reaction at the fracture site was changed from a minor to a major feature. The association with specific diseases and drug exposures was removed from the minor features, because it was considered that these associations should be sought rather than be included in the case definition. Studies with radiographic review consistently report significant associations between AFFs and BP use, although the strength of associations and magnitude of effect vary. Although the relative risk of patients with AFFs taking BPs is high, the absolute risk of AFFs in patients on BPs is low, ranging from 3.2 to 50 cases per 100,000 person‐years. However, long‐term use may be associated with higher risk (∼100 per 100,000 person‐years). BPs localize in areas that are developing stress fractures; suppression of targeted intracortical remodeling at the site of an AFF could impair the processes by which stress fractures normally heal. When BPs are stopped, risk of an AFF may decline. Lower limb geometry and Asian ethnicity may contribute to the risk of AFFs. There is inconsistent evidence that teriparatide may advance healing of AFFs.

Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein

Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD) is a dominant progressive disorder that maps to chromosome 9p21.1–p12. We investigated 13 families with IBMPFD linked to chromosome 9 using a candidate-gene approach. We found six missense mutations in the gene encoding valosin-containing protein (VCP, a member of the AAA-ATPase superfamily) exclusively in all 61 affected individuals. Haplotype analysis indicated that descent from two founders in two separate North American kindreds accounted for IBMPFD in ∼50% of affected families. VCP is associated with a variety of cellular activities, including cell cycle control, membrane fusion and the ubiquitin-proteasome degradation pathway. Identification of VCP as causing IBMPFD has important implications for other inclusion-body diseases, including myopathies, dementias and Paget disease of bone (PDB), as it may define a new common pathological ubiquitin-based pathway.

Mutations in TNFRSF11A, affecting the signal peptide of RANK, cause familial expansile osteolysis

Familial expansile osteolysis (FEO, MIM 174810) is a rare, autosomal dominant bone disorder characterized by focal areas of increased bone remodelling. The osteolytic lesions, which develop usually in the long bones during early adulthood, show increased osteoblast and osteoclast activity. Our previous linkage studies mapped the gene responsible for FEO to an interval of less than 5 cM between D18S64 and D18S51 on chromosome 18q21.2–21.3 in a large Northern Irish family. The gene encoding receptor activator of nuclear factor-κ B (RANK; ref. 5), TNFRSF11A, maps to this region. RANK is essential in osteoclast formation. We identified two heterozygous insertion mutations in exon 1 of TNFRSF11A in affected members of four families with FEO or familial Paget disease of bone (PDB). One was a duplication of 18 bases and the other a duplication of 27 bases, both of which affected the signal peptide region of the RANK molecule. Expression of recombinant forms of the mutant RANK proteins revealed perturbations in expression levels and lack of normal cleavage of the signal peptide. Both mutations caused an increase in RANK-mediated nuclear factor-κB (NF-κB) signalling in vitro, consistent with the presence of an activating mutation.

Alkaline Phosphatase Knock-Out Mice Recapitulate the Metabolic and Skeletal Defects of Infantile Hypophosphatasia†

Hypophosphatasia is an inborn error of metabolism characterized by deficient activity of the tissue‐nonspecific isoenzyme of alkaline phosphatase (TNSALP) and skeletal disease due to impaired mineralization of cartilage and bone matrix. We investigated two independently generated TNSALP gene knock‐out mouse strains as potential models for hypophosphatasia. Homozygous mice (–/–) had < 1% of wild‐type plasma TNSALP activity; heterozygotes had the predicted mean of ∼50%. Phosphoethanolamine, inorganic pyrophosphate, and pyridoxal 5′‐phosphate are putative natural substrates for TNSALP and all were increased endogenously in the knock‐out mice. Skeletal disease first appeared radiographically at ∼10 days of age and featured worsening rachitic changes, osteopenia, and fracture. Histologic studies revealed developmental arrest of chondrocyte differentiation in epiphyses and in growth plates with diminished or absent hypertrophic zones. Progressive osteoidosis from defective skeletal matrix mineralization was noted but not associated with features of secondary hyperparathyroidism. Plasma and urine calcium and phosphate levels were unremarkable. Our findings demonstrate that TNSALP knock‐out mice are a good model for the infantile form of hypophosphatasia and provide compelling evidence for an important role for TNSALP in postnatal development and mineralization of the murine skeleton.

Carbonic Anhydrase II Deficiency in 12 Families with the Autosomal Recessive Syndrome of Osteopetrosis with Renal Tubular Acidosis and Cerebral Calcification

Osteopetrosis with renal tubular acidosis and cerebral calcification was identified as a recessively inherited syndrome in 1972. In 1983, we reported a deficiency of carbonic anhydrase II, one of the isozymes of carbonic anhydrase, in three sisters with this disorder. We now describe our study of 18 similarly affected patients with this syndrome in 11 unrelated families of different geographic and ethnic origins. Virtual absence of the carbonic anhydrase II peak on high-performance liquid chromatography, of the esterase and carbon dioxide hydratase activities of carbonic anhydrase II, and of immunoprecipitable isozyme II was demonstrated on extracts of erythrocyte hemolysates from all patients studied. Reduced levels of isozyme II were found in obligate heterozygotes. These observations demonstrate the generality of the findings that we reported earlier in one family and provide further evidence that a deficiency of carbonic anhydrase II is the enzymatic basis for the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. We also summarize the clinical findings in these families, propose mechanisms by which a deficiency of carbonic anhydrase II could produce this metabolic disorder of bone, kidney, and brain, and discuss the clinical evidence for genetic heterogeneity in patients from different kindreds with this inborn error of metabolism.

Physiological role of alkaline phosphatase explored in hypophosphatasia

Hypophosphatasia (HPP) is the instructive rickets or osteomalacia caused by loss‐of‐function mutation(s) within TNSALP, the gene that encodes the “tissue nonspecific” isoenzyme of alkaline phosphatase (TNSALP). HPP reveals a critical role for this enzyme in skeletal mineralization. Increased extracellular levels of pyridoxal 5′‐phosphate and inorganic pyrophosphate (PPi) demonstrate that TNSALP is a phosphomonoester phosphohydrolase and a pyrophosphatase that hydrolyzes much lower concentrations of natural substrates than the artificial substrates of laboratory assays. Clearly, TNSALP acts at physiological pH and “alkaline phosphatase” is a misnomer. Aberrations of vitamin B6 metabolism in HPP revealed that TNSALP is an ectoenzyme. PPi excesses cause chondrocalcinosis and sometimes arthropathy. The skeletal disease is due to PPi inhibition of hydroxyapatite crystal growth extracellularly so that crystals form within matrix vesicles but fail to enlarge after these structures rupture. Trials of alkaline phosphatase replacement therapy for HPP suggest that TNSALP functions at the level of skeletal tissues.

Enzyme Replacement Therapy for Murine Hypophosphatasia

Introduction: Hypophosphatasia (HPP) is the inborn error of metabolism that features rickets or osteomalacia caused by loss‐of‐function mutation(s) within the gene that encodes the tissue‐nonspecific isozyme of alkaline phosphatase (TNALP). Consequently, natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5′‐phosphate (PLP), a co‐factor form of vitamin B6. Babies with the infantile form of HPP often die with severe rickets and sometimes hypercalcemia and vitamin B6‐dependent seizures. There is no established medical treatment.

Postmenopausal osteoporosis: A heterogeneous disorder as assessed by histomorphometric analysis of iliac crest bone from untreated patients

Twenty-six women with untreated postmenopausal osteoporosis underwent iliac crest biopsy following tetracycline-labeling and mineral metabolism studies. Histomorphometric assessment of their bone remodeling rates, including formation determined by the tetracycline-labeling technique, revealed considerable variation. Eight women had no evidence of active bone formation (inactive remodeling osteoporosis), whereas the others showed a spectrum of bone formation rates (active remodeling osteoporosis). Clinical and biochemical studies failed to predict the histomorphometric findings. Postmenopausal osteoporosis is a histologically heterogeneous disorder with morphologic expression in bone that cannot be predicted by single or combined routine clinical and laboratory parameters. Bone biopsy, necessary to identify the histologic lesion and assess skeletal dynamics, may prove to be important for optimal therapy of osteoporosis, as a variety of agents--with different effects on bone remodeling--are available.

Marrow Cell Transplantation for Infantile Hypophosphatasia

An 8‐month‐old girl who seemed certain to die from the infantile form of hypophosphatasia, an inborn error of metabolism characterized by deficient activity of the tissue‐nonspecific isoenzyme of alkaline phosphatase (TNSALP), underwent the first trial of bone marrow cell transplantation for this heritable type of rickets. After cytoreduction, she was given T‐cell‐depleted, haplo‐identical marrow from her healthy sister. Chimerism in peripheral blood and bone marrow became 100% donor. Three months later, she was clinically improved, with considerable healing of rickets and generalized skeletal remineralization. However, 6 months post‐transplantation, worsening skeletal disease recurred, with partial return of host hematopoiesis. At the age of 21 months, without additional chemotherapy or immunosuppressive treatment, she received a boost of donor marrow cells expanded ex vivo to enrich for stromal cells. Significant, prolonged clinical and radiographic improvement followed soon after. Nevertheless, biochemical features of hypophosphatasia have remained unchanged to date. Skeletal biopsy specimens were not performed. Now, at 6 years of age, she is intelligent and ambulatory but remains small. Among several hypotheses for our patients survival and progress, the most plausible seems to be the transient and long‐term engraftment of sufficient numbers of donor marrow mesenchymal cells, forming functional osteoblasts and perhaps chondrocytes, to ameliorate her skeletal disease.

Mutations in AP2S1 cause familial hypocalciuric hypercalcemia type 3.

Adaptor protein-2 (AP2), a central component of clathrin-coated vesicles (CCVs), is pivotal in clathrin-mediated endocytosis, which internalizes plasma membrane constituents such as G protein–coupled receptors (GPCRs). AP2, a heterotetramer of α, β, μ and σ subunits, links clathrin to vesicle membranes and binds to tyrosine- and dileucine-based motifs of membrane-associated cargo proteins. Here we show that missense mutations of AP2 σ subunit (AP2S1) affecting Arg15, which forms key contacts with dileucine-based motifs of CCV cargo proteins, result in familial hypocalciuric hypercalcemia type 3 (FHH3), an extracellular calcium homeostasis disorder affecting the parathyroids, kidneys and bone. We found AP2S1 mutations in >20% of cases of FHH without mutations in calcium-sensing GPCR (CASR), which cause FHH1. AP2S1 mutations decreased the sensitivity of CaSR-expressing cells to extracellular calcium and reduced CaSR endocytosis, probably through loss of interaction with a C-terminal CaSR dileucine-based motif, whose disruption also decreased intracellular signaling. Thus, our results identify a new role for AP2 in extracellular calcium homeostasis.