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Nesbit M.A.,University of Oxford | Hannan F.M.,University of Oxford | Howles S.A.,University of Oxford | Reed A.A.C.,University of Oxford | And 12 more authors.
Nature Genetics | Year: 2013

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. © 2013 Nature America, Inc. All rights reserved.

Whyte M.P.,Center for Metabolic Bone Disease and Molecular Research | Whyte M.P.,University of Washington
Annals of the New York Academy of Sciences | Year: 2010

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. © 2010 New York Academy of Sciences.

Whyte M.P.,Center for Metabolic Bone Disease and Molecular Research | Whyte M.P.,Washington University
Nature Reviews Endocrinology | Year: 2016

Hypophosphatasia is the inborn error of metabolism characterized by low serum alkaline phosphatase activity (hypophosphatasaemia). This biochemical hallmark reflects loss-of-function mutations within the gene that encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). TNSALP is a cell-surface homodimeric phosphohydrolase that is richly expressed in the skeleton, liver, kidney and developing teeth. In hypophosphatasia, extracellular accumulation of TNSALP natural substrates includes inorganic pyrophosphate, an inhibitor of mineralization, which explains the dento-osseous and arthritic complications featuring tooth loss, rickets or osteomalacia, and calcific arthopathies. Severely affected infants sometimes also have hypercalcaemia and hyperphosphataemia due to the blocked entry of minerals into the skeleton, and pyridoxine-dependent seizures from insufficient extracellular hydrolysis of pyridoxal 5'-phosphate, the major circulating form of vitamin B 6, required for neurotransmitter synthesis. Autosomal recessive or dominant inheritance from ∼300 predominantly missense ALPL (also known as TNSALP) mutations largely accounts for the remarkably broad-ranging expressivity of hypophosphatasia. High serum concentrations of pyridoxal 5'-phosphate represent a sensitive and specific biochemical marker for hypophosphatasia. Also, phosphoethanolamine levels are usually elevated in serum and urine, though less reliably for diagnosis. TNSALP mutation detection is important for recurrence risk assessment and prenatal diagnosis. Diagnosing paediatric hypophosphatasia is aided by pathognomic radiographic changes when the skeletal disease is severe. Hypophosphatasia was the last type of rickets or osteomalacia to await a medical treatment. Now, significant successes for severely affected paediatric patients are recognized using asfotase alfa, a bone-targeted recombinant TNSALP. © 2016 Macmillan Publishers Limited.

Millan J.L.,Sanford Burnham Institute for Medical Research | Whyte M.P.,Center for Metabolic Bone Disease and Molecular Research | Whyte M.P.,University of Washington
Calcified Tissue International | Year: 2015

Hypophosphatasia (HPP) results from ALPL mutations leading to deficient activity of the tissue-non-specific alkaline phosphatase isozyme (TNAP) and thereby extracellular accumulation of inorganic pyrophosphate (PPi), a natural substrate of TNAP and potent inhibitor of mineralization. Thus, HPP features rickets or osteomalacia and hypomineralization of teeth. Enzyme replacement using mineral-targeted TNAP from birth prevented severe HPP in TNAP-knockout mice and was then shown to rescue and substantially treat infants and young children with life-threatening HPP. Clinical trials are revealing aspects of HPP pathophysiology not yet fully understood, such as craniosynostosis and muscle weakness when HPP is severe. New treatment approaches are under development to improve patient care. © 2015 The Author(s)

Guanabens N.,University of Barcelona | Mumm S.,Center for Metabolic Bone Disease and Molecular Research | Mumm S.,University of Washington | Moller I.,Institute Poal of Rheumatology | And 5 more authors.
Journal of Bone and Mineral Research | Year: 2014

Hypophosphatasia (HPP) is the inborn error of metabolism that features low serum alkaline phosphatase (ALP) activity caused by loss-of-function mutation(s) within the gene for the tissue nonspecific isoenzyme of ALP (TNSALP). In HPP, extracellular accumulation of inorganic pyrophosphate (PPi), a TNSALP substrate and inhibitor of mineralization, leads frequently to premature tooth loss and often to rickets or osteomalacia. In affected adults, the excess PPi sometimes also causes calcium pyrophosphate dihydrate (CPPD) deposition, PPi arthropathy, or pseudogout, or seemingly paradoxical deposition of hydroxyapatite crystals in ligaments or around joints when the condition is called calcific periarthritis (CP). We report three middle-aged sisters with CP as the only clinical manifestation of HPP. Each presented during early adult life with recurrent episodes of pain principally around the shoulders, elbows, wrists, hips, or Achilles tendon. Otherwise, they were in good health, including no history of unusual dental disease, fractures, or pseudofractures. Calcific deposits were identified in symptomatic areas principally by ultrasonographic assessment but also confirmed radiographically. All three sisters had low serum levels of total and bone-specific ALP, hyperphosphatemia, and increased serum concentrations of the TNSALP substrate pyridoxal 5′-phosphate together characteristic of HPP. Mutation analysis revealed that each carried a single unique 18-bp duplication within TNSALP (c.188-205dup18, p.Gly63-Thr68dup) as did two of their healthy sons and their mother, who was without signs of CPPD deposition or CP but had knee osteoarthritis. We find that CP can be the only complication of HPP in adults. Thus, multiple juxta-articular deposits of hydroxyapatite causing CP may be a useful sign of HPP, especially when the CP is familial. © 2014 American Society for Bone and Mineral Research.

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