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M Andrew Nesbit,
Fadil M Hannan,
Sarah A Howles,
Anita A C Reed,
Treena Cranston,
Clare E Thakker,
Lorna Gregory,
Andrew J Rimmer,
Nigel Rust,
Una Graham,
Patrick J Morrison,
Steven J Hunter, Michael P Whyte,
Gil McVean,
David Buck,
Rajesh V Thakker
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ABSTRACT: 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.
Nature Genetics 12/2012; · 35.53 Impact Factor
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ABSTRACT: Familial hypocalciuric hypercalcemia (FHH) is a genetically heterogenous disorder that consists of three defined types, FHH1, FHH2, and FHH3 whose chromosomal locations are 3q21.1, 19p, and 19q13, respectively. FHH1, caused by mutations of the calcium-sensing receptor (CASR), occurs in more than 65% of patients, whereas the abnormalities underlying FHH2 and FHH3, which have each been described in single North American kindreds, are unknown.
The aim of this study was to determine the basis of FHH in a proband, who did not have CASR mutations, and her kindred.
The proband was a 43-yr-old woman who presented with a corrected serum calcium of 2.74 mmol/liter (normal = 2.15-2.55 mmol/liter), a serum PTH of 47 pg/ml (normal = 10-65 pg/ml), and a urinary calcium clearance:creatinine clearance of 0.006. She did not have a CASR mutation within the coding region and splice sites, and 24 members from three generations of her kindred were ascertained and investigated for serum abnormalities and cosegregation with polymorphic loci from chromosomes 3q21.1 and 19q13 using leukocyte DNA.
Sixteen members were hypercalcemic with normal or elevated serum PTH concentrations and mild hypophosphatemia, features consistent with FHH3. Use of microsatellite and single nucleotide polymorphic loci from chromosome 19q13.3 demonstrated cosegregation with FHH in the kindred, with a peak LOD score = 5.98 at 0% recombination with D19S412. Analysis of recombinants mapped FHH to a 3.46-Mbp interval flanked centromerically by single nucleotide polymorphism rs1990932 and telomerically by D19S604.
FHH3 may explain the calcium homeostasis disorder in those FHH patients who do not have CASR mutations.
The Journal of clinical endocrinology and metabolism 04/2010; 95(4):1947-54. · 6.50 Impact Factor
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ABSTRACT: Familial benign hypocalciuric hypercalcaemia (FBHH) is a genetically heterogeneous disorder that consists of three designated types, FBHH1, FBHH2 and FBHH3, whose chromosomal locations are 3q21.1, 19p and 19q13, respectively. FBHH1 is caused by mutations of a calcium-sensing receptor (CaSR), but the abnormalities underlying FBHH2 and FBHH3 are unknown. FBHH3, also referred to as the Oklahoma variant (FBHH(Ok)), has been mapped to a 12cM interval, flanked by D19S908 and D19S866. To refine the location of FBHH3, we pursued linkage studies using 24 polymorphic loci. Our results establish a linkage between FBHH3 and 17 of these loci, and indicate that FBHH3 is located in a 4.1 Mb region flanked centromerically by D19S112 and telomerically by rs245111, which in the syntenic region on mouse chromosome 7 contains four Casr-related sequences (Gprc2a-rss). However, human homologues of these Gprc2a-rss were not found and a comparative analysis of the 22.0 Mb human and 39.3 Mb mouse syntenic regions showed evolutionary conservation of two segments that were inverted with loss from the human genome of 11.6 Mb that contained the four Gprc2a-rss. Thus, FBHH3 cannot be attributed to Gprc2a-rss abnormalities. DNA sequence analysis of 12 other genes from the interval that were expressed in the parathyroids and/or kidneys did not detect any abnormalities, thereby indicating that these genes are unlikely to be the cause of FBHH3. The results of this study have refined the map location of FBHH3, which will facilitate the identification of another CaSR or a mediator of calcium homeostasis.
European journal of human genetics: EJHG 10/2009; 18(4):442-7. · 3.56 Impact Factor
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Michael R Bowl,
M Andrew Nesbit,
Brian Harding,
Elaine Levy,
Andrew Jefferson,
Emanuela Volpi,
Karine Rizzoti,
Robin Lovell-Badge,
David Schlessinger, Michael P Whyte,
Rajesh V Thakker
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ABSTRACT: X-linked recessive hypoparathyroidism, due to parathyroid agenesis, has been mapped to a 906-kb region on Xq27 that contains 3 genes (ATP11C, U7snRNA, and SOX3), and analyses have not revealed mutations. We therefore characterized this region by combined analysis of single nucleotide polymorphisms and sequence-tagged sites. This identified a 23- to 25-kb deletion, which did not contain genes. However, DNA fiber-FISH and pulsed-field gel electrophoresis revealed an approximately 340-kb insertion that replaced the deleted fragment. Use of flow-sorted X chromosome-specific libraries and DNA sequence analyses revealed that the telomeric and centromeric breakpoints on X were, respectively, approximately 67 kb downstream of SOX3 and within a repetitive sequence. Use of a monochromosomal somatic cell hybrid panel and metaphase-FISH mapping demonstrated that the insertion originated from 2p25 and contained a segment of the SNTG2 gene that lacked an open reading frame. However, the deletion-insertion [del(X)(q27.1) inv ins (X;2)(q27.1;p25.3)], which represents a novel abnormality causing hypoparathyroidism, could result in a position effect on SOX3 expression. Indeed, SOX3 expression was demonstrated, by in situ hybridization, in the developing parathyroid tissue of mouse embryos between 10.5 and 15.5 days post coitum. Thus, our results indicate a likely new role for SOX3 in the embryonic development of the parathyroid glands.
Journal of Clinical Investigation 11/2005; 115(10):2822-31. · 15.39 Impact Factor
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Ann M Kennedy,
Masaki Inada,
Stephen M Krane,
Paul T Christie,
Brian Harding,
Carlos López-Otín,
Luis M Sánchez,
Anna A J Pannett,
Andrew Dearlove,
Claire Hartley,
Michael H Byrne,
Anita A C Reed,
M Andrew Nesbit, Michael P Whyte,
Rajesh V Thakker
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ABSTRACT: MMPs, which degrade components of the ECM, have roles in embryonic development, tissue repair, cancer, arthritis, and cardiovascular disease. We show that a missense mutation of MMP13 causes the Missouri type of human spondyloepimetaphyseal dysplasia (SEMD(MO)), an autosomal dominant disorder characterized by defective growth and modeling of vertebrae and long bones. Genome-wide linkage analysis mapped SEMD(MO) to a 17-cM region on chromosome 11q14.3-23.2 that contains a cluster of 9 MMP genes. Among these, MMP13 represented the best candidate for SEMD(MO), since it preferentially degrades collagen type II, abnormalities of which cause skeletal dysplasias that include Strudwick type SEMD. DNA sequence analysis revealed a missense mutation, F56S, that substituted an evolutionarily conserved phenylalanine residue for a serine in the proregion domain of MMP13. We predicted, by modeling MMP13 structure, that this F56S mutation would result in a hydrophobic cavity with misfolding, autoactivation, and degradation of mutant protein intracellularly. Expression of wild-type and mutant MMP13s in human embryonic kidney cells confirmed abnormal intracellular autoactivation and autodegradation of F56S MMP13 such that only enzymatically inactive, small fragments were secreted. Thus, the F56S mutation results in deficiency of MMP13, which leads to the human skeletal developmental anomaly of SEMD(MO).
Journal of Clinical Investigation 11/2005; 115(10):2832-42. · 15.39 Impact Factor
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ABSTRACT: X-linked hypoparathyroidism (HPT) has been mapped to a 988-kb region on chromosome Xq27 that contains three genes, MCF2/DBL, SOX3, and U7snRNA homologue, and a partial cDNA, AS6. We isolated the full-length AS6 cDNA, determined its genomic organization, and sought for abnormalities in HPT patients. AS6 was identified as the 3' UTR of ATP11C, a novel member of the P-type ATPases, which consists of 31 exons with alternative transcripts. The colocalization of ATP11C with SOX3 and MCF2/DBL on Xq27 mirrors that of ATP11A with SOX1 and MCF2L on 13q34 and ATP11B with SOX2 on 3q26. These colocalizations are evolutionarily conserved in mouse, and analyses indicate that SOX2 divergence likely occurred before the separation of SOX1 and SOX3. Analyses of ATP11C, MCF2, SOX3, and U7snRNA in HPT patients did not reveal mutations, implicating regulatory changes or mutation of an as yet unidentified gene in the etiology of X-linked hypoparathyroidism.
Genomics 01/2005; 84(6):1060-70. · 3.02 Impact Factor
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ABSTRACT: Rickets and osteomalacia are consequences of impaired mineralization of the paediatric and adult skeleton, respectively. Softened bone leads to deformity during growth and fractures during adult life. Radiographs show widened growth plates in rickets, whereas fractures and pseudofractures characterize osteomalacia. Vitamin D deficiency from impaired biosynthesis of cholecalciferol (vitamin D3) from insufficient exposure to sunlight, without dietary supplementation using cholecalciferol or ergocalciferol (vitamin D2), is the most common cause of rickets, but calcium malabsorption from hepatobiliary, pancreatic or gastrointestinal disease can be an additional explanation for these disorders. Rarely, patients have heritable impairment of vitamin D bioactivation or resistance to its hormonal form, 1,25-dihydroxyvitamin D (calcitriol). All of these conditions feature secondary hyperparathyroidism from hypocalcaemia causing hypophosphataemia from renal phosphate wasting. Other patients manifest renal loss of phosphate from kidney tubule defects or damage, or circulating ‘phosphatonins’ engendered by certain genetic diseases or sometimes elaborated by benign neoplasms. In these disorders, suppression of calcitriol formation with inappropriately normal or low (rather than high) circulating levels is a typical biochemical finding. Hypophosphatasia features paradoxically low levels of serum alkaline phosphatase activity because of deactivating mutation of the gene encoding the isoenzyme expressed in bone, and accumulation of pyrophosphate – an inhibitor of mineralization. Therapy of rickets or osteomalacia requires clinical expertise and close patient monitoring, because potent vitamin D pharmaceuticals and mineral supplements can be effective, but excessive dosing can cause hypercalciuria and hypercalcaemia with renal damage from nephrocalcinosis or nephrolithiasis.
Medicine.
