Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2009) 19 OC28

SFEBES2009 Oral Communications Bone and Calcium (8 abstracts)

Novel Glial Cells Missing B (GCMB) mutations (Arg39Stop and Arg110Trp) that result in loss of subcellular localization and DNA binding, respectively, are associated with autosomal recessive hypoparathyroidism

MR Bowl 1 , S Mirczuk 1 , T Cranston 2 , S Bahl 3 , J Allgrove 4 , MA Nesbit 1 & RV Thakker 1

1Academic Endocrine Unit, OCDEM, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK; 2Oxford Medical Genetics Laboratories, Churchill Hospital, Oxford, UK; 3Childrens Unit, St Peter’s Hospital, Surrey, UK; 4Endocrinology, The Royal London Hospital, London, UK.

GCMB, which is the mammalian homologue of the Drosophila gene Glial cells missing, encodes a 506 amino acid parathyroid-specific transcription factor that contains: a GCM DNA-binding domain at residues 19–176; a predicted nuclear localization signal (NLS) at residues 176–193; an inhibitory domain at residues 258–347; and two transactivation domains at residues 174–263, and residues 428–506. To date only six different GCMB mutations have been reported in 10 kindreds with hypoparathyroidism. We therefore investigated additional families with autosomal recessive hypoparathyroidism for GCMB mutations, after obtaining informed consent, using guidelines approved by the local ethical committee. Two novel homozygous mutations, consisting of a nonsense mutation (Arg39Stop) and a missense mutation (Arg110Trp) were identified. The Arg39Stop predicts a truncated protein lacking the GCM DNA-binding domain, NLS, and transactivation domains. The Arg110Trp results in the substitution of an evolutionarily conserved basic Arginine residue by an aromatic Tryptophan (Arg110Trp) within the GCM DNA-binding domain. Functional studies, including transient transfections of GCMB wild-type and mutant tagged constructs into COS7 cells, demonstrated that the Arg39Stop mutant did not localize to the nucleus unlike the wild-type and Arg110Trp mutant proteins. Electrophoretic mobility shift assays (EMSAs), showed that the Arg110Trp mutant failed to bind the GCM-consensus recognition motif, thereby demonstrating a loss of DNA-binding ability. In order to gain further mechanistic insights we undertook a three-dimensional modelling analysis, based on the Gcm1-DNA crystal structure. This revealed that the Arg110 is important for forming a structural stabilizing polar interaction with Ala104 and that the Trp110 would lead to a loss of this hydrogen bonding. Thus, our results, which have identified the first nonsense GCMB mutation, help to increase our understanding of the mechanism underlying DNA binding that is required for the function of this parathyroid gland-specific transcription factor.

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