Identification and characterization of novel parathyroid-specific transcription factor Glial Cells Missing Homolog B (GCMB) mutations in eight families with autosomal recessive hypoparathyroidism

• Published in: Human molecular genetics 2010-05, Vol.19 (10), p.2028-2038
• Main Authors: Bowl, Michael R., Mirczuk, Samantha M., Grigorieva, Irina V., Piret, Sian E., Cranston, Treena, Southam, Lorraine, Allgrove, Jeremy, Bahl, Shailini, Brain, Caroline, Loughlin, John, Mughal, Zulf, Ryan, Fiona, Shaw, Nick, Thakker, Yogini V., Tiosano, Dov, Nesbit, M. Andrew, Thakker, Rajesh V.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; Biological and medical sciences; Cell Nucleus - metabolism; DNA - metabolism; Endocrinopathies; Enzyme Assays; Family; Female; Fundamental and applied biological sciences. Psychology; Genes, Recessive - genetics; Genes, Reporter; Genetics of eukaryotes. Biological and molecular evolution; Humans; Hypoparathyroidism - genetics; Luciferases - metabolism; Male; Medical sciences; Models, Molecular; Molecular and cellular biology; Molecular Sequence Data; Mutant Proteins - chemistry; Mutation - genetics; Non tumoral diseases. Target tissue resistance. Benign neoplasms; Nuclear Proteins - chemistry; Nuclear Proteins - genetics; Organ Specificity - genetics; Parathyroid Glands - metabolism; Parathyroid Glands - pathology; Parathyroids. Parafollicular cells. Cholecalciferol. Phosphocalcic homeostasis (diseases); Pedigree; Protein Binding; Protein Transport; Transcription Factors - chemistry; Transcription Factors - genetics
• ISSN: 0964-6906; 1460-2083
• DOI: 10.1093/hmg/ddq084

GCMB is a member of the small transcription factor family GCM (glial cells missing), which are important regulators of development, present in vertebrates and some invertebrates. In man, GCMB encodes a 506 amino acid parathyroid gland-specific protein, mutations of which have been reported to cause both autosomal dominant and autosomal recessive hypoparathyroidism. We ascertained 18 affected individuals from 12 families with autosomal recessive hypoparathyroidism and have investigated them for GCMB abnormalities. Four different homozygous germline mutations were identified in eight families that originate from the Indian Subcontinent. These consisted of a novel nonsense mutation R39X; a missense mutation, R47L in two families; a novel missense mutation, R110W; and a novel frameshifting deletion, I298fsX307 in four families. Haplotype analysis, using polymorphic microsatellites from chromosome 6p23-24, revealed that R47L and I298fsX307 mutations arose either as ancient founders, or recurrent de novo mutations. Functional studies including: subcellular localization studies, EMSAs and luciferase-reporter assays, were undertaken and these demonstrated that: the R39X mutant failed to localize to the nucleus; the R47L and R110W mutants both lost DNA-binding ability; and the I298fsX307 mutant had reduced transactivational ability. In order to gain further insights, we undertook 3D-modeling of the GCMB DNA-binding domain, which revealed that the R110 residue is likely important for the structural integrity of helix 2, which forms part of the GCMB/DNA binding interface. Thus, our results, which expand the spectrum of hypoparathyroidism-associated GCMB mutations, help elucidate the molecular mechanisms underlying DNA-binding and transactivation that are required for this parathyroid-specific transcription factor.