Endocrine Abstracts

A novel GATA3 mutation, Tyr345Cys, in hypoparathyroidism, deafness and renal dysplasia (HDR) syndrome results in abolished DNA binding

Irina Grigorieva¹, Katie Gaynor¹, Treena Cranston¹, Catherine McWilliam², Faisal Ahmed², M Andrew Nesbit¹ & Rajesh Thakker¹

¹University of Oxford, Oxford, UK; ²University of Glasgow, Glasgow, UK.

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GATA3 mutations cause the congenital autosomal dominant hypoparathyroidism, Deafness and Renal dysplasia (HDR) syndrome. GATA3 belongs to a family of dual zinc-finger transcription factors that recognise the consensus (A/T)GATA(A/G) motif and are involved in vertebrate embryonic development. We investigated a HDR proband for GATA3 abnormalities. Venous blood was obtained after informed consent, as approved by the local ethical committee, and leukocyte DNA extracted. GATA3 specific primers were used for PCR amplification and the DNA sequence of both strands determined. This revealed a novel missense mutation, Tyr345Cys, in the basic region C-terminal to zinc finger 2 (ZnF2) of GATA3. Sub-cellular localisation studies revealed that this mutation did not interfere with correct localisation of the mutant GATA3 protein to the nucleus. However, luciferase reporter assays using a construct containing a functional GATA3 binding site in the promoter region, revealed that the Tyr345Cys GATA3 mutant was unable to transactivate expression of the reporter gene, and electrophoretic mobility shift assays confirmed that this was due to loss of DNA binding. The effect of the Tyr345Cys is in contrast to a previously characterised Leu348Arg mutation which is located in close proximity but did not cause loss of DNA binding or abolish transactivation activity. Three-dimensional modelling revealed that the Tyr345 residue lies in close contact with the major groove of the DNA and the mutation results in a polar neutral, tyrosine, being substituted for a non-polar neutral, cysteine. This subtle change in charge may affect the protein–DNA interactions, with the change in electrostatic interactions affecting the specificity and strength of binding. These results elucidate further the molecular mechanisms of the altered function of this zinc finger transcription factor and its role in causing this developmental anomaly.

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Endocrine Abstracts (2010) 21 P16