Characterization of GATA3 Mutations in the Hypoparathyroidism, Deafness, and Renal Dysplasia (HDR) Syndrome

Abstract

The hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome is an autosomal dominant disorder caused by mutations of the dual zinc finger transcription factor, GATA3. The C-terminal zinc finger (ZnF2) binds DNA, whereas the N-terminal finger (ZnF1) stabilizes this DNA binding and interacts with other zinc finger proteins, such as the Friends of GATA (FOG). We have investigated seven HDR probands and their families for GATA3 abnormalities and have identified two nonsense mutations (Glu-228 3 Stop and Arg-367 3 Stop); two intragenic deletions that result in frameshifts from codons 201 and 355 with premature terminations at codons 205 and 370, respectively; one acceptor splice site mutation that leads to a frameshift from codon 351 and a premature termination at codon 367; and two missense mutations (Cys-318 3 Arg and Asn-320 3 Lys). The functional effects of these mutations, together with a previously reported GATA3 ZnF1 mutation and seven other engineered ZnF1 mutations, were assessed by electrophoretic mobility shift, dissociation, yeast twohybrid and glutathione S-transferase pull-down assays. Mutations involving GATA3 ZnF2 or adjacent basic amino acids resulted in a loss of DNA binding, but those of ZnF1 either lead to a loss of interaction with specific FOG2 ZnFs or altered DNA-binding affinity. These findings are consistent with the proposed three-dimensional model of ZnF1, which has separate DNA and protein binding surfaces. Thus, our results, which expand the spectrum of HDR-associated GATA3 mutations and report the first acceptor splice site mutation, help to elucidate the molecular mechanisms that alter the function of this zinc finger transcription factor and its role in causing this developmental anomaly.

Molecular analysis of the GATA3 gene in five Japanese patients with HDR syndrome

“…Patient 4 had a previously reported acceptor splice site mutation, G>T transversion at the boundary of intron 5/exon 6 (Fig. 2D), generating a new open reading frame encoding a missense peptide with a premature termination at codon 367 [10]. Finally, a second missense mutation in the second zinc finger region (C318S) was identified in patient 5 and his affected family members (Figs.…”

“…WT-GATA3 and MT-GATA3 constructs were subcloned into the mammalian expression vector pEG-FP-C1 (BD Biosciences Clontech, Mountain View, CA) as previously described [10]. COS-7 cells were transfected with either WT-GATA3-GFP or MT-GATA3-GFP, using Lipofectamine 2000, and the cells were placed onto glass coverslips and fixed in 4% (vol/vol) formaldehyde/PBS at 48hr after transfection.…”

“…GATA3 encodes a 444-amino-acid protein that contains two transactivating domains (TA1 and TA2) and two zinc finger domains (ZnF1 and ZnF2) [3,8,9]. ZnF2 is highly conserved and essential for binding of its consensus response element, whereas ZnF1 is thought to stabilize this binding and to interact physically with the multi-type zinc finger Friends of GATA (FOG) proteins, which were cloned as GATA-specific factors [9,10].…”

Parafibromin is a nuclear protein with a functional monopartite nuclear localization signal

“…Mutations were introduced by use of site-directed mutagenesis (Quikchange, Stratagene, CA, USA) (Kennedy et al, 2005). The DNA sequences of all constructs were verified using a semi-automated detection system (ABI 377 sequencer; PE Applied Biosystems, CA, USA), as described previously (Nesbit et al, 2004).…”

Critical YxKxHxxxRP Motif in the C-Terminal Region of GATA3 for Its DNA Binding and Function

“…GATA3 haploinsufficiency is reported to be associated with the hypoparathyroidism, deafness, and renal dysplasia syndrome (52). A mutation in the splicing acceptor site around the GATA3 intron 5/exon 6 boundary was found in familial hypoparathyroidism, deafness, and renal dysplasia syndrome patients (53). This mutation results in a frame shift that produces mis-sense polypeptides from aa 351 to 367 with a premature termination at position 367.…”