In the present case, we identified a novel heterozygous missense mutation of GATA3 in a Japanese family affected with HDR syndrome. This R299Q mutation induced an orientation change of the 2 zinc fingers and abolished its physiological function as a transcription factor.
The clinical manifestation of HDR syndrome is heterogeneous . A triad of HDR symptoms is observed in 62.3 % of patients; 28.6 % of patients show only hypoparathyroidism and deafness, and 2.6 % of patients show only deafness and renal disease . Hypoparathyroidism and hearing loss is the most common combination. Many different renal anomalies, with variable penetrance, are observed, including renal dysplasia, hypoplasia, aplasia, and vesico-ureteral reflux [3, 10, 11]. In addition, similar to our case, patients with HDR syndrome present with heterogeneous clinical manifestation even among individuals having the same mutation [10, 11]. The mechanism of heterogeneous symptoms has not been fully elucidated. In animal study, Grote et al. showed that the nephric duct-specific inactivation of Gata3 leads to a wide spectrum of urogenital malformations through the glial cell line-derived neurotrophic factor/Ret signaling pathway . Gata3 prevents ectopic ureter budding and premature differentiation of nephric duct cells. On the other hand, Gata3, which is also expressed in spiral ganglion neurons throughout their development, is essential for formation of the intricately patterned connections in the cochlea .
GATA3 belongs to a family of dual zinc finger transcription factors. Zinc fingers of the mammalian GATA proteins (GATA 1–6) have a Cys-X2-Cys-×17-Cys-X2-Cys structure (with X representing any amino acid residue), in which a single zinc ion is coordinated by 4 cysteine residues . Its tertiary structure reveals 2 anti-parallel β-sheets, an α-helix, and a long loop. The α-helix binds into the major groove of DNA. In GATA3, ZnF2 is essential to the binding of the transcription factor to the consensus sequence (A/T)GATA(A/G) on the target gene promoter region, whereas ZnF1 is thought to stabilize this binding through its interaction with another cofactor, Friends of GATA (FOG) 2 [21, 22].
In this study, the 3-dimensional conformation of wild-type and mutant GATA3 was analyzed using SFAS, which runs several external programs for sequence alignment and structural modeling and organizes their results. Protein structure comparison was presented using another web application, UCSF Chimera, to construct high-resolution images. The structure of residues 263–347, ranging from ZnF1 to ZnF2, is displayed in Fig. 3. The configuration of the linker between the zinc fingers was affected severely by the mutation R299Q, which is located between the 2 zinc fingers. In addition, the mutation created a greater conformational change in ZnF1 than in ZnF2, probably because Arg-299 is located near the zinc ion of ZnF1. This Arg residue may stabilize a coordinate bond between the zinc ion and Cys residues, which are the crucial components of ZnF1. Substituting the basic amino acid (Arg) with neutral amino acid (Gln) may affect this stabilization and cause a change in the orientation in the protein structure. Furthermore, this conformational change spread into ZnF2. These results suggest that R299Q mutation impairs the function of both ZnF1 and ZnF2.
To date, the conformation of GATA3 mutant proteins has been investigated only in 3 cases of HDR syndrome [10, 11, 23]. Gaynor et al. showed that Thr272Ile, located in ZnF1, resulted in the loss of a polar side chain interaction between Thr-272 and Leu-274 . This mutation changed the ZnF1 structure, leading to loss of DNA binding and FOG2 interactions. Ali et al. also showed that Leu348Arg could alter the transcriptional activity of GATA3 because of the substitution of a non-polar hydrophobic Leu residue for a positively charged Arg residue; this Leu 348 residue is located in ZnF2 and lies at the end of an α-helix which makes contact with DNA at the DNA major groove within the GATA motif of the promoter region . Moreover, Chiu et al. analyzed the conformation change caused by Arg353Ser, which is located at the C-terminal DNA-binding region following ZnF2 (residues 318–342), using NNPredict software. This mutation was predicted to disrupt the helix turn composed of residues 355–358, because of the substitution of a basic for a polar, uncharged residue, which changed the angle between ZnF2 and the adjacent C-terminal tail . However, our case is the first report of a conformation change caused by a mutation at the junction between the 2 zinc fingers; the amino acid substitution affected the conformation of both Zn fingers. Structure prediction model obtained by PDBj provides useful information about GATA3 mutant. Amino acids are classified into 4 groups such as nonpolar, polar, acidic, and basic amino acids. Substitution between amino acids belonging to different groups is likely to cause critical conformation change.
We also confirmed that R299Q was a loss-of-function mutation using an in vitro reporter assay system. GATA3/R299Q overexpression failed to increase the transcription activity of the GATA responsive element. In addition, our data showed that GATA3/WT activated PTH transcriptional activity, whereas GATA3/R299Q did not (Fig. 4b). This result is of interest, as there has been no previous direct evidence that GATA3 is a positive regulator of PTH expression. The consensus GATA3 binding element is not present in the 5′-promoter region of human PTH, suggesting that the observed enhancing effect of GATA3 is caused by an indirect pathway, rather than by the direct binding of GATA3. Further investigations are required to elucidate this molecular mechanism. Our results suggest that GATA3 plays an important role not only in the development of the parathyroid gland in fetus, but also in PTH regulation after birth.