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Functional Analysis of a Novel GATA3 Mutation in a Family with the Hypoparathyroidism, Deafness, and Renal Dysplasia Syndrome

Divison of Nephrology (A.Z., K.W., N.H., Y.P.), Department of Medicine, University Health Network and University of Toronto, Toronto, Ontario, Canada M5G2C4; Academic Endocrine Unit (A.M.N., A.A., R.V.T.), Nuffield Department of Medicine, University of Oxford, Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Oxford OX3 7LJ, United Kingdom; and Renal Unit of General Regional Hospital "G. Papanikolaou" (M.S., G.B., K.S.), Thessaloniki, Greece

Address all correspondence and requests for reprints to: York Pei, M.D., 13 EN-228, 200 Elizabeth Street, Toronto, Ontario, Canada M5G2C4. E-mail: york.pei{at}uhn.on.ca.

	Abstract

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The hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome is an autosomal dominant disorder caused by mutations of a member of the GATA-binding family of transcription factors, GATA3. This dual zinc finger transcription factor binds DNA with its C-terminal zinc finger (ZnF2) and stabilizes this binding with its N-terminal zinc finger (ZnF1). ZnF1 also interacts with other zinc finger proteins, notably Friend of GATA (FOG). The HDR syndrome has been described in patients with mutations affecting both ZnF1 and ZnF2 domains; the former result in inefficient interaction with FOG, and the latter result in disruption of DNA binding. We report a patient with renal failure, hypoparathyroidism, and bilateral hearing loss. Assessment of family members indicated that the disease arose as a de novo mutation in her mother. Analysis of GATA3 in the family revealed a heterozygous missense mutation resulting in a nonconservative change of a single amino acid (R276P) in the ZnF1 domain. Functional analysis using dissociation electrophoretic mobility shift and yeast two-hybrid assays showed reduced binding affinity to the GATA motifs but normal interaction with FOG in vitro. These results are consistent with the predicted functions of human GATA3-ZnF1 from three-dimensional molecular modeling and with HDR being a result of GATA3 haploinsufficiency.

	Introduction

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THE HYPOPARATHYROIDISM, SENSORINEURAL deafness, and renal dysplasia (HDR; MIM 146255) syndrome is an autosomal dominant disorder (1, 2). Clinically, the HDR syndrome is characterized by low plasma calcium concentrations as a result of a deficiency of PTH secretion. PTH levels can vary from low normal to undetectable. The sensorineural hearing loss is bilateral and most pronounced at higher frequencies. Hearing loss is typically moderate to severe and present at birth (1, 3, 4). Numerous renal anomalies have been observed with variable penetrance, including renal dysplasia, hypoplasia, aplasia, and vesico-ureteral reflux (1, 3, 5, 6, 7, 8). Since its initial recognition, major advancements have been made in elucidating the pathophysiology of this syndrome. Deletion-mapping studies in two HDR families with chromosomal rearrangement and deletion refined the critical interval of the disease gene to approximately 200 kb on chromosome 10p14-pter (8). This region contains GATA3, a protein that is expressed in developing parathyroid glands, inner ears, and kidneys (9, 10). Using a candidate gene approach, the causal role of GATA3 was established in HDR by documenting pathogenic mutations in patients without cytogenetic abnormalities (8).

GATA3 belongs to a highly conserved family of dual zinc finger transcription factors. All six members of the mammalian GATA proteins share amino acid homology in their zinc finger binding domains and bind to the consensus motif 5'-(A/T) GATA (A/G)-3' (11). The N-terminal zinc finger (ZnF1) interacts with other zinc finger proteins, notably Friend of GATA (FOG). It also stabilizes DNA binding by the C-terminal zinc finger (ZnF2) in vitro (12). To date, GATA3 mutations in both ZnF2 and ZnF1 have been reported in families with HDR syndrome (5, 7, 12). Mutations affecting ZnF2 result in loss of DNA binding. In contrast, all reported mutations in ZnF1 have resulted in inefficient interaction between GATA3 and other zinc finger transcription factors, namely FOG2 (12). Using GATA1 as a model, it was predicted that specific ZnF1 mutations with inefficient DNA binding may exist (12). To test this hypothesis, GATA3 constructs with ZnF1 mutations were engineered, and DNA binding assays showed that, indeed, four of the seven mutant constructs had reduced binding affinity (12). The manner in which these GATA3 mutations cause HDR is likely to be haploinsufficiency, which is consistent with the role of GATA3 as a transcription factor (8, 13, 14). This is further supported by the observation that some HDR patients have deletions that result in a complete loss of a GATA3 allele indicating that HDR is not a result of a dominant-negative effect of a mutated GATA3 or non-sense-mediated decay of mutant GATA3 mRNA (8, 12, 14). We report here a family with a novel GATA3 mutation in the ZnF1 domain, which is associated with reduced DNA binding affinity.

	Subjects and Methods

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Clinical and biochemical analysis

All eight members of this family were evaluated both clinically and biochemically. Plasma creatinine, albumin, total calcium, and phosphorus concentrations were measured using standard Technicon methods on a SMAC II analyzer (Technicon Corp., Tarrytown, NY). Intact 1–84 PTH concentrations were measured using a two-site immunochemiluminometric assay [Advantage Bio-Intact PTH (1–84) assay; Nichols Institute, San Juan Capistrano, CA]. Formal audiogram was performed to evaluate the high frequency deafness of the affected individuals.

Mutation analysis

Venous blood was collected from all family members and 50 ethnically matched unrelated control subjects after informed consent, as approved by the local ethical research committee. Genomic DNA from leukocytes was extracted using a commercial kit (FlexiGene DNA kit, QIAGEN Inc., Mississauga, Ontario, Canada). To detect gross gene deletion within members of the family, we genotyped four microsatellite markers (D105189, D1051751, D1051779, and D105226) that spanned the GATA3 genomic region (8). To screen for intragenic mutations, nine pairs of GATA3-specific primers were used to amplify all six GATA3 exons and intron-exon junctions from the proband and an unaffected control subject (8). Both strands of the PCR products were then sequenced by cycle sequencing and resolved on a semiautomated sequencer (ABI 377, Applied Biosystems, Foster City, CA). Restriction enzyme genotyping using AatII and HaeIII was performed to determine the disease segregation pattern of C480G (amino acid D160E) and G827C (amino acid R276P) gene variants, respectively, within the family as well as their frequencies in 100 normal chromosomes.

Similarly, single-stranded conformational polymorphism analysis was used to determine the frequency of the synonymous amino acid substitution (P191P) in the control population and its segregation within the study family.

EMSA

Dissociation EMSA was performed using COS-1 cells that were transiently transfected with either a wild-type GATA3 construct prepared in pcDNA 3.1 or a construct harboring the R276P mutation that was introduced by site-directed mutagenesis (QuikChange, Stratagene, La Jolla, CA) (8, 12). Forty-eight hours after transfection, cells were harvested and nuclear extracts prepared for use in binding reactions that used a ³²P-labeled double-stranded (ds) oligonucleotide containing a palindromic GATA3 site as described (8, 12, 13). Unlabeled competitor DNA was added to a 100-fold excess to the binding reactions, and aliquots were removed after 0, 10, 30, and 60 min for nondenaturing 6% PAGE. Western blot analysis using HG3–31 monoclonal antibody against GATA3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used to detect the presence of GATA3 protein in the nuclear extracts (12).

Yeast two-hybrid assay

As described previously (12), in vivo interactions between the GATA3 ZnF1 (see Fig. 2) and FOG2 ZnF1, -5, -6, and -8 were studied using a yeast two-hybrid system (BD Biosciences Clontech, Palo Alto, CA) (15, 16). The GATA3 mutation was introduced into this construct by site-directed mutagenesis (QuikChange, Stratagene) (12). Competent AH109 yeast cells were transformed sequentially with the appropriate GATA3 and FOG2 ZnF plasmid constructs using the LiAc/single-stranded DNA/polyethylene glycol procedure (17). The transformants were selected on Leu^–Trp^– (double dropout) minimal media plates by growth at 30 C for 3 d. Transformants were then patched onto His^–Ade^–Leu^–Trp^– (quaternary dropout) media plates and monitored for growth for up to 3 d. Expression of GATA3 and FOG2 Gal4 fusion proteins was confirmed by preparing protein extracts from each clone according to the manufacturer’s instructions (BD Biosciences Clontech) and analyzing them by SDS-PAGE in Tris-glycine-SDS buffer (Bio-Rad, Hercules, CA) and electrotransference onto PolyScreen polyvinylidene difluoride transfer membrane (PerkinElmer Life Sciences, Boston, MA) in CAPS buffer (10 mM, pH 11; Sigma Chemical Co., St. Louis, MO). Western blot analysis was performed with antibodies to either the Gal4-activating domain (AD) (FOG2-pGADT7 constructs) or the Gal4 DNA-binding domain (BD) (GATA3-pGBKT7 constructs), according to the manufacturer’s instructions (BD Biosciences Clontech) except that Gal4 DNA-BD antibody was used at 50 ng/ml and Gal4-AD antibody at 100 ng/ml (15). A secondary antibody, goat antimouse horseradish peroxidase (Bio-Rad) was used at 1/5000 and detected by using an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ).

View larger version (31K): [in this window] [in a new window]   FIG. 2. Gene structure and predicted amino acid sequence of GATA3. A, The human GATA3 is located on chromosome 10p14-pter and consists of 6 exons (188, 610, 537, 146, 126, and 806 bp) spanning 20 kb of genomic DNA. GATA3 encodes a 444-amino-acid transcription factor that contains two zinc finger (ZnF1 and ZnF2) and two transactivating (TA1 and TA2) domains. The zinc finger domains consist of a C-X2-C-X17-C-X2-C consensus sequence, where X represents any amino acid. The zinc moiety interacts with the four cysteine residues. The location of the R276P mutation (shown in bold) is located in a highly conserved region of the N-terminal zinc finger domain, ZnF1. The site of another previously reported adjacent mutation W275R in an HDR patient is also shown in bold. B, The amino acid sequence of ZnF1 in eight eukaryotic species is shown and demonstrates the highly conserved 4-amino-acid sequence, LWRR (amino acids 274–277).

	Results

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The proband was a 22-yr-old female (III:1 in Table 1 and Fig. 1) with a history of hearing loss and polycystic ovarian disease who presented to medical attention with hypertension and renal failure. She was diagnosed with polycystic ovarian disease at the age of 20 on the basis of irregular menstrual cycles and obesity. Bilateral high-frequency sensorineural hearing loss was diagnosed since childhood by a formal audiogram. She had no known family history of renal disease. Her initial investigations also showed evidence of hypoparathyroidism with inappropriately low intact 1–84 PTH in the presence of chronic renal insufficiency (Table 1). Family history was significant for a 19-yr-old younger sister who had bilateral high-frequency sensorineural hearing loss since childhood. She had presented earlier with hypocalcemic seizures at age 14 yr and was diagnosed with primary hypoparathyroidism. She also had mild mental retardation. The proband’s mother had mild mental retardation and bilateral sensorineural hearing loss since childhood. All the relevant laboratory investigations in the family members are summarized in Table 1. Based on the clinical findings and family history, a presumptive diagnosis of HDR syndrome was made.

View larger version (34K): [in this window] [in a new window]   FIG. 1. R276P GATA3 mutation. A, Electropherogram showing C827G transversion in exon 4 of the proband. The mutation (MT) results in a nonconservative amino acid substitution, R276P. The sequence tracing of a normal subject (WT) is shown below. B, The R276P missense mutation also produces a new HaeIII restriction site. C, HaeIII digestion of exon 4 PCR product in the proband (III:1) and her family members. The R276 missense mutation cosegregated with the disease in this family and arose as a de novo mutation in the proband’s mother (II:3). Males and females are denoted by squares and circles, respectively. Affected individuals are denoted by the black symbols. D, The wild-type exon 4 sequence is cleaved by HaeIII to produce two DNA fragments (209 and 88 bp), whereas the heterozygous R276P missense mutation is associated with two extra DNA fragments (137 and 72 bp).

We genotyped all family members with four polymorphic microsatellite markers and found no evidence of gross genomic deletion in the GATA3 region (data not shown). Direct sequencing of all six GATA3 exons and their splice junctions in the proband revealed three heterozygous gene variants. The first was a single base change (nucleotide C480G) located in exon 3B that results in a conservative amino acid substitution (D160E). The second sequence variant (nucleotide C573T), also located in exon 3B, results in synonymous amino acid substitution (P191P). The third sequence variant (nucleotide G827C) is located in exon 4 and results in a nonconservative amino acid substitution (R276P) (Fig. 1).

Restriction enzyme analysis of exon 3B with AatII (not shown) revealed that the missense variant, D160E, did not segregate with the disease within the family. Furthermore, this gene variant was found in two of 100 normal chromosomes, thus likely representing a rare polymorphism. Similarly, single-stranded conformational polymorphism analysis showed the synonymous amino acid substitution (P191P) was a rare polymorphism occurring in 2% of the control population and did not segregate with the disease. By contrast, restriction enzyme analysis of exon 4 with HaeIII showed that the missense variant (R276P) segregated with the disease within the family and was not found in 100 normal chromosomes. Because both maternal grandparents of the proband did not carry the R276P variant, it arose as a de novo mutation in the proband’s mother (Fig. 1). Additionally, the R276P variant is located within a highly conserved 4-amino-acid (LWRR) motif of the ZnF1 domain, suggesting that it may be a disease-causing mutation (Fig. 2).

Functional analysis of the R276P mutant

We performed functional studies to evaluate the effects of the R276P mutation on GATA3 DNA binding and protein interaction. By dissociation EMSA (Fig. 3A), we found DNA binding by both wild-type (WT) and R276P GATA3 at time 0 min. The R276P GATA3 yielded an additional larger band that was observed previously with the engineered GATA3 mutant R276Q (8) as well as the equivalent GATA1 mutant, R216Q, which is associated with X-linked thrombocytopenia and thalassemia (18). The same larger band can also be occasionally seen in association with WT GATA3 and GATA1 (8). The nature of these different GATA3-DNA species is unknown, but they may represent binding of more than one GATA3 molecule. Such multimeric forms of GATA3 may be a result of a higher GATA3 protein concentration in the reaction, and this may help to explain the inconstant occurrence of these larger bands in different experiments. Another possibility is that the larger bands may represent complexes in which GATA3 cofactors are also bound, and again this may depend upon the amount of GATA3 concentration in the reaction. It is important to note that although equal amounts of total nuclear protein are loaded, it is nevertheless possible that the amount of GATA3 varies because of variability in expression in each individual WT or mutant GATA3 transfectant. However, all the labeled dsDNA that was bound to the mutant GATA3 was displaced by unlabeled dsDNA by 30 min. In contrast, the wild-type GATA3 bound the labeled DNA stably over the entire time course of the experiment. These findings thus indicate that the R276P mutation is associated with reduced DNA binding affinity in vitro.

View larger version (86K): [in this window] [in a new window]   FIG. 3. Functional analysis of GATA3 R276P mutant. A, Dissociation EMSA. COS-1 cells were transfected with either the WT or mutant (R276P) GATA3 constructs and nuclear extracts prepared for binding reactions that used a radiolabeled (32P) double-stranded oligonucleotide containing a palindromic GATA consensus DNA sequence. The time interval after the addition of unlabeled competitor DNA is indicated in minutes. Control binding reactions using untransfected (UT) cells were performed. Initially, both WT and mutant GATA3 bound labeled dsDNA (0 min). However, by 30 min, the labeled dsDNA to the R276P mutant was completely displaced by unlabeled DNA. By contrast, the WT GATA3 retained DNA avidly for the entire duration of the experiment (60 min). These data support the conclusion that R276P GATA3 has decreased DNA binding affinity. B, Yeast two-hybrid analysis. The interactions between GATA3 ZnF1 and FOG2 ZnF1, -5, -6, and -8 were studied in the yeast reporter strain AH109 after transformation with vectors containing GATA3ZnF1 (pGBKT7) and each FOG2 ZnF (pGADT7) in turn. Yeast growth was monitored 48 h after streaking and incubation at 30 C, and the results using quaternary dropout (Leu–Trp–Ade–His–) media in which growth is dependent on the physical interaction between GATA3-Gal4 DNA-BD and FOG-2-Gal4-AD fusion proteins (12 ) are shown. All colonies grew on control double-dropout (Leu–Trp–) J media (data not shown). The WT GATA3 fusion protein (positive control) interacted with FOG2 ZnF1, -5, -6, and -8 fusion proteins (results with ZnF1 shown), whereas the W275R mutant fusion protein (negative control) did not interact with FOG2 ZnF1, -5, and -8 (results with ZnF1 shown), as previously reported (12 ). The GATA3 mutant R276P fusion protein interacted with FOG2 and ZnF1, -5, -6, and -8 and yielded results similar to that of the WT GATA3 fusion protein. These results observed with the HDR-associated mutant R276P fusion protein are similar to those previously reported (12 ) with the engineered mutant R276Q fusion protein.

GATA3 and FOG2 have been shown to interact in mouse embryos (6). In addition GATA1 ZnF1 has also been shown to interact with four of the nine zinc-finger binding domains (ZnF1, -5, -6, and -9) of FOG and four of the eight zinc-finger binding domains (ZnF1 -5, -6, and -8) of FOG2 (19). Human GATA3 ZnF1 has also been discovered to interact with FOG2 ZnF1, -5, -6, and -8 (12). A GATA3 W275R mutant was previously shown to have normal DNA binding affinity but inefficient interaction with FOG2 ZnF1, -5, and -8 (12). Using a yeast two-hybrid assay we found that the R276P mutant behaved like the WT GATA3, effectively interacting with all four FOG2 zinc-finger binding domains (Fig. 3B). Coexpression of the GATA3 and FOG2 Gal4 fusion proteins in the yeast colonies was confirmed in each case by Western blot analysis (data not shown). These results observed with the HDR-associated R276P mutant are identical with those observed with the engineered mutant R276Q (12) and are consistent with the reported three-dimensional model of GATA3-ZnF1 in which R276 is located at the DNA binding surface but not on the surface that participates in binding to FOG2 ZnF1 (12).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
In the family presented in this report, three members exhibited clinical and laboratory findings that were consistent with the diagnosis of the HDR syndrome (1, 2, 3, 5, 6, 12). Although the sensorineural deafness and hypoparathyroidism were present in all these individuals, the renal phenotype was more variable. Although the proband has right renal agenesis and moderate chronic renal insufficiency (creatinine clearance estimated to be 40–50 ml/min·1.73 m²) at age 22 yr, her affected mother has no apparent renal abnormality at age 44 yr. These findings indicate variable expressivity of the renal phenotype. Additionally, the renal manifestations of HDR syndrome, which may be asymmetrical, can include renal agenesis, renal dysplasia, and vesico-ureteric reflux in different affected members of the same family (1, 2, 3, 5, 6). These findings, taken together, suggest that additional genetic, environmental, or stochastic factors are likely involved in determining the clinical expression of the HDR syndrome.

A total of 19 GATA3 mutations have been reported to date (5, 8, 12). Gross genomic deletion, frameshift, and non-sense mutations in 16 of them are predicted to result in either the complete loss of GATA3 or the production of a truncated protein lacking one or both of the zinc finger domains (5, 8, 12). Thus, most of the reported GATA3 mutations are predicted to result in haploinsufficiency (8, 14). On the other hand, the effects of the reported missense mutations (W275R, C318R, and N320K) on GATA3 function cannot be easily predicted (5, 12). Using a number of function assays, we have recently shown that the W275R mutation, which is located in the ZnF1 domain, was associated with disrupted interaction with specific FOGs but retained normal DNA binding. By contrast, the C318R and N320K missense mutations, which are located in the ZnF2 domain, were associated with loss of DNA binding (12). The R276P mutation identified in our family is located within a highly conserved 4-amino-acid (LWRR) motif of the ZnF1 domain (Fig. 2). In contrast to the W275R mutant, we found the R276P mutation was associated with normal protein interaction with FOGs but reduced DNA binding affinity. These results are in complete agreement with those from an engineered GATA3 mutant (R276Q) (12) and the naturally occurring GATA1 mutation (R216Q) (18). The R276P GATA3 mutant is likely to translocate to the nucleus because the equivalent R276Q GATA3 mutant has previously been shown to translocate as efficiently as the WT GATA3 to the nucleus (8). Taken together, our findings suggest that the GATA3 ZnF1 residue, R276, is critical for stabilizing the DNA binding by ZnF2. These findings are in keeping with a previously proposed three-dimensional structure of GATA3 ZnF1 that places R276 in a DNA binding surface, whereas W275 is involved in zinc finger protein interaction (12). The observation that the LWRR (274–277) sequence in GATA3 ZnF1 is so highly conserved among different species (Fig. 2) further supports the conclusion that the preservation of this sequence is essential for protein function.

Electronic database information

Accession numbers and URL for data in this article are as follows: Online Mendelian Inheritance of Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ for GATA3 (MIM 131320) and HRD (MIM 146255).

	Footnotes

 
First Published Online February 10, 2005

¹ R.V.T. and Y.P. contributed equally to this work.

Abbreviations: AD, Activating domain; BD, binding domain; ds, double-stranded; FOG, Friend of GATA; HDR, hypoparathyroidism, deafness, and renal dysplasia; WT, wild type; ZnF1, N-terminal zinc finger; ZnF2, C-terminal zinc finger.

Received October 5, 2004.

Accepted January 27, 2005.

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