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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-1783
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 4 1426-1432
Copyright © 2008 by The Endocrine Society

Analysis of the GCM2 Gene in Isolated Hypoparathyroidism: A Molecular and Biochemical Study

Alexander Maret1, Changlin Ding1, Sara Levine Kornfield and Michael A. Levine

Division of Pediatric Endocrinology (A.M.), University Hospital of Geneva, CH-1211 Geneva, Switzerland; Division of Pediatrics and Department of Biomedical Engineering (C.D., M.A.L.), Lerner Research Institute and the Children’s Hospital, Cleveland Clinic, Cleveland, Ohio 44195; and Department of Psychology (S.L.K.), Drexel University, Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: Michael A. Levine, M.D., The Children’s Hospital of Philadelphia, Division of Endocrinology, 34th Street and Civic Center Boulevard, Philadelphia, Pennsylvania 19104. E-mail: levinem{at}email.chop.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Hypoparathyroidism is characterized by hypocalcemia, hyperphosphatemia, and absent or markedly reduced serum levels of intact PTH. The transcription factor GCM2 is critical for the development of parathyroid glands in mice and humans.

Objective: We sought to determine the prevalence of GCM2 gene mutations in patients with familial or sporadic forms of isolated hypoparathyroidism (IH).

Design and Setting: We used PCR to analyze the promoter, the exons, and flanking intronic sequences in 10 IH families with 17 affected members and in 10 patients with sporadic IH. Wild-type and mutant GCM2 proteins were expressed in HEK293 cells and characterized.

Results: We identified nine single nucleotide changes, three in the 5' untranslated region and six in exon 5, that led to nonsynonymous changes in the GCM2 protein (G203S, I227V, Y282D, N315D, Q330L, and M354V). Variant GCM2 proteins had normal size, nuclear localization, and transactivational function when expressed in HEK293 cells. Similar analyses of two previously described GCM2 missense mutations, R47L and G63S, revealed decreased nuclear expression and markedly reduced (5–20% of normal) transactivational activity. The variant alleles did not segregate with inheritance of IH, and many of the single nucleotide substitutions were present in DNA from unrelated normal subjects, suggesting that these base changes were polymorphisms.

Conclusion: Our study describes nine single nucleotide changes in the GCM2 gene that represent polymorphisms. Although GCM2 mutations appear to be an uncommon cause of IH, the wide variety of GCM2 polymorphisms suggests that variant alleles may have a role in determining parathyroid function.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
Hypoparathyroidism is characterized by hypocalcemia and hyperphosphatemia due to deficient or absent secretion of PTH. Hypoparathyroidism may occur due to genetic defects that result in complex developmental disorders [e.g. DiGeorge syndrome, MIM 188400 (1, 2, 3, 4, 5) or the hypoparathyroidism, deafness, renal anomaly (GATA3 gene, MIM 146255) syndrome (6)] or which lead to widespread autoimmune consequences [e.g. type 1 autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome (MIM 240300) (7, 8, 9)]. Hypoparathyroidism can also occur as an isolated endocrinopathy termed isolated hypoparathyroidism (IH) and can be sporadic or familial, with autosomal (9, 10) or X-linked inheritance (11). Molecular genetic studies indicate that IH is caused by defects in a variety of genes (10). Rare mutations in the PTH gene (MIM 168450) have been associated with both autosomal dominant (12) and recessive (9, 13) forms of familial IH. More commonly, gain-of-function mutations in the CASR gene encoding the calcium-sensing receptor (MIM 601199) have been identified in subjects with autosomal dominant hypocalcemia, a syndrome associated with low but not absent levels of serum PTH and relative hypercalciuria (14, 15). Recently, homozygous mutations in the glial cells missing B (GCMB or GCM2, MIM 603716) gene have been identified as a cause of autosomal recessive IH (16, 17). GCM2 encodes a human ortholog of the Drosophila glial cells missing (gcm) gene, which is predominantly, if not exclusively, expressed in developing (18) and mature parathyroid cells (19) and is required for the development of parathyroid glands in mice (20). The gene is located on the short arm of chromosome 6 (6p23–24) and consists of five exons that encode a protein of 506 amino acids (21). GCM2 is a transcription factor with an evolutionarily conserved DNA-binding domain in the amino-terminal region, known as the GCM domain, and two transactivation domains at the carboxyl-terminal end (22). The GCM motif spans approximately 150 amino acids and is a unique Zn-containing DNA binding domain that consists of a large and a small arm (β sheets) tethered together by the Zn2+ ion to form a clamp that seizes the major groove of the DNA from two sides (23).

The goal of the present study was to analyze the GCM2 gene in a large series of patients with familial or sporadic IH to determine genotypic variants and assess the prevalence of mutations in this disorder.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

We obtained blood samples from 27 patients with IH. Seventeen subjects were members of 10 IH kindreds (four with autosomal dominant and six with autosomal recessive patterns of inheritance), whereas 10 patients had sporadic IH. In addition, we collected blood samples from 10 unaffected members of three families and from both parents of three sporadic cases. All subjects underwent evaluation to exclude the presence of a more generalized metabolic, autoimmune, or developmental disorder. This protocol included measurement of serum levels of intact PTH, vitamin D metabolites (25-hydroxyvitamin D and 1,25-dihydroxyvitamin D), free T4 and TSH, and minerals (calcium, phosphorus, magnesium); renal calcium clearance was determined to identify subjects with autosomal dominant hypocalcemia due to defects in the CASR gene; fluorescence in situ hybridization analysis for a deletion on chromosome 22q11 was performed to exclude DiGeorge syndrome. Genetic analyses of the PTH (9, 24) and CASR (14) genes were performed to exclude mutations in these genes. The diagnosis of IH was based on 1) absence of somatic, developmental, or other physical defects; 2) low or absent levels of serum intact PTH in the presence of hypocalcemia, hyperphosphatemia, and normal serum levels of magnesium and 25-hydroxyvitamin D; 3) absence of other endocrinopathy; and 4) absent karyotypic defect or loss of function mutation of the PTH or CASR genes. The protocol was approved by the Johns Hopkins Hospital Joint Committee on Clinical Investigation, and written informed consent/assent was obtained from all subjects.

Normal DNA was obtained from a set of genomic DNA samples from 48 unrelated disease-free individuals (Oncomatrix, San Diego, CA) plus two unrelated healthy individuals from our center.

5' Rapid amplification of cDNA ends (RACE)

To determine the 5' end of GCM2 mRNA, we used the SMART RACE cDNA amplification kit from Clontech (Palo Alto, CA) for RACE. We extracted total RNA from a human parathyroid adenoma using TRIzol (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions and used the RNA as a template to generate 5' cDNA fragments. RACE was performed using four exonic antisense oligonucleotide primers [(a) 5' untranslated region (UTR) of exon 1: 5'-TGCATCCCGTAGGAGCACACGCCGA; (b) coding region of exon 1: 5'-TGCATCCCGTAGGAGCACACGCCGA; (c) exon 3: 5'-TCCGCTGTGCCCTCGACAAGGAATCAA; (d) exon 5: 5'-TGGTGACTGTCACCGGAGGACCCCAGG], 1 µg of total parathyroid RNA and Clontech’s universal primer mix according to manufacturer’s instructions (25). The amplified products were isolated from agarose gel slices (Mini Elute Gel Extraction Kit, QIAGEN, Valencia, CA) and subjected to direct sequence analysis using 1 pmol/µl of the antisense primer and the fluorescent dideoxy terminator method of cycle sequencing (Applied Biosystems Division 3700 DNA analyzer; Perkin-Elmer, Lincoln, CA).

In silico analysis of the GCM2 promoter

The genomic sequence upstream of the transcription start site (TSS) was analyzed for consensus binding sites of known transcription factors using Matinspector (Genomatix Software, München, Germany) and the Mapper platform for the computational identification of transcription factor binding sites (26).

Mutation detection and analysis

DNA was extracted from peripheral blood leukocytes by standard methods. To analyze the GCM2 gene for mutations, we used sets of oligonucleotide primers to amplify the five exons including exon-intron junctions, as well as the 5'-UTR, from genomic DNA (16). PCR was performed with 2.5 mM magnesium chloride, 0.2 mM deoxynucleotide triphosphates, 0.5 µM of each primer, 1 U of Taq polymerase (Amplitaq, Perkin-Elmer Applied Biosystems, Foster City, CA), and 200 ng of genomic DNA as template. PCR products were purified using a 1% agarose gel and sequenced directly.

For restriction fragment length polymorphism analysis of the 5' UTR, PCR was used to amplify a 318-bp fragment that contains the TSS which, if mutated (G to C transversion), creates a new restriction site for NlaIII yielding a 234- and 84-bp fragment. PCR conditions for the 318-bp product of the 5' UTR included forward primer: 5'-GATTGGTACCAAAGGATACATTCCTACTTACCCTC-3'; and reverse primer: 5'-CATGCTCGAGAAGCTGGGGACAATGGTTAT-3', with 35 cycles of PCR with an annealing temperature 61 C for 30 sec. Aliquots of PCR products were digested for 1 h at 37 C using NlaIII (New England Biolabs, Beverly, MA) and subjected to electrophoresis through 1% agarose gels.

We used the NNPredict program (http://www.cmpharm.ucsf.edu/~nomi/nnpredict.html) to predict the effect of amino acid substitutions on human GCM2 structure.

We also examined conservation of amino acid substitutions within GCM orthologs because the biological importance of a residue often correlates with its level of evolutionary conservation within the protein family. Specifically, amino acids that are important for maintaining the three-dimensional structure of a protein and/or its function are often under strong evolutionary constrains. ConSurf (http://consurf.tau.ac.il/) (27, 28) is a web-based tool that automatically calculates evolutionary conservation scores and maps them on protein structures. We analyzed changes using default parameters and a PDB file that was acquired from CPHmodels 2.0 server (http://www.cbs.dtu.dk/services/CPHmodels/) (29).

Analysis of variant GCM2 proteins

Site-directed mutagenesis was performed to introduce single nucleotide replacements into the wild-type GCM2 cDNA using the Quickchange site-directed mutagenesis kit from Stratagene (La Jolla, CA). All changes were verified by sequence analysis. We examined expression of recombinant GCM2 proteins in the embryonal kidney cell line HEK293T. HEK293T cells were cultured in DMEM (Life Technologies, Rockville, MD) supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 µg/ml) in a humidified (95%) atmosphere of 5% CO2 at 37 C and passaged as necessary. Cells were plated at 60–80% confluence in six-well dishes, and 24 h later they were transfected with 1.0 µg of the expression vector per well using Fugene 6 or Lipofectamine Plus. Approximately 48 h after transfection the recombinant GCM2 protein was extracted from the cells by the addition of Laemmeli buffer. Cell extracts were subjected to SDS-PAGE on 8% gels, transferred to polyvinyl difluoride membranes, and incubated with an affinity-purified rabbit antihuman GCM2 polyclonal antiserum (1:3000) that had been raised against a GST-GCM2 fusion protein. This antibody is reactive against human, mouse, and bovine GCM2 protein, but not against GCM1 protein. Antibody binding was detected by use of peroxidase-labeled secondary antibody, and was visualized using enhanced chemiluminescence following the manufacturer’s instructions (Pierce Chemical Co., Rockford, IL) and exposure to autoradiographic film. Densitometric image analysis was performed using Quantity One software (Bio-Rad, Hercules, CA).

Immunofluorescence

To determine cellular distribution of GCM2 proteins, we transfected HEK293T cells that had been seeded on coverslips in six-well plates. After 72 h, cells were fixed with 1% paraformaldehyde for 6 min, permeated by 0.05% Triton X-100 for 10 min, and blocked with 5% BSA for 1 h. Recombinant protein was detected with an affinity-purified rabbit anti-GCM2 antibody (1:400 dilution) and a secondary goat antirabbit antibody linked to Cy3 (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) (1:400 dilution). Nuclei were counterstained with DAPI, and cells were examined by fluorescent microscopy (Leica Mikroskopie und Systeme GmbH, Wetzlar, Germany) with a TRITC filter (Chroma Technology Corp., Brattleboro, VT).

Transactivational studies

To analyze transcriptional activity of GCM2 variants, we generated an HEK293 cell line that stably expressed pTATAluc-6xgbs (a generous gift of Dr. Michael Wegner, Universität Hamburg, Hamburg, Germany), a reporter gene consisting of the luciferase reporter gene under the transcriptional control of six tandemly arranged GCM consensus binding sites (5'-ATGCGGGT-3') (30, 31). This cell line was transiently transfected with 1.0 µg of the expression vector per well of six-well plates using Fugene 6. To control for background luciferase activity and to control for transfection efficiency, 0.05 µg/well of a Renilla luciferase reporter vector DNA, driven by a minimally active thymidine kinase promoter (pRL-TK; Promega, Madison, WI), was cotransfected with all GCM2 constructs. At 48 h after transfection, cells were gently rinsed with PBS (pH 7.4) and lysed using the Passive Lysis Buffer from Promega. Luciferase activity was measured using Promega’s Dual-Luciferase Reporter Assay kit, and the ratio of firefly to Renilla luciferase activity was calculated. The data shown here represent the mean ± SEM for five separate experiments and are expressed as relative activity compared with relative light units generated by the empty plasmid vector, which was assigned a value of 1.0.

Statistical analysis

All values are expressed as the mean ± SEM. Statistical significance for transfection experiments was assessed by univariant ANOVA using the Statistical Package for the Social Sciences (version 15; SPSS Inc., Chicago, IL) to determine whether differences were present between any of the alleles tested. When the ANOVA proved statistically significant, we performed post hoc Sheffe, Tukey, HSD, and Bonferonni tests to determine which specific alleles were different. Luciferase activity and relative luciferase activity were expressed as fold stimulation over empty vector, and data from all experiments were combined and analyzed. A P value of 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Characterization of the promoter and 5' UTR of GCM2 mRNA

5' RACE of GCM2 mRNA generated a single amplicon for each of the gene-specific oligonucleotide antisense primers, indicating the existence of a single TSS that is located about 250 bp upstream of the translation initiation site (+1). Direct sequence analysis of the amplicons showed that the complete 5' UTR contains 248 nucleotides (Fig. 1Go), which is longer than previously estimated from cDNA clones. There was a thymidine instead of cytosine at position –74 bp in the 5' UTR (Fig. 1Go and see below). Analysis of the 5' region revealed consensus sequences corresponding to TATA and GC boxes that are signatures of a core promoter sequence, as well as binding sites for embryonic transcription factors Pax1 and Pax 9 (Fig. 1Go).


Figure 1
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  		FIG. 1. Analysis of the 5' flanking region of GCM2. The TSS determines the 5' end of the 248 bp long UTR of exon 1. Two common single nucleotide polymorphisms are located at the TSS (G>C; carrier frequency: 22%) and at position –149 (G>T; carrier frequency: 8%). The cytosine at position –74 bp represents an error or a rare polymorphism in the published genomic sequence of GCM2 (NT_007592;gi:29804415) because all alleles in 50 normal subjects show a thymidine at this position. Analysis of the sequence for transcription factor consensus binding sites using Matinspector indicates an AP2 and a TATA- and GC-box site shortly upstream of the TSS that are signature of a core promoter. Moreover, there is a proximal binding site for the homeobox transcription factors Pax1 and Pax 9, which are critical for parathyroid gland development.

 
Mutational analysis of GCM2

We found nine different single nucleotide sequence changes after analysis of genomic DNA from 17 affected patients from 10 families and 10 patients with sporadic IH. Two changes were upstream of the initiator ATG codon in the 5' UTR of exon 1, a G to A transition at –149 from initiator ATG and a G to C transversion at –248 from initiator start site, which corresponded to the TSS (Fig. 1Go). There were six changes in exon 5, all of which led to amino acid replacements (Fig. 2Go and Table 1Go). Amino acid changes G203S and I227V occur in transactivating domain 1, whereas Y282D, N315D, and Q330L are located in the inhibitory domain. Amino acid substitution M354V is located immediately beyond the inhibitory domain. All of these changes are in amino acid residues that are not highly conserved in GCM2 proteins from other species (32, 33, 34), suggesting that they are noncritical sites. Moreover, based on molecular modeling, none of these amino acid changes are predicted to alter the secondary or tertiary structure of the GCM2 protein (data not shown).


Figure 2
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  		FIG. 2. Organization of the GCM2 gene and protein. The upper panel shows the various functional domains of the human GCM2 protein [after Wegner and Riethmacher (40 )], whereas the lower panel shows the position of each nucleotide or amino acid change that was identified in this study or as previously described (see text). The clear boxes represent coding sequences of exons, and the number above each box represents the first amino acid residue for that exon.

 

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  		TABLE 1. Allele frequency of GCM2 single nucleotide polymorphisms

 
None of the affected subjects were homozygous for a specific single-nucleotide change, and the variant GCM2 alleles did not segregate with IH in informative families (Fig. 3Go, and data not shown). There were three different single nucleotide changes in members of one family with autosomal dominant IH (Fig. 3Go), two of which resulted in amino acid changes (Y282D and N315D). One affected member (5) carries both amino acid changes (i.e. he is a compound heterozygote for Y282D and N315D in exon 5), and two other affected family members (subjects 4 and 7) are heterozygous for either the Y282D or N315D amino acid change. In addition, one unaffected member (subject 1) is heterozygous for N315D. An unrelated subject with sporadic IH was compound heterozygous for G203S and I227V.


Figure 3
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  		FIG. 3. Pedigree of family with autosomal dominant IH. The three polymorphic nucleotides and the two corresponding amino acid changes are shown to the left of the figure. The G/A at position –149 represents a G to A transition; Y282D indicates a nonsynonymous nucleotide change in codon 282 that replaces amino acid tyrosine (Y) with aspartic acid (D), and N315D indicates a nonsynonymous nucleotide change in codon 315 that replaced amino acid asparagine (N) with aspartic acid (D). Squares represent males, and circles represent females; solid figures indicate affected subjects. The proband is subject 4. The pedigree illustrates discordance between IH and either of the affected mother’s GCM2 alleles.

 
With the exception of I227V, all the single nucleotide changes that were present in subjects with IH were also present in at least one GCM2 allele from 50 unrelated normal subjects. The two single nucleotide changes in the 5' UTR, –248 (G to C transversion) and –149 (G to A transition), were the most prevalent, with allelic frequencies of 22 and 8%, respectively, including some normal individuals who were homozygous. All 50 healthy individuals had a thymidine instead of cytosine at position –74 in the 5' UTR (Fig. 1Go), indicating that the published genomic sequence of GCM2 (NT_007592; gi:29804415) contains either an error or a rare polymorphism. Restriction fragment length polymorphism analysis was effective for distinguishing hetero- and homozygous forms of the G to C transversion at the TSS (Fig. 4Go).


Figure 4
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  		FIG. 4. NlaIII restriction digest of GCM2 exon 1 amplicons. Restriction fragment length polymorphism distinguishes the C allele for the TSS polymorphism from the more common G allele. PCR generates a 318-bp fragment; the G allele is not cut by NlaIII, whereas the C allele is digested into two fragments of 234 and 84 bp.

 
Analysis of GCM2 protein variants

We used site-directed mutagenesis to create unique GCM2 cDNAs that contained the amino acid variants that we had identified (I227V, Y282D, G203S, N315D, Q330L, and M354V) as well as two GCM2 mutations [R47L (17) and G63S (2)] within the DNA binding domain that had been previously identified in patients with autosomal recessive familial IH. We expressed these cDNAs in HEK293 cells and found that the variant recombinant GCM2 proteins migrated with apparent molecular masses (65 kDa) that were similar to that for the wild-type protein (Fig. 5BGo). Transfection efficiency of all GCM2 cDNAs was comparable, and all variant GCM2 proteins were expressed at levels that were similar to that of the wild-type protein. By contrast, the R47L and G63S mutants were expressed at levels that were 34 and 60% of the level of wild-type GCM2 protein, respectively. The wild-type and variant GCM2 proteins were exclusively localized to the nucleus, with the R47L and G63S mutants showing much weaker expression (Fig. 6Go).


Figure 5
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  		FIG. 5. Analysis of GCM2 protein expression. Panel A shows functional analysis of the GCM2 protein and its variants using a luciferase assay. Y282D and N315D are slightly but not significantly more active than wild-type GCM2, whereas R47L and G63S are significantly (P < 0.001) less active than wild-type GCM2. Error bars indicate SEM. Panel B shows the corresponding immunoblot of cell lysates. All GCM2 proteins migrated with apparent molecular masses of 65-70 kDa, despite a predicted molecular mass for GCM2 that is 56.6 kDa.

 

Figure 6
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  		FIG. 6. Subcellular localization of GCM2 protein. HEK293 cells were transiently transfected with cDNAs encoding wild-type or mutant GCM2 and were stained with anti-GCM2 antibody plus antirabbit IgG-conjugated Alexa Fluor 488 (Molecular Probes, Eugene, OR) and DAPI nuclear stain. Panel A shows wild-type; panel B shows I227V as representative of mutants identified in this work (G203S, I227V, Y282D, N315D, Q330L, and M354V); panel C shows R47L (17 ); and panel D shows G63S (2 ). Panels A, B, and D show indirect immunofluorescent images at 20x, and panel C is at 40x. GCM2 staining in wild-type cells and the mutants identified in this work showed compact and intense nuclear staining; staining for R47L is very weak (C) and staining for G63S is reduced.

 
Functional analyses showed that the wild-type GCM2 stimulated a 91-fold increase in activity of the luciferase hybrid reporter gene compared with the empty vector control plasmid. Variants Y282D and N315D were about 125% as active as the wild-type GCM2, whereas the other variants ranged from 79 to 122% of the wild-type activity (Fig. 5AGo), but there were no significant differences between these variants and the wild-type GCM2 sequence. By contrast, the R47L and G63S mutants were only 5 and 20% as active as wild-type GCM2, respectively (P < 0.001; Fig. 5AGo). The relative activities of these two mutants corresponded to the respective level of protein expression (Fig. 5BGo).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
IH is a heterogeneous group of disorders with autosomal dominant, autosomal recessive, and X-linked modes of inheritance. Mutations in the PTH and CASR genes primarily affect PTH secretion, whereas defects in GCM2 are associated with parathyroid dysembryogenesis. The goal of the present study was to analyze a large series of patients with sporadic or familial IH to determine the prevalence of GCM2 mutations. Although all previous GCM2 mutations have been recessive, we included families in which IH was transmitted in an autosomal dominant manner to screen for potential dominant inhibitor mutations.

We first identified the TSS of GCM2, and analysis of the nucleotide sequence upstream of the TSS revealed several classical promoter elements, such as TATA- and GC-boxes, as well as consensus binding sites for the two homeobox transcription factors Pax1 and Pax9, both of which are essential, but not specific, for parathyroid gland development (35, 36). Pax1 and Pax9 typically act in concert to regulate tissue specific genes that induce cell differentiation (37). Importantly, Gcm2 expression is reduced in mouse embryos in which Pax1 or Pax9 has been genetically inactivated, an observation that establishes a direct link between these two transcription factors (35, 36). Thus, the presence of Pax1 and Pax9 binding sites in the GCM2 promoter strongly suggests that the action of the two embryonic transcription factors on parathyroid development is mediated at least in part through transactivation of GCM2.

Our analysis of the DNA sequences of the GCM2 gene in 20 unrelated individuals with IH revealed nine single nucleotide changes. None of the single nucleotide changes that we discovered could fully account for IH because inheritance of hypoparathyroidism did not cosegregate with any of the variant alleles, and/or the allelic variant was also found in unrelated healthy individuals. Interestingly, one of the three 5' UTR single nucleotide changes is a G to C transversion that resides precisely at the TSS. This allele variant is also found in healthy individuals with a high frequency (22%), including homozygous forms that suggest that gene function is not significantly altered. The TSS variant introduces a new restriction site for NlaIII that can be used for linkage studies for the GCM2 locus.

The six exonic single nucleotide changes that we identified were all located in exon 5 and led to amino acid substitutions. Amino acid changes G203S and I227V occur in transactivating domain 1, whereas Y282D, N315D, and Q330L are located in the inhibitory domain. Amino acid substitution M354V is located immediately beyond the inhibitory domain. Functional analysis showed that none of these changes had a significant effect on transcriptional activity. By contrast, the previously reported GCM2 mutations R47L (17) and G63S (2), which occur within the DNA binding domain, were both associated with markedly reduced transactivating activity that could be accounted for in part by the reduced level of expression of each mutant protein. Previous studies had shown that both R47L (17) and G63S (2) possessed markedly reduced transactivating potential, but whereas R47L was unable to bind to the GCM binding site, the G63S mutant had normal DNA binding activity. We therefore propose that the primary molecular mechanism for decreased activity of both of these mutations is reduced intracellular accumulation of the corresponding GCM2 proteins. Importantly, our studies allowed direct comparison of these two mutant proteins and show that the G63S GCM2 protein is expressed at greater levels than the R47L protein and also retains considerably greater transactivating activity (approximately 20% vs. 5% of normal), which could account for the residual secretion of PTH in affected patients who were homozygous for the G63S mutation (2). Although these interactions were tested in a heterologous system that might not fully reproduce the environment of the parathyroid cell (or its precursor), taken in the context of our prior observation that heterozygous patients with one active GCM2 allele (i.e. 50% of normal GCM2 activity) have normal parathyroid function (16), these results suggest that normal development of the parathyroid glands requires greater than 20% of wild-type GCM2 activity.

In conclusion, mutation of the GCM2 gene is an important, albeit uncommon, cause of IH, and mutations that lead to different levels of residual activity of this transcription factor may account for phenotypic variability in the severity of hypoparathyroidism. Moreover, our studies indicate that there is considerable allelic variability for GCM2 within the general population. Although it is currently unknown whether any of these different alleles play a role in modifying prenatal development or postnatal function of the parathyroid glands, several recent studies have suggested that expression of GCM2 is altered in parathyroid adenomas (38, 39). Future studies will be required to address the possible association of these variants with various forms of parathyroid dysfunction.


   Acknowledgments
 
The authors gratefully acknowledge support for this work through grants from the Lawson Wilkins Pediatric Endocrine Society (Lilly research fellowship, to A.M.) and from the Alyssa Center for Cellular and Molecular Endocrinology of Johns Hopkins, and by the Cleveland Clinic Lerner Research Institute.


   Footnotes
 
Disclosure Statement: The authors have nothing to disclose.

First Published Online January 8, 2008

1 * These authors have contributed equally. Back

Abbreviations: IH, Isolated hypoparathyroidism; RACE, rapid amplification of cDNA ends; TSS, transcription start site; UTR, untranslated region.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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M. Mannstadt, G. Bertrand, M. Muresan, G. Weryha, B. Leheup, S. R. Pulusani, B. Grandchamp, H. Juppner, and C. Silve
Dominant-Negative GCMB Mutations Cause an Autosomal Dominant Form of Hypoparathyroidism
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