Gcm2 is a transcription factor that is crucial for parathyroid gland homeostasis (16
). In transgenic mice, loss of Gcm2 results in absence of the parathyroids, and in humans spontaneous mutations of GCM2
are associated with congenital hypoparathyroidism (17
). We report a novel point mutation in exon 3 of the GCM2
gene that is associated with a null-protein defect and severe early onset hypoparathyroidism. The proband presented with tetany and a hypocalcemic seizure at 7 days of age. She has, thus far, had normal developmental and cognitive milestones throughout early childhood on calcium and calcitriol supplementation. Her two affected siblings were also ascertained within the first few days of life, and have similarly done well with calcitriol therapy. The three affected subjects had undetectable levels of serum intact PTH, which differs from reports of low serum levels of PTH in previously reported patients who carried small mutations that lead to Gcm2 variants with reduced function or that mislocalize within the cell (8
). Thus, it is conceivable that residual Gcm2 action allows development of small amounts of parathyroid tissue that can secrete detectable but physiologically insignificant amounts of PTH into the circulation.
The c.408C>A null-protein mutation follows a predictable autosomal recessive mode of inheritance with three siblings who each have a homozygous GCM2
mutation and two heterozygous, non-consanguineous parents. The parents are both of Guatemalan ethnicity, which is similar to the first reported human cases of isolated hypoparathyroidism caused by a different, large intragenic deletion of GCM2
). In both the previous family and the family reported here, molecular analysis demonstrated that the mutant alleles shared similar haplotypes and thus likely arose within common founders. By contrast, two other GCM2
mutations – the R47L missense (9
) and the I298fsX307 frameshift (11
) – have each been identified in three unrelated kindreds, and thus may represent potential mutational ‘hotspots’.
Mutational hotspots are often associated with repetitive sequences, such as homonucleotide runs, direct and inverted repeats and microsatellite repeats (18
). For these mutation hotspots, the exact DNA sequence is not critical but only the fact that a sequence motif is repeated. Alternatively, mutation hotspots can depend on nucleotide sequence context (mutable motifs, subsequences) nucleotide substitutions or deletions within homonucleotide repeats, or during the repair of mismatches caused by heteroduplex formation between imperfect direct repeats (18
). Although our haplotype analysis suggests identity by descent for the c.408C>A mutation that we have identified, this substitution occurs within a four cytosine homonucleotide repeat, and it is conceivable that the C to A transversion could have arisen independently.
We expressed the c.408C>A Gcm2 cDNA in mammalian cells to assess the biochemical consequences of the mutation. The mutation introduces a premature stop codon (p.Try136Ter) within the DNA-binding domain or GCM domain that is proximal to the nuclear localization signal and transactivation domain 1 () (9
GCM2 is a member of a small family of GCM transcription factors that are present in vertebrates and some invertebrates (11
), and which share a conserved zinc-coordinating DNA-binding domain (20
) that binds to the consensus motif 5-ATGCGGGT-3 (21
). Mutations in the GCM domain are predicted to cause structurally significant effects that have been shown to interfere with DNA binding or transactivational activity (10
To date, previously described small mutations in GCM2 have been described in exons 2 or 5 and have disrupted or eliminated sequences required for DNA binding, nuclear localization or transactivation. Accordingly, mutations in exon 2 have been associated with hypomorphic alleles and recessive hypoparathyroidism, while mutations in exon 5 have been associated with inhibitory function and dominant hypoparathyroidism ().
Theoretically, the p.Tyr136Ter mutation could lead to nonsense-mediated decay of transcripts, a truncated protein that lacks the nuclear localization signal as well as the transcriptional activation domains, or an unstable protein that is rapidly degraded. Biochemical studies performed in our laboratory showed that Gcm2 transcripts but not protein were expressed in HEK293T cells that had been transfected with the Gcm2 p.Try136Ter cDNA.
Hence, we suggest that severe hypoparathyroidism occurred in the three affected subjects we studied because the premature termination reduced stability of the Gcm2 protein, further enhancing the rapid rate of degradation (23
) and leading to a null-protein phenotype.
In conclusion, this is the first report of a mutation in exon 3 of the GCM2 gene and a mutation that leads to a null-protein phenotype. All three affected children presented with profound hypocalcemia and hyperphosphatemia during the neonatal period, and intact PTH was undetectable in the circulation. These results increase our understanding of the spectrum of GCM2 mutations that cause autosomal recessive isolated hypoparathyroidism and suggest that minimal GCM2 activity may allow development of sufficient parathyroid tissue to account for small amounts of circulating PTH.