Endocrine Abstracts

Glial cells missing B (GCMB), which is the mammalian homologue of the Drosophila GCM gene, encodes a 506 amino acid parathyroid-specific transcription factor that contains: a DNA-binding domain (residues 21–174); a predicted nuclear localization signal (residues 176–193); an inhibitory domain (residues 258–347); and two transactivation domains (residues 174–263, and residues 428–506). To date only two different GCMB mutations have been reported in three kindreds with autosomal dominant hypoparathyroidism (AD–HPT). We therefore investigated an additional family with AD–HPT for GCMB mutations, after obtaining informed consent, using guidelines approved by the national ethical committee. Leukocyte DNA was used with GCMB-specific primers for PCR amplification of the five exons. DNA sequence analysis of the PCR products revealed an A to C transversion at codon 502, which altered the wild-type asparagine (Asn) to histidine (His). The DNA sequence abnormality was demonstrated to be absent in 110 alleles of 55 unrelated normal individuals, thereby demonstrating that it is not a common polymorphism. Functional studies, utilizing transient transfections of COS7 cells with GCMB wild-type and mutant (Asn502His) tagged constructs, demonstrated that: the wild-type and mutant GCMB Asn502His proteins localized to the nucleus; and, electrophoretic mobility shift assays (EMSAs) showed that the mutant protein retained the ability to bind the GCM-consensus DNA recognition motif. However, a luciferase-reporter assay, demonstrated that the GCMB Asn502His mutation resulted in a reduction in gene transactivation. Moreover, co-transfection of the wild-type with mutant GCMB Asn502His did not lead to an increase in luciferase activity, thereby demonstrating a dominant-negative effect of the Asn502His mutant which would be consistent with its autosomal dominant inheritance. Thus, our results, which have identified the first dominant missense GCMB mutation, help to increase our understanding of the mechanism underlying gene transactivation, that is a prerequisite for the function of this parathyroid gland-specific transcription factor.