Albright hereditary osteodystrophy (AHO) is an inherited disorder associated with deficient activity of the α-subunit of the guanine nucleotide-binding regulatory protein (Gsα) that couples receptors to adenylyl cyclase. To identify mutations that lead to Gsα deficiency, we isolated genomic DNA from patients with AHO and used the polymerase chain reaction to amplify exons of the Gsα genes. DNA was amplified using intron-specific oligonucleotide primers flanking exons of the Gsα gene. To optimize our ability to detect mutations, one oligonucleotide from each primer pair was synthesized with a 5′ GC-clamp. Amplified Gsα gene fragments were analyzed by denaturing gradient gel electrophoresis in order to detect mutations that alter the melting point of the double-stranded DNA fragment. Using this technique, we have identified and characterized three mutations and one neutral polymorphism. The polymorphism, located in exon 5, consisted of a T → C substitution that conserves the isoleucine residue at codon 131 (ATT → ATC). Two mutations were missense mutations, which in one family consisted of a nucleotide substitution (T → C) in exon 4 that results in replacement of Leu by Pro at codon 99 of the Gsα molecule. Affected subjects in a second family had a single base (C → T) mutation in exon 6 that resulted in replacement of Arg by Cys at codon 165. A 4-base pair deletion (GTGG) in exon 8 at position +214 was identified in one Gsα allele from each affected subject in the third family. This mutation causes a frameshift after the codon for Gln213 that results in a premature stop codon 81 base pair after the deletion. Immunoblot analysis of plasma membranes prepared from cultured fibroblasts or erythrocytes indicated that levels of immunoactive Gsα protein were decreased in all affected subjects. We conclude that heterogeneous mutations in the gene encoding Gsα, including deletions and single amino acid substitutions, are responsible for Gsα deficiency in AHO.
|Original language||English (US)|
|Number of pages||9|
|Journal||Journal of Clinical Endocrinology and Metabolism|
|State||Published - Jun 1993|
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism