Heterogeneous mutations in the gene encoding the 1 α-subunit of the stimulatory G protein of adenylyl cyclase in albright hereditary osteodystrophy

Albright hereditary osteodystrophy (AHO) is an inherited disorder associated with deficient activity of the α-subunit of the guanine nucleotide-binding regulatory protein (G_sα) that couples receptors to adenylyl cyclase. To identify mutations that lead to G_sα deficiency, we isolated genomic DNA from patients with AHO and used the polymerase chain reaction to amplify exons of the G_sα genes. DNA was amplified using intron-specific oligonucleotide primers flanking exons of the G_sα gene. To optimize our ability to detect mutations, one oligonucleotide from each primer pair was synthesized with a 5′ GC-clamp. Amplified G_sα 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 G_sα 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 G_sα 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 G_sα protein were decreased in all affected subjects. We conclude that heterogeneous mutations in the gene encoding G_sα, including deletions and single amino acid substitutions, are responsible for G_sα deficiency in AHO.