IDENTIFICATION BY MOLECULAR-CLONING OF AN AUTOANTIGEN ASSOCIATED WITHADDISONS-DISEASE AS STEROID 17-ALPHA-HYDROXYLASE

Idiopathic Addison's disease is characterised by a progressive failure in the synthesis of all classes of steroid hormones and by an immune response against the steroid-producing cells of the adrenal cortex; th e nature of the adrenal autoantigens is not known. We have used molecu lar cloning and sequencing to identify the target antigens. We screene d a human fetal adrenal cDNA expression library in lambda-gt11 vector with serum samples from patients with Addison's disease as part of the type 1 polyendocrine autoimmunity syndrome. Samples from 3 patients, which had precipitating antibodies against two adrenal proteins detect ed by immunodiffusion and against five adrenal proteins of molecular m ass 55, 48, 43, 39, and 19 kDa as judged by immunoblotting, were used to identify 60 immunoreactive clones 39 of these were subcloned, inser ted into the M13mp10 vector, and sequenced by the dideoxy method or id entified by Southern and dot-blot hybridisation. All but 1 of the inse rts showed more than 98.8% homology with the published sequence of ste roid 17-alpha-hydroxylase. This protein was expressed by insertion of 1 of the clones into the pGEMEX-1 vector. Only serum from patients wit h Addison's disease and type 1 polyendocrine autoimmunity syndrome tha t reacted with the 55 kDa adrenal protein recognised the recombinant 1 7-alpha-hydroxylase protein on immunoblotting. Our results show that o ne of the key enzymes in steroid biosynthesis, 17-alpha-hydroxylase, i s an autoantigen involved in the pathogenesis of adrenocortical failur e.