Inactivation of the luteinizing hormone/chorionic gonadotropin receptor by an insertional mutation in Leydig cell hypoplasia.

We previously identified a nonsense mutation (Cys545Stop) in the paternal human LH/CG receptor (hLHR) allele in a family with two 46,XY children afflicted with Leydig cell hypoplasia. This mutation abolished the signal transduction capability of the affected hLHR. We have now examined all coding exons and the transcript of both alleles of the hLHR gene of the affected children. A 33-bp in-frame insertion was found in the maternal hLHR allele. This insertion occurred between nucleotide 54 and 55 and might be the result of a partial gene duplication. Genomic DNA-PCR showed that this defective maternal hLHR allele was inherited by the two affected children. However, examination of the inheritance of the 935-A/G polymorphism of the hLHR by genomic- and RT-PCR indicated that the maternal hLHR allele was not expressed in cultured fibroblasts of the patients. The effect of the in-frame insertion on the biological activity of the hLHR was examined by expressing the mutated hLHR construct, generated by site-directed mutagenesis, in HEK 293 cells. The expression of the mRNA for the mutant hLHR in HEK 293 cells was not affected. Response of cells expressing the mutated hLHR to hCG stimulation was impaired as demonstrated by reduced intracellular cAMP biosynthesis. This change in signal transduction was the result of a profound reduction in hormone binding at the cell surface due to altered expression and processing of the mutated receptor. We conclude that Leydig cell hypoplasia in this family is the result of compound heterozygous loss-of-function mutations of the hLHR gene.

Genetic heterogeneity of adrenocorticotropin (ACTH) resistance syndromes: identification of a novel mutation of the ACTH receptor gene in hereditary glucocorticoid deficiency.

Hereditary primary adrenal insufficiency syndromes due to ACTH resistance include hereditary glucocorticoid deficiency (HGD) and Allgrove's syndrome (AS). Patients with both conditions present in childhood with failure to thrive, weakness, and fatigue or adrenal crisis; patients with AS in addition have alacrima and achalasia (triple A syndrome). We studied four kindreds with HGD and four kindreds with AS for abnormalities of the ACTH receptor (ACTHR) gene. The ACTHR coding sequence in all AS kindreds and two HGD kindreds was normal. Analysis of the ACTHR gene of the proband in one of the HGD kindreds showed him to be homozygous for the previously described G221T transition causing a Ser74Ile substitution of the protein, which has been shown to inactivate the ACTHR in signal transduction. The proband in another HGD kindred was found to be a compound heterozygote with the G221T transition in one allele and a novel C818A transition in the other allele of ACTHR. The C818A transition caused the substitution of the highly conserved Pro273 by His in the receptor protein. In vitro expression of the mutated ACTHR in mouse melanoma M3 cells showed that at a medium ACTH concentration of 3 nM, cells transfected with the wild-type ACTHR produced twofold and threefold, respectively, of the amount of intracellular cAMP when compared to cells transfected with the ACTHR carrying the Pro273His and the Ser74Ile mutation, respectively, confirming that HGD in this kindred is caused by loss-of-function mutations of the ACTHR. These results showed that the genetic cause of the ACTH-resistant syndromes is heterogeneous.