Genomic DNA from 32 unrelated families with male-limited precocious puberty was examined for the previously described Asp-578 --Gly,

Leydig cell hypoplasia (LCH) is a form of male pseudohermaphroditism in which Leydig cell differentiation and testosterone production are impaired. This report describes the first case of a nonsense mutation (A1635C) in exon 11 of the human luteinizing hormone receptor (hLHR) gene in two sisters with LCH. This mutation causes loss of function of the receptor by introducing a stop codon at residue 545 in transmembrane helix 5 of the hLHR. Surface expression of the truncated hLHR (hLHR-t545) in human embryonic kidney cells stably transfected with cDNA encoding hLHR-t545 was diminished compared to the wild-type hLHR and hCG-induced cAMP accumulation was impaired. These results establish that single base mutations in exon 11 of the hLHR gene can produce inactivation as well as activation of the hLHR. Furthermore, they demonstrate that functional domains between transmembrane helix 5 and the C-terminal cytoplasmic tail of the hLHR are required for normal cell surface expression of the receptor and signal transduction.

The human LH receptor (hLHR) is a member of the G protein-coupled receptors characterized by the presence of seven-transmembrane (TM) helices. Inactivating mutations of the hLHR lead to Leydig cell hypoplasia (LCH), a form of male pseudohermaphroditism resulting from the failure of fetal testicular Leydig cell differentiation. We have identified three mutations of the hLHR in a patient with LCH: deletion of exon 8 (delta Exon 8), A872G transition resulting in Asn291Ser substitution in the extracellular domain, and C1847A transversion resulting in Ser616Tyr substitution in the seventh TM helix. Nucleotide sequencing, gene dosage, and allele-specific amplification analyses revealed that exon 8 deletion and the two missense mutations are present in different alleles of the hLHR. Constructs of mutated hLHR (hLHR-delta Exon8, hLHR-872/1847, hLHR-1847, and hLHR-872) were used to transfect 293 cells, and the properties of the hLHR expressed were examined. Ligand-binding assays failed to detect the expression of hLHR-delta Exon8. Transfectants expressing hLHR-872/1847 demonstrated greatly reduced ligand binding and ligand-induced cAMP accumulation in comparison to those expressing wild type hLHR. Similar reduction in cAMP accumulation was observed in transfectants expressing hLHR-1847, but not hLHR-872 alone. These findings suggest that, in addition to the 7-TM helices, the polypeptide encoded by exon 8 plays an important role in LHR expression and signal transduction. On the other hand, glycosylation of Asn291 may not be critical for these activities. These results also establish that LCH can result from impaired signal transduction due to compound heterozygous mutations. Implications of these mutations on structure-function relationship of the hLHR and the genotype-phenotype correlation in LCH are discussed.

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Computational analyses have identified the widespread occurrence of antisense transcripts in the human and the mouse genome. However, the structure and the origin of the majority of the antisense transcripts are unknown. The presence of antisense transcripts for 19 of 64 differentially expressed genes during mouse spermatogenesis was demonstrated with orientation-specific RT-PCR. These antisense transcripts were derived from a wide variety of origins, including processed sense transcripts, intronic and exonic sequences of a single gene or multiple genes, intergenic sequences, and pseudogenes. They underwent normal and alternative splicing, 5' capping, and 3' polyadenylation, similar to the sense transcripts. There were also antisense transcripts that were not capped and/or polyadenylated. The testicular levels of the sense transcripts were higher than those of the antisense transcripts in all cases, while the relative expression in nontesticular tissues was variable. Thus antisense transcripts have complex origins and structures and the sense and antisense transcripts can be regulated independently.

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.

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