CHARACTERIZATION OF HETEROGENEOUS MUTATIONS CAUSING CONSTITUTIVE ACTIVATION OF THE LUTEINIZING-HORMONE RECEPTOR IN FAMILIAL MALE PRECOCIOUS PUBERTY

Familial male precocious puberty (FMPP) is a gonadotropin-independent disorder that is inherited in an autosomal dominant, male-limited pattern. A heterozygous mutation encoding substitution of Asp(578) with Gly in transmembrane helix 6 of the G protein-coupled receptor for luteinizing hormone (LHR) has been found in affected males from nine American FMPP families. Cells expressing the mutant LHR exhibit markedly increased cyclic adenosine monophosphate (cAMP) production in the absence of agonist, suggesting that autonomous Leydig cell activity in FMPP is caused by a constitutively activated LHR. We have now analyzed genomic DNA from affected males from six additional FMPP families. PCR was used to amplify a fragment of the LHR gene encoding amino acid residues 441-594. None of the six new samples contained the Asp(578)-->Gly mutation, as indicated by absence of digestion with Mspl. PCR products were then screened for heterozygous mutations using temperature-grad lent gel electrophoresis. DNA fragments from two of the patients migrated abnormally. Direct sequencing of PCR product from one affected German male revealed a heterozygous mutation (ATG-->ATA) encoding Met(571)-->Ile at the cytoplasmic end of helix 6, the same mutation that has been reported in another European FMPP kindred. Affected males in the second family had a novel Thr(577)-->Ile mutation (ACC-->ATC). Mutations in different portions of the LHR or in a different gene may be responsible for disease in the other FMPP kindreds. Agonist binding and functional coupling of the mutant receptors to the cAMP and inositol phosphate pathways were studied by transiently expressing them in COS-7 cells. Agonist affinity was unaffected by the mutations. Like the Asp(578)-->Gly mutant receptor, the two newly identified mutant receptors triggered agonist-independent production of cAMP, but not of inositol phosphates, suggesting that autonomous testosterone production in FMPP can be explained by constitutive activation of the cAMP pathway alone.