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

Familial male precocious puberty (FMPP) is a gonadotropin-independent disorder that is inherited in an autosomal dominant, male-limited patt ern. A heterozygous mutation encoding substitution of Asp(578) with Gl y in transmembrane helix 6 of the G protein-coupled receptor for lutei nizing hormone (LHR) has been found in affected males from nine Americ an FMPP families. Cells expressing the mutant LHR exhibit markedly inc reased cyclic adenosine monophosphate (cAMP) production in the absence of agonist, suggesting that autonomous Leydig cell activity in FMPP i s caused by a constitutively activated LHR. We have now analyzed genom ic DNA from affected males from six additional FMPP families. PCR was used to amplify a fragment of the LHR gene encoding amino acid residue s 441-594. None of the six new samples contained the Asp(578)-->Gly mu tation, as indicated by absence of digestion with Mspl. PCR products w ere then screened for heterozygous mutations using temperature-grad le nt gel electrophoresis. DNA fragments from two of the patients migrate d abnormally. Direct sequencing of PCR product from one affected Germa n male revealed a heterozygous mutation (ATG-->ATA) encoding Met(571)- ->Ile at the cytoplasmic end of helix 6, the same mutation that has be en reported in another European FMPP kindred. Affected males in the se cond family had a novel Thr(577)-->Ile mutation (ACC-->ATC). Mutations in different portions of the LHR or in a different gene may be respon sible for disease in the other FMPP kindreds. Agonist binding and func tional coupling of the mutant receptors to the cAMP and inositol phosp hate pathways were studied by transiently expressing them in COS-7 cel ls. Agonist affinity was unaffected by the mutations. Like the Asp(578 )-->Gly mutant receptor, the two newly identified mutant receptors tri ggered agonist-independent production of cAMP, but not of inositol pho sphates, suggesting that autonomous testosterone production in FMPP ca n be explained by constitutive activation of the cAMP pathway alone.