Gonadotropin-independent precocious puberty due to luteinizing hormone receptor mutations in Brazilian boys: A novel constitutively activating mutation in the first transmembrane helix

Naturally occurring activating mutations in the human LH receptor (hLHR) ge ne are the cause of sporadic or familial male gonadotropin-independent prec ocious puberty. We have previously reported three different activating muta tions of the hLHR gene in four unrelated Brazilian boys with male-limited p recocious puberty. In the current study, we examined three other Brazilian boys, two brothers and one unrelated boy, with gonadotropin-independent pre cocious puberty. Direct sequencing of the entire exon 11 of the hLHR gene i n the two brothers revealed a heterozygous substitution of T for C at nucle otide 1103, resulting in the substitution of leucine at position 368 by pro line in the first transmembrane helix. Their mother carried the same mutati on, establishing the familial nature of this mutation. Human embryonic 293 cells expressing hLHR(L368P) bound hCG with the same high affinity as cells expressing the wild-type hLHR. Cells expressing the novel L368P mutation d isplayed up to a 12-fold increase in basal cAMP production compared with ce lls expressing the same number of cell surface wild-type hLHR, indicating c onstitutive activation of the mutant receptor. In addition, the cAMP levels in cells expressing the hLHR mutant were further augmented by hCG. Molecul ar dynamics simulations suggest that substitution of L368 of the hLHR by pr oline results in lack of a salt bridge interaction between D405 and R464 (d istance 9.0 Angstrom vs. 4.7 Angstrom in wild-type hLHR) as well as by the opening of a crevice between the second and third intracellular loops, whic h may allow G proteins greater accessibility. These structural features wer e shared by other activating mutants of the hLHR. Sequencing of exon 11 of the hLHR gene of the unrelated boy revealed that h e carried a homozygous nucleotide substitution causing an A568V mutation in the third cytoplasmic loop of the receptor. This mutation was previously f ound in two unrelated Brazilian boys, but in heterozygous state. Clinical a nd hormonal data of the patient with the homozygous A568V were not differen t from those individuals with the Ala568Val mutation in a heterozygous stat e. Furthermore, the phenotype caused by dominant activating mutations of th e hLHR gene are not altered when both alleles carry a mutant sequence. Our studies show that the A568V is the most frequent cause of male-limited prec ocious puberty in Brazilian boys. Lastly, the identification of a novel act ivating L368P mutation in the first transmembrane helix of two Brazilian bo ys with familial male-limited precocious puberty provides further insights into the mechanism of activation of the hLHR.