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) gene are the cause of sporadic or familial male gonadotropin-independent precocious puberty. We have previously reported three different activating mutations of the hLHR gene in four unrelated Brazilian boys with male-limited precocious puberty. In the current study, we examined three other Brazilian boys, two brothers and one unrelated boy, with gonadotropin-independent precocious puberty. Direct sequencing of the entire exon 11 of the hLHR gene in the two brothers revealed a heterozygous substitution of T for C at nucleotide 1103, resulting in the substitution of leucine at position 368 by proline in the first transmembrane helix. Their mother carried the same mutation, 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 displayed up to a 12-fold increase in basal cAMP production compared with cells expressing the same number of cell surface wild-type hLHR, indicating constitutive activation of the mutant receptor. In addition, the cAMP levels in cells expressing the hLHR mutant were further augmented by hCG. Molecular dynamics simulations suggest that substitution of L368 of the hLHR by proline results in lack of a salt bridge interaction between D405 and R464 (distance 9. 0 A vs. 4.7 A in wild-type hLHR) as well as by the opening of a crevice between the second and third intracellular loops, which may allow G proteins greater accessibility. These structural features were shared by other activating mutants of the hLHR. Sequencing of exon 11 of the hLHR gene of the unrelated boy revealed that he carried a homozygous nucleotide substitution causing an A568V mutation in the third cytoplasmic loop of the receptor. This mutation was previously found in two unrelated Brazilian boys, but in heterozygous state. Clinical and hormonal data of the patient with the homozygous A568V were not different from those individuals with the Ala568Val mutation in a heterozygous state. Furthermore, the phenotype caused by dominant activating mutations of the 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 precocious puberty in Brazilian boys. Lastly, the identification of a novel activating L368P mutation in the first transmembrane helix of two Brazilian boys with familial male-limited precocious puberty provides further insights into the mechanism of activation of the hLHR.