Abstract 22

GnRH-independent isosexual precocious puberty in girls may be caused by estrogen secreting ovarian cyst or tumor, by adrenal neoplasm or by inadvertent exposure to estrogen. Autonomous ovarian follicular cysts are the most common childhood estrogen ovarian condition. Because FSH is the major hormone controlling folliculogenesis in females, we hypothesized that activating mutations of the FSH receptor gene (FSHR) could be involved in the development of autonomous ovarian follicular cysts in girls with precocious puberty. In the present study, we investigated 5 girls with gonadotropin-independent precocious puberty for abnormalities in FSHR gene. All patients had signs of secondary sexual maturation before age 8. prepubertal basal and stimulated LH and FSH levels, multiple ovarian cysts in pelvic ultrasound and advanced bone age. Clinical features of McCune-Albright syndrome, such as cafe au lait spots and abnormal bone surveys were absent in all 5 patients. Genomic DNA was extracted from peripheral blood. The entire exon 10 of FSHR gene, which encodes the seven transmembrane and the intracellular domains of FSHR, was amplified by PCR using 4 pairs of oligonucleotide primers, generating small overlapping fragments. The PCR products were screened for mutations by denaturing gradient gel electrophoresis (DGGE) and sequenced using nucleotide chain termination method. DGGE analysis of PCR fragments revealed heteroduplex formation in fragment A and fragment C of exon 10. Direct sequencing demonstrated a heterozygous substitution of G for A at nucleotide position 919 in three and one patient, respectively. Direct sequencing revealed a heterozygous or homozygous substitution of G for A at nucleotide position 2039 in four and one patient, respectively. The point mutation at position 919 determined the substitution of threonine for alanine at amino-acid position 307 in the amino-terminal region of the FSHR. The point mutation at position 2039 determined the substitution of asparagine for serine at amino-acid position 680 in the carboxyterminal domain of the FSHR. We analyzed these two mutations in 42 unrelated normal controls. Both mutations, Ala307Thr and Ser680Asn were identified a heterozygous in 57% and as homozygous in 31% of the normal population. In conclusion, FSHR gene is polymorphic and germline activating mutations are not found in girls with gonadotropin-independent isosexual precocious puberty. Other studies will be required to exclude the presence of somatic mutations in FSHR of affected ovarian cells in autonomous ovarian follicular cysts.