SUN-159 A Novel Splice-site Mutation In Lhcgr Causing Leydig Cell Hypoplasia In 46,xy Sisters: Clinical, Molecular And In Silico Analysis

Abstract Disclosure: M.G. Meirelles: None. R.D. Scalco: None. J.M. Moreira: None. S. Domenice: None. R.L. Batista: None. J. Mesia: None. N. Scalissi: None. J.P. Batatinha: None. J.V. Junior: None. C.B. Bueno: None. A. Narciso: None. A. Reis: None. A. Latronico: None. I.J. Arnhold: None. B.B. Mendonca: None. The luteinizing hormone/choriogonadotropin receptor (LHCGR) is essential for gonadal steroidogenesis and sexual differentiation. Loss-of-function variants in LHCGR lead to a spectrum of 46,XY disorders of sex development (DSD), with phenotypic variability depending on chromosomal sex and mutation type. We report two 46,XY sisters, from a consanguineous family, presenting with primary amenorrhea and absent secondary sexual characteristics. Both, aged 31 and 27, showed tall stature (184 cm and 188 cm; target height: 173 cm), and the elder sister also had obesity and metabolic comorbidities. Hormonal evaluation showed elevated gonadotropins (LH: 49–60 IU/L; FSH: 46–50.7 IU/L), low testosterone (<10 ng/dL), and undetectable estradiol (<17 pg/mL). Pelvic ultrasound and MRI confirmed absence of uterus and presence of bilateral intra-abdominal testes (3.5–4.0 cm AP). Histopathological analysis revealed Leydig cell hypoplasia and severe seminiferous tubule hyalinization. Genetic testing identified a novel homozygous LHCGR splice-site variant (c.384-2A>G; NM_000233.4), classified as pathogenic by ACMG guidelines (PM2, PVS1, PP4). In silico prediction tools indicated complete loss of the canonical AG acceptor site at intron 4. SpliceAI showed high probability of splice site loss (Δ score: 0.99), and MaxEntScan indicated a collapse in splice strength, suggesting exon skipping or intron retention. RNA secondary structure modeling (ViennaRNA Fold) revealed reduced thermodynamic stability (ΔG: –30.05 vs. –32.62 kcal/mol), lower frequency of the minimum free energy structure (0.60% vs. 1.96%), and increased structural diversity (35.3 vs. 23), supporting impaired splicing efficiency. This variant likely disrupts LHCGR expression through aberrant splicing and RNA instability, leading to Leydig cell hypoplasia and testosterone deficiency. The disproportionately high FSH in subject with LHCGR loss-of-function longstanding Sertoli cell damage due to undescended testes. These findings expand the mutational and phenotypic spectrum of LHCGR-related DSD and emphasize the relevance of considering LHCGR defects in 46,XY individuals with hypergonadotropic hypogonadism and female phenotype. Keywords:LHCGR variant, Leydig cell hypoplasia, 46,XY DSD, hypergonadotropic hypogonadism, LH resistance. Presentation: Sunday, July 13, 2025