ESPE Abstracts

Background: Mutations in HESX1, SOX3 responsible for combined pituitary hormone deficiency (CPHD) have been identified in a small number of hypogonadotropic hypogonadism (HH), suggesting that the genetic overlap between CPHD and HH.

Case presentation: A 2-month-old boy was referred because of micropenis (stretched penile length 1.0 cm) with intrascrotal testes (1 ml). Hormone assays revealed very-low plasma testosterone levels (0.06 ng/ml). LHRH stimulating test performed at the age of 3 months revealed LH peak 7.3 mIU/ml, and FSH peak 20.7 mIU/ml, suggesting pre-pubertal response. Plasma concentrations of thyroxine and insulin-like growth factor-1 were within normal ranges. No episode of hypoglycemia was noted. Clinical diagnosis was HH. Brain MRI showed a normal size anterior pituitary with a visible stalk.

Method: Using a next-generation sequencing strategy, we sequenced 9 genes implicated in CPHD, including POU1F1, PROP1, LHX3, HESX1, OTX2, SOX3, SOX2, GLI2, LHX4, and 12 genes implicated in HH, including CHD7, FGFR1, FGF8, GNRH1, GNRHR, KAL1, KISS1, KISS1R, PROK2, PROKR2, TAC3, and TACR3. Transcriptional activity of identified LHX4 variant was evaluated by luciferase reporter assays constructed by inserting the POU1F1 or αGSU promoters. We also performed subcellular localization analyses and EMSA assays.

Results: We identified a novel heterozygous LHX4 sequence variation, c.661C>T (p.R221W). R221W LHX4 had reduced transactivation and no dominant negative effect. Subcellular localization revealed no significant difference between wild-type and mutant LHX4. EMSA experiments showed that the R221W LHX4 abrogated DNA-binding ability.

Conclusion: The R221W was proven to be loss-of function mutation. We showed for the first time that LHX4 mutation is associated with HH, not CPHD. This study extends our understanding of the phenotypic features, molecular mechanism, and developmental course associated with mutations in LHX4.

Funding: This work was supported by a Grant-in-Aid for the Health Science Research Grant for Research on Applying Health Technology (Jitsuyoka (Nanbyo)-Ippan-014 (23300102)) from the Ministry of Health, Labour and Welfare of Japan.