ESPE Abstracts

Background: An activation cascade of specific genes sets up the initiation of sex determination leading in males to testes formation and synthesis of testicular hormones. Disruption of this gene cascade may cause a spectrum of 46,XY disorders/differences of sex development (DSD) phenotypes. Here we describe for the first time two sisters suffering from 46,XY DSD, who by whole exome sequencing revealed to carry a X-linked mutation in the StAR-related lipid transfer domain protein 8 (STARD8) gene. STARD8, also known as deleted in liver cancer 3 (DLC-3) is a functional Rho-specific GAP protein, the loss of which enhances perinuclear Ras homolog gene family member A (RhoA) activity. Simultaneously, RhoA is known to play a role downregulating the expression of SOX9 and, thus, inducing the female sexual development pathway. On the other hand, available literature also linked STARD8 with the targeting of focal adhesions and the stimulation of the enzymatic activity of Phospholipase C δ1 (PLCδ1), which is known to lower the levels of active-β-catenin. Moreover, β-catenin has been identified as a key pro-ovarian and anti-testis signaling molecule.

Objectives: To gain new insights in human sex development mechanisms, we aimed to analyse the functional consequences of STARD8 mutations as a putative novel 46,XY DSD- related gene by testing the RhoA phosphorylation-dependent SOX9 expression. Since the STARD8 knockout NMRI mouse model we generated did not recapitulate the human clinical picture, we chose to test the mutation in a cell system.

Methods: COS1 cells were transfected with wild type and mutant STARD8. Quantification of activated RhoA (RhoA-GTP) levels in cell lysates was performed by a pull-down assay that specifically binds the active form of RhoA. Subsequently, the RhoA-GTP, total RhoA, SOX9 and STARD8 protein expression levels were analyzed by western blot. Quantitative RT-PCR expression profiling of STARD8, SOX9, and PLCD1 was also performed.

Results: Preliminary results showed an increase in activated RhoA levels and consequent reduced SOX9 protein expression levels with the STARD8 mutant when compared to the wild type. Also, an increase in the STARD8 protein expression levels was observed in the mutant when compared to the wild type.

Conclusions: The mentioned results suggest a STARD8-dependent Sox9 expression mediated by RhoA phosphorylation that could be triggering sex reversal in the 2 affected patients. Therefore, STARD8 resembles a strong novel candidate gene for 46,XY DSD which might have an important role in sexual differentiation.