Introduction: 3--hydroxysteroid dehydrogenase (3-HSD) deficiency is a rare cause of congenital adrenal hyperplasia caused by inactivating mutations in the HSD3B2 gene. Most mutations are located within domains regarded crucial for enzyme function. The function of the c-terminus of the 3-HSD protein is not known.

Objective: We studied the functional consequences of three novel c-terminal mutations in the 3-HSD protein (p.P341L, p.R335X and p.W355X), detected in unrelated 46,XY neonates with classical 3-HSD type II deficiency showing different degrees of under-virilization.

Methods and Results: In vitro expression of the two truncated mutant proteins yielded absent conversion of pregnenolone and dehydroepiandrosterone (DHEA), whereas the missense mutation p.P341L showed a residual DHEA conversion of 6% of wild-type activity. Additional analysis of p.P341L, including three dimensional protein modeling, revealed that the mutant's inactivity predominantly originates from a putative structural alteration of the 3-HSD protein and is further aggravated by increased protein degradation. The stop mutations cause truncated proteins missing the final G-helix what abolishes enzymatic activity irrespective of an augmented protein degradation. Genital appearance did not correlate with the mutants' residual in vitro activity.

Conclusions: Three novel c-terminal mutants of the HSD3B2 gene are responsible for classical 3-HSD deficiency. The c-terminus is essential for the enzymatic activity. However, further studies are needed to clarify the exact function of this part of the protein. Our results indicate that the genital phenotype in 3-HSD deficiency cannot be predicted from in vitro 3-HSD function alone.