17β-Hydroxysteroid dehydrogenase type 3 (encoded by HSD17B3) catalyses the conversion of Δ4-androstene-3.17-dione to testosterone and has a key role in male sexual development. Mutations in the HSD17B3 gene can result in reduced enzyme activity and decreased testosterone synthesis, leading to a rare autosomal recessive aetiology of 46, XY Disorders of Sex Development (46, XY DSD) named 17β-HSD3 deficiency. Here, we characterised three Tunisian patients from non-consanguineous families with a 46, XY karyotype and impaired virilisation of the external genitalia. The hormonal analysis and family history pointed to 17β-HSD3 deficiency. Genetic analysis revealed novel compound heterozygous mutations, i.e. a missense mutation (G133R) and a premature stop codon (C206X) in the HSD17B3 gene. Using site-directed mutagenesis, an expression plasmid for 17β-HSD3 G133R was constructed. Additionally, expression plasmids for substitutions of glycine 133 to alanine (G133A), to phenylalanine (G133F), and to glutamine (G133Q) were created. WT and mutant enzymes were expressed in HEK-293 cells, followed by assessment of the conversion of radiolabeled Δ4-androstene-3.17-dione to testosterone. Mutants G133R, G133Q and G133F were almost completely inactive, whereas G133A retained more than 80% of WT activity. A homology model of 17β-HSD3 predicted that the loss of activity is due to a disruption of the cofactor-binding site. While an alanine at position 133 was still tolerated, more bulky side-chains led to steric hindrance thus preventing cofactor binding. The glycine at position 133 is highly conserved among the short-chain dehydrogenases. The functional analysis and homology modelling revealed an important role of this residue in the structural arrangement of the cofactor binding pocket. The results provide an improved mechanistic understanding of the 17β-HSD3 structure-function relationship and explained the 17β-HSD3 deficiency observed in the patients.
Disclosure: Swiss National Science Foundation 31003A_140961.
16 - 20 May 2015
European Society of Endocrinology