Biochemical Analysis of Four Missense Mutations in the HSD17B3 Gene Associated With 46,XY Disorders of Sex Development in Egyptian Patients

Background: Mutations in the HSD17B3 gene are associated with a 46, XY disorder of sexual development (46, XY DSD) as a result of low testosterone production during embryogenesis. Aim: To elucidate the molecular basis of the disorder by chemically analyzing four missense mutations in HSD17B3 (T54A, M164T, L194P, G289S) from Egyptian patients with 46, XY DSD. Methods: Expression plasmids for wild-type 17 beta-hydroxysteroid hydrogenase type 3 (17 beta-HSD3) and mutant enzymes generated by site-directed mutagenesis were transiently transfected into human HEK-293 cells. Protein expression was verified by western blotting and activity was determined by measuring the conversion of radiolabeled Delta(4)-androstene-3,17-dione to testosterone. Application of a homology model provided an explanation for the observed effects of the mutations. Outcomes: Testosterone formation by wild-type and mutant 17 beta-HSD3 enzymes was compared. Results: Mutations T54A and L194P, despite normal protein expression, completely abolished 17 beta-HSD3activity, explaining their severe 46, XY DSD phenotype. Mutant M164T could still produce testosterone, albeit with significantly lower activity compared with wild-type 17 beta-HSD3, resulting in ambiguous genitalia or a microphallus at birth. The substitution G289S represented a polymorphism exhibiting comparable activity to wild-type 17 beta-HSD3. Sequencing of the SRD5A2 gene in three siblings bearing the HSD17B3 G289S polymorphism disclosed the homozygous Y91Hmutation in the former gene, thus explaining the 46, XY DSD presentations. Molecular modeling analyses supported the biochemical observations and predicted a disruption of cofactor binding by mutations T54A and M164T and of substrate binding by L196P, resulting in the loss of enzyme activity. In contrast, the G289S substitution was predicted to disturb neither the three-dimensional structure nor enzyme activity. Clinical Translation: Biochemical analysis of mutant 17 beta-HSD3 enzymes is necessary to understand genotype-phenotype relationships. Strengths and Limitations: Biochemical analysis combined with molecular modeling provides insight into disease mechanism. However, the stability of mutant proteins in vivo cannot be predicted by this approach. Conclusion: The 17 beta-HSD3 G289S substitution, previously reported in other patients with 46, XY DSD, is a polymorphism that does not cause the disorder; thus, further sequence analysis was required and disclosed a mutation in SRD5A2, explaining the cause of 46, XY DSD in these patients. Copyright (C) 2017, International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved.