In humans, androgen synthesis crucially depends on the enzyme CYP17A1 expressed in adrenals and gonads. The 17,20 lyase activity of CYP17A1 catalyses the key step in human androgen biosynthesis, the conversion of 17-hydroxypregnenolone to the universal sex steroid precursor dehydroepiandrosterone (DHEA). For its catalytic activity, CYP17A1 requires electron transfer from P450 oxidoreductase (POR). Mutations in CYP17A1 and POR are known to disrupt human androgen synthesis and causing 46,XY DSD. Cytochrome B5 (CYB5) is thought to enhance allosteric interaction of CYP17A1 and POR proteins required for normal 17,20 lyase function.
We have identified a disease-causing missense CYB5 mutation in a large consanguineous family including three children with 46,XY DSD. The index case, a 46,XY female presented with lack of pubertal development at 13 years of age. She was fully pre-pubertal and her genitalia appeared predominantly female except for mild clitoromegaly; the gonads were not palpable. Serum DHEAS was undetectable; urinary steroid profiling revealed low or undetectable androgen metabolites with elevated excretion of 17-hydroxypregnenolone, indicative of isolated 17,20 lyase deficiency. No mutations were found in CYP17A1 nor POR. However, we detected a homozygous CYB5 missense mutation (g.2510A>T) replacing highly conserved histidine with leucine at amino acid position 44 (p.H44L). Segregation analysis revealed parental heterozygosity for p.H44L and homozygosity for p.H44L in two younger brothers who presented perineal hypospadias and bifid scrotum. We performed functional in vitro characterisation by co-expressing wild-type and mutant CYB5 with CYP17A1 in HEK293 cells. Kinetic analysis revealed that p.H44L CYB5 decreases 17,20 lyase activity of CYP17A1 to 38% of wild-type catalytic activity.
We have identified the first human CYB5 missense mutation, a novel co-factor mutation causing androgen deficiency. The considerable phenotypic variability in the degree of male undermasculinisation indicates variable penetrance, e.g. due to genetic differences in other determinants of 17,20 lyase activity such as CYP17A1 and POR.