Petr Rubtsov1, Alexander Nizhnik2, Ivan Dedov2, Natalia Kalinchenko2, Vasily Petrov2, Anna Orekhova1, Pavel Spirin2, Vladimir Prassolov1, Anatoly Tiulpakov3. 1. Department and Laboratory of Inherited Endocrine DisordersEndocrinology Research Centre, Ulitsa Dmitriya Ulianova, 11, Moscow 117036, Russian FederationEngelhardt Institute of Molecular BiologyMoscow 119991, Russian FederationInstitute of Physics and TechnologyMoscow Region 141700, Russian FederationInstitute of General Pathology and PathophysiologyMoscow 125315, Russian Federation Department and Laboratory of Inherited Endocrine DisordersEndocrinology Research Centre, Ulitsa Dmitriya Ulianova, 11, Moscow 117036, Russian FederationEngelhardt Institute of Molecular BiologyMoscow 119991, Russian FederationInstitute of Physics and TechnologyMoscow Region 141700, Russian FederationInstitute of General Pathology and PathophysiologyMoscow 125315, Russian Federation. 2. Department and Laboratory of Inherited Endocrine DisordersEndocrinology Research Centre, Ulitsa Dmitriya Ulianova, 11, Moscow 117036, Russian FederationEngelhardt Institute of Molecular BiologyMoscow 119991, Russian FederationInstitute of Physics and TechnologyMoscow Region 141700, Russian FederationInstitute of General Pathology and PathophysiologyMoscow 125315, Russian Federation. 3. Department and Laboratory of Inherited Endocrine DisordersEndocrinology Research Centre, Ulitsa Dmitriya Ulianova, 11, Moscow 117036, Russian FederationEngelhardt Institute of Molecular BiologyMoscow 119991, Russian FederationInstitute of Physics and TechnologyMoscow Region 141700, Russian FederationInstitute of General Pathology and PathophysiologyMoscow 125315, Russian Federation ant@endocrincentr.ru.
Abstract
BACKGROUND: Deficiency of 17α-hydroxylase/17,20-lyase is a rare cause of 46,XY disordered sex development. OBJECTIVE: We characterize in vitro and in vivo effects of two novel CYP17A1 gene mutations identified in a patient with a mild phenotype of CYP17A1 deficiency. SUBJECTS AND METHODS: A 46,XY patient presented with ambiguous genitalia. CYP17A1 deficiency was suspected at 2 months on the basis of steroid analysis performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mutational analysis of the CYP17A1 gene was performed by PCR and Sanger sequencing. To characterize the effect of CYP17A1 mutation on 17α-hydroxylase and 17,20-lyase activities in vitro, HEK293 cells were transiently transfected with CYP17A1 expression plasmids, incubated with progesterone or 17-OH-pregnenolone and concentrations of 17-OH-progesterone or DHEA were then measured in the cell culture medium by LC-MS/MS. RESULTS: Clinical and hormonal findings in the patient were consistent with partial combined deficiency of 17α-hydroxylase/17,20-lyase. The sequencing of the CYP17A1 gene in the patient revealed compound heterozygosity for two novel mutations: c.107delT p.R36fsX107 and p.W121R. After 6-h in vitro culture of transfected HEK293 cells in the presence of 1 μM progesterone, 17α-hydroxylase activity of p.W121R mutant was 60.5±16.3%, while 17,20-lyase activity of mutant measured from the amount of DHEA produced in the presence of 1 μM of 17-OH-pregnenolone was 15.8±2.6% compared with the WT. CONCLUSIONS: p.W121R substitution, affecting the first residue in the conserved heme-interacting WXXXR motif of CYP17A1, is associated with partial combined deficiency of 17α-hydroxylase/17,20-lyase.
BACKGROUND: Deficiency of 17α-hydroxylase/17,20-lyase is a rare cause of 46,XY disordered sex development. OBJECTIVE: We characterize in vitro and in vivo effects of two novel CYP17A1 gene mutations identified in a patient with a mild phenotype of CYP17A1 deficiency. SUBJECTS AND METHODS: A 46,XY patient presented with ambiguous genitalia. CYP17A1 deficiency was suspected at 2 months on the basis of steroid analysis performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mutational analysis of the CYP17A1 gene was performed by PCR and Sanger sequencing. To characterize the effect of CYP17A1 mutation on 17α-hydroxylase and 17,20-lyase activities in vitro, HEK293 cells were transiently transfected with CYP17A1 expression plasmids, incubated with progesterone or 17-OH-pregnenolone and concentrations of 17-OH-progesterone or DHEA were then measured in the cell culture medium by LC-MS/MS. RESULTS: Clinical and hormonal findings in the patient were consistent with partial combined deficiency of 17α-hydroxylase/17,20-lyase. The sequencing of the CYP17A1 gene in the patient revealed compound heterozygosity for two novel mutations: c.107delT p.R36fsX107 and p.W121R. After 6-h in vitro culture of transfected HEK293 cells in the presence of 1 μM progesterone, 17α-hydroxylase activity of p.W121R mutant was 60.5±16.3%, while 17,20-lyase activity of mutant measured from the amount of DHEA produced in the presence of 1 μM of 17-OH-pregnenolone was 15.8±2.6% compared with the WT. CONCLUSIONS:p.W121R substitution, affecting the first residue in the conserved heme-interacting WXXXR motif of CYP17A1, is associated with partial combined deficiency of 17α-hydroxylase/17,20-lyase.
Authors: Min Sun; Jonathan W Mueller; Lorna C Gilligan; Angela E Taylor; Fozia Shaheen; Anna Noczyńska; Guy T'Sjoen; Louise Denvir; Savitha Shenoy; Piers Fulton; Timothy D Cheetham; Helena Gleeson; Mushtaqur Rahman; Nils P Krone; Norman F Taylor; Cedric H L Shackleton; Wiebke Arlt; Jan Idkowiak Journal: Eur J Endocrinol Date: 2021-10-11 Impact factor: 6.664