title: Functional and Structural Consequences of NineCYP21A2Mutations Ranging from Very Mild to Severe Effects.
abstract: We present the functional and structural effects of seven novel (p.Leu12Met, p.Arg16Cys, p.Ser101Asn, p.Ser202Gly, p.Pro267Leu, p.Gln389_Ala391del, and p.Thr450Met) and two previously reported but not studied (p.Ser113Phe and p.Thr450Pro)CYP21A2mutations. Functional analyses were complemented within silicoprediction of mutation pathogenicity based on the recently crystallized human CYP21A2 structure. Mutated proteins were transiently expressed in COS-1 cells and enzyme activities towards 17-hydroxyprogesterone and progesterone were determined. Residual enzyme activities between 43% and 97% were obtained for p.Arg16Cys, p.Ser101Asn, p.Ser202Gly, p.Pro267Leu, and p.Thr450Met, similar to the activities of the well-known nonclassic mutations p.Pro453Ser and p.Pro482Ser, whereas the p.Leu12Met variant showed an activity of 100%. Conversely, the novel p.Ser113Phe, p.Gln389_Ala391del, and p.Thr450Pro mutations drastically reduced the enzyme function below 4%. TheKmvalues for all novel variants were in the same order of magnitude as for the wild-type protein except for p.The450Met. The maximum velocity was decreased for all mutants except for p.Leu12Met. We conclude that p.Leu12Met is a normal variant; the mutations p.Arg16Cys, p.Ser101Asn, p.Ser202Gly, p.Pro267Leu, and p.Thr450Met could be associated with very mild nonclassic CAH, and the mutations p.Ser113Phe, p.Gln389_Ala391del, and p.Thr450Pro are associated with classic CAH. The obtained residual activities indicated a good genotype-phenotype correlation.
authors.affiliation: Laboratório de Genética Molecular Humana, Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Av. Cândido Rondon 400, 13083-875 Campinas, SP, Brazil; Department of Women's and Children's Health, Karolinska Institutet, Pediatric Endocrinology Unit (Q2:08), Karolinska University Hospital, 171 76 Stockholm, Sweden.
authors.forename: Débora
authors.identifier: 0000-0001-9956-0611
authors.initials: D
authors.lastname: de Paula Michelatto
authors.affiliation: Department of Women's and Children's Health, Karolinska Institutet, Pediatric Endocrinology Unit (Q2:08), Karolinska University Hospital, 171 76 Stockholm, Sweden.
authors.forename: Leif
authors.identifier: 0000-0001-6274-9330
authors.initials: L
authors.lastname: Karlsson
authors.affiliation: Laboratório de Genética Molecular Humana, Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Av. Cândido Rondon 400, 13083-875 Campinas, SP, Brazil.
authors.forename: Ana Letícia Gori
authors.identifier: 0000-0001-7034-3031
authors.initials: AL
authors.lastname: Lusa
authors.affiliation: Departamento de Pediatria, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Rua Tessália Vieira de Camargo 126, 13083-887 Campinas, SP, Brazil.
authors.forename: Camila D'Almeida Mgnani
authors.initials: CD
authors.lastname: Silva
authors.affiliation: Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 171 77 Stockholm, Sweden.
authors.forename: Linus Joakim
authors.initials: LJ
authors.lastname: Östberg
authors.affiliation: Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 171 77 Stockholm, Sweden; Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, 751 24 Uppsala, Sweden.
authors.forename: Bengt
authors.initials: B
authors.lastname: Persson
authors.affiliation: Departamento de Pediatria, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Rua Tessália Vieira de Camargo 126, 13083-887 Campinas, SP, Brazil.
authors.forename: Gil
authors.initials: G
authors.lastname: Guerra-Júnior
authors.affiliation: Departamento de Pediatria, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Rua Tessália Vieira de Camargo 126, 13083-887 Campinas, SP, Brazil.
authors.forename: Sofia Helena Valente
authors.identifier: 0000-0002-8289-5703
authors.initials: SH
authors.lastname: de Lemos-Marini
authors.affiliation: Department of Molecular Medicine and Surgery, Karolinska Institutet and Center for Inherited Metabolic Diseases (CMMS L7:05) Karolinska University Hospital, 171 76 Stockholm, Sweden.
authors.forename: Michela
authors.initials: M
authors.lastname: Barbaro
authors.affiliation: Laboratório de Genética Molecular Humana, Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Av. Cândido Rondon 400, 13083-875 Campinas, SP, Brazil.
authors.forename: Maricilda Palandi
authors.identifier: 0000-0003-0521-9100
authors.initials: MP
authors.lastname: de Mello
authors.affiliation: Department of Women's and Children's Health, Karolinska Institutet, Pediatric Endocrinology Unit (Q2:08), Karolinska University Hospital, 171 76 Stockholm, Sweden.
authors.forename: Svetlana
authors.identifier: 0000-0002-4398-0948
authors.initials: S
authors.lastname: Lajic