title: Functional studies of p.R132C, p.R149C, p.M283V, p.E431K, and a novel c.652-2A>G mutations of the CYP21A2 gene.
abstract: Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism and accounts for 90-95% of CAH cases. In the present work, we analyzed the functional consequence of four novel previously reported point CYP21A2 mutations -p.R132C, p.R149C, p.M283V, p.E431K- found in Argentinean 21-hydroxylase deficient patients. In addition, we report an acceptor splice site novel point mutation, c.652-2A>G, found in a classical patient in compound heterozygosity with the rare p.R483Q mutation. We performed bioinformatic and functional assays to evaluate the biological implication of the novel mutation. Our analyses revealed that the residual enzymatic activity of the isolated mutants coding for CYP21A2 aminoacidic substitutions was reduced to a lesser than 50% of the wild type with both progesterone and 17-OH progesterone as substrates. Accordingly, all the variants would predict mild non-classical alleles. In one non-classical patient, the p.E431K mutation was found in cis with the p.D322G one. The highest decrease in enzyme activity was obtained when both mutations were assayed in the same construction, with a residual activity most likely related to the simple virilizing form of the disease. For the c.652-2A>G mutation, bioinformatic tools predicted the putative use of two different cryptic splicing sites. Nevertheless, functional analyses revealed the use of only one cryptic splice acceptor site located within exon 6, leading to the appearance of an mRNA with a 16 nt deletion. A severe allele is strongly suggested due to the presence of a premature stop codon in the protein only 12 nt downstream.
authors.affiliation: Centro Nacional de Genética Médica, Administración Nacional de Laboratorios e Institutos de Salud (ANLIS), Dr. Carlos G. Malbrán, Buenos Aires, Argentina; Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (IBYME-CONICET), Buenos Aires, Argentina.
authors.forename: Melisa
authors.initials: M
authors.lastname: Taboas
authors.affiliation: Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires y Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.
authors.forename: Luciana
authors.initials: L
authors.lastname: Gómez Acuña
authors.affiliation: Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires y Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.
authors.forename: María Florencia
authors.initials: MF
authors.lastname: Scaia
authors.affiliation: Centro Nacional de Genética Médica, Administración Nacional de Laboratorios e Institutos de Salud (ANLIS), Dr. Carlos G. Malbrán, Buenos Aires, Argentina; Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (IBYME-CONICET), Buenos Aires, Argentina.
authors.forename: Carlos D
authors.initials: CD
authors.lastname: Bruque
authors.affiliation: Centro Nacional de Genética Médica, Administración Nacional de Laboratorios e Institutos de Salud (ANLIS), Dr. Carlos G. Malbrán, Buenos Aires, Argentina.
authors.forename: Noemí
authors.initials: N
authors.lastname: Buzzalino
authors.affiliation: División Endocrinología, Hospital Durand, Buenos Aires, Argentina.
authors.forename: Mirta
authors.initials: M
authors.lastname: Stivel
authors.affiliation: Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires y Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.
authors.forename: Nora R
authors.initials: NR
authors.lastname: Ceballos
authors.affiliation: Centro Nacional de Genética Médica, Administración Nacional de Laboratorios e Institutos de Salud (ANLIS), Dr. Carlos G. Malbrán, Buenos Aires, Argentina; Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (IBYME-CONICET), Buenos Aires, Argentina.
authors.forename: Liliana
authors.initials: L
authors.lastname: Dain
mesh_terms: D019326:17-alpha-Hydroxyprogesterone / Q000378:metabolism*; D000312:Adrenal Hyperplasia, Congenital / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D015153:Blotting, Western; D019295:Computational Biology; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D006801:Humans; D017354:Point Mutation / Q000235:genetics*; D011374:Progesterone / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013255:Steroid 21-Hydroxylase / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D012333:RNA, Messenger; D011374:Progesterone; D019326:17-alpha-Hydroxyprogesterone; C531510:CYP21A2 protein, human; D013255:Steroid 21-Hydroxylase