title: Molecular CYP21A2 diagnosis in 480 Brazilian patients with congenital adrenal hyperplasia before newborn screening introduction.
abstract: BACKGROUND
  Most congenital adrenal hyperplasia (CAH) patients carry CYP21A2 mutations derived from conversion events involving the pseudogene, and the remaining carry new mutations.
  
  
  OBJECTIVE
  To review causal mutations and genotype-phenotype correlation in 480 Brazilian patients.
  
  
  METHODS
  DNA was extracted from 158 salt-wasters (SWs), 116 simple virilizing (SV), and 206 nonclassical (NC) patients. Fourteen point mutations were screened by allele-specific PCR, large rearrangements by Southern blotting/MLPA, and sequencing was performed in those with incomplete genotype. The gene founder effect was analyzed by microsatellite studies. Patients were divided into six genotypes (Null; A: <2%; B: 3-7%; C: >20% of residual enzymatic activity (EA); D: unknown EA; E: incomplete genotype).
  
  
  RESULTS
  Targeted methodologies defined genotype in 87.6% of classical and in 80% of NC patients and the addition of sequencing in 100 and 83.5%, respectively. The most frequent mutations were p.V281L (26.6% of alleles), IVS2-13A/C>G (21.1%), and p.I172N (7.5%); seven rare mutations and one novel mutation (p.E351V) were identified. Gene founder effect was observed in all but one (p.W19X) mutation. Null, A, B, and C genotypes correlated with SW (88%), SW (70%), SV (98%), and NC forms (100%), respectively. In group D, the p.E351V mutation correlated with classical form and group E comprised exclusively NC-patients. ACTH-stimulated 17OHP level of 44.3ng/mL was the best cutoff to identify NC-patients carrying severe mutations.
  
  
  CONCLUSIONS
  We identified a good genotype-phenotype correlation in CAH, providing useful data regarding prediction of disease's severity; moreover, we suggest that ACTH-stimulated 17OHP levels could predict carrier status for severe mutations. Sequencing is essential to optimize molecular diagnosis in Brazilian CAH patients.
authors.affiliation: Laboratório de Hormônios e Genética Molecular- LIM/42Unidade de Adrenal, Disc. de Endocrinologia e Metabologia dacarv89@gmail.com.
authors.forename: Daniel F
authors.initials: DF
authors.lastname: de Carvalho
authors.affiliation: Laboratório de Hormônios e Genética Molecular- LIM/42Unidade de Adrenal, Disc. de Endocrinologia e Metabologia.
authors.forename: Mirela C
authors.initials: MC
authors.lastname: Miranda
authors.affiliation: Laboratório de Hormônios e Genética Molecular- LIM/42Unidade de Adrenal, Disc. de Endocrinologia e Metabologia.
authors.forename: Larissa G
authors.initials: LG
authors.lastname: Gomes
authors.affiliation: Laboratório de Hormônios e Genética Molecular- LIM/42Unidade de Adrenal, Disc. de Endocrinologia e Metabologia.
authors.forename: Guiomar
authors.initials: G
authors.lastname: Madureira
authors.affiliation: Laboratório de Hormônios e Genética Molecular- LIM/42Unidade de Adrenal, Disc. de Endocrinologia e Metabologia.
authors.forename: José A M
authors.initials: JA
authors.lastname: Marcondes
authors.affiliation: Laboratório de Hormônios e Genética Molecular- LIM/42Unidade de Adrenal, Disc. de Endocrinologia e Metabologia.
authors.forename: Ana Elisa C
authors.initials: AE
authors.lastname: Billerbeck
authors.affiliation: Laboratório de Hormônios e Genética Molecular- LIM/42Unidade de Adrenal, Disc. de Endocrinologia e Metabologia.
authors.forename: Andresa S
authors.initials: AS
authors.lastname: Rodrigues
authors.affiliation: Unidade de Endocrinologia PediátricaInstituto da Criança, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Paula F
authors.initials: PF
authors.lastname: Presti
authors.affiliation: Unidade de Endocrinologia PediátricaInstituto da Criança, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Hilton
authors.initials: H
authors.lastname: Kuperman
authors.affiliation: Unidade de Endocrinologia PediátricaInstituto da Criança, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Durval
authors.initials: D
authors.lastname: Damiani
authors.affiliation: Laboratório de Hormônios e Genética Molecular- LIM/42Unidade de Adrenal, Disc. de Endocrinologia e Metabologia.
authors.forename: Berenice B
authors.initials: BB
authors.lastname: Mendonca
authors.affiliation: Laboratório de Hormônios e Genética Molecular- LIM/42Unidade de Adrenal, Disc. de Endocrinologia e Metabologia.
authors.forename: Tania A S S
authors.initials: TA
authors.lastname: Bachega
mesh_terms: D000293:Adolescent; D000312:Adrenal Hyperplasia, Congenital / Q000175:diagnosis* / Q000235:genetics; D000483:Alleles; D001938:Brazil; D002648:Child; D002675:Child, Preschool; D005260:Female; D056726:Genetic Association Studies; D005838:Genotype; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D025202:Molecular Diagnostic Techniques; D010641:Phenotype; D017354:Point Mutation; D013255:Steroid 21-Hydroxylase / Q000235:genetics*
chemical_list: C531510:CYP21A2 protein, human; D013255:Steroid 21-Hydroxylase