title: Novel deletion alleles carrying CYP21A1P/A2 chimeric genes in Brazilian patients with 21-hydroxylase deficiency.
abstract: BACKGROUND
  Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is caused by deletions, large gene conversions or mutations in CYP21A2 gene. The human gene is located at 6p21.3 within a locus containing the genes for putative serine/threonine Kinase RP, complement C4, steroid 21-hydroxylase CYP21 tenascin TNX, normally, in a duplicated cluster known as RCCX module. The CYP21 extra copy is a pseudogene (CYP21A1P). In Brazil, 30-kb deletion forming monomodular alleles that carry chimeric CYP21A1P/A2 genes corresponds to ~9% of disease-causing alleles. Such alleles are considered to result from unequal crossovers within the bimodular C4/CYP21 locus. Depending on the localization of recombination breakpoint, different alleles can be generated conferring the locus high degree of allelic variability. The purpose of the study was to investigate the variability of deleted alleles in patients with 21-hydroxylase deficiency.
  
  
  METHODS
  We used different techniques to investigate the variability of 30-kb deletion alleles in patients with 21-hydroxylase deficiency. Alleles were first selected after Southern blotting. The composition of CYP21A1P/A2 chimeric genes was investigated by ASO-PCR and MLPA analyses followed by sequencing to refine the location of recombination breakpoints. Twenty patients carrying at least one allele with C4/CYP21 30-kb deletion were included in the study.
  
  
  RESULTS
  An allele carrying a CYP21A1P/A2 chimeric gene was found unusually associated to a C4B/C4A Taq I 6.4-kb fragment, generally associated to C4B and CYP21A1P deletions. A novel haplotype bearing both p.P34L and p.H62L, novel and rare mutations, respectively, was identified in exon 1, however p.P30L, the most frequent pseudogene-derived mutation in this exon, was absent. Four unrelated patients showed this haplotype. Absence of p.P34L in CYP21A1P of normal controls indicated that it is not derived from pseudogene. In addition, the combination of different approaches revealed nine haplotypes for deleted 21-hydroxylase deficiency alleles.
  
  
  CONCLUSIONS
  This study demonstrated high allelic variability for 30-kb deletion in patients with 21-hydroxylase deficiency indicating that a founder effect might be improbable for most monomodular alleles carrying CYP21A1P/A2 chimeric genes in Brazil.
authors.affiliation: Laboratório de Genética Molecular Humana, Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas, SP, Brasil.
authors.forename: Fernanda B
authors.initials: FB
authors.lastname: Coeli
authors.forename: Fernanda C
authors.initials: FC
authors.lastname: Soardi
authors.forename: Renan D
authors.initials: RD
authors.lastname: Bernardi
authors.forename: Marcela
authors.initials: M
authors.lastname: de Araújo
authors.forename: Luciana C
authors.initials: LC
authors.lastname: Paulino
authors.forename: Ivy F
authors.initials: IF
authors.lastname: Lau
authors.forename: Reginaldo J
authors.initials: RJ
authors.lastname: Petroli
authors.forename: Sofia H V
authors.initials: SH
authors.lastname: de Lemos-Marini
authors.forename: Maria T M
authors.initials: MT
authors.lastname: Baptista
authors.forename: Gil
authors.initials: G
authors.lastname: Guerra-Júnior
authors.forename: Maricilda P
authors.initials: MP
authors.lastname: de-Mello
mesh_terms: D000312:Adrenal Hyperplasia, Congenital / Q000235:genetics*; D000483:Alleles; D015139:Blotting, Southern; D001938:Brazil; D005091:Exons; D005784:Gene Amplification; D017353:Gene Deletion; D005808:Genes, Recessive; D006801:Humans; D050506:Mutant Chimeric Proteins / Q000235:genetics; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D011544:Pseudogenes; D017384:Sequence Deletion; D013255:Steroid 21-Hydroxylase / Q000235:genetics*
chemical_list: D050506:Mutant Chimeric Proteins; C531510:CYP21A2 protein, human; D013255:Steroid 21-Hydroxylase