title: Junction site analysis of chimeric CYP21A1P/CYP21A2 genes in 21-hydroxylase deficiency.
abstract: BACKGROUND
  Chimeric CYP21A1P/CYP21A2 genes, caused by homologous recombination between CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide 2) and its highly homologous pseudogene CYP21A1P (cytochrome P450, family 21, subfamily A, polypeptide 1 pseudogene), are common in patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD). A comprehensive junction site analysis of chimeric CYP21A1P/CYP21A2 genes is needed for optimizing genetic analysis strategy and determining clinical relevance.
  
  
  METHODS
  We conducted a comprehensive genetic analysis of chimeric CYP21A1P/CYP21A2 genes in a cohort of 202 unrelated 21-OHD patients. Targeted CYP21A2 mutation analysis was performed, and genotyping of chimeric CYP21A1P/CYP21A2 genes was cross-confirmed with Southern blot, RFLP, and multiplex ligation-dependent probe amplification analyses. Junction sites of chimera genes were determined by sequencing the long-PCR products amplified with primers CYP779f and Tena32F. An updated bioinformatics survey of Chi-like sequences was also performed.
  
  
  RESULTS
  Of 100 probands with a chimeric allele, 96 had a chimera associated with the severe classic salt-wasting form of CAH, and the remaining 4 carried an uncommon attenuated chimera with junction sites upstream of In2G (c.293-13A/C>G), which is associated with a milder phenotype. In addition to 6 of 7 reported chimeras, we identified a novel classic chimera (CH-8) and a novel attenuated chimera (CH-9). Attenuated chimeras explained prior genotype-phenotype discrepancies in 3 of the patients. Sequencing the CYP779f/Tena32F amplicons accurately differentiated between classic and attenuated chimeras. The bioinformatics survey revealed enrichment of Chi-like sequences within or in the vicinity of intron 2.
  
  
  CONCLUSIONS
  Junction site analysis can explain some genotype-phenotype discrepancies. Sequencing the well-established CYP779f/Tena32F amplicons is an unequivocal strategy for detecting attenuated chimeric CYP21A1P/CYP21A2 genes, which are clinically relevant.
authors.affiliation: Laboratory of Clinical Investigation, National Institute on Aging, Baltimore, MD 21224, USA.
authors.forename: Wuyan
authors.initials: W
authors.lastname: Chen
authors.forename: Zhi
authors.initials: Z
authors.lastname: Xu
authors.forename: Annie
authors.initials: A
authors.lastname: Sullivan
authors.forename: Gabriela P
authors.initials: GP
authors.lastname: Finkielstain
authors.forename: Carol
authors.initials: C
authors.lastname: Van Ryzin
authors.forename: Deborah P
authors.initials: DP
authors.lastname: Merke
authors.forename: Nazli B
authors.initials: NB
authors.lastname: McDonnell
mesh_terms: D000312:Adrenal Hyperplasia, Congenital / Q000235:genetics*; D001483:Base Sequence; D015331:Cohort Studies; D019295:Computational Biology; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D050506:Mutant Chimeric Proteins / Q000235:genetics*; D009154:Mutation; D011544:Pseudogenes; D013255:Steroid 21-Hydroxylase / Q000235:genetics*
chemical_list: C570632:CYP21A1P pseudogene, human; D050506:Mutant Chimeric Proteins; C531510:CYP21A2 protein, human; D013255:Steroid 21-Hydroxylase