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. 2012 Feb;58(2):421-30.
doi: 10.1373/clinchem.2011.174037. Epub 2011 Dec 7.

Junction site analysis of chimeric CYP21A1P/CYP21A2 genes in 21-hydroxylase deficiency

Wuyan Chen  1 , Zhi Xu, Annie Sullivan, Gabriela P Finkielstain, Carol Van Ryzin, Deborah P Merke, Nazli B McDonnell
Affiliations

Junction site analysis of chimeric CYP21A1P/CYP21A2 genes in 21-hydroxylase deficiency

Wuyan Chen et al. Clin Chem. 2012 Feb.

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.

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Conflict of interest statement

Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Figures

Fig. 1
Fig. 1. Schematic diagram of the RCCX module with Southern blotting and TaqI digestion analysis
Shown are a common bimodular RCCX (A) and a monomodular RCCX with a chimeric CYP21A1P/CYP21A2 gene (B) in which junction sites can vary. Functional genes are in gray. Sizes and locations of the TaqI restriction fragments at CYP21 genes are annotated with open squares. C4 is a generalized symbol for the C4A, C4B, C4L, and C4S genes. A C4 gene can be C4A or C4B regarding protein isotype, and it can also be long (C4L) or short (C4S) regarding gene size. Only a C4L gene with unknown isotype is shown in the schematic. The size of the sequence in the dashed frame is approximately 30 kb. An 8515-bp PCR product amplified by primer pair CYP779f/Tena32F was digested with TaqI for genotyping of the chimeric gene. (C), Genotyping of chimeric gene by Southern blotting after TaqI digestion of genomic DNA. Probands 1 and 2 do not have a CYP21A2 band. Three parents (1M, 2M, and 2F) showed a reduced CYP21A2:CYP21A1P ratio, indicating a chimera allele. The relatively weak band of proband 2 was due to a low yield of extracted DNA. (D), Genotyping of a chimeric gene by TaqI digestion of the 8515-bp PCR product. Probands 1 and 2 do not have a CYP21A2 band. Four parents (1M, 1F, 2M, and 2F), proband 6, and 2 siblings from family 7 (proband 7 and patient 7S) are heterozygous for the chimera gene. Among 3 controls of known genotype, proband 3 is homozygous for the In2G (c.293−13A/C>G) mutation and presents structurally intact CYP21A2 genes. Probands 4 and 5 are homozygous and heterozygous, respectively, for a classic chimera gene. M, molecular-size markers (from top: 4.0 kb, 3.5 kb, and 3.0 kb); 21A2, functional gene CYP21A2; 21A1P, pseudogene CYP21A1P.
Fig. 2
Fig. 2. Junction site analysis of attenuated chimeric CYP21A1P/CYP21A2 by DNA sequencing
(A), Junction site of CH-4 is between c.138 and c.292+45 (highlighted in light blue). Probands 1 and 2 demonstrated a CYP21A1P-like sequence in exon 1 (homozygous for c.92, c.118, and c.138) and a CYP21A2-like sequence at the beginning of intron 2 (homozygous for c.292+45). Participants 1F, 2M, and proband 6 are heterozygous for 3 sites at exon 1, showing that they are carriers of the uncommon attenuated chimeric gene. Proband 7 and her sister (patient 7S) are heterozygous for both exon 1 sites and c.292+45, indicating that they carry a distinct chimeric allele with a junction site downstream of c.292+45. (B), Junction site of CH-9 is between c.293−74 and c.293−67 (highlighted in light green). CH-9 and CYP21A2 alleles, which share a CYP21A2-like sequence from c.293−67, were distinguished with TA cloning and sequencing for proband 7. 21A2, functional gene CYP21A2; 21A1P, pseudogene CYP21A1P.
Fig. 3
Fig. 3. Sequence alignment of CYP21 genes from promoter to exon 3
Located in this region, junction sites of 3 chimeric CYP21A1P/CYP21A2 genes (CH-4, −6, and −9) are demonstrated. CH-4, between c.138 and c.292 + 45 (highlighted in light blue); CH-6, between c.293−13 and c.332 (highlighted in pink); CH-9, between c.293−74 and c.293−67 (highlighted in light green). Conserved CYP21A1P sites, which are highlighted in yellow with the corresponding CYP21A2 sequence, are annotated. The coding sequence is presented in red. The hats (ˆ) denote nonexisting nucleotides, and dashed lines represent consensus sequence. Six sites of Chi-like sequence with only a 1-bp mismatch to 5′-GCTGGTGG-3′ or its complementary sequence (5′-CCACCAGC-3′) are framed in black.
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