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.

Molecular genetic study of congenital adrenal hyperplasia in Serbia: novel p.Leu129Pro and p.Ser165Pro CYP21A2 gene mutations

“…The MutPred probability of deleterious mutation was 0.595 for p.L129P (with p = 0.0394 gain of disorder) and 0.633 for p.S165P confirming pathogenic effect. Additional defected alleles were not determined precisely due to lack of segregation family analysis c Enzyme activities were described in Krone and Arlt [8] d Variability of CH-Ex3 enzyme activity is described in Chen et al [40]. If CH-Ex3 chimera contains pseudogene-specific mutation c.290-13A/C>G enzyme activity is totally abolished, but if the junction site is located between exon 1 and intron 2, upstream of c.290-13A/C>G, chimeric enzyme retains partial activity and is related to milder phenotype (depicted in green) and Leu128 (depicted in dark blue).…”

Management of adolescents with congenital adrenal hyperplasia

“…9,11,12,15 Variations in large deletions can result in a partly functional gene and thus a milder phenotype, or a contiguous gene-deletion syndrome associated with hypermobility type Ehlers-Danlos syndrome. 16,17 Genetic sequencing is necessary to identify rare mutations that are estimated to occur in 10% of affected alleles. 9 Although the CYP21A2 gene was identified more than 30 years ago, novel mutations and new aspects of the genetics of CYP21A2 are continually being defined.…”

“…9 Although the CYP21A2 gene was identified more than 30 years ago, novel mutations and new aspects of the genetics of CYP21A2 are continually being defined. 9,16–18 Thus, molecular diagnosis should only be undertaken by a laboratory familiar with the genetic complexities of CYP21A2 and able to offer a combination of diagnostic strategies.…”

Congenital Adrenal Hyperplasia

“…10 Another example of misalignment is a CYP21A1P / CYP21A2 chimera in which a portion of the CYP21A1P gene is fused to a portion of the CYP21A2 gene. 11 Rarely, CAH can be associated with uniparental disomy. 12 The de novo mutation rate is very low and is approximately 1%.…”