A new CYP21A1P/CYP21A2 chimeric gene identified in an Italian woman suffering from classical congenital adrenal hyperplasia form

Abstract

Background: More than 90% of Congenital Adrenal Hyperplasia (CAH) cases are associated with mutations in the 21-hydroxylase gene (CYP21A2) in the HLA class III area on the short arm of chromosome 6p21.3. In this region, a 30 kb deletion produces a non functional chimeric gene with its 5' and 3' ends corresponding to CYP21A1P pseudogene and CYP21A2, respectively. To date, five different CYP21A1P/CYP21A2 chimeric genes have been found and characterized in recent studies. In this paper, we describe a new CYP21A1P/CYP21A2 chimera (CH-6) found in an Italian CAH patient.

Methods: Southern blot analysis and CYP21A2 sequencing were performed on the patient. In addition, in order to isolate the new CH-6 chimeric gene, two different strategies were used.

Results: The CYP21A2 sequencing analysis showed that the patient was homozygote for the g.655C/A>G mutation and heterozygote for the p.P30L missense mutation. In addition, the promoter sequence revealed the presence, in heterozygosis, of 13 SNPs generally produced by microconversion events between gene and pseudogene. Southern blot analysis showed that the woman was heterozygote for the classic 30-kb deletion producing a new CYP21A1P/CYP21A2 chimeric gene (CH-6). The hybrid junction site was located between the end of intron 2 pseudogene, after the g.656C/A>G mutation, and the beginning of exon 3, before the 8 bp deletion. Consequently, CH-6 carries three mutations: the weak pseudogene promoter region, the p.P30L and the g.655C/A>G splice mutation.

Conclusion: We describe a new CYP21A1P/CYP21A2 chimera (CH-6), associated with the HLA-B15, DR13 haplotype, in a young Italian CAH patient.

Figure 1

Southern blot analysis. Taq I restriction patterns of genomic DNA hybridized to a mixture of the CYP21A2 and C4 cDNA probes. Fa: father, Mo: mother. For pattern analysis see the text in results section.

Figure 2

Sequence of the new chimeric gene. Sequence of CH-6 chimera from +1 nucleotide (exon 1) to +721 nucleotide (exon 3). Nucleotides were numbered starting from ATG. The lines showed: the amino acid sequence of CYP21A2 gene (M13936), the CYP21A2 nucleotide sequence (M13936), the CYP21A1P nucleotide sequence (M13935) and the CH-6 nucleotide sequence. Only nucleotide differences between CYP21A1P or CH-6 and CYP21A2 active gene are shown under corresponding nucleotides in CYP21A2 gene.

Figure 3

CYP21A1P/CYP21A2 chimeric genes. CH-1, CH-2, CH-3, CH-4, CH-5 and CH-6 represent six distinct chimeric CYP21A1P/CYP21A2 genes as described in the text. The black and white regions represent the non-functional CYP21A1P and the functional CYP21A2 sequences, respectively. The eight nucleotide deletion in exon 3 is marked by an asterisk.