Abstract
BackgroundCongenital 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.MethodsWe 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.ResultsAn 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.ConclusionsThis 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.
Highlights
Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is caused by deletions, large gene conversions or mutations in CYP21A2 gene
The present study describes the variability of such alleles in Brazilian patients (n = 20) with 21-hydroxylase deficiency using the conventional Southern blot, ASO-PCR and sequencing techniques combined to recently developed Multiplex Ligation-dependent Probe Amplification (MLPA) technique
This study reports two novel deletion alleles in Brazilian patients with 21-hydroxylase deficiency and describes the variability of C4/CYP21 monomodular alleles evaluated by combining Southern blot, ASO-PCR, MLPA and sequencing techniques
Summary
Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is caused by deletions, large gene conversions or mutations in CYP21A2 gene. 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. The CYP21A2 gene and CYP21A1P, its pseudogene, are located within the class III locus of the human major histocompatibility complex at the short arm of chromosome 6 [4,5]. Both have 10 exons, except that CYP21A1P is inactive due to deleterious mutations [6,7]. Monomodular (16%), and trimodular (14%) alleles with long and short C4B gene variants are not uncommon [12]
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