Novel deletion alleles carrying CYP21A1P/A2 chimeric genes in Brazilian patients with 21-hydroxylase deficiency

Abstract

Background: Congenital 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.

Methods: We 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.

Results: An 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.

Conclusions: This 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.

Figure 1

Schematic representation illustrating the hybridization sites for C4 and CYP21 probes. a) Southern blot: bars indicate hybridization regions for each probe; T denote Taq I restriction sites; numbers indicate sizes in kb of fragments recognized by each probe; b) MLPA experiment: arrows indicate the location where each probe hybridizes. Ex = exon, Int - intron.

Figure 2

Taq I Southern blots showing patients carrying monomodular alleles. Hybridizations with CYP21 (pC21/3c) and C4 (C4B550) probes are shown. a) Homozygous genotypes for monomodular alleles; b) compound heterozygous genotypes with mono- and bimodular alleles carrying large gene conversion; c) compound heterozygous genotypes with mono- and bimodular alleles carrying pseudogene-derived mutations; d) compound heterozygous genotype with mono- and trimodular alleles carrying pseudogene-derived mutations; e) compound heterozygous genotype with mono- and tetramodular alleles (lane 3); maternal genotype showing compound heterozygosis with mono- and bimodular alleles (lane 2); and paternal genotype with tetramodular and bimodular alleles (lane 1). Numbers below each lane depict patient numbers.

Figure 3

Integrated and normalized MLPA data. C4A, C4B, CYP21A1P, CYP21A2, TNXB, CREB1 are shown. Numbers 1-15 correspond to: (1) C4A exon 17, (2) C4B exon 19, (3-5) CYP21A1P 5' promoter region, intron 2, 3'UTR; (6-10) CYP21A2 5' promoter region, exon 3, exon 4, exon 6, exon 8; (11-13) TNXB exon 32, 15, 1; (14) CREB1 probe. (15) Y-chromosome probe. Horizontal black bars denote recombination breakpoints (RB); red bar, for patient 8. Columns correspond to integrated and normalized electropherogram peak areas, values between 0.8 and 1.2 indicate two copies, below and above correspond to one or more than two copies, respectively. Patients' numbers are on the right. Green arrows indicate heterozygosis for p.I172N and p.Q318X (patient 18). Black arrows indicate 2:1 ratio for two copies of 3'UTR pseudogene (5) and one copy of exon 8 (10). (a-g) Upper panels - MLPA results; lower panels - schematic genotypes. a) Monomodular homozygosis; red circles denote different C4 5'-end; b) Mono- and bimodular alleles carrying chimeric genes; red arrows indicate 1:2 ratio (C4A:C4B); c) heterozygosis for mono- and bimodular alleles; red arrows indicate three copies of C4B; d) heterozygosis for mono- and tri- or tetramodular alleles; red arrows indicate 2:2 ratio for 3'-end CYP21A1P (5) to exon 8 CYP21A2 (10); patient 10 - trimodular allele bearing normal p.Q318 in the two pseudogenes; e) heterozygosis for alleles mono- and bimodular with CYP21A1P/A2 and C4A/C4B, respectively; f-g) heterozygosis for mono- and bimodular alleles; RB between exons 3-4 and 1-3, respectively; red arrow - null C4B hybridization signal (patient 20).