Novel deletion alleles carrying CYP21A1P/A2chimeric genes in Brazilian patients with 21-hydroxylase deficiency

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.

Junction Site Analysis of Chimeric CYP21A1P/CYP21A2 Genes in 21-Hydroxylase Deficiency

“…No sequencing analysis was presented to narrow down the precise junction sites, however. In addition, haplotype IV in the Brazilian study might represent the same kind of chimera as the CH-8 chimera identified and so designated in our study (31). …”

“…Proband 7 and sibling patient 7S have NC CAH, findings that are in accord with their carrying CH-9 on one allele and V281L, a common NC mutation, on the other. Interestingly, 2 patients with moderate SV phenotypes in the Brazilian study carried a second chimera allele (haplotype VIII) that seems to have a junction site similar to CH-9 (31). No sequencing analysis was presented to narrow down the precise junction sites, however.…”

“…A recent report described a Brazilian patient who carried a similar chimera allele and also demonstrated a moderate SV phenotype in the presence of SW mutation In2G at the other allele. The junction site of the chimera in this patient was probably located at the beginning of intron 2, but no detailed sequencing data were provided (31). Similarly, our patients’ phenotypes were most consistent with the SV type of CAH.…”

“…Both the MLPA methodology and PCR-based strategies, such as using primer pair C/E, have unavoidable limitations in their ability to detect chimeric CYP21A1P/CYP21A2 genes. MLPA is an efficient method for detecting large gene deletions and duplications and has been widely used in previous studies of CAH (9, 31, 33, 34). Current MLPA probes, however, are not able to distinguish attenuated chimeras CH-4 and CH-9 from classic chimera CH-6, owing to the lack of a probe for In2G, which is the crucial site for classifying chimeric CYP21A1P/CYP21A2 genes into the classic and attenuated types.…”

Sonic Hedgehog mutations are not a common cause of congenital hypopituitarism in the absence of complex midline cerebral defects

“…Normalized relative values ranging from 0·7 to 1·3 corresponded to two gene copies in the genotype (normal). This confidence interval was established by data obtained with five controls in each MLPA assay (Supporting information).…”