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

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. © 2009 Concolino et al; licensee BioMed Central Ltd

High Frequency of Copy Number Variations and Sequence Variants at CYP21A2 Locus: Implication for the Genetic Diagnosis of 21-Hydroxylase Deficiency

BACKGROUND: The systematic study of the human genome indicates that the inter-individual variability is greater than expected and it is not only related to sequence polymorphisms but also to gene copy number variants (CNVs). Congenital Adrenal Hyperplasia due to 21-hydroxylase deficiency (21OHD) is the most common autosomal recessive disorder with a carrier frequency of 1:25 to 1:10. The gene that encodes 21-hydroxylase enzyme, CYP21A2, is considered to be one of the most polymorphic human genes. Copy number variations, such as deletions, which are severe mutations common in 21OHD patients, or gene duplications, which have been reported as rare events, have also been described. The correct characterization of 21OHD alleles is important for disease carrier detection and genetic counselling METHODOLOGY AND FINDINGS: CYP21A2 genotyping by sequencing has been performed in a random sample of the Spanish population, where 144 individuals recruited from university students and employees of the hospital were studied. The frequency of CYP21A2 mutated alleles in our sample was 15.3% (77.3% were mild mutations, 9% were severe mutations and 13.6% were novel variants). Gene dosage assessment was also performed when CYP21A2 gene duplication was suspected. This analysis showed that 7% of individuals bore a chromosome with a duplicated CYP21A2 gene, where one of the copies was mutated. CONCLUSIONS: As far as we know, the present study has shown the highest frequency of 21OHD carriers reported by a genotyping analysis. In addition, a high frequency of alleles with CYP21A2 duplications, which could be misinterpreted as 21OHD alleles, was found. Moreover, a high frequency of novel genetic variations with an unknown effect on 21-hydroxylase activity was also found. The high frequency of gene duplications, as well as novel variations, should be considered since they have an important involvement in carrier testing and genetic counseling

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

<p>Abstract</p> <p>Background</p> <p>Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is caused by deletions, large gene conversions or mutations in <it>CYP21A2 </it>gene. The human gene is located at 6p21.3 within a <it>locus </it>containing the genes for putative serine/threonine Kinase <it>RP</it>, complement <it>C4</it>, steroid 21-hydroxylase <it>CYP21 </it>tenascin <it>TNX</it>, normally, in a duplicated cluster known as RCCX module. The <it>CYP21 </it>extra copy is a pseudogene (<it>CYP21A1P</it>). In Brazil, 30-kb deletion forming monomodular alleles that carry chimeric <it>CYP21A1P/A2 </it>genes corresponds to ~9% of disease-causing alleles. Such alleles are considered to result from unequal crossovers within the bimodular <it>C4/CYP21 locus</it>. 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.</p> <p>Methods</p> <p>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 <it>CYP21A1P/A2 </it>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 <it>C4/CYP21 </it>30-kb deletion were included in the study.</p> <p>Results</p> <p>An allele carrying a <it>CYP21A1P/A2 </it>chimeric gene was found unusually associated to a <it>C4B/C4A </it><it>Taq </it>I 6.4-kb fragment, generally associated to <it>C4B </it>and <it>CYP21A1P </it>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 <it>CYP21A1P </it>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.</p> <p>Conclusions</p> <p>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 <it>CYP21A1P/A2 </it>chimeric genes in Brazil.</p