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Microconversion between CYP21A2 and CYP21A1P Promoter Regions Causes the Nonclassical Form of 21-Hydroxylase Deficiency

Unidade de Endocrinologia do Desenvolvimento e Laboratorio de Hormonios e Genetica Molecular (R.S.A., B.B.M., C.J.L., J.A.M.M., A.E.C.B., T.A.S.S.B.), LIM/42, Disciplina de Endocrinologia, Hospital das Clinicas da Faculdade de Medicina da Universidade de Sao Paulo, SP 05403-900, Brazil; and Centro de Biotecnologia (A.S.B.), Instituto Butantan, 05504-900 Sao Paulo, SP, Brazil

Address all correspondence and requests for reprints to: T. A. S. S. Bachega, M.D., Hospital das Clínicas, Faculdade de Medicina da Universidade de Sao Paulo, Disciplina de Endocrinologia, Av Dr Enéas de Carvalho 155, 2 andar, Bloco 6, São Paulo, SP, CEP 05403-900, Brasil. E-mail: tbachega{at}usp.br.

Context: Most mutations causing 21-hydroxylase deficiency originate from microconversions between CYP21 pseudogenes and active genes. However, around 20% of the alleles in the nonclassical form (NC-21OHD) remain without identified mutations, suggesting the involvement of regulatory regions. The pseudogene promoter is 80% less active than the CYP21A2 due to the presence of –126C>T, –113G>A, –110T>C, and –103A>G mutations. Additionally, mutations in the steroidogenic factor-1 binding sites of the CYP21 distal regulatory region, located at 4676 bases upstream from the cap site of the CYP21A2 gene, decrease its transcription to 35%.

Objective: The objective of the study was to investigate the CYP21A2 promoter/regulatory regions in NC-21OHD patients with undetermined genotype.

Subjects: The study included 17 NC-21OHD patients and 50 controls.

Methods: Promoter/regulatory regions were sequenced from peripheral leukocytes’ genomic DNA. The identified substitutions were evaluated through EMSA using –132/–97 wild-type and mutant probes and nuclear extracts from NCI-H295A cells. Transcriptional activity studies were performed with wild-type and mutant constructions transfected in NCI-H295A cells.

Results: No mutations were identified in the distal regulatory regions. The –126C>T, –113G>A, –110T>C promoter mutations were found in compound heterozygosity with the V281L mutation in one patient and the –126C>T mutation in compound heterozygosity with the I2 splice in another. The –126T mutation decreases the transcriptional activity to 52%, compatible with the patient’s nonclassical phenotype. EMSA demonstrated that the –132/–121 region is important for the DNA interaction with the specificity protein-1 transcription factor.

Conclusion: Microconversions between CYP21A2 and CYP21A1P promoters could be involved in the nonclassical phenotype. Therefore CYP21A2 promoter analysis should be included in genetic studies of 21OHD.

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