Microconversion between CYP21A2 and CYP21A1P promoter regions causes the nonclassical form of 21-hydroxylase deficiency.
BACKGROUND
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.


METHODS
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.


CONCLUSIONS
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.
Araújo
Rogério S
RS
Unidade de Endocrinologia do Desenvolvimento e Laboratorio de Hormonios e Genetica Molecular, Disciplina de Endocrinologia, Hospital das Clinicas da Faculdade de Medicina da Universidade de Sao Paulo, SP 05403-900, Brazil.
Mendonca
Berenice B
BB
Barbosa
Angela S
AS
Lin
Chin J
CJ
Marcondes
José A M
JA
Billerbeck
Ana Elisa C
AE
Bachega
Tânia A S S
TA
D000293:Adolescent; D000312:Adrenal Hyperplasia, Congenital / Q000235:genetics*; D000328:Adult; D001483:Base Sequence; D002648:Child; D015342:DNA Probes; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D006801:Humans; D007438:Introns / Q000235:genetics; D008297:Male; D010641:Phenotype; D017354:Point Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011544:Pseudogenes / Q000235:genetics; D013255:Steroid 21-Hydroxylase / Q000235:genetics*
D015342:DNA Probes; D013255:Steroid 21-Hydroxylase