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.
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.
Rogério S
RS
Araújo
Berenice B
BB
Mendonca
Angela S
AS
Barbosa
Chin J
CJ
Lin
José A M
JA
Marcondes
Ana Elisa C
AE
Billerbeck
Tânia A S S
TA
Bachega
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