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

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.