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.