Mutational analysis of CYP21A2 gene and CYP21A1P pseudogene: long-range PCR on genomic DNA

CYP21A2, the gene that codes for P450c21 (Steroid 21-hydroxylase), has a duplicated pseudogene called CYP21A1P. The gene and the pseudogene share 98 % and 96 % sequence homology in exons and in noncoding sequences, respectively, and are located 30 kb apart within the HLA class III human histocompatibility complex locus on chromosome 6p21.3. CYP21A1P is inactive due to the presence of 11 deteriorated mutations in its coding region. These mutations can be transferred to the functional CYP21A2 through intergenic recombination during meiosis or mitosis and lead to the congenital adrenal hyperplasia (CAH) resulting from 21-hydroxylase deficiency. Conversely, portions of CYP21A2 sequence can be transferred to CYP21A1P, modifying the haplotype. Here, we describe a well-established protocol that can be used to unambiguously study the mutational profile of CYP21A2 gene and CYP21A1P pseudogene. The protocol is based on long-range PCR amplification with allele-specific primers, followed by DNA sequencing of smaller fragments.