Four sets of primer pairs amplify all possible recombinant forms of CYP21A2, CYP21A1P (the pseudogene), and their hybrids 5'-CYP21A2/CYP21A1P-3' and 5'-CYP21A1P/CYP21A2-3', via PCR to determine whether there are large rearrangements between the gene and pseudogene. Fluorescent DNA sequence analysis is then performed on all exons of the active form of CYP21A2 and any presumed active hybrid to test for the presence of sequencing mutations (GenBank accession number: NM_000500.5). If necessary, further analysis may be performed on nonexpressed copies of CYP21A2 or hybrids to gain insight into possible rearrangements. In addition, multiplex ligation-dependent probe amplification (MLPA) is performed to determine exact copy numbers of the active gene (CYP21A2), its inactive pseudogene (CYP21A1P), and any rearrangements. However, this technology cannot determine the cis/trans status (cis=same chromosome, trans=opposite chromosomes) of the identified genes, rearrangements, or mutations. Family studies of blood relatives might assist in determination of the cis/trans status.(Cradic KW, Grebe SK: A diagnostic sequencing assay for Cyp21 based on promoter activity provides better understanding of gene rearrangements. Abstract. Endocrine Society Annual Meeting, ENDO 2005)
Whole blood 60 days; extracted DNA indefinitely, patient must opt-out.