Genotyping of CYP21A2 for Congenital Adrenal Hyperplasia Screening using Allele-Specific Primer Extension followed by Bead Array Hybridization

Abstract

A novel CAH mutation screening method has been developed to detect ten point mutations and the 8 bp deletion in CYP21A2, as well as large deletions between CYP21A1P and CYP21A2. This novel genotyping strategy is superior to PCR-RFLP-based methods and equally as accurate as sequencing.

Novel method to characterize CYP21A2 in Florida patients with congenital adrenal hyperplasia and commercially available cell lines

“…The importance of determining what nucleotide positions are specific to either CYP21A2 or the pseudogene is underscored by the variation that was seen in the normal population controls at nucleotide positions c.*440C > T and c.*443T > C. Differences in the reference sequences for these sites in CYP21A2 ( C GC T ) and CYP21A1P ( T GC C) have been utilized as the basis of locus-specific PCR amplification in multiple studies as well as determining the CYP21A2 copy number analysis by MLPA [6], [17], [21], [23], [37], [39], [40]. In the normal population controls characterized in this study, 15/60 individuals had aberrant PCR profiles for the CYP21A2, CYP21A1P, 30 kb deletion and large scale conversion amplifications when using locus specific primers to this site.…”

“…Thus, the dependency on the use of clinically relevant mutations for PCR specificity will lead to additional false positives and complicated genotypic analysis. Several methods for mutation analysis of CYP21A2, including allele specific PCR, primer extension and oligo ligation assays [17], [18], [20], [23], [38], [41], [42], [43], [44] are based upon either the Exon 10 3′ UTR region, or the Exons 3 and 6 mutations, raising the question of analytical specificity for these CYP21A2 mutation screening approaches. The single long-range PCR amplification method described in this study overcomes this lack of specificity in the 3′UTR region by exploiting the deletion region in the TNXA and TNXB genes downstream of CYP21A1P and CYP21A2 and also simplifies downstream analysis because it is not dependent on what mutations may be present in the CYP21A2 gene.…”

“…For molecular analysis to be practical for CAH NBS, the method must have the specificity required to target only the CYP21A2 gene without detecting related CYP21A1P pseudogene sequences and be able to detect large chromosomal rearrangements that lead to 21OHD. CYP21A2 genotyping methods from CAH samples have been described that use either oligo-ligation reactions, allele specific primer extension or reverse-hybridization assays [17], [18], [20], [21], [22], [23], [24]. These methods include a primary PCR reaction which is then used as a template for targeted genotyping assays or in the case of the reverse-hybridization assay, the PCR products are directly assayed on mutation-specific probes immobilized on test strips.…”