Validation and Clinical Application of a Locus-Specific Polymerase Chain Reaction- and Minisequencing-Based Assay for Congenital Adrenal Hyperplasia (21-Hydroxylase Deficiency)

Abstract

Congenital adrenal hyperplasia is an autosomal recessive disorder caused by defective adrenal steroid biosynthesis, resulting in reduced glucocorticoid and increased androgen production. The majority of cases are due to inactivation of the 21-hydroxylase gene (CYP21A2), most commonly caused by genomic rearrangements with the adjacent, highly homologous pseudogene CYP21A. The most common deletions and gene conversion events have been defined and are typically detected by Southern hybridization detection of CYP21 rearrangements and/or polymerase chain reaction (PCR). However, Southern hybridization is laborious, and allele-specific PCR results may be difficult to interpret. We have therefore developed a locus-specific, PCR-based, minisequencing method for detecting the 12 most common CYP21A2 mutations. We validated the assay using a panel of 20 previously genotyped samples obtained from individuals who collectively have a broad spectrum of mutations causing 21-hydroxylase deficiency. We also used 19 control samples having no CYP21 mutations. All validation samples were correctly typed, and we identified haplotypes that may be useful for clinical diagnosis. Results from 102 clinical samples demonstrate that this assay is a rapid, reliable, and robust method for locus-specific identification of mutations and is suitable for routine clinical use and prenatal diagnosis. (J Mol Diagn 2005, 7:236 -246)

Recent advances in diagnosis, treatment, and outcome of congenital adrenal hyperplasia due to 21-hydroxylase deficiency

“…However, compared to LC-MSMS steroid profiling as second-tier test, the minisequencing strategy is more expensive and slower. Keen-Kim et al later expanded this latter method to detect 12 common mutations [45].…”

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

“…1). An internal positive amplification control of AVPR2, located on the X chromosome (primers ME0004, ME0027) was duplexed in the reactions [21]. The expected sizes of the PCR products are as follows: CYP21A2—5.6 kb, CYP21A1P—6.1 kb, 30 kb deletion (CYP21A1P/CYP21A2 gene chimera)—6.2 kb, large-scale conversion (CYP21A2/CYP21A1P gene chimera)—5.5 kb and AVPR2—1.1 kb.…”

“…Each PCR product was prepared for DNA sequencing with 5 μl of Exo SAP-IT (Affymetrix, Santa Clara, CA) and incubated at 37 °C for 30 min and heat-inactivated at 80 °C for 20 min. Products were diluted 1:50 in molecular biology grade water for Sanger cycle sequencing using 1:8 dilutions of BigDye Terminator V1.1 (Life Technologies) and sequencing primers as previously described (Table 1) [21]. Cycle sequencing products were purified using BigDye Xterminator and run on the 3730 DNA Analyzer (Life Technologies).…”

“…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.…”

Prenatal diagnosis of congenital adrenal hyperplasia owing to 21-hydroxylase deficiency

“…A number of strategies have been developed that utilize polymerase chain reaction (PCR)-based techniques to specifically amplify the CYP21A2, not the pseudogene [8][9][10]. Techniques for molecular diagnosis of point mutations include allele-specific oligonucleotide hybridization [11], amplification-created restriction sites [12], single-stranded conformation polymorphisms [13], allele-specific PCR [10], ligation detection reactions [14] and multiplex minisequencing [15].…”