Reverse-hybridization assay for rapid detection of common CYP21A2 mutations in dried blood spots from newborns with elevated 17-OH progesterone

Abstract

Background: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder most commonly caused by defects in the CYP21A2 gene. Neonatal CAH-screening based on 17-hydroxyprogesterone (17-OHP) measurements prevents life-threatening salt wasting conditions in newborns, but results in a considerable false-positive rate. Therefore, efficient second tier tests are required.

Methods: We developed a reverse-hybridization test strip-based assay (CAH StripAssay) covering the most prevalent CYP21A2 point mutations/small insertions/deletions occurring in Middle European populations. Assay specificity was validated using plasmid clones, and wild-type and mutant reference DNAs. Its practicability was evaluated in 271 samples from patients with CAH, suspected CAH, and dried blood spots from screening-positive newborns.

Results: All eleven point mutations and 51% of large deletions/conversions could be unambiguously identified when compared to reference methods (DNA sequencing, MLPA). After exclusion of rare mutations (6.4%) not covered by the StripAssay, the overall detection rate was 85%. Undetected heterozygous deletions/conversions caused a lack of information, but did not result in an incorrect prediction of phenotypes.

Conclusions: Our novel CAH StripAssay proved to be a fast (7h) and reliable method for detection of common CYP21A2 mutations. Implemented as a second-tier test in CAH newborn screening, it has the potential to significantly reduce recall rates.