title: 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.
authors.affiliation: ViennaLab Diagnostics GmbH, Gaudenzdorfer Gürtel 43-45, 1120 Vienna, Austria. nemeth@viennalab.co.at
authors.forename: Stefan
authors.initials: S
authors.lastname: Németh
authors.forename: Stefan
authors.initials: S
authors.lastname: Riedl
authors.forename: Gernot
authors.initials: G
authors.lastname: Kriegshäuser
authors.forename: Sabina
authors.initials: S
authors.lastname: Baumgartner-Parzer
authors.forename: Paola
authors.initials: P
authors.lastname: Concolino
authors.forename: Vassos
authors.initials: V
authors.lastname: Neocleous
authors.forename: Leonidas A
authors.initials: LA
authors.lastname: Phylactou
authors.forename: Maryla
authors.initials: M
authors.lastname: Borucka-Mankiewicz
authors.forename: Hüseyin
authors.initials: H
authors.lastname: Onay
authors.forename: Ajlan
authors.initials: A
authors.lastname: Tukun
authors.forename: Christian
authors.initials: C
authors.lastname: Oberkanins
mesh_terms: D019326:17-alpha-Hydroxyprogesterone / Q000097:blood*; D000312:Adrenal Hyperplasia, Congenital / Q000097:blood / Q000235:genetics*; D059788:Dried Blood Spot Testing; D006801:Humans; D007231:Infant, Newborn; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013255:Steroid 21-Hydroxylase / Q000235:genetics*
chemical_list: D019326:17-alpha-Hydroxyprogesterone; C531510:CYP21A2 protein, human; D013255:Steroid 21-Hydroxylase