Multiplex minisequencing of the 21-hydroxylase gene as a rapid strategy to confirm congenital adrenal hyperplasia.
BACKGROUND
Congenital adrenal hyperplasia (CAH) is a frequent autosomal recessive disease, with a wide range of clinical manifestations, most commonly attributable to mutations in the 21-hydroxylase gene (CYP21). Large gene deletions, large gene conversions, a small 8-basepair deletion, and eight point mutations in CYP21 account for approximately 95% of all enzyme deficiencies. We developed a new strategy for a rapid CYP21 analysis.


METHODS
DNA samples from 40 CAH patients previously genotyped by direct DNA sequencing were reanalyzed by allele-specific amplification of the functional CYP21 gene followed by a multiplex minisequencing reaction using 13 primers. In addition, a second PCR that amplified a part of exon 3 was used to demonstrate the presence or absence of at least one functional gene.


RESULTS
The assay detected the P453S mutation and nine of the most common mutations (P30L, intron 2 splice, Delta 8bp, I172N, exon 6 cluster, V281L, F306+t, Q318X, and R356W) caused by microconversions from the CYP21P pseudogene. The concordance was 100% for detecting these mutations, including gene deletions and large gene conversions. The 40 patient DNA samples were analyzed in 1.5 working days by one technician (actual hands-on time, 3.5 h). The material cost for analyzing one sample was approximately 10.00 Euros (US $9.00).


CONCLUSIONS
This novel mutation screening strategy rapidly detects 90-95% of all mutations associated with CAH and appears applicable as a tool for confirmation of increased 17-hydroxyprogesterone found in neonatal CAH screening.
Krone
Nils
N
Laboratory of Molecular Endocrinology, Division of Pediatric Endocrinology, Department of Pediatrics, Universitätsklinikum Kiel, Schwanenweg 20, D-24105 Kiel, Germany.
Braun
Andreas
A
Weinert
Stefanie
S
Peter
Michael
M
Roscher
Adelbert A
AA
Partsch
Carl-Joachim
CJ
Sippell
Wolfgang G
WG
D000312:Adrenal Hyperplasia, Congenital / Q000175:diagnosis* / Q000201:enzymology; D015139:Blotting, Southern; D004252:DNA Mutational Analysis / Q000191:economics / Q000379:methods; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000191:economics / Q000379:methods; D013255:Steroid 21-Hydroxylase / Q000235:genetics*
D013255:Steroid 21-Hydroxylase