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.
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.
Authors: Gabriela P Finkielstain; Wuyan Chen; Sneha P Mehta; Frank K Fujimura; Reem M Hanna; Carol Van Ryzin; Nazli B McDonnell; Deborah P Merke Journal: J Clin Endocrinol Metab Date: 2010-10-06 Impact factor: 5.958
Authors: Nils Krone; Felix G Riepe; Joachim Grötzinger; Carl-Joachim Partsch; Jürgen Brämswig; Wolfgang G Sippell Journal: J Mol Med (Berl) Date: 2005-04-14 Impact factor: 4.599
Authors: Wuyan Chen; Mimi S Kim; Sujata Shanbhag; Andrew Arai; Carol VanRyzin; Nazli B McDonnell; Deborah P Merke Journal: Am J Med Genet A Date: 2009-12 Impact factor: 2.802
Authors: I Milacic; M Barac; T Milenkovic; M Ugrin; K Klaassen; A Skakic; M Jesic; I Joksic; K Mitrovic; S Todorovic; S Vujovic; S Pavlovic; M Stojiljkovic Journal: J Endocrinol Invest Date: 2015-08-02 Impact factor: 4.256