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Reverse Dot-blot Hybridization as an Improved Tool for the Molecular Diagnosis of Point Mutations in Congenital Adrenal Hyperplasia Caused by 21-Hydroxylase Deficiency


Background: More than 90% of cases of congenital adrenal hyperplasia (CAH) are caused by mutations of the CYP21 gene that result in deficiencies of the enzyme 21-hydroxylase. Allele-specific PCR, allele-specific oligonucleotide hybridization, and Southern blot analysis are the most common methods to detect point mutations and deletions in the CYP21 gene.

Methods and Results: This report is the first application of the reverse dot-blot (RDB) assay for diagnosis of the nine most common point mutations in the CYP21 gene associated with CAH (P30L, g.659A>G or g.659C>G, I172N, I236N-V237E-M239K, V281L, g.l767-1768insT, Q318X, R356W, P453S). Normal and mutant oligonucleotides spanning these nine mutation sites were spotted onto a nylon membrane. DNA was extracted from dried blood spots, and exons encompassing mutations from samples to be tested were amplified and labeled with biotin-dUTP by PCR. These exons then were hybridized to membrane strips. Signal detection was achieved by chemiluminescence. Thirty clinically confirmed cases that were identified by the Texas Newborn Screening Program were tested. All mutations were subsequently confirmed by automated DNA sequencing.

Conclusion: The RDB method has the advantages of being accurate and cost-effective for the molecular diagnosis of CYP21 point mutations in CAH. It permits simultaneous detection of a panel of point mutations with only one hybridization per sample and could be automated to study many samples.

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  1. 1.

    Pang S, Clark A: Congenital adrenal hyperplasia due to 21-hydroxylase deficiency: Newborn screening and its relationship to the diagnosis and treatment of the disorder. Screening 1993;2:105–139

  2. 2.

    Speiser PW, Dupont J, Zhu D, et al.: Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Invest 1992;90:584–595

  3. 3.

    Strachan T: Molecular pathology of 21-hydrozylase deficiency. J Inherit Metab Dis 1994;17:430–441

  4. 4.

    Wedell A: Molecular approaches for the diagnosis of 21-hydroxylase deficiency and congenital adrenal hyperplasia. DNA Technol 1996;16:125–137

  5. 5.

    Higashi Y, Tanae A, Inoue H, Hiromasa T, Fujii-Kuriyama Y: Aberrant splicing and missense mutations cause steroid 21-hydroxylase [P-450(C21)] deficiency in humans: Possible gene conversion products. Proc Natl Acad Sci U S A 1988;85:7486–7490

  6. 6.

    White PC, New MI, Dupont B: Structure of human steroid 21-hydroxylase genes. Proc Natl Acad Sci U S A 1986;83:5111–5115

  7. 7.

    Wedell A, Luthman H: Steroid 21-hydroxylase deficiency: Two additional mutations in salt-wasting disease and rapid screening of disease-causing mutations. Hum Mol Genet 1993;2:499–504

  8. 8.

    Day DJ, Speiser PW, Schulze E, et al.: Identification of non-amplifying CYP21 genes when using PCR-based diagnosis of 21-hydroxylase deficiency in congenital adrenal hyperplasia (CAH) affected pedigrees. Hum Mol Genet 1996;5:2039–2048

  9. 9.

    Ordonez-Sanchez ML, Ramirez-Jimenez S, Lopez-Gutierrez AU, et al.: Molecular genetic analysis of patients carrying steroid 21-hydroxylase deficiency in the Mexican population: Identification of possible new mutations and high prevalence of apparent germ-line mutations. Hum Genet 1998;102; 170–177

  10. 10.

    Saiki RK, Walsh PS, Levenson CH, Erlich HA: Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc Natl Acad Sci U S A 1989;86:6230–6234

  11. 11.

    Cai SP, Wall J, Kan YW, Chehab FF: Reverse dot blot probes for the screening of beta-thalassemia mutations in Asians and American blacks. Hum Mutat 1994;3:59–63

  12. 12.

    Maggio A, Giambona A, Cai SP, Wall J, Kan YW, Chehab FF: Rapid and simultaneous typing of hemoglobin S, hemoglobin C, and seven Mediterranean beta-thalassemia mutations by covalent reverse dot-blot analysis: Application to prenatal diagnosis in Sicily. Blood 1993;81:239–242

  13. 13.

    Winichagoon P, Saechan V, Sripanich R, et al.: Prenatal diagnosis of beta-thalassaemia by reverse dot-blot hybridization. Prenat Diagn 1999;19:428–435

  14. 14.

    Grody WW, Dunkel-Schetter C, Tatsugawa ZH, et al.: PCR-based screening for cystic fibrosis carrier mutations in an ethnically diverse pregnant population. Am J Hum Genet 1997;60:935–947

  15. 15.

    Schollen E, Vandenberk P, Cassiman JJ, Matthijs G: Development of reverse dot-blot system for screening of mitochondrial DNA mutations associated with Leber hereditary optic atrophy. Clin Chem 1997;43:18–23

  16. 16.

    Yang T, Yuan L, Huang S, Zhao S: Screening mutations in phenylketonuria by means of nonradioactive reverse dot blot hybridization. Chung Hua I Hsueh I Chuan Hsueh Tsa Chih 1998;15:307–309

  17. 17.

    Romano V, Lio D, Cali F, et al.: A methodological strategy for PAH genotyping in population with a marked molecular heterogeneity of hyperphenylalaninemia. Mol Cell Probes 2001;15:13–19

  18. 18.

    Lau J, Tolan DR: Screening for hereditary fructose intolerance mutations by reverse dot-blot. Mol Cell Probes 1999;13:35–40

  19. 19.

    Lee HH, Chao HT, Lee YJ, et al.: Identification of four novel mutations in the CYP21 gene in congenital adrenal hyperplasia in the Chinese. Hum Genet 1998;103:304–310

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Correspondence to Y.-P. Yang PhD.

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Yang, Y., Corley, N. & Garcia-Heras, J. Reverse Dot-blot Hybridization as an Improved Tool for the Molecular Diagnosis of Point Mutations in Congenital Adrenal Hyperplasia Caused by 21-Hydroxylase Deficiency. Molecular Diagnosis 6, 193–199 (2001).

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Key words

  • reverse dot-blot (RDB)
  • congenital adrenal hyperplasia (CAH)
  • DNA diagnosis
  • CYP21 point mutation detection