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Detection of Mutations in the CYP21 Gene Using the DCode System

Gita Ohlsson and Marianne Schwartz, Department of Clinical Genetics, Juliane Marie Center, University Hospital, Rigshospitalet, Copenhagen, Denmark

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is caused by mutations in the gene CYP21 encoding the enzyme steroid 21-hydroxylase. In addition to deletions, approximately 20 different point mutations have been reported14 and novel mutations are still detected.58 This makes genetic diagnosis as well as carrier detection of 21-hydroxylase deficiency a complicated matter. We have used the denaturing gradient gel electrophoresis method (DGGE) using the DCode universal mutation detection system to detect mutations in the coding sequence and intron-exon junctions of CYP21.9, 10

Materials and Methods
DNA from healthy individuals and patients with 21-hydroxylase deficiency was isolated from peripheral blood lymphocytes using the salting-out method by Miller et al.11

Polymerase Chain Reaction (PCR)
For amplification of CYP21 and selection against CYP21P, PCR was performed according to Wedell.12 The resulting two PCR products were purified and subsequently used as template for the amplification of DNA fragments for DGGE. The PCR reactions were performed with GC-clamped primers. The GC-density of almost all CYP21 exons are very high requiring long GC-clamps to obtain a single melting domain of the sequence of interest.

Site-Directed Mutagenesis
In order to obtain mutant control samples for all exons it was necessary to introduce mutations i n some DNA fragments. This was done by PCR-based site-directed mutagenesis.13, 14 The mutant PCR product was mixed with the corresponding wild type PCR product; the mixture was placed at 96 C for 10 minutes to denature the PCR products and subsequently left at room temperature for gradual renaturation, thereby generating heteroduplex molecules.

Denaturing Gradient Gel Electrophoresis
Denaturing gradient gel electrophoresis was carried out using the DCode system (Bio-Rad). Fifteen l of PCR product was loaded on a gel containing a polyacrylamide gradient ranging from 612% and a gradient of urea and formamide. The gels were run in 1x TAE buffer, at 80 V overnight. After electrophoresis, the gels were stained in TAE buffer containing ethidium bromide and subsequently the resolved bands were visualized by ultraviolet (UV) transillumination.

The results of the DGGE analysis of exons 2, 5, 6, and 7 are presented in Figure 1. The presented DGGE analysis was carried out using a denaturing gradient of 3070%. To validate the method, one wild type control sequence and one heterozygous mutant sequence was analyzed for each exon. For exons 2 and 5 the mutant controls were generated by site-directed mutagenesis at the following nucleotide positions: exon 2: 2011 (A→C) and exon 5: 2851 (T→G). Nucleotide positions are given in accordance with CYP21, GenBank: accession numbers M12792; M23280. The mutant control sample for exon 6 contains the cluster-E6 mutation and for exon 7 the Val281Leu mutation.

To perform complete genotyping of 21-hydroxylase disease alleles and reliable carrier diagnosis, it is essential to use a technique that not only detects the most frequent previously identified point mutations but also both undefined and rare point mutations. The DGGE analysis fulfills these criteria, and therefore constitutes a fast and reliable procedure for genetic analysis of 21-hydroxylase deficiency.

1. Speiser, P. W., Dupont, J., Zhu, D., Serrat, J., Buegeleisen, M., Tusie-Luna, M. T., Lesser, M., New, M. I., and White, P. C., Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency, J. Clin. Invest., 90, 58495 (1992).

2. Barbat, B., Bogyo, A., Raux-Demay, M. C., Kuttenn, F., Boue, J., Simon- Bouy, B., Serre, J. L., and Mornet, E., Screening of CYP21 gene mutations in 129 French patients affected by steroid 21-hydroxylase deficiency, Hum. Mutat., 5, 12630 (1995).

3. Wedell, A., Thilen, A., Ritzen, E. M., Stengler, B., and Luthman, H., Mutational spectrum of the steroid 21-hydroxylase gene in Sweden: implications for genetic diagnosis and association with disease manifestation, J. Clin. Endocrinol. Metab., 78, 114552 (1994).

4. Ezquieta, B., Oliver, A., Gracia, R., and Gancedo, P. G., Analysis of steroid 21-hydroxylase gene mutations in the Spanish population, Hum. Genet., 96, 198204 (1995).

5. Lajic, S., and Wedell, A., An intron 1 splice mutation and a nonsense mutation (W23X) in CYP21 causing severe congenital adrenal hyperplasia, Hum. Genet., 98(2):1824 (1996).

6. Levo, A., and Partanen, J., Novel nonsense mutation (W302X) in the steroid 21-hydroxylase gene of a Finnish patient with the salt-wasting form of congenital adrenal hyperp lasia, Hum. Mutat., 9(4):3635 (1997).

7. Kirby Keyser, L., Porter, C. C., and Donohoue, P. A., E380D: a novel point mutation of CYP21 in an HLA-homozygous patient with salt-losing congenital adrenal hyperplasia due to 21-hydroxylase deficiency, Hum. Genet., 9(2):1812 (1997).

8. Lajic, S., Levo, A., Nikoshkov, A., Lundberg, Y., Partanen, J., and Wedell, A., A cluster of missense mutations at Arg356 of human steroid 21-hydroxylase may impair redox partner interaction, Hum. Genet., 99(6):7049 (1997).

9. Guldberg, P., and Guttler, F., A simple method for identification of point mutations using denaturing gradient gel electrophoresis, Nucleic Acids Res., 21(9):22612 (1993).

10. Sheffield, V. C., Cox, D. R., Lerman, L. S., and Myers, R. M., Attachment of a 40-base-pair G+C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of singlebase changes, Proc. Natl. Acad. Sci. U S A, 86(1):23269 (1989).

11. Miller, S. A., Dykes, D. D., and Polesky, H. F., A simple salting out procedure for extracting DNA from human nucleated cells, Nucleic Acids Res., 16(3):1215 (1988).

12. Wedell, A, and Luthman, H., Steroid 21-hydroxylase deficiency: two additional mutations in saltwasting disease and rapid screening of disease-causing mutations, Hum. Mol. Genet., 2:499504 (1993).

13. Kuipers, O. P., Boot, H. J., and de Vos, W. M., Improved site-directed mutagenesis method using PCR, Nucleic Acids Res.,19(16):4558 (1991).

14. Landt, O., Grunert, H. P., and Hahn, U., A general method for rapid sitedirected mutagenesis using the polymerase chain reaction, Gene, 96(1):1258 (1990).

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