mutation analysis

tech note 2341

Detection of Mutations in the CYP21 Gene Using the DCode™ System
Gita Ohlsson and Marianne Schwartz, Department of Clinical Genetics, JulianeMarie Center, University Hospital, Rigshospitalet, Copenhagen, Denmark

Introduction
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 reported(Barbat et al. 1995, Ezquieta et al. 1995, Speiser et al. 1992,Wedell et al. 1994) and novel mutations are still detected(Kirby-Keyser et al. 1997, Lajic and Wedell 1996, Lajic et al.
1997, Levo and Partanen 1997). This makes genetic diagnosis
as well as carrier detection of 21-hydroxylase deficiency acomplicated matter. We have used the denaturing gradient gelelectrophoresis method (DGGE) using the DCode universal
mutation detection system to detect mutations in the coding
sequence and intron-exon junctions of CYP21 (Guldberg andGüttler 1993, Sheffield et al. 1989).

mixed with the corresponding wild-type PCR product; themixture was placed at 96°C for 10 min to denature thePCR products and subsequently left at room temperaturefor gradual renaturation, thereby generating heteroduplex
molecules.
DGGE

DGGE was carried out using the DCode system (Bio-RadLaboratories, Inc.). PCR product (15 μl) was loaded on a gel
containing a polyacrylamide gradient ranging from 6–12% anda gradient of urea and formamide. The gels were run in 1x
TAE buffer, at 80 V overnight. After electrophoresis, the gelswere stained in TAE buffer containing ethidium bromide, andsubsequently, the resolved bands were visualized by ultraviolet(UV) transillumination.
12345678910Methods
Samples

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. (1988).
Polymerase Chain Reaction (PCR)

For amplification of CYP21 and selection against CYP21P, PCR
was performed according to Wedell and Luthman (1993). 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 wasnecessary to introduce mutations in some DNA fragments. Thiswas done by PCR-based site-directed mutagenesis (Kuiperset al. 1991, Landt et al. 1990). The mutant PCR product wasFig. 1. DGGE analysis of CYP21. Each exon is represented by a mutant anda normal control sample. Lanes 1–2, exon 2; lanes 3–4, exon 5; lanes 5–6,exon 6; lanes 7–8, exon 7. For exon 2 and 5, the mutant control samples wereobtained by site-directed mutagenesis at the following nucleotide positions:exon 2: 2011 (AÆC) and exon 5: 2851 (TÆG). Nucleotide positions are given inaccordance with CYP21, GenBank accession numbers: M12792; M23280. For
exon 6, the mutant control sample represents the cluster-E6 mutation (lane 5)
and for exon 7, the Val281Leu mutation (lane 7).

Results
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 30–70%. 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 bysite-directed mutagenesis at the following nucleotide positions:exon 2: 2011 (AÆC) and exon 5: 2851 (TÆG). 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 forexon 7 the Val281Leu mutation.

References

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

Lajic S et al., A cluster of missense mutations at Arg356 of human steroid
21-hydroxylase may impair redox partner interaction, Hum Genet 99, 704–709(1997)

Barbat B et al., Screening of CYP21 gene mutations in 129 French patientsaffected by steroid 21-hydroxylase deficiency, Hum Mutat 5, 126–30 (1995)
Ezquieta B et al., Analysis of steroid 21-hydroxylase gene mutations in theSpanish population, Hum Genet 96, 198–204 (1995)
Guldberg P and Güttler F, A simple method for identification of point mutations
using denaturing gradient gel electrophoresis, Nucleic Acids Res 21,2261–2262 (1993)
Kirby-Keyser L et al., E380D: a novel point mutation of CYP21 in an HLAhomozygous patient with salt-losing congenital adrenal hyperplasia due to
21-hydroxylase deficiency, Hum Mutat 9, 181–182 (1997)
Kuipers OP et al., Improved site-directed mutagenesis method using PCR,
Nucleic Acids Res 19, 4558 (1991)
Lajic S and Wedell A, An intron 1 splice mutation and a nonsense mutation(W23X) in CYP21 causing severe congenital adrenal hyperplasia, Hum Genet98, 182–184 (1996)

Landt O, Grunert HP, and Hahn U, A general method for rapid site-directedmutagenesis using the polymerase chain reaction, Gene 96, 125–128 (1990)
Levo A and Partanen J, Novel nonsense mutation (W302X) in the steroid 21hydroxylase gene of a Finnish patient with the salt-wasting form of congenital
adrenal hyperplasia, Hum Mutat 9, 363–365 (1997)
Miller SA et al., A simple salting out procedure for extracting DNA from humannucleated cells, Nucleic Acids Res 16, 1215 (1988)
Sheffield VC et al., Attachment of a 40-base-pair G + C-rich sequence (GCclamp) to genomic DNA fragments by the polymerase chain reaction resultsin improved detection of single-base changes, Proc Natl Acad Sci USA 86,232–236 (1989)
Speiser PW et al., Disease expression and molecular genotype in congenital
adrenal hyperplasia due to 21-hydroxylase deficiency, J Clin Invest 90,584–595 (1992)
Wedell A et al., Mutational spectrum of the steroid 21-hydroxylase gene in
Sweden: implications for genetic diagnosis and association with disease
manifestation, J Clin Endocrinol Metab 78, 1145–1152 (1994)
Wedell A and Luthman H, Steroid 21-hydroxylase deficiency: two additional
mutations in salt–wasting disease and rapid screening of disease-causing
mutations, Hum Mol Genet 2, 499–504 (1993)
Practice of the polymerase chain reaction (PCR) may require a license.
Information in this tech note was current as of the date of writing (1998) andnot necessarily the date this version (rev B, 2008) was published.

Bio-RadLaboratories, Inc.
Web site www.bio-rad.com USA 800 4BIORAD Australia 61 02 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 21 3237 9400
Canada 905 364 3435 China 86 21 6426 0808 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 69 65
Germany 089 318 84 0 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 455 8800 India 91 124 4029300 Israel 03 963 6050
Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 0508 805 500
Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3188 South Africa 27 861 246 723
Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 061 717 95 55 Taiwan 886 2 2578 7189 United Kingdom 020 8328 2000

Life ScienceGroup

Bulletin 2341 Rev B

US/EG06-0373

0408

Sig 0308