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Articles |
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Servei d'Hormonologia, Hospital Clínic i Provincial de Barcelona, and
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Secció d'Endocrinologia, Hospital Universitari de Sant Joan de Deu, Spain.
a Address for correspondence: Servei d'Hormonologia, Hospital Clínic, Villarroel 170 08036, Barcelona, Spain. Fax 34-3-227.54.54; e-mail labhor{at}medicina.ub.es
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Key Words: indexing terms: congenital adrenal hyperplasia • heritable disorders • adrenal steroid hormones • amplification-created restriction site analysis
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Because 21-hydroxylase deficiency is one of the most common inborn errors of metabolism in humans (4)(5), procedures are needed that will detect known mutations in the 21-hydroxylase gene to support a rapid diagnosis. We have designed a coordinated strategy to detect nine common 21-hydroxylase mutations, identifying some of them with the amplification-created restriction site (ACRS) method (6). ACRS, a PCR-based method, can identify previously known allelic mutations in nucleic acid sequences.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Preparation of human DNA.
Human DNA was prepared from
peripheral white blood cells. 10 mL of heparinized blood was mixed with
40 mL of isotonic saline (9 g/L NaCl) and centrifuged at
1500g. The pellet was resuspended in 30 mL of erythrocytes
lysis buffer (5 mmol/L MgCl2 and 20 mmol/L Tris-HCl, pH
7.8) and centrifuged at 3000g. Leukocytes were lysed by
adding to 10 mL of lysis buffer (0.2 mol/L NaCl, 1 mmol/L EDTA, and 10
mmol/L Tris-HCl, pH 7.8) containing 10 mg of sodium dodecyl sulfate and
0.4 mg of proteinase K per milliliter (Merck, Darmstadt, Germany).
After having shaken this mixture for 2 h at room temperature, we
extracted it once with an equal volume of phenol and then twice with
chloroform/isoamyl alcohol (24/1 by vol). The DNA was precipitated from
the combined extracts by adding 2.5 volumes of isopropanol as described
(7). The two-wavelength absorbance ratio
(A260/A280) of the DNA
preparations was >1.7. We checked the integrity of the DNA samples by
electrophoresis of 2 µg of DNA in a 0.9% agarose gel.
Methodology.
Primers used were synthesized in our hospital in
a DNA synthesizer (Model 392; Applied Biosystems, Foster City, CA);
subsequent purification by HPLC was not required. The oligonucleotide
probes used for allele-specific oligonucleotide hybridization were
essentially those described by Owerbach et al. (5); for
the R356W mutation, we used oligonucleotides described by Speiser et
al. (8). PCR-amplified DNA was blotted onto Hybond-N Nylon
membranes and hybridized to specific oligonucleotides with use of
Rapid-hyb buffer (both from Johnson & Johnson, Amersham, UK).
Deletion/conversion analysis was performed by Southern blot with
restriction enzymes BglII and TaqI
(8). Diagnosis of CAH was based on clinical and hormonal
criteria (9)(10).
PCR analysis.
Nine 21-hydroxylase gene mutations were analyzed
by means of the following strategy: Genomic DNA was amplified in two
segments by PCR with primers that selectively amplify CYP21.
Fragment 1 (Fig. 1
, upper left scheme) represents a 1339-bp segment extending from
exon 1 to exon 6. Fragment 2 (Fig. 1
, lower right scheme) is a 2.22-kb
fragment extending from the 8-bp deletion in exon 3 to beyond exon 10.
These two primary PCR reactions were performed as follows: After a hot
start of 2 min at 96 °C, we used 35 cycles, each consisting of
denaturation for 30 s at 94 °C, annealing for 30 s at
60 °C, and extension for 2 min (P1/P2) or 10
min (P3/P4) at 72 °C. For the second PCR
round we used a 100-fold dilution from each first amplification—with
the adequate primers listed in Table 1
and under similar conditions to the primary PCR, except that
the extension time was only 1 min. Using fragment 1 as template, we
performed a second PCR with the appropriate primers (Fig. 1
, upper
left) to detect P30L, intron 2 (A,C
G), and exon 3 (8-bp deletion)
mutations. Fragment 2 was digested to detect both V281L and R339H
mutations at the same time. Fragment 2 was also used as template to
perform a second PCR with the appropriate primers (Fig. 1
, lower right)
to detect I172N, Q318X, R356W, and P453S mutations.
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The method used to detect point mutations in some of these secondary amplifications involves the gain or loss of at least a single restriction site in the PCR product (R356W, Q318X). Other secondary amplifications were performed with the ACRS method, which enables the detection of any new restriction sites generated by a single-base mutation and of a second mutation generated in vitro by the use of a modified oligonucleotide as a primer (P30L, intron 2, I172N, and P453S).
After the secondary PCR reaction, products were of sufficient quality
for restriction analysis. PCR products were digested with the
appropriate restriction enzyme and subjected to electrophoresis through
an agarose gel (1%), agarose/Nu-Sieve gel (0.5%/2.5%) (FMC
Bioproducts, Rockland, ME), or polyacrylamide gel (10%), according to
the expected length of bands (Table 2
). All reactions were set up and analyzed in separate areas to
prevent contamination from previously amplified material and taking
special care during the second PCR reaction. Pipettes with aerosol
barrier tips were used, and controls with no genomic DNA template were
included in the analyses at all times.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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). Families pictured in Fig. 2
e, g
, and h
were diagnosed as nonclassical CAH; those in Fig. 2a
, d, and f as simple virilizing; and those in Fig. 2b
and c as
salt-wasting forms.
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experimental design for pcr amplification and mutation detection
P30L mutation (11) was detected by performing the
second PCR with P5 and P6 primers.
P5 primer is modified from its original sequence to give
rise to an HhaI restriction site when no P30L mutation
is present (Fig. 2a
). The P30L mutation also destroys an
AcyI restriction site but this restriction enzyme often
yielded partial digestions in our hands, leading to wrong results (data
not shown). Intron 2 mutation was detected by using a second PCR with
P7 and P8 primers. The intron 2 site has three
possible nucleotide substitutions, two neutral, A or C, and one mutated
G, which causes an alternative splice site to be used
[12]. P8 primer is modified to give rise
to an HhaI restriction site only when the G nucleotide
is present (Fig. 2b
). With this methodology, only one reaction is
required, whereas allele-specific PCR, in contrast, requires the use of
three different reactions.
Exon 3 (8-bp deletion) was detected by means of a second PCR with
P9 and P10 primers. The amplified product of a
nonmutated gene is a 89-bp fragment, whereas exon 3 (8-bp deletion)
gives an 81-bp fragment (Fig. 2c
). I172N mutation (13) was
detected by means of a second PCR with P11 and
P2 primers. P11 primer is modified to generate
a TaqI restriction site when this mutation is present (Fig. 2d
).
V281L (14) mutation was detected by means of digestion
with Alw 44I restriction enzyme from the
P3/P4-specific CYP21-amplified
product (Fig. 2e
), but the R339H mutation (15) was not
detected in any of our CAH families. Although we do not include a
positive control in our assay, our approach should still detect any
R339H mutation present because both V281L and R339H mutations produce a
loss of the Alw 44I restriction site.
Q318X mutation (16), which produces a codon stop, was
detected by means of a second PCR with P12 and
P13 primers (neither of them is modified). When the Q318X
mutation is present, a PstI restriction site is lost (Fig. 2f
). R356W mutation (17) was detected by means of a second
PCR with P14 and P15 primers (neither of them
is modified) and subsequent digestion with BsofI restriction
enzyme (this mutation produces a loss of a BsofI restriction
site) (Fig. 2g
). P453S mutation (15) was detected
also by with a second PCR using P16 and P17
primers. P17 primer was modified to give rise to an
HhaI restriction site when P453S mutation is not present
(Fig. 2h
).
considerations for the pcr strategy
The number of common mutations and the presence of a highly
homologous pseudogene complicate the routine use of molecular
techniques for diagnosis of 21-hydroxylase deficiency. Nonetheless, we
have developed a simple strategy to determine nine of the most common
mutations in the CYP21 gene. Amplification at two
independent sites of this gene allows an accurate genotyping of
subjects. In contrast to previous methods, e.g., dot-blot
analysis (with radioactive probes) or allele-specific PCR (which
amplifies normal and mutant alleles in different tubes), this strategy
can characterize nine CYP21 gene mutations by using ethidium
bromide-stained agarose or polyacrylamide gel electrophoresis and five
common restriction enzymes. Moreover, normal and mutant alleles are
both amplified in the same reaction tube, thus allowing determination
of the zygosity of the mutations.
The ligase chain reaction has also been described as an alternative to dot-blot analysis and allele-specific PCR (18). Although robust, however, the ligase chain reaction is more complex (fluorescently labeled oligonucleotides, asymmetric PCR, and automated sequencing) than our ACRS method. Moreover, the ACRS strategy can be applied to other CYP21 gene mutations in the initial amplified products, leading a more accurate mutational screening of this disease.
In theory, this strategy can also be used to detect CYP21 deletions/conversions. If homozygous deletions/conversions extend to exons 3, 4, or 5, then P30L, intron 2, and exon 3 (8-bp deletion) mutations would appear in the same allele in a homozygous state and fragment 2 would fail to be amplified. If homozygous deletions/conversions extend to exon 6 or further, neither of the fragments would be amplified. If heterozygous deletions/conversions extend to exons 3, 4, or 5, then P30L, intron 2, and exon 3 (8-bp deletion) mutations would appear in the same allele in a heterozygous state. If, in these last cases, the other allele also carried a P30L, intron 2 or exon 3 (8-bp deletion) mutation, the subject tested would incorrectly be considered a homozygote. Therefore, this methodology does not replace the Southern transfer assay with BglII and TaqI, but it can help to show how far the deletion/conversion extends. Genotyping of parents could also be used to infer the existence of a chromosome so affected.
We have used the described strategy to study three of our families having one deleted allele detected by Southern transfer, but found none of the mutations (data not shown). We suggest that in these three cases, the deletions extend beyond exon 6. Exon 6 (x3 cluster) mutation cannot be detected in this strategy because of P2 primer matches at this point. "Back-conversions" of CYP21 into CYP21P have been described (18). In our strategy, a back-mutation around the primer sites for P2 or P3 could be amplified, so in these cases we would find several CYP21P mutations in the same allele, suggesting a conversion. Nevertheless, the most common back-conversions are not at either of these two sites.
As a way to take advantage of this strategy, one can use the same PCR
amplification products to detect polymorphic sites in the
CYP21 gene (Table 4
) so as to perform a linkage analysis when one or both mutations
are not found.
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In conclusion, our PCR strategy has a complete concordance with allele-specific oligonucleotide hybridization, requires simple diagnostic tools, avoids the use of radioactivity, and can determine CYP21 mutations rapidly and accurately. In addition, other CYP21 mutations can be enclosed through use of this strategy.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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