Clinical Chemistry 43: 2121-2127, 1997;
(Clinical Chemistry. 1997;43:2121-2127.)
© 1997 American Association for Clinical Chemistry, Inc.
Rapid detection of 21-hydroxylase deficiency mutations by allele-specific in vitro amplification and capillary zone electrophoresis
Paola Carrera1,
Anna Maria Barbieri1,
Maurizio Ferrari1,
Pier Giorgio Righetti2,
Marilena Perego2 and
Cecilia Gelfi2,a
1
I.R.C.C.S., H.S. Raffaele, Via Olgettina 60, I-20132 Milano, Italy.
2
University of Verona, Department of Agricultural and
Industrial Biotechnologies, Strada Le Grazie, Cà Vignal, 37134
Verona, Italy.
3
ITBA, CNR, Via Ampère 56, Milano, Italy.
a Author for correspondence. Fax Int +39-2-26423364;
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Abstract
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A quick diagnosis of the classic form of 21-hydroxylase deficiency
(simple virilizing and salt wasting) is of great importance, especially
for prenatal diagnosis and treatment in pregnancies at risk. A method
for simultaneous detection of common point mutations in the P450c21 B
gene is here proposed by combining a nested PCR amplification
refractory mutation system (ARMS) with capillary zone electrophoresis
(CZE) in sieving liquid polymers. In the first PCR, B genes are
selectively amplified. In the nested reaction, ARMS-detected wild-type
and mutated alleles are separately pooled and resolved by CZE. CZE is
performed in coated capillaries in the presence of 30 g/L hydroxyethyl
cellulose in the background electrolyte for size separation of the DNA
analytes. For high-sensitivity detection the electrophoresis buffer
contains the fluorescent dye SYBR Green I. Laser-induced fluorescence
detection is obtained by excitation at 488 nm and signal collection at
520 nm. Specificity and reproducibility of the protocols were
established by using samples from 75 Italian families with
21-hydroxylase deficiency already genotyped by allele-specific
oligonucleotide hybridization or direct sequencing. Whereas dot-blot is
time consuming because of the high number of hybridizations with
radioactive probes, this present protocol is more rapid, giving
sufficient separation on CZE after PCR reactions without
preconcentration or desalting of samples.
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Introduction
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Steroid 21-hydroxylase deficiency is a recessive inherited disease
accounting for ~90% of congenital adrenal hyperplasia
(CAH).1
Lack of 21-hydroxylation results in the
accumulation of 17-hydroxyprogesterone, and stimulates excessive
androgen production (1). The severe form of
this disease occurs with a frequency of 1:14 000 live births
(2). In this form prenatal virilization in females is
observed (simple virilizing, SV) and in 70% of cases it also causes
salt wasting (SW). In the classical form, prevention of virilization in
females is feasible by prenatal diagnosis and treatment with
dexamethasone starting at early stages of pregnancy (3).
The milder nonclassical form (late onset, LO), with postnatal
virilization, is more common, with variable frequencies in different
ethnic groups (1:27–1:2000). The LO form may also be asymptomatic,
called cryptic form (4). The 21-hydroxylase enzyme is
encoded by the P450c21-B gene, located within the HLA complex, on
chromosome 6p21.3. The B gene and a 98% homologous pseudogene form a
tandem repeat adjacent to C4B and C4A genes, respectively. A certain
heterogeneity of mutations in the active gene has been described, such
as whole gene deletion, gene conversion
(5)(6), and, more frequently, several common
point mutations. The majority of small rearrangements, which are also
present in the pseudogene, are probably the result of small-scale gene
conversions (7). We recently described the distribution of
different classes of mutations in the Italian population
(8)(9). In particular, the distribution of
gene rearrangements has been compared with that found in other ethnic
groups and the rearrangements have been correlated with clinical
phenotypes, including classic, nonclassic, and cryptic forms of the
disease.
Molecular diagnosis of the disease can be performed with direct methods
for identifying the different classes of mutations. Conventional
Southern transfer hybridization allows detection of gene deletion and
large-scale gene conversion, whereas point gene conversions can be
scored by a variety of PCR-based methods. Several protocols in which
selective amplification of the active gene is performed before
screening for different pathological mutations (10)(11)(12)(13)(14)
have been proposed. The development of rapid methods for direct
detection of mutations is of great importance, especially for prenatal
diagnosis of 21-hydroxylase deficiency, because prenatal treatment can
reduce or avoid virilization in affected female fetuses.
In this work we describe a new protocol for the simultaneous detection
of the most common point mutations in the 21-hydroxylase active gene,
by combining the PCR-based amplification-refractory mutation system
(ARMS) with capillary zone electrophoresis (CZE).
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Materials and Methods
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A protocol was established by using samples from 75 Italian
families with 21-hydroxylase deficiency already genotyped for gross
rearrangements by restriction mapping analysis and for point mutations
by PCR and either hybridization with allele-specific oligonucleotides
(ASOs) or direct sequencing (8)(9).
selective amplification of p450c21b genes
PCR was carried out on genomic DNAs isolated from peripheral blood
lymphocytes by standard procedures. To minimize allele drop-out, B
genes were selectively amplified in two overlapping fragments by using
the forward B-specific primer 606 with the reverse primer 2197 for the
first fragment and the forward primer -280 with the reverse
B-specific primer 1394R for the second one (Table 1
). Overlapping fragments were designed to share a quite large
region (606–1394) containing the mutations in intron 2 and
in exon 4. The PCR reaction was performed in the presence of 20 pmol of
each oligonucleotide, either 250 ng of genomic DNA or 5 ng of
constructs obtained by cloning into TA vectors (Invitrogen) patients'
amplified B gene sequences, and 2.5 U of Taq polymerase in a final
volume of 50 µL containing 10 mmol/L Tris-HCl pH 8.3, 50 mmol/L KCl,
1.5 mmol/L MgCl2, and 0.2 mmol/L each deoxynucleotide.
A first round of amplification was carried out for 30 cycles
(denaturation, 30 s at 94 °C; annealing, 30 s at 60 °C;
extension, 2 min at 72 °C). The PCR reaction was ended by incubating
for 5 min at 72 °C. A Gene Amp PCR system 9600 thermal cycler was
used (Perkin-Elmer). The amplified products were analyzed by 1%
agarose gel electrophoresis, and specificity of the system was
confirmed in patients with P450c21B homozygous deletion.
arms system
In the second round of PCR, 0.5 µL of the B gene-amplified
fragments, diluted 1:20, were amplified with primers specific for each
allele. Amplification reactions were identical to that described above,
except for use of 1 U of Taq polymerase. Amplifications were performed
by using a two-step protocol with 30 cycles of denaturation at 94 °C
for 30 s, followed by annealing/extension for 1 min at
temperatures indicated in Table 1
plus a 1-s extension/cycle. The final
annealing/extension step was 5 min followed by 15 min at 37 °C to
facilitate reannealing of complementary chains. In Table 1
the primer
sequences, the mutations detected, the annealing temperatures, and the
length of PCR products are listed. As positive and negative controls in
each set of reactions, DNA samples from subjects previously genotyped
as homozygous normal or homozygous mutant or heterozygous were
included.
Amplified products were analyzed in 2% agarose gels and by CZE. Before
CZE, PCR products with primers specific for the normal and mutated
sequences were pooled into the wild-type and mutant set, respectively
(see Table 1
).
capillary electrophoresis
CZE analyses were performed with a Beckman P/ACE System 5000
equipped with a laser-induced fluorescence (LIF) detector providing 488
nm excitation with a 520 nm bandpass filter. We used a 27 cm (20 cm to
the detector window) x 100 (i.d.) capillary coated by a slight
modification of Hjertén's protocol (15) with a
novel monomer (N-acryloyl amino propanol) that combines high
hydrolytic stability with high hydrophilicity
(15)(16)(17). The capillary was filled with a
sonicated solution of 30 g/L hydroxyethyl cellulose (HEC; 27 000
Mr) from Polysciences dissolved in 89 mmol/L
Tris–borate buffer with 1 mmol/L EDTA (TBE), pH 8.3, and a
1:30 000-diluted solution of SYBR Green I (FMC Bio Products) as
fluorescent dye. After 3 min of purging, the samples were injected
either by pressure or electrokinetically, depending on whether they
were previously desalted by a Centricon 30 (Amicon) or not. After each
run the capillary was washed for 5 min in water and then in TBE buffer.
The separation was performed at 100 V/cm at 25 °C external
temperature.
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Results
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The ARMS is a rapid and reliable method for detecting any mutation
involving single base changes or small deletions. Initially, 20-mer
sequence-specific primers were designed for all mutations. Only the
ARMS for the V281L substitution gave specific results by amplifying
target alleles. To increase the specificity of the other mutants, we
designed longer primers (30-mer), and a mismatched residue at the
penultimate position of primers was introduced. Mismatches were
selected as previously described (18). The calculated
melting temperature (Tm) of the oligonucleotides
in the nested ARMS was ~90–95 °C, allowing us to use a
two-temperature amplification profile. With those primers we observed a
good reproducibility by testing this system in CAH patients and the
previously genotyped parents. In all the samples analyzed by this
protocol, we have excellent agreement with the results obtained by ASO
or sequencing. In Fig. 1
are shown all the possible peaks that we can obtain in the
wild-type and mutant sets, after resolution in a 27-cm coated capillary
in the presence of 30 g/L HEC as sieving liquid polymer in TBE buffer.
We can see that also the polymorphism A/C in the same 655 position of
intron 2 mutation is well resolved. Easy identification of the two
systems of fragments can be achieved with acceptable and reproducible
separations (though not to baseline) of similar-size fragments (the
couples 172/180 bp and 243/252 bp).
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Figure 1. CZE detection of nested ARMS products.
CZE of the set of five mutant (MUT, upper tracing) and six
wild-type (WT, lower profile) ARMS-amplified DNA fragments
for detection of 21-hydroxylase deficiency. The corresponding mutations
are listed in Table 1
. CZE conditions are as described in
Materials and Methods. RFU, relative fluoresence units.
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We next report a series of family profilings, showing how CZE can
identify carrier parents and affected children in families with steroid
21-hydroxylase deficiency. In Figs. 2
through 5 the results of CZE of nested ARMS with the
wild-type-specific and with the mutant-specific primers are shown in
parallel for each subject in these families. In Figs. 2
and 3
, two families with the classic form of the disease in the
offspring and the cryptic form in the parents are analyzed. In Figs. 4
and
5, two additional families with the SW form of the disease are
shown. In each family study, the differential analysis of the spectrum
of bands produced by each member of the family when amplifying the
wild-type and mutant regions allows a precise diagnosis.
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Figure 2. Family profiling by CZE for detection of 21-hydroxylase
deficiency.
In this family the affected child, with the SW form, was homozygous for
the mutation in intron 2. The father was heterozygous for the intron 2
mutation; on the left part of the electropherogram the
normal peak is present, as is the mutated one on the right
panel. The mother, having the cryptic form, was found to be
double-heterozygous for the severe mutation in intron 2 and also for
the milder V281L missense mutation. In her second pregnancy, early
prenatal diagnosis, by direct detection of mutations, revealed that she
transmitted to the fetus the mutation V281L, which was not transmitted
to her previous affected child. Left panel: wild type;
right panel: mutant type.
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Figure 3. Family profiling by CZE.
In this family the father, with the cryptic form, was
double-heterozygous for the I172 N and the Q318X mutations, both
associated with the classic form of the disease. The affected daughter,
with the SV form, inherited from her father the I172 N mutation and
from the mother the 8-bp deletion in exon 3. All experimental CZE
conditions are as in Materials and Methods.
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Figure 4. Family profiling by CZE.
In this family the SW patient was double- heterozygous for Q318X and
intron 2 mutations, transmitted from the mother and the father,
respectively. The carrier brother inherited the mutation Q318X from the
mother and the healthy gene from the father. All experimental CZE
conditions are as in Materials and Methods.
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Discussion
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We had previously reported the use of CZE in sieving liquid
polymers for the analysis of 21-hydroxylase deficiency
(19). In that paper detection of a single mutation,
namely, an 8-bp deletion in exon 3 of the B gene, was reported. The two
products (normal, 135-bp, and disease-linked, 127-bp, fragments) were
easily separated in 6% T liquid linear polyacrylamide by exploiting
the natural UV absorbance of DNA fragments at 254 nm.
In the present report we developed a protocol that presents the
advantage of rapidly and specifically detecting five common
mutations in steroid 21-hydroxylase deficiency by using a nested
ARMS coupled with detection of amplified products by CZE. In comparison
with dot-blot technique, which is time consuming because of the high
number of hybridizations with specific oligonucleotides, this protocol
is more rapid, nonradioactive, and does not require ethidium bromide
staining.
The major problem of this method is to differentiate between failed PCR
and homozygosity. In our protocol, we included as external controls DNA
samples from subjects who had been previously genotyped, either
homozygous or heterozygous. The control PCR reactions were set up in
parallel with the DNA samples to be tested and amplifications were run
simultaneously. In addition, by amplifying DNA samples in 11 different
reactions we had indirect evidence about the quality of templates.
Finally, a very important point is family genotyping, which is
advisable to confirm transmission of mutations. Moreover, direct
genotyping is crucial in families in which a cryptic parent is present;
in this case, indirect methods do not allow identification of
cryptogenic alleles. Therefore, the development of a rapid method for
direct detection of mutations in the P450c21-B gene is an important
goal, especially for the prenatal diagnosis of the disease when
considering the possibility of performing, in pregnancies at risk,
prenatal treatment.
The introduction in CZE of fluorescent labels (the SYBR Green I,
recently reported in CZE by Skeidsvoll and Ueland (20)),
with detection at 520 nm, allows an extraordinary increment of
sensitivity (by at least 103 to 105) in sample
analysis. Additionally, concentration and desalting steps are not
required when using LIF detection (21)(22)(23). As the sieving
liquid polymer we used a commercially available HEC, which has low
viscosity and can be pumped automatically into the capillary, giving
maximum run-to-run reproducibility. Finally, all the electrophoretic
runs can be manipulated with computer algorithms, which allows the
overlay of several runs for easy comparison among different samples and
consequent easy assessment of correct genotypes in families.
In conclusion, CZE in sieving liquid polymers of PCR-amplified DNA
fragments is rapidly becoming the technique of choice in genetic
analysis, viral and microbial infection detection, molecular biology,
and forensic medicine. Several reviews have already appeared,
highlighting the technical aspects of this methodology and the
achievements in these various fields (24)(25)(26)(27)(28).
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Figure 5. Family profiling by CZE.
In this family, the affected children (SW1,
SW2) were both homozygous for the mutation in intron 2. The
daughter and the parents were heterozygous for the same mutation,
having the C polymorphism on the normal allele. All experimental CZE
conditions are as in Materials and Methods.
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Acknowledgments
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P.G.R. is supported by the European Community, Bio Med II (no.
BMH4-CT96–1158), by Associazione Italiana Ricerca sul Cancro (AIRC),
by Telethon-Italy (no. E-555), and by CNR (Roma, Italy) Comitato
Tecnologico.
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Footnotes
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1 Nonstandard abbreviations: CAH, congenital adrenal hyperplasia; SV, simple virilizing; SW, salt wasting; LO, late onset; ARMS, amplification-refractory mutation system; CZE, capillary zone electrophoresis; ASO, allele-specific oligonucleotide; LIF, laser-induced fluorescence; HEC, hydroxyethyl cellulose; and TBE, Tris–borate–EDTA buffer. 
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Abstract
Introduction
