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Articles |
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Department of Medical Research, Division of Molecular Medicine, China Medical College Hospital, No. 2, Yue-Der Road, Taichung 404, Taiwan, Republic of China.
2
Department of Obstetrics and Gynecology, Veterans
General Hospital-Taipei, Taipei 11217, Taiwan, Republic of
China.
3
Institute of Molecular Biology, Academia Sinica,
Nankang, Taipei 115, Taiwan, Republic of China.
4
King Car Food Industrial Co., Ltd., Yuan-Shan Research
Institute, No. 86, Chen-Hsiang Rd., Yuan-Shan I-Lan 264, Taiwan,
Republic of China.
a Author for correspondence. Fax 886-3-9228030; e-mail hhlee{at}Kingcar.com.tw
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We developed a rapid and direct method to detect a chimeric CYP21P/CYP21 gene that uses a 3'-specific primer for the CYP21 gene and two different 5' primers for both CYP21 and CYP21P to amplify the wild-type CYP21 and the chimeric CYP21P/CYP21 genes. A secondary PCR that can differentiate the chimeric from the wild-type gene was also performed. The PCR product was directly analyzed on agarose gel.
Results: After careful titration, we found that earlier failure to detect the chimeric CYP21P/CYP21 gene could be caused by unequal concentrations of two independent alleles as the PCR template or by the lack of primers to amplify chimeric molecules. We successfully amplified the chimeric gene using our improved method.
Conclusions: The chimeric CYP21P/CYP21 is present in a large portion of congenital adrenal hyperplasia patients. By adding a CYP21P/CYP21-specific primer, we were able to amplify and detect both homozygous and heterozygous chimeric genes. Therefore, our new PCR-based assay is a more effective way to analyze congenital adrenal hyperplasia mutations.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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More than 90% of CAH cases are caused by mutations of the CYP21 gene (2)(4)(5). Approximately 75% of the defective CYP21 genes are generated through intergenic recombination so that CYP21 carries one or more deleterious mutations usually found in the neighboring CYP21P (6). These mutations are termed "apparent gene conversion" (7)(8). Approximately 20% of alleles have a 30-kb deletion that includes the 3' end of CYP21P, all of C4B, and the 5' end of CYP21. This allele carries a single nonfunctional chimeric gene with its 5' and 3' ends corresponding to CYP21P and CYP21, respectively (9). This is presumably related to unequal crossover during meiosis (9).
The CYP21 and CYP21P genes both are 3.2 kb long
and share 98% homology in exons (10)(11). The
mutation of the aberrant splicing site at nucleotide (nt) 656 of intron
2 (A/C
G) is the most common mutation in CAH in Taiwanese
(12)(13). In addition, there is a seemingly high
rate (~10–30%) of apparent gene deletions
(12)(13). During molecular analysis using the
differential PCR that we developed (12), we noticed that we
could preferentially amplify the allele containing the intron 2 nt 656
mutation, which sometimes is also combined with an 8-bp deletion at
exon 3 (codons 111–113). The unknown mutated allele, however, could
not be amplified. This allele dropout may result from the formation of
the chimeric CYP21P/CYP21, which lacks a specific
primer for amplification. Because our primers were designed
specifically for the CYP21 gene, they would not be able to
amplify the chimeric mutant CYP21P/CYP21 gene.
Apparently, we need another analysis method in these cases.
To circumvent the above problem, and as a step toward accurate clinical diagnosis, we describe here a mixed-primer PCR that allows the amplification of both the wild-type CYP21 and the chimeric CYP21P/CYP21 genes. This approach was the first attempt to detect gene fusion in steroid 21-hydroxylase in Chinese CAH patients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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pcr and molecular analysis of the cyp21 gene
For CYP21 gene amplification, a differential PCR was
used as described previously (12). To amplify both the
chimeric CYP21P/CYP21 and CYP21
molecules, primers BF1, AF1, and 21BR (Table 1
) (12) were mixed in a 50-µL reaction. A secondary
PCR was then performed as described previously (12). The
secondary PCR products were routinely run in 1.5% agarose gels, and if
necessary, 2.5% Metaphor (FMC BioProducts) agarose gels were used. To
detect the three most common mutations in Chinese at codons 172 and 356
of intron 2, we first amplified the CYP21 gene (primers
BF1/21BR) (12), followed by the amplification-created
restriction site (ACRS) method (12). If no mutation was
found, single-strand conformation polymorphism analysis using 11 pairs
of primers designed for quick screening of the CYP21 gene,
including 10 exons and the 5'-untranslated region, was applied
(13).
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primers
The primers used for detecting the chimeric
CYP21P/CYP21 molecule and secondary PCR for
determining the extent of gene conversion and crossover by
size-differential amplification are listed in Table 1
. The forward
(C3B) and the reverse (C4) primers for ACRS detection of intron 2 nt
656 are also listed in Table 1
.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A site at nt 656 of intron 2 (A/CG) of the CYP21 gene was
tested using the specific primers C3B and C4 (Table 1
) (12).
A 132-bp (Fig. 1
, lane 1) or a 124-bp (Fig. 1
, lane 2) PCR product was derived
from the CYP21 and CYP21P genes, respectively, of
a healthy individual. The mutant CYP21P (124 bp) was
digested into a 93-bp and a small 31-bp fragment by SacI,
whereas the 132-bp wild-type CYP21 gene (Fig. 1
, lane 6)
could not be digested (Fig. 1
, lane 7). To demonstrate whether template
concentration during the first PCR affects amplification in the second
round, equal concentrations of the 3.3-kb primary PCR products from
both CYP21 and CYP21P were mixed and subjected to
a secondary PCR amplification. The same amounts of the 132- and 124-bp
PCR products were obtained (Fig. 1
, lane 3). Likewise, after
SacI digestion, an equal concentration of wild-type (132 bp)
and mutant (93 bp) bands (Fig. 1
, lane 8) was also present. When the
template ratio was changed to 5:1 for CYP21 and
CYP21P, the mutant secondary PCR product was present as a
faint band compared with the wild-type band (Fig. 1
, lane 4). This was
better seen when secondary PCR products were digested with
SacI (Fig. 1
, lane 9). Conversely, amplification of a 5x
concentration of CYP21P to a 1x concentration of
CYP21 (Fig. 1
, lane 5) followed by SacI digestion
produced a clear mutant band (93 bp) and a faint wild-type one (132 bp;
Fig. 1
, lane 10). This lack of amplification attributable to the low
concentration of the template was because PCR amplification is a
competitive reaction. The unequal amplification during the primary PCR
reaction led to failure to detect the intron 2 mutation. In addition,
the same phenomenon was also observed for other common mutations such
as at codon 172 (IleAsn) and codon 356 (ArgTrp; data not shown).
The analysis indicates that allele dropout might occur when
amplification of both alleles in the primary PCR is unequal.
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chimeric cyp21p/cyp21 gene leads to
ambiguous mutation identification
The CYP21 and CYP21P genes have 90% (31 of
284 nt) (10) sequence homology in the intron 2 region, and
in particular, there is almost complete sequence identity for exons
4–10 (10)(11). The region between intron 2 and
the 3' end of exon 3 is considered the hotspot of recombination. This
is where microconversion frequently occurs (15). In
screening 94 CAH families with 21-hydroxylase deficiency, we detected
15 individuals with intron 2 mutations in one allele; however, the
mutation in the other allele was not detected by the ACRS method. In
addition, single-strand conformation polymorphism analysis in this
region failed to obtain a precise result. Therefore, it is possible
that a chimeric CYP21P/CYP21 gene, which cannot
be detected by the original ACRS method, may be present. The strategy
for verifying the existence of a chimeric
CYP21P/CYP21 gene is described in Fig. 2
A. We used a 3'-specific primer (primer 21BR) for the
CYP21 gene and two different 5' primers for both the
wild-type CYP21 (primer BF1) and the CYP21P
(primer AF1) in the chimeric CYP21P/CYP21 genes
during the primary PCR amplification (Table 1
). A 3.3-kb PCR product
was produced from both the CYP21 and the chimeric
CYP21P/CYP21 genes. To distinguish between
wild-type and chimeric genes, we performed a secondary PCR reaction
using a common 3' primer (IN3R) and two specific 5' primers for the
wild-type CYP21 gene (B1) and the chimeric gene (2HP),
respectively (Fig. 2B
). Secondary PCR products of 307 bp (B1/IN3R) and
281 bp (2HP/IN3R) could be amplified simultaneously (Table 1
).
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One example of this analysis is shown in Fig. 3
. The conventional PCR amplification for the primary PCR product
by BF1/21BR primers (12) followed by SacI
digestion of the secondary PCR product can detect only the wild-type
132-bp allele from both parents (Fig. 3A
, lanes M and F), whereas the
proband had only a 93-bp defective allele (Fig. 3A
, lane P). Similarly,
secondary amplification with the B1/2HP/IN3R primers detected only the
wild-type 307-bp allele from the parents (Fig. 3B
, lanes M and F). The
same procedure detected a single 281-bp allele from the proband (Fig. 3B
, lane P). When the same family members were analyzed by primary
amplification with mixed primers (BF1/AF1/21BR), both parents had the
wild-type 132-bp and the defective 93-bp bands (Fig. 3C
, lanes M and
F). Similarly, the analysis of size-differential amplification by mixed
primers (B1/2HP/IN3R) showed the parents (Fig. 3D
, lanes M and F) had
both the 281-p and 307-bp fragments. The proband (Fig. 3D
, lane P) had
only a 281-bp fragment. The results clearly demonstrated that both
parents had the chimeric CYP21P/CYP21 gene in one
of their chromosomes.
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Analysis of another CAH patient showed similar results (Fig. 4
A). A single 103-bp band representing the ACRS mutant in the
proband (Fig. 4A
, lane P) appeared when a pair of primers (BF1/21BR)
were used. In this case, the father (Fig. 4A
, lane F) had both the
wild-type (132 bp) and mutant (103 bp, without the 8-bp deletion)
bands, whereas the mother (Fig. 4A
, lane M) had an intense wild-type
band (132 bp) and a weak mutant band (93 bp, with the 8-bp deletion).
The presence of the weak mutant band probably is attributable to
mispriming of the primary PCR by the supposedly
CYP21-specific BF1/21BR primer pair. When three primers were
used for primary PCR, the 93-bp defective allele from the mother became
apparent (Fig. 4B
). It indicated that the proband (Fig. 4B
, lane P)
carried two mutant bands (103 and 93 bp), one of which was inherited
from the father (103-bp; Fig. 4B
, lane F) and one from the mother
(93-bp; Fig. 4B
, lane M), who had a chimeric gene in one allele. When
we used size-differential amplification (B1/2HP/IN3R) of the primary
PCR product amplified with two primers (BF1/21BR), both the parents
(Fig. 4C
, lanes M and F) and the proband (Fig. 4C
, lane P) had only a
307-bp wild-type band. Size-differential amplification using the
primary PCR product with mixed-primer amplification (BF1/AF1/21BR)
indicated that the proband (Fig. 4D
, lane P) had not only a 307-bp
wild-type band but also carried a 281-bp chimeric molecule, which was
inherited from the mother (Fig. 4D
, lane M). The father had one 307-bp
wild-type band. It indicated that there was no chimeric gene in the
father’s alleles. Our analysis established that the father carries the
mutation nt 656G on one chromosome and that the other is unaffected.
The mother has one wild-type chromosome, and the other is a chimera
containing the intron 2 nt 656G mutation plus the 8-bp deletion. This
analysis for the chimeric molecule of
CYP21P/CYP21 in CYP21 deficiency
demonstrated that in this case allele dropout from the analysis of the
chimeric CYP21P/CYP21 gene was attributable to
the lack of specific primers for this molecule in PCR amplification.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). It
demonstrated that when unequal amounts of primary PCR products are used
as a template for secondary amplification, allele dropout occurs. This
shows that a smaller amount of product from one allele cannot compete
with abundant product from another allele. This phenomenon is similar
to the amplification of trisomy 21 by homologous gene quantitative PCR
(24). The amplification of an unequal copy of chromosome 21
leads to an uneven amplification product.
In analyzing 94 unrelated CAH families, we detected 15 CAH patients
carrying a mutation at nt 656 of intron 2 (A/CG) without a combined
8-bp deletion at codons 111–113 in one allele. The cause of mutation
in the other allele was uncertain with differential PCR combined with
ACRS (12). Using the improved method, in which three primers
were added, we showed the presence of a chimeric
CYP21P/CYP21 gene in 9 CAH families. Only 6 of
the 15 patients had one gene deletion. The presence of a chimeric
CYP21P/CYP21 gene often is mistaken for gene
deletion. Thus, the frequency of gene deletion in CYP21
deficiency may not be as high as reported
(16)(17). As described earlier
(20)(22), allele dropout was attributable to
preferential amplification of DNA segments and was an artifact caused
by polymerase (25). This led to an excessively high
frequency of apparent homozygosity and genotyping discordance between
parents and probands. Our cases showed that the allele dropout was
related to the presence of chimeric molecules. The PCR product for the
CYP21 gene would be the major product, and because of the
PCR competition reaction, there is very little chimeric
CYP21P/CYP21 product. This study indicated that
the small amount of chimeric CYP21/CYP21P primary
PCR product cannot compete with the CYP21 PCR product if its
concentration is only one-fifth that of the more abundant product (Fig. 1
). This leads to the failure of the ACRS method to detect the
intron 2 mutation in the secondary amplification. The improved method
that uses three primers for primary PCR amplification could
successfully detect the chimeric gene in the detection of the intron 2
mutation. Therefore, we suggest that both unequal concentrations of two
independent alleles as a template for PCR amplification and the lack of
a primer that targets the chimeric molecule may lead to allele dropout
in the detection of CAH mutations.
We have successfully amplified two independent chromosomes that contain one of the chimeric genes. Combined with size differentiation, this leads to accurate genotyping. Our ability to demonstrate CAH allele dropout was based on the methodology of molecular techniques and intensive multiple-mutation typing and clinical information about this disease. When screening for other genetic traits containing homologous genes such as CYP11B1 and CYP11B2 hydroxylases, mixed primer amplification combined with size differentiation would help ensure that both chromosomes amplify.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  S. Parajes, C. Quinterio, F. Dominguez, and L. Loidi A Simple and Robust Quantitative PCR Assay to Determine CYP21A2 Gene Dose in the Diagnosis of 21-Hydroxylase Deficiency Clin. Chem., September 1, 2007; 53(9): 1577 - 1584. [Abstract] [Full Text] [PDF]
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 H.-H. Lee, S.-F. Chang, F.-J. Tsai, L.-P. Tsai, and C.-Y. Lin Mutation of IVS2 -12A/C>G in Combination with 707-714delGAGACTAC in the CYP21 Gene Is Caused by Deletion of the C4-CYP21 Repeat Module with Steroid 21-Hydroxylase Deficiency J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2726 - 2729. [Abstract] [Full Text] [PDF]
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 P. F.J. Koppens, T. Hoogenboezem, and H. J. Degenhart Carriership of a defective tenascin-X gene in steroid 21-hydroxylase deficiency patients: TNXB -TNXA hybrids in apparent large-scale gene conversions Hum. Mol. Genet., October 2, 2002; 11(21): 2581 - 2590. [Abstract] [Full Text] [PDF]
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N. Krone, A. Braun, S. Weinert, M. Peter, A. A. Roscher, C.-J. Partsch, and W. G. Sippell Multiplex Minisequencing of the 21-Hydroxylase Gene as a Rapid Strategy to Confirm Congenital Adrenal Hyperplasia Clin. Chem., June 1, 2002; 48(6): 818 - 825. [Abstract] [Full Text] [PDF]
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