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Molecular Diagnostics and Genetics |
1
Department of Pathology, University of Michigan, 1301 Catherine Street, Ann Arbor, MI 48109-0602.
2
Department of Pediatrics, University of Minnesota,
Minneapolis, MN 55455.
a Author for correspondence. Fax 734-936-2756; e-mail akilleen{at}umich.edu.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Most mutations in CYP21 appear to be attributable to gene
conversion events that transfer deleterious sequences from
CYP21P (6). Approximately 20–30% of
CYP21 mutations are caused by deletions of the gene, usually
in conjunction with a deletion of the neighboring C4B gene
(Fig. 1
). This deletion pattern is often seen as part of an extended
MHC haplotype that includes the HLA markers A3, B47, and DR7. The most
frequent gene conversion event, "i2g" (A,C656G), which leads to a
premature 3' splice site, occurs in intron 2. This intron also contains
the largest number of sequence differences from the corresponding
intron in CYP21P. Published sequences of CYP21
show polymorphisms at several nucleotides within intron 2, including
nt395, nt419, nt453, and nt601, although the frequencies of these
polymorphisms are unknown in wild-type alleles
(2)(7)(8)(9). The numbering of nucleotides is
based on the published sequence of Higashi et al. (2). A
rare polymorphism at the PvuII restriction site near nt623
in this intron, originally detected by Southern analysis, has been
reported to occur at a frequency of 2% (10).
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Frequent intragenic polymorphisms could function as useful markers for determining the segregation of wild-type and mutant alleles in genetic studies of CYP21 and could facilitate identification of CYP21 gene deletions. We examined the sequence of intron 2 of CYP21 in seven clinically healthy subjects known to be heterozygous for deletions of the gene and determined the frequencies of the nt395, nt453, and nt601 polymorphisms, which alter restriction sites, in 48 wild-type CYP21 alleles.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A nested PCR strategy was used to amplify portions of CYP21. The use of a nested technique was found to yield PCR products that were purer and quantitatively more consistent than those attainable with single PCR reactions. In addition, several of the DNA samples had been stored for up to 10 years and showed some degradation. These could be successfully amplified using a nested approach.
The first-round reaction used primers 1F and 832R, which amplify
sequences from both CYP21 and CYP21P; the
second-round reaction used primers 335F and b73R. Primer b73R
recognizes the 8 bp in exon III of CYP21 that are deleted in
CYP21P, and therefore can be used to selectively amplify the
former gene. This primer has been described previously by Day et al.
(13). For experiments involving cleavase fragment length
polymorphism (CFLP) analysis, a 5'-biotinylated version of primer 335F
was used for subsequent chemiluminescent detection of CFLP bands. The
sequences and locations of hybridization of these primers are shown in
Table 1
. First-round PCR reactions were performed for 30 cycles with
the following reagent concentrations: 20 mmol/L Tris-HCl, pH 8.4, 50
mmol/L KCl, 1.5 mmol/L MgCl2, 200 µmol/L each dNTP, 200
nmol/L each primer, 50–100 ng genomic DNA, and 2.5 U of Taq polymerase
(Life Technologies) in a reaction volume of 50 or 100 µL. For the
second-round reactions, PCR products from the first round were diluted
1000-fold with DNase-free water (Sigma Chemical Co.), and 1 µL of
this dilution was used as template for the second round (volume, 100
µL), using the same reagent concentrations for 25 cycles. Annealing
conditions were 60 °C for round 1 and 69 °C for round 2
reactions, both for 30 s. Extension conditions were 72 °C for 2
min (round 1) or 1 min (round 2). The latter reactions used a manual
"hot-start" technique.
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cflp
For experiments involving CFLP analysis, exonuclease I digestion
was performed to hydrolyze unincorporated primers and other
single-stranded DNA present in the second-round PCR products. The
magnesium concentration was increased to 5 mmol/L, and then 10 U of
exonuclease I (Amersham Life Sciences) was added per microliter of PCR
solution. The solution was incubated for 30 min at 37 °C; the
exonuclease I was then inactivated by heating to 70 °C for 30 min.
Finally, the PCR products were purified on High Pure PCR purification
columns from Boehringer Mannheim, using the manufacturer's protocol
except that the final elution of DNA was with DNase-free water rather
than the supplied buffer.
Cleavase I reactions were performed using kits from Third Wave Technologies. Reaction conditions were as follows: 10 mmol/L 2-(N-morpholino)propanesulfonic acid, pH 7.5, 0.5 g/L Tween 20, 0.5 g/L Nonidet P-40, 0.4 mmol/L MnCl2, and 50–100 fmol of DNA in a 10-µL reaction volume. The DNA was denatured by heating to 95 °C for 20 s, and then cooled to 55 °C. After 15 s, 2.5 µL (62.5 U) of cleavase I was added, and the reaction was allowed to proceed for 1 min, at which time it was stopped by the addition of 8 µL of stop solution consisting of 950 mL/L deionized formamide, 10 mmol/L EDTA, pH 8.0, 0.5 g/L xylene cyanol, and 0.5 g/L bromphenol blue.
Electrophoresis of CFLP fragments was performed in 8% denaturing polyacrylamide gels, 15 cm x 17 cm x 0.5 mm, containing 0.5x Tris-borate-EDTA buffer and 8 mol/L urea. Gels were prerun at 20 W for 30–60 min. CFLP products were denatured at 90 °C for 90 s, and then 9 µL was loaded in each lane. GelMarker I biotinylated molecular weight markers (Research Genetics) in the range of 50–700 bp were diluted 10-fold in loading dye, and 5 µL was loaded after heat denaturation. Gels were electrophoresed for 55 min at 20 W so that the xylene cyanol tracking dye had migrated ~90% of the length of the gel. Transfer of DNA to Nytran Plus nylon membrane (Schleicher & Schuell) was performed by capillary transfer overnight.
After DNA transfer, the membrane was blocked twice for 15 min using 100 mL of blocking solution (Amersham) on a rocking platform. Streptavidin-alkaline phosphatase conjugate [4 µL of 1000 U/mL stock solution (Amersham)] was diluted in 100 mL of blocking solution and applied to the membrane for 15 min with rocking. The membrane was then washed three times with SAAP buffer (100 mmol/L NaCl, 50 mmol/L Tris base, pH 10.0) containing 1 g/L sodium dodecyl sulfate for 5 min, and then three times with SAAP buffer containing 1 mmol/L MgCl2 for 5 min. After the final wash, the membrane was placed in a clear polythene bag (Oncor). SAAP buffer (3 mL) containing 30 µL of 100x CDP-Star (Boehringer Mannheim) was added and allowed to coat the membrane for 3 min. The excess buffer was discarded, and the bag was sealed. Chemiluminescent bands were then visualized by placing the bag directly against Kodak XOMAT-AR film in the dark for 30 min, after which the film was developed.
dna sequencing
DNA sequencing of the purified PCR products prepared for the CFLP
reactions was performed with an ABI 373 sequencer using standard
methodologies.
pcr/restriction enzyme digestion
Second-round PCR products (10 µL) were digested with either 2.5
U of HaeIII (Life Technologies) or 2 U of BbvI
(New England Biolabs) after addition of 1 µL of the manufacturer's
10x restriction buffer. After incubation for 2 h at 37 °C,
restriction products were electrophoresed in 2% agarose gels
containing 1 mg/L ethidium bromide and photographed on an ultraviolet
transilluminator.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The first was the presence or absence of a doublet of CFLP
bands located ~60 bp from the 5' end of the sense strand. A second
CFLP polymorphism, characterized by the absence of a CFLP band ~190
bp from the 5' end of the sense strand, was seen in one subject.
Sequence analysis demonstrated that the first CFLP polymorphism
correlated with a pair of nucleotides that are in linkage
disequilibrium in the samples studied: C/T at nt395 and A/C at nt419,
the two bands being present when the nucleotides at these positions
are, respectively, C and A, and absent when they are T and C. The
second CFLP polymorphism correlated with two nucleotide substitutions
at nt601 and nt605.
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Sequence analysis showed several additional polymorphisms. Of these,
one was seen in more than two subjects: C/T at nt453 (Fig. 3
). A deletion of G at nt564 was seen in two subjects. Of all the
different polymorphisms, three occur at restriction enzyme recognition
sites: nt395 and nt601 are in HaeIII restriction sites; and
nt453 is in a BbvI recognition site. There are also
additional restriction sites for both of these enzymes within the
amplified region: one HaeIII site is near nt614; and one
BbvI recognition site is near nt623. Restriction with
BbvI gives rise to a constant fragment of 125 bp and
polymorphic fragments of 278 bp when nt453 is T or 169 and 109 bp when
nt453 is C. It should be noted that the restriction cutting site for
BbvI is offset from the recognition site. The
BbvI recognition site partially overlaps the previously
described PvuII recognition site, which is rarely
polymorphic (10). Restriction with HaeIII
produces a constant fragment of 118 bp and polymorphic fragments of
either 265 bp when nt395 is T or 206 and 59 bp when nt395 is C. If the
polymorphic HaeIII site at nt601 is also mutated, the latter
fragments are 15 bp longer.
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frequencies of polymorphisms
The frequencies of the HaeIII and BbvI
polymorphisms, which fall on restriction sites, were determined in DNA
samples from 24 Centre d'Étude du Polymorphisme Humain (CEPH)
subjects in the parental generation (48 chromosomes). The frequency of
each polymorphism was as follows: 395C, 8 of 48; 395T, 40 of 48; 453C,
34 of 48; and 453T, 14 of 48. The frequencies of the genotypes at these
positions are shown in Table 2
. Values of 
for the observed and expected
distributions of genotypes for the nt395 and nt453 polymorphisms were
3.83 and 0.23, respectively, (P >0.05 with 2 degrees of
freedom for each polymorphism). The distributions are consistent with
Hardy-Weinberg equilibrium. The nt601A polymorphism was seen in only 5
of 48 chromosomes.
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Inheritance of the nt395 and nt453 polymorphic markers was studied in
families with known deletions of CYP21. An illustrative
analysis of these markers in an informative family with deletions of
CYP21 is shown in Fig. 4
. Each parent is heterozygous for the CYP21 deletion
associated with the HLA-A3, B47, DR7 haplotype. The affected child, who
is known to be homozygous for the deletion haplotypes, has not
inherited markers of a CYP21 gene from either parent. The
presence of available template DNA for the second-round PCR reaction
was confirmed by gel analysis. The unaffected child has inherited
polymorphic markers from each parent, demonstrating the presence of two
distinct CYP21 alleles.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). It is therefore of value
to test both these markers when screening for polymorphisms. The C/T
transition at nt395 occurs at a CG dinucleotide, a frequent context for
this kind of mutation (14). The linked polymorphisms at nt395/nt419 and those at nt601/nt605 were initially observed by CFLP analysis. The polymorphism at nt453 did not alter the CFLP patterns under the experimental conditions used, nor did the single nucleotide polymorphism (A/G) at nt683, which produces a lysine/arginine (AAG/AGG) codon substitution in exon III. The detection of polymorphisms by CFLP depends on the recognition and cleavage of sequence-specific secondary structure in the target DNA (15). It may be possible to detect these polymorphisms by CFLP analysis of the antisense strand or with the use of other cleavase enzymes. However, this issue was not investigated further because the frequent nt395 and nt453 polymorphisms can be detected easily with commercially available restriction enzymes, using a PCR/restriction enzyme digestion technique. For both of these enzymes, there are also constant restriction sites in the amplified region of CYP21, which can serve as internal controls for restriction endonuclease cutting.
These polymorphisms are sufficiently frequent to be of use in following the segregation of CYP21 alleles in genetic studies. They can also be used to follow the segregation of mutant CYP21 alleles in families affected by 21-hydroxylase deficiency. In general, direct mutation detection is preferred for molecular diagnostic studies because methods based on the linkage of polymorphic markers are subject to errors arising from recombination events, new mutations, non-paternity, and mistaken diagnosis in a proband, and cannot be applied if a pedigree is uninformative for the markers tested. However, in the case of CYP21 mutation analysis, the presence of the highly homologous CYP21P complicates the routine application of direct mutation testing. To selectively amplify the CYP21 gene for analysis by PCR, gene-specific primers must be used. Because of the high frequency of gene conversions from CYP21P to CYP21, critical sequences for CYP21 gene-specific amplification may be altered, preventing amplification of the expected gene. Furthermore, frequent "back conversions" of CYP21 sequences to CYP21P have been reported (13). Selection of a primer sequence that coincides with such a mutation might lead to undesired amplification of portions of CYP21P and thus, theoretically at least, to the possibility of mistaken identification of a mutation. In addition, it may not always be possible to identify a mutation in affected individuals by direct mutation testing, and several investigators have used other polymorphic markers within the MHC, such as HLA typing or MHC microsatellites, when evaluating relatives of a known patient with congenital adrenal hyperplasia (16). Analysis of the segregation pattern of the polymorphisms described in the study may provide additional useful genetic information when evaluating relatives of an affected patient.
These polymorphisms may also be useful in identifying deletions of CYP21, which account for ~20–30% of all mutations (6). The currently preferred method to detect deletions of CYP21 is to perform Southern analysis on TaqI- and BglII-restricted DNA, using a 21-hydroxylase probe. Relative gene dosages of CYP21 and CYP21P can then be assessed by densitometry. If a family is informative for the polymorphic markers described in this study, a gene deletion leads to the absence of parental markers in an affected offspring. However, one must bear in mind that detection of these markers depends on the successful amplification of the CYP21 alleles. The downstream primer b73R is specific for the 8 bp in CYP21 that are deleted in CYP21P. Gene conversion events that transfer this deletion to CYP21 produce a mutant allele that would not be expected to amplify when the PCR approach described here is used. This pattern might be confused with a CYP21 deletion. An alternative CYP21-specific primer could be used if the family was known or suspected to have a gene deletion, for example, from HLA typing or Southern analysis. Other investigators have reported CYP21-specific primers (13)(17). Furthermore, the occasional dropout of alleles during PCR amplification has been observed in intron 2 of CYP21 (18). For these reasons, the markers described here must be used cautiously, and in conjunction with other molecular genetic studies, for diagnostic purposes.
The data presented in this study confirm the high frequency of polymorphism found in intron 2 of CYP21. These markers may be of use in studying the mechanisms of gene conversion and in determining the extent of gene conversion events, which appear to give rise to the majority of mutations in 21-hydroxylase deficiency alleles. In addition, because of their location in the class III region of the HLA complex, these markers may be of use in studies of the genetics of the human MHC.
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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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  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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R. W.K. Chiu, T. K. Lau, P. T. Cheung, Z. Q. Gong, T. N. Leung, and Y.M. D. Lo Noninvasive Prenatal Exclusion of Congenital Adrenal Hyperplasia by Maternal Plasma Analysis: A Feasibility Study Clin. Chem., May 1, 2002; 48(5): 778 - 780. [Full Text] [PDF]
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E. Vilain CYPs, SNPs, and Molecular Diagnosis in the Postgenomic Era Clin. Chem., December 1, 1998; 44(12): 2403 - 2404. [Full Text] [PDF]
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indicates a base deletion. HaeIII and BbvI
restriction sites are shown with single and double
underlining, respectively.