JMD 2001, Vol. 3, No. 2
Copyright © 2001 American Society for Investigative Pathology & Association for Molecular Pathology
An Overview of Molecular Diagnosis of Steroid 21-Hydroxylase Deficiency
Catherine E. Keegan* and
Anthony A. Killeen
From the Departments of Pediatrics
*
and Pathology,
University of Michigan, Ann Arbor, Michigan
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Introduction
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Steroid 21-hydroxylase deficiency is the most frequent
cause of congenital adrenal hyperplasia (CAH), a group of inborn errors
of adrenal steroid biosynthesis.1
Approximately 1 in
14,000 newborns is affected with the classic form of the disease. A
milder, late-onset or non-classic form that is clinically evident in
approximately 1 in 1000 females also exists. Both forms are the result
of mutations at the same locus and are inherited as autosomal recessive
traits. Very rarely, 21-hydroxylase deficiency can result from
uniparental disomy.2
The primary laboratory means of
diagnosis of this disease is by measurement of serum
17-hydroxyprogesterone, which is elevated in subjects with deficiency
of this enzyme. This testing is also used in newborn screening programs
that screen for 21-hydroxylase deficiency. Although biochemical testing
remains the mainstay of diagnosis, molecular methods have acquired an
essential role for identification of mutations and for assessment of
at-risk pregnancies. This article provides an overview of published
analytical methods for molecular diagnosis of steroid 21-hydroxylase
deficiency.
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Biochemical Abnormalities in CAH
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Approximately 90% of CAH result from deficiency of adrenal
steroid 21-hydroxylase. Owing to a deficiency of this enzyme activity,
there is a reduction in the ability of the adrenal cortices to produce
physiological quantities of adrenal glucocorticoids, primarily
cortisol. Synthesis of mineralocorticoids is also reduced, but because
of the relatively smaller physiological requirement for
mineralocorticoids, primarily aldosterone, impairment of enzyme
function that does not abolish all activity may not be manifest as
overt salt-wasting. Salt-wasting results from mineralocorticoid
insufficiency at the distal renal tubule where sodium is reabsorbed
from the glomerular filtrate and potassium and hydrogen ions are
excreted. This syndrome results in loss of body sodium and water,
metabolic acidosis, and the hyponatremia and hyperkalemia
characteristic of adrenal insufficiency. Clinically, the disorder is
characterized by life-threatening dehydration, vomiting, and diarrhea
in the first few weeks of life. Approximately one-third of patients
with classic CAH are able to produce enough mineralocorticoid to
prevent salt-wasting.The other major biochemical feature of CAH is increased production of
adrenal androgens. These compounds, which do not require 21-hydroxylase
activity for their synthesis, provide the outlet for steroid synthesis
in the adrenal gland. Owing to the lack of negative feedback that
cortisol normally provides to the pituitary and hypothalamus, ACTH
levels are elevated, which, in turn, stimulates the adrenal cortices.
This stimulation causes hyperplasia of the glands and, combined with
the metabolic block, results in high levels of production of adrenal
androgens. This effect occurs in utero and results in
variable degrees of virilization of the external genitalia of affected
female fetuses and newborns. Affected males generally appear normal at
birth.
The mainstay of therapy is replacement of the missing steroids,
electrolytes, and fluids. Surgical correction of the external genital
abnormalities is commonly required in affected females.
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Molecular Genetics of Steroid 21-Hydroxylase
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The functional CYP21 gene and the non-functional
pseudogene, CYP21P, are located on the short arm of
chromosome 6 in the class III region of the major histocompatibility
complex (MHC).3, 4
The functional gene is a member of the
cytochrome P450 family that specifically hydroxylates C21 on steroid
precursors in the adrenal cortex. Both genes are normally in tandem
arrangement with the genes encoding complement proteins 4A and 4B
(Figure 1)
. It is generally believed that this pattern results from an ancient
gene duplication event. Similar arrangements of 21-hydroxylase and
C4 genes are found in several other mammalian species,
suggesting that the duplication event preceded the radiation of
mammals in evolution and implying that it occurred at least 60 million
years ago.
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Figure 1. Arrangement of the 21-hydroxylase and C4 genes in the
class III region of the MHC. CYP21 is the functional
steroid 21-hydroxylase, CYP21P is a pseudogene. Both
C4A and C4B can encode functional
complement 4 proteins.
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The CYP21P pseudogene usually contains a number of
deleterious mutations that render it non-functional. These include a
premature splice mutation in intron 2, a 8-bp deletion in exon 3, a
series of point mutations that cause several missense mutations in exon
6, and a non-sense mutation in exon 8. Mutations that are commonly
found in CYP21 are shown in Table 1
. The majority of mutations that appear in CYP21 are either
gene conversions of deleterious sequences normally found in
CYP21P, or they are deletions. The severity of the phenotype
generally depends on the mutations present on both alleles, but the
phenotype is not entirely consistent among unrelated patients with the
same mutations, or even among siblings of the same genotype. In
general, gene deletions, the 8-bp deletion in exon 3, the exon 6
cluster of mutations, and Arg356Trp are associated with salt-wasting.
The intron 2 mutation can be found in either salt-wasting or simple
virilizing disease. Ile172Asn is associated with simple virilization,
whereas Pro30Leu and Val281Leu are commonly seen in patients with
non-classic disease. Based on consideration of the expected enzyme
activity of the less severely impaired allele, it is possible to
predict the phenotype with reasonable, but not complete,
certainty.5
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Linkage
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The initial report of linkage of human leukocyte antigen (HLA)
serotypes with 21-hydroxylase deficiency indicated the approximate
position of the locus.6
Linkage-based diagnostic methods
have been reported using various markers in the MHC. HLA
serotyping7
provided a mechanism to track mutant alleles
within families but could not be easily applied to prenatal samples
obtained by amniocentesis. However, serotyping revealed the existence
of important linkage disequilibrium between certain HLA haplotypes and
21-hydroxylase deficiency. One of these that involves the HLA haplotype
A3, B47, DR7, is associated with a 30-kb deletion that has removed most
of the CYP21 functional gene and the adjacent C4B
gene, and left a non-functional hybrid gene composed of 5' sequence
from CYP21P and 3' sequence from
CYP21.8
Molecular approaches to performing HLA genotyping signaled the first
DNA methods suitable for linkage-based prenatal diagnosis. These
methods involved the use of various class I DNA probes, a HLA-B
specific probe, and HLA class II probes.9, 10, 11, 12
These probes
were used to test restriction fragment length polymorphisms (RFLPs) on
Southern blots and took at least several days to perform. As with any
linkage-based diagnostic strategy, errors can arise from recombination
events between the marker locus and the disease locus, non-paternity,
new mutation or back mutation, or mistaken clinical diagnosis in the
proband. In addition, linkage-based approaches are only applicable to
families in which an informative polymorphism can be identified. More
recently, diagnostic approaches based on linkage have used polymorphic
microsatellite markers in the region that can be typed by polymerase
chain reaction (PCR).13, 14, 15
The principal advantage of
testing these is the ability to type the marker more rapidly than is
possible by using Southern blot analysis for RFLPs.
Our laboratory has identified several intragenic single nucleotide
polymorphisms (SNPs) in intron 2 and in intron 6 of CYP21
and CYP21P.16, 17
These can provide information
on the inheritance of alleles in families and are also useful in
identifying deletions that appear as apparent non-transmission of an
allele by a parent.
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Southern Blots and Deletions
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The most widely used method to detect the relatively common
deletion of CYP21 in affected families is Southern blot
analysis. The use of restriction enzymes that generate separate
fragments that contain CYP21 and CYP21P allows
for estimation of the relative dosage of CYP21. Standard
restriction enzymes used to digest genomic DNA include TaqI
and BglII. The expected sizes of restriction fragments
generated from these digestions are shown in Table 2
. It is also useful to probe Southern blots for the neighboring
C4 genes because deletions of CYP21 frequently
include C4B. TaqI-restricted DNA can be simultaneously
probed for both the C4 genes and the 21-hydroxylase genes
because of the non-overlapping RFLPs generated. DNA probes for Southern
blot analysis for the 21-hydroxylase and C4 genes are
available from the American Type Culture Collection (Manassas, VA).
In TaqI-restricted genomic DNA, CYP21 and
CYP21P are located on 3.7-kb and 3.2-kb restriction
fragments, respectively. Subjects with heterozygous deletions of
CYP21 can be identified by decreased intensity of the 3.7-kb
fragment relative to that of the 3.2-kb fragment. Subjects with
homozygous CYP21 deletion show absence of the 3.7-kb
fragment.
Difficulties with interpretation of Southern blots can arise from some
of the duplication and deletion patterns that can be found at the
21-hydroxylase loci. Deletions of CYP21P are fairly common,
being present in 9 to 14% of Caucasian chromosomes.17, 18
Because the dosage of CYP21 is determined by comparing its
signal strength to that of CYP21P on Southern blots, it
follows that a subject with deletions of both CYP21 and
CYP21P could be mistakenly thought to have a normal gene
dosage.13
Subjects with a duplication of either
21-hydroxylase gene might also give unexpected results on gene dosage
studies by Southern blot analysis. These issues emphasize the need for
careful family studies when evaluating a subject for gene
deletions.1
Pulsed-field gel electrophoresis may provide
additional information on gene dosage in unusual cases.19
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PCR
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Considerations in PCR Amplification of CYP21
As with many genetic diseases, PCR has been widely adopted for
amplification of the locus before specific mutation identification.
Owing to the presence of the highly homologous pseudogene,
amplification of CYP21 is not straightforward. Primers must
be selected that recognize sequences in CYP21 that are not
present in CYP21P. These primer binding sites must be
sufficiently specific for CYP21 such that CYP21P
cannot be amplified in the PCR through promiscuous priming. These
requirements severely limit the number of CYP21-specific
primer targets. Most authors have exploited two regions in which
CYP21 differs from CYP21P. These are the 8-bp
sequence in exon 3 that is present in CYP21 but deleted in
CYP21P, and the exon 6 cluster of mutations that is composed
of four nucleotide differences between the functional gene and
pseudogene. Using this approach, CYP21 can be amplified in
two overlapping fragments as shown in Figure 2
. After amplification, a variety of methods to detect the mutations
present can be used.
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Figure 2. Typical PCR method for amplification of CYP21 in two
overlapping fragments. The numbered boxes represent exons in
CYP21. The lines below represent the two PCR
fragments, which are approximately 1.3 kb and 2.2 kb in length,
respectively. Approximate primer locations and 5'-3' orientations of
the primers are shown with arrows at the end of the
fragments. To provide specificity for amplification of
CYP21, the primer that hybridizes to sequences in exon 6
(right end of the top PCR
fragment) recognizes a cluster of nucleotides
that differ from the corresponding nucleotides in CYP21P.
The primer that hybridizes to exon 3 (left end
of the bottom PCR fragment) recognizes the 8 bp
present in that exon, but which are absent from CYP21P. The
other primers can hybridize to either gene.
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Several caveats involving PCR amplification of CYP21 need to
be considered. First, several studies have reported the inability to
amplify both CYP21 alleles, particularly around intron 2,
the site of the important premature splice mutation, A655G or C655G.
This can result in subjects who are heterozygous for this mutation
appearing to be homozygous.20
A report indicates that this
artifact may be overcome through the use of a blend of DNA polymerases
that includes both Taq polymerase and Pwo
polymerase.21
Careful examination of the mutational status
of family members and of the segregation of linked microsatellites may
be useful in identifying this artifact.
Second, it is clear that CYP21 sequences can be
back-converted to CYP21P.22
This brings
sequences from CYP21 into the pseudogene. If
CYP21 primer binding sequences were introduced into
CYP21P, such conversion could conceivably lead to unintended
PCR priming of CYP21P and mistaken identification of
mutations that could be assumed to represent gene conversion events in
CYP21. Additional useful information may be obtained by
analysis of overlapping PCR products and by probing Southern blots with
gene-specific oligonucleotide probes for the primer-binding
regions.23
Detection of Mutations after PCR
Following gene-specific amplification of CYP21, some
method for detection of mutations is required. Various schemes have
been used, all of which have been used for mutation detection in other
genetic disorders. These include allele-specific oligonucleotide
hybridization, restriction fragment length polymorphism analysis,
ligase chain reaction, single-strand conformational polymorphism
(SSCP), cleavase fragment length polymorphism analysis, and
heteroduplex analysis. Some of these techniques can indicate only the
presence of a sequence variant and must be followed by DNA sequencing
for identification of a mutation.
Allele-Specific Oligonucleotide Probing
Allele-specific oligonucleotides have been used to probe
PCR-amplified DNA in a dot blot format.24
In this
approach, overlapping fragments of CYP21 are amplified,
immobilized on a membrane and probed with labeled probes for 9 common
mutations. This approach was used to identify mutations in 24 at-risk
pregnancies using DNA obtained by chorionic villous
biopsy.25
Mutations were identified in 95% of these
cases, and the molecular diagnosis was clinically confirmed in 96%.
Southern blot analysis was performed to detect gene deletions.
Allele-Specific PCR
The use of allele-specific PCR to detect CYP21
mutations has been reported.26
In this approach, primers
that are specific for eight common mutations and the corresponding
primers for the wild-type sequences are used to perform PCR. Gene
specificity is conferred by the use of primers for the exon 3 or exon 6
cluster of nucleotides that are characteristic of CYP21. Of
160 alleles typed using this method, mutations could be identified in
148. Deletions were detected by use of Southern blot analysis. Because
absence of amplification is a possible result, it is good laboratory
practice to include primers for an unrelated target to confirm the
presence of both amplifiable DNA and the reagents necessary for
amplification.
PCR with Restriction Enzyme Digestion
In this approach, CYP21 is amplified with gene-specific
PCR primers. Nested PCR reactions are performed using mutagenic primers
to introduce restriction sites as needed.27, 28
Restriction
of the resulting PCR products is used to detect the presence of
mutations. This approach has advantages and disadvantages. The use of
nested PCR reactions requires additional steps and, more importantly,
increases the possibility of PCR contamination because of the
additional manipulations required. An advantage of this technique is
that if additional restriction sites are present in the amplified
product, they can serve as important internal controls of restriction
enzyme activity.
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Ligase Chain Reaction (LCR)
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A few papers have reported the use of LCR for detection of
CYP21 mutations. The strategy used involves initial PCR
using the exon 3, 8-bp, and exon 6 cluster positions to provide targets
for gene-specific priming.15, 22
A total of 4 PCR products
are generated from CYP21 in overlapping segments. These PCR
products are subjected to multiplex LCR using oligonucleotides specific
for the mutations to be tested. By adding poly(A) tails to the LCR
oligonucleotides, LCR products of different length for each mutation
can be generated. Size separation of these allows for identification of
the mutation present. The advantages of this approach are that much of
the analysis can be multiplexed and, by using fluorescent labels on the
oligonucleotides, the LCR products can be rapidly analyzed using
sequencing-type apparatus.
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Screening for Mutations
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Cleavase Fragment Length Polymorphism
Cleavase fragment length polymorphism analysis was used to detect
the presence of several frequent polymorphisms in intron 2 of
CYP21.16
Subsequently, the utility of this
technique for screening for common salt-wasting mutations in
CYP21 was reported.29
These included the intron
2 splice mutation, exon 3 8-bp deletion, exon 6 cluster, and R356W. A
conclusion of the referenced study was that although the technique was
able to detect the mutations, a clear distinction between homozygosity
and heterozygosity was not always possible, which limits the usefulness
of the method for clinical diagnostic work.
SSCP
SSCP analysis has been used to detect mutations in
CYP21 after gene-specific PCR.30, 31
Unlike most
methods, SSCP does not identify the specific mutation, and subsequent
characterization is required. If samples containing known mutations are
run in parallel with an unknown sample, it may be possible to infer the
presence of the known mutation in the unknown sample if the SSCP
conformers have identical mobility. Given the technical complexities of
running SSCP gels that require rigorously controlled temperatures and
possibly multiple gels involving the use of glycerol, the advantages of
SSCP are not obvious, and the technique is not widely used in practice.
Heteroduplex Analysis
Heteroduplex analysis has not been extensively applied to
diagnosis of 21-hydroxylase deficiency, although it is a very widely
used technique for mutation screening in other disorders. Heteroduplex
analysis has been used to detect the T insertion
mutation32
and the value of heteroduplex analysis in
distinguishing 2 mutations in cis versus in
trans has been reported.33
As with SSCP
analysis, heteroduplex analysis does not provide characterization of
the mutation present; for this, sequencing or some other mutation
identification scheme is necessary.
Denaturing Gradient Gel Electrophoresis
Denaturing gradient gel electrophoresis of restricted genomic DNA
followed by Southern blot analysis using a 21-hydroxylase probe
demonstrated a striking degree of polymorphism of CYP21
among normal individuals.34
These polymorphisms were shown
to be inherited as Mendelian traits and could be used to track mutant
CYP21 alleles in families affected by CAH. Presumably these
polymorphisms observed on denaturing gradient gel electrophoresis are
due to the known polymorphisms in the exons and introns of
CYP21 and CYP21P; however, a complete
characterization of multiple alleles from normal individuals may reveal
additional polymorphic sites.
Prenatal Screening
Steroid 21-hydroxylase deficiency can be treated in
utero, and several papers have reported a benefit of prenatal
hormonal therapy on the severity of virilization in affected newborn
females.1
The usual situation in which prenatal diagnosis
is considered is that of a family with a previously affected child.
Prenatal diagnosis of 21-hydroxylase deficiency has been available for
several decades based on analysis of 17-hydroxyprogesterone in amniotic
fluid. This approach is of limited value because virilization of
affected females begins much earlier in pregnancy, probably around the
middle of the first trimester. Prenatal hormonal therapy, which
consists of administration of dexamethasone to the mother, needs to be
initiated as soon as pregnancy is detected and this should not await
the results of genetic testing.26
Genetic testing based on
DNA analysis can be performed on chorionic villi or on amniocytes. It
is very helpful to have DNA available from a previously affected child,
any other children, and both parents for both mutation identification
and for testing chromosomal inheritance using microsatellites. The
expected fraction of fetuses that can be expected to benefit from
prenatal therapy is 1/8 (ie, affected females). This treatment is not
without possible risks to the mother or the 7 of every 8 fetuses who
will not benefit from in utero exposure to
dexamethasone.1
Therapy can be stopped if genetic testing
indicates the fetus is a male, or is an unaffected
female.35
Newborn Screening
In many countries and States, congenital adrenal hyperplasia is on
the schedule of diseases that are tested in newborn screening programs.
Interest in laboratory identification of the disease is therefore
substantially greater than might be expected for a relatively uncommon
disease. DNA based diagnosis has not replaced routine biochemical
screening using 17-hydroxyprogesterone as the test analyte, and is
unlikely to do so in the near future. Reasons for this include the
relative cost of DNA testing compared with immunoassay, the complexity
of DNA testing for multiple mutations, particularly the common gene
deletions, and locus heterogeneity (approximately 10% of cases of CAH
are due to mutations in other genes, most often steroid
11ß-hydroxylase. Moreover, since 
2% of the population are
carriers, it is unclear what counseling or further testing should be
offered to parents of carrier newborns, who should provide this advice,
and what such interventions might involve in terms of personnel and
financial costs. Of course, these considerations are not unique to CAH
but are applicable to a number of other inborn errors of metabolism.
In most cases, DNA analysis is performed for prenatal assessment of
at-risk pregnancies or for mutation identification in affected
families. In the laboratory evaluation of families with CAH, several
different techniques are necessary. Southern analysis remains the most
reliable method to detect gene deletions. PCR followed by one of the
specific mutation detection schemes listed above is needed to identify
non-deletional mutations. The choice of which mutation detection system
is used depends on the laboratory; no method is clearly superior, and
no PCR method is free from the potentially confounding effects of gene
conversion at primer binding sites. Occasionally, DNA sequencing is
needed. Amplification of overlapping regions of CYP21 is the
standard method to amplify this gene. PCR of closely-linked
microsatellite markers still has a role in family studies.
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Footnotes
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Address reprint requests to Dr. A. A. Killeen, Department of Pathology, University of Michigan, 1301 Catherine St., Ann Arbor, MI 48109-0602. E-mail: akilleen{at}umich.edu
Accepted for publication March 2, 2001.
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D. Keen-Kim, J. B. Redman, R. U. Alanes, M. M. Eachus, R. C. Wilson, M. I. New, J. M. Nakamoto, and R. G. Fenwick
Validation and Clinical Application of a Locus-Specific Polymerase Chain Reaction- and Minisequencing-Based Assay for Congenital Adrenal Hyperplasia (21-Hydroxylase Deficiency)
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S. F. Dobrowolski, R. A. Banas, J. G. Suzow, M. Berkley, and E. W. Naylor
Analysis of Common Mutations in the Galactose-1-Phosphate Uridyl Transferase Gene: New Assays to Increase the Sensitivity and Specificity of Newborn Screening for Galactosemia
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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
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Top
Introduction
Biochemical Abnormalities in CAH
