Transcript Molecular diagnostics in congenital adrenal hyperplasia

MOLECULAR
DIAGNOSTICS IN
CONGENITAL
ADRENAL
HYPERPLASIA (21HYDROXYLASE
DEFICIENCY)
Introduction
Congenital adrenal hyperplasia (CAH), the inherited inability •
to synthesize cortisol, is one of the most common inherited
endocrine disorders. Cortisol is normally synthesized in the
zona fasciculata of the adrenal cortex in five enzymatic steps:
cleavage of the cholesterol side chain to convert cholesterol
to pregnenolone, dehydrogenation at the 3 β position to
yield progesterone and a series of hydroxylations at the 17α,
21 and 11 β positions to form 17α-hydroxyprogesterone, 11deoxycortisol and cortisol respectively. Typical signs of
androgen excess include masculinization of female external
genitalia and precocious pseudopuberty in both sexes.
Patients undergo rapid somatic growth with premature
epiphyseal closure resulting in short adult stature. Moreover,
elevated metabolites with mineralocorticoid activity, such as
deoxycorticosterone and its derivatives, cause hypertension
in about two thirds of patients. Patients are treated with
glucocorticoid replacement and with antihypertensive
therapy if necessary.
Congenital adrenal hyperplasia (CAH) is inherited as an •
autosomal recessive disorder and maps within the MHC locus
on chromosome 6. Indeed, linkage analysis using HLA Class I
and or Class II markers is often used for genetic studies of
suitable families. About 95% of the cases of CAH are due to a
limited number of mutations in the 21-hydroxylase gene (21OH or CYP21 - also known as P450C21B). Molecular analysis of
CAH is complicated due to the presence of a non-functional
pseudogene, CYP21P - also known as P450C21A) adjacent to
the functional 21-OH gene. Most of the pathogenic CYP21
mutations are found in the pseudogene, hence, it is assumed
that gene conversion is a common mutational mechanism in
CAH (see below). The other significant cause of CAH is gross
deletion of the functional 21-OH gene. Presumably the
presence of the tandem homologous CYP21 and CYP21P genes
provides the opportunity for unequal crossing over events
during meiosis.
CompGene's strategy for •
molecular diagnosis of
CAH first uses a Southern
blot analysis with a 21-OH
cDNA probe and the
restriction endonucleases
Taq I and a double digest
with Eco RI/Bgl II. As
shown in the cartoon
above, distinct fragments
and fragment dosage
patterns will be revealed
in the presence of
deletions and gene
conversions. An example
of Southern blot analysis
of the 21-hydroxylase
locus using the
restriction enzyme Taq I
is shown below.
A normal individual's DNA hybridization pattern is shown in •
lane 1. All other lanes show individuals affected by CAH and
illustrates the limited information that is obtained by this
diagnostic approach alone. Although the patient in lane 2
obviously has homozygous deletion of the 21-hydroxylase
locus as the cause of his disease, the other patients show
subtle or no obvious changes. For example, the patients in
lanes 5, 7 and 8 appear completely normal, the patient in lane
4 shows a half dose of the 3.7 Kbp Taq I fragment consistent
with gene deletion/conversion, while the patient in lane 6
shows an increased copy number of the pseudogene as
detected by increased intensity of the 3.2 Kbp Taq I
fragment.
Our method of evaluation of 21-hydroxylase mutations uses •
either "amplification refractory mutation analysis" where a
PCR product is obtained when a specific nucleotide is present
(either normal or mutant) in the 21-hydroxylase gene (P30L,
A/C656G, exon 3 deletion, I172N, exon 6 cluster, R356W) or, in
some cases, uses a specific restriction endonuclease to
detect the sequence change that results in the loss of a
restriction endonuclease site (Q318X, V281L). Examples of
direct mutation analysis are shown in the figures below. n
this first figure, the upper panel shows PCR results with no
DNA controls in lanes 1 and 6, and test samples in the rest of
the lanes. Lanes 1- 5 test for the wild type 172 T nucleotide
while lanes 6 - 10 test for the mutant 172 A nucleotide that
results in the I172N mutation. As can be seen by the results
depicted in lanes 5 and 10, this individual is a carrier of the
I172 mutation. All other individuals are wild type at this site
(although they might be deletion carriers on the other
chromosome).
The lower panel
illustrates the use of a
restriction
endonuclease to
detect a point
mutation. Specific
restriction fragments
appear in the presence
of the mutation and
are indicated by the
red arrows. Lane 4
shows an individual
who carries the V281L
mutation on one
chromosome while
the individuals in lanes
7 and 9 carry the
Q318X mutation
Similar analysis •
shows in the upper
portion of the
figure shown
below, detection of
the R356W
mutation in the
samples in lanes 8,
9 and 10. The lower
portion of the
figure shows
detection of the
common A/C656G
splice mutation in
the samples of
lanes 7, 8 and 9.
In certain families, it may be necessary to consider a linkage •
analysis study. An example of this type of analysis is shown
below.
In this fully informative situation, the proband has inherited the
paternal C allele and maternal A allele at D6S276 and the paternal
C allele and maternal A allele at D6S1568. DNA prepared from the
CVS obtained from the mother showed a recurrence of the
predicted affected haplotypes. In addition, the evaluation of
these flanking markers confirmed fetal cell sampling by the CVS
with no evidence of maternal cell contamination. Wherever
possible, CompGene performs evaluation of flanking markers to
complement direct mutation analysis studies. These and our
other in-house flanking and physically mapped STR markers
provide >99% accuracy in linkage analysis situations. Please
consult with Celtek when linkage studies are being considered.
We are often asked about genotype/phenotype correlations. This
is a difficult question to answer because CAH can present in a
variety of clinical forms presumably due to the nature of the
CYP21 mutations present and compound heterozygosity
These clinical presentations •
range from the lifethreatening "classic" or salt
wasting presentations in
infancy to late-onset "nonclassic" forms that may
present simply as mild
hirsutism in females (for
examples of mutation
analysis in the latter group,
see Blanche et al. (1997)
Hum. Genet. 101:56-60. It is
also possible that other
genotypic factors not at the
21-hydroxylase locus can
modify clinical severity. In a
general sense, the figure
below modified from
Wedell et al. (1996; Clin.
Lab. Med. 16:125-137)
illustrates the spectrum of
phenotypes associated with
particular 21-hydroxylase
mutations.