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Max Delbrück Centrum für Molekulare Medizin (M.H.), 13125 Berlin, Germany; National Center for Natural Science and Technology, Institute of Biotechnology (N.T.N.D.), Cau Giay-Hanoi, Vietnam; National Institute of Pediatrics (N.T.H.), Vietnam; and Universität des Saarlandes, FB 8.8 Biochemie (R.B.), 66041 Saarbrücken, Germany
Address all correspondence and requests for reprints to: Dr. Rita Bernhardt, Universität des Saarlandes, FR 8.8 Biochemie, P.O. Box 15 11 50, D-66041 Saarbrucken, Germany. E-mail: ritabern{at}mx.uni-saarland.de
Abstract
Congenital adrenal hyperplasia is one of the most frequently
inherited diseases. It is characterized by a severe decline in cortisol
secretion, which results in a compensatory increase in ACTH and
consequent adrenal growth (hyperplasia). Here we describe the first
case of 11ß-hydroxylase deficiency that is caused by an unequal
cross-over of the genes encoding aldosterone synthase (CYP11B2) and
11ß-hydroxylase (CYP11B1). CYP11B1 and
CYP11B2 are located on chromosome 8q24 approximately 45
kb apart from each other. The investigated genetic recombination
deleted the normal alleles of the two genes and created a chimeric
fusion gene, which consists of the promotor and exons 1 through 4 of
the aldosterone synthase gene plus intron 4 through exon 9 of the
11ß-hydroxylase gene. This recombination event subordinates any
remaining 11ß-hydroxylase activity of the chimeric enzyme to the
control mechanisms of CYP11B2, the expression of which is
mainly regulated by angiotensin II and K+. Normally the
11ß-hydroxylase activity is controlled by ACTH. The existence of the
CYP11B2/CYP11B1 chimera was discovered by means of a PCR method and was
confirmed with a Southern blot. Furthermore, by applying a minigene
expression method we demonstrated a point mutation in intron 3
(IVS3+16G
T) of the patient’s second 11ß-hydroxylase allele that
radically diminishes proper splicing of the pre-mRNA by giving rise to
a new, highly preferred donor splice site.
MORE THAN 90% of the cases of congenital adrenal hyperplasia (CAH) are due to 21-hydroxylase (CYP21B) deficiency, whereas 5–8% arise from 11ß-hydroxylase (CYP11B1) deficiency (1, 2). Previously reported mutations on CYP11B1 are distributed over the entire coding region, but cluster in exons 2, 6, 7, and 8. In addition to missense mutations, nonsense and splice site mutations of the CYP11B1 gene were shown to occur (3, 4, 5, 6, 7).
11ß-Hydroxylase (CYP11B1) is expressed in the zona
fasciculata/reticularis (8), is regulated by ACTH, and
catalyzes the synthesis of cortisol from 11-deoxycortisol. Aldosterone
synthase (CYP11B2) is expressed in the zona glomerulosa
(9), is mainly regulated by serum levels of angiotensin II
(ANGII) as well as K+, and catalyzes the
synthesis of aldosterone from deoxycorticosterone via corticosterone
and 18-hydroxycorticosterone (6, 10, 11, 12). In humans
CYP11B1 and CYP11B2 are encoded by two genes, which lie tandemly
arranged approximately 45 kb apart from each other on chromosome 8q24.
The genes share 95% sequence homology within their nine exons and
about 90% in their introns. The most significant difference between
the two structural genes is a 442-bp insertion in intron 5 of
CYP11B2 (10, 13, 14, 15, 16). The presence of a
chimeric CYP11B1/CYP11B2 gene has been shown as the cause of
glucocorticoid-remediable aldosteronism (GRA) (14, 17, 18, 19). The chimeric gene contains the 5'-regulatory sequence
of CYP11B1 and the 3'-structural sequence of CYP11B2, so that
expression of a protein with aldosterone synthase activity is under
control of the CYP11B1 promotor and, thus, ACTH. This chimeric gene is
located on the same chromatid as the normal genes for CYP11B1 and
CYP11B2 (Fig. 1
). Published cross-over
break points in GRA patients range from intron 2 to intron 4 (14, 18, 20). The consequent high expression of aldosterone synthase
results in primary aldosteronism, which can be inhibited by
glucocorticoids. The opposite case of a chimeric gene containing the
CYP11B2 promotor and the CYP11B1 structural gene has never been
observed. In that case, one chromatid would only contain the
CYP11B2/CYP11B1 chimera, with no normal genes for CYP11B1 and
CYP11B2.
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Subject and Methods
Patient
The following data were obtained at the first examination of the 31-month-old Vietnamese male: Na+, 141 mEq/liter; K+, 3.4 mEq/liter, Cl-, 105 mEq/liter; estradiol, 37 pmol/liter; progesterone, 5.2 nmol/liter (elevated); and testosterone, 2.2 nmol/liter (elevated). The patient’s blood pressure was 160–170/80–90 mm Hg, pulse was 110/min, weight was 15 kg, and height was 90 cm. Symptoms of sexual precocity occurred at the age of 24 months. The administration of prednisolone was started (5 mg/d) after diagnosing a hypertensive form of CAH. Within 6 wk of medical treatment blood pressure dropped to 110/70 mm Hg. The medication was changed to hydrocortisone (20 mg/d) when progesterone and testosterone levels remained elevated and concomitant hyperpigmentation of the skin occurred. At the age of 9 yr, the patient was 135 cm tall and weighed 29 kg, and his bone age was 13 yr, 9 months. Blood pressure was 100/60 mm Hg, progesterone was 5.43 nmol/liter, testosterone was 0.05 nmol/liter, and cortisol was 309 nmol/liter (medication was not ceased). The patient was in a low cortisol level (91 nmol/liter) situation (for an unknown reason) when another determination of the serum steroid concentrations was performed at the age of 9 yr. The concentrations of the precursors 11-deoxycortisol (49.8 nmol/liter) and 11-deoxycorticosterone (10.33 nmol/liter, 17-deoxy pathway) were elevated. The corticosterone concentration was 5.8 nmol/liter, 17-hydroxyprogesterone was 3.6 nmol/liter, 17-hydroxypregnenolone was 2.73 nmol/liter, 21-desoxycortisol was 0.29 nmol/liter, dehydroepiandrosterone was 3.4 nmol/liter, and androstenedione was 5.27 nmol/liter.
PCR and sequencing
PCR A, B, and C (Fig. 2) were
performed as described previously (3) with primer pairs
24/25, 26/27, and 28/29 (Table 1),
respectively. Primer pairs 18/6, 17/27, 26/29, and 19/20 (Table 1) were
used for PCRs A ext., chimera, B+C, and the entire gene, respectively.
The incubation time at 72 C was 3 min for reactions A, B, and C; 4 min
for reactions A ext., and B+C; 5 min for PCR chimera; and 7 min for PCR
of the entire gene. The cycle program was run for 30 cycles as
described below in RT-PCR. The CombiPol PCR kit (InViTek) containing 3'
to 5' proofreading activity was used for PCR chimera (Fig. 2). We
purified the amplified DNA by using QIAEX II gel extraction kit
(QIAGEN). Big Dye terminator cycle sequencing kit
(PE Applied Biosystems) and primers 1–16 (Table 1) were
used to sequence both strands of the exons and adjoining intronic parts
directly. Electrophoresis was performed on the ABI PRISM 377 DNA
sequencer and analyzed with Sequencing Analysis software version
3.3.
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Both the wild-type (from healthy control) and mutant PCR product
spanning the complete coding part of CYP11B1 (Fig. 2
; PCR
entire gene) were subcloned into pCR-XL-Topo (Invitrogen,
San Diego, CA), excised with NotI/XbaI
(restriction sites in primers 19 and 20, respectively; Table 1), and
cloned into NotI and XbaI sites of pRc/CMV
(Invitrogen). All exons and splice sites were sequenced to
verify the integrity of the inserts.
Cell culture and transfection
COS-1 cells were cultured as previously described (21, 22). One day before transfection cells were plated at a density of 4 x 105/6-cm dish. Four micrograms of each of the CYP11B1 mutant, wild-type, and control (pRc/CMV without insert) plasmids were transfected as previously described (22). Twenty-four hours after transfection, total RNA was extracted from the cells using the RNAesy kit (QIAGEN, Chatsworth, CA).
RT-PCR
Two micrograms of total RNA and oligo(deoxythymidine) primer
were used for RT, which was performed according to the Superscript II
kit protocol (Life Technologies, Inc., Grand Island, NY).
The PCR was performed with primers 19 and 20 (Table 1) and the CombiPol
PCR kit (InViTek). The reaction conditions were denaturation at 95 C
for 2 min, followed by 50 cycles at 94 C for 1 min, annealing at 64 C
for 2 min, extension at 68 C for 2 min, and final extension for 5 min.
The PCR products were purified from an agarose gel and ligated to pCR
2.1 (Invitrogen). Inserts were sequenced with primers 19,
20, 21, 22, 23, and 28 (Table 1).
Southern blot
Five micrograms of genomic DNA each from the patient, a normal
control, and a patient with GRA (previously described in Ref.
23) were digested with the restriction endonuclease
BamHI for 16 h. The resulting fragments were separated
on a 0.8% agarose gel and blotted onto a Nylon membrane (Hybond-N+,
Amersham Pharmacia Biotech, Arlington Heights, IL) with a
vacuum blotting apparatus. The Gene Images random prime labeling
module (Amersham Pharmacia Biotech, RPN3540) was used to
generate a fluorescein-labeled probe from 100 ng product of PCR A (see
Fig. 2). The blot was hybridized overnight and washed twice at 68 C
with 0.1 x SSC (standard saline citrate)/0.1% SDS for 30 min,
and the hybrids were visualized with the Gene Images CDP-Star detection
module (Amersham Pharmacia Biotech, RPN3510) on x-ray
film. The image was scanned, and band intensities were determined with
the program TINA 2.08e (Raytest Isotopenmessgeraete GmbH,
Straubenhardt, Germany).
Results
The patient, the son of a Vietnamese couple, showed typical symptoms of 11ß-hydroxylase deficiency when he was taken to a pediatrician at the age of 31 months because of abdominal pain. Besides signs of sexual precocity, a strikingly high blood pressure was observed (see Subject and Methods).
PCR amplification and DNA sequencing
A set of three overlapping PCRs (Fig. 2) was performed to amplify
the complete structural gene of the 11ß-hydroxylase. Sequencing of
the obtained products revealed heterozygosity at all positions in exon
3, intron 3, and exon 4, which are different in the highly similar
11ß-hydroxylase and aldosterone synthase genes (Fig. 1). In all cases
the CYP11B1 sequence was replaced by the corresponding
sequence of CYP11B2, which gave rise to the three amino acid
exchanges, E147D, N152K (Fig. 3), and
T248I. These changes were very unlikely to destroy the
11ß-hydroxylase activity, as the corresponding mutants investigated
in our laboratory (data not shown) and by others (T248I)
(24) had been found to exhibit unchanged or increased
11ß-hydroxylase activity.
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To investigate whether a gene conversion, an unequal cross-over,
or a PCR artifact was the reason for the observed mutations, PCR A was
performed with a different 3'-end primer (Fig. 2; PCR A ext.) which
bound downstream of exon 3 in CYP11B1 as well as in
CYP11B2. The sequence of the PCR product showed a loss of
the heterozygous positions. This indicated that the forward primer
binding in the CYP11B1 promoter, which is only 48% similar
to that of CYP11B2 (10), discriminated between
an apparently normal CYP11B1 allele and the mutated allele,
which was not amplified in this reaction. For amplification of the
mutant allele, a long range PCR (Fig. 2; PCR chimera), using a forward
primer binding in the CYP11B2 promoter and a reverse primer
binding specifically a sequence in exon 6 of CYP11B1, was
performed. The successful PCR (Fig. 4)
supported the theory that an unequal cross-over created the chimeric
allele. The PCR product (Fig. 2; PCR chimera) showed the sequence of
CYP11B2 up to intron 4, where it switched to that of
CYP11B1.
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).
The mother and sister of the patient were found to be carriers of the
chimeric gene when all available family members were tested by PCR
(Fig. 4, A and B).
Southern blot
To demonstrate that the proposed unequal cross-over had created
the observed chimeric gene, a Southern blot was performed (Fig. 5). The digestion of genomic DNA with
BamHI would yield a characteristic band at 6.5 kb when
hybridized with an exon 1-intron 2 probe. Furthermore, quantitation of
the respective fragments should show a 1:1:1 ratio of
CYP11B2, the chimera, and CYP11B1.
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). It was shown that this chromosome contains one
copy each of CYP11B2, the chimera accounting for GRA, and
CYP11B1 in a GRA patient (14). In the
investigated patient with 11ß-hydroxylase deficiency, it is supposed
to contain just the CYP11B2/CYP11B1 chimera. Hybridization of the exon 1-intron 2 probe to the blot detected, besides the normal bands for CYP11B1 (8.5 kb) and CYB11B2 (4.4 kb), the predicted 6.5-kb fragment in the DNA of our patient and the 6.3-kb fragment in the DNA of the GRA patient. Quantitation (not shown) confirmed a ratio of CYP11B1:chimera:CYP11B2 genes of 1:1:1 for the 11ß-hydroxylase-deficient patient and 2:1:2 for the patient with GRA. This indicates a single additional gene (CYP11B2/CYP11B1 chimera) and the deletion of one copy each of CYP11B1 and CYP11B2 within the diploid genome of our patient.
Polymorphisms and minigene expression of CYP11B1
The chimera, however, explained the disruption of one allele. As 11ß-hydroxylase deficiency is inherited as an autosomal recessive trait, a mutation was hidden in the allele inherited from the father.
Besides the five polymorphisms R43Q, D82D (GATGAC),
L362L (CTGCTC), A386V, and C494F no other differences were found
within the exons and the splice sites (data not shown) when the
sequence was compared with that reported by Mornet et al.
(13). Therefore, an intron mutation disrupting right
splicing or a cumulative effect of the polymorphisms was considered to
diminish the enzyme’s activity.
A minigene expression of the complete CYP11B1 structural gene (5.5 kb) was performed in COS-1 cells to detect any mutations causing wrong splicing of the primary transcript and to obtain the corresponding cDNA for subsequent activity tests.
RT-PCR with the isolated total RNA amplified a fragment of the expected
size (
1550 bp), which was ligated to a cloning vector. When the
identity of the insert was checked, an intronic insertion consisting of
the first 14 bases of intron 3 was observed between exon 3 and exon 4
in all three investigated cDNA clones. A GT transversion at position
16 of intron 3 (IVS3+16GT) was found when sequence data of the exon
3/intron 3 boundary of the patient and the father were reexamined (Fig. 6). The mutation created the typical GT
consensus of a donor splice site.
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, in normal splicing of exons 3
and 4. Calculation of the scores (or consensus values) according to
Senapathy et al. (26, 27) showed the new site
to have a higher score (85.1) than the normal donor splice site of
intron 3 (80.2), which is still present and unmutated. Krawczak
et al. (28) calculated a mean consensus value
of 83 when they analyzed 406 donor splice sites from a random selection
of 29 human genes. Values below 70 were found in less than 15% of the
donor splice sites, although the score can theoretically range from
0–100. As a splice site with a higher score is not always associated
with the exclusive recognition by the spliceosome (29),
another RT-PCR was performed to compare the frequencies of the splice
site utilization in COS-1 cells. Thirty-three of 33 investigated clones
contained the 14-bp insert, whereas 4 of 4 clones lacking the intronic
mutation were found to be normally spliced (Fig. 6). Discussion
Molecular genetic analysis of a male Vietnamese patient with CAH revealed two new mutations of the 11ß-hydroxylase gene. One allele had been formed by an unequal crossing-over, which created a chimeric fusion gene composed of the promoter and exon 1 to exon 4 of CYP11B2 and intron 4 to exon 9 of CYP11B1. The second allele comprises a mutation in intron 3, which leads to a wrong splicing of the primary mRNA transcript.
Former studies from our laboratory showed that the enzyme encoded by
the chimeric gene is most likely an active 11ß-hydroxylase
(21). The amount of the protein formed under control of
the aldosterone synthase promoter, however, will be considerably less
than the amount formed under the original 11ß-hydroxylase promotor,
as human aldosterone synthase is expressed in the adrenal cortex at a
level much lower than 11ß-hydroxylase (30). The
expression is probably driven by ANGII and K+
instead of ACTH and is limited to the adrenal zona glomerulosa, which
lacks the expression of 17
-hydroxylase, an enzyme acting further
upstream in the biosynthetic pathway of cortisol. As adrenal
circulation is mainly centripetal (from the capsule/zona glomerulosa to
the medulla) (31), only minimal amounts of the
11ß-hydroxylase substrate 11-deoxycortisol produced by the zona
fasciculata would be expected to reach zona glomerulosa cells
expressing the chimeric 11ß-hydroxylase. Thus, the changed spatial
expression within the adrenal cortex, the changed level of expression,
and the switch of the main expression stimulus from ACTH to ANGII and
K+ result in a reduction of the cortisol
synthesis being low enough to cause CAH.
The discovered chimeric gene showed all of the features proposed by Lifton et al. (14) for a reciprocal product of the unequal cross-over between CYP11B1 and CYP11B2, when he published his findings about the genetic reason for GRA, a dominant inherited form of hypertension.
Our clinical and genetic data showed that the discovered deletion chimera and the intronic mutation behaved as null alleles. Heterozygotes were phenotypically normal. Compound heterozygotes (as in our case) for the chimeric allele in combination with an 11ß-hydroxylase deficiency allele show the phenotype of 11ß-hydroxylase deficiency (see Subject and Methods).
The existence of the deletion chimera was demonstrated by Southern blot. The additional band at 6.5 kb and quantitation of the gene copies ruled out the possibility that a gene conversion or a PCR artifact generated the anomalous PCR fragments that had been observed at the first screening for mutations.
The intronic mutation within the second 11ß-hydroxylase allele of the patient was revealed by a minigene expression of the structural gene of CYP11B1 in COS-1 cells. All investigated cDNA clones contained a 14-bp insert between exons 3 and 4. This insert causes a shift of the reading frame, and the result of this is a premature translation stop in exon 4 (codon 226 of the mutated mRNA). The 11ß-hydroxylase, a cytochrome P450 enzyme, uses heme as a prosthetic group for the hydroxylation of its substrates. The heme-binding domain of the human 11ß-hydroxylase is thought to be located between codons 443 and 463 (32). The loss of this functional domain will abolish the activity of our truncated mutant completely. Furthermore, earlier in vitro studies showed that the mere deletion of the eight C-terminal residues 496–503 rendered the enzyme inactive (22).
Our experiments showed that COS-1 cells can be used to obtain complete
cDNAs from genomic DNA of steroid hydroxylases. To our knowledge this
is the second publication after that by Higashi et al.
(33) that describes the in vitro expression of
an adrenal steroid hydroxylase gene for the detection of intronic
mutations. The method, originally published in the late 1970s
(34, 35), could be a helpful tool for studying frequent
polymorphisms and mutations in short genes of the entire P450
superfamily, which includes the adrenal steroid hydroxylases, the exons
of which are mostly arranged within a relatively short DNA section
[CYP21B, 2.7 kb (36); 17-hydroxylase (CYP17), 6.4 kb
(37)].
To date, all defects in the CYP11B1 gene have been found to be due to nonsense, missense, or splice site mutations (5). That 11ß-hydroxylase deficiency can result from unequal crossing-over raises the possibility that such mutations may be more common than is presently recognized. The reciprocal GRA chimera, which emerges from the same recombination event and consists of the 11ß-hydroxylase promoter and the aldosterone synthase-coding region, is being detected with increasing frequency (38, 39). Therefore, it is advisable that patients with CAH in whom no mutations are found within the coding regions of the 21-hydroxylase and 11ß-hydroxylase (40, 41) should be tested for the deletion chimera consisting of aldosterone synthase promoter and 11ß-hydroxylase-coding region. The same applies for aldosterone synthase deficiency. The chimera would definitely behave as a complete CYP11B2 null allele as long as the cross-over point is upstream of exon 5, which codes together with exon 6 for the amino acids, which confer aldosterone synthase activity to the enzyme (21, 22, 24, 42). The finding of the cross-over allele, the performed long range PCRs for the detection of biallelic amplification, and the described minigene expression method will hopefully unveil secrets about the "phenotype does not match genotype" cases and lead to deeper insight into the molecular genetic basis of adrenal steroid hydroxylase deficiencies.
Acknowledgments
We thank Christiane Maser-Gluth (University of Heidelberg, Heidelberg, Germany), Tran Van Khan, and Do Thi Tuyen (IBT, Hanoi, Vietnam) for technical support, and Tomas Seeman (University Hospital V, Prague, Czech Republic) for providing the genomic DNA of the GRA patient for the Southern blot. We also thank Rick Gardner and Pierre Debs for critical comments as native speakers.
Footnotes
This work was supported by a scholarship from the Verbund Klinische Pharmakologie Berlin-Brandenburg (to M.H.) and by the Fonds der Chemischen Industrie (to R.B.).
Abbreviations: ANGII, Angiotensin II; CAH, congenital adrenal hyperplasia; GRA, glucocorticoid-remediable aldosteronism.
Received August 29, 2000.
Accepted May 7, 2001.
References
His) associated
with steroid 11ß-hydroxylase deficiency in Jews of Moroccan origin. J Clin Invest 87:1664–1667
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-DNA-digested
EcoRI/HindIII) was loaded onto the gel.
Numbers indicate the lengths of the marker fragments in base pairs.
