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Original Articles |
INSERM, U-342, Laboratoire de Biochimie Endocrinienne, Hopital Debrousse (S.P., Y.M.), 69322 Lyon, France; Hypertension Unit, University of Torino (P.M.), 10132 Torino, Italy; Baker Medical Research Institute (K.M.C.), Prahran 3181, Australia; Pediatric Endocrinology, Hopital Saint Vincent de Paul (J.-L.C.), 75674 Paris, France; and Fondation Jean Dausset CEPH (L.P.), 75010 Paris, France
Address all correspondence and requests for reprints to: Dr. Leigh Pascoe, Fondation Jean Dausset CEPH, 27 rue Juliette Dodu, 75010 Paris, France. E-mail: leigh{at}cephb.fr
Abstract
Chromosomal rearrangements are natural experiments that can provide unique insights into in vivo regulation of genes and physiological systems. We have studied a patient with congenital adrenal hyperplasia and steroid 11ß-hydroxylase deficiency who was homozygous for a deletion of the CYP11B1 and CYP11B2 genes normally required for cortisol and aldosterone synthesis, respectively. The genes were deleted by unequal recombination between the tandemly arranged CYP11B genes during a previous meiosis, leaving a single hybrid gene consisting of the promoter and exons 1–6 of CYP11B2 and exons 7–9 of CYP11B1. The hybrid gene also carried an I339T mutation formed by intracodon recombination at the chromosomal breakpoint. The mutant complementary DNA corresponding to this gene was expressed in COS-1 cells and was found to have relatively unimpaired 11ß-hydroxylase and aldosterone synthase activities. Apparently the 11ß-hydroxylase deficiency and the adrenal hyperplasia are due to the lack of expression of this gene in the adrenal zona fasciculata/reticularis resulting from replacement of the CYP11B1 promoter and regulatory sequences by those of CYP11B2.
THE TERMINAL STEPS in the synthesis of cortisol and aldosterone in the human adrenal cortex are catalyzed by the cytochrome P450 enzymes CYP11B1 and CYP11B2, respectively (1, 2). CYP11B2 is expressed uniquely in the zona glomerulosa of the human adrenal and has the three activities, 11ß-hydroxylase, 18-hydroxylase, and 18-oxidase, required for the successive hydroxylation of 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone, and aldosterone. Expression of the enzyme is principally controlled by serum concentrations of angiotensin II, acting through its type 1 receptor, and potassium ions. CYP11B1 also has 11ß-hydroxylase activity and is expressed in the zona fasciculata/reticularis, where it catalyzes the conversion of 11-deoxycortisol to cortisol under the control of ACTH, and in the zona glomerulosa, where it can contribute to the catalytic conversion of 11-deoxycorticosterone to corticosterone (3). The limitation of the expression of CYP11B2 to the zona glomerulosa and of CYP17 (whose 17-hydroxylase activity is required for cortisol synthesis) to the zona fasciculata, is the mechanism by which aldosterone and cortisol syntheses are limited to the zona glomerulosa and the zona fasciculata/reticularis, respectively. The two genes are 95% similar in sequence (4) and lie in tandem about 45 kb apart (5, 6) on the long arm of chromosome 8 (7, 8).
Gametes are formed by unequal crossing over between the CYP11B genes. The normal synthesis of aldosterone is perturbed in the dominantly inherited hypertensive syndrome, glucocorticoid-suppressible hyperaldosteronism (GSH), caused by the presence of a duplication hybrid CYP11B gene (5, 6, 9). The hybrid gene consists of CYP11B1 sequences at the 5'-end fused to CYP11B2 sequences at the 3'-end of the gene. The presence of the CYP11B1 promoter and regulatory elements ensures that the gene is expressed in the zona fasciculata/reticularis under the control of ACTH and the 3' CYP11B2 coding sequences lead to the encoded enzyme having the three activities required for aldosterone synthesis (6). Consequently, aldosterone is inappropriately synthesized and secreted in excess by the zona fasciculata/reticularis under the control of ACTH.
The duplication forming the CYP11B hybrid gene that causes
GSH arises from unequal crossing over between the CYP11B
genes during meiosis (Fig. 1
). The
complementary gamete formed during this process also contains a hybrid
gene, this time with a CYP11B2 5'-end and a
CYP11B1 3'-end. This gamete also lacks the normal copies of
CYP11B1 and CYP11B2, which are deleted by the
unequal cross-over. The deletion hybrid gamete is expected to be found
with the same frequency as the duplication allele, assuming equal
success in fertilizing an ovum and no deleterious viability
effects.
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Materials and Methods
Molecular analysis
Peripheral blood samples of the index case were obtained with
informed consent for this study according to our institutional
guidelines. Preparation of genomic DNA (14) and PCR
amplification and sequencing of CYP11B exons (15, 16) were carried out as previously described. The PCR
amplifications shown in Fig. 2 were
performed with oligonucleotides 5'-ACCCAGAGAGTAGAGGAACACG-3'
(CYP11B1 antisense exon 8) and 5'-CAGCACCAAAGTCTGAGGGC-3'
(CYP11B1 sense intron 4) or 5'-CCAAGATCTAGGGCTGTCCCCT-3'
(CYP11B2 sense intron 4). Reactions were denatured at 95 C
for 5 min, followed by 30 cycles of 15 s at 95 C, 15 s at 56
C, and 20 s at 72 C, with a final extension at 72 C for 10 min in
the last cycle.
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Mutant hybrid and wild-type complementary DNAs (cDNAs) were constructed and subcloned into the expression vector pCMV4 and transfected into COS-1 cells using Lipofectamine (Life Technologies, Inc., Gaithersburg, MD) as previously described (17). Transfected cells were incubated with radioactively labeled 11-deoxycortisol or 11-deoxycorticosterone (DuPont de Nemours, Brussels, Belgium), and the resulting products were resolved by TLC as previously described (18). Steroid products were identified by their comigration with equivalent unlabeled steroids.
Results
An initial diagnosis of pseudo-precocious puberty was made in a child from Cote d’Ivoire, with a normal male karyotype after the development of pubic hair at 2 yr of age. The diagnosis of 11ß-hydroxylase deficiency was confirmed at 3 yr, 2 months of age by hormonal studies. The child exhibited severe manifestations of congenital adrenal hyperplasia, including penile enlargement (9.5 cm), increased growth velocity (statural age of 7 yr, 9 months) with advanced bone age (11 yr, 6 months), and elevated blood pressure for age (120/80 mm Hg). Hormonal data included increased 11-deoxycortisol (638 nmol/L) and androstenedione (125 nmol/L) with undetectable PRA and aldosterone. Compliance with treatment was poor, and true precocious puberty, treated by cyproterone acetate (100 mg), began at 10 yr of age. The final attained height was 166 cm. The parents were first cousins.
After initial attempts to amplify exons from the 5'-end of the
CYP11B1 gene by PCR had failed (not shown), we investigated
the possibility of a major chromosomal deletion. The presence of a
hybrid gene was shown by the successful amplification of
CYP11B exons by PCR using a mixed oligonucleotide strategy
(Fig. 2
). PCR amplification was carried out with an antisense
oligonucleotide corresponding to exon 8 of CYP11B1 and a
sense oligonucleotide corresponding to intron 4 of either
CYP11B2 or common to both CYP11B1 and
CYP11B2. The first reaction will uniquely amplify a hybrid
gene segment and was successful using DNA from our patient, but not
with other DNA samples. The second reaction will amplify both a
CYP11B1 gene segment and a hybrid gene segment, if they are
present. The two segments can be distinguished by their size due to the
presence of a small insertion in intron 5 of CYP11B2. The
results shown in Fig. 2 show the exclusive presence of a hybrid gene in
patient DNA and of a CYP11B1 gene in normal DNA.
Southern blot analysis (Fig. 3) and
sequencing (not shown) revealed the presence of a single hybrid gene in
homozygous form containing exons 1–6 of CYP11B2 and exons
7–9 of CYP11B1. Both parents were predicted to be
heterozygous for the deletion hybrid gene, but were not available for
study. The exact breakpoint occurred after the first base of codon 339
(GCC and ATC for CYP11B1 and CYP11B2,
respectively), leading to the formation of a novel triplet (ACC)
encoding threonine (I339T), which was found on both copies of the
allele. These data are consistent with a common origin for the alleles
inherited by the patient from each of his parents, who were first
cousins.
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). The steroid products produced by
cells transfected with the hybrid mutant construct were identical with
those produced by cells transfected with normal CYP11B2
cDNA. The hybrid mutant enzyme appears capable of catalyzing complete
conversion of 11-deoxycortisol to cortisol and to have a near-normal
aldosterone synthase activity, as might be predicted from the presence
of the first six exons of CYP11B2 in the clone. The I339T
mutation has little apparent effect on the activity of the encoded
enzyme in the in vitro assay. We conclude therefore that the
impaired cortisol synthesis in this patient probably results from a
lack of expression of the hybrid gene in the adrenal zona
fasciculata/reticularis due to the replacement of the
CYP11B1 promoter and regulatory sequences by those of
CYP11B2.
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Deletion hybrid CYP11B alleles are expected to be present in the general population with a frequency comparable to that of the duplication alleles that cause glucocorticoid-suppressible hyperaldosteronism. However, unlike this allele, which causes detectable effects in heterozygous form, the deletion allele is most likely to pass undetected. Although heterozygote carriers would lack the normal CYP11B1 and CYP11B2 genes on this allele, the lack would be compensated for by the presence of normal copies of both genes on the homologous allele. In homozygous form they would be expected to cause congenital adrenal hyperplasia due to steroid 11ß-hydroxylase deficiency, as observed in our patient. This deficiency is expected despite the fact that the encoded enzyme should, in general, be capable of catalyzing 11ß-hydroxylation. The defect lies rather in a failure of expression of the hybrid gene in the adrenal zona fasciculata/reticularis. The hybrid gene is apparently expressed in a similar manner as the CYP11B2 gene, whose expression is limited to the adrenal zona glomerulosa (3). The effect of this lack of expression of the hybrid gene is identical to that of an inactivating mutation in the CYP11B1 gene: a deficit of 11ß-hydroxylase activity in the zona fasciculata. In both cases the result is adrenal hyperplasia and excess androgen production from the accumulating steroid hormone precursors. The accumulation of 11-deoxycorticosterone and its metabolites, which act as mineralocorticoids, leads to salt and water retention and consequently to down-regulation of the adrenal zona glomerulosa, which is unable to compensate for the defect in the fasciculata.
The failure of expression of the hybrid gene could be due to the presence of sequence from the CYP11B2 gene that normally acts as a zona fasciculata-specific suppressor of expression or because it lacks sequence from the CYP11B1 gene that normally ensures its expression in that zone. All patients with glucocorticoid-suppressible hyperaldosteronism who have been studied to date have breakpoints between the 3'-end of intron 2 and intron 4, suggesting that the sequence 5' of exon 3 of CYP11B1 is sufficient to ensure expression in the zona fasciculata and a normal response to ACTH. However, these data are also consistent with the hypothesis that there is a suppressor in or 5' of intron 2 of CYP11B2 that prevents expression in the zona fasciculata. Recent studies using the promoter of CYP11B2 in front of a reporter gene showed that this gene has functional cAMP response elements in the promoter (19). Nevertheless studies of cultured adrenal cortex cells from a patient with glucocorticoid-suppressible hyperaldosteronism showed undetectable expression of CYP11B2 and no response to incubation with ACTH. These same cells displayed significantly increased expression of both the CYP11B1 and the CYP11B1/CYP11B2 hybrid gene messenger ribonucleic acids in response to incubation with ACTH (3). Taken together these results suggest that CYP11B2 expression may be actively suppressed in the normal adrenal zona fasciculata.
Whether the deletion hybrid allele encodes an enzyme with all of the activities necessary for aldosterone synthesis would depend on how much of the CYP11B2 sequence was retained in the hybrid. It has been shown that amino acid residues encoded in exons 5 and 6 of CYP11B2 are particularly important to these additional activities (17, 18). Thus, the hybrid enzyme may lack aldosterone synthase activity (if the breakpoint is 5' of exon 6) and cause a combined 11ß-hydroxylase deficiency and aldosterone synthase deficiency when in homozygous form. Aldosterone synthesis is predicted to be normal in our patient, in whom the breakpoint was at codon 339 in exon 6; however, both plasma aldosterone and PRA were undetectable. This result is probably due to negative feedback in the renin-angiotensin system resulting from the increased sodium reabsorption induced by the metabolic disorder.
The deletion hybrid allele could also be found in heterozygous form in patients with either 11ß-hydroxylase deficiency or aldosterone synthase deficiency depending on whether a mutation was present in the homologous copy of CYP11B1 or CYP11B2, respectively. Patients presenting with either or both of these clinical phenotypes should be studied for the presence of a CYP11B deletion hybrid gene by the methods used in this study.
Footnotes
1 This work was supported by grants from the Fondation IPSEN pour la
Recherche Therapeutique (to L.P.) and the Australian Foundation for
High Blood Pressure Research (to K.M.C.). Parts of this work were
carried out while P.M., K.M.C., and L.P. were at INSERM, U-36 (Paris
France). 
2 Present address: Pharmacia Australia, Rydalmere, New South Wales
2116, Australia.
Received November 16, 2000.
Revised March 12, 2001.
Accepted April 1, 2001.
References
This article has been cited by other articles:
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  L. J. Mullins, M. A. Bailey, and J. J. Mullins Hypertension, Kidney, and Transgenics: A Fresh Perspective Physiol Rev, April 1, 2006; 86(2): 709 - 746. [Abstract] [Full Text] [PDF]
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