0021-972X/01/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2001 by The Endocrine Society
Vol. 86, No. 7 Printed in U.S.A.
Deletion Hybrid Genes, due to Unequal Crossing Over between CYP11B1 (11-Hydroxylase) and CYP11B2 (Aldosterone Synthase) Cause Steroid 11-Hydroxylase Deficiency and Congenital Adrenal Hyperplasia* STEPHANIE PORTRAT, PAOLO MULATERO, KATHLEEN M. CURNOW†, JEAN-LOUIS CHAUSSAIN, YVES MOREL, AND LEIGH PASCOE 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 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 11-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
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HE 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, 11-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 11-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 Received November 16, 2000. Revision received March 12, 2001. Accepted April 1, 2001. Address all correspondence and requests for reprints to: Dr. Leigh Pascoe, Fondation Jean Dausset CEPH, 27 rue Juliette Dodu, 75010 Paris, France. E-mail:
[email protected]. * 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). † Present address: Pharmacia Australia, Rydalmere, New South Wales 2116, Australia.
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 11-hydroxylase and aldosterone synthase activities. Apparently the 11-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. (J Clin Endocrinol Metab 86: 3197–3201, 2001)
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
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FIG. 1. Recombinant gametes formed by unequal crossing over between the CYP11B1 (steroid 11-hydroxylase) and CYP11B2 (aldosterone synthase) genes. Crossing over between the mispaired CYP11B genes results in an allele with both wild-type genes deleted, leaving only a hybrid gene containing CYP11B2 sequence at the 5⬘-end and CYP11B1 sequence at the 3⬘-end. The other allele contains both genes as well as a duplication resulting in the formation of the complementary hybrid gene.
frequency as the duplication allele, assuming equal success in fertilizing an ovum and no deleterious viability effects. It has previously been predicted that such deletion hybrid alleles may be found in patients with either steroid 11hydroxylase deficiency or aldosterone synthase deficiency, based on consideration of the activities and expression patterns of each gene (6). As both CYP11B1 and CYP11B2 encode enzymes with 11-hydroxylase activity, the encoded hybrid enzyme is also expected to have this activity. However, the hybrid enzyme may not be expressed in the adrenal zona fasciculata/reticularis, because it lacks the promoter sequence and control elements of CYP11B1 and could hence lead to a deficiency of the 11-hydroxylase activity that is required for cortisol synthesis in that zone. A deficit of 11hydroxylase activity normally leads to a hypertensive form of congenital adrenal hyperplasia, a distinctive phenotype characterized by ambiguous genitalia in female infants and precocious growth with ultimate short stature in both sexes (10). To date, all reported cases of this inherited disease have been caused by microdeletions or point mutations in the CYP11B1 gene (11–13). Here we report the first demonstration of a patient who is homozygous for the deletion hybrid gene described above and who has severe 11-hydroxylase deficiency. 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
FIG. 2. Ethidium bromide-stained gel of DNA from PCR amplifications of CYP11B gene segments. Reactions containing 1) DNA from the patient, 2) DNA from a normal individual, and 3) no DNA were amplified with an antisense oligonucleotide corresponding to CYP11B1 and a sense oligonucleotide corresponding to CYP11B2 (mixed strategy) or common to both CYP11B1 and CYP11B2 (common strategy). The results show amplification of a single CYP11B1 fragment from normal DNA. A single larger fragment, indicating the presence of the larger CYP11B2 intron 5, was amplified from the patient DNA using either the common or the mixed amplification strategy. The positions of the oligonucleotides are indicated on the diagram of the hybrid gene. 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.
Mutant construction and expression 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 11-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 in-
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creased 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. From the sequence data alone, it was not possible to decide whether the deficiency was due to the absence of the CYP11B1 promoter or to the change in the coding sequence. We therefore constructed the cDNA corresponding to this gene and expressed it in COS-1 cells to determine the activity
FIG. 3. Autoradiogram of Southern blot of genomic DNA that has been digested with BamHI and EcoRI and hybridized with a radioactively labeled CYP11B cDNA. Lane 1, Radioactive size standard; lanes 2– 6, DNA digested with BamHI from three normal individuals, the patient with a deletion hybrid gene, and a patient with GSH carrying a CYP11B gene duplication, respectively; lanes 7–10, DNA from two normal individuals, from the patient, and from a patient with GSH digested with EcoRI. Note the absence of normal CYP11B bands (white arrows) in DNA from our patient as well as the presence of BamHI bands and a unique 8.5-kb EcoRI band corresponding to the deletion hybrid gene restriction map, as shown in the diagram.
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of the encoded enzyme. The transfected cells were subsequently incubated with radioactively labeled steroid hormone precursors, and the resulting products were identified by TLC (Fig. 4). 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. Discussion
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 11-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 11-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 11hydroxylase activity in the zona fasciculata. In both cases the
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FIG. 4. Autoradiography of a thin layer chromatogram of steroids produced by COS-1 cells that were transfected with CYP11B cDNAs and incubated with radioactively labeled steroid hormone precursors. Cells were transfected with vector DNA (lane 1), CYP11B1 (lane 2), and CYP11B2 (lane 3) cDNAs and with a CYP11B2/B1 hybrid cDNA with a T339 M mutation (lane 4). The radioactive precursor 11-deoxycortisol (S; upper panel) was converted to cortisol (F) and cortisone (E), whereas 11-deoxycorticosterone (DOC; lower panel) was converted to corticosterone (B), 11-dehydrocorticosterone (A), 18-hydroxycorticosterone (18-OHB), and aldosterone (Aldo). Other steroid products were not specifically identified. The steroids produced by the mutant hybrid CYP11B2/B1 cDNA were similar to those produced by CYP11B2 cDNA.
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 11hydroxylase 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 11-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. References 1. Curnow KM, Tusie-Luna MT, Pascoe L, Natarajan R, Gu JL, Nadler JL, White PC. 1991 The product of the CYP11B2 gene is required for aldosterone biosynthesis in the human adrenal cortex. Mol Endocrinol. 5:1513–1522. 2. Kawamoto T, Mitsuuchi Y, Ohnishi T, et al. 1990 Cloning and expression of a cDNA for human cytochrome P-450aldo as related to primary aldosteronism. Biochem Biophys Res Commun. 173:309 –316. 3. Pascoe L, Jeunemaitre X, Lebrethon MC, et al. 1995 Glucocorticoid-suppressible hyperaldosteronism and adrenal tumors occurring in a single French pedigree. J Clin Invest. 96:2236 –2246. 4. Mornet E, Dupont J, Vitek A, White PC. 1989 Characterization of two genes encoding human steroid 11 beta-hydroxylase (P-450(11)). J Biol Chem. 264:20961–20967. 5. Lifton RP, Dluhy RG, Powers M, et al. 1992 Hereditary hypertension caused by chimaeric gene duplications and ectopic expression of aldosterone synthase. Nat Genet. 2:66 –74. 6. Pascoe L, Curnow KM, Slutsker L, Connell JM, Speiser PW, New MI, White PC. 1992 Glucocorticoid-suppressible hyperaldosteronism results from hybrid genes created by unequal crossovers between CYP11B1 and CYP11B2. Proc Natl Acad Sci USA. 89:8327– 8331. 7. Chua SC, Szabo P, Vitek A, Grzeschik KH, John M, White PC. 1987 Cloning of cDNA encoding steroid 11 beta-hydroxylase (P450c11). Proc Natl Acad Sci USA. 84:7193–7197. 8. Brand E, Chatelain N, Mulatero P, et al. 1998 Structural analysis and evaluation of the aldosterone synthase gene in hypertension. Hypertension. 32:198 –204. 9. Lifton RP, Dluhy RG, Powers M, Rich GM, Cook S, Ulick S, Lalouel JM.
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1992 A chimaeric 11-hydroxylase/aldosterone synthase gene causes glucocorticoid-remediable aldosteronism and human hypertension. Nature. 355:262–265. Zachmann M, Tassinari D, Prader A. 1983 Clinical and biochemical variability of congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency. A study of 25 patients. J Clin Endocrinol Metab. 56:222–229. White PC, Dupont J, New MI, Leiberman E, Hochberg Z, Rosler A. 1991 A mutation in CYP11B1 (Arg448-His) associated with steroid 11-hydroxylase deficiency in Jews of Moroccan origin. J Clin Invest. 87:1664 –1667. Curnow KM, Slutsker L, Vitek J, et al. 1993 Mutations in the CYP11B1 gene causing congenital adrenal hyperplasia and hypertension cluster in exons 6, 7, and 8. Proc Natl Acad Sci USA. 90:4552– 4556. Geley S, Kapelari K, Johrer K, et al. 1996 CYP11B1 mutations causing congenital adrenal hyperplasia due to 11-hydroxylase deficiency. J Clin Endocrinol Metab. 81:2896 –2901. Morel Y, Andre J, Uring-Lambert B, et al. 1989 Rearrangements and point mutations of P450c21 genes are distinguished by five restriction endonuclease haplotypes identified by a new probing strategy in 57 families with congenital adrenal hyperplasia. J Clin Invest. 83:527–536.
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15. Chabre O, Portrat-Doyen S, Vivier J, et al. 2000 Bilateral laparoscopic adrenalectomy for congenital adrenal hyperplasia with severe hypertension, resulting from two novel mutations in splice donor sites of CYP11B1. J Clin Endocrinol Metab. 85:4060 – 4068. 16. Portrat-Doyen S, Tourniaire J, Richard O, et al. 1998 Isolated aldosterone synthase deficiency caused by simultaneous E198D and V386A mutations in the CYP11B2 gene. J Clin Endocrinol Metab. 83:4156 – 4161. 17. Curnow KM, Mulatero P, Emeric-Blanchouin N, Aupetit-Faisant B, Corvol P, Pascoe L. 1997 The amino acid substitutions Ser288Gly and Val320Ala convert the cortisol producing enzyme, CYP11B1, into an aldosterone producing enzyme. Nat Struct Biol. 4:32–35. 18. Mulatero P, Curnow KM, Aupetit-Faisant B, et al. 1998 Recombinant CYP11B genes encode enzymes that can catalyze conversion of 11-deoxycortisol to cortisol, 18-hydroxycortisol, and 18-oxocortisol. J Clin Endocrinol Metab. 83:3996 – 4001. 19. Clyne CD, Zhang Y, Slutsker L, Mathis JM, White PC, Rainey WE. 1997 Angiotensin II and potassium regulate human CYP11B2 transcription through common cis-elements. Mol Endocrinol. 11:638 – 649.
