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Original Studies |
Service d’Endocrinologie (O.C., J.V.), Chirurgie Générale et Thoracique (P.C.), and Laboratoire de Pathologie Cellulaire (F.L.-M.), Centre Hospitalier Universitaire, 38043 Grenoble, France; INSERM U-329, Hôpital Debrousse (S.P.-D., Y.M.), 69322 Lyon, France; INSERM U-244, Biochimie des Regulations Cellulaires Endocrines, Département de Biologie Moléculaire et Structurale, Commissariat à l’Energie Atomique (P.L., E.C., G.D.), 38054 Grenoble, France
Address all correspondence and requests for reprints to: Dr. Olivier Chabre, Service d’Endocrinologie, Centre Hospitalier Universitaire, 38043 Grenoble, France. E-mail: olivier.chabre{at}ujf-grenoble.fr
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Abstract |
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 Top Abstract IntroductionSubject and MethodsResultsDiscussionReferences |
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C transversion at the
last base of exon 5 (T318T), and a IVS8 + 4AG transition in intron
8. In addition, two polymorphisms were found in exons 1 and 2. The
genetically female patient was raised as a male because of severe
pseudohermaphroditism. Glucocorticoid-suppressive treatment encountered
difficulties in equilibration and compliance, resulting in uncontrolled
hypertension with pronounced hypertrophic cardiomyopathy. At 42 yr of
age the occurrence of central retinal vein occlusion with permanent
loss of left eye vision led to the decision to perform bilateral
laparoscopic adrenalectomy. Surgery was followed by normalization of
blood pressure and good compliance with glucocorticoid and androgen
substitutive therapies. In vitro, adrenal cells in
culture and isolated mitochondria showed extremely low
11ß-hydroxylase activity. Analysis of adrenal CYP11B1
messenger ribonucleic acid (mRNA) by RT-PCR and sequencing showed the
expression of a shorter mRNA that lacked exon 8 and did not contain
either the exon 5 mutation or the exon 1 and 2 polymorphisms. This
suggested that one CYP11B1 allele carried the intron 8
mutation, responsible for skipping exon 8. The other allele carried the
exon 5 mutation, and its mRNA was not detectable. Western blot analysis
showed weak expression of a shorter CYP11B immunoreactive band of 43
kDa, consistent with truncation of exon 8. Thus, bilateral
adrenalectomy in this patient allowed effective treatment of severe
hypertension and helped in understanding the mechanisms and
physiopathological consequences of two novel mutations of
CYP11B1. |
Introduction |
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TopAbstractIntroductionSubject and MethodsResultsDiscussionReferences |
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The established treatment of steroid 11ß-hydroxylase deficiency is similar to that of steroid 21-hydroxylase deficiency and consists of glucocorticoid-suppressive therapy and surgical correction of the ambiguous external genitalia in virilized female patients. Suppressive therapy seems at first glance perfectly adapted to the physiopathology of steroid 11ß-hydroxylase deficiency. Glucocorticoids substitute for F deficiency and inhibit ACTH oversecretion, which is the drive for excessive androgen and mineralocorticoid production. However, in CAH, effective suppression of ACTH requires supraphysiological doses of glucocorticoid, which explains why it is often difficult to maintain satisfactory adrenal suppression without producing an unacceptable degree of hypercortisolism (7). Compliance is also a major concern, especially in adolescence and adulthood. As steroid 11ß-hydroxylase deficiency is relatively rare, large follow-up studies are not available. Nevertheless, complications of hypertension have been reported in both male and female patients (1, 4). By comparison, patients with congenital adrenal insufficiency, which are only faced with substitution therapy, are generally believed to fare well, with normal stature, normal fertility, no androgen excess, and no hypertension (New, M. I., commentary in Ref. 8). Therefore, in CAH, a controversy has arisen as to whether some patients with steroid 21-hydroxylase deficiency might benefit from bilateral adrenalectomy to be raised as Addisonians (7). By contrast, in the less frequent steroid 11ß-hydroxylase deficiency, the option of bilateral adrenalectomy has rarely been discussed, and to our knowledge there has been only one report that illustrated its interest in the management of a 14-yr-old girl of unknown genetics, facing difficulties in suppressing androgen production (8).
In the present work we report the use of laparoscopic bilateral adrenalectomy to treat severe hypertension in a 42-yr-old adult patient with steroid 11ß-hydroxylase deficiency, who had experienced long-term difficulties with equilibrium and compliance with suppressive therapy. Bilateral adrenalectomy allowed normalization of blood pressure and was followed by, to date, good compliance with the substitutive therapy. This patient carried two germline CYP11B1 base substitutions whose signification had remained speculative, as one was a conservative exonic mutation and the other was an intronic mutation. Analysis of the patient macronodular adrenals showed that they had very low 11ß-hydroxylase activity and only expressed a CYP11B1 messenger ribonucleic acid (mRNA), which lacked one exon, and a low amount of a truncated CYP11B1 protein. Thus, this study allowed us to understand the significance of two previously undescribed CYP11B1 mutations and raises the possibility of bilateral adrenalectomy for the treatment of some patients carrying severe mutations in CYP11B1.
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Subject and Methods |
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TopAbstractIntroductionSubject and MethodsResultsDiscussionReferences |
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The patient was born in 1953 and was raised as a male. At 20 months of age, development of pubic hair lead to the diagnosis of pseudoprecocious puberty in an XX female with pseudohermaphroditism (stage IV of Prader). Bone age was 6.5 yr, and systolic blood pressure was 105 mm Hg. An iv pyelogram with retropneumoperitoneum showed bilateral hyperplastic adrenals. Urinary 17-ketosteroid levels were elevated (7.6 mg/day; normal, <2.3 mg/day). The patient had a brother who had presented with signs of precocious puberty and died at 6.5 yr of age, probably of brain hemorrhage. Suppressive treatment was initiated at 21 months of age with 30 mg hydrocortisone, followed by various dosages of prednisone. At 4 yr of age, after psychological evaluation, the decision was made to follow the parents’ desire to raise the child as a boy. Bilateral ovariectomy was performed. In childhood, suppressive treatment was difficult to equilibrate, with periods of high blood pressure (up to 150/90 mm Hg, with headaches and blurred vision) and hyperandrogenism and periods of good control of blood pressure, but hypoandrogenism and development of Cushingoid features. At 14 yr, chronological age reached bone age. Androgen substitutive therapy was added, allowing completion of virilization. Final height was only 146 cm. At 16 yr of age, explorations performed after interruption of treatment and on a regular salt intake showed suppressed renin activity (5 ng/L·min; normal, 90 ± 30) and a very high DOC secretion rate (22.9 mg/day). Both were normalized by glucocorticoid-suppressive treatment, confirming the diagnosis of 11ß-hydroxylase deficiency. In adolescence, suppressive treatment was complicated by weight gain of up to 70 kg (body mass index, 33), with development of Cushingoid features, including striaes. The patient was married at 24 yr of age, but divorced 2 yr later and then abandoned all treatment for 18 yr even though he knew his systolic blood pressure was chronically around 180 mm Hg. He was also a heavy smoker, and he later proved to have a high salt intake.
At 42 yr of age, sudden loss of vision of the left eye lead to the
diagnosis of central retinal vein occlusion. Blood pressure was 200/100
mm Hg, and kaliemia was 2.4 mmol/L (n = 3.5–5). Weight was 51 kg
(body mass index, 24), and there was no dyslipidemia or diabetes.
Echocardiogram showed pronounced concentric ventricular hypertrophy
(septum wall, 16–20 mm; posterior wall, 12–14 mm; n = 10 mm)
consistent with uncontrolled hypertension of long duration. Serum
creatinine was normal. An abdominal computed tomography scan showed
large bilateral macronodular adrenal hyperplasia (left gland, 55
x 50 x 30 mm; right gland, 48 x 25 x 12 mm; Fig. 1A
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Because of the difficulties with suppressive treatment, the poor
control of blood pressure, and the risk of more ophthalmological and
cardiovascular complications, the option of bilateral adrenalectomy was
then discussed with the patient. Special emphasis was placed on the
postoperative life-time requirement for both a vital substitutive
therapy with hydrocortisone and an androgen substitutive therapy. The
patient volunteered for surgery, which was performed at 44 yr of age,
using an abdominal laparoscopic approach with a lateral decubitus
position. The two adrenals were removed successively in the same
operation, which lasted 3 h, including repositioning of the
patient after the first adrenalectomy. No complications occurred,
intestinal mobility was restored on the second postoperative day, and
the patient was discharged from the hospital on the fourth
postoperative day with hydrocortisone (20 mg/day) and testosterone
heptylate (250 mg/3 weeks). Nifedipine (30 mg/day) was maintained for 1
yr, and then all antihypertensive medications were stopped (Table 1).
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The patient did not require mineralocorticoid replacement therapy to
maintain normal renin and potassium levels. This is probably due to the
fact that the patient was used to a high salt intake (which might have
explained in part the severity of his hypertension) and that his DOC
levels remained slightly elevated. Eighteen months after surgery a new
echocardiogram showed regression of the concentric ventricular
hypertrophy. Compliance with the substitutive therapy remained
excellent, and the patient expressed a very high degree of satisfaction
with the adrenal surgery. Despite quitting smoking, the patient’s
weight was stabilized at 58 kg.
On macroscopic examination, the total weight of the glands was 30
g, and their usual shape was deformed by numerous nodules with
diameters up to 2 cm (Fig. 1B). Pathological examination confirmed the
diffuse and nodular hyperplastic aspects, with some nodules showing
morphological features indistinguishable from those of an
adrenocortical adenoma (Fig. 1C). Most cells had features of
hyperstimulated, lipid-depleted fasciculo-reticular cells, whereas some
cells located at the inner border of the capsula showed features of
glomerula cells (Fig. 1D).
Materials
All chemicals were purchased from Roche Molecular Biochemicals (Mannheim, Germany) or Sigma (St. Louis, MO). Silica gel plates 60F 254 were obtained from Merck & Co., Inc. (Darmstadt, Germany). Synthetic ACTH-(1–24) (Synacthen) was provided by Ciba (Basel, Switzerland). [1,2,6,7-3H]Dehydroepiandrosterone (DHEA; 64.5 Ci/mmol), [7-N-3H]pregnenolone (25 Ci/mmol), [1,2,6,7-3H]aldosterone, [4-14C]DOC (60 mCi/mmol), and [1,2-N-3H]S (41.7 Ci/mmol) were obtained from NEN Life Science Products (Boston MA); [1,2,6,7-3H]corticosterone ([1,2,6,7-3H]B; 60 Ci/mmol) and [1,2,6,7-3H]F (100 Ci/mmol) were purchased from the Radiochemical Center (Amersham Pharmacia Biotech, Aylesbury, UK). Rabbit polyclonal antibodies to bovine 3ß-hydroxysteroid dehydrogenase (3ßHSD), CYP11A1 (9), and CYP11B (10) were developed in the laboratory using purified proteins from bovine adrenal cortex.
Cell isolation and culture
Adrenocortical cells were obtained as described previously (11) by enzymatic digestion of 5 g of one hyperplastic adrenal followed by purification on a Percoll gradient. Cells (3 x 106) were obtained, seeded in multiwell dishes, and cultured for 24 h in Ham’s F-12/DMEM containing 10% FCS with antibiotics, 10 mg/ml transferrin, 10 mg/ml insulin, and 10-4 mol/L vitamin C. After 24 h of culture, the medium was changed for serum-free Ham’s F-12/DMEM. Normal human adrenocortical cells were prepared using the same procedure with a fragment of histologically normal adrenal cortex adjacent to a nonsecreting adrenocortical adenoma.
Mitochondria
A piece of adrenal gland (2 g) was homogenized with a Teflon Potter-Elvejhem apparatus in 5 mmol/L Tris-HCl buffer (pH 7.4) containing 250 mmol/L sucrose. The supernatant resulting from an 800 x g, 15-min centrifugation was centrifuged at 10,000 x g for 15 min. The resulting mitochondrial pellet was washed in the same buffer and centrifuged at 10,000 x g for 15 min.
Immunoblot analyses
Mitochondrial proteins were subjected to SDS-PAGE (10% polyacrylamide gel) and then transferred to nitrocellulose membranes. After transfer, the sheets were washed in phosphate-buffered saline (PBS) containing 0.1% (wt/vol) Tween-20 (TPBS), then incubated (1–12 h) with TPBS containing 5% (wt/vol) skim milk. Incubations with the antisera (10 µl in 15 ml TPBS) were run for 2 h. After washing, the sheets were incubated with antirabbit IgG-horseradish peroxidase conjugate (Bio-Rad Laboratories, Inc., Ivry sur Seine, France) for 1 h. The antigen-antibody complex was revealed by enhanced chemiluminescence (Renaissance, NEN Life Science Products) or was visualized by incubation in 10 ml Tris-HCl buffer (50 mmol/L; pH 7.6) containing 6 mg 3,3'-diaminobenzidine tetrachloride and 10 µl H2O2 (30%).
Hormone measurements
18-Hydroxycorticosterone, DOC, 18-hydroxydeoxycorticosterone, and aldosterone were measured in the patient’s serum by RIA (12), using antisera that were gifts from Dr. Aupetit (Hopital Pitie-Salpetriere, Paris, France). Cortisol and S were measured by RIA either in the patient’s serum or in the cell medium after separation on a Celite column to protect against cross-reactivity of F antiserum with S. Cortisol antiserum was obtained from Endocrine Sciences, Inc. (Calabasas Hills, CA); S antiserum was developed in the laboratory. Plasma ACTH-(1–39) and active renin were measured by immunoradiometric assay with commercial kits from Nichols Institute Diagnostics (San Juan Capistrano, CA) and Cis-Bio (Les Ulis, France), respectively.
Enzymatic activities
Cells. Cells were incubated for 2 h in fresh Ham’s F-12 medium in the presence or absence of ACTH (1 nmol/L). Deoxycortisol and F secreted in the medium were measured by RIA. Aldosterone production was obtained after incubation of the cells for 2 h in fresh Ham’s F-12 medium with 10 µmol/L B . Aldosterone secreted in the medium was measured by RIA.
Mitochondria: 11- and 18-hydroxylase activities. Thawed mitochondrial proteins (250 µg) were incubated with substrates [14C]DOC (2.5 nmol, 0.025 µCi) and [3H]S (2.5 nmol, 0.25 µCi) in 10 mmol/L Tris, 20 mmol/L KCl, 5 mmol/L MgCl2, 250 mmol/L saccharose, 10 mmol/L KH2PO4 (pH 7.4) buffer (buffer A) at 37 C during 15 min in the presence of 120 nmol NADPH (13). The reaction was stopped by the addition of methanol, then the steroids were extracted with chloroform. The organic phase was evaporated. The steroids were dissolved in 50 µl methanol containing unlabeled steroids corresponding to the substrate and metabolites and were spotted on TLC plates. The solvent system used was methylene chloride/methanol/water (300:20:1, vol/vol/vol). Plates containing 3H-labeled compounds were sprayed with En3Hance spray (NEN Life Science Products) before analysis by autoradiography. For plates containing 14C-labeled steroids, radiolabeled bands were visualized on a ß-imager (PhosphorImager, Molecular Dynamics, Inc., Sunnyvale, CA). Side-chain cleavage activity was measured by incubation of mitochondria proteins (250 µg) for 15 min at 37 C with 200 µmol/L 25-hydroxycholesterol in the presence of 10 µmol/L trilostane as a blocker of 3ßHSD activity and 250 µmol/L NADPH in a final volume of 500 µL buffer A. After extraction of the steroids by dichloromethane, the pregnenolone content was quantified by RIA. 3ßHSD/isomerase activity (14) was measured by incubation of 20 µmol/L [3H]DHEA in the presence of 2 µmol/L metyrapone and 200 µmol/L NAD+. DHEA and androstenedione were separated on TLC using chloroform-acetone (9:1, vol/vol).
DNA amplification and sequencing
Peripheral blood samples of the index case were obtained with informed consent according to our institutional guidelines. On the contrary, the living relatives (mother and three sisters) of the index case refused to give blood samples, which prevented us from performing a familial genetic analysis. Genomic DNA was prepared as previously described (15). Selective amplification of the CYP11B1 gene was performed in five fragments by PCR in 100 µL reactions containing 750 ng genomic DNA, 500 µmol/L of each primer, 200 µmol/L of each deoxy-NTP, 2.5 U Taq polymerase, and 1 x Taq reaction buffer (Eurobio, Les Ulis, Paris). The concentration of MgCl2 was 1.5 mmol/L, except for the exon 6–7 fragment (2 mmol/L). The PCR program on a GeneAmp 9600 Perkin-Elmer Corp. thermocycler (Norwalk, CT) was 95 C for 5 min, followed by 30 cycles of 15 s at 95 C, 15 s at 56 C (60 C for exons 7–9), and 20 s at 72 C, with a 72 C final extension for 10 min in the last cycle. The oligonucleotide primers were located in the introns, except for the reverse primer used to amplify exons 7–9, and their sequences as follows: exons 1–2, 5'-TGACGTGATCCCTCTCGAAG-3' (forward) and 5'-AAGCTTTTCCTGCCTCTCTCGCC-3' (reverse); exons 3–5, 5'-TAGACCTGAGTGGCCTTTGTC-3' (forward) and 5'-TCACATCACAATCCCAAGTAAG-3' (reverse); exons 6 and 7, 5'-GGGGTTTGGATGGGCATTAGGAT-3' (forward) and 5'- ACCCAGAGAGTAGAGGAACACG-3' (reverse); and exons 7–9, 5'- CGACCTGGGCTTCCCATGGATCT-3'(forward), 5'-CTGGGACCCTGGGTGCAGAGACG-3' (reverse). Mutation detection was performed by fluorescent sequencing with dye primer chemistry as described previously (16).
Mutations and polymorphisms are designated according to the convention of Beaudet and Tsui (17) as modified by Antonarakis (18). Numbering of the nucleotides is based on the gene sequence reported by Mornet et al. (19), excluding the introns and using as 1 the first nucleotide of the initiating ATG codon.
RNA extraction and RT-PCR
Total RNAs were purified from adrenal tissue as previously
described (11). The first complete cDNA of the CYP11B1 gene has been
generated using the Titan one-tube RT-PCR method (Roche Molecular Biochemicals), using primers located in the translated region of
exons 1 and 9: 5'-TTTGGATCCACCATGGCACTCAGGGCAAAGGCA-3' (forward) and
5'-GGGGAATCTAGAGACGTGATTAGTTGATGGC-3' (reverse). Complete sequencing of
this cDNA was performed using six primers, including the two described
above. To demonstrate the presence of one mRNA, a partial cDNA
encompassing the two polymorphisms, 225GA and 246TC, was
generated for sequencing by RT-PCR methods using the same forward
primer and a reverse primer overlapping the border between exons 2 and
3.
Ribonuclease protection assay
Probes for human P450aldo, P450c11, and actin were cloned in
pCRII and were a gift from W. Freige and W. E. Rainey (20, 21).
The linearized plasmids were used in a transcription reaction with
[
-32P]CTP and T7 RNA polymerase using the
MAXIscript in vitro transcription kit (Ambion, Inc., Austin, TX). The ribonuclease protection assay was
performed using the RPA II Ribonuclease Protection Assay kit
(Ambion, Inc.). Protected RNA species were resolved by
electrophoresis on a denaturing acrylamide (5%)-urea (8 mol/L) gel. A
32P-labeled DNA ladder was used to determine the
size of the protected fragments. pUC19 was digested with
Sau3A1, then end labeled with
[32P]deoxy-CTP and 1 U Klenow DNA polymerase.
Radiolabeled bands were visualized and quantitated by direct scanning
with a ß-imager using ImageQuant software (PhosphorImager, Molecular
Dynamics), with the actin signal as the reference.
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Results |
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TopAbstractIntroductionSubject and MethodsResultsDiscussionReferences |
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Production of F and S was measured in the cells under basal
conditions or during a 2-h stimulation by ACTH. As expected, the
patient’s cells showed no measurable production of F, but had high
basal and ACTH-stimulated productions of S. The cells were also
incubated in the presence of B. Aldosterone production was detectable,
although weaker, than the production of normal adrenocortical cells
(Fig. 2A). Incubation of the patient’s
adrenocortical mitochondria with [3H]S or
[14C]DOC also showed no production of F and B,
respectively (Fig. 2, B and C), demonstrating the absence of detectable
11ß-hydroxylase activity. However, the patient’s mitochondria showed
weak production of 18-hydroxycorticosterone (Fig. 2C). Both
mitochondrial side-chain cleavage activity (patient, 2 ± 0.3
pmol/min·mg protein; normal adrenal, 1.15 ± 0.3 pmol/min·mg
protein) and 3ßHSD/isomerase activity (patient, 49 ± 5
pmol/min·mg protein; normal, 46 ± 3 pmol/min·mg protein) were
similar to those of normal adrenal mitochondrias.
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Sequencing of the patient’s DNA revealed two new base
substitutions in the CYP11B1 genomic sequence: 954 GC and
IVS8 + 4AG (Fig. 3). The G to C
substitution at the last nucleotide of exon 5 does not change the
predicted amino acid, threonine 318. The A to G substitution at the
fourth nucleotide of intron 8, termed either 1398 + 4AG or IVS8 +
4AG, is located two nucleotides downstream of the 5'-GT splice donor
site. These substitutions, the last nucleotide G of an exon and the
fourth nucleotide A of an intron, have two properties in common: they
are highly conserved in higher eukaryotes (80.3% and 72%,
respectively), and they belong to the consensus sequence for the
5'-splicing donor site (exon G GUAA intron) (22). In
addition, two frequent polymorphisms, 225GA (exon 1) and 246TC
(exon 2), were found in the patient’s DNA. As the family members
refused to participate in a genetic analysis, it was not possible to
study the segregation of the two CYP11B1 alleles.
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To understand the mechanisms by which the two genomic DNA base
substitutions could account for the lack of 11ß-hydroxylase activity,
mRNA was extracted from the patient’s adrenal and reverse transcribed
into cDNA, and CYP11B1 cDNA were amplified by PCR. A normal adrenal and
an adrenal originating from a patient with ACTH-dependent Cushing’s
syndrome were used as controls. Separation of the PCR products on an
agarose gel showed that both the normal adrenal and the Cushing adrenal
had three cDNA bands, including one of the expected size for the full
CYP11B1 cDNA and two smaller ones. In contrast, the patient’s adrenal
showed only the smallest band (Fig. 4A).
Sequence analysis showed that this band corresponded to a CYP11B1 cDNA
that lacked exon 8 and did not bear the 954 GC base substitution at
exon 5 (Fig. 4B). To demonstrate the presence of one mRNA, a partial
cDNA encompassing the two polymorphisms, 225GA and 246TC, was
generated for sequencing by RT-PCR methods using the same forward
primer and a reverse primer overlapping the border between exons 2 and
3. Neither of the two polymorphisms was found in the patient’s cDNA.
Ribonuclease protection assay was then performed using specific CYP11B1
and CYP11B2 probes located in the untranslated region of exon 9, in
which there is less homology between the two sequences (20). Expression
of CYP11B1 mRNA was detected in the patient’s adrenals (
30% of the
expression in normal cells), confirming expression of a CYP11B1 allele
containing exon 9. Expression of CYP11B2 mRNA, which was high in an
aldosterone-producing tumor used as a control, was below the limit of
detection in the patient’s cells, as it was in normal cells (Fig. 5).
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To test the hypothesis that the two genomic DNA mutations
resulted in the expression of only a truncated protein lacking exon 8,
analysis of CYP11B1 immunoreactivity was then performed on the
patient’s adrenal mitochondria using a bovine anti-CYP11B serum that
also recognizes human CYP11B1 and CYP11B2. Cytochrome
P450scc (CYP11A1) and 3ßHSD immunoreactivities
were assayed as controls. The patient’s mitochondria showed normal
cytochrome CYP11A1 and 3ßHSD immunoreactivities, but almost no CYP11B
immunoreactivity at the normal apparent size (51 kDa). There was
instead weak CYP11B immunoreactivity corresponding to a protein of
about 43 kDa, consistent with the size of an exon 8 truncated CYP11B1
protein (Fig. 6). The very weak CYP11B
immunoreactive band that could be detected at the normal size might be
related to the very low expression of a normal CYP11B1 protein, to the
expression of CYP11B2, or to the low cross reactivity of the
anti-CYP11B serum toward CYP11A1.
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Discussion |
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TopAbstractIntroductionSubject and MethodsResultsDiscussionReferences |
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The patient reported here illustrates the difficulties with suppressive therapy and the potential interest of adrenalectomy. In childhood, suppressive therapy was marked by periods of either undertreatment with poor control of hypertension or overtreatment and appearance of Cushingoid features. The very short final stature is the combined result of androgen-related precocious bone maturation, supraphysiological glucocorticoid administration, and the female genetic sex. During adulthood, compliance was very poor, as suppressive treatment was dropped because it was felt to be responsible for weight gain. As a result the patient developed pronounced left ventricular concentric hypertrophy and finally presented with central retinal vein occlusion, for which hypertension is a well established major risk factor and the only one present in this patient (25). Restoration of suppressive treatment was again rapidly confronted with the fact that effective suppression of mineralocorticoid secretion required doses of glucocorticoid that induced Cushingoid features, especially weight gain. It should be stressed that the macronodular hyperplasia that had developed during the years of unsuppressed ACTH secretion probably made suppressive treatment even more difficult even though no truly autonomous secretion had developed.
A comment is necessary on the decision, made in the early 1950s, to raise this pseudohermaphroditic female patient as a male and perform ovariectomy. This strategy, which resulted in infertility and had major consequences on the patient’s social and sexual life, is now generally abandoned in favor of surgical reconstruction of the abnormal female external genitalia (5, 26). Here, one can assume that the male phenotype helped the patient to drop suppressive treatment, as it allowed him to escape the symptoms of unsuppressed adrenal androgen production.
This report and others (8, 23, 24, 27) question the feasibility and effectiveness of suppressive treatment in preventing the long-term consequences of exposure to androgen and mineralocorticoid excess. Hence, the question of adrenalectomy as an alternative treatment deserves the same attention in steroid 11ß-hydroxylase deficiency as in 21-hydroxylase deficiency (7). Regarding this debate there are some differences between the two main causes of CAH. In steroid 11ß-hydroxylase deficiency the patients are not naturally deficient in mineralocorticoid, so noncompliant patients are generally not exposed to the threat of acute adrenal insufficiency; salt loss is rare and has been described mainly in patients after suppressive treatment, who can develop atrophy of the fascicula-reticularis zone (29, 30, 31). This point would argue against adrenalectomy in poorly compliant 11ß-hydroxylase-deficient patients. However, poorly compliant 11ß-hydroxylase-deficient patients are precisely those who may benefit more from adrenalectomy; most of them will eventually develop moderate to severe hypertension, and because of long-term poor suppression of ACTH, they will develop a marked degree of nodular hyperplasia that makes suppressive therapy even more difficult. Therefore, in discussing adrenalectomy in a poorly compliant, steroid 11ß-hydroxylase-deficient patient, one needs to thoroughly analyze the reason for poor compliance. Here, we believed that the main reason was the responsibility of suppressive therapy for the appearance of Cushingoid features, a problem unlikely to happen with substitutive therapy. So we hypothesized that there will not be compliance problems with substitutive therapy. This proved, to date, to be correct.
Molecular genetics aspects of congenital hyperplasia are relevant to
the decision for adrenalectomy. In steroid 21-hydroxylase deficiency,
Ritzen and Wedell advocate surgical treatment instead of suppressive
therapy for girls harboring mutations that are known to annihilate
21-hydroxylase activity (7). Patients with the classical form of
steroid 11ß-hydroxylase deficiency are also believed to bear
mutations that totally destroy the expression or activity of CYP11B1
(5). However, in the patient reported here, this was unclear, as
analysis of genomic DNA had shown two previously undescribed base
substitutions whose significance remained elusive, as the 954 GC
transversion in exon 5 is conservative, and the IVS8 + 4AG mutation
is intronic (intron 8). Adrenal tissue obtained at surgery showed that
these substitutions were deleterious. Sequencing of adrenal
CYP11B1 cDNA showed that it was shorter due to the absence
of exon 8 and that it lacked exon 5 954 GA mutation and the two
polymorphisms. This indicated that the intron 8 mutation (IVS8 +
4AG) and the exon 5 954 GA mutation were on different alleles.
One CYP11B1 allele carried the intron 8 mutation (IVS8 +
4AG) in the 5'-splice donor site, and this mutation should be
responsible for the skipping of exon 8. The other allele carried exon 5
954 GA mutation and the two polymorphisms, and this allele was not
expressed. As exon 8 encodes for the binding region of the heme
prosthetic group necessary for the enzymatic activity (32), the
truncated P450c11 protein is not functional, as suggested by in
vitro studies. The reason for the loss of the other allele remains
somewhat speculative. Codon 318 has been reported to be the site of
different mutations, T318M (953CT), T318R (953TG) (6, 33). In our
patient, the 954 GC transversion is conservative (T318T), but it is
located on the last base of exon 5, which is part of the splicing donor
site. A homozygous conservative mutation in the same codon, 954GA,
has actually been described in a patient with steroid 11ß-hydroxylase
deficiency, and it was suggested to affect the correct splicing of
CYP11B1mRNA. However, this patient also carried a
nonconservative homozygous mutation C494F in exon 9 that could be
sufficient to explain his phenotype (34). Several mutations of the last
nucleotide have been reported as 5'-splice site mutations (35). The
major consequence of these mutations was often the skipping of the
entire preceding exon and sometimes the use of cryptic donor sites
downstream in the exon. Sequencing of CYP11B1 cDNA seems to rule out
these hypotheses. Nevertheless, as skipping of exon 5 should create a
frame shift with a codon stop at 329, it is possible that the aberrant
splicing due to this 954 GC mutation generates an unstable mRNA
undetectable by RT-PCR methods. However, we cannot exclude the
possibility that the allele carrying the exon 5 mutation might also
carry another mutation in the promoter region of CYP11B1 that would be
responsible for the loss of expression of this allele.
In conclusion, we have described two new mutations in CYP11B1 in a patient with severe steroid 11ß-hydroxylase deficiency. These mutations are presumably responsible for the loss of 11ß-hydroxylase activity, although they do not change, at first glance, the predicted amino acid sequence coded by the mutated alleles. These severe mutations were described in a patient who illustrates the difficulties in maintaining an effective suppressive therapy until adulthood and raises the question of adrenalectomy as an alternative. It should be stressed that adrenalectomy in adulthood for CAH is a decision that in a way negates the previous therapeutic choices made in infancy by the medical team and endorsed by the patient for many years. Therefore, one can hope that further clinical and genetic studies will help define which patients with steroid 11ß-hydroxylase deficiency will most benefit from adrenalectomy in early infancy.
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Acknowledgments |
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Received March 4, 2000.
Revised June 29, 2000.
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References |
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TopAbstractIntroductionSubject and MethodsResultsDiscussionReferences |
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G splice-donor-site mutation
at position +3 of the collagenlike-tail-subunit gene (COLQ): how does G
at position +3 result in aberrant splicing? Am J Hum Genet. 65:635–644.[CrossRef][Medline]
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  J. J. Van Wyk and E. M. Ritzen The Role of Bilateral Adrenalectomy in the Treatment of Congenital Adrenal Hyperplasia J. Clin. Endocrinol. Metab., July 1, 2003; 88(7): 2993 - 2998. [Abstract] [Full Text] [PDF]
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S. Portrat, P. Mulatero, K. M. Curnow, J.-L. Chaussain, Y. Morel, and L. Pascoe Deletion Hybrid Genes, due to Unequal Crossing Over between CYP11B1 (11{beta}-Hydroxylase) and CYP11B2(Aldosterone Synthase) Cause Steroid 11{beta}-Hydroxylase Deficiency and Congenital Adrenal Hyperplasia J. Clin. Endocrinol. Metab., July 1, 2001; 86(7): 3197 - 3201. [Abstract] [Full Text] [PDF]
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