The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 12 4391-4393
Copyright © 1998 by The Endocrine Society
The Codon 213 of the 11ß-Hydroxysteroid Dehydrogenase Type 2 Gene Is a Hot Spot for Mutations in Apparent Mineralocorticoid Excess1
Daniela Rogoff,
Zuzana Smolenicka,
Ignacio Bergadá,
Graciela Vallejo,
Marta Barontini,
Juan Jorge Heinrich and
Paolo Ferrari
Division of Endocrinology (D.R., I.B., M.B., J.J.H.), Nephrology
Unit (G.V.), Hospital de Niños "Ricardo Gutiérrez",
Buenos Aires 1425, Argentina; and Division of Nephrology (Z.S., P.F.),
Inselspital Bern 3010, Switzerland
Address all correspondence and requests for reprints to: Juan Jorge Heinrich, Division of Endocrinology, Hospital de Niños "Ricardo Gutiérrez", Buenos Aires 1425, Argentina. E-mail:
cedie{at}exito.pccp.com.ar
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Abstract
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In the kidney, the 11ß-hydroxysteroid dehydrogenase type 2 enzyme
(11ßHSD2) inactivates glucocorticoids to their inactive ketoforms and
thus prevents endogenous glucocorticoids from occupying the
nonselective mineralocorticoid receptor in epithelial tissues. Several
mutations have been described in the 11ßHSD2 gene in the congenital
syndrome of apparent mineralocorticoid excess. These mutations
generate partially or completely inactive 11ßHSD2 enzymes.
In the present work, we describe an already known mutation in a new
patient affected by apparent mineralocorticoid excess, which results in
an arginine-to-cysteine mutation (R213C) in the 11ßHSD2 enzyme. This
mutation has been found in two other independent families. In
vitro expression studies of this mutant provide evidence that
the mutant protein is normally expressed, but its activity is
abolished. The CGC-to-TGC (C-toT) transition at codon 213 can be
considered a typical CpG-consequence mutation.
The present finding suggests that the codon R213 of 11ßHSD2 is a hot
spot for mutations in this gene, as shown by the occurrence of an R213C
point-mutation in several families unrelated to each other.
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Introduction
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THE SYNDROME of apparent mineralocorticoid
excess (AME) is a rare disease characterized by childhood onset
hypertension, hypokalemic alkalosis, and low renin and aldosterone
secretion (1). It is the consequence of the inadequate conversion of
cortisol to cortisone caused by a defect in the activity of the
11ß-hydroxysteroid dehydrogenase type 2 enzyme (11ßHSD2). The
11ßHSD2 enzyme is predominantly found in mineralocorticoid target
organs such as kidney, colon, and salivary glands, as well as in the
placenta and some fetal tissues (2, 3). Because, in vitro,
the mineralocorticoid receptor has a similar affinity to aldosterone
and cortisol and, in vivo, cortisol is found in
concentrations 100-1000 times that of aldosterone, a mechanism of
selectivity for aldosterone is needed. This selectivity is provided by
the 11ßHSD2, a unidirectional enzyme that catalyzes the conversion of
cortisol into cortisone through oxidation of the 11-hydroxyl residue
(4, 5). Thus, because cortisone is biologically inactive, the
nonselective mineralocorticoid receptor is protected from the
occupation by cortisol (6, 7, 8).
Mutations in the 11ßHSD2 gene generate a compromised 11ßHSD2 enzyme
activity (9, 10), leading to an overstimulation of the
mineralocorticoid receptor by cortisol and, thus, sodium retention,
hypokalemia, and high blood pressure (1). To date, approximately 60
cases of AME syndrome have been reported in the literature. An
autosomic recessive transmission has been proposed (6). Since the
cloning of the human 11ßHSD2 gene in 1994 (11), at least 13 different
mutations have been described (12) and the associated functional
impairment demonstrated (9, 13, 14). One of the already published
mutations is located on codon 213 of exon 3 (9, 14).
In this study, we report another patient with severe hypertension,
secondary to an AME syndrome with a mutation at the codon 213 of the
11ßHSD2 gene, and the impairment of the activity of the expressed
enzyme.
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Subjects and Methods
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History, clinical, and other findings
C.P. is an 11-yr-old prepubertal Caucasian boy, born at term,
from nonconsanguineous Argentinean parents, after an uneventful
pregnancy, and referred to our Hospital because of moderate arterial
hypertension and hypokalemic alkalosis. His birth weight was 2050
g. During infancy, many studies had been performed because of failure
to thrive, but no specific diagnosis was provided. Arterial
hypertension was detected a few months before admission, after an
episode of epistaxis. Subsequent controls showed a permanent arterial
hypertension. At admission, his height was 132.0 cm (Standard Deviation
Score -1.9), weight was 23 kg (below 3rd percentile), and blood
pressure was 130/90 mm Hg. Other findings, on physical examination,
were unremarkable. An electrocardiogram revealed left ventricular
hypertrophy, and a fundoscopic examination was normal. A renal
ultrasound revealed normal-sized kidneys but with marked signs of
nephrocalcinosis. Results of biochemical studies are shown in Table 1
. The hypokalemic alkalosis, the
polyuria (5.6 mL/k·h), and the hypercalciuria were remarkable.
Gas chromatography-mass spectrometry of urinary steroid metabolites,
kindly performed by Prof. Dr. M. Zachmann (University of Zurich,
Switzerland) showed an abnormally high tetrahydrocortisol (5ßTHF) +
allotetrahydrocortisol (5
THF)/tetrahydrocortisone (THE) ratio. All
these data confirmed the diagnosis of AME syndrome (Table 1
) (6). To
continue with studies, informed consent was obtained from both parents
and the patient.
Molecular studies
DNA of the index subject was analyzed by PCR amplification of
exons 3, 4, and 5 of the 11ß-hydroxysteroid dehydrogenase type 2
gene, carried out in a GeneAmp 9600 thermal cycler (Perkin-Elmer Corp., Oak Brook, IL) using standard protocols. Primers were
derived from the intronic sequences flanking exons published elsewhere
(9). PCR products were analyzed on 12% acrylamide gels, containing
7.25% glycerol, using a two-buffer system; 4 µL of the PCR sample
were loaded, and DNA was visualized by silver staining (15). This
showed a band shift in the PCR product of exon 3, whereas exons 4 and 5
were normal. The variant was purified using QIA-quick PCR purification
columns (Quiagen, Chatsworth, CA), according to the
supplier’s instructions and sequenced on an ABI 373 A automated
sequencer (PE Applied Biosystems, Foster City, CA) using
the same primers as for PCR. The obtained sequence was compared with
the 11ßHSD2 sequence and showed a homozygous point mutation from CGC
to TGC at codon 213, which resulted in the substitution of an arginine
for cysteine. Molecular analysis of both parents confirmed the presence
of the mutation in the heterozygous state.
Full-length human 11ßHSD2 complementary DNA (cDNA) was subcloned into
the pALTER vector (Promega Corp., Madison, WI) and
mutagenized with the appropriate oligonucleotides, as described
previously (10). The construct was sequenced throughout the
corresponding coding regions to verify the mutation. Mutant cDNA was
subcloned into pcDNA1 to generate the pR213C expression plasmid and was
transfected into mammalian CHOP cells. The 11ßHSD2 activity was
analyzed by incubating transfected CHOP cells with [3H]-cortisol for
2 h. Steroids were extracted and analyzed as previously described
(10). This showed a mutated enzyme with completely abolished enzymatic
activity, as compared with wild-type enzyme (Fig. 1). Western blot analysis was performed
with an immunopurified polyclonal antibody (HUH21), using
chemiluminescent detection (16), and it identified the presence of
immunoreactive mutated protein of identical molecular weight as the
normal enzyme (Fig. 1).
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Figure 1. A, Metabolism of cortisol by CHOP cells
transfected with plasmids expressing the vector (V, open
bar), wild-type (WT, solid bar), and R213C
mutant (Mut, hatched bar) enzymes. Mean ±
SD, n = 3. B, Western blot analysis of homogenates
from CHOP cells transfected with plasmid V, WT, and Mut constructs.
Molecular weight markers are indicated on the left; the
sizes of the WT and Mut proteins are both 41K.
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Discussion
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The clinical features of this patient, homozygous for the R213C
mutation, are characteristic of a markedly defective 11ßHSD2 activity
causing AME (1, 7). In a previous study, incubation for up to 20 h
showed 16% activity of the R213C mutant for cortisol (9), whereas in
the present study, incubation of the mutant for 2 h did not show
any significant conversion of cortisol into cortisone, as compared with
vector. Thus, this mutation generates an enzyme with nearly completely
abolished dehydrogenase activity. To date, this mutation has been
described previously in two siblings from a Caucasian/Native South
American ethnic background (6, 9), and it has been identified in
another Caucasian sibship in France (14). Even when we can not exclude
a founder effect, as was suspected in three families with the R 337 H
mutation (17), it seems that the R213 codon of the HSD11B2 gene is a
hot spot for mutations in this gene, occurring in several families
unrelated to each other. This is not surprising, because the C-to-T
transition can be considered a typical CpG-consequence mutation (18, 19). In fact, C-to-T transitions happen relatively frequently, because
C is spontaneously deaminated to uracil. In cells, there are specific
enzymes that remove deoxyuracil; and therefore, the C-to-T transition
is slower than the deamination frequency. However, if C is methylated
at the 5 position, then its deaminated form is no longer a U but
a T; thus, it is not so efficiently removed, although the mismatch is
usually efficiently repaired. Dinucleotides of the sequence 5'-CG-3'
are small palindromes that, in vertebrates, are recognized by the
methyltransferase that methylates C residues at the 5' position. The
methylation occurs in transcriptionally inactive DNA segments. Thus, CG
dinucleotides are preferred substrates for the C-to-T conversion and on
the complementary strand G-to-A conversion (18, 19).
The AME syndrome is a rare autosomal disorder that, similarly to Liddle
Syndrome (20) and glucocorticoid remediable aldosteronism (21), belongs
to a group of endocrine diseases that causes hypokalemic hyporeninemic
arterial hypertension secondary to specific well-characterized gene
defects. It is clinically characterized by polydipsia, polyuria,
intrauterine growth retardation, failure to thrive, and hypertension,
with hypokalemia that usually develop during infancy or childhood (1, 7, 22). Nephrocalcinosis is usually found in this syndrome, as well as
in other hyperaldosteronism states (23). Polyuria is caused by the
hypokalemia and by the extracellular volume expansion, with an
increased glomerular filtration rate typical of all
hypermineralocorticisms. As a consequence, a reduced absorption of
sodium and (secondary) of calcium in the proximal tubule occurs,
resulting in hypercalciuria (24). The persistent alkalosis produces a
reduction of ionic calcium that, in conjunction with the
hypercalciuria, induces a secondary hyperparathyroidism and osteopenia
(25). In our case, the prominent clinical features were the growth
impairment, hypertension, hypokalemia, and nephrocalcinosis. The
biochemical diagnosis was made, detecting the abnormally high ratio of
tetrahydro urine metabolites of cortisol-to-cortisone (5ßTHF +
5THF/THE). (1, 6).
In summary, we report a new case of AME syndrome, with an already known
mutation at codon 213, previously reported in two unrelated families.
This mutation generated an 11ßHSD2 enzyme with abolished function.
Thus, the arginine-to-cysteine substitution at codon 213 could possibly
be a frequent site of mutation in the 11ßHSD2 gene, potentially
accounting for some severe cases of childhood hypertension.
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Acknowledgments
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Our thanks to H. Domené (Ph.D.) and G. Sansó
(Ph.D.), who collaborated in the processing of the samples.
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Footnotes
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1 This work was supported by grants (to P.F.) from the Cloetta
Foundation and Swiss National Foundation for Scientific Research (NR
3200–049835). 
Received April 22, 1998.
Revised August 13, 1998.
Accepted August 18, 1998.
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Top
Abstract
Introduction
