The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 11 3525-3528
Copyright © 1997 by The Endocrine Society
Hereditary Defect in Biosynthesis of Aldosterone: Aldosterone Synthase Deficiency 1964–19971
Michael Peter,
Lubna Fawaz,
Stenvert L. S. Drop,
Hendrik K. A. Visser and
Wolfgang G. Sippell
Divisions of Pediatric Endocrinology, Departments of Pediatrics,
Christian-Albrechts-University of Kiel (M.P., L.F., W.G.S.), Kiel,
Germany and Sophia Children’s Hospital (S.L.S.D., H.K.A.V.), Erasmus
University of Rotterdam, The Netherlands
Address all correspondence and requests for reprints to: W.G. Sippell, Professor of Pediatrics, Division of Pediatric Endocrinology, Department of Pediatrics, Universitäts-Kinderklinik, D-24105 Kiel, Schwanenweg 20, Germany.
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Abstract
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We studied two of the three patients with a hereditary defect in the
biosynthesis of aldosterone originally described by Visser and Cost in
1964. All three presented as newborns with salt-losing syndrome and
failure to thrive. The original biochemical studies showed a defect in
the 18-hydroxylation of corticosterone. According to the nomenclature
proposed by Ulick, this defect would be termed corticosterone methyl
oxidase deficiency type I. We measured plasma steroids in the untreated
adult patients and performed molecular genetic studies. Aldosterone and
18-OH-corticosterone were decreased, whereas corticosterone and
11-deoxycorticosterone were elevated, thus confirming the diagnosis of
corticosterone methyl oxidase deficiency type I. Cortisol and its
precursors were in the normal range. Genetic defects in the gene
CYP11B2 encoding aldosterone synthase (P450c11Aldo) have been described
in a few cases. We identified a homozygous single base exchange (G to
T) in codon 255 (GAG) causing a premature stop codon E255X (TAG). This
mutation destroys a Aoc II restriction site. Digestion of a PCR
fragment containing exon 4 of CYP11B2 (261 bp) with this restriction
enzyme revealed in the two patients homozygous for the E255X mutation
only a 261-bp fragment, whereas the heterozygous parents had three
fragments (261 bp from the mutant allele and 194 and 67 bp from the
wild-type allele). The mutant enzyme had lost the five terminal exons
containing the heme binding site, and thus there was a loss of function
enzyme. We conclude that the biochemical phenotype of these prismatic
cases of congenital hypoaldosteronism can be explained by the patients
genotype.
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Introduction
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IN 1964, Visser and Cost (1) and Ulick
et al. (2) described contemporaneously and independently a
biosynthetic defect with autosomal recessive inheritance causing
selective hypoaldosteronism caused by deficient 18-hydroxylation of
corticosterone (3) and 18-oxidation of 18-hydroxycorticosterone
(2), respectively. Ulick (4) suggested the two biochemically different
forms of selective aldosterone deficiency be termed corticosterone
methyl oxidase (CMO) deficiency type I and type II. In both CMO types,
aldosterone biosynthesis is impaired, whereas corticosterone of zona
glomerulosa origin, under the primary control of the renin angiotensin
system, is excessively produced. The two defects differ biochemically
in that 18-hydroxycorticosterone is deficient in CMO I but overproduced
in CMO II. Both disorders are characterized clinically by salt-wasting,
failure to thrive, and growth retardation. The clinical spectrum of the
defects has been summarized by Veldhuis and Melby (5), Rösler
(6), Ulick (7), and Drop et al. (8). The molecular basis of
terminal aldosterone biosynthesis has been studied in detail in recent
years. It is now clear that the terminal steps of aldosterone
biosynthesis in the zona glomerulosa are catalyzed by a single
cytochrome P450 enzyme termed P450c11Aldo (9). Humans have a distinct
cytochrome P450 isoenzyme that catalyzes hydroxylation at position
11ß in the zona fasciculata termed P450c11. The genes encoding for
P450c11 and P450c11Aldo are termed CYP11B1 and CYP11B2, respectively.
The human P450c11 and P450c11Aldo enzymes have been predicted to be
93% identical in their amino acid sequence (10). Both genes are
located on chromosome 8q22 (11). So far, the molecular basis of CMO I
and CMO II deficiencies has been explained in a few cases by
identifying mutations on the CYP11B2 gene destroying the enzyme
activity (12, 13, 14, 15, 16). There exists one report showing no mutations in the
CYP11B2 gene in a patient with CMO deficiency type II (17). The
disorders of steroid 11ß-hydroxylase isoenzymes have been reviewed
previously by White et al. (18). In this study, we report
results of a biochemical and molecular genetic reexamination of the two
prismatic cases originally described by Visser and Cost in 1964
(1).
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Subjects and Methods
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Patients
Three infants with a salt-losing syndrome in the neonatal period
were originally described by Visser and Cost (1) and Degenhart et
al. (3). They presented with dehydration, poor feeding, occasional
vomiting, failure to gain weight, and intermittent febrile temperature.
The main clinical features are summarized in Table 1
. The pedigree of the family is shown in
Fig. 1.
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Table 1. Summary of main clinical features of patients with
congenital hypoaldosteronism caused by a defect in 18-hydroxylation
published by Visser and Cost (1)
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Figure 1. Pedigree of family published by Visser and
Cost (1) (upper). Point mutation in CYP11B2 gene
detected by direct sequencing of exon 4 in patient 1 and her mother
(lower right). Patient is homozygous for a stop mutation
(G to T transversion) in codon 255. Codon 255 (GAG) encodes for
glutamic acid in wild-type sequence. Mutation causes a premature stop
codon (TAG) in exon 4 (E255X). Mother is a heterozygous carrier of
mutation. Same sequencing data were obtained in patient 3 and both
parents (lower left).
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Methods
Steroid determinations.
The diagnosis of corticosterone
methyl oxidase deficiency was reconfirmed by measurement of adrenal
plasma steroids. Blood samples for steroid determination in the
untreated adult patients were drawn in the morning, collected in
prechilled heparinized tubes, and immediately centrifuged at +4 C.
Plasma was kept frozen at -20 C until assayed. Plasma steroids were
measured using a previously described method for the simultaneous
determination of multiple steroids in a small plasma volume of 1–2 mL
developed in our laboratory (19, 20). Normal values for the different
steroids determined by our method of multisteroid analysis have been
published elsewhere (21).
Nucleotide sequences of exons and exon/intron boundaries.
Blood samples for molecular genetic studies were drawn after informed
consent was obtained from all family members. Genomic DNA was extracted
from peripheral blood leukocytes, and the CYP11B2 gene was specifically
amplified in two fragments containing the 9 exons by PCR. Regions of
the CYP11B2 gene having extensive mismatches with CYP11B1 were used for
synthesis of primers (Table 2). PCR
products were treated before sequencing using exonuclease I and shrimp
alkaline phosphatase. The nucleotide sequence of both strands of the
PCR products was directly determined by thermocycle sequencing using
the Thermo Sequenase radiolabeled terminator cycle sequencing kit
following the manufacturer’s instructions (Amersham Life Sciences,
Cleveland, OH).
Restriction endonuclease digestion to confirm mutation.
Using the 3.5-kilobase PCR product containing exons 1–4, exon 4 was
reamplified using primers shown in Table 2
. The 261-bp PCR product was
then digested with the restriction endonuclease Aoc II (Amersham Life
Sciences). Digestion products were visualized after electrophoresis in
a 3% agarose gel and staining with ethidium bromide.
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Results
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Steroid determinations
Results of the plasma steroid determinations are shown in Table 3. The two adult patients studied in an
untreated state showed the typical steroid pattern of a defect in
18-hydroxylation of corticosterone. Aldosterone and
18-OH-corticosterone were decreased, whereas
corticosterone and 11-deoxycorticosterone were increased. The
corticosterone/18-OH-corticosterone ratio was elevated, and the
18-OH-corticosterone/aldosterone ratio was decreased. According to the
nomenclature proposed by Ulick, this defect is termed CMO type I (CMO
I).
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Table 3. Basal plasma steroids in two adult
aldosterone-deficient patients (CMO I) determined by multisteroid
analysis
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Nucleotide sequences of exons and exon/intron boundaries
Direct sequencing of the patients’ DNA (patients 1 and 3) showed
that both were homozygous for a single base exchange in their CYP11B2
genes. We identified a homozygous G to T transversion in codon 255
(Fig. 1). Codon 255 encodes glutamic acid (GAG) in the wild-type
enzyme. The mutation causes a premature stop codon E255X (TAG). Direct
sequencing of exon 4 showed that the mother from patient 1 and both
parents from patient 3 were heterozygous for the E255X mutation (Fig. 1).
Restriction endonuclease digestion to confirm mutation
The mutation detected by direct sequencing of the PCR-amplified
CYP11B2 gene was confirmed by restriction endonuclease analysis. The
E255X mutation destroys a Aoc II restriction site (GAGCA 
C).
Digestion of a PCR fragment containing exon 4 of CYP11B2 (261 bp) with
this restriction enzyme revealed in the two patients homozygous for the
E255X mutation only a 261-bp fragment, whereas the heterozygous parents
had three fragments of 261 bp from the mutant allele and 194 and 67 bp
from the wild-type allele (data not shown).
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Discussion
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In the early 1960s, Visser and Cost (1) and Ulick et
al. (2) were contemporaneous and independent observers of a new
biosynthetic defect in the terminal aldosterone biosynthesis. We had
the opportunity to perform biochemical and molecular genetic studies in
two of the patients reported in the original publication by Visser and
Cost (1). The plasma steroid pattern in the now-adult patients still
shows the same striking features as originally reported (1, 3).
According to the nomenclature proposed by Ulick (4) and to the plasma
steroid profiles in the large series of patients reported by our group
(22), this defect is termed CMO deficiency type I. In comparison with
patients with CMO deficiency type II, patients with CMO deficiency type
I seem to have the more severely decreased enzyme activity of
P450c11Aldo (12, 13, 14, 15, 16, 17). With regard to clinical severity, there seems to
be no difference between the two biochemical types (23). We made the
same observation as Rösler (6) that the clinical severity of the
disease decreased with age even though the biochemical abnormalities
continue.
Amino acid residues have been identified in a recent study using
transfection experiments with complementary DNAs that encode hybrids
between the highly homologous cytochrome P450 enzymes, CYP11B1
(11ß-hydroxylase) and CYP11B2 (aldosterone synthase), determining the
different catalytic activities of both enzymes. Efficient
18-hydroxylation requires a glycine residue at position 288, and
subsequent sufficient 18-oxidation requires an alanine at position 320
(24). The stop codon in exon 4 identified in the two patients reported
in this study might explain the biochemical phenotype, because the
truncated enzyme lost the five terminal exons. Homology alignment of
the mitochondrial P450 enzymes to the bacterial P450BM-3 (CP102 from
Bacillus megaterium) for which x-ray diffraction crystallographic data
are available, suggests that these enzymes may consist of a conserved
core that is built up around the prosthetic heme molecule, and to which
variable loops are attached (25). Exons 5–9 encode for several
-helices and ß-strands containing important residues for proton
transfer, accessory protein binding, heme binding, and substrate
binding (26). Thus, one can imagine that the P450c11Aldo expressed in
the adrenals of the patients is a loss of function enzyme. The progress
in molecular biology and steroid determination techniques permit a
better understanding of the etiology and pathophysiology of this very
rare disorder in steroid metabolism.
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Acknowledgments
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We thank Mrs. Sabine Stein and Mrs. Susanne Neumann-Olin for
their technical assistance in the multisteroid analyses and Mrs. Gisela
Hohmann for her expert technical assistance in molecular biology
techniques. We also thank Mr. W. Deelen for preparation of genomic DNA.
We are grateful to Mrs. Joanna Voerste for linguistic editing of the
manuscript.
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Footnotes
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1 This work was supported by Grant Pe 589/1–1 from the Deutsche
Forschungsgemeinschaft (DFG). 
Received March 28, 1997.
Revised July 11, 1997.
Accepted August 15, 1997.
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References
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Top
Abstract
Introduction
