The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 10 3506-3508
Copyright © 1997 by The Endocrine Society
Evidence for Endocrinological Abnormalities in Heterozygotes for Adrenal 11ß-Hydroxylase Deficiency of a Family with the R448H Mutation in the CYP11B1 Gene1
Michael Peter and
Wolfgang G. Sippell
Division of Pediatric Endocrinology, Department of Pediatrics,
Christian-Albrechts University, Kiel, Germany
Address all correspondence and requests for reprints to: W. G. Sippell, M.D., Division of Pediatric Endocrinology, Department of Pediatrics, Universitäts-Kinderklinik, Schwanenweg 20, D-24105 Kiel, Germany.
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Abstract
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In about 5% of cases of classical congenital adrenal hyperplasia,
steroid 11ß-hydroxylase deficiency is the underlying defect. In two
publications, no biochemical abnormalities have been reported in
obligate heterozygotes for 11ß-hydroxylase deficiency. We found the
typical plasma steroid pattern of 11ß-hydroxylase deficiency and
identified the R448H mutation in the CYP11B1 gene in a boy presenting
with pseudoprecocious puberty at age 2 yr. Both parents and an older
sister were genotyped and were heterozygous carriers for the R448H
mutation in CYP11B1. In contrast to the data reported in the
literature, we found increased responses of plasma 11-deoxycortisol and
11-deoxycorticosterone in the short term ACTH test in the three family
members heterozygous for the R448H mutation.
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Introduction
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CONGENITAL adrenal hyperplasia (CAH) caused
by a deficiency of steroid 11ß-hydroxylase (P450c11) is an autosomal
recessive inherited disorder of steroid metabolism. The molecular basis
of this disorder is a defective gene (CYP11B1) encoding adrenal
microsomal cytochrome P450c11 (1, 2). 11ß-Hydroxylase deficiency
comprises about 5% of cases of congenital adrenal hyperplasia,
occurring in about 1 of 100,000 births in the general Caucasian
population (3). In CAH due to 21-hydroxylase (P450c21) deficiency,
hormonal studies in obligate heterozygous parents demonstrated a mild
21-hydroxylase deficiency. Family studies have shown that heterozygous
carriers of a defective gene for P450c21 have a higher
17-hydroxyprogesterone response after ACTH stimulation than the control
population. There is, however, an overlap of about 20% in the range of
response between heterozygotes and people without the defect,
particularly among pubertal girls and women (4). In an own study, we
showed that after ACTH stimulation, the ratio of 17-hydroxyprogesterone
to 11-deoxycorticosterone, determined by our method of multisteroid
analysis (5), can discriminate almost all heterozygotes for
21-hydroxylase deficiency from the normal population (6). There are
only 2 reports on heterozygotes for 11ß-hydroxylase deficiency. Pang
et al. reported that hormonal measurements, including
ACTH-stimulated serum levels of 11-deoxycorticosterone and
11-deoxycortisol, are not useful for detecting heterozygotes for
11ß-hydroxylase deficiency (7). Rösler studied 3 families with
an index case for 11ß-hydroxylase deficiency and the R448H mutation
in exon 8 of CYP11B1, which is the common molecular finding in
11ß-hydroxylase deficiency among Jews of Moroccan origin (2). He did
not find any demonstrable abnormalities in steroid plasma levels in his
sample (8). We here report a contrasting study with clear hormonal
abnormalities in the heterozygous parents and a sister of a boy with
proven 11ß-hydroxylase deficiency and the R448H mutation in exon 8 of
CYP11B1.
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Subjects and Methods
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Subjects
The index case (A.T.) presented with premature
pubarche, penile enlargement, acne, increased growth velocity, and
advanced bone age of 6 yr (Greulich and Pyle) at the chronological age
of 1.8 yr. Pubertal stage at presentation was Tanner stage 2 pubic
hair, Tanner stage 3 genitalia, and a 1-mL testicular volume on both
sides. Height was 100 cm (+4.9 SD score), growth velocity
was 22 cm/yr (+6.3 SD score), weight was 17 kg, and the
weight for height index was 107%. The diagnosis of CAH due to
11ß-hydroxylase deficiency in the index case was established on the
basis of elevated plasma levels of 11-deoxycorticosterone and
11-deoxycortisol (Table 1
). PRA was
suppressed (0.1 ng/mL·h). Plasma androgens were elevated
(testosterone, 6.1 nmol/L; androstenedione, 217 nmol/L). With
hydrocortisone treatment of 18 mg/m2·day for 5 yr, the
patient is doing well, his growth is at 90th percentile, he has lost
the signs of pseudoprecocity, and bone age advancement has decreased.
The parents and sister were studied after informed consent was
obtained. A short term ACTH test was performed in all family members
with an iv bolus injection of 250 µg ACTH-(1–24) (Synacthen,
Ciba-Geigy, Wehr, Germany) between 0800–1000 h. Blood samples were
drawn immediately before and 60 min after ACTH injection.
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Table 1. Basal and ACTH-stimulated plasma steroids determined
by multisteroid analysis in the index case, the parents, and a sister
genotyped to have the R448H mutation in the CYP11B1 gene in both or one
allele, respectively
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Steroid determinations
Blood samples were 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, developed in our laboratory (5), for the simultaneous
determination of multiple adrenal steroids in a small plasma volume of
1–2 mL.
To 0.5–1.0 mL plasma, approximately 2000 dpm radiolabeled
steroids (dissolved in 150 µL 
-globulin buffer) were added to as
an internal standard. After thorough mixing and an equilibration period
of at least 2 h, plasma was extracted twice with ice-cold
methylene chloride and washed once. Plasma extracts were redissolved
and then submitted to Sephadex LH-20 automated multicolumn
chromatography (9). The fractions containing one of the isolated
steroids were evaporated to dryness, redissolved in 2.0 mL absolute
ethanol (15 C), and then rapidly divided into two aliquots at a
constant temperature at 15 C. Internal tracer recovery was determined
in one aliquot. The average recovery of radioactive steroids added to
plasma varied from 52–68.2% after extraction and one or two Sephadex
LH-20 chromatographies. The other aliquot was used in duplicate for
steroid quantification by RIA using specific antisera. For the plasma
steroids determined, intra- and interassay coefficients of variation
ranged from 6.9–14.5% and from 11.9–16.3%, respectively.
The results are expressed in nanomoles per L; to convert to
nanograms per mL, divide by the following factors: aldosterone, 2.774;
18-hydroxycorticosterone, 2.759; corticosterone, 2.886;
18-hydroxy-11-deoxycorticosterone, 2.886; 11-deoxycorticosterone,
3.026; progesterone, 3.18; pregnenolone, 3.16; 17-hydroxypregnenolone,
3.008; 17-hydroxyprogesterone, 3.026; 11-deoxycortisol, 2.887;
cortisol, 2.759; and cortisone, 2.774.
Normal ranges for different age groups using our method of multisteroid
analysis have been reported previously (10, 11).
A similar method, using extraction, chromatography, and RIA, was used
for plasma testosterone and androstenedione measurements (12).
Nucleotide sequences of exons and exon/intron boundaries
Genomic DNA was extracted from peripheral blood
leukocytes, and the CYP11B1 gene was specifically amplified as
described previously (13). 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 Science, Cleveland, OH).
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Results
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Steroid determinations
The results of the plasma steroid determinations are given
in Table 1
. The index patient studied before substitution therapy with
hydrocortisone was initiated showed the typical steroid pattern of a
classical defect in 11ß-hydroxylation. The plasma steroid
levels measured in the parents and the older sister are also shown in
Table 1. The mother and the sister had increased plasma levels of
11-deoxycorticosterone after ACTH stimulation. All three had increased
plasma levels of 11-deoxycortisol after ACTH stimulation compared to
those in normal age- and sex-matched controls (10, 11). The increase in
plasma 11-deoxycorticosterone compared to the mean plasma level after
ACTH stimulation in controls was normal in the father, 354% in the
mother, and 247% in the sister. The increase in plasma
11-deoxycortisol compared to the mean plasma level after ACTH in
controls was 353% in the father, 712% in the mother, and 894% in the
sister.
Nucleotide sequences of exons and exon/intron boundaries
Direct sequencing of the patients DNA showed that the index
patient was homozygous for a single base exchange in his CYP11B1 gene.
We identified a homozygous G to A transversion in codon 448, which has
been published in a previous series of patients (13). The mutation,
leading to the substitution of arginine (CGC) 448 by histidine (CAC),
has previously been shown to abolish 11ß-hydroxylase activity in
in vitro expression studies (14). Direct sequencing of exon
8 of the CYP11B1 gene showed that both parents and the older sister
were heterozygous for the R448H mutation (Fig. 1).
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Discussion
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The two reports existing to date dealing with hormonal
changes in heterozygous carriers for 11ß-hydroxylase deficiency did
not find any demonstrable changes in plasma steroids in these subjects
(7, 8). Pang et al. found in a few subjects elevated ratios
of 17-hydroxyprogesterone to cortisol and of 11-deoxycortisol to
cortisol, but these results were too inconsistent to be significant.
Similarly, Rösler and Cohen did not find any demonstrable
hormonal deficiency in six parents and two brothers heterozygous for
11ß-hydroxylase deficiency carrying in one allele the same mutation
as that in our family. Heterozygote individuals may be considered to
have 50% of normal 11ß-hydroxylase enzyme activity. In contrast to
the data reported to date in heterozygotes for classical
11ß-hydroxylase deficiency, in CAH due to 21-hydroxylase (P450c21)
deficiency, hormonal studies in obligate heterozygous parents have
demonstrated a mild 21-hydroxylase deficiency. Family studies have
shown that heterozygous carriers of a defective gene for P450c21 have a
higher 17-hydroxyprogesterone response after ACTH stimulation than the
control population. There is, however, an overlap of about 20% in the
ranges of the 17-hydroxyprogesterone response after ACTH stimulation
between heterozygotes and subjects without the defect, particularly
among pubertal girls and women (4).
In the family reported here we demonstrated for the first time that
heterozygous carriers of the R448H mutation have reduced
11ß-hydroxylase activity. In this family, ACTH-stimulated plasma
levels of 11-deoxycortisol and, to a lesser extent,
11-deoxycorticosterone were increased compared to age- and sex-matched
control levels. Our normal ranges for the different steroids measured
in this study are in good agreement with those reported by Lashansky
et al. (15, 16), Pang et al. (7), and
Rösler and Cohen (8). An explanation for the different results in
family studies of heterozygous carriers for 11ß-hydroxylase
deficiency might be the existence of other intragenic (e.g.
promoter activity and splicing variants) or extragenic effects that
modulate the activity of CYP11B1 gene expression in homozygotes as well
as in heterozygotes for 11ß-hydroxylase deficiency.
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Acknowledgments
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The authors thank Mrs. Jutta Biskupek-Sigwart and Mrs. Susanne
Neumann-Olin for their expert technical assistance in the multisteroid
analyses, and Mrs. Gisela Hohmann for her skillful technical assistance
with the molecular biology techniques. 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. 
Received April 14, 1997.
Revised June 19, 1997.
Accepted June 26, 1997.
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References
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Abstract
Introduction
