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Journal of Clinical Endocrinology and Metabolism
Copyright © 1997 by The Endocrine Society
Vol. 82, No. 7
Printed in U.S.A.
Evidence for a Heterozygote Advantage in Congenital
Adrenal Hyperplasia due to 21-Hydroxylase Deficiency*
SELMA F. WITCHEL, PETER A. LEE, MAKIKO SUDA-HARTMAN,
MASSIMO TRUCCO, AND ERIC P. HOFFMAN
Divisions of Endocrinology (S.F.W., P.A.L., M.S.-H.) and Immunogenetics (M.T.), Department of
Pediatrics, Children’s Hospital of Pittsburgh, and the Department of Molecular Genetics and
Biochemistry (E.P.H.), University of Pittsburgh, Pittsburgh, Pennsylvania 15213
ABSTRACT
21-Hydroxylase deficiency is one of the most common inherited
disorders, with carrier frequencies of approximately 10% in all world
populations studied to date. The high prevalence of the mutant gene
is probably due to a flanking pseudogene serving as a reservoir for
mutations. Despite the potential for a high rate of de novo mutations,
a founder effect for specific gene conversions is observed in most
populations. We hypothesized that there was a survival advantage to
21-hydroxylase heterozygotes, and here we report endocrinological
and molecular investigations to test this hypothesis. We defined 28
carriers and 22 mutation-negative controls by molecular genotyping
and determined ACTH-stimulated adrenal hormone responses. We
found significantly elevated cortisol responses in the carriers compared to controls (30 min cortisol levels: normal, 24.2 6 4.6 mg/dL;
carrier, 28.1 6 4.2 mg/dL; P , 0.005). Cortisol has a crucial role in
maintaining homeostasis, influencing differentiation, suppressing inflammation, and effecting cross-talk among the immune, nervous,
and endocrine systems. The brisk cortisol response we have documented in carriers of 21-hydroxylase may enable a rapid return to
homeostasis in response to infectious, inflammatory, or other environmental stresses and may protect from inappropriate immune responses, such as autoimmune diseases. (J Clin Endocrinol Metab 82:
2097–2101, 1997)
C
the nonclassical form with hirsutism, menstrual irregularity,
and infertility (7). Nonclassical 21-hydroxylase deficiency
occurs in both sexes, but the clinical manifestations create an
ascertainment bias favoring the detection of affected women.
The high prevalence, mild symptoms, negligible effect on life
span, and only partial infertility of nonclassical 21-hydroxylase deficiency has led some to suggest that this disorder
may instead be a variant of normal (8).
The frequency of heterozygotic carriers for 21-hydroxylase
deficiency appears to be high in all populations studied to
date. Based on 17-hydroxyprogesterone responses to pharmacological ACTH stimulation, approximately 1 in 16 Caucasians was classified as a probable carrier of 21-hydroxylase
deficiency (9). Linkage of nonclassical congenital adrenal
hyperplasia with HLA-B14 and, subsequently, to the
Val2813 Leu CYP21 mutation in conjunction with ACTHstimulated hormone responses suggested that one of three
persons of Ashkenazi Jewish descent was probably a carrier
for nonclassical 21-hydroxylase deficiency (10). Based on
ACTH-stimulated 17-OHP responses, heterozygote frequencies have been estimated as one in four for Hispanics, one in
five for Yugoslavs, one in eight for Yupik Eskimos, and one
in 10 for Italians (10 –13). Despite the potential for a high rate
of de novo mutations, homozygosity for the intron 2 splicing
mutation and a distinct HLA-extended haplotype (DR4;
DRw53;DQw3) in the Yupik Eskimos suggest a founder effect in this isolated population (13).
Clearly, cortisol and mineralocorticoid deficiencies leading to lethal salt loss and/or genital ambiguity in affected
patients cannot impart selective or reproductive advantages.
However, as with other common autosomal recessive gene
disorders, the high prevalence of CYP21 heterozygosity sug-
ORTISOL has a crucial role in maintaining homeostasis,
influencing differentiation, suppressing inflammation, and effecting cross-talk among the immune, nervous,
and endocrine systems. Diurnal variations in cortisol concentrations and stress-induced cortisol secretion are tightly
regulated through feedback inhibition by the hypothalamicpituitary-adrenal axis to maintain optimal cortisol concentrations (1).
Disorders of cortisol biosynthesis are known as the congenital adrenal hyperplasias. The most common is 21-hydroxylase deficiency, an autosomal recessive disorder due to
mutations in the 21-hydroxylase gene (CYP21) located on the
short arm of chromosome 6 (Fig. 1) where it lies in close
proximity to a nonfunctional pseudogene (CYP21P) (2– 6). In
affected homozygotes, decreased adrenal 21-hydroxylase activity interferes with cortisol and aldosterone biosynthesis,
but leaves the adrenal androgen biosynthetic pathway intact.
Loss of negative feedback inhibition by cortisol leads to increased secretion of ACTH by the pituitary, with subsequent
excessive adrenal 17-hydroxyprogesterone (17-OHP) and
adrenal androgen production. The clinical consequences of
these allelic variants range from the classical salt-wasting
form with glucocorticoid and mineralocorticoid deficiencies
to the simple virilizing form with premature pubic hair and
Received February 10, 1997. Revision received March 21, 1997.
Accepted March 25, 1997.
Address all correspondence and requests for reprints to: Selma F.
Witchel, M.D., Division of Endocrinology, Children’s Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, Pennsylvania 15213.
* This work was supported by NIH Grants HD-00965 (to S.F.W.) and
5M01-RR-00084 (to the General Clinical Research Center). The data were
presented in part at the ICE/Endocrine Society Meeting, San Francisco,
CA, June 1996.
2097
2098
WITCHEL ET AL.
JCE & M • 1997
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FIG. 1. Structure of the CYP21 locus and location of the mutations tested. The upper panel illustrates the C4-CYP21 gene modules in the context
of the tenascin-X genes (adapted and redrawn from Ref. 46). The scale is in kilobases. The direction of gene transcription is denoted by arrows.
C4A and C4B code for the fourth component of complement, 21B codes for CYP21, and XB codes for tenascin-X (47). Bristow et al. identified
only YA transcripts originating from the CYP21P (21A) promoter (48). The lower panel illustrates the intron/exon structure of CYP21 and the
locations of the mutations tested..
gests a survival advantage for carriers (14). In considering
possible heterozygotic advantages, we hypothesized that if
there is elevated adrenal androgen (androstenedione with
peripheral conversion to testosterone) production, it could
potentially advance the onset of puberty and increase assertive behavior; this could be advantageous during times of
severe environmental or social stress. Alternatively, partial
infertility in carrier females could increase the interval between pregnancies, thereby decreasing the birth rate and
improving maternal and infant survival. Perhaps most intriguing is the possible effects of the carrier state on cortisol
homeostasis. One would predict decreased cortisol production in carriers. However, decreases in cortisol cause a loss
of negative feedback inhibition to the hypothalamus and
pituitary. This, in turn, could increase pituitary ACTH secretion, leading to up-regulation of adrenal steroidogenic
enzymes (15). This “turned-on” or “primed” adrenal cortex
could secrete cortisol more robustly in response to physio-
logical stimulation and offer a selective advantage by preventing excessive or inappropriate immune responses, such
as autoimmune disease (16 –18). Thus, we hypothesized that
a paradoxical increase in stimulated cortisol concentrations
could ensue.
One way to distinguish among these possibilities is to
measure basal and stimulated cortisol responses in heterozygous carriers. We have previously shown that cortisol concentrations in both carriers and control subjects increase
within minutes following acute ACTH stimulation (19). In
this previous study, the carriers and control subjects showed
similar cortisol responses, but none of the subjects was genotyped, leading to the potential inclusion of carriers in the
control population. Here, we carefully compare steroid hormone responses between genotyped healthy controls and
heterozygotic carriers. We document a remarkably vigorous
cortisol response in carriers and discuss the implications of
this finding as a possible heterozygote advantage.
HETEROZYGOTE ADVANTAGE IN CONGENITAL ADRENAL HYPERPLASIA
Materials and Methods
2099
Subjects
were used to define the upper limit of the normal hormone concentrations. Student’s independent t test was used to compare hormone concentrations between the two groups.
First degree and second degree adult relatives of children with 21hydroxylase deficiency (n 5 31; 15 men and 16 women) and normal
volunteers (n 5 19; 9 men and 10 women) participated in this study. The
protocol was approved by the Human Rights Committee of Children’s
Hospital of Pittsburgh. Informed consent was obtained from all
participants.
Results
Molecular diagnostic studies
Molecular genotype analysis
Blood samples were obtained for HLA haplotype analysis and for
DNA extraction from peripheral blood lymphocytes. Standard serologic
and molecular methods were used to determine the extended HLA
haplotypes for the A, B, C, DR, and DQ loci (20, 21). DNA was extracted
from peripheral blood lymphocytes (22).
PCR amplification and allele-specific oligonucleotide hybridization
were performed as previously described; at least one primer of each primer
pair was specific for the functional 21-hydroxylase gene (23). Additional
oligonucleotide probes used were: Arg339-WT, 59-TACAAGGACCGTGCACGGCT-39; His339-MUT, 59-TACAAGGACCATGCACGGCT-39; exon
6-WT, 59-CTCAGCTGCATCTCCACGA-39; exon 6-MUT, 59-CTCAGCTGCTTCTCCTCGT-39; GT1769-WT, 59-ACCCTGAGGTGCGTCCTG-39;
CT1769-MUT, 59-ACCCTGAGCTGCGTCCTG-39; Pro453-WT, 59-CGCTGCTGCCCTCCGGGG-39; Ser453, 59-CGCTGCTGTCCTCCGGGG-39;
Arg484-WT, 59-ATCCCCCGGGGCTGCAG-39; Pro484-MUT, 59-ATCCCCGGGGGCTGCAG-39; STOP484-MUT, 59-ATCCCGGGGGCTGCAG39; 1761-WT, 59-CGTGAAGCAAAAAAACCACGG-39; and i1761-MUT,
59-CGTGAAGCAAAAAAAACCACGG-39.
Single strand conformational polymorphism analysis was used to
detect the splicing mutation in intron 2, V281L in exon 7, Q318X in exon
8, and R356W in exon 8 as previously described (24, 25). Primers 694F
(59-ACCTGTCCTTGGGAGACTAC-39) and 1122R (59-TCGTCCTGCCAGAAAAGGAG-39) were used to detect the I172N mutation on a 5%
acrylamide gel prepared with 10% glycerol, which was electrophoresed
at 40 watts at 4 C for 11 h. After electrophoresis, the gels were dried and
autoradiographed at 280 C.
Hormonal analysis
ACTH stimulation tests were performed on relatives (16 women and
15 men) and 19 healthy adults (10 women and 9 men). All women had
regular menstrual cycles and were tested during the follicular phase of
the menstrual cycle (basal plasma progesterone, ,45 ng/dL). None was
hirsute or taking oral contraceptives. Samples for progesterone, 17hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione (D4), and cortisol were obtained before the
administration of Cortrosyn (0.25-mg iv infusion over 1 min). Subsequent blood samples were obtained 10 (n 5 46) and 30 (n 5 50) min
postinfusion. Plasma steroid hormones were measured as previously
described (26). For all subjects, the basal cortisol level was less than 20
mg/dL. All hormone determinations for each individual were performed in duplicate within single assays.
Statistical analysis
Statistical analysis was performed using AbSTAT statistical software
(Anderson-Bell, Arvada, CO). The 99% confidence intervals (mean 6
2.57 sd) of hormone concentrations measured in the healthy individuals
Genotypes were ascertained by allele-specific oligonucleotide hybridization and single strand conformational polymorphism analyses. Twenty-seven of the 31 family members
studied were found to be heterozygotic carriers of 21-hydroxylase deficiency. Deleterious mutations detected were
gene conversion/gene deletion (n 5 8), intron 2 splicing
mutation (n 5 7), I172N (n 5 2), exon 6 triple codon mutation
(n 5 1), V281L (n 5 1), i1761 (n 5 2), Q318X (n 5 4), and
R356W (n 5 2). Four family members carried no deleterious
mutations by both mutation studies and HLA linkage data
and are included with the healthy controls for statistical
analysis of their ACTH-stimulated hormone responses.
Genotype analysis and ACTH stimulation tests were performed in 19 healthy volunteers. The intron 2 splicing mutation was detected in 1 of these 19 volunteers. The ACTHstimulated hormone responses obtained in this volunteer are
included with the heterozygotic carrier group for statistical
analysis. Thus, based on molecular genotype analysis, 28
heterozygotic carriers and 22 genotyped normal subjects
were identified. We screened for 13 previously identified
mutations, but cannot exclude the possibility that control
subjects carry unidentified mutations.
Comparison of ACTH stimulation tests
Comparison of ACTH-stimulated hormone responses between the 28 heterozygotic carriers of 21-hydroxylase deficiency and 22 genotyped normal controls showed similar
mean basal 17-hydroxyprogesterone levels (106.1 6 63.4 vs.
105.3 6 65.5 ng/dL, respectively). As anticipated (27), the
heterozygotic carriers had greater mean 17-hydroxyprogesterone responses at 10 and 30 min than the genotyped normal
controls (Table 1 and Fig. 2; P , 0.0001).
Mean basal cortisol concentrations (Table 1) were not different between the heterozygotic carriers and the healthy
controls (11.1 6 3.8 vs. 11.3 6 3.2 mg/dL, respectively). Ten
minutes after iv ACTH stimulation (Fig. 2), the heterozygotic
carriers had a greater cortisol response (22.0 6 4.7 vs. 19.1 6
3.8 mg/dL; P , 0.05). The 30-min cortisol concentrations (Fig.
2) were also greater among the heterozygotic carriers (28.1 6
4.2 vs. 24.2 6 4.6 mg/dL; P , 0.005).
Among the 50 individuals (22 genotype normal and 28
genotype heterozygotes), there was a positive correlation
TABLE 1. Basal and ACTH stimulated 17-OHP (ng/dL) and cortisol (mg/dL) concentrations in normal and heterozygotic carriers
OHP-0
OHP-10
OHP-30
F-0
F-10
F-30
Normal
Heterozygotic
carriers
P value
t statistic
105 6 65 (22)
171 6 79 (22)
207 6 89 (22)
11.3 6 3.2 (22)
19.1 6 3.8 (22)
24.2 6 4.6 (22)
106 6 63 (28)
365 6 208 (24)
468 6 243 (28)
11.1 6 3.8 (28)
22.0 6 4.7 (24)
28.1 6 4.2 (28)
NS
0.0001
0.0001
NS
0.0254
0.0027
0.04
4.08
4.78
0.175
2.315
3.158
Mean, standard deviation, and number of subjects are provided. NS, not significant.
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WITCHEL ET AL.
FIG. 2. ACTH-stimulated cortisol responses. The graph depicts the
cortisol responses (mean 6 SEM) in healthy controls (ç) and heterozygotic carriers (M) before and after ACTH stimulation. Blood samples
were obtained 10 and 30 min after the iv Cortrosyn bolus. Mean basal
cortisol responses are not significantly different. At both 10 and 30
min, mean cortisol concentrations were greater in heterozygotic carriers than controls (P , 0.05 and P , 0.005, respectively).
between stimulated 17-OHP and cortisol concentrations. At
10 min, the correlation coefficient was 0.61 (P , 0.0001). At
30 min, the correlation coefficient was 0.45 (P 5 0.001). There
was no correlation between specific CYP21 mutation and
peak cortisol response.
Discussion
Common recessive disorders, such as sickle cell anemia
and cystic fibrosis, are frequently thought to impart a selective survival advantage to heterozygotic carriers (28, 29).
Although the concept of heterozygote advantage has been
entertained for 21-hydroxylase deficiency, direct evidence
delineating the specific mechanism of the survival benefit has
proven difficult to obtain. The CYP21 gene is a frequent
target of mutations, with carrier frequency between 1 in 3 and
1 in 16 in all populations studied despite the crucial role of
the 21-hydroxylase enzyme in adrenal steroidogenesis. This
high carrier frequency suggests that heterozygotes could
have a survival advantage.
Cortisol secretion is regulated through the hypothalamicpituitary-adrenal axis by negative feedback inhibition of
ACTH secretion. ACTH acutely increases cortisol secretion
and chronically maintains transcription rates of the steroidogenic enzymes and optimal steroidogenic capacity (17).
Our interpretation of the more vigorous cortisol response
that we observed in heterozygotic carriers was that the adrenal gland of heterozygotic carriers was up-regulated or
primed to secrete cortisol, because the clinically imperceptible decrease in cortisol secretion had led to increased ACTH
secretion (30).
Comparison of the inbred histocompatible Lewis and Fischer rat strains provides an example of the physiological
consequences of subtle alterations in cortisol concentrations.
Lewis rats are extraordinarily susceptible to both experimentally induced autoimmune and inflammatory disorders,
whereas Fischer rats are resistant to the same challenges (31).
JCE & M • 1997
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Investigation into the basis for the difference in susceptibility
has shown that the Lewis rat shows hypothalamic-pituitaryadrenal axis hyporesponsiveness in contrast to the hyperresponsiveness of the Fischer rat (32, 33). Both strains show
comparable target tissue sensitivity to glucocorticoids (34,
35). Clinical studies of cortisol concentrations during human
cardiac arrest or severe illness suggest that too low or too
high cortisol concentrations are associated with increased
mortality (36). These data, obtained from animal and clinical
studies, indicate that small differences in glucocorticoid concentrations may be physiologically significant.
Three previous studies have looked at ACTH-stimulated
cortisol responses in 21-hydroxylase carriers. All were performed without mutation detection, and thus, it is likely that
the control populations included carriers. Two studies using
less specific assays to measure cortisol (37, 38) found no
significant differences in cortisol responses. A third study,
using HLA haplotypes to infer carrier status, showed carriers
to have slightly greater cortisol responses, although these
differences were deemed not significant (36). None of these
studies showed the hypersensitization of the adrenal cortex
that we have documented here or speculated on a possible
heterozygote advantage.
Through secretion of peptide and steroid hormones, i.e.
ACTH and cortisol, the hypothalamic-pituitary-adrenal axis
restores homeostasis after stress (39). Glucocorticoids function as potent antiinflammatory and immunosuppressive
agents. Indeed, the widespread therapeutic use of glucocorticoids in the treatment of immune disorders often overshadows their physiological functions. Specific glucocorticoid actions include trafficking of circulating leukocytes,
suppression of accessory immune cells, inhibition of cytokine
production, and induction of resistance to cytokines (40 – 42).
The production of nitric oxide, prostanoids, and plateletactivating factor, three major mediators of the inflammatory
response, is decreased by glucocorticoids (42). Glucocorticoids enhance the synthesis of acute phase proteins, which
scavenge the toxic superoxide radicals generated to kill invading microorganisms and tumor cells (43). The net result
is the prevention of DNA and tissue damage that thwart
self-destruction by cytokines and other effectors of immune
reactions (18). These restraining actions are thought to hinder
the development of autoimmune disorders.
Glucocorticoids also play a role in energy metabolism by
maintaining the liver enzymes involved in gluconeogenesis,
increasing substrate availability, and inducing phenylethanolamine N-methyl transferase in the adrenal medulla (44).
The venous efflux of the adrenal cortex exposes the adrenal
medulla to high glucocorticoid levels, which may be important in epinephrine biosynthesis. Thus, cortisol acts by maintaining a state of readiness. Maintenance of epinephrine biosynthesis impacts on the fight or flight response and
probably promotes survival (45).
We postulate that the brisk cortisol secretion observed
among heterozygotic carriers could be a genetically favorable trait, providing greater survival fitness. If so, these data
support speculation that heterozygotic advantage has led to
the high gene frequency of CYP21 mutations.
HETEROZYGOTE ADVANTAGE IN CONGENITAL ADRENAL HYPERPLASIA
Acknowledgments
We gratefully acknowledge the assistance of Tamara Johnston, R.N.;
Amy Jones, R.N.; Janet Bell, R.N.; and Debbie Cleary.
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