Evidence for a Heterozygote Advantage in Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency*

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 Downloaded from https://academic.oup.com/jcem/article/82/7/2097/2865904 by guest on 02 October 2021 Biochemistry (E.P.H.), University of Pittsburgh, Pittsburgh, Pennsylvania 15213

ABSTRACT found significantly elevated cortisol responses in the carriers com- 21-Hydroxylase deficiency is one of the most common inherited pared to controls (30 min cortisol levels: normal, 24.2 Ϯ 4.6 ␮g/dL; disorders, with carrier frequencies of approximately 10% in all world carrier, 28.1 Ϯ 4.2 ␮g/dL; P Ͻ 0.005). Cortisol has a crucial role in populations studied to date. The high prevalence of the mutant gene maintaining homeostasis, influencing differentiation, suppressing in- is probably due to a flanking pseudogene serving as a reservoir for flammation, and effecting cross-talk among the immune, nervous, mutations. Despite the potential for a high rate of de novo mutations, and endocrine systems. The brisk cortisol response we have docu- a founder effect for specific gene conversions is observed in most mented in carriers of 21-hydroxylase may enable a rapid return to populations. We hypothesized that there was a survival advantage to homeostasis in response to infectious, inflammatory, or other envi- 21-hydroxylase heterozygotes, and here we report endocrinological ronmental stresses and may protect from inappropriate immune re- and molecular investigations to test this hypothesis. We defined 28 sponses, such as autoimmune diseases. (J Clin Endocrinol Metab 82: carriers and 22 mutation-negative controls by molecular genotyping 2097–2101, 1997) and determined ACTH-stimulated adrenal hormone responses. We

ORTISOL has a crucial role in maintaining homeostasis, the nonclassical form with hirsutism, menstrual irregularity, C influencing differentiation, suppressing inflamma- and infertility (7). Nonclassical 21-hydroxylase deficiency tion, and effecting cross-talk among the immune, nervous, occurs in both sexes, but the clinical manifestations create an and endocrine systems. Diurnal variations in cortisol con- ascertainment bias favoring the detection of affected women. centrations and stress-induced cortisol secretion are tightly The high prevalence, mild symptoms, negligible effect on life regulated through feedback inhibition by the hypothalamic- span, and only partial infertility of nonclassical 21-hydrox- pituitary-adrenal axis to maintain optimal cortisol concen- ylase deficiency has led some to suggest that this disorder trations (1). may instead be a variant of normal (8). Disorders of cortisol biosynthesis are known as the con- The frequency of heterozygotic carriers for 21-hydroxylase genital adrenal hyperplasias. The most common is 21-hy- deficiency appears to be high in all populations studied to droxylase deficiency, an autosomal recessive disorder due to date. Based on 17-hydroxyprogesterone responses to phar- mutations in the 21-hydroxylase gene (CYP21) located on the macological ACTH stimulation, approximately 1 in 16 Cau- short arm of chromosome 6 (Fig. 1) where it lies in close casians was classified as a probable carrier of 21-hydroxylase proximity to a nonfunctional pseudogene (CYP21P) (2–6). In deficiency (9). Linkage of nonclassical congenital adrenal affected homozygotes, decreased adrenal 21-hydroxylase ac- hyperplasia with HLA-B14 and, subsequently, to the tivity interferes with cortisol and aldosterone biosynthesis, Val2813Leu CYP21 mutation in conjunction with ACTH- but leaves the adrenal androgen biosynthetic pathway intact. stimulated hormone responses suggested that one of three Loss of negative feedback inhibition by cortisol leads to in- persons of Ashkenazi Jewish descent was probably a carrier creased secretion of ACTH by the pituitary, with subsequent for nonclassical 21-hydroxylase deficiency (10). Based on excessive adrenal 17-hydroxyprogesterone (17-OHP) and ACTH-stimulated 17-OHP responses, heterozygote frequen- adrenal androgen production. The clinical consequences of cies have been estimated as one in four for Hispanics, one in these allelic variants range from the classical salt-wasting form with glucocorticoid and mineralocorticoid deficiencies five for Yugoslavs, one in eight for Yupik Eskimos, and one to the simple virilizing form with premature pubic hair and 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; Received February 10, 1997. Revision received March 21, 1997. DRw53;DQw3) in the Yupik Eskimos suggest a founder ef- Accepted March 25, 1997. Address all correspondence and requests for reprints to: Selma F. fect in this isolated population (13). Witchel, M.D., Division of Endocrinology, Children’s Hospital of Pitts- Clearly, cortisol and mineralocorticoid deficiencies lead- burgh, 3705 Fifth Avenue, Pittsburgh, Pennsylvania 15213. ing to lethal salt loss and/or genital ambiguity in affected * This work was supported by NIH Grants HD-00965 (to S.F.W.) and patients cannot impart selective or reproductive advantages. 5M01-RR-00084 (to the General Clinical Research Center). The data were presented in part at the ICE/Endocrine Society Meeting, San Francisco, However, as with other common autosomal recessive gene CA, June 1996. disorders, the high prevalence of CYP21 heterozygosity sug-

2097 2098 WITCHEL ET AL. JCE&M 1997 Vol 82 •No 7 • Downloaded from https://academic.oup.com/jcem/article/82/7/2097/2865904 by guest on 02 October 2021

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 logical stimulation and offer a selective advantage by pre- possible heterozygotic advantages, we hypothesized that if venting excessive or inappropriate immune responses, such there is elevated adrenal androgen (androstenedione with as autoimmune disease (16–18). Thus, we hypothesized that peripheral conversion to testosterone) production, it could a paradoxical increase in stimulated cortisol concentrations potentially advance the onset of puberty and increase asser- could ensue. tive behavior; this could be advantageous during times of One way to distinguish among these possibilities is to severe environmental or social stress. Alternatively, partial measure basal and stimulated cortisol responses in heterozy- infertility in carrier females could increase the interval be- gous carriers. We have previously shown that cortisol con- tween pregnancies, thereby decreasing the birth rate and centrations in both carriers and control subjects increase improving maternal and infant survival. Perhaps most in- within minutes following acute ACTH stimulation (19). In triguing is the possible effects of the carrier state on cortisol this previous study, the carriers and control subjects showed homeostasis. One would predict decreased cortisol produc- similar cortisol responses, but none of the subjects was geno- tion in carriers. However, decreases in cortisol cause a loss typed, leading to the potential inclusion of carriers in the of negative feedback inhibition to the hypothalamus and control population. Here, we carefully compare steroid hor- pituitary. This, in turn, could increase pituitary ACTH se- mone responses between genotyped healthy controls and cretion, leading to up-regulation of adrenal steroidogenic heterozygotic carriers. We document a remarkably vigorous enzymes (15). This “turned-on” or “primed” adrenal cortex cortisol response in carriers and discuss the implications of could secrete cortisol more robustly in response to physio- this finding as a possible heterozygote advantage. HETEROZYGOTE ADVANTAGE IN CONGENITAL ADRENAL HYPERPLASIA 2099

Materials and Methods were used to define the upper limit of the normal hormone concentra- Subjects tions. 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 21- hydroxylase deficiency (n ϭ 31; 15 men and 16 women) and normal Results volunteers (n ϭ 19; 9 men and 10 women) participated in this study. The protocol was approved by the Human Rights Committee of Children’s Molecular diagnostic studies Hospital of Pittsburgh. Informed consent was obtained from all participants. Genotypes were ascertained by allele-specific oligonucleotide hybridization and single strand conformational poly- Molecular genotype analysis morphism analyses. Twenty-seven of the 31 family members studied were found to be heterozygotic carriers of 21-hy- Blood samples were obtained for HLA haplotype analysis and for DNA extraction from peripheral blood lymphocytes. Standard serologic droxylase deficiency. Deleterious mutations detected were

and molecular methods were used to determine the extended HLA gene conversion/gene deletion (n ϭ 8), intron 2 splicing Downloaded from https://academic.oup.com/jcem/article/82/7/2097/2865904 by guest on 02 October 2021 haplotypes for the A, B, C, DR, and DQ loci (20, 21). DNA was extracted mutation (n ϭ 7), I172N (n ϭ 2), exon 6 triple codon mutation from peripheral blood lymphocytes (22). (n ϭ 1), V281L (n ϭ 1), i1761 (n ϭ 2), Q318X (n ϭ 4), and PCR amplification and allele-specific oligonucleotide hybridization were performed as previously described; at least one primer of each primer R356W (n ϭ 2). Four family members carried no deleterious pair was specific for the functional 21-hydroxylase gene (23). Additional mutations by both mutation studies and HLA linkage data oligonucleotide probes used were: Arg339-WT, 5Ј-TACAAGGACCGTG- and are included with the healthy controls for statistical CACGGCT-3Ј; His339-MUT, 5Ј-TACAAGGACCATGCACGGCT-3Ј; exon analysis of their ACTH-stimulated hormone responses. 6-WT, 5Ј-CTCAGCTGCATCTCCACGA-3Ј; exon 6-MUT, 5Ј-CTCAGCT- Genotype analysis and ACTH stimulation tests were per- GCTTCTCCTCGT-3Ј; GT1769-WT, 5Ј-ACCCTGAGGTGCGTCCTG-3Ј; CT1769-MUT, 5Ј-ACCCTGAGCTGCGTCCTG-3Ј; Pro453-WT, 5Ј-CGCT- formed in 19 healthy volunteers. The intron 2 splicing mu- GCTGCCCTCCGGGG-3Ј; Ser453,5Ј-CGCTGCTGTCCTCCGGGG-3Ј; tation was detected in 1 of these 19 volunteers. The ACTH- Arg484-WT, 5Ј-ATCCCCCGGGGCTGCAG-3Ј; Pro484-MUT, 5Ј-ATC- stimulated hormone responses obtained in this volunteer are 484 CCCGGGGGCTGCAG-3Ј; STOP -MUT, 5Ј-ATCCCGGGGGCTGCAG- included with the heterozygotic carrier group for statistical 3Ј; 1761-WT, 5Ј-CGTGAAGCAAAAAAACCACGG-3Ј; and i1761-MUT, 5Ј-CGTGAAGCAAAAAAAACCACGG-3Ј. analysis. Thus, based on molecular genotype analysis, 28 Single strand conformational polymorphism analysis was used to heterozygotic carriers and 22 genotyped normal subjects detect the splicing mutation in intron 2, V281L in exon 7, Q318X in exon were identified. We screened for 13 previously identified 8, and R356W in exon 8 as previously described (24, 25). Primers 694F mutations, but cannot exclude the possibility that control (5Ј-ACCTGTCCTTGGGAGACTAC-3Ј) and 1122R (5Ј-TCGTCCTGC- subjects carry unidentified mutations. CAGAAAAGGAG-3Ј) 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 Comparison of ACTH stimulation tests autoradiographed at Ϫ80 C. Comparison of ACTH-stimulated hormone responses be- Hormonal analysis tween the 28 heterozygotic carriers of 21-hydroxylase defi- ACTH stimulation tests were performed on relatives (16 women and ciency and 22 genotyped normal controls showed similar 15 men) and 19 healthy adults (10 women and 9 men). All women had mean basal 17-hydroxyprogesterone levels (106.1 Ϯ 63.4 vs. regular menstrual cycles and were tested during the follicular phase of 105.3 Ϯ 65.5 ng/dL, respectively). As anticipated (27), the the menstrual cycle (basal plasma progesterone, Ͻ45 ng/dL). None was heterozygotic carriers had greater mean 17-hydroxyproges- hirsute or taking oral contraceptives. Samples for progesterone, 17- terone responses at 10 and 30 min than the genotyped normal hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandros- terone, androstenedione (⌬4), and cortisol were obtained before the controls (Table 1 and Fig. 2; P Ͻ 0.0001). administration of Cortrosyn (0.25-mg iv infusion over 1 min). Subse- Mean basal cortisol concentrations (Table 1) were not dif- quent blood samples were obtained 10 (n ϭ 46) and 30 (n ϭ 50) min ferent between the heterozygotic carriers and the healthy postinfusion. Plasma steroid hormones were measured as previously controls (11.1 Ϯ 3.8 vs. 11.3 Ϯ 3.2 ␮g/dL, respectively). Ten described (26). For all subjects, the basal cortisol level was less than 20 ␮g/dL. All hormone determinations for each individual were per- minutes after iv ACTH stimulation (Fig. 2), the heterozygotic formed in duplicate within single assays. carriers had a greater cortisol response (22.0 Ϯ 4.7 vs. 19.1 Ϯ 3.8 ␮g/dL; P Ͻ 0.05). The 30-min cortisol concentrations (Fig. Statistical analysis 2) were also greater among the heterozygotic carriers (28.1 Ϯ ␮ Statistical analysis was performed using AbSTAT statistical software 4.2 vs. 24.2 Ϯ 4.6 g/dL; P Ͻ 0.005). (Anderson-Bell, Arvada, CO). The 99% confidence intervals (mean Ϯ Among the 50 individuals (22 genotype normal and 28 2.57 sd) of hormone concentrations measured in the healthy individuals genotype heterozygotes), there was a positive correlation

TABLE 1. Basal and ACTH stimulated 17-OHP (ng/dL) and cortisol (␮g/dL) concentrations in normal and heterozygotic carriers

Heterozygotic Normal P value t statistic carriers OHP-0 105 Ϯ 65 (22) 106 Ϯ 63 (28) NS 0.04 OHP-10 171 Ϯ 79 (22) 365 Ϯ 208 (24) 0.0001 4.08 OHP-30 207 Ϯ 89 (22) 468 Ϯ 243 (28) 0.0001 4.78 F-0 11.3 Ϯ 3.2 (22) 11.1 Ϯ 3.8 (28) NS 0.175 F-10 19.1 Ϯ 3.8 (22) 22.0 Ϯ 4.7 (24) 0.0254 2.315 F-30 24.2 Ϯ 4.6 (22) 28.1 Ϯ 4.2 (28) 0.0027 3.158 Mean, standard deviation, and number of subjects are provided. NS, not significant. 2100 WITCHEL ET AL. JCE&M 1997 Vol 82 •No 7 • Investigation into the basis for the difference in susceptibility has shown that the Lewis rat shows hypothalamic-pituitary- adrenal axis hyporesponsiveness in contrast to the hyperre- sponsiveness 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 Downloaded from https://academic.oup.com/jcem/article/82/7/2097/2865904 by guest on 02 October 2021 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 FIG. 2. ACTH-stimulated cortisol responses. The graph depicts the significant differences in cortisol responses. A third study, ç cortisol responses (mean Ϯ SEM) in healthy controls ( ) and heterozy- using HLA haplotypes to infer carrier status, showed carriers gotic carriers (Ⅺ) before and after ACTH stimulation. Blood samples were obtained 10 and 30 min after the iv Cortrosyn bolus. Mean basal to have slightly greater cortisol responses, although these cortisol responses are not significantly different. At both 10 and 30 differences were deemed not significant (36). None of these min, mean cortisol concentrations were greater in heterozygotic car- studies showed the hypersensitization of the adrenal cortex riers than controls (P 0.05 and P 0.005, respectively). Ͻ Ͻ that we have documented here or speculated on a possible heterozygote advantage. between stimulated 17-OHP and cortisol concentrations. At Through secretion of peptide and steroid hormones, i.e. 10 min, the correlation coefficient was 0.61 (P Ͻ 0.0001). At ACTH and cortisol, the hypothalamic-pituitary-adrenal axis 30 min, the correlation coefficient was 0.45 (P ϭ 0.001). There restores homeostasis after stress (39). Glucocorticoids func- was no correlation between specific CYP21 mutation and tion as potent antiinflammatory and immunosuppressive peak cortisol response. agents. Indeed, the widespread therapeutic use of glucocorticoids in the treatment of immune disorders often over- Discussion shadows their physiological functions. Specific glucocorti- Common recessive disorders, such as sickle cell anemia coid actions include trafficking of circulating leukocytes, and cystic fibrosis, are frequently thought to impart a selec- suppression of accessory immune cells, inhibition of cytokine tive survival advantage to heterozygotic carriers (28, 29). production, and induction of resistance to cytokines (40–42). Although the concept of heterozygote advantage has been The production of nitric oxide, prostanoids, and platelet- entertained for 21-hydroxylase deficiency, direct evidence activating factor, three major mediators of the inflammatory delineating the specific mechanism of the survival benefit has response, is decreased by glucocorticoids (42). Glucocorti- proven difficult to obtain. The CYP21 gene is a frequent coids enhance the synthesis of acute phase proteins, which target of mutations, with carrier frequency between 1 in 3 and scavenge the toxic superoxide radicals generated to kill in- 1 in 16 in all populations studied despite the crucial role of vading microorganisms and tumor cells (43). The net result the 21-hydroxylase enzyme in adrenal steroidogenesis. This is the prevention of DNA and tissue damage that thwart high carrier frequency suggests that heterozygotes could self-destruction by cytokines and other effectors of immune have a survival advantage. reactions (18). These restraining actions are thought to hinder Cortisol secretion is regulated through the hypothalamic- the development of autoimmune disorders. pituitary-adrenal axis by negative feedback inhibition of Glucocorticoids also play a role in energy metabolism by ACTH secretion. ACTH acutely increases cortisol secretion maintaining the liver enzymes involved in gluconeogenesis, and chronically maintains transcription rates of the steroidogenic enzymes and optimal steroidogenic capacity (17). increasing substrate availability, and inducing phenyleth- Our interpretation of the more vigorous cortisol response anolamine N-methyl transferase in the adrenal medulla (44). that we observed in heterozygotic carriers was that the ad- The venous efflux of the adrenal cortex exposes the adrenal renal gland of heterozygotic carriers was up-regulated or medulla to high glucocorticoid levels, which may be impor- primed to secrete cortisol, because the clinically impercep- tant in epinephrine biosynthesis. Thus, cortisol acts by main- tible decrease in cortisol secretion had led to increased ACTH taining a state of readiness. Maintenance of epinephrine bio- secretion (30). synthesis impacts on the fight or flight response and Comparison of the inbred histocompatible Lewis and Fi- probably promotes survival (45). scher rat strains provides an example of the physiological We postulate that the brisk cortisol secretion observed consequences of subtle alterations in cortisol concentrations. among heterozygotic carriers could be a genetically favor- Lewis rats are extraordinarily susceptible to both experi- able trait, providing greater survival fitness. If so, these data mentally induced autoimmune and inflammatory disorders, support speculation that heterozygotic advantage has led to whereas Fischer rats are resistant to the same challenges (31). the high gene frequency of CYP21 mutations. HETEROZYGOTE ADVANTAGE IN CONGENITAL ADRENAL HYPERPLASIA 2101

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