THE GORDON WILSON LECTURE

CONGENITAL ADRENAL HYPERPLASIA* MARIA I. NEW NEW YORK

Introduction and Pathophysiology

Congenital adrenal hyperplasia (CAH) is a family of genetic disorders of adrenal steroidogenesis. The adrenal cortex synthesizes the glucocorticoid cortisol, the mineralocorticoid aldosterone, and sex steroids (testosterone and estrogen precursors) via a balanced network of parallel but interconnecting enzymatic pathways (Figure 1). Synthesis of cortisol by the hypothalamic-pituitary-adrenal axis is regulated directly by pituitary release of adrenocorticotropic hormone (ACTH) and negative product feedback of cortisol. A defect in the activity of an enzyme in one pathway will result in shunting of precursor and intermediate compounds to an alternate pathway. Biochemically, CAH arises from reduced enzyme activity in cortisol synthesis, which leads to increased production of androgens. There are two forms of CAH: classical and nonclassical. Classical CAH produces a total or near-total block in the activity of one of the five enzymes active in cortisol synthesis (21-hydroxylase, llfl-hydroxylase, 3f-hydroxysteroid dehydrogenase, 17a-hydroxylase, and cholesterol desmolase). In 90% of cases, it is the enzyme 21-hydroxylase which is impaired. The result is genital ambiguity in affected females and salt wasting in 75% of affected males and females. Improved biochemical assessment of adrenal function revealed the existence of partial enzyme deficiencies, confirmed for 21-hydroxylase (1, 2), 113-hydroxylase (3), and 3f-hydroxysteroid dehydrogenase (4, 5) which constitute nonclassical CAH (NC CAH). While they involve the same enzymes, classical and NC CAH are genetically distinct; they are associated with different alleles, as will be discussed below. The expression of symptoms of hyperandrogenism in NC CAH varies greatly. There is no genital ambiguity, but such symptoms as hirsutism, severe acne, or reduced fertility eventually appear in affected persons within a wide range of severity. As it is the most important cause of CAH, this paper will discuss * From: The Division of Pediatric Endocrinology, Department of Pediatrics, The New York Hospital-Cornell Medical Center, Pediatrics Bldg N-236, 525 East 68th Street, New York, NY 10021.

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exclusively the clinical features, screening and epidemiology, treatment, and prenatal diagnosis of 21-hydroxylase deficiency (21OHD).

Screening and Epidemiology: 21-Hydroxylase Deficiency 21-Hydroxylase deficiency CAH is diagnosed by clinical assessment, hormonal evaluation, HLA typing, DNA analysis, or combinations of these. Screening for salt wasting CAH in newborns, using a dried capillary blood specimen on filter paper obtained by heel prick, first became available in 1977 (6), and its utility as the basis for population screening was proven in a pilot program conducted in Alaska in 1981 (7). The obvious direct patient benefits to be obtained by newborn screening coupled with its economic feasibility has prompted the development of similar programs worldwide (see Table 1) (8, 9, 10). Recently, it has been demonstrated that 17-hydroxyprogesterone, the index compound for cortisol, can be measured in saliva to screen for NC CAH (11). Salivary 17-OHP correlates excellently with serum 17-OHP

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levels, and early morning collection of 3 mL of saliva suffices to obtain the diagnosis. The simplicity and practicality of the assay make this method suitable for population studies. Both classical and NC CAH occur with increased frequency in certain ethnic groups. The incidence of classical CAH (12), particularly the saltwasting kind, is strikingly high in the Yu'pik Eskimos of southwestern Alaska (1:282) and, to a lesser extent, in the inhabitants of La R6union, an island in the Indian Ocean (1:3147). Screening for CAH in the general population in eight countries revealed incidences ranging from 1:5600 in Rome, Italy, to between 1:11,000 and 1:18,000 in France, Illinois, Sweden, Portugal, Emilia-Romagna, Scotland, Washington State, and New Zealand (12). The lowest incidence was in Japan (1:20,000). NC CAH is far more common than classical CAH. In an analytic study (13), confirmed by a later computer-aided independent study (14), it was shown to be the most common autosomal recessive disease in man. The disease occurs with a mean frequency of 1:100 in a heterogeneous, predominantly caucasoid population and with increased frequency in some ethnic groups, most notably, Ashkenazic Jews, in whom it occurs with a frequency of 1:27, Hispanics (1:40), Yugoslavs (1:50), and Italians (1:300). The heterozygote frequencies are 1:3 in Ashkenazic Jews, 1:4 in Hispanics, 1:5 in Yugoslavs, 1:9 in Italians, and 1:14 for other caucasians.

Clinical Features Classical CAH Classical CAH occurs in two forms: simple virilizing and salt-wasting. Simple virilizing 21-hydroxylase deficiency (21OHD) is found in only 25% of cases. It is characterized by progressive virilization with accelerated somatic development. Because adrenocortical cell differentiation and active adrenal steroid synthesis occur early in embryogenesis, the excess androgens secreted by the fetal adrenal owing to steroid 21OHD will result in some degree of genital ambiguity in genetic females. The ambiguity ranges from mild clitoral enlargement through various degrees of fusion of the labioscrotal folds to the profound morphological anomaly of a penile urethra. The internal female reproductive organs are unaffected because stabilization of the wolfflan (mesonephric) ducts which develop into the epididymides, vasa deferentia, seminal vesicles, and ejaculatory ducts requires high local concentrations of testosterone provided only by male gonads. Further, in the genetic female without a fetal testis there is no production of antimullerian hormone, the glycoprotein synthesized by Sertoli cells which suppresses the development of the mullerian ducts into the fallopian tubes, uterus, cervix, and upper vagina. Thus, the internal genitalia

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of the genetic female are normal. Hyperandrogenism may cause infertility, as well as hirsutism, severe acne, polycystic ovary disease, malepattern baldness, and premature pubarche/adrenarche. Male genitalia will be normal, and the syndrome often goes unrecognized in boys until signs of salt wasting or androgen excess such as accelerated height and precocious sexual hair appear later in childhood. In males, continued androgen excess may suppress the pituitary-gonadal axis, preventing maturation of the gonads and causing infertility. Without treatment, both males and females have precocious physical development, with an early growth spurt followed by premature fusion of the epiphyses resulting in short stature. The untreated patients are therefore tall children but short adults. In 75% of cases of classical CAH, low serum levels of aldosterone lead to renal sodium loss with hyponatremia/hyperkalemia; plasma renin activity is concomitantly high. Salt wasting symptoms, sometimes crises, may not appear until the 7th to 10th day of life. The presence or absence of salt wasting is generally consistent within a family, although recently discordance in this regard has been reported in sibs who are HLA identical (15). Nonclassical Congenital Adrenal Hyperplasia The expression of hyperandrogenism in NC CAH varies widely in individuals. Some patients have had no history of symptoms when identified, but it is now believed that overt hyperandrogenism is almost always to be expected. Untreated, NC21OHD may be the cause of androgen excess syndrome including polycystic ovarian disease, hirsutism, irregular menses, and severe acne in females. Premature pubarche/ adrenarche (16), short stature, and reduced fertility may occur in both sexes. NC21OHD may cause premature pubarche in children as young as 5 months of age (1). Gynecomastia has been identified in one prepubertal boy (17). Menarche may be advanced in girls. In adolescent and adult women hirsutism is a very common manifestation, not infrequently in association with irregular menses or secondary amenorrhea. In different patient series, the prevalence of NC21OHD in hirsute, oligomenorrheic women has ranged from 1.2 to 30% (5, 18, 19, 20, 21, 22, 23); this wide range probably reflects different ethnic populations studied. Cystic acne (24, 25, 26) may be accompanied by hirsutism or irregular menses or amenorrhea. In some young women male-pattern baldness has been the sole presenting symptom. Testing of the adrenal axis reveals NC21OHD in a percentage of women with PCO (5, 27). Elevated serum androgens of adrenal origin could act centrally to alter gonadotropin release or could have direct

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effects on the ovary; once formed, ovarian cysts can autonomously maintain hyperandrogenism. Abnormal responses to luteinizing hormone-releasing hormone have been noted in NC21OHD (28). In boys early growth spurt, beard development and acne may occur. In a case of more significant androgen excess causing pubic hair growth and phallic enlargement, relatively small testicular size signals an adrenal rather than a testicular origin. Untreated, such suppression of the hypothalamic-pituitary-gonadal axis results in incomplete testicular maturation, with oligospermia and diminished fertility (29, 30). It is not known how often affected adult men who are otherwise asymptomatic may have disturbed gonadal function (31, 32). Androgens affect timing of the growth spurt and fusion of the epiphyseal plates of the bones. This results in slightly reduced stature relative to midparental height in males and females (33). Genetic predisposition to short stature may also be a factor, since the height of NC21OHD parents is below secular controls.

Treatment Treatment with glucocorticoids is effective in suppressing adrenal androgen production, and clinical improvement will follow with time, depending on the sign of androgen excess. Cystic acne may respond in 4 to 5 months. Remission of hirsutism may take 1 to 2 years, given the 9month life expectancy of established hair follicles. Return of regular menses may be accomplished in 2 months. Glucocorticoid treatment is effective in reversing infertility in a significant number of cases in both men and women, either by inducing ovulation resulting in pregnancy (11, 34, 35, 36, 37, 38, 39), or by improving sperm count and motility resulting in the ability to conceive (37, 40, 41). Low-dose treatment is currently being maintained in some asymptomatic NC21OHD pediatric patients with the expectation of improvement in their final adult height. Surgical correction of the genital ambiguity of females with classical CAH requires an experienced surgeon. It is often a two-step procedure, the first step being clitoroplasty, and the second, vaginoplasty, which is usually delayed until regular sexual intercourse is anticipated. Molecular Genetics The 210H enzyme is an adrenal microsomal cytochrome P450c21, coded for by the gene CYP21. This gene was duplicated at some time during evolution, so that the haploid chromosome normally contains two copies, one active (CYP21) and one inactive, a pseudogene (CYP21P). These genes are located within the human major histocompatibility complex (MHC), HLA (human leukocyte antigens), on the short arm of

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chromosome 6 in the class III region of MHC antigens (42). CYP21 and CYP21P are each paired with a copy of the gene for component C4 of serum complement, called C4A and C4B, apparently as a result of tandem duplication (43). These gene pairs have retained a remarkable degree of homology: C4A and C4B are >99% identical, and the exons of CYP21 and CYP21P are 98% identical (44). Deletions of the pseudogene produce no endocrinopathy. Characterization of the gene pair as one active and one pseudogene was made by determining the hormonal types of individuals who had deletions of one of the pairs. Sample DNA of normal subjects digested with TaqI showed two bands, one of 3.7 kg and one of 3.2 kb. Subjects with null expression of protein C4B were missing the 3.7-kb fragment and had salt-wasting CAH (43). Null expression of C4A and absence of the 3.2-kb fragment were associated with normal endocrine status. The differences between CYP21P and CYP21 consist of two shifts of reading frame arising from an 8-base pair deletion and a single base pair insertion, as well as one point mutation introducing a termination codon and several point mutations causing nonconservative amino acid substitutions. The tandem C4A-CYP21P-C4B-CYP21 arrangement makes misalignment of homologous chromosomal segments possible during meiosis and mitosis. Unequal crossing over during gametogenesis could result in one or in three C4-CYP21 sets on a haplotype. The HLA-A1,B8,DR3 and -A3,Bw47,DR7 haplotypes have a single set of C4-CYP21 (45), and the HLA-B14,DR1 haplotype, which is associated with NC21OHD, carries a third C4-CYP21 set consisting of an extra C4B gene and an extra CYP21P or CYP21P-like gene (based on the size of the extra restriction fragments) (46, 47). This may be a pseudogene like CYP21A, but its identity has not been definitively established. Genetic linkage between HLA and the 210H gene was first shown by Dupont et al. in 1977 (48). Confirming reports from the same (49) and other groups (50) and compilation of data from studies of persons with intra-HLA recombinations strongly indicated a locus for 210H between HLA-B and -D (51). Linkage to the HLA-B locus was established with peak lod scores as high as 9.5 and 15.65 (at recombinant fraction 0 = 0.00) (13). In addition to localizing the 210H gene, we have found that its alleles are in linkage disequilibrium with alleles of other genes within the HLAB and -DR locus. Salt-wasting 21OHD shows an increased association with Bw60 and Bw47; the latter is a rare antigen (0.9, 0.4, and 0.4 frequency in European caucasian, North American caucasian, and Japanese) within the haplotype HLA-A3;Bw47;DR7, which also carries a null allele for either C4A or C4B (52, 53). The incidence of Bw5l is

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increased with simple virilizing disease in some ethnic groups (53). Negative association with 21OHD has been observed for HLAA1;B8;DR3 which carries a null allele for C4A. Nonclassical disease is associated with HLA-B14;DR1 in haplotypes which typically also carry a third copy of the C4 isotype (54, 55, 56). This association has been found in all ethnic groups examined so far except Yugoslavs (50). Almost all Ashkenazic Jews affected with NC 21OHD are positive for antigen B14 and often for DR1 (13, 57). This suggests the transmission of a single mutation in the Ashkenazic population. Since the characteristic Ashkenazic NC21OHD allele and associated B14 antigen are not restricted to any Ashkenazic subgroup and since the frequency of B14 is not as high in Sephardic and Oriental Jews (58), a stem mutation is postulated to have occurred after the Second Diaspora (A.D. 70) and by the time of the consolidation of the major Jewish communities in northern and eastern Europe (probably before A.D. 1100) (Figure 2). Molecular Characterization The B14,DR1-associated CYP21 molecular mutation causing NC CAH was characterized by Speiser et al. (59). They identified three deviations from normal of which only one proved significant: (1) a single base change (GTC to CTG) in codon 211 causing a conservative substitution of valine to leucine (both are nonpolar amino acids); (2) three base changes between positions 8 and 13 of intron 6 (CTGTAC to ATGTGT); and (3) a single base change (GTC to TTG) in codon 281 resulting in a second valine to leucine substitution. Mutation 2 was excluded from consideration because intronic base changes known to affect 5'-end splicing have all started before position 5 of the intron. Mutations 1 and 3 involve the same conservative substitution, but its significance depends on context: amino acids other than valine occur at the position corresponding to residue 211 in the P450 protein of other species, whereas valine-281 is uniformly conserved, which suggests a more critical requirement (perhaps having to do with determination of molecular volume). Thus, the third mutation was selected as the most likely to be of significance, and a 21-mer oligonucleotide probe was synthesized to recognize the change in the base sequence. Ten individuals (nine unrelated NC21OHD patients and one normal control) were screened. Eight of the NC21OHD patients were B14,DR1positive (three homozygotes and five heterozygotes) and of Ashkenazic Jewish descent. The ninth patient, who was Yugoslav, typed negative for B14,DR1, as did the control. DNA analysis with the probe identified the mutation in all eight Ashkenazic Jewish patients and not in the B14-

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PALESTINIAN JEWS FIG. 2 First Diaspora, A.D. 61; Second Diaspora, A.D. 70. Because the characteristic Ashkenazic NC21OHD allele and associated B14 antigen are not restricted to any Ashkenazic subgroup and the frequency of B14 is not as high in Sephardic and Oriental Jews, a stem mutation is postulated to have occurred after the Second Diaspora.

negative patient nor the unaffected control. A second probe specific for the active B gene was used to screen the positive samples to confirm that the extra (third) C4-CYP21 sequence that is known to be carried on the B14,DR1 haplotype is a duplicated CYP21P or CYP21P-like pseudogene. Since altered protein structure might not be the only change causing expression of the NC21OHD phenotype, transcription assays were per-

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formed using mouse Y1 adrenal cells transfected with the mutant CYP21. It was determined that the gene sequence is transcribed to mRNA and a protein synthesized that exhibits reduced enzymatic activity. Stability of mRNA transcripts cannot be assessed in this cell expression system, nor can changes in the rate of transcription be detected (perhaps there is an undetected mutation in a regulatory region). The single base change causing missense mutation Val 281 to Leu appears to be a consistent molecular genetic marker for the NC21OHD allele associated with HLAB14,DR1.

Heterogeneity According to the enzyme function provided by the alleles present in a subject with CAH, there will be characteristic serum/urinary biochemical values and, in combination with other individual factors, characteristic clinical manifestations. However, the alleles for classical disease correlate with both simple virilizing and salt-wasting CAH. It has also been noted that family members who are HLA identical may be discordant in clinical expression of their disease. In NC CAH affected individuals may show the same biochemical characteristics and be either symptomatic or asymptomatic. Classical and nonclassical mutations are currently being studied to determine their specific effects, and it is hoped that laboratory synthesis of normal and defective enzymes in quantity will permit kinetic and physicochemical study of human 210H protein for the first time. Prenatal Detection and Management Prenatal diagnosis has been available for two decades for pregnancies known to be at risk. Hormonal diagnosis has been possible since 1975 based on elevated levels of amniotic fluid 17-OHP in the second trimester (60). A recent report suggests that measurement of 21-deoxycortisol is useful as a confirmatory test (61). Genetic diagnosis until recently was performed by HLA serotyping of fetal cells cultured from the amniotic fluid, specifying the class I and II transplantation antigens (HLA-A, -B, -C, and -DR), whose genes are closely linked to the 210H genes (62, 63, 64). These tests are accurate in most cases. [False-negative 17-OHP levels in non-salt-losing cases and intra-HLA recombination were among the causes of imprecision in some prenatal diagnostic studies (65).] But as a diagnostic modality amniocentesis has the disadvantage that it is performed in the second trimester, when genital ambiguity in the female has already been established. With the advent of chorionic villus sampling, evaluation of the fetus at risk is possible during the first trimester (at 8 to 10 weeks' gestation).

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DNA can be extracted from cultured villus tissue for analysis by molecular genetic techniques. With the use of appropriate cDNA probes, restriction fragment length polymorphisms signal the allelic genes for class I (HLA-A, -B, or -C) (66), class II (HLA-DR, -DP, and -DQ) (67), C4 (C4A/C4B) (68), and 210H (69) transmitted from the parents and present in the genotype. Diagnosis by this approach, thus, can be made in some instances even before amniocentesis can be scheduled and early enough to consider treating the fetus to prevent ambiguous genitalia in affected females. Prenatal treatment has recently been introduced using dexamethasone, a zA'-steroid administered to the pregnant woman which crosses the placental barrier to suppress the fetal adrenal (70, 71, 72). The procedure is as follows (Figure 3): when a pregnancy is confirmed in a family known to be at risk, dexamethasone treatment of the mother (20 Ag/kg/day given in 3 divided doses) is started immediately. Chorionic villus sampling (CVS) is performed at 7 to 10 weeks' gestation. Karyotyping results are available 2 to 5 days later: if the fetus is male, dexamethasone is discontinued; if female, it is prolonged. One to two weeks later, DNA analysis of the CVS is complete and the normal, carrier, or nonclassical/ classical affected status of the fetus at risk can be known. If a female fetus is shown to be unaffected or to have NC CAH, dexamethasone is stopped at this early point in the pregnancy. Treatment should be continued throughout the pregnancy for a classically affected female fetus. As shown in Figure 3, diagnosis by amniocentesis can also be used to determine treatment strategy, but because it is performed later, when genital ambiguity has already evolved, it is not the diagnostic modality of choice, particularly since CVS is now comparable in safety and efficacy to amniocentesis (73, 74). The safe gestational use of glucocorticoids has been established (75), and in the author's experience to date, treatment begun as early as possible and continued uninterrupted at the proper dosage can produce a favorable outcome, sparing an affected female corrective genital surgery. Treated infants should be followed for evidence of previously unrecognized long-term side effects of prenatal dexamethasone treatment. Conclusion 21-Hydroxylase deficiency is the main cause of both classical and nonclassical CAH, which are congenital disorders of adrenal steroidogenesis found with increased frequency in certain ethnic groups. Nonclassical CAH is the most common autosomal recessive disease in man. Screening at birth is available. In the case of the salt-losing form of classical CAH,

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which is present in 75% of CAH-affected individuals, screening can be lifesaving. A salivary screening assay is effective in detecting nonclassical CAH, which may underlie a wide range of androgen excess syndromes that can appear at any age. Prenatal diagnosis by DNA analysis after chorionic villus sampling in the first trimester of a pregnancy permits prenatal treatment that can prevent the development of ambiguous genitalia in classically affected females.

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