Congenital Adrenal Hyperplasia

Reproductive Medicine Review 1993; 2: 1-13

Articles

Congenital adrenal hyperplasia

Phyllis W Speiser, Perrin C White and Maria I New The New York Hospital-Cornell Medical Center, New York, USA

Introduction c) 17a-hydroxylase deficiency and/or 17,20-lyase deficiency; Congenital adrenal hyperplasia (CAH) is a group d) 3p-ol dehydrogenase deficiency (classical and of diseases which result from reduced or absent nonclassical); activity of one of the five enzymes of cortisol e) cholesterol desmolase deficiency (lipoid synthesis in the adrenal cortex. Each enzyme hyperplasia). deficiency produces characteristic alterations in the levels of adrenal steroid hormones and their The 21-hydroxylase and ll^-hydroxylase precursors. The particular hormonal imbalances deficiencies, occurring late in cortisol synthe- cause a spectrum of abnormalities includ- sis, cause shunting of accumulating precursor ing abnormal fetal genital development and steroids into androgen pathways, resulting in pseudohermaphroditism, disturbances in sodium genital ambiguity in newborn females and homeostasis and blood pressure regulation, and later hyperandrogenic effects in both sexes. specific metabolic disturbances. Accompanying the virilizing effects, imbalances in salt metabolism distinguish these two The following enzymatic defects of steroido- forms: in 21-hydroxylase deficiency, deficient genesis and their associated clinical syndromes 1 2 aldosterone synthesis causes salt-wasting and have been described ' : hypovolaemia in 75% of cases; whereas excess a) 21-hydroxylase deficiency: classical (salt- secretion of the mineralocorticoid agonist wasting and simple virilizing) and non- deoxycorticosterone (DOC) or its metabolites classical; in llp-hydroxylase deficiency causes sodium b) lip-hydroxylase deficiency (hypertensive retention and hypertension. CAH) with corticosterone methyl oxidase In the more proximal 17a-hydroxylase/17,20- (CMO) types I and II (salt-wasting); lyase deficiency, blocked production both of 17a-hydroxy (glucocorticoid) and of C19/C18 steroids causes pseudohermaphroditism in males Address for correspondence: Phyllis W Speiser, Department of Pediatrics, Division of Pediatric Endocrinology, The New and sexual infantilism in females. Shunting of York Hospital-Cornell Medical Center, 525 East 68th Street, P450cl7 precursor steroids into the 17-deoxy New York, NY 10021, USA. pathway produces mineralocorticoid excess and © Edward Arnold 1993 2 PW Speiser et al. hypertension. The 17,20-lyase deficiency is a 21-hydroxylase gene to HLA was utilized in variant of 17a-hydroxylase deficiency in which genotyping sibs in pedigrees with an affected glucocorticoid and mineralocorticoid levels are index case. Thus, a sib sharing both HLA relatively unaffected and a block in adrenal and haplotypes with the index case is predicted to gonadal C21 to C19 steroid conversion results in a be affected, one who shares a single haplotype specific clinical phenotype. is predicted to be a heterozygote, and one In the 3p-ol dehydrogenase defect, poor or who shares no HLA haplotype is predicted absent conversion to A4-steroids allows produc- to be unaffected. Although this system of tion only of the A5-steroid precursors which genetic counselling was imperfect, with a ~1% are relatively inactive, causing salt wasting and recombination rate between the HLA-B or -DR cortisol insufficiency. While lack of potent A4- and the 21-hydroxylase locus, serotyping for androgens produces hypospadias in the male, HLA served as a useful adjunct to amniotic enormously high levels of the weak androgen fluid hormonal measurements for the purposes DHEA may cause limited virilization (clitoral of prenatal diagnosis before the advent of more enlargement) in females. specific molecular genetic testing. Cholesterol desmolase deficiency blocks all steroid production, with build-up of choles- Linkage disequilibrium terol substrate (lipoid adrenal hyperplasia), and In addition to linkage of the 21-hydroxylase accordingly, among its very serious effects, results locus with the neighbouring HLA-B and -DR in pseudohermaphroditism in genetic males. antigen loci, 21-hydroxylase deficiency alleles Sexual ambiguity is not a feature of the are found in linkage disequilibrium with HLA 18-hydroxylase (CMO I) or 18-dehydrogenase antigen genes or haplotypic combinations9 that (CMO II) deficiencies causing hypotension, since may include specific alleles of C4 of serum these distal blocks in aldosterone synthesis do complement.10 The two most prominent such not alter the normal synthesis of cortisol, and cases are linkage disequilibrium of the extended thus cause no disturbance of the hypothalamic- haplotype HLA-A3,Bw47,DR7 with the salt- pituitary-adrenal axis. wasting form of 21-hydroxylase deficiency, and of the haplotype HLA-A1, B14,DR1 with nonclassical 21-hydroxylase deficiency.11 Classical genetics in CAH Epidemiology of classical 21-hydroxylase The autosomal recessive mode of genetic trans- deficiency mission of various forms of adrenal steroidogenic A reliable and valid screening test for 21- defects was suspected in the early 1950s,3-5 but it hydroxylase deficiency CAH using a heel-prick was not until the late 1970s that the genetics of capillary blood specimen impregnated on filter the most common form of CAH, 21-hydroxylase paper first became available in 1977.12 A deficiency, began to be unravelled. Linkage pilot newborn screening programme among the between the human major histocompatibility Alaskan Yup'ik Eskimos at high-risk for 21- complex (MHC), or HLA, located on the short hydroxylase deficiency CAH first demonstrated arm of chromosome 6 (between subregions 6p21.1 the feasibility of an effective newborn screen- and 6p21.3) and 21-hydroxylase deficiency was ing programme for 21-hydroxylase deficiency first shown by Dupont et al.6 Initial calculated CAH.13 The direct benefit of 21-hydroxylase LOD scores demonstrating linkage were only deficiency CAH screening from this programme slightly higher than the requisite 3.00, but — such as avoidance of adrenal crisis, shock, its recent calculated LOD scores are in excess of sequelae and death - promted further devel- 22. Compiled data on intra-HLA recombinations opment of newborn screening programmes strongly indicated a gene locus for 21-hydroxylase for 21-hydroxylase deficiency CAH in various between HLA-B and -DR.7-8 The more recent nations.14 The world-wide incidence of 21- molecular studies have confirmed this location in hydroxylase deficiency CAH as determined by the class III region of the MHC. these screening programmes is 1:14 554 live The knowledge of close genetic linkage of the births, of which approximately 75% are of the Congenital adrenal hyperplasia 3 salt-wasting phenotype.14 Applying the Hardy- with 17,20-lyase deficiency (reviewed in19). Of Weinberg Law, the heterozygote frequency for all the genetic males reported with this enzyme classical 21-hydroxylase gene defects is thus 1:61 deficiency, several are from the same three persons. kindreds.20"27 One genetic female has been detected.28 Population genetics of nonclassical 21-hydroxylase First described by Bongiovanni in 1962,29 3(3- deficiency HSD deficiency seemed most likely to have a A high frequency of occurrence for nonclassical monogenic autosomal recessive mode of trans- 21-hydroxylase deficiency has been determined mission based on pedigree analysis.29"31 The in a number of ethnic groups.11 In this analysis, exact frequency of this disorder is unknown; heterozygote (carrier) and (homozygous) affec- it is conceivable that affected individuals with ted status were established in family members early steroidogenic defects which severely by HLA typing, correlating HLA-B types with impair cortisol synthesis, for example, in 3(3- known HLA-B associations, in conjunction with hydroxysteroid dehydrogenase and side-chain ACTH testing using criteria provided by refer- cleavage, are poorly viable. ence hormone data.15 By counting the incidence Deficiency of side-chain cleavage or choles- of nonclassical deficiency genes relative to the terol desmolase first described by Prader32-33 presumed normal genes among allowed paren- is extremely rare. A recent clinical case report tal haplotypes, the frequency of nonclassical describes a patient diagnosed in the newborn 21-hydroxylase deficiency was calculated. The period and successfully treated for 18 years, gene frequency for nonclassical 21-hydroxylase and also reviews 32 cases from the literature34; deficiency was highest in Ashkenazic Jews and cholesterol desmolase deficiency seems to occur was also high in Hispanics, Yugoslavs and with less severity and somewhat more frequently Italians. Disease frequencies were 0.037 (1/27) among Japanese. for Ashkenazic Jews, 0.019 (1.53) for Hispanics, 0.016 (1/63) for Yugoslavs, 0.003 (1/333) for Italians, and 0.001 (1/1000) for other caucasoids Molecular genetics in CAH (40% of whom had Anglo-Saxon background).11 Confirmation of these data was obtained employ- Gene structure and molecular pathology ing the statistical method of commingling distribu- 21-hydroxylase deficiency is inherited as a tions of an expanded database including the same 16 monogenic autosomal recessive trait closely families. Nonclassical 21-hydroxylase deficiency linked to the HLA major histocompatibility is thus among the most frequent autosomal reces- complex on chromosome 6p23. The structural sive disorders in man. gene encoding P450c21 (CYP21 or CYP21B) and a 98% identical pseudogene (CYP21P or Epidemiology of other defects of steroidogenesis CYP21A) are located in the HLA complex Classical lip-hydroxylase (P450cll) deficiency adjacent to and alternating with the C4B and C4A comprises 5-8% of cases, occurring in about genes encoding the fourth component of serum 1/100 000 births in the general Caucasian popula- complement.3536 The CYP21P pseudogene has tion.17 A large number of cases have been accumulated a number of mutations that render reported in Israel among Jewish immigrants the putative gene product completely inactive. from Morocco, a relatively inbred population. The close proximity between CYP21 and CYP21P The incidence in this group is currently estimated (30 Kb) appears to generate frequent mutations to be 1/5000-1/7000 births, with a gene frequency in CYP21 by two mechanisms: unequal crossing- of 1.2-1.4%.18 CMO II deficiency, representing a over during meiosis results in a complete deletion defect in the terminal step of aldosterone synthe- of a DNA segment of about 30 kilobases (kb) in sis, is apparently rare in the general population, length including the C4B and CYP21 genes C4B but it has been found at an increased frequency and CYP21; gene conversion events result in the among Jews of Iranian origin. transfer of small, deleterious mutations, often 37 Over 120 cases have been reported of 17a- single base changes, from CYP21P to CYP21. hydroxylase deficiency, mostly in combination Early efforts to genotype 21-hydroxylase 4 PW Speiser et al. deficient patients relied on Southern blotting, Analysis of the remaining 65-90% of disease in which relatively large gene-specific DNA haplotypes without obvious gene deletions has probes are hybridized against genomic DNA revealed small mutations attributable to gene digested with restriction endonucleases capable of conversion. There are eleven missense mutations discriminating between the CYP21 and CYP21P in CYP21P which have been observed in patients genes. This approach is potentially inaccurate with 21-hydroxylase deficiency. Most of these are when hybrid CYP21/CYP21P genes are formed presumed to have arisen from gene conversion as a result of crossover events. Careful studies events (Figure 1). These mutations differ in employing multiple informative restriction digests their associations with phenotypic forms of 21- (such as Taq I and Bgl II) and oligonucleotide hydroxylase deficiency. hybridizations can resolve such ambiguities.38 The availability of pulse field gel electrophoresis used in conjunction with rare-cutting endonucleases Mutations associated with nonclassical 21- such as Bss HII has permitted long range mapping hydroxylase deficiency of the MHC in the vicinity of the C4 and CYP21 Val-281—»Leu, a single base change (G—»T) genes.39 Based on a number of studies in differ- in a highly conserved subregion of the seventh ent ethnic populations, the frequency of gene exon resulting in a conservative amino acid deletion causing 21-hydroxylase deficiency ranges substitution, has been identified in 75-80% from 11-35% (reviewed in40), and is highest in of nonclassical haplotypes carrying the HLA- northern European populations which carry the B14;DR1 haplotype.4142 A change of a proline to HLA-B47;DR7 haplotype at a high frequency. leucine at residue 30 in exon 1 was recently found

t t T t t Tt t T A B C D E FG H I i i i I

Figure 1 Top; Diagram of the chromosomal region containing the CYP21 (21Aand21B) 21-hydroxylase genes. HLA-B is about 600 kb to the left and HLA-DR about 400 kb to the right of the diagrammed region.96 Other genes in this region are BF (properdin factor B), C2, C4A and C4B (second and fourth serum complement components). These genes are all transcribed in the same direction. Additional genes of unknown function are transcribed from the opposite chromosomal strand: RD, XA and XB. A scale is marked every 20 kb. The bracket indicates the region deleted in about 20% of classic 21-hydroxylase deficiency alleles. Bottom; A CYP21 gene is diagrammed. A scale is marked every 500 bp. Numbered bars represent exons which are sequences found in mRNA. Full bars are protein-coding sequences whereas half-height bars are untranslated sequences. Nine deleterious mutations normally found only in theCYP21Apseudogeneare marked: A, mutation of codon 30 from CCG, encoding proline, to CTG, leucine; B, A or C to G mutation in intron 2 causing aberrant splicing; C, 8-basepair deletion in codons 110-112; D, mutation of codon 172 from ATC, isoleucine, to AAC, asparagine; E, cluster of three mutations in codons 235-238 changing the amino acid sequence from isoleucine-valine-glutamate-methionine to asparagine-glutamate-glutamate- lysine; F, mutation of codon 281 from GTG, valine, toTTG, leucine; G, single-base insertion between codons 306-307; H, nonsense mutation, CAG to TAG, in codon 318; I, mutation of codon 356 from CGG, arginine, toTGG, tryptophan. Two additional point mutations have been found: one in exon 8, the other in exon 10. (Reproduced from White and New.97) Congenital adrenal hyperplasia 5 in 16% of haplotypes in nonclassical patients who showed that the cluster of mutations in codons carried at least one non-B14 haplotype.43 235-238 and the Arg-356—>Trp mutation resulted in enzymes with no detectable activity, whereas Mutations associated with simple virilizing 21- trace activity was detected in the enzyme carry- hydroxylase deficiency ing the Ile-172—>Asn mutation. However, these Ile-172—»Asn, a single base change (T—>A) in studies did not attempt quantitation of activity, exon 4, results in substitution of a polar amino possibly because levels of expression were low. acid for a highly conserved nonpolar residue. This In order to address this problem, several mutation has been described in patients with the mutant enzymes were synthesized at higher simple virilizing form44 of the disease, and in levels in cultured cells using recombinant vaccinia 6-14% of classical disease alleles. virus.4349 The enzyme with the cluster of substitutions in codons 235-238 again had no activity Mutations associated with salt-wasting 21- even when expressed at higher levels. When hydroxyiase deficiency 17-hydroxyprogesterone was the substrate, the An A—>G substitution near the 3' end of the enzyme carrying Ile-172—>Asn had an activity second intron activates a novel splice acceptor site of 0.6% of normal as measured by the first- and shifts the reading frame of translation. The order rate constant, V^JY^. The Val-281^Leu erroneously processed mRNA yields a truncated mutation resulted in an enzyme with 50% of protein with no enzymatic activity. In one study normal activity when 17-hydroxyprogesterone this mutation accounted for 57% of nondeletional was the substrate but only 20% of normal alleles.45 It appears that mRNA splicing with this activity for progesterone. The Pro-30—»Leu mutation is variable, however, since the mutation mutation had 60% of wild type activity for has been found in patients with the severe salt- 17-hydroxyprogesterone and 30% activity for wasting as well as with the less severe simple progesterone when assayed in intact cells. The virilizing pheno types. activity of enzyme expressed after transfection of A cluster of mutations, Ile-Val-Glu-Met-235- a CYP21 gene carrying a substitution of Thr for 238—>Asn-Glu-Glu-Lys,46 and a single substi- Ser-268 was no different from that of the wild tution, Arg-356—»Trp,47 have been described type enzyme, thus confirming earlier reports that the latter represents a normal polymorphism of in patients with salt-wasting disease. A stop 50 codon introduced into the eighth exon by CYP21. The latter mutation has been detected virtue of C—>T substitution48 is seen in 4-7% incidentally in CAH haplotypes carrying a second of classical 21-hydroxylase deficiency haplotypes, severe mutation, the A—>G mutation in the and the 8-basepair deletion in exon 3, also a second intron which introduces a novel splice acceptor site, and disrupts the reading frame of salt-wasting mutation, is found in 3-10% of 51 disease haplotypes. The only gene conversion translation. which has not as yet been detected in a patient The main conclusion to be drawn from the with 21-hydroxylase deficiency is a T insertion in foregoing studies is that mutant P450c21 enzymes the seventh exon, expected to cause a disruption carrying specific amino acid substitutions seen in of the translational reading frame. patients with 21-hydroxylase deficiency exhibit activities that usually correlate with the clinical Functional analysis of mutations severity of the disease when present in the Because synthesis of P450c21 is restricted to the homozygous or hemizygous (i.e. with deletion adrenal gland, the effect of missense mutations of CYP21 in trans) state, and with biochemical on the enzymatic activity of P450c21 cannot be abnormalities such as 17-hydroxyprogesterone directly tested in affected patients. Therefore, a levels after ACTH (corticotropin) stimulation. number of attempts have been made to measure the activities of mutant enzymes in cultured Further correlation of mutation with phenotype: cells. In one such study, transfected normal salt-wasting versus simple virilizing phenotypes and mutated CYP21 genes or P450c21 cDNA An important facet of the clinical variability in plasmid vectors containing a strong promoter of 21-hydroxylase deficiency concerns the to allow expression in COS cells.46 These studies ability to synthesize adequate amounts of the 6 PW Speiser et al. mineralocorticoid hormone aldosterone. Because excess. A heterozygous carrier of a salt- aldosterone is normally secreted at a rate wasting mutation might also be expected to 100-1000 times lower than that of cortisol, it is have about 50% of normal 21-hydroxylase apparent that 21-hydroxylase activity would have activity, but such individuals are asymptomatic to decrease to very low levels before it became and have minimal biochemical abnormalities. rate-limiting. Apparently, as little as 0.6% of This suggests that in vivo 21-hydroxylase normal activity, as seen in the enzyme carry- deficiency must actually be less than 50% ing the Ile-172—»Asn mutation, allows adequate of normal. This apparent paradox is resolved aldosterone synthesis to prevent significant salt by the finding that progesterone at physiologic wasting, thus resulting in the 'simple virilizing' intra-adrenal concentrations (2-4 /JM)57 acts as phenotype. In contrast, mutations that completely a powerful competitive inhibitor of the mutant destroy enzymatic activity are associated with salt- 21-hydroxylase enzyme for its main substrate, wasting. It should be noted, however, that the 17-hydroxyprogesterone. Thus, relatively small distinction between the simple virilizing and salt- differences in intra-adrenal progesterone concen- wasting phenotypes is not absolute. One patient tration could account for much of the clinical with the Ile-172—»Asn mutation has been reported variability that is a hallmark of nonclassical to have an elevated ratio of plasma renin to 21-hydroxylase deficiency. Another contributing aldosterone, consistent with mild salt-wasting,14 factor to phenotypic variability is undoubtedly and HLA-identical sib pairs have been reported pseudosubstrate inhibition of other steroidogenic in which one sib has salt-wasting disease whereas enzymes by accumulated precursors of 21- the other can synthesize adequate amounts of hydroxylase; this phenomenon probably accounts aldosterone.52 for reports of multiple enzyme deficiencies.58 The mechanism of occasional recovery from salt-wasting52-55 is not known. A study Further phenotype-genetype correlations correlating clinical, biochemical and molecu- We have genotyped a group of patients lar genetic findings in five patients who had followed at our institution who have been severe salt-wasting 21-hydroxylase deficiency in thoroughly clinically characterized. Presence of infancy showed that all but one patient had one or more of ten mutations in the CYP21 persistent impairment of aldosterone synthe- gene was performed using Southern blot analysis sis associated with homozygous deletion or to detect CYP21 deletions or large gene conver- equivalently severe mutations in CYP21B.56 sions, and allele specific hybridizations with DNA The fact that one patient had amelioration of amplified by the polymerase chain reaction aldosterone deficiency with age and the variable to detect smaller mutations. Mutations were responses to sodium deprivation in three patients detected on 95% of chromosomes examined. with identical genotypes indicate that nongenetic The most common mutations were: an A—*G factors contribute to phenotype in this form of change in the second intron affecting pre- congenital adrenal hyperplasia. Based on data mRNA splicing (26%), large deletions (21%), gleaned from radiolabelled progesterone infusion, Ile-172-^Asn (16%), and Val-281->Leu (11%). it appears that recovery from salt-wasting may be Patients were classified into three mutation explained by activity of an adrenal P450 enzyme groups based on degree of predicted enzymatic other than P450c21.56 compromise. Mutation groups were correlated with clinical diagnosis and specific measures Nonclassical phenotype of in vivo 21-hydroxylase activity such as 17- As mentioned, the Val-281—»Leu mutation hydroxyprogesterone, aldosterone and sodium results in an enzyme with about 50% of balance. Mutation group A (no enzymatic activ- normal activity when 17-hydroxyprogesterone is ity) consisted principally of salt-wasting (severely the substrate but only about 20% of normal activ- affected) patients, group B (2% activity) of simple ity for progesterone. An individual homozygous virilizing patients, and group C (10-20% activity) for this mutation has nonclassical 21-hydroxylase of nonclassical (mildly affected) patients, but each deficiency with significant biochemical abnor- group contained patients with phenotypes either malities and variable symptoms of androgen more or less severe than predicted. (Nonclassic Congenital adrenal hyperplasia 7 patients were deliberately under-represented in and after actual purification from aldos- this study.) These data suggest that most terone secreting tumours.66 The isozyme but not all of the phenotypic variability in encoded by CYP11B2, termed P450XIB2 21-hydroxylase deficiency results from allelic (or P450cmo or P450aldo), lip-hydroxylates variation in CYP21.59 Accurate prenatal diagnosis 11-deoxycorticosterone to corticosterone and is now possible in most cases using the described 11-deoxycortisol to cortisol. It 18-hydroxylates strategy. corticosterone and cortisol, and further oxidizes 18-hydroxycorticosterone to aldosterone. In Steroid lip-hydroxylase contrast, the product of CYP11B1, termed There are two human genes60 on chromo- P450XIB1 (or P450cll), has a strong lip- some 8q21-q226162 that encode lip-hydroxylase hydroxylase activity but 18-hydroxylates only (P450cll) isozymes with predicted amino acid about one-tenth as well as P450XIB2. P450XIB1 sequences that are 93% identical. One gene, does not synthesize detectable amounts of aldos- CYP11B1, is expressed at high levels in normal terone from 18-hydroxycorticosterone. adrenal glands,60 and transcription of this gene These data suggest that P450XIB1 synthesizes is appropriately regulated by cAMP (the second cortisol in the zona fasciculata whereas P450XIB2 messenger for ACTH).63 Transcripts of the synthesizes aldosterone in the zona glomerulosa. other gene, CYP11B2, cannot be detected by This hypothesis has been confirmed by studying hybridization to Northern blots of normal adrenal individuals with defective cortisol or aldosterone RNA,60 but such transcripts have been detected synthesis due to respective deficiencies in lip- by hybridization to RNA from an aldosterone- hydroxylase and corticosterone methyloxidase II secreting tumour.64 (CMOII) activities. CYP11B2 transcripts have been detected in Patients with 11-hydroxylase deficiency are normal adrenal RNA using a more sensitive assay unable to convert 11-deoxycortisol to cortisol. wherein RNA was reverse-transcribed and then Elevated levels of ACTH cause steroid precur- amplified using the polymerase chain reaction sors to accumulate proximal to the blocked step. (RT-PCR).65 Whereas an amplified product Many of these precursors are shunted into the corresponding to transcripts of CYP11B1 was pathway for androgen biosynthesis as occurs in detected after 20 PCR cycles in samples from 21-hydroxylase deficiency. Thus, female patients normal adrenal glands, the CYP11B2 product with this disorder are born with masculinized was visible after 30 cycles by staining with external genitalia, and affected individuals of ethidium bromide. When an RNA sample of both sexes undergo rapid somatic growth with an aldosterone-secreting adrenal tumour was premature epiphyseal closure, resulting in short examined in this manner, it contained a adult stature. concentration of CYP11B1 transcripts slightly A parallel defect usually exists in the synthesis lower than that of the normal adrenal, but of 17-deoxy steroids, so that deoxycorticosterone CYP11B2 transcripts were increased fivefold over is not converted to corticosterone and instead the normal gland. accumulates. Because deoxycorticosterone and To determine if levels of CYP11B2 transcripts some of its metabolites have mineralocorticoid were appropriately regulated, the zone glo- activity, elevated levels may cause hypertension merulosa was dissected out of human adrenal and hypokalaemia. About two-thirds of untreated surgical specimens and cultured in the presence patients become hypertensive, sometimes early of angiotensin II or corticotropin (ACTH) in life.67 This clinical feature distinguishes before preparing RNA. Angiotensin II markedly lip-hydroxylase deficiency from 21-hydroxylase increased levels of both CYP11B1 and B2 deficiency, in which poor aldosterone synthesis transcripts. ACTH increased CYP11B1 mRNA causes renal salt-wasting in the majority of levels more effectively than angiotensin II, but it patients. had no effect on CYP11B2 transcription. In an analysis of mutations causing this disor- The enzymes encoded by the CYP11B1 and der,68 six families carrying an allele for lip- CYP11B2 genes have been studied by expressing hydroxylase deficiency were studied; all were the corresponding cDNAs in cultured cells64-65 Jews originating from Morocco. Eleven of twelve 8 PW Speiser et al. mutant alleles carried the same mutation, a single (tryptophan) (R181W), a substitution of an amino base change in exon 8 of CYP11B1. Codon 448, acid with a large nonpolar group for a basic amino CGC, encoding arginine, was changed to CAC, acid, whereas the second in exon 7, codon 386, histidine (R448H). is GTG (valine) -» GCG (alanine) (V386A), The sulfhydryl of Cys-450 in P450cll is a more conservative substitution of one amino presumed to constitute the fifth ligand to the acid with a nonpolar side-chain for another. iron atom of the heme prosthetic group. This All individuals affected with CMO II deficiency residue is completely conserved in all cytochrome were homozygous for both of these mutations, P450 enzymes and the surrounding 'heme-binding whereas no unaffected individuals carried both peptide' is also highly conserved. In particular, mutations; individuals homozygous for either one Arg-448 is conserved in all eukaryotic P450 of the mutations alone were asymptomatic. enzymes examined thus far (see69) suggesting When normal and mutant P450cmo were that substitutions at this position are poorly expressed in cultured cells, the R181W mutant tolerated. Thus, it is reasonable to speculate had normal lip-hydroxylase activity, decreased that the Arg-448—»His mutation interferes with 18-hydroxylase activity and undetectable 18- binding or functioning of the heme functional oxidase activity. The V386A mutant had slightly group. In unpublished studies, we found that decreased activity as compared with the normal this mutation indeed abolished normal enzymatic enzyme. No differences could be demonstrated activity. between the enzymes carrying R181W alone and Although patients in five out of six families in in combination with V386A, but the studies on this study were presumably genotypically identi- patients suggest that the double mutant enzyme cal, there were significant differences in signs must have even more severely compromised 18- and symptoms of androgen and mineralocorticoid oxidase activity than the enzyme carrying R181W excess, even within families. For example, all alone. affected females were born virilized, but only It is likely that the results presented here for five out of seven males had an abnormally large both lip-hydroxylase and CMO II deficiencies penis in infancy. Only eight out of eleven patients reflect founder effects. There was relatively were hypertensive when untreated. Thus, as is little intermarriage in many relatively small the case with 21-hydroxylase deficiency, other Sephardic (nonEuropean) Jewish communities epigenetic or nongenetic factors probably influ- prior to emigration to Israel, and so genetic ence the clinical phenotype of the disorder. heterogeneity at certain loci may be limited.

Corticosterone methyloxidase II deficiency Types of mutations observed in the CYP11B genes CMO II deficiency is an inherited defect of Steroid 21-hydroxylase deficiency, the most aldosterone biosynthesis.70 Patients with this common cause of congenital adrenal hyper- disorder are subject to potentially fatal electrolyte plasia, is due to mutations in the CYP21 gene abnormalities as neonates and a variable degree encoding the enzyme P450c21 (P450XXI). CYP21 of hyponatraemia and hyperkalaemia combined and a 98% identical pseudogene, CYP21P, are with poor growth in childhood, but they may closely linked on chromosome 6p21.3 in the have no symptoms as adults. Asymptomatic major histocompatibility complex. Most reported adults with this disorder have been ascertained mutations causing 21-hydroxylase deficiency are in the course of family studies because affected apparently the result of recombinations between individuals invariably have an elevated ratio of CYP21 and CYP21P. These are either deletions 18-hydroxycorticosterone to aldosterone, which of CYP21 due to unequal meiotic crossing-over has been presumed to reflect a block in the final (approximately 20% of alleles), or apparent step of the biosynthetic pathway.71 gene conversions in which deleterious mutations normally present in CYP21P are transferred to CMO II deficiency was found to be geneti- 74 cally linked to a unique Msp I polymorphism CYP21 (reviewed in ). in CYPIIB1, whereas two missense mutations Like CYP21 and CYP21P, CYP11B1 and were identified in CYP11B2.73 The first of these, CYP11B2 are closely linked homologs, but in exon 3, codon 181, is CGG (arginine) —» TGG CYP11B1 and CYP11B2 both encode active Congenital adrenal hyperplasia 9 enzymes. Thus, gene conversions that trasnfer Analysis of two additional patients showed polymorphic sequences between CYP11B1 and a four basepair duplication in exon 8 which CYP11B2 might not be expected to have major caused abolition of enzymatic activity for both adverse effects on enzymatic activity, in which 17a-hydroxylase and 17,20-lyase in transfection case genetic deficiencies of the encoded enzymes studies. Homozygous deletion of three basepairs (P450cll and P450cmo) should be the result of in exon 1 in another case resulted in a gene mutations that are not gene conversions. Indeed, product with partial activity, the 17,20-lyase we have characterized three CYP11B1 mutations being more severely compromised. Interestingly, causing lip-hydroxylase deficiency in addition to this patient was a genetic and phenotypic R448H, and all are de novo point mutations female with sexual infantilism, as one would or small insertions and not large deletions or predict from the inability to synthesize sex gene conversions (unpublished observations). steroids. Finally, one genetic male with ambigu- Similarly, the R181W mutation in CYP11B2 ous genitalia had a different mutation on each associated with CMO II deficiency is also a chromosome: a stop codon introduced by a simple point mutation (like R448H, it is a single base substitution in exon 4; and a mutation of CpG to TpG, the most common nonconservative proline—»threonine substitution type of point mutation in higher eukaryotes75). In in exon 6. Expression studies in this last case contrast, V386A is normally present in CYP11B1 showed equivalently decreased activity of both and thus its presence in the mutant CYP11B2 17a-hydroxylase and 17,20-lyase. genes of CMO II deficiency patients may be the result of an ancestral gene conversion, although 3|i-ol dehydrogenase an independent mutation is also possible. As Deficiency of 3f3-ol dehydrogenase is not predicted, V386A itself has a minimal effect on linked to the HLA complex.85 In contrast with enzymatic activity. the other adrenal steroidogenic enzymes, the 3p-ol dehydrogenase enzyme, a dehydrogenase Steroid 17a-hydroxylase/17,20-lyase typically requiring NAD+ as a cofactor, is not The P450cl7 structural gene (CYP17) has been a cytochrome P450. The enzyme is classed as a located on chromosome 10,76 but thus far has short-chain dehydrogenase which is similar to not been regionally localized. Apparently, the the genes encoding the 170- and llp-hydroxy- same gene is expressed in both the adrenal steroid dehydrogenases.86 Closely associated with and the testis.77 Initial hybridization studies of 33-ol dehydrogenase is the enzyme activity DNA samples from patients with 17a-hydroxylase 3-ketosteroid A5-4 isomerase, which requires deficiency did not disclose the presence of any NAD+ or NADH.87 In mammalian species gross deletions or rearrangements of this gene.78 these two functions appear to reside within the Further molecular characterization of specific same protein, but the enzyme generally has not mutations in the structural gene coding for been well characterized. the P450cl7 enzyme have been reported in a Two genes encoding human 3p-ol dehydro- number of patients.79-84 Details concerning these genase/ A5-^ isomerase have been cloned.88-90 The molecular genetic studies are reviewed in19. In deduced amino acid sequence is 94% homologous brief, no deletions or major gene rearrangements between the Type I gene expressed in placenta were seen on southern blots of affected patients. and skin, and the Type II gene expressed in In all cases, mutations in the structural gene have adrenal and gonads. Structural gene mutations been found. These have included: a stop codon have recently been described in patients with introduced in the first exon by a single base clinically apparent classical 3p-ol dehydrogenase substitution, and in a separate case, a seven deficiency: one patient with familial hypospadias basepair duplication in exon 2 which shifts the and urethral diverticula91 had a Type II gene reading frame of translation and introduces a paternal missense mutation, Tyr-253—>Asn, and stop codon downstream. These patients were a maternal frameshift mutation, a C insertion homozygous for these two mutations, respec- between codons 186-187. In a second patient tively, and thus they had complete combined with salt-wasting and hypospadias,92 only the 17a-hydroxylase and 17,20-lyase deficiencies. frameshift could be detected in the Type 10 PW Speiser et al.

II maternally inherited gene. This suggests Berlin: Springer-Verlag, 1984: 660. that perhaps the second mutation involves 5' 10 Awdeh ZL, Raum D, Yunis EJ, Alper CA. Extended regulatory elements, which have not yet been HLA complement allele haplotypes: evidence for well-characterized.91 T/t-like complex in man. Proc NatlAcad Sci USA 1983; 80: 259-63. 11 Speiser PW, Dupont B, Rubinstein P etal. High Cholesterol desmolase (P450scc) frequency of nonclassical steroid 21-hydroxylase The gene (CYP11A) for this mitochondrial deficiency. Am J Hum Genet 1985; 37: 650. P450 enzyme (P450scc) has been isolated, cloned 12 Pang S, Hotchkiss J, Drash AL, Levine LS, New and localized to chromosome 15.93 Mutation of MI. Microfilter paper method for 17a-progesterone the structural gene has not yet been identified in radioimmunoassay: its application for rapid screening 94 95 for congenital adrenal hyperplasia. J Clin Endocrinol lipoid adrenal hyperplasia ' although in vitro Metab 1977; 45: 1003-1008. studies suggest that the 20a-hydroxylase function 13 Pang S, Murphey W, Levine LS et al. A pilot is deficient in at least one patient with the newborn screening for congenital adrenal hyperplasia syndrome. Remote lesions affecting other cellular (CAH) in Alaska. / Clin Endocrinol Metab 1982; 55: components fundamental to early steroidogenesis 413-20. could have similar effects. 14 Pang SP, Wallace MA, Hofman L etal. Worldwide experience in newborn screening for classical congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Pediatrics 1988; 81: 866-74. 15 New MI, Lorenzen F, Lerner AJ etal. Genotyping References steroid 21-hydroxylase deficiency: hormonal reference data. J Clin Endocrinol Metab 1983; 57: 320-26. 1 New MI, White PC, Pang S, Dupont B, Speiser PW. 16 Sherman SL, Aston CE, Morton NE, Speiser PW, The adrenal hyperplasias. In: Scriver CR, Beaudet New MI. A segregation and linkage study of classical AL, Sly WS, Valle D eds. The metabolic basis of and nonclassical 21-hydroxylase deficiency. Am J inherited disease, sixth edition. New York: McGraw- Hum Genet 1988; 42: 830-38. Hill, 1989:1881-917. 17 Zachmann M, Tassinari D, Prader A. Clinical 2 White PC, New MI, Dupont B. Congenital adrenal and biochemical variability of congenital adrenal hyperplasia. N EnglJ Med 1987; 316: 1519 and 1580. hyperplasia due to HB-hydroxylase deficiency. / Clin 3 Knudson AG Jr. Mixed adrenal disease of infancy. J Endocrinol Metab 1983; 56: 222-29. Pediatr 1951; 39: 408-23. 18 Rosier A. Classic and nonclassic congenital adrenal 4 Bentinck RC, Hinman F Sr, Lisser H et al. The hyperplasia among non-Askenazi Jews. In: Bonne- familial congenital adrenal syndrome: report of two Tamir B, Adams A eds. Genetic diversity among Jews. cases and review of the literature. Postgrad Med 1952; Diseases and markers at the DNA level. London: 11: 301-12. Oxford University Press, 1992: 283-97. 5 Childs B, Grumbach MM, Van Wyk JJ. Virilizing 19 Yanase T, Simpson ER, Waterman MR. adrenal hyperplasia: a genetic and hormonal study. J 17ot-hydroxylase/17,20-lyase deficiency: from clinical Clin Invest 1956; 35: 213-22. investigation to molecular definition. Endocr Rev 6 Dupont B, Oberfield SE, Smithwick EM etal. Close 1991; 12: 91-108. genetic linkage between HLA and congenital adrenal 20 Zachmann M, Vollmin JA, Hamilton W, Prader A. hyperplasia (21-hydroxylase deficiency). Lancet 1977; Steroid 17,20-desmolase deficiency: a new cause of ii: 1309-12. male pseudohermaphroditism. Clin Endocrinol 1972; 7 Levine LS, Zachmann M, New MI etal. Genetic 1: 369-85. mapping of the 21-hydroxylase deficiency gene within 21 Zachmann M, Werder EA, Prader A. Two types of the HLA linkage group. N EnglJ Med 1978; 299: male pseudohermaphroditism due to 17,20-desmolase 911-15. deficiency. J Clin Endocrinol Metab 1982; 55: 487-90. 8 Dupont B, Pollack MS, Levine LS etal. Congenital 22 Forest MG, Lecornu M, dePeretti E. Familial male adrenal hyperplasia and HLA: joint report from the pseudohermaphroditism due to 17,20-desmolase Eighth International Histocompatibility Workshop. deficiency. I: In vivo endocrine studies. / Clin In: Terasaki PI ed. Histocompatibility testing 1980. Endocrinol Metab 1980; 50: 826. Los Angeles: HLA Tissue Typing Laboratory, 1981: 23 Goebelsmann U, Zachmann M, Davajan V, 693. Israel R, Mestman JH, Mishell DR. Male 9 Dupont B, Virdis R, Lerner AJ etal. Distinct HLA-B pseudohermaphroditism consistent with 17-20 antigen associations for the salt-wasting and simple desmolase deficiency. Gynecol ObstetInvest 1976; 7: virilizing forms of congenital adrenal hyperplasia due 138-56. to 21-hydroxylase deficiency. In: Albert ED, Baur 24 David M, Forest MG, Zachmann M, DePeretti MP, Mayr WR eds. Histocompatibility testing 1984. E. 17,20-Desmolase deficiency in two unrelated Congenital adrenal hyperplasia 11

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