Scholarly article on topic 'Congenital adrenal hyperplasia'

Congenital adrenal hyperplasia Academic research paper on "Biological sciences"

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Reproductive Medicine Review
OECD Field of science

Academic research paper on topic "Congenital adrenal hyperplasia"

Reproductive Mediane Review 1993 ; 2: 1-13


Congenital adrenal hyperplasia

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


Congenital adrenal hyperplasia (CAH) is a group of diseases which result from reduced or absent activity of one of the five enzymes of Cortisol synthesis in the adrenal cortex. Each enzyme deficiency produces characteristic alterations in the levels of adrenal steroid hormones and their precursors. The particular hormonal imbalances cause a spectrum of abnormalities including abnormal fetal genital development and pseudohermaphroditism, disturbances in sodium homeostasis and blood pressure regulation, and specific metabolic disturbances.

The following enzymatic defects of steroidogenesis and their associated clinical syndromes have been described1-2:

a) 21-hydroxylase deficiency: classical (salt-wasting and simple virilizing) and non-classical;

b) lip-hydroxylase deficiency (hypertensive CAH) with corticosterone methyl oxidase (CMO) types I and II (salt-wasting);

Address for correspondence: Phyllis W Speiser, Department of Pediatrics, Division of Pediatric Endocrinology, The New York Hospital-Cornell Medical Center, 525 East 68th Street, New York, NY 10021, USA.

© Edward Arnold 1993

c) 17a-hydroxylase deficiency and/or 17,20-lyase deficiency;

d) 3p-ol dehydrogenase deficiency (classical and nonclassical) ;

e) cholesterol desmolase deficiency (lipoid hyperplasia).

The 21-hydroxylase and 11 ^-hydroxylase deficiencies, occurring late in Cortisol synthesis, cause shunting of accumulating precursor steroids into androgen pathways, resulting in genital ambiguity in newborn females and later hyperandrogenic effects in both sexes. Accompanying the virilizing effects, imbalances in salt metabolism distinguish these two forms: in 21-hydroxylase deficiency, deficient aldosterone synthesis causes salt-wasting and hypovolaemia in 75% of cases; whereas excess secretion of the mineralocorticoid agonist deoxycorticosterone (DOC) or its metabolites in llp-hydroxylase deficiency causes sodium retention and hypertension.

In the more proximal 17a-hydroxylase/17,20-lyase deficiency, blocked production both of 17a-hydroxy (glucocorticoid) and of C19/C18 steroids causes pseudohermaphroditism in males and sexual infantilism in females. Shunting of P450cl7 precursor steroids into the 17-deoxy pathway produces mineralocorticoid excess and

hypertension. The 17,20-lyase deficiency is a variant of 17a-hydroxylase deficiency in which glucocorticoid and mineralocorticoid levels are relatively unaffected and a block in adrenal and gonadal C21 to C19 steroid conversion results in a specific clinical phenotype.

In the 3p-ol dehydrogenase defect, poor or absent conversion to A4-steroids allows production only of the A5-steroid precursors which are relatively inactive, causing salt wasting and Cortisol insufficiency. While lack of potent A4-androgens produces hypospadias in the male, enormously high levels of the weak androgen DHEA may cause limited virilization (clitoral enlargement) in females.

Cholesterol desmolase deficiency blocks all steroid production, with build-up of cholesterol substrate (lipoid adrenal hyperplasia), and accordingly, among its very serious effects, results in pseudohermaphroditism in genetic males.

Sexual ambiguity is not a feature of the 18-hydroxylase (CMO I) or 18-dehydrogenase (CMO II) deficiencies causing hypotension, since these distal blocks in aldosterone synthesis do not alter the normal synthesis of Cortisol, and thus cause no disturbance of the hypothalamic-pituitary-adrenal axis.

Classical genetics in CAH

The autosomal recessive mode of genetic transmission of various forms of adrenal steroidogenic defects was suspected in the early 1950s,3-5 but it was not until the late 1970s that the genetics of the most common form of CAH, 21-hydroxylase deficiency, began to be unravelled. Linkage between the human major histocompatibility complex (MHC), or HLA, located on the short arm of chromosome 6 (between subregions 6p21.1 and 6p21.3) and 21-hydroxylase deficiency was first shown by Dupont et al.6 Initial calculated LOD scores demonstrating linkage were only slightly higher than the requisite 3.00, but recent calculated LOD scores are in excess of 22. Compiled data on intra-HLA recombinations strongly indicated a gene locus for 21-hydroxylase between HLA-B and -DR.7-8 The more recent molecular studies have confirmed this location in the class III region of the MHC.

The knowledge of close genetic linkage of the

21-hydroxylase gene to HLA was utilized in genotyping sibs in pedigrees with an affected index case. Thus, a sib sharing both HLA haplotypes with the index case is predicted to be affected, one who shares a single haplotype is predicted to be a heterozygote, and one who shares no HLA haplotype is predicted to be unaffected. Although this system of genetic counselling was imperfect, with a ~1% recombination rate between the HLA-B or -DR and the 21-hydroxylase locus, serotyping for HLA served as a useful adjunct to amniotic fluid hormonal measurements for the purposes of prenatal diagnosis before the advent of more specific molecular genetic testing.

Linkage disequilibrium

In addition to linkage of the 21-hydroxylase locus with the neighbouring HLA-B and -DR antigen loci, 21-hydroxylase deficiency alleles are found in linkage disequilibrium with HLA antigen genes or haplotypic combinations9 that may include specific alleles of C4 of serum complement.10 The two most prominent such cases are linkage disequilibrium of the extended haplotype HLA-A3,Bw47,DR7 with the salt-wasting form of 21-hydroxylase deficiency, and of the haplotype HLA-A1, B14,DR1 with non-classical 21-hydroxylase deficiency.11

Epidemiology of classical 21-hydroxylase deficiency

A reliable and valid screening test for 21-hydroxylase deficiency CAH using a heel-prick capillary blood specimen impregnated on filter paper first became available in 1977.12 A pilot newborn screening programme among the Alaskan Yup'ik Eskimos at high-risk for 21-hydroxylase deficiency CAH first demonstrated the feasibility of an effective newborn screening programme for 21-hydroxylase deficiency CAH.13 The direct benefit of 21-hydroxylase deficiency CAH screening from this programme - such as avoidance of adrenal crisis, shock, its sequelae and death - promted further development of newborn screening programmes for 21-hydroxylase deficiency CAH in various nations.14 The world-wide incidence of 21-hydroxylase deficiency CAH as determined by these screening programmes is 1:14 554 live births, of which approximately 75% are of the

salt-wasting phenotype.14 Applying the Hardy-Weinberg Law, the heterozygote frequency for all classical 21-hydroxylase gene defects is thus 1:61 persons.

Population genetics of nonclassical 21-hydroxylase deficiency

A high frequency of occurrence for nonclassical 21-hydroxylase deficiency has been determined in a number of ethnic groups.11 In this analysis, heterozygote (carrier) and (homozygous) affected status were established in family members by HLA typing, correlating HLA-B types with known HLA-B associations, in conjunction with ACTH testing using criteria provided by reference hormone data.15 By counting the incidence of nonclassical deficiency genes relative to the presumed normal genes among allowed parental haplotypes, the frequency of nonclassical 21-hydroxylase deficiency was calculated. The gene frequency for nonclassical 21-hydroxylase deficiency was highest in Ashkenazic Jews and was also high in Hispanics, Yugoslavs and Italians. Disease frequencies were 0.037 (1/27) 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 (40% of whom had Anglo-Saxon background).11 Confirmation of these data was obtained employing the statistical method of commingling distributions of an expanded database including the same families.16 Nonclassical 21-hydroxylase deficiency is thus among the most frequent autosomal recessive disorders in man.

Epidemiology of other defects of steroidogenesis

Classical ll(3-hydroxylase (P450cll) deficiency comprises 5-8% of cases, occurring in about 1/100 000 births in the general Caucasian population.17 A large number of cases have been reported in Israel among Jewish immigrants from Morocco, a relatively inbred population. The incidence in this group is currently estimated to be 1/5000-1/7000 births, with a gene frequency of 1.2-1.4%.18 CMO II deficiency, representing a defect in the terminal step of aldosterone synthesis, is apparently rare in the general population, but it has been found at an increased frequency among Jews of Iranian origin.

Over 120 cases have been reported of 17a-hydroxylase deficiency, mostly in combination

with 17,20-lyase deficiency (reviewed in19). Of the genetic males reported with this enzyme deficiency, several are from the same three kindreds.20-27 One genetic female has been detected.28

First described by Bongiovanni in 1962,29 3(3-HSD deficiency seemed most likely to have a monogenic autosomal recessive mode of transmission based on pedigree analysis.29"31 The exact frequency of this disorder is unknown; it is conceivable that affected individuals with early steroidogenic defects which severely impair Cortisol synthesis, for example, in 3(3-hydroxysteroid dehydrogenase and side-chain cleavage, are poorly viable.

Deficiency of side-chain cleavage or cholesterol desmolase first described by Prader32-33 is extremely rare. A recent clinical case report describes a patient diagnosed in the newborn period and successfully treated for 18 years, and also reviews 32 cases from the literature34; cholesterol desmolase deficiency seems to occur with less severity and somewhat more frequently among Japanese.

Molecular genetics in CAH

Gene structure and molecular pathology

21-hydroxylase deficiency is inherited as a monogenic autosomal recessive trait closely linked to the HLA major histocompatibility complex on chromosome 6p23. The structural gene encoding P450c21 (CYP21 or CYP21B) and a 98% identical pseudogene (CYP21P or CYP21A) are located in the HLA complex adjacent to and alternating with the C4B and C4A genes encoding the fourth component of serum complement.35-36 The CYP21P pseudogene has accumulated a number of mutations that render the putative gene product completely inactive. The close proximity between CYP21 and CYP21P (30 Kb) appears to generate frequent mutations in CYP21 by two mechanisms: unequal crossing-over during meiosis results in a complete deletion of a DNA segment of about 30 kilobases (kb) in length including the C4B and CYP21 genes C4B and CYP21; gene conversion events result in the transfer of small, deleterious mutations, often single base changes, from CYP21P to CYP21.37

Early efforts to genotype 21-hydroxylase

deficient patients relied on Southern blotting, in which relatively large gene-specific DNA probes are hybridized against genomic DNA digested with restriction endonucleases capable of discriminating between the CYP21 and CYP21P genes. This approach is potentially inaccurate when hybrid CYP21/CYP21P genes are formed as a result of crossover events. Careful studies employing multiple informative restriction digests (such as Taq I and Bgl II) and oligonucleotide hybridizations can resolve such ambiguities.38 The availability of pulse field gel electrophoresis used in conjunction with rare-cutting endonucleases such as Bss HII has permitted long range mapping of the MHC in the vicinity of the C4 and CYP21 genes.39 Based on a number of studies in different ethnic populations, the frequency of gene deletion causing 21-hydroxylase deficiency ranges from 11-35% (reviewed in40), and is highest in northern European populations which carry the HLA-B47;DR7 haplotype at a high frequency.

Analysis of the remaining 65-90% of disease haplotypes without obvious gene deletions has revealed small mutations attributable to gene conversion. There are eleven missense mutations in CYP21P which have been observed in patients with 21-hydroxylase deficiency. Most of these are presumed to have arisen from gene conversion events (Figure 1). These mutations differ in their associations with phenotypic forms of 21-hydroxylase deficiency.

Mutations associated with nonclassical 21-hydroxylase deficiency

Val-281—»Leu, a single base change (G—>T) in a highly conserved subregion of the seventh exon resulting in a conservative amino acid substitution, has been identified in 75-80% of nonclassical haplotypes carrying the HLA-B14;DR1 haplotype.4142 A change of a proline to leucine at residue 30 in exon 1 was recently found

Figure 1 7op; 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 the CYP21A pseudogene are 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)

in 16% of haplotypes in nonclassical patients who carried at least one non-B14 haplotype.43

Mutations associated with simple virilizing 21-hydroxylase deficiency

Ile-172—>-Asn, a single base change (T—>A) in exon 4, results in substitution of a polar amino acid for a highly conserved nonpolar residue. This mutation has been described in patients with the simple virilizing form44 of the disease, and in 6-14% of classical disease alleles.

Mutations associated with salt-wasting 21-hydroxylase deficiency

An A—>G substitution near the 3' end of the second intron activates a novel splice acceptor site and shifts the reading frame of translation. The erroneously processed mRNA yields a truncated protein with no enzymatic activity. In one study this mutation accounted for 57% of nondeletional alleles.45 It appears that mRNA splicing with this mutation is variable, however, since the mutation has been found in patients with the severe salt-wasting as well as with the less severe simple virilizing pheno types.

A cluster of mutations, Ile-Val-Glu-Met-235-238—>Asn-Glu-Glu-Lys,46 and a single substitution, Arg-356—»Trp,47 have been described in patients with salt-wasting disease. A stop codon introduced into the eighth exon by virtue of C—>T substitution48 is seen in 4-7% of classical 21-hydroxylase deficiency haplotypes, and the 8-basepair deletion in exon 3, also a salt-wasting mutation, is found in 3-10% of disease haplotypes. The only gene conversion which has not as yet been detected in a patient with 21-hydroxylase deficiency is a T insertion in the seventh exon, expected to cause a disruption of the translational reading frame.

Functional analysis of mutations

Because synthesis of P450c21 is restricted to the adrenal gland, the effect of missense mutations on the enzymatic activity of P450c21 cannot be directly tested in affected patients. Therefore, a number of attempts have been made to measure the activities of mutant enzymes in cultured cells. In one such study, transfected normal and mutated CYP21 genes or P450c21 cDNA in plasmid vectors containing a strong promoter to allow expression in COS cells.46 These studies

showed that the cluster of mutations in codons 235-238 and the Arg-356—>Trp mutation resulted in enzymes with no detectable activity, whereas trace activity was detected in the enzyme carrying the Ile-172—>Asn mutation. However, these studies did not attempt quantitation of activity, possibly because levels of expression were low.

In order to address this problem, several mutant enzymes were synthesized at higher levels in cultured cells using recombinant vaccinia virus.43-49 The enzyme with the cluster of substitutions in codons 235-238 again had no activity even when expressed at higher levels. When 17-hydroxyprogesterone was the substrate, the enzyme carrying Ile-172—>Asn had an activity of 0.6% of normal as measured by the firstorder rate constant, V^/Kj,,. The Val-281—>Leu mutation resulted in an enzyme with 50% of normal activity when 17-hydroxyprogesterone was the substrate but only 20% of normal activity for progesterone. The Pro-30—»Leu mutation had 60% of wild type activity for 17-hydroxyprogesterone and 30% activity for progesterone when assayed in intact cells. The activity of enzyme expressed after transfection of a CYP21 gene carrying a substitution of Thr for Ser-268 was no different from that of the wild type enzyme, thus confirming earlier reports that the latter represents a normal polymorphism of CYP21.50 The latter mutation has been detected incidentally in CAH haplotypes carrying a second severe mutation, the A—>G mutation in the second intron which introduces a novel splice acceptor site, and disrupts the reading frame of translation.51

The main conclusion to be drawn from the foregoing studies is that mutant P450c21 enzymes carrying specific amino acid substitutions seen in patients with 21-hydroxylase deficiency exhibit activities that usually correlate with the clinical severity of the disease when present in the homozygous or hemizygous (i.e. with deletion of CYP21 in trans) state, and with biochemical abnormalities such as 17-hydroxyprogesterone levels after ACTH (corticotropin) stimulation.

Further correlation of mutation with phenotype: salt-wasting versus simple virilizing phenotypes

An important facet of the clinical variability of 21-hydroxylase deficiency concerns the ability to synthesize adequate amounts of the

mineralocorticoid hormone aldosterone. Because aldosterone is normally secreted at a rate 100-1000 times lower than that of Cortisol, it is apparent that 21-hydroxylase activity would have to decrease to very low levels before it became rate-limiting. Apparently, as little as 0.6% of normal activity, as seen in the enzyme carrying the Ile-172—»Asn mutation, allows adequate aldosterone synthesis to prevent significant salt wasting, thus resulting in the 'simple virilizing' phenotype. In contrast, mutations that completely destroy enzymatic activity are associated with salt-wasting. It should be noted, however, that the distinction between the simple virilizing and salt-wasting phenotypes is not absolute. One patient with the Ile-172—»Asn mutation has been reported to have an elevated ratio of plasma renin to aldosterone, consistent with mild salt-wasting,14 and HLA-identical sib pairs have been reported in which one sib has salt-wasting disease whereas the other can synthesize adequate amounts of aldosterone.52

The mechanism of occasional recovery from salt-wasting52-55 is not known. A study correlating clinical, biochemical and molecular genetic findings in five patients who had severe salt-wasting 21-hydroxylase deficiency in infancy showed that all but one patient had persistent impairment of aldosterone synthesis associated with homozygous deletion or equivalently severe mutations in CYP21B.56 The fact that one patient had amelioration of aldosterone deficiency with age and the variable responses to sodium deprivation in three patients with identical genotypes indicate that nongenetic factors contribute to phenotype in this form of congenital adrenal hyperplasia. Based on data gleaned from radiolabeled progesterone infusion, it appears that recovery from salt-wasting may be explained by activity of an adrenal P450 enzyme other than P450c21.56

Nonclassical phenotype

As mentioned, the Val-281—»Leu mutation results in an enzyme with about 50% of normal activity when 17-hydroxyprogesterone is the substrate but only about 20% of normal activity for progesterone. An individual homozygous for this mutation has nonclassical 21-hydroxylase deficiency with significant biochemical abnormalities and variable symptoms of androgen

excess. A heterozygous carrier of a salt-wasting mutation might also be expected to have about 50% of normal 21-hydroxylase activity, but such individuals are asymptomatic and have minimal biochemical abnormalities. This suggests that in vivo 21-hydroxylase deficiency must actually be less than 50% of normal. This apparent paradox is resolved by the finding that progesterone at physiologic intra-adrenal concentrations (2-4 /xM)57 acts as a powerful competitive inhibitor of the mutant 21-hydroxylase enzyme for its main substrate, 17-hydroxyprogesterone. Thus, relatively small differences in intra-adrenal progesterone concentration could account for much of the clinical variability that is a hallmark of nonclassical 21-hydroxylase deficiency. Another contributing factor to phenotypic variability is undoubtedly pseudosubstrate inhibition of other steroidogenic enzymes by accumulated precursors of 21-hydroxylase; this phenomenon probably accounts for reports of multiple enzyme deficiencies.58

Further phenotype-genetype correlations

We have genotyped a group of patients followed at our institution who have been thoroughly clinically characterized. Presence of one or more of ten mutations in the CYP21 gene was performed using Southern blot analysis to detect CYP21 deletions or large gene conversions, and allele specific hybridizations with DNA amplified by the polymerase chain reaction to detect smaller mutations. Mutations were detected on 95% of chromosomes examined. The most common mutations were: an A—*G change in the second intron affecting pre-mRNA splicing (26%), large deletions (21%), Ile-172—»Asn (16%), and Val-281->Leu (11%). Patients were classified into three mutation groups based on degree of predicted enzymatic compromise. Mutation groups were correlated with clinical diagnosis and specific measures of in vivo 21-hydroxylase activity such as 17-hydroxyprogesterone, aldosterone and sodium balance. Mutation group A (no enzymatic activity) consisted principally of salt-wasting (severely affected) patients, group B (2% activity) of simple virilizing patients, and group C (10-20% activity) of nonclassical (mildly affected) patients, but each group contained patients with phenotypes either more or less severe than predicted. (Nonclassic

patients were deliberately under-represented in this study.) These data suggest that most but not all of the phenotypic variability in 21-hydroxylase deficiency results from allelic variation in CYP21.59 Accurate prenatal diagnosis is now possible in most cases using the described strategy.

Steroid lip-hydroxylase

There are two human genes60 on chromosome 8q21-q226162 that encode ll(3-hydroxylase (P450cll) isozymes with predicted amino acid sequences that are 93% identical. One gene, CYP11B1, is expressed at high levels in normal adrenal glands,60 and transcription of this gene is appropriately regulated by cAMP (the second messenger for ACTH).63 Transcripts of the other gene, CYP11B2, cannot be detected by hybridization to Northern blots of normal adrenal RNA,60 but such transcripts have been detected by hybridization to RNA from an aldosterone-secreting tumour.64

CYP11B2 transcripts have been detected in normal adrenal RNA using a more sensitive assay wherein RNA was reverse-transcribed and then amplified using the polymerase chain reaction (RT-PCR).65 Whereas an amplified product corresponding to transcripts of CYP11B1 was detected after 20 PCR cycles in samples from normal adrenal glands, the CYP11B2 product was visible after 30 cycles by staining with ethidium bromide. When an RNA sample of an aldosterone-secreting adrenal tumour was examined in this manner, it contained a concentration of CYP11B1 transcripts slightly lower than that of the normal adrenal, but CYP11B2 transcripts were increased fivefold over the normal gland.

To determine if levels of CYP11B2 transcripts were appropriately regulated, the zone glo-merulosa was dissected out of human adrenal surgical specimens and cultured in the presence of angiotensin II or corticotropin (ACTH) before preparing RNA. Angiotensin II markedly increased levels of both CYP11B1 and B2 transcripts. ACTH increased CYP11B1 mRNA levels more effectively than angiotensin II, but it had no effect on CYP11B2 transcription.

The enzymes encoded by the CYP11B1 and CYP11B2 genes have been studied by expressing the corresponding cDNAs in cultured cells64 65

and after actual purification from aldosterone secreting tumours.66 The isozyme encoded by CYP11B2, termed P450XIB2 (or P450cmo or P450aldo), lip-hydroxylates 11-deoxycorticosterone to corticosterone and 11-deoxycortisol to Cortisol. It 18-hydroxylates corticosterone and Cortisol, and further oxidizes 18-hydroxycorticosterone to aldosterone. In contrast, the product of CYP11B1, termed P450XIB1 (or P450cll), has a strong 11(3-hydroxylase activity but 18-hydroxylates only about one-tenth as well as P450XIB2. P450XIB1 does not synthesize detectable amounts of aldosterone from 18-hydroxycorticosterone.

These data suggest that P450XIB1 synthesizes Cortisol in the zona fasciculata whereas P450XIB2 synthesizes aldosterone in the zona glomerulosa. This hypothesis has been confirmed by studying individuals with defective Cortisol or aldosterone synthesis due to respective deficiencies in 11(3-hydroxylase and corticosterone methyloxidase II (CMOII) activities.

Patients with 11-hydroxylase deficiency are unable to convert 11-deoxycortisol to Cortisol. Elevated levels of ACTH cause steroid precursors to accumulate proximal to the blocked step. Many of these precursors are shunted into the pathway for androgen biosynthesis as occurs in 21-hydroxylase deficiency. Thus, female patients with this disorder are born with masculinized external genitalia, and affected individuals of both sexes undergo rapid somatic growth with premature epiphyseal closure, resulting in short adult stature.

A parallel defect usually exists in the synthesis of 17-deoxy steroids, so that deoxycorticosterone is not converted to corticosterone and instead accumulates. Because deoxycorticosterone and some of its metabolites have mineralocorticoid activity, elevated levels may cause hypertension and hypokalaemia. About two-thirds of untreated patients become hypertensive, sometimes early in life.67 This clinical feature distinguishes 11 ^-hydroxylase deficiency from 21-hydroxylase deficiency, in which poor aldosterone synthesis causes renal salt-wasting in the majority of patients.

In an analysis of mutations causing this disorder,68 six families carrying an allele for 11(3-hydroxylase deficiency were studied; all were Jews originating from Morocco. Eleven of twelve

mutant alleles carried the same mutation, a single base change in exon 8 of CYP11B1. Codon 448, CGC, encoding arginine, was changed to CAC, histidine (R448H).

The sulfhydryl of Cys-450 in P450cll is presumed to constitute the fifth ligand to the iron atom of the heme prosthetic group. This residue is completely conserved in all cytochrome P450 enzymes and the surrounding 'heme-binding peptide' is also highly conserved. In particular, Arg-448 is conserved in all eukaryotic P450 enzymes examined thus far (see69) suggesting that substitutions at this position are poorly tolerated. Thus, it is reasonable to speculate that the Arg-448—»His mutation interferes with binding or functioning of the heme functional group. In unpublished studies, we found that this mutation indeed abolished normal enzymatic activity.

Although patients in five out of six families in this study were presumably genotypically identical, there were significant differences in signs and symptoms of androgen and mineralocorticoid excess, even within families. For example, all affected females were born virilized, but only five out of seven males had an abnormally large penis in infancy. Only eight out of eleven patients were hypertensive when untreated. Thus, as is the case with 21-hydroxylase deficiency, other epigenetic or nongenetic factors probably influence the clinical phenotype of the disorder.

Corticosterone methyloxidase II deficiency

CMO II deficiency is an inherited defect of aldosterone biosynthesis.70 Patients with this disorder are subject to potentially fatal electrolyte abnormalities as neonates and a variable degree of hyponatraemia and hyperkalemia combined with poor growth in childhood, but they may have no symptoms as adults. Asymptomatic adults with this disorder have been ascertained in the course of family studies because affected individuals invariably have an elevated ratio of 18-hydroxycorticosterone to aldosterone, which has been presumed to reflect a block in the final step of the biosynthetic pathway.71

CMO II deficiency was found to be genetically linked to a unique Msp I polymorphism in CYPIIB1, whereas two missense mutations were identified in CYP11B2.73 The first of these, in exon 3, codon 181, is CGG (arginine) —» TGG

(tryptophan) (R181W), a substitution of an amino acid with a large nonpolar group for a basic amino acid, whereas the second in exon 7, codon 386, is GTG (valine) GCG (alanine) (V386A), a more conservative substitution of one amino acid with a nonpolar side-chain for another. All individuals affected with CMO II deficiency were homozygous for both of these mutations, whereas no unaffected individuals carried both mutations; individuals homozygous for either one of the mutations alone were asymptomatic.

When normal and mutant P450cmo were expressed in cultured cells, the R181W mutant had normal 11 (3-hydroxylase activity, decreased 18-hydroxylase activity and undetectable 18-oxidase activity. The V386A mutant had slightly decreased activity as compared with the normal enzyme. No differences could be demonstrated between the enzymes carrying R181W alone and in combination with V386A, but the studies on patients suggest that the double mutant enzyme must have even more severely compromised 18-oxidase activity than the enzyme carrying R181W alone.

It is likely that the results presented here for both lip-hydroxylase and CMO II deficiencies reflect founder effects. There was relatively little intermarriage in many relatively small Sephardic (nonEuropean) Jewish communities prior to emigration to Israel, and so genetic heterogeneity at certain loci may be limited.

Types of mutations observed in the CYP11B genes

Steroid 21-hydroxylase deficiency, the most common cause of congenital adrenal hyperplasia, is due to mutations in the CYP21 gene encoding the enzyme P450c21 (P450XXI). CYP21 and a 98% identical pseudogene, CYP21P, are closely linked on chromosome 6p21.3 in the major histocompatibility complex. Most reported mutations causing 21-hydroxylase deficiency are apparently the result of recombinations between CYP21 and CYP21P. These are either deletions of CYP21 due to unequal meiotic crossing-over (approximately 20% of alleles), or apparent gene conversions in which deleterious mutations normally present in CYP21P are transferred to CYP21 (reviewed in74).

Like CYP21 and CYP21P, CYP11B1 and CYP11B2 are closely linked homologs, but CYP11B1 and CYP11B2 both encode active

enzymes. Thus, gene conversions that trasnfer polymorphic sequences between CYP11B1 and CYP11B2 might not be expected to have major adverse effects on enzymatic activity, in which case genetic deficiencies of the encoded enzymes (P450cll and P450cmo) should be the result of mutations that are not gene conversions. Indeed, we have characterized three CYP11B1 mutations causing lip-hydroxylase deficiency in addition to R448H, and all are de novo point mutations or small insertions and not large deletions or gene conversions (unpublished observations). Similarly, the R181W mutation in CYP11B2 associated with CMO II deficiency is also a simple point mutation (like R448H, it is a mutation of CpG to TpG, the most common type of point mutation in higher eukaryotes75). In contrast, V386A is normally present in CYP11B1 and thus its presence in the mutant CYP11B2 genes of CMO II deficiency patients may be the result of an ancestral gene conversion, although an independent mutation is also possible. As predicted, V386A itself has a minimal effect on enzymatic activity.

Steroid 17a-hydroxylase/17,20-lyase

The P450cl7 structural gene (CYP17) has been located on chromosome 10,76 but thus far has not been regionally localized. Apparently, the same gene is expressed in both the adrenal and the testis.77 Initial hybridization studies of DNA samples from patients with 17a-hydroxylase deficiency did not disclose the presence of any gross deletions or rearrangements of this gene.78 Further molecular characterization of specific mutations in the structural gene coding for the P450cl7 enzyme have been reported in a number of patients.79-84 Details concerning these molecular genetic studies are reviewed in19. In brief, no deletions or major gene rearrangements were seen on southern blots of affected patients. In all cases, mutations in the structural gene have been found. These have included: a stop codon introduced in the first exon by a single base substitution, and in a separate case, a seven basepair duplication in exon 2 which shifts the reading frame of translation and introduces a stop codon downstream. These patients were homozygous for these two mutations, respectively, and thus they had complete combined 17a-hydroxylase and 17,20-lyase deficiencies.

Analysis of two additional patients showed a four basepair duplication in exon 8 which caused abolition of enzymatic activity for both 17a-hydroxylase and 17,20-lyase in transfection studies. Homozygous deletion of three basepairs in exon 1 in another case resulted in a gene product with partial activity, the 17,20-lyase being more severely compromised. Interestingly, this patient was a genetic and phenotypic female with sexual infantilism, as one would predict from the inability to synthesize sex steroids. Finally, one genetic male with ambiguous genitalia had a different mutation on each chromosome: a stop codon introduced by a single base substitution in exon 4; and a nonconservative proline—»threonine substitution in exon 6. Expression studies in this last case showed equivalently decreased activity of both 17a-hydroxylase and 17,20-lyase.

3(i-ol dehydrogenase

Deficiency of 3f3-ol dehydrogenase is not linked to the HLA complex.85 In contrast with the other adrenal steroidogenic enzymes, the 3p-ol dehydrogenase enzyme, a dehydrogenase typically requiring NAD+ as a cofactor, is not a cytochrome P450. The enzyme is classed as a short-chain dehydrogenase which is similar to the genes encoding the 170- and llp-hydroxy-steroid dehydrogenases.86 Closely associated with 33-ol dehydrogenase is the enzyme activity 3-ketosteroid A5-4 isomerase, which requires NAD+ or NADH.87 In mammalian species these two functions appear to reside within the same protein, but the enzyme generally has not been well characterized.

Two genes encoding human 3p-ol dehydrogenase/ A5-^ isomerase have been cloned.88-90 The deduced amino acid sequence is 94% homologous between the Type I gene expressed in placenta and skin, and the Type II gene expressed in adrenal and gonads. Structural gene mutations have recently been described in patients with clinically apparent classical 3p-ol dehydrogenase deficiency: one patient with familial hypospadias and urethral diverticula91 had a Type II gene paternal missense mutation, Tyr-253—>Asn, and a maternal frameshift mutation, a C insertion between codons 186-187. In a second patient with salt-wasting and hypospadias,92 only the frameshift could be detected in the Type

II maternally inherited gene. This suggests that perhaps the second mutation involves 5' regulatory elements, which have not yet been well-characterized.91

Cholesterol desmotase (P450scc)

The gene (CYP11A) for this mitochondrial P450 enzyme (P450scc) has been isolated, cloned and localized to chromosome 15.93 Mutation of the structural gene has not yet been identifiéd in lipoid adrenal hyperplasia94-95 although in vitro studies suggest that the 20a-hydroxylase function is deficient in at least one patient with the syndrome. Remote lesions affecting other cellular components fundamental to early steroidogenesis could have similar effects.


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