Steroid 21-hydroxylase Deficiency Research Articles

MOLECULAR PATHOLOGY OF 21-HYDROXYLASE DEFICIENCY

Pathological mutations in steroid 21-hydroxylase deficiency appear to derive exclusively from sequence exchange between the 21-hydroxylase gene (CYP21B) and a closely related pseudogene (CYP21A), located 30 kb telomeric to CYP21B and functionally inactive because of several mutations distributed along its length. Three basic classes of disease chromosome are seen: (a) deletion chromosomes where about 30 kb of DNA has been eliminated and a single 21-hydroxylase gene, often a CYP21A/CYP21B fusion gene, remains; (b) chromosomes which have 2 or more CYP21 genes, but without an apparent evidence for CYP21B-specifisce quences; (c) chromosomes which have 2 or more CYP21 genes, in which there is at least one gene with CYP21B-specific sequences, either a fusion gene or a mosaic, so-called “converted” gene. In the latter type of gene a central segment which may be very small or substantial shows CYP21A-specifics equences. Data will be presented on de novo mutations, showing how disease chromosomes can arise byunequal crossover and gene conversion-like events. The frequencies of different pathological mutations and genotype-phenotype correlations will also be discussed.

PRENATAL TREATMENT OF CONGENITAL ADRENAL HYPERPLASIA

Prenatal treatment was given to 67 fetuses at risk for classical steroid 21-hydroxylase deficiency (21OHD) congenital adrenal hyperplasia (CAH). The mothers were given dexamethasone (DEX) 1-1.5 mg/d po, computed at 20 μg/kg (pre-pregnancy weight)/day, in three divided doses. Treatment began as early as 4 weeks, continuing until birth for affected female fetuses. The diagnosis of 21OHD was established or excluded by DNA analysis using HLA-linked markers or 21OHD gene probes on the tissue provided by chorionic villus sampling (CVS). When amniocentesis was performed the diagnosis was based on the concentration of 17OHP in the supernatant and occasionally DNA was analyzed from amniotic cells. Seventeen fetuses were predicted to be affected, 10 of which were females. In every case the virilization of the genitalia of the unborn prenatally-treated females was significantly less than in the index case. No complications were observed in those fetus treated briefly and who were predicted to be unaffected. Maternal complications were observed in 6 pregnancies out of 67. These included increased appetite, weight gain, mood swings, pedal edema, and mild hypertension. None were permanent. One fetus was spontaneously aborted at 33 wks. It appears unlikely that dexamethasone was causative of the abortion since treatment with dexamethasone occurred from 10-19 weeks.In conclusion, prenatal treatment of fetuses at risk for CAH is effective and safe.

Problems in diagnosis and management of congenital adrenal hyperplasia due to 21-hydroxylase deficiency

A number of biochemical tests have been utilized to assist the diagnosis of steroid 21-hydroxylase deficiency. The specificity and accuracy of plasma 17-hydroxyprogesterone assays are important. A profile of steroids in urine by gas chromatography and mass spectrometry is the definitive test. Molecular biology is not practical for the diagnosis of a new case. The ACTH stimulation test for detection of heterozygotes is a poor discriminant. Fertility in patients with congenital adrenal hyperplasia may be due to excess of progesterone as well as of androgens. Gene amplification offers the best approach in molecular biology for the prenatal diagnosis of 21-hydroxylase deficiency.

Prenatal diagnosis of congenital adrenal hyperplasia by direct detection of mutations in the steroid 21‐hydroxylase gene

Our aim was to develop a rapid and accurate method for the prenatal diagnosis of congenital adrenal hyperplasia using the polymerase chain reaction to detect mutations in the steroid 21-hydroxylase gene. These procedures will help to minimize exposure to dexamethasone treatment of either affected males or unaffected females. Chorionic villus biopsy samples were obtained between 10 and 11 weeks gestation from three females carrying fetuses at risk of steroid 21-hydroxylase deficiency. Blood samples were taken from parents and the index case in each family. Three common mutations in the 21-hydroxylase B gene were detected following DNA amplification. Prenatal diagnosis of congenital adrenal hyperplasia was successful in all three cases. One affected female was treated with dexamethasone to term. In the other two cases, one affected male and one carrier also male, dexamethasone was withdrawn at an early stage. First trimester prenatal diagnosis of steroid 21-hydroxylase deficiency was achieved in three pregnancies with a strategy based on direct detection of gene mutations.

Steroid 21-hydroxylase deficiency: two additional mutations in salt-wasting disease and rapid screening of disease-causing mutations.

A method for genetic diagnosis of steroid 21-hydroxylase deficiency was developed based on allele-specific PCR. With this approach, genotyping of fourteen mutations and diagnosis of homozygous gene deletions can be performed within hours from tissue sampling. One patient with salt-wasting disease had normal genotype at all positions screened. DNA sequencing revealed two novel mutations, a G to C transversion at the conserved splice donor site of intron 7 and a TGG to TAG nonsense mutation at Trp 406 in exon 9. Allele-specific PCR was established also for these mutations and used to screen for their presence in the pseudogene. However, the two novel mutations were not found in at least 34 pseudogenes.

Insulin insensitivity in adrenal hyperplasia due to nonclassical steroid 21-hydroxylase deficiency.

To determine whether hyperandrogenism caused by an inborn error of adrenal steroidogenesis could produce insulin resistance, we examined insulin sensitivity in females with 21-hydroxylase deficiency. Minimal modelling was used to analyze the results of tolbutamide-modified, frequently sampled, iv glucose tolerance testing. Insulin sensitivity [Si; (min-1) (microU/mL)-1] was plotted against body mass index (BMI; defined as kilograms per m2). Six patients with nonclassical 21-hydroxylase deficiency (mean age, 27 yr; mean BMI, 23.2) underwent testing. None of these patients was in active puberty, nor was any patient being treated with glucocorticoids at the time of the study. Twelve eumenorrheic nonhyperandrogenic young adult female control subjects (mean age, 27 yr; mean BMI, 22.4) were also tested. The basal 17-hydroxyprogesterone concentration, but not the total serum testosterone level, was significantly different in the two groups (mean +/- SEM, 11,987 +/- 2,761 vs. 4,059 +/- 802 pmol/L; P < 0.05). As a group the patients' Si values were significantly lower than those of the controls (mean +/- SEM, 4.1 +/- 0.6 vs. 9.7 +/- 1.2; P < 0.05). There was no correlation between Si and basal serum 17-hydroxyprogesterone, testosterone, delta 4-androstenedione, or dehydroepiandrosterone. We conclude that chronic hypersecretion of androgen precursors due to an inborn error of metabolism can induce a reduction in insulin sensitivity.

Insulin insensitivity in adrenal hyperplasia due to nonclassical steroid 21-hydroxylase deficiency

Insulin insensitivity in adrenal hyperplasia due to nonclassical steroid 21-hydroxylase deficiency

Pro-453 to Ser mutation in CYP21 is associated with nonclassic steroid 21-hydroxylase deficiency.

Steroid 21-hydroxylase deficiency is the leading cause of impaired cortisol synthesis in congenital adrenal hyperplasia (CAH), with the nonclassic form (NC) comprising approximately 1% of the Caucasian population. The structure of the CYP21 gene was studied in 13 unrelated NC-CAH patients, three affected siblings, and 55 blood donors using polymerase chain reaction. In addition to the Leu-281 and Leu-30 mutations previously associated with NC-CAH, the finding of a Pro-453 to Ser mutation in exon-10 of CYP21 in the NC-CAH patients is reported. Ser-453 was found in 46.2% of unrelated NC-CAH patients, but only 7.7% and 3.6% of salt-wasting CAH patients and blood donors, respectively. In contrast to the Leu-281 and Leu-30 mutations, Ser-453 has not been previously detected in the CYP21 pseudogene (CYP21P) and, therefore, has not likely arisen by gene conversion.

Steroid 21-hydroxylase deficiency: three additional mutated alleles and establishment of phenotype-genotype relationships of common mutations.

Lesions in the gene encoding steroid 21-hydroxylase [steroid hydrogen-donor: oxygen oxidoreductase (21-hydroxylating), EC 1.14.99.10] result in defective adrenal steroid synthesis; the severe forms are known as congenital adrenal hyperplasia. To facilitate complete characterization of mutations in this region of tandemly repeated genes, we have developed selective PCR amplification and direct sequencing of full-length nonpseudogene steroid 21-hydroxylase genes. This technique identifies known mutations, characterizes or excludes unknown mutations, and determines the gene-copy number. Three additional defective alleles were found. A Gly-292----Ser mutation and a frameshift mutation at Arg-484 (GG----C) were identified in patients with severe steroid 21-hydroxylase deficiency. An allele with three additional sequence variations--C----T at 4 bases upstream of translation initiation, Pro-106----Leu, and Pro-454----Ser--were identified in two siblings with late-onset deficiency. Pro-454 is conserved in four species, indicating its importance for normal enzyme function. Functional consequences of individual alleles have been determined in vivo by studying individuals with only one steroid 21-hydroxylase gene. Detailed analyses of clinical data revealed that genotyping could predict the clinical course of the disease. The locations of disease-causing mutations on different haplotypes of the steroid 21-hydroxylase gene region are described.

Pro-453 to Ser mutation in CYP21 is associated with nonclassic steroid 21-hydroxylase deficiency

Pro-453 to Ser mutation in CYP21 is associated with nonclassic steroid 21-hydroxylase deficiency

Prenatal diagnosis of 21-hydroxylase deficiency congenital adrenal hyperplasia using the polymerase chain reaction.

We present an improved method for the prenatal diagnosis of congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency. The polymerase chain reaction (PCR) was used to analyze DNA from an affected index case, the parents, and a cultured chorionic villus sample, for point mutations in the steroid 21-hydroxylase (CYP21) gene. We can predict that the fetus is an unaffected carrier.

Prenatal diagnosis/treatment in families at risk for infants with steroid 21-hydroxylase deficiency (congenital adrenal hyperplasia)

The most common enzymatic defect of steroid synthesis is adrenal steroid 21-hydroxylase deficiency. Inhibited formation of cortisol causes increased pituitary release of ACTH, driving the adrenal cortex to overproduce androgens, whose synthesis does not involve the 21-hydroxylase enzyme. This hormonal setting is established in the embryonic period and affects development of genetic females, misdirecting differentiation of the external genitalia toward male type. At birth, the genitalia are visibly ambiguous (enlarged clitoris, fused labia) or in some cases even male in appearance {phallus with urethral opening, rugated scrotal sac), leading to wrong sex assignment. Adrenal steroid 21-hydroxylase deficiency is the most common basis of female pseudohermaphroditism. These females, however, have normal fertility and potential for gestation (gonads are functional and the internal duct-derived structures are well-formed), thus the sex of rearing should always be female. Management is by life-long hormonal (glucocorticoid) replacement, with surgical correction of the genital ambiguity. Prenatal diagnosis of 21-hydroxylase deficiency, first possible by steroid assay of the amniotic fluid, has utilized HLA typing for identification of loci (antigens B and DR) in close linkage with the 21-hydroxylase gene, and now increasingly relies on DNA analysis for linked HLA or C4 genes or for mutant 21-hydroxylase alleles directly by molecular genetic techniques. The most recent clinical advance is a program of combined prenatal diagnosis with karyotyping and suppression of fetal androgen production in genetic females by steroid administration to the mother. This is the first instance of an inborn metabolic error to be prenatally treated. A series of 85 managed pregnancies is reported on, including accuracy of diagnosis, response of the mother to steroid treatment, and outcome for treated and untreated male and female fetuses (of 77 born by 6/91). Prenatal diagnosis by current techniques is accurate. Normal growth and development patterns postnatally suggest that dexamethasone treatment is safe.

Genotype of Yupik Eskimos with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

An A-to-G transition in the second intron was the sole mutation detected in four Yupik Eskimo patients with salt-wasting congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency. Allele-specific hybridization should be an efficient means of performing prenatal diagnosis of the disease in this highly inbred population.

A method for specific amplification and PCR sequencing of individual members of multigene families: application to the study of steroid 21-hydroxylase deficiency.

Mutations at the human HLA-linked CYP21B locus are responsible for 21-hydroxylase deficiency, a recessively inherited disorder of steroidogenesis. The scope for PCR-based analysis of the CYP21B gene has been restricted by the very high sequence homology between CYP21B and a closely related pseudogene, CYP21A. Here we describe a novel PCR sequencing strategy that allows the independent amplification of the entire CYP21B coding sequence and the subsequent enzyme-mediated conversion of the PCR product to a single-stranded form for dideoxy sequencing. We have used this approach to characterize the 21-hydroxylase deficiency allele associated with HLA-B55, the most frequent HLA marker associated with a CYP21B point mutation in the British population, and also an HLA-B35 associated allele of Asian origin. Allele-specific oligonucleotide (ASO) hybridization analyses have confirmed the selective amplification of CYP21B genes and the identity of the pathological mutations. The method can be adapted to permit selective amplification and PCR sequencing of individual closely related members of other multigene families and small-copy-number repetitive DNA families.

Urinary excretion of 17-hydroxypregnanolones in patients with different forms of congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency

To improve diagnostic criteria in different (classical salt-wasting (SW), classical simple virilizing (SV) and non classical late onset (LO)) forms of congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase deficiency, we investigated the urinary excretion of 17-hydroxypregnanolones (17OH-PO(5 beta) and (5 alpha)), 15 beta-hydroxypregnanolone(15 beta OH-PO), pregnanetriol(PT) and 11-oxo-pregnanetriol (11-O-PT) compared to hydrocortisone metabolities. During the 1st month of life newborn infants with CAH-SW excreted from barely detectable to very large amounts of 17OH-PO(5 beta), 15 beta OH-PO and PT, and, in 12 of 14 cases, also 11-O-PT in their urines. From the 1st to the 28th day of life, cortisol metabolites were virtually absent in urines of CAH-SW infants. This was in contrast of 36 healthy newborn infants. We measured the excretion of 17OH-PO(5 alpha) in children with CAH of whom 19 patients with CAH-SV had a median 17OH-PO(5 alpha) excretion of 1110 micrograms/day (range: 152-5515). In 21 patients with CAH-LO, median excretion of 17OH-PO(5 alpha) was 294 micrograms/day (range: 66-1273). Besides the conventional metabolites of 17-hydroxyprogesterone (17OH-PO(5 beta), PT and 11-O-PT), no 17OH-PO(5 alpha) was detected in the urines of 14 patients with precocious pubarche, in 14 patients with virilization of unknown origin and in 94 healthy children of comparable age. The ratio of 17OH-PO(5 alpha) to tetrahydrocortisone (THE) discriminated between CAH-SV and CAH-LO from the 1st to the 18th year of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Haplotypes of the steroid 21-hydroxylase gene region encoding mild steroid 21-hydroxylase deficiency.

Haplotypes of the complement 4 (C4) and steroid 21-hydroxylase [21-OHase; steroid hydrogen-donor: oxygen oxidoreductase (21-hydroxylating), EC 1.14.99.10] repeated gene complex were studied in nine families with at least one member affected with a mild form of 21-OHase deficiency. DNA probes from different parts of the repeated C4/21-OHase unit were used to follow the segregation of hybridization patterns in the families. Ten structurally distinct haplotypes of the C4/21-OHase gene region were identified, and the encoded phenotype was assigned to 34 of the 36 C4/21-OHase haplotypes. Four structurally different haplotypes with three C4/21-OHase repeat units were found. Eight of the nine haplotypes found with triplications of the C4/21-OHase repeat unit encoded the mild form of 21-OHase deficiency, whereas one particular triplicated haplotype encoded a severe form of the disease. In one case the mild form of 21-OHase deficiency was encoded by a haplotype with a single C4/21-OHase repeat unit. Mild 21-OHase deficiency was predicted in a patient by the presence of a triplicated haplotype. The finding of deranged 21-OHase genes on all triplicated C4/21-OHase haplotypes indicate that most of these common haplotypes carry mutated 21-OHase genes, and thus may cause functional polymorphism of general importance in the population.

Mutations of P450c21 (steroid 21-hydroxylase) at Cys428, Val281, and Ser268 result in complete, partial, or no loss of enzymatic activity, respectively.

Steroid 21-hydroxylase deficiency is the major cause of congenital adrenal hyperplasia (CAH), a common genetic disease. To define the relationship between gene mutations and enzyme deficiency, we generated missense mutations of the 21-hydroxylase cDNA at three different sites and characterized the mutant proteins after expressing them in cultured mammalian and yeast cells. Among them, Ser268 and Val281 have been found to be mutated in CAH patients, whereas Cys428 has been implicated as the heme ligand. Our results show mutations at these sites result in complete, partial, or no loss of the enzymatic activity. All the Cys428 mutants had neither enzymatic activity nor P450 absorption, thus supporting the notion that Cys428 is the heme ligand. All the 268-mutants exhibited the same activity as normal 21-hydroxylase, demonstrating that the clinically observed Ser268----Thr change represents a polymorphism rather than the cause of the enzyme deficiency. The 281-mutants had normal Km but greatly reduced Vmax values that also paralleled the reduction in the heme content, in the order Val281 (normal, 100%) greater than Ile281 (50%) greater than Leu281 (20%) greater than Thr281 (10%). Our findings suggest that the methyl group at the beta-carbon of Val281 is required for heme incorporation and consequently enzymatic activity.

A Mutation (Pro-30 to Leu) inCYP21 Represents a Potential Nonclassic Steroid 21-Hydroxylase Deficiency Allele

The mild nonclassic form of steroid 21-hydroxylase deficiency is one of the most common autosomal recessive disorders in humans, occurring in almost 1% of caucasians and about 3% of Ashkenazi Jews. Many patients with this disorder carry a Val-281----Leu missense mutation in the CYP21 gene. This and most other mutations causing 21-hydroxylase deficiency are normally present in the CYP21P pseudogene and have presumably been transferred to CYP21 by gene conversion. To identify other potential nonclassic alleles, we used recombinant vaccinia virus to express two mutant enzymes carrying the mutations Pro-30----Leu (normally present in CYP21P) and Ser-268----Thr (considered a normal polymorphism of CYP21). Whereas the activity of the protein carrying the Ser----Thr mutation was indeed indistinguishable from the wild type, the enzyme with the Pro----Leu substitution had 60% of wild-type activity for 17-hydroxyprogesterone and about 30% of normal activity for progesterone when assayed in intact cells. When kinetic analysis of the latter mutant enzyme was performed in cellular lysates, the first order rate constants (maximum velocity/dissociation constant) for both substrates were reduced 10- to 20-fold compared with those for the wild-type enzyme. Pro-30 is conserved in many microsomal P450 enzymes and may be important for proper orientation of the enzyme with respect to the aminoterminal transmembrane segment. The Pro----Leu mutation was present in 5 of 18 patients with nonclassic 21-hydroxylase deficiency, suggesting that this mutation indeed acts as a nonclassic deficiency allele.

Steroid 21-Hydroxylase Deficiency in Mice. An Animal Model for Congenital Adrenal Hyperplasia.

The classical form of congenital adrenal hyperplasia (CAH) in humans is caused by a deficiency in steroid 21-hydroxylase (21-OHase) . The enzyme 21-OHase plays a key role in adrenal steroidogenesis. Duplicated genes for the enzyme are located in the class III region of the major histocompatibility complex (MHC), HLA. In the mouse, the genes encoding 21-OHase have been mapped to the homologous region of the H-2 complex. We have previously obtained an intra-H-2 recombinant haplotype aw18 from a cross between H-2 congenic strains of B10. A (H-2a) and B10. MOL-SGR (H-2wm7), which carries the H-2 complex derived from the Japanese wild mouse. When mice that were heterozygous for aw18 were intercrossed, no mice that were homozygous for the aw18 haplotype were detected among live offspring of more than 15 days of age, which suggested presence of a recessive lethal gene in the H-2aw18 haplotype. Molecular analysis of the H-2aw18 chromosome has revealed a deletion in the H-2 class III region encompassing the gene for the complement component C4 and one of two genes for 21-OHase. We now report that newborn aw18 homozygous mice are deficient in 21-OHase activity, and that homozygosity of the aw18 haplotype directly causes death at the early postnatal stage. Morphological changes in the adrenal glands of newborn aw18 homozygotes are also observed. The aw18 recombinant haplotype is expected to serve as a useful animal model for the inherited human disease of CAH.

Molecular pathology of steroid 21-hydroxylase deficiency

The molecular pathology of steroid 21-hydroxylase deficiency is attributable to unequal crossover-mediated gene deletion or to large- or small-scale replacement of the functional CYP21B gene sequence by a copy of the analogous CYP21A pseudogene sequence. Because the pathological point mutations originate from the pseudogene which shows only a small number of differences from the functional CYP21B gene sequence, the total number of different pathological point mutations is likely to be small. Mutant P450c21 enzymes carrying specific amino acid substitutions seen in patients with 21-hydroxylase deficiency exhibit activities that correlate with the clinical severity of the disease and with biochemical abnormalities such as 17-hydroxyprogesterone levels after ACTH (corticotropin) stimulation.