High frequency of nonclassical steroid 21-hydroxylase deficiency.

Nonclassical steroid 21-hydroxylase deficiency is an autosomal recessive disorder that is defined by clinical and hormonal criteria that distinguishes it from the classical 21-hydroxylase deficiency. No estimates of the gene frequency of nonclassical 21-hydroxylase deficiency, also called attenuated, late-onset, acquired, and cryptic adrenal hyperplasia, have been published thus far. Here, we have used HLA-B genotype data in families containing multiple members affected with nonclassical 21-hydroxylase deficiency together with the results of quantitative hormonal tests to arrive at estimates of gene and disease frequencies for this disorder. We found nonclassical 21-hydroxylase deficiency to be a far more common disorder than classical 21-hydroxylase deficiency, which occurs in 1/8,000 births. The prevalence of the disease in Ashkenazi Jews was 3.7%; in Hispanics, 1.9%; in Yugoslavs, 1.6%; in Italians, 0.3%; and in the diverse Caucasian population, 0.1%. The gene for nonclassical 21-hydroxylase deficiency is in genetic linkage disequilibrium with HLA-B14 in Ashkenazi Jews, Hispanics, and Italians, but not in Yugoslavs or in a diverse, non-Jewish, Caucasian group. The penetrance of nonclassical 21-hydroxylase deficiency gene in the HLA-B14 containing haplotypes was incomplete. Thus, nonclassical 21-hydroxylase deficiency is probably the most frequent autosomal recessive genetic disorder in man and is especially frequent in Ashkenazi Jews, Hispanics, Italians, and Yugoslavs.     

Associations between restriction fragment length polymorphisms detected with a probe for human 21-hydroxylase (21-OH) and two clinical forms of 21-OH deficiency

Summary DNAs from unrelated healthy individuals and unrelated individuals affected with 21-hydroxylase deficiency (congenital and late-onset adrenal hyperplasia) were digested with seven restriction enzymes and hybridized with a cDNA probe specific for human 21-hydroxylase genes. Associations were found between restriction fragments and the two forms of the disease: (i) The late onset form is associated with a double dose of a 14 kb fragment generated by Eco RI and with a triple dose of a 3.2 kb fragment generated by TaqI in patients with HLA B14 haplotypes; (ii) The classical congenital form is negatively associated with the 14 kb fragment and with a 3.7 kb fragment generated by TaqI in patients with HLA Bw47 haplotypes. A 3.2 kb TaqI fragment is negatively associated with the HLA B8 haplotypes. The other five enzymes tested give no polymorphisms or polymorphisms without correlation with the two forms of the disease.     

Late-onset 21-hydroxylase deficiency is an allelic variant of congenital adrenal hyperplasia characterized by attenuated clinical expression and different HLA haplotype associations

Three families with late-onset 21-hydroxylase deficiency were studied. Homozygous females presented with symptoms of mild hyperandrogenism such as acne, hirsutism, oligomenorrhea and menometrorrhagia. A homozygous male was asymptomatic and had reached normal adult height. The diagnosis of 21-hydroxylase deficiency was based upon markedly elevated responses of plasma 17-hydroxyprogesterone during a short (30-min) ACTH infusion test. The propositi of two of the families were diagnosed despite long-standing glucocorticoid therapy and adrenal suppression by using a prolonged (48-hour) ACTH infusion. Heterozygotes of late-onset 21-hydroxylase deficiency had mildly elevated 17-hydroxy-progesterone responses to ACTH. Late-onset 21-hydroxylase deficiency was inherited as an autosomal recessive trait with close linkage to the histocompatibility leukocyte antigens. The B14 haplotype was present in all affected members. One affected female had a daughter with classic, salt-losing 21-hydroxylase deficiency. Mixed heterozygosity of this patient for a classic and a late-onset 21-hydroxylase deficiency allele may have caused the classic phenotype in her daughter (homozygote for 2 classic alleles).     

The immunological detection of a 21-OH deficiency mutation HLA supratype.

Previous studies have shown that the late-onset and cryptic forms of 21-hydroxylase deficiency are highly associated with the HLA supratype HLA-B14,C4A2,C4B1/2,DR1. Since cells from a number of unrelated normal individuals from different ethnic backgrounds expressing the DR1 associated with this supratype failed to stimulate two different DR1-restricted T-cell clones that proliferated in the presence of most other DR1 cells, we decided to test the hypothesis that cells with this supratype express "abnormal" DR1 molecules that have been affected in some way by the chromosomal mutation responsible for B14,DR1-associated 21-hydroxylase deficiency (21-OH-defL). The results showed an association between "abnormal" DR1 and 21-OH-defL (elevated rates of 17 alpha-hydroxyprogesterone [17-OHP] increase and elevated peak 17-OHP values following ACTH stimulation). The presence of the B14,DR1 supratype can be used to predict the presence of "abnormal" DR1 and the clinical status of individuals not previously known to be 21-OH-defL carriers.     

Molecular basis of 17α-hydroxylase/17,20-lyase deficiency

17α-Hydroxylase deficiency is characterized by a defect in either or both of 17α-hydroxylase and 17,20-lyase activities, based on the fact that a single polypeptide P450c17 can catalyze both reactions. The clinical manifestations of 17α-hydroxylase/17,20-lyase deficiency seem to be more heterogeneous than expected, varying from the classical type to less symptomatic forms as also observed in 21-hydroxylase deficiency. We have sequenced all eight exons of the CYP17 (P450c17) gene in DNA from several patients, reconstructed the mutations in a human P450c17 cDNA and expressed the mutant P450c17 in COS 1 cells to characterize the kinetic properties of 17α-hydroxylase and 17,20-lyase activities. The molecular bases of cases clinically reported as 17α-hydroxylase deficiency have turned out to be complete or partial combined deficiencies of 17α-hydroxylase/17,20-lyase. The elucidation of the molecular basis generally explains the patient's clinical profiles including the sexual phenotype of the external genitalia. In one case clinically reported as isolated 17,20-lyase deficiency, the molecular basis was found to be partial combined deficiency of both activities, somewhat discordant with the patient's clinical profile. Based on the results obtained so far we can predict that those 17α-hydroxylase deficient individuals having a homozygous stop codon in the CYP17 gene positioned at the amino terminal side of the P450c17 heme-binding cysteine (442) will all have the same phenotype. However those individuals having homozygous missense mutations or those who are compound heterozygotes having a missense mutation in at least one CYP17 allele will display their own unique phenotype which clinically will be subtly different from all others.     

HLA linkage and B14, DR1, BfS haplotype association with the genes for late onset and cryptic 21-hydroxylase deficiency.

Classical congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21-OH-def) has been established to be an HLA-linked, recessive monogenetic disease. However, two nonclassical forms of 21-OH-def have also been described: "cryptic" 21-OH-def, which has been shown to be HLA-linked, and "late onset" 21-OH-def, for which the status of linkage to HLA has been less certain. We now describe studies of eight additional unrelated probands with symptomatic, "late onset" 21-OH-def, and conclude that this form is also HLA-linked. Both "late onset" and "cryptic" 21-OH-def are highly associated with the same HLA antigens and markers (HLA-B14, HLA-DR1, and Bf type S) in individuals from different ethnic and geographical backgrounds. Since both "late onset" and "cryptic" 21-OH-def appear to occur in individuals with one classical 21-OH-def (21-OHCAH) allele who in addition have another 21-OH-def allele, as well as in individuals who appear to be homozygous for variant 21-PH-def alleles, and since both late onset and cryptic 21-OH-def appear to occur in the same families, our data suggest that these syndromes may represent different clinical expressions of similar or identical nonclassical 21-OH-def alleles.     

Pre-pubertal gynaecomastia as the presenting feature of late-onset 21-hydroxylase deficiency

We describe an 8-year-old boy with pre-pubertal gynaecomastia as the presenting feature of late-onset 21-hydroxylase deficiency, an association not previously reported. Although absolute oestrogen levels were not higher than previously described in 21-hydroxylase deficiency, the gynaecomastia may have arisen through a relative disproportion of the C18 to C19 steroids.     

Basic and clinical aspects of congenital adrenal hyperplasia

Defective steroid 21-hydroxylation is the most common of the biochemical defects causing hyperplasia of the adrenal cortex. The genetic mode of transmission of all enzyme abnormalities seen in cortisol biosynthesis is autosomal recessive. Steroid 21-hydroxylase deficiency has three currently accepted forms: the simple virilizing and salt-wasting variants of the classical deficiency, and the nonclassical (attenuated) form, which shows a wide clinical range of effects and whose characterization emerged from co-ordinated hormonal testing and family studies. More recent molecular genetic studies have started to identify specific mutations altering 21-hydroxylase activity. Defects in the other enzymes occur more rarely and are less well known, although initial work with abnormal 11 beta-hydroxylase and 3 beta-hydroxylase indicates that allelic gene defects may be correlated with different clinical phenotypes seen for these disorders also. The gene for the enzyme steroid 21-hydroxylase, a cytochrome P-450, is situated within the major histocompatibility complex on the p arm of human chromosome 6, proximal to the HLA-B antigen locus. Linkage disequilibria between certain B and DR alleles and classical and nonclassical 21-hydroxylase deficiency permit the use of HLA genotyping in conjunction with hormonal evaluation for diagnosis of this disorder and for identification of carrier haplotypes in population studies. Test programs have shown the feasibility of neonatal screening for 21-hydroxylase deficiency by blood-spot hormonal assay for elevated 17-hydroxyprogesterone. Prenatal detection of disease currently depends on HLA serotyping of cultured aminocytes jointly with measurement of amniotic 17-hydroxyprogesterone (13-18 week gestation); molecular genetic techniques with more specific nuclear probes will improve the specificity of this test and will in addition permit even earlier definitive fetal genotyping by chorionic villus biopsy (6-10 week gestation).     

17α-Hydroxylase/17,20-lyase defects

Congential adrenal hyperplasia due to 17α-hydroxylase/17/20-lyase deficiency is caused by genetic defects in the gene encoding P450c17 (CYP17). To date, 18 different mutations in 27 individuals have been identified and all of them are located in the coding region of CYP17. Several mutations have been reconstructed in human P450c17 cDNA and expressed in COS cells to characterize the kinetic properties of 17α-hydroxylase and 17,20-lyase activities. The molecular bases of cases clinically reported as 17α-hydroxylase deficiency have turned out to result from complete or partial combined deficiencies of 17α-hydroxylase/17,20-lyase. The elucidation of the molecular bases generally explains the patient's clinical profiles including the sexual phenotype of the external genitalia. In one case initially reported as isolated 17,20-lyase deficiency, the molecular basis was found to be partial combined deficiency of both activities, somewhat discordant with the patient's clinical profile. However, the patient was subsequently found to have 17α-hydroxylase deficiency, suggesting involvements of age-dependent unknown factors affecting P450c17 activity.     

Congenital adrenal hyperplasia: update on prenatal diagnosis and treatment

The diagnostic term congenital adrenal hyperplasia (CAH) applies to a family of inherited disorders of steroidogenesis caused by an abnormality in one of the five enzymatic steps necessary in the conversion of cholesterol to cortisol. The enzyme defects are translated as autosomal recessive traits, with the enzyme deficient in more than 90% of CAH cases being 21-hydroxylase. In the classical forms of CAH (simple virilizing and salt wasting), owing to 21-hydroxylase deficiency (21-OHD), androgen excess causes external genital ambiguity in newborn females and progressive postnatal virilization in males and females. Non-classical 21-OHD (NC21OHD) refers to the condition in which partial deficiencies of 21-hydroxylation produce less extreme hyperandrogenemia and milder symptoms. Females do not demonstrate genital ambiguity at birth.

The gene for adrenal 21-hydroxylase, CYP21, is located on chromosome 6p in the area of HLA genes. Specific mutations may be correlated with a given degree of enzymatic compromise and the clinical form of 21-OHD. NC21OHD patients are predicted to have mild mutations on both alleles or one severe and one mild mutation of the 21-OH locus (compound heterozygote). In most cases the mutation groups represent one diagnosis (e.g., Del/Del with SW CAH), however we have found several non-correlations of genotype to phenotype. Non-classical and classical patients were found within the same mutation group. Phenotypic variability within each mutation group has important implications for prenatal diagnosis and treatment.

Prenatal treatment of 21-OHD with dexamethasone has been utilized for a decade. An algorithm has been developed for prenatal diagnosis and treatment, which, when followed closely, has been safe for both the mother and the fetus, and has been effective in preventing ambiguous genitalia in the affected female newborn. This is an instance of an inborn metabolic error successfully treated prenatally.

Since 1986, prenatal diagnosis and treatment of congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21-OHD) has been carried out in 403 pregnancies in The New York Hospital–Cornell Medical Center. In 280, diagnoses were made by amniocentesis, while 123 were diagnosed using chorionic villus sampling. Of the 403 pregnancies evaluated, 84 babies were affected with classical 21-OHD. Of these, 52 were females, 36 of whom were treated prenatally with dexamethasone. Dexamethasone administered at or before 10 weeks of gestation (23 affected female fetuses) was effective in reducing virilization. Thirteen cases had affected female sibs (Prader stages 1–4); 6 of these fetuses were born with entirely normal female genitalia, while 6 were significantly less virilized (Prader stages 1–2) than their sibs, and one was Prader stage 3. Eight newborns had male sibs; 4 were born with normal genitalia, 3 were Prader stages 1–2, and 3 were born Prader stages 3–4. No significant or enduring side effects were noted in either the mothers or the fetuses, indicating that dexamethasone treatment is safe. Prenatally treated newborns did not differ in weight, length, or head circumference from untreated, unaffected newborns.

Based on our experience, proper prenatal diagnosis and treatment of 21-OHD is effective in significantly reducing or eliminating virilization in the newborn female. This spares the affected female the consequences of genital ambiguity of genital surgery, sex misassignment, and gender confusion.     

Analysis of steroid 21-hydroxylase gene mutations in the Spanish population

Steroid 21 -hydroxylase deficiency is the major cause of congenital adrenal hyperplasia. Genotyping for deletions and nine point mutations in the CYP21 gene has been performed in 38 Spanish patients and their relatives by Southern blot analysis and allele-specific oligonucleotide hybridization. Three clinical variants were included in this study, viz., salt-wasting (SW, 21 patients), simple virilizer (SV, two patients), and late-onset (LO, 15 patients) forms. Twenty-three patient genotypes (16 SW, two SV, and five LO) were fully characterized. In both alleles, all but one of these severe forms (SW and SV) presented mutations that abolished or severely affected enzymatic activity. Patients with LO forms showed mutations that moderately impaired enzymatic activity in both alleles, or severe mutations in only one chromosome. Of 46 chromosomes from severe forms, 41 were characterized in this study (89%). The most frequent mutation was an aberrant splicing site (655 A or C to G) in intron 2, in 30% of these chromosomes. Deletions were found in 20%, and large gene conversions in 13% of these alleles. This screening allowed the characterization of 18 out of 30 LO chromosomes, the most frequent mutation being Val281Leu (37%). Severe mutations were found, in heterozygosis, in one third of LO patients.     

Frequent deletion and duplication of the steroid 21-hydroxylase genes

Congenital adrenal hyperplasia due to 21-hydroxylase (21-OHase) deficiency is an HLA-linked disorder resulting from a mutation in the 21-OHase B gene encoding the adrenal cytochrome P450 specific for steroid 21-hydroxylation. To identify polymorphisms associated with 21-OHase deficiency, DNA samples from 22 unrelated patients with this disorder were examined with a human cDNA clone encoding the enzyme. Deletions of the active 21-OHase gene were found in almost one-fourth of classical 21-OHase deficiency alleles. In contrast, mild, "nonclassical" 21-OHase deficiency is associated with a duplicated 21-OHase gene.     

Plasma Globotriaosylsphingosine and α-Galactosidase A Activity as a Combined Screening Biomarker for Fabry Disease in a Large Japanese Cohort

Fabry disease is an X-linked disorder of α-galactosidase A (GLA) deficiency. Our previous interim analysis (1 July 2014 to 31 December 2015) revealed plasma globotriaosylsphingosine as a promising primary screening biomarker for Fabry disease probands. Herein, we report the final results, including patients enrolled from 1 January to 31 December 2016 for evaluating the potential of plasma globotriaosylsphingosine and GLA activity as a combined screening marker. We screened 5691 patients (3439 males) referred from 237 Japanese specialty clinics based on clinical findings suggestive of Fabry disease using plasma globotriaosylsphingosine and GLA activity as primary screening markers, and GLA variant status as a secondary screening marker. Of the 14 males who tested positive in the globotriaosylsphingosine screen (≥2.0 ng/mL), 11 with low GLA activity (<4.0 nmol/h/mL) displayed GLA variants (four classic, seven late-onset) and one with normal GLA activity and no pathogenic variant displayed lamellar bodies in affected organs, indicating late-onset biopsy-proven Fabry disease. Of the 19 females who tested positive in the globotriaosylsphingosine screen, eight with low GLA activity displayed GLA variants (six classic, two late-onset) and five with normal GLA activity displayed a GLA variant (one classic) and no pathogenic variant (four late-onset biopsy-proven). The combination of plasma globotriaosylsphingosine and GLA activity can be a primary screening biomarker for classic, late-onset, and late-onset biopsy-proven Fabry disease probands.     

Genetic analysis of the steroid 21-hydroxylase gene following in vitro amplification of genomic DNA.

The 5′ end of the steroid 21-hydroxylase B gene encompassing putative control regions and the first 3 exons, has been selectively amplified in vitro from a number of patients with congenital adrenal hyperplasia caused by a deficiency of this enzyme. Sequence analysis has revealed a number of isolated instances of gene conversion to the 21-hydroxylase A sequence. One mutation, a C to G transversion at the 3′ end of the second intron, thought to lead to incorrect splicing of the mRNA, was found in 11 subjects all with the classical form of the disease.     

Development of plasma 21-deoxycortisol radioimmunoassay and application to the diagnosis of patients with 21-hydroxylase deficiency

Specific 21-deoxycortisol (21-DF) antiserum was raised in New Zealand white rabbits using a 21-DF-3,20-oxime-bovine serum albumin complex. Plasma radioimmunoassay of 21-DF was developed and used together with a radioimmunoassay of 17-hydroxyprogesterone (17-OH-P) for diagnosis of patients with 21-hydroxylase deficiency of congenital and postpubertal forms. The assays were performed in plasma extracts after isolation by paper chromatography. The response of plasma 21-DF and 17-OH-P to i.v. ACTH (25 IU) was studied in 15 adult controls and compared to 8 women with the late onset form of 21-hydroxylase deficiency and 23 women with idiopathic hirsutism. Normal 21-DF values for women were 6.9 +/- 3.6 ng/dl and for men 9.71 +/- 2.73 ng/dl. Newborn children (age: 3-10 days) had a value of 8.3 +/- 4.8 ng/dl. These values are definitely lower than the lowest value ever published. This is possibly due to the specificity of the antibody. During the menstrual cycle the 21-DF values did not change. The baseline and post-stimulated concentrations of hormone were similar in controls and women with hirsutism but were significantly higher in women with the late onset form of 21-hydroxylase deficiency. In the congenital form of 21-hydroxylase deficiency the 21-DF values (baseline) were high. In general, the 21-DF and 17-OH-P values have shown parallel changes. However, one case of 21-hydroxylase deficiency with elevated 21-DF but normal 17-OH-P was observed. The use of 21-DF for the diagnosis of 21-hydroxylase deficiency is suggested.     

TaqI HLA-B and -DRB RFLP analysis can predict disease in siblings of affected children with 21-hydroxylase deficiency

Summary The possibility of using TaqI restriction fragment length polymorphism (RFLP) analysis of the HLA-B locus and the HLA-DR-DQ subregions, flanking the 21-hydroxylase genes, for predicting disease in siblings of children with 21-hydroxylase deficiency was analyzed in 12 nuclear families with at least one affected child and a total of 18 at-risk off-spring. As part of the study allelic TaqI HLA-B RFLP patterns were determined in homozygous cell lines and families. The frequencies of individuals homozygous for TaqI allelic patterns of the different investigated HLA loci, each locus alone and in various combinations, were determined in 100 random controls. In all 12 families it was possible to make correct genetic diagnosis by the use of only one restriction enzyme, TaqI, and two locus-specific HLA cDNA probes, HLA-B and -DRB. In all families four haplotypes were obtained. Thus, affected siblings as well as carriers could be identified. Seven of the eight sibling pairs concordant for 21-hydroxylase deficiency had pairwise identical TaqI HLA-B-DRB-DQA-DQB haplotypes. The last disease-concordant sibling pair had inherited different haplotypes from their mother, who had nonclassical 21-hydroxylase deficiency. None of the ten healthy children shared both haplotypes with their affected sibling(s). Early prenatal suppression of the fetal adrenal cortex with fluorinated corticosteroids can prevent virilization of female fetuses with 21-hydroxylase deficiency. In most cases RFLP analysis of the 21-hydroxylase genes is not informative enough for prenatal diagnosis. Our results from the present retrospective family study indicate that TaqI HLA-B and -DRB RFP analysis will be a valuable tool for first trimester assessment of 21-hydroxylase deficiency. TagI HLA-B and -DRB RFLP analysis can be performed on DNA from chorionic villi biopsies obtained in the 8th week of pregnancy. Supplemented with sex determination, early withdrawal of prophylactic steroid therapy will thus be feasible when the mother carries a male or an unaffected female fetus.     

Genetic mapping of the 21-hydroxylase locus: estimation of small recombination frequencies

The locus for 21-hydroxylase (CA21HB) has been mapped to the interval between the HLA-B and HLA-DR loci on chromosome 6. Several methods of estimating genetic distance were used to determine whether CA21HB is closer to HLA-B or HLA-DR based on data collected on 157 families ascertained through a proband with the classical form of 21-hydroxylase deficiency (CA12Hd). The results were inconclusive but serve to highlight the limitations of present methods of estimating genetic distance when recombination frequencies are of the order of .005.     

Determination of functional effects of mutations in the steroid 21-hydroxylase gene (CYP21) using recombinant vaccinia virus

Steroid 21-hydroxylase (P450c21) is absent or defective in more than 90% of patients with congenital adrenal hyperplasia. This disorder of cortisol biosynthesis occurs in a wide spectrum of clinical severity; specific mutations in the 21-hydroxylase gene (CYP21) have been found in association with particular clinical phenotypes. To determine the functional effects of mutations causing amino acid substitutions, normal P450c21 and three mutagenized P450c21 enzymes were expressed at high levels in cultured COS-1 cells using recombinant vaccinia virus. A single amino acid substitution (Val281----Leu) present in patients with mild "nonclassical" 21-hydroxylase deficiency resulted in an enzyme with 20-50% of normal activity. A mutation (Ile172----Asn) identified in patients with the "simple virilizing" form (poor cortisol synthesis but adequate aldosterone synthesis) resulted in an enzyme with less than 2% of normal activity. Finally, a cluster mutation (Ile-Val-Glu-Met234-238----Asn-Glu-Glu-Lys) found in a patient with severe "salt wasting" 21-hydroxylase deficiency (inadequate aldosterone synthesis) results in an enzyme with no detectable activity. These data indicate that the severity of 21-hydroxylase deficiency correlates with the degree of enzymatic compromise.     

Late-onset adrenal hyperplasia in north Indian hirsute women

The occurrence of late-onset congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency was studied in 60 consecutive hirsute women by means of adrenocorticotrophin (ACTH)-stimulated serum 17-hydroxyprogesterone (17-OHP) levels. Five (8.3%) women had an exaggerated response (ACTH-stimulated 17-OHP 3,160 +/- 560 ng/dl). All of them had regular periods and 3 were virilized. The other 2 were indistinguishable from those with idiopathic hirsutism or polycystic ovarian disease.