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Mutational Spectrum of the Steroid 21-Hydroxylase Gene in Austria: Identification of a Novel Missense Mutation

Department of Internal Medicine III, Division of Endocrinology and Metabolism, and Department of Pediatrics (H.F., F.W.), University of Vienna, A-1090 Vienna, Austria; and Institute of Pharmacology, University of Heidelberg (E.S., S.R., K.M.), Heidelberg, Germany

Address all correspondence and requests for reprints to: Sabina M. Baumgartner-Parzer, Ph.D., Department of Internal Medicine III, Division of Endocrinology and Metabolism, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail: sabina.baumgartner-parzer{at}akh-wien.ac.at

Abstract

This study attempted an analysis of the mutational spectrum of 21-hydroxylase deficiency in 79 unrelated Austrian patients with classical and nonclassical forms of congenital adrenal hyperplasia and their respective 112 family members. Apparent large gene deletions/conversions were present in 31% of the 158 unrelated congenital adrenal hyperplasia alleles, whereas the most frequent point mutations were intron 2 splice (22.8%), I172N (15.8%), V281L (12%), and P30L (7.6%), in line with the frequencies reported for other countries. In 5 of the 12 congenital adrenal hyperplasia alleles carrying a P30L mutation the aberration is based on a single base substitution, whereas the remaining 7 represent part of a CYP21B conversion (1 allele) or CYP21B/21A hybrid gene (6 alleles), the latter characterized by a junction site before intron 2 as indicated by Southern blot, PCR, and sequence analyses.

Previously described mutations were not present in 1.2% of unrelated congenital adrenal hyperplasia alleles, including one female patient presenting with severe genital virilization. Sequence analysis of the complete functional 21-hydroxylase gene revealed an as yet undescribed mutation in exon 10-Arg⁴²⁶His, which has not yet been described to represent a common pseudogene sequence. In vitro expression experiments showed the Arg⁴²⁶His mutant to exhibit only low enzyme activity toward the natural substrate 17-hydroxyprogesterone corresponding to the degree of disease manifestation in the patient in whom it was found.

CONGENITAL ADRENAL hyperplasia (CAH) is an autosomal recessive disorder with impaired cortisol secretion from the adrenal (1, 2). More than 90% of CAH cases are caused by lesions in the steroid 21-hydroxylase (21-OH) gene, encoding a microsomal cytochrome P450 enzyme (P450c21). The latter normally converts 17-hydroxyprogesterone into 11-deoxycortisol and progesterone into 11-deoxycorticosterone; these steroids are subsequently converted to cortisol and aldosterone, respectively (3, 4).

21-OH deficiency exhibits a wide range of clinical manifestations, from life-threatening neonatal salt-wasting crises and genital virilization (classical forms of CAH) to rather inconspicuous symptoms [nonclassical (NC) and late-onset CAH], such as growth acceleration in childhood, menstrual disorders, and/or hirsutism in the adult (5).

This clinical diversity reflects varying degrees of enzyme inactivation caused by combinations of a panel of different mutations of the CYP21 gene (6, 7, 8). The 21-OH locus exists as an active CYP21B gene and a 98% homologous functionally inactive pseudogene CYP21A, arranged in tandem repeat with complement C4B and C4A genes in the human leukocyte antigen (HLA) class III region on the short arm of chromosome 6 (3, 9). The majority of mutations accounting for 21-OH deficiency result from the transfer of deleterious microsequences normally present in the CYP21A pseudogene into the functional CYP21B gene (2, 9, 10). Together with apparent gene deletions and large gene conversions, nine pseudogene-derived mutations are responsible for approximately 90–95% of all CAH alleles (11). The remaining 5–10% are due to rare or yet unidentified genetic lesions (2, 12), which need characterization by direct sequencing of the CYP21B or C4B gene.

The present work aimed at analysis and determination of frequencies of different types of mutations of the active CYP21B gene in unrelated Austrian CAH patients. Thereby we identified three sisters with severe CAH (Prader stage IV) exhibiting hemizygosity for an as yet unknown mutation (exon 10, Arg⁴²⁶His), which to date has not been described to represent a pseudogene sequence.

Subjects and Methods

Patients/subjects

Informed consent for mutation analysis was obtained from all patients and family members. We studied 79 unrelated CAH patients (158 unrelated CAH alleles) registered at the Departments of Internal Medicine III and Pediatrics, University Hospital (Vienna, Austria). For 45 index patients (but not the remaining 34), analysis of respective family members was possible and included 67 parents (42 mothers and 25 fathers) as well as 45 siblings, 15 of the latter known to suffer from CAH. As 2 families of the respective index patients were found to be related (13), only 1 of the family was included in the study.

CAH diagnosis was based on clinical manifestation and the detection of the relevant steroid metabolites in plasma and urine (14).

Mutation analysis of the CYP 21 gene

Genomic DNA was extracted from peripheral blood leukocytes according to standard procedures. Genotyping for disease causing gene deletions and large gene conversions was by Southern blot analysis (15). In brief, TaqI- and BglII (Roche Molecular Biochemicals, Mannheim, Germany)-digested DNAs, immobilized on Hybond N nylon membranes (Amersham Pharmacia Biotech, Little Chalfont, UK), were hybridized with [³²P]deoxy-CTP (Amersham Pharmacia Biotech)-labeled (Megaprime DNA labeling system, Amersham Pharmacia Biotech) probes specific for CYP21 and C4 genes (American Type Culture Collection, Manassas, VA).

The novel mutation R426H and the common mutations P30L, I2Splice, I172N, Cluster E6, V281L, F307insT, G291S, Q318X, R356W, G424S, and P453S were detected by direct sequencing (using [-³³P]dideoxy-NTP terminators and the radiolabeled terminator cycle sequencing kit from Amersham Pharmacia Biotech) of three fragments of the CYP21B gene specifically amplified by PCR. Selective PCR primers differentiating CYP21B from CYP21A by the 8-bp deletion located in exon 3 of CYP21A as well as primers for sequencing reactions were described previously (16, 17, 18). In addition, a PCR fragment of CYP21B was amplified independently of the 8-bp deletion in exon 3 with the primers CYP-5 (5'-AGCTATAAGTGGCACCTCAGG-3') and CYP-14 (5'-GCATCTCCACGATGTGA-3'). The primer CYP-14 (exon 6 of CYP21B) is specific for CYP21B. The reference sequence reported by White et al. (19) was used for numbering of nucleotides and amino acids.

In vitro expression experiments (20, 21, 22, 23)

To determine the effects of the missense mutation on enzymatic function, wild-type and P450c21 mutants (Arg⁴²⁶His and Val²⁸¹Leu) were expressed in vitro in COS-7 cells as described previously (20, 21, 22) with some modifications. The Val²⁸¹Leu mutant was used as a control for mild impaired 21-hydroxylase activity (23). The complete CYP21A and CYP21B genes [from bp 1639–4440 of the sequence previously reported (19)] were amplified by PCR using the unspecific (for CYP21B or CYP21A) primers CYP21-forward (5'-GTAAGCTTGCTATAAGTGGCACCTCAGG-3') and CYP21-reverse (5'-GCTCTAGAGTACGGGAGCAATAAAGGAG-3'). In the two primer sequences restriction sites for the restriction enzymes HindIII (CYP21-up) and XbaI (CYP21-down) were included (the primer sequences were underlined). The PCR was carried out in a total volume of 50 µl containing 200 ng genomic DNA, 50 µmol/liter of each primer, 200 µM of each deoxynucleotide triphosphate, 1 x reaction buffer proof 4 (AGS, Heidelberg, Germany), 2.5 mmol/liter MgCl₂, and 1 U of a Taq/Pwo polymerase mixture (AGS). The PCR program was 95 C for 5 min, followed by 35 cycles of 45 sec at 95 C, 45 sec at 60 C, and 3 min at 72 C, with a 72 C final extension for 10 min in the last cycle. The PCR products of the appropriate size were digested with the restriction enzymes HindIII and XbaI and ligated into the HindIII/XbaI-digested vector pcDNA3 (Invitrogen, Groningen, The Netherlands). Electroporation into Escherichia coli XL-I blue cells yielded single colonies from which the plasmid DNA was extracted with a Miniprep Kit (QIAGEN, Hilden, Germany). The nucleotide sequences of the inserts were determined by cycle sequencing with the cycle sequencing kit from Amersham Pharmacia Biotech and IRD-800-labeled primers (MWG Biotech, Ebersberg, Germany) on a Licor 4200 DNA Sequencer (LICOR, Lincoln, NE). Primer sequences were previously described (16, 17, 18). Plasmids containing the wild-type CYP21B sequence, the Arg⁴²⁶His, or the Val²⁸¹Leu mutation and no other mutations were transfected into COS-7 cells by lipofection with TransFast (Promega Corp., Madison, WI) according to the manufacturer’s recommended procedure. After 2 d of incubation in DMEM containing 15% FCS, 2 µmol/liter 17-hydroxyprogesterone, and 200,000 cpm 17-hydroxy-[³H]progesterone were added to the culture medium. Samples from the culture medium were collected after 1, 2, and 8 h. Steroids from the culture medium were extracted with dichloromethane and chromatographed on chloroform/acetone (70:30, vol/vol). The separated products on the paper were subjected to scintillation counting as described previously (24). Steroid products (17-hydroxyprogesterone and 11-deoxycortisol) were identified by comigration with authentic standards. Enzyme activities were expressed as percentage of substrate conversion from 17-hydroxyprogesterone to 11-deoxycortisol after correction for total protein content.

Results

Genotyping in Austrian CAH patients

In total we genotyped 79 unrelated Austrian CAH index patients (158 unrelated CAH alleles) with classical and nonclassical forms of CAH as well as 112 family members (42 mothers, 25 fathers, and 45 siblings, 15 of the latter known to suffer from CAH). Apparent gene deletions/large gene conversions were present in 31% of 158 unrelated CAH alleles, the most frequent point mutations were Intron2 splice (22.8%), I172N (15.8%), V281L (12%), and P30L (7.6%). Other mutations were rarely (R356W, Cluster, Q318x, P453S) or never (Ins T, G291S) detected (Table 1).

P30L mutation

In only 5 (41.7%) of 12 unrelated CAH alleles carrying a P30L mutation did the aberration seem to represent a single base transfer from the pseudogene to the functional CYP21B gene (2). For the majority of Pro³⁰Leu alleles, however, Southern blot (Table 2), and sequence analyses show the mutations to be part of a larger gene conversion (1 allele) or a chimeric CYP21B/CYP21A gene with a junction site before intron 2 differing from the functional 17-hydroxycorticosterone (21-OHB) gene only by the P30L mutation in exon 1 and the promoter sequences from CYP21A, as only recently described for one CAH patient by L’Àllemands (25). Whereas patients with the single base substitution present with the NC phenotype, all patients carrying the larger P30L aberration exhibit the classical form of CAH.

Two of 168 unrelated CAH alleles were negative for previously described mutations, suggesting the presence of new mutations or other defects (12). One female index patient and her 2 sisters presenting with classical (without salt-wasting crisis) CAH (14) and severe genital virilization (Prader stage IV) were compound heterozygous for a large gene deletion of the CYP21B (paternal) and CYP21A (maternal allele) genes, but were negative for other known mutations of the functional 21-OH gene.

Sequencing the complete CYP21B genes revealed all three sisters to exhibit hemizygosity for a yet unknown mutation CGCCAC in exon 10, leading to an ArgHis exchange in codon 426 of the maternal 21-OHB gene. One of the three sisters has given birth to a daughter who is, in turn, a clinically asymptomatic carrier of the Arg⁴²⁶His mutation (Fig. 1). Her heterozygosity for the Arg⁴²⁶His mutation (grandmaternal allele) is in line with inheritance of the respective HLA haplotypes (Fig. 1).

View larger version (18K): [in this window] [in a new window]   Figure 1. Pedigree of the CAH family bearing a novel 21-OHB Arg426His mutation. Genotypes of the functional CYP21B, of the presumably inactive pseudogene CYP21A, and of the respective HLA haplotypes are shown below each individual. Arrowheads indicate the hemizygous nucleotide mutation from the normal sequence CGC (codon 426) to CAC leading to an arginine to histidine exchange. As shown, the three sisters (index patients) exhibit hemizygosity for this R426H missense mutation (genotype a/c), which was inherited from the mother (c/d), whereas the father (a/b) transmitted a chromosome heterozygous for a CYP21B deletion. The asymptomatic daughter (e/c) of one of the index patients is heterozygous for the novel missense mutation, in line with inheritance of the respective HLA haplotype.

Functional activity of in vitro expressed wild-type P450c21 and the mutants Arg⁴²⁶His and Val²⁸¹Leu toward the natural substrate 17-hydroxyprogesterone is shown in Fig. 2. The wild-type enzyme converted 89% of 17-hydroxyprogesterone to 11-deoxycortisol within 8 h. The 50% conversion rate of the Val²⁸¹Leu mutant that was used as an internal control for the expression system is in good agreement with published data (23). The enzyme activity of the Arg⁴²⁶His mutant was strongly decreased to 5.2% after 8 h. This result corresponds to the degree of disease manifestation (classical CAH) in the patients in whom it was found.

View larger version (13K): [in this window] [in a new window]   Figure 2. Enzyme activity of steroid 21-OH (P450c21) wild-type enzyme and the mutants V281L and R426H after expression in intact COS-7 cells as described in Materials and Methods. Activities are expressed as a percentage of synthesis of 11-deoxycortisol from 17-hydroxyprogesterone, added to the culture medium. Values are means from two separate transfections.

We genotyped 79 unrelated Austrian CAH index patients with classical and nonclassical forms of CAH and 112 respective family members. The most frequent 21-OH defects were large gene deletions/conversions and point mutations present in the pseudogene, similar to previous reports (2, 6, 7, 8, 16, 26). In general, there is a good correlation between severe and mild phenotypes of CAH caused by 21-OH deficiency and the respective genotypes (2). Patients are, however, usually compound heterozygotes for mutations of different categories of enzymatic compromise, and thus there is a high degree of overlap in moderate and mild forms of CAH, reflected by the wide and heterogeneous spectrum of clinical manifestation. Evaluation of genotype/phenotype correlation has been extensively performed by others (6, 7, 8, 16, 27, 28) and was thus not attempted in this study. In addition, roughly estimated, 50% of our index patients were first referred to our department after they had reached adulthood. In these patients the precise determination of phenotype would be arbitrary at best.

We found that in the minority of CAH alleles carrying the P30L mutation, the aberration is based on a single base substitution. In the majority of cases, however, the P30L mutation is part of a larger gene deletion/conversion event with a junction site before intron 2, as only recently described for one patient by Morel (25). Such differences of P30L aberrations may escape identification unless Southern blot analysis (15, 26, 25), which is no longer routinely used (2), or specific PCR and sequence analyses (25, 29) are employed. Such differences might account for the nonconformity of P30L genotype/phenotype correlations (26, 28) and might be of relevance for a considerable number of patients. With the PCR amplification of exons 1–6, as described in this study, all cases of P30L mutations could be detected and resolved by DNA sequencing (index patients as well as the respective parents were studied).

Some 1.2% of the 168 unrelated CAH alleles did not carry previously described mutations, in line with the findings of earlier studies (2, 6, 7, 8, 26). Complete 21-OH sequence analysis in 1 index patient and her 2 affected sisters [all 3 of them presenting with classical CAH (Prader stage IV)] revealed that they carried a large gene deletion on the paternal and an as yet undescribed exchange mutation (Arg⁴²⁶His) on the maternal 21-OHB gene, leaving 0.6% of the 168 CAH alleles uncharacterized. Mutations of CG dinucleotides are known to be overrepresented in genetic disorders, through methylation of the cytosine followed by deamination, resulting in a CG to CT or CA transversion. This relates to the P450c21 mutations R339H, R341W, R356W, and R356Q as well as to the new one (R426H), all representing CG to CT or CG to CA substitutions. In summary, there is a clear clustering of missense mutations at the arginine residues, which are hypothesized to be involved in redox partner interaction (21).

When introduced into the enzyme and expressed in cultured cells, the Arg⁴²⁶His mutation completely abolished enzymatic activity toward 17-hydroxyprogesterone compared with the wild-type, set at 100%. The three sisters diagnosed with classical CAH after birth in 1959, 1965, and 1961, respectively, presenting with severe virilization of external genitalia (Prader stage IV, without salt-wasting crisis), have inherited the paternal allele carrying a large deletion of the 21-OHB gene and do not exhibit any other defect on the common maternal 21-OHB gene than the Arg⁴²⁶His mutation. The mutation is not associated with infertility, as one of the three sisters has given birth to a clinically asymptomatic daughter, who is, in turn, carrier of the Arg⁴²⁶His mutation.

Severe impairment of enzymatic function by this new mutation, as observed in in vitro expression experiments, is in line with the clinical manifestation observed in the patients in whom it was found. Such a marked effect of this base substitution and the implicated amino acid exchange was not unexpected, as Arg⁴²⁶His lies within a relatively highly conserved heme-binding peptide (residues 421–437) near the C-terminus. Mutation of the completely conserved Cys⁴²⁸, which is a ligand for the heme prosthetic group, has previously been found to destroy enzymatic activity (22). The R426H exchange has not yet been described by others as a disease causing CYP21B mutation or as a common CYP21A pseudogene sequence. Thus, it is tempting to speculate that its presence in the patients’ mutant CYP21B gene is not the result of a gene conversion. Such a hypothesis, however, remains to be verified by sequence analysis of the CYP21A pseudogene in a larger number of individuals.

It remains to be clarified, whether the R426H exchange mutation represents a gene conversion event and is responsible for a significant number of as yet uncharacterized 21-OH deficiency alleles.

Acknowledgments

We thank Angelika Freudenthaler, Karin Pölzler, and Hans-Peter Gensheimer for expert technical assistance.

Footnotes

Abbreviations: CAH, Congenital adrenal hyperplasia; HLA, human leukocyte antigen; NC, nonclassical; 21-OH, 21-hydroxylase; 17-OHP, 17-hydroxyprogesterone.

Received March 26, 2001.

Accepted June 16, 2001.

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