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Other entities represented in this entry:
HGNC Approved Gene Symbol: CYP21A2
Cytogenetic location: 6p21.33 Genomic coordinates (GRCh38) : 6:32,038,415-32,041,644 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 6p21.33 | Adrenal hyperplasia, congenital, due to 21-hydroxylase deficiency | 201910 | Autosomal recessive | 3 |
| Hyperandrogenism, nonclassic type, due to 21-hydroxylase deficiency | 201910 | Autosomal recessive | 3 |
The CYP21A2 gene encodes the 21-hydroxylase enzyme (EC 1.14.99.10), which is essential for adrenal steroidogenesis (summary by Araujo et al., 2007).
White et al. (1986) found that cDNA corresponding to 21-hydroxylase is 2 kb long. The encoded protein is predicted to contain 494 amino acids with a molecular weight of 55,000. The enzyme is at most 28% homologous to other cytochrome P450 enzymes that have been studied.
The gene encoding 21-hydroxylase contains 10 exons; the genes for other P450 enzymes contain 7, 8, or 9 exons. The inactive A gene has an 8-base deletion in codons 110 through 112, resulting in a frameshift that brings a stop codon into the reading frame at codon 130; a second frameshift and a nonsense mutation occur farther downstream. The two P450C21 genes have 9 introns and are about 3.4 kb long (Higashi et al., 1986).
Carroll et al. (1985) identified two 21-hydroxylase genes situated in the following relationship to C4A and C4B: 5-prime--C4A--21-OHA--C4B--21-OHB- -3-prime. White et al. (1985) presented evidence for the existence of 2 genes encoding steroid 21-hydroxylase in the C4 gene region, i.e., among the MHC class III genes. The order appears to be: centromere--GLO--DP--DQ--DR--C2--BF--C4A--21OHA--C4B--2 1OHB--B--C--A. The 21-hydroxylase B gene and the adjacent C4B gene appear to be deleted on the chromosome carrying HLA-Bw47 and the allele for salt-wasting 21-hydroxylase deficiency. In contrast, the chromosome carrying the HLA-A1;B8;DR3 haplotype is not associated with 21-hydroxylase deficiency and in the conclusions of White et al. (1985) based on restriction enzyme analysis may have a deletion of the C4A and 21OHA genes. This suggests that the latter is not functional. (In the human, the 21-hydroxylase B gene is functional; the A gene is missing 8 basepairs from exon 2. In the mouse, the 21-hydroxylase A gene is functional; the B gene is missing 215 basepairs from exon 2.)
The 21-hydroxylase pseudogene, symbolized CYP21P or CYP21A, is situated on 6p, close to the functional gene, CYP21. Higashi et al. (1986) suggested that this particular genomic anatomy predisposes the functional gene to mutation through gene conversion or through deletion by homologous recombination and unequal crossing-over.
Higashi et al. (1986) confirmed close linkage to C4 by finding that the cloned P450C21 genes hybridized with the 5-prime or 3-prime end regions of human C4 DNA. The P450C21 gene that is nonfunctional is identical to the other except for 3 mutations, each of which is capable of causing premature termination: a 1-base insertion, an 8-base deletion, and a transition mutation. Higashi et al. (1986) suggested that tandem arrangement of the highly homologous pseudo- and genuine genes in close proximity could account for the high incidence of P450C21 gene deficiency or defect through nonhomologous pairing and unequal crossing-over.
White et al. (1984) demonstrated that the mutations in the several forms of congenital adrenal hyperplasia due to 21-hydroxylase deficiency involve the structural gene for the adrenal microsomal cytochrome P450 specific for steroid 21-hydroxylation (EC 1.14.99.10). Rodrigues et al. (1987) pointed out that 21-hydroxylation was the first enzymatic activity ascribed to any cytochrome P450 (Cooper et al., 1965).
Rodrigues et al. (1987) determined the nucleotide sequence of the 21-hydroxylase B gene in a patient with congenital adrenal hyperplasia. Eleven nucleotide differences from the normal were found: 2 in the 5-prime flanking region, 4 in introns, 1 in the 3-prime untranslated region, and 4 in exons. Two of the differences in exons caused codon changes: serine-269 to threonine and asparagine-494 to serine. Rodrigues et al. (1987) confirmed that the 21-hydroxylase A gene is a pseudogene due to 3 deleterious mutations in the exons. Comparison of published sequences with those they determined suggested that the 21-hydroxylase B gene is polymorphic. They suggested, as had others, that the 4 distinct clinical forms of 21-hydroxylase deficiency (simple virilizing, salt-wasting, late-onset, and cryptic) may be the consequence of different allelic mutations in the 21-hydroxylase B gene.
Jospe et al. (1987) performed genomic restriction analysis of 14 unrelated patients with salt-losing congenital adrenal hyperplasia, identifying 3 patterns of mutation in the CA21HB gene: in 16 of the 28 chromosomes (or haplotypes) analyzed, there was no detectable restriction fragment abnormality suggesting that these were point mutations or small deletions or insertions. Complete deletion of CA21HB was found in 9 of 28 haplotypes (32%). In 3 of 28 haplotypes (11%), apparent conversion of CA21HB to the pseudogene CA21HA had occurred. Jospe et al. (1987) described how apparent gene conversion could be detected in the restriction fragment patterns. An alternative explanation to conversion is that unequal crossing-over occurred between a haplotype of 2 CA21HA genes and 1 CA21HB gene, and a normal haplotype to produce loss of the CA21HB gene from the first haplotype but retention of 2 CA21HA genes. CA21HB deletion was associated with HLA-Bw47 in 6 haplotypes and with absent C4B expression in 7 haplotypes of the 9.
In studies of DNA from 20 patients with 21-hydroxylase deficiency, Rumsby et al. (1986) found one homozygous for a deletion encompassing the C4B and 21-hydroxylase genes. They presented evidence that this originated by recombination between homologous regions of 21-hydroxylase A and B. No alteration in the 21-hydroxylase gene was detected in 12 patients. Seven patients appeared to be heterozygous for the above deletion; i.e., they were genetic compounds.
In molecular studies of the C4/21-hydroxylase genes in patients with the classic salt-wasting form, Schneider et al. (1986) found deletion of C4B and 21-hydroxylase B genes in some. In 2, only the 21-hydroxylase B gene was deleted. Werkmeister et al. (1986) found deletion of the active CA21H gene in almost one-fourth of classic cases of 21-hydroxylase deficiency, whereas mild 'nonclassic' 21-hydroxylase deficiency was associated with a duplicated CA21H gene.
Using multiple restriction enzymes in the analysis of the 21-hydroxylase gene in 10 families, each of which included 2 or more affected persons, Matteson et al. (1987) concluded that the 'deletions' that have been reported as a frequent finding in CAH patients probably represent gene conversions, unequal crossovers, and polymorphisms rather than simple gene deletions. Miller (1987) challenged the interpretation of a high frequency of gene deletion underlying 21-hydroxylase deficiency. White et al. (1987) defended their interpretation. They reiterated their view that probes for the closely linked and highly polymorphic HLA genes should be used for prenatal diagnosis, not CYP21 probes (Mornet et al., 1986).
By Southern blot analysis of genomic DNA using a 21-hydroxylase DNA probe, Harada et al. (1987) found an apparent absence of restriction fragments corresponding to the 21-hydroxylase B gene. They found that this apparent absence was not due to deletion of the gene but rather to a conversion of the functional 21-hydroxylase B gene into the nonfunctional 21-hydroxylase A pseudogene. In 2 patients studied, the affected HLA haplotypes were different, suggesting that conversion had occurred as independent events in the 2 instances. Harada et al. (1987) suggested that gene conversion-like events may be a relatively common cause of 21-hydroxylase deficiency in Japanese. They suggested that this mechanism might also account in part for the predominance of congenital adrenal hyperplasia due to 21-hydroxylase deficiency over that due to deficiency of other steroidogenic P450 enzymes. There may be other examples of gene conversion-like events that are responsible for monogenic disorders when related homologous genes reside in tandem array.
Baumgartner-Parzer et al. (2001) studied the mutational spectrum of 21-hydroxylase deficiency in 79 unrelated Austrian patients with classic and nonclassic forms of CAH and their respective 112 family members. Apparent large gene deletions/conversions were present in 31% of the 158 unrelated CAH alleles, whereas the most frequent point mutations were intron 2 splice (613815.0006; 22.8%), I172N (613815.0001; 15.8%), V281L (613815.0002; 12%), and P30L (613815.0004; 7.6%), in line with the frequencies reported for other countries. Previously described mutations were not present in 1.2% of unrelated CAH alleles, including those of one female patient presenting with severe genital virilization. Sequence analysis of the complete functional 21-hydroxylase gene revealed a novel mutation in exon 10, arg426 to his (R426H; 613815.0026). In vitro expression experiments showed that the R426H mutant exhibited only low enzyme activity toward the natural substrate 17-hydroxyprogesterone.
Olney et al. (2002) developed an assay using real-time quantitative PCR to detect deletions of CYP21A2. This assay was able to detect heterozygous gene deletions with an alpha error rate of less than 5% and with a power greater than 95%. When combined with allele-specific PCR, genotyping for the 9 most common mutations could be completed within hours of blood sampling. This technique was used to study subjects with 21-hydroxylase deficiency in north Florida. Twenty-eight subjects with CAH, 7 first-degree relatives, and 13 normal subjects were characterized. Of 96 chromosomes, 69 abnormal alleles were identified. Among unrelated abnormal alleles, the frequency of specific mutations was 28% for a gene deletion (613815.0011), 24% for the intron 2 splice mutation, 10% for I172N, 8% each for V281L and the exon 6 cluster (613815.0016), and 6% for gln318 to ter (Q318X; 613815.0020). These frequencies, as well as the genotype/phenotype correlation, were similar to those found in comparable populations.
Tukel et al. (2003) performed allele-specific PCR for the 8 most frequently reported CYP21 point mutations in 31 Turkish families having at least 1 21-hydroxylase-deficient individual. The allele frequencies of the point mutations were as follows: P30L (613815.0004), 0%; IVS2 (613815.0006), 22.5%; G110-delta-8nt (613815.0015) , 3.2%; I172N (613815.0001), 11.4%; exon 6 cluster (613815.0016), 3.2%; V281L (613815.0002), 0%; Q318X (613815.0020), 8%; and R356W (613815.0003), 9.6%. Large deletions and gene conversions were detected by Southern blot analysis, with allele frequencies of 9.6% and 22.5%, respectively. Sequence analysis of CYP21, performed on patients with only 1 mutant allele, revealed 2 missense mutations, R339H (613815.0021) and P453S (613815.0010). A semiquantitative PCR/enzyme digestion-based method for the detection of large-scale deletions/conversions of the gene was developed for routine diagnostic purposes, and its accuracy was shown by comparison with the results of Southern blot analysis.
To determine the mutational spectrum in the Tunisian CAH population, Kharrat et al. (2004) analyzed the CYP21 active gene in 51 unrelated patients using a strategy of digestion by restriction enzyme and sequencing. All patients had a classical form of 21-hydroxylase deficiency. Mutations were detected in over 94% of the chromosomes examined. The most frequent mutation in the Tunisian CAH population was Q318X (613815.0020), with large prevalence (35.3%), in contrast to the 0.5-13.8% described in other series. Incidence of other mutations did not differ, as had been described: large deletions (e.g., 613815.0011) (19.6%), mutation in intron 2 (613815.0006) (17.6%), and I172N (613815.0001) (10.8%). Four novel mutations were found in 4 patients with the salt-wasting form.
Sido et al. (2005) reported molecular analysis of 43 Romanian patients with classical CAH, 38 with 21-hydroxylase deficiency and 5 with 11-beta-hydroxylase deficiency. The most frequent mutation in patients with 21-hydroxylase deficiency was I2G (613815.0006) (43.9%), followed by deletions and large conversions (16.7%). Genotypes were categorized in 3 mutation groups according to their predicted functional consequences and compared with clinical phenotype. Overall genotype-phenotype correlation was 87.88%. In the 5 patients with 11-beta-hydroxylase deficiency, 3 homozygous mutations were identified.
Origin of Mutations
Mornet et al. (1991) estimated that gene conversions involving small DNA segments probably account for 74% of cases of 21-hydroxylase deficiency. Complete deletion of the CYP21B gene (613815.0011) accounted for about 20% of cases of the classic form of the disease. Complete deletion of CYP21B was associated with the salt-wasting form, as was an 8-bp deletion in the third exon (613815.0015). A G-to-T transversion in the seventh exon (613815.0002) was associated with the late-onset form of the disease. Ghanem et al. (1990) concluded that about 70% of the mutations in the CYP21B gene causing classic and nonclassic CAH are point mutations, because the defective gene was indistinguishable from its structurally intact corresponding gene in Southern blot analysis. Due to the presence of a varying number of C4/21-hydroxylase repeat units, this gene region varies in length among haplotypes. Haplotypes carrying one C4/21-hydroxylase repeat unit with a CYP21P gene transmit the severe form of 21-hydroxylase deficiency. Haglund-Stengler et al. (1991) found association between triplication of the C4/21-hydroxylase repeat unit and the mild form of 21-hydroxylase deficiency.
Gene conversion, a nonreciprocal exchange of homologous genetic information, has been studied extensively in lower eukaryotes in which all the products of a single meiosis can be recovered and analyzed. Because the latter is not possible in mammals, nonreciprocality of the genetic exchange cannot formally be demonstrated. Despite this limitation, the designation 'gene conversion' has been applied to exchanges observed in mammalian genomes that involve an alteration of an allele at a specific locus in such a way as to suggest that an internal portion of its sequence has been replaced by a homologous segment copied from another allele or locus. Gaucher disease (230800) is another example of a disorder in which conversion events occur between the functional gene and a neighboring pseudogene. Gene conversion has been postulated in other clustered gene families, including those for globins (e.g., 142200), immunoglobulins (e.g., 147070), red-green visual pigments (300822, 300821), and others. With the notable exception of the HLA genes, in which many of the presumed gene conversion events involve allelic exchanges, the postulated gene conversion events in the other systems involve interlocus exchange. The evidence for gene conversion in the human genome had been circumstantial until the description by Collier et al. (1993) of a de novo mutation which permitted the direct comparison of the 'converted' allele with its original form. They observed the de novo introduction of a CYP21A pseudogene-specific mutation into a CYP21B allele. Despite extensive investigations, not a single mutant CYP21B allele has been reported to lack pseudogene-specific mutations that are incompatible with normal gene expression. Consequently, the pathogenesis of 21-hydroxylase deficiency appears to be due almost exclusively to gene-pseudogene exchanges. Tajima et al. (1993) concluded that approximately 90% of the genes in patients with 21-hydroxylase deficiency are accounted for either by a causative mutation from the pseudogene or by a deletion and suggested that the remaining 10% may represent new mutations that do not exist in the pseudogene. Tajima et al. (1993) described a de novo mutation of the CYP21B gene causing CAH. HLA-identical affected and unaffected sibs were observed. Both inherited a missense mutation in exon 4 from the father, but only the affected sib received an intron 2 mutation that caused aberrant RNA splicing from the mother, who was homozygous normal.
White et al. (1994) reviewed mutations in the CYP21 gene which are responsible for more than 90% of cases of the inherited inability to synthesize cortisol. Most of the mutations in CYP21 causing CAH are generated by recombinations between CYP21 and CYP21P which either delete CYP21 or transfer deleterious mutations from CYP21P to CYP21.
Miller (1988) discussed gene conversion in relation to the monogenic form of adrenal hyperplasia. Higashi et al. (1988) presented evidence for either unequal intragenic or intergenic recombination and/or gene conversion events taking place between the pseudogene and the functional gene.
In 4 steroid 21-hydroxylase B mutations from three 21-hydroxylase-deficient patients, Higashi et al. (1988) observed several base changes as compared with the functional B gene. Many of these base changes were identical to those in the CYP21A pseudogene. Two of them were shown to have a point mutation in the second intron, causing aberrant splicing. A third carried 3 clustered missense mutations in the sixth exon, which impaired 21-hydroxylase activity. Since all of these critical mutations could be seen in the corresponding site of the CYP21A pseudogene, the data strongly suggested the involvement of gene conversion in this genetic disease.
Using a genomic probe, Morel et al. (1989) defined 5 haplotypes that identified the mutations in 57 families. Specifically, of 116 CAH-bearing chromosomes, 114 could be sorted into 1 of these 5 haplotypes, based on blots of DNA digested with TaqI and BglII. Haplotype 1, present in 65.6%, was indistinguishable from the normal, and therefore bore very small lesions, presumably point mutations. Haplotype 2, present in 3.4%, and haplotype 3, present in 6.9%, had deletions and duplications of the CYP21 pseudogene but had especially intact functional genes, presumably bearing point mutations. Thus, point mutation was the genetic defect in 75.9% of the chromosomes. Haplotypes 4 and 5, present in 11.2%, appeared to represent a gene that had undergone a gene conversion event. Haplotype 5, present in 11.2%, appeared to have a deletion of about 30 kb of DNA, resulting in a single hybrid CYPA/B gene.
Donohoue et al. (1989) concluded that a single unequal crossing-over between the CYP21A and CYP21B genes yields deletion of the latter active gene to result in salt-losing CAH; furthermore, these crossovers do not occur randomly within the complex. In a patient with 21-hydroxylase deficiency, Sinnott et al. (1990) demonstrated a maternally inherited haplotype that carried a de novo deletion of an approximately 30-kb segment including the CYP21B gene and the associated C4B gene. The disease haplotype appeared to have been generated through meiotic unequal crossing-over. One of the maternal haplotypes was the frequently occurring HLA-DR3,B8,A1 haplotype that normally carries a deletion of an approximately 30-kb segment including the CYP21A gene and C4A gene. Haplotypes of this type may act as premutations, increasing the susceptibility to development of a 21-hydroxylase deficiency mutation by facilitating unequal chromosome pairing. Mutations in the pseudogene CYP21A include a C-to-T change that leads to a termination codon, TAG, in the eighth exon. Urabe et al. (1990) found that same change in a mutant CYP21B gene isolated from a patient with 21-hydroxylase deficiency. Furthermore, a reciprocal change, i.e., a T-to-C change in the eighth exon of the CYP21A gene, was observed in the Japanese population. This was considered evidence for gene conversion.
Wu and Chung (1991) studied the effects of induced missense mutations at cysteine-428, valine-281, and serine-268 of the 21-hydroxylase gene. A ser268-to-thr mutation (613815.0005) had been found in a patient suffering from CAH and a val281-to-leu mutation (613815.0002) was identified in a patient with nonclassic CAH characterized by partial enzyme deficiency. Cysteine-428 is the invariant cys among all cytochrome P450s and is presumed to be the heme ligand. Wu and Chung (1991) mutated ser268 to thr, cys, and met to see if these changes altered the function of 21-hydroxylase. They changed val281 to leu, ile, and thr, similarly, to study the effects on structure and function of 21-hydroxylase. Val, leu, and ile share properties; therefore, substituting one with another should not drastically disturb the structure of the protein. Wu and Chung (1991) changed cys428 to thr, met, and ser to study the effects of these mutations. They found that the cys428, val281, and ser268 mutations resulted in complete, partial, or no loss of enzymatic activity, respectively. 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-to-thr change represents a polymorphism rather than the cause of enzyme deficiency.
Tusie-Luna and White (1995) pointed out that steroid 21-hydroxylase deficiency is unusual among genetic diseases in that approximately 95% of the mutant alleles have apparently been generated by recombination between a normally active gene (CYP21) and a closely linked pseudogene (CYP21P). Approximately 20% of mutant alleles carry DNA deletions of 30 kb that have presumably been generated by unequal meiotic crossing-over, whereas 75% carry one or more mutations in CYP21 that are normally found in the CYP21P pseudogene. These latter mutations are termed 'gene conversions.' To assess the frequency at which these different recombination events occur, Tusie-Luna and White (1995) used PCR to detect de novo deletions and gene conversions in matched sperm and peripheral blood leukocyte DNA samples from normal persons. Deletions with breakpoints in a 100-bp region in intron 2 and exon 3 were detected in sperm DNA samples with frequencies of approximately 1 in 10(5)-10(6) genomes but were not detected in the matching leukocyte DNA. Gene conversions in the same region occurred in approximately 1 in 10(3)-10(5) genomes in both sperm and leukocyte DNA. These data suggested to the authors that whereas deletions occur exclusively in meiosis, gene conversions occur during both meiosis and mitosis, or perhaps only during mitosis. Thus, the authors concluded that gene conversions must occur by a mechanism distinct from unequal crossing-over.
Araujo et al. (2007) studied the CYP21A2 promoter/regulatory regions in 17 patients with the nonclassic form of 21-hydroxylase deficiency with undetermined genotype and 50 controls. Promoter mutations were found in compound heterozygosity with the V281L mutation in 1 patient and with the I2 splice mutation in another. The authors concluded that microconversions between CYP21A2 and CYP21A1P promoters could be involved in the nonclassic form and that CYP21A2 promoter analysis should be included in genetic studies of the disorder.
CYP21A2, the adjacent complement C4A gene, and parts of the flanking genes serine/threonine protein kinase-19 (STK19; 604977) and tenascin-X (TNXB; 600985) constitute a tandemly duplicated arrangement. The typical number of repeats of the CYP21/C4 region is 2, with 1 repeat carrying CYP21A2 and the other carrying the highly homologous pseudogene CYP21A1P (see 613815.0012). Koppens et al. (2002) determined that apparent large-scale conversions accounted for the defect in 9 of 77 chromosomes in a group of patients with CAH due to steroid 21-hydroxylase deficiency. They further showed that 4 of the 9 'conversions' extended into the flanking TNXB gene. This implies that 1 in every 10 steroid 21-hydroxylase deficiency patients is a carrier of tenascin-X deficiency, which is associated with a recessive form of the Ehlers-Danlos syndrome (606408). Koppens et al. (2002) stated that data on the structure of 'deletion' and 'large-scale conversion' chromosomes strongly suggest that both are the result of the same mechanism, namely unequal meiotic crossover.
Baumgartner-Parzer et al. (2007) identified 2 unrelated female patients with CAH who inherited the intron 2 splice mutation (613815.0006) from their father and harbored a de novo gene aberration on their maternal haplotype, a large deletion in one and the I172N mutation (613815.0001) in the other. Both mothers were found to be carriers of rare duplicated CYP21A2 haplotypes, which were not detected in the daughters. Baumgartner-Parzer et al. (2007) hypothesized that duplicated CYP21A2 genes could predispose for de novo mutations in offspring, which is relevant for prenatal CYP21 genotyping and genetic counseling.
Lopez-Gutierrez et al. (1998) studied 47 Mexican families with 21-hydroxylase deficiency. In 9 families they failed to detect the mutation found in the proband in either parent; paternity was established in all cases. In 1 individual, paternal uniparental disomy for 6p was established, and Lopez-Gutierrez et al. (1998) hypothesized that germline mutations might explain the segregation pattern in the remaining 8 families.
Corticosteroids have specific effects on cardiac structure and function mediated by mineralocorticoid and glucocorticoid receptors (MR and GR (138040), respectively). Aldosterone and corticosterone are synthesized in rat heart. To see whether they might also be synthesized in the human cardiovascular system, Kayes-Wandover and White (2000) examined the expression of genes for steroidogenic enzymes as well as genes for GR, MR, and 11-hydroxysteroid dehydrogenase (HSD11B2; 614232), which maintains the specificity of MR. Human samples were from left and right atria, left and right ventricles, aorta, apex, intraventricular septum, and atrioventricular node, as well as whole adult and fetal heart. Using RT-PCR, mRNAs encoding CYP11A, CYP21, CYP11B1 (610613), GR, MR, and HSD11B2 were detected in all samples except ventricles, which did not express CYP11B1. CYP11B2 (124080) mRNA was detected in the aorta and fetal heart, but not in any region of the adult heart, and CYP17 was not detected in any cardiac sample. Levels of steroidogenic enzyme gene expression were typically 0.1% those in the adrenal gland. The authors concluded that these findings are consistent with autocrine or paracrine roles for corticosterone and deoxycorticosterone, but not cortisol or aldosterone, in the normal adult human heart.
In each of 2 women with hyperandrogenism (see 201910), Lajic et al. (2002) identified a novel missense mutation in the CYP21 gene (613815.0031 and 613815.0032). The women were predicted to carry mutations by hormonal evaluation, but did not display any of the genotypes commonly associated with congenital adrenal hyperplasia. The authors studied the functional and structural consequences of the mutations, and their results emphasized the importance of genetic evaluation and counseling in hyperandrogenic women who are predicted to carry CAH-causing mutations by biochemical tests.
The CYP21 pseudogene has 3 main defects: an 8-bp deletion in exon 3, a T insertion in exon 7, and a stop codon in exon 8. Kawaguchi et al. (1992) demonstrated that the 8-bp deletion is present in the chimpanzee also, whereas the other 2 defects are not found in the chimpanzee, gorilla, or orangutan. In the gorilla and orangutan, however, extra CYP21 copies are inactivated by other defects so that the number of functional copies is reduced in each species. Comparison of the sequences revealed evidence for intraspecific homogenization (concerted evolution) of the CYP21 genes, presumably through an expansion-contraction process effected by relatively frequent unequal but homologous crossing-over.
Most mutations in the CYP21 gene causing congenital adrenal hyperplasia (201910) are deletions. Amor et al. (1988) reported the cloning and characterization of a nondeleted mutant CYP21B gene. Codon 172 of the mutant gene was found to be changed from ATC, encoding isoleucine, to AAC, encoding asparagine. This mutation (I172N) is normally present in the CYP21A pseudogene, so that it may have been transferred to the mutant CYP21B gene by gene conversion. Hybridization of oligonucleotide probes corresponding to this and 2 other mutations normally present in CYP21A demonstrated that 4 out of 20 patients carried the codon 172 mutation; in 1 of these patients, the mutation was present as part of a larger gene conversion involving at least exons 3-6. Gene conversion may be a frequent cause of 21-hydroxylase deficiency alleles due to the presence of 6 chi-like sequences (GCTGGGG) in the CYP21 genes and the close proximity of the CYP21A pseudogene, which has several potentially deleterious mutations. Chiou et al. (1990) also found this mutation on 1 allele in a compound heterozygote. Partanen and Campbell (1991) amplified the full-length genomic P450C21 gene by PCR. The ile172-to-asn mutation in exon 4 was demonstrated. This mutation was observed also by Wedell et al. (1992), who referred to it as ILE173ASN. Speiser et al. (1992) found this mutation in 16% of 88 families with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. The mutation falls into their group B with 2% enzyme activity and a simple virilizing phenotype. Among 127 patients with 21-hydroxylase deficiency in Sweden, Wedell et al. (1994) found that the ile173-to-asn mutation accounted for 20.8% of 186 unrelated chromosomes. (In the same study, the CYP21 gene was completely absent in 29.8% of chromosomes, the val281-to-leu mutation accounted for 5.4%, and the arg356-to-trp mutation accounted for 3.8%. The most frequent nondeletional mutation was the splice mutation in intron 2, which accounted for 27.7% of the chromosomes.) This mutation is found in 28% of all the cases of simple virilizing type (White et al., 1994). To clarify the molecular basis of nonclassic CAH detectable by neonatal screening in Japan, Tajima et al. (1997) identified 2 sibs and 2 unrelated newborns who were diagnosed with probable nonclassic steroid 21-hydroxylase deficiency. The 2 sibs were found to have 1 allele that had 2 mutations, ile172 to asn and arg356 to trp.
In 9 patients with nonclassic 21-hydroxylase deficiency (201910) associated with HLA-B14;DR1, Speiser et al. (1988) found a change in codon 281 from GTG, encoding valine, to TTG, encoding leucine. Speiser et al. (1989) concluded that this codon 281 mutation is a consistent change in nonclassic 21-hydroxylase deficiency associated with HLA-B14;DR1. The val281-to-leu mutation (V281L), found in association with the HLA-B14;DR1 haplotype, accounts for 75 to 80% of nonclassic 21-hydroxylase deficiency (Mornet et al., 1991). This mutation was observed in several patients by Wedell et al. (1992), who referred to it as VAL282LEU.
In an analysis of steroid 21-hydroxylase gene mutations in the Spanish population, Ezquieta et al. (1995) found that the most frequent mutation causing the late onset form of disease (present in 15 of 38 patients) was val281 to leu, found in 18 of 30 chromosomes (37%). This mutation is found in 34% of all cases of the nonclassic type (White et al., 1994).
In samples from 2 patients (1 with a cortisol-producing adenoma and 1 with an androgen-secreting adrenocortical carcinoma), Beuschlein et al. (1998) detected the heterozygous germline mutation val281 to leu in exon 7.
In a patient with simple virilizing CAH (201910) who was a compound heterozygote for CYP21A2 mutations, Chiou et al. (1990) found a CGG-to-TGG change in 1 allele resulting in substitution of a tryptophan residue for arginine-356 (R356W). Mutants corresponding to this and the ile172-to-asn (I172N; 613815.0001) allele were constructed from the normal CYP21 cDNA by site-directed mutagenesis. Both mutations failed to produce active enzyme. This mutation was also observed by Wedell et al. (1992), who referred to it as ARG357TRP. Tajima et al. (1997) analyzed CYP21 genes for nonclassic steroid 21-hydroxylase deficiency. The 4 patients tested (2 sibs and 2 unrelated newborns) carried the R356W mutation.
The mild nonclassic form of steroid 21-hydroxylase deficiency (201910) 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 val281-to-leu (V281L) 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, Tusie-Luna et al. (1991) used recombinant vaccinia virus to express 2 mutant enzymes carrying the mutations pro30 to leu (normally present in CYP21P) and ser268 to thr (considered a normal polymorphism of CYP21; see 613815.0005). Whereas the activity of the protein carrying the ser-to-thr mutation was indeed indistinguishable from the wildtype, the enzyme with the pro-to-leu substitution had 60% of the wildtype activity for 17-hydroxyprogesterone and about 30% of normal activity for progesterone when assayed in intact cells. Proline-30 is conserved in many microsomal P450 enzymes and may be important for proper orientation of the enzyme with respect to the amino-terminal transmembrane segment. The pro30-to-leu mutation was present in 5 of 18 patients with nonclassic 21-hydroxylase deficiency. Tajima et al. (1997) observed the P30L mutation in 1 allele in 3 of 4 patients (2 sibs and 2 unrelated newborns) with nonclassic CAH in Japan.
Rodrigues et al. (1987) identified a substitution of threonine for serine-268 in 21-hydroxylase (S268T) in a patient with congenital adrenal hyperplasia. Wu and Chung (1991) reported studies of induced mutations changing ser268 to thr, cys, and met. All of these 268-mutants exhibited the same activity as normal 21-hydroxylase, demonstrating that the ser268-to-thr change clinically represents a polymorphism rather than the cause of the enzyme deficiency.
The most frequent nondeletional mutation found in patients with classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency (201910) is an A-to-G transition at position -2 in the acceptor splice site of intron 2. As a result of the mutation, an aberrant splice acceptor site is activated 7 bases upstream of the mutation (Higashi et al., 1988). As pointed out by Miller (1996), this mutation, located in intron 2, is 13 bases (not 2) from the splice acceptor site of exon 3. According to the nucleotide numbering system of Higashi et al. (1988), it is residue 655. Miller (1996) noted that this base is normally polymorphic, being either C or A with roughly equal frequency in the normal population. Either a C-to-G or A-to-G mutation at nucleotide -13 causes the severe 21-OH deficiency.
This mutation has been detected in patients affected with either the salt-wasting or simple virilizing forms of the disorder (Owerbach et al., 1990; Mornet et al., 1991). White et al. (1994) reported that this mutation represents 22% of the salt-wasting cases, 25% of the simple virilizing cases, and 12% of the nonclassic cases.
As reported by Hirschfeld and Fleshman (1969) and Pang et al. (1982), the Yupik Eskimos of western Alaska have the world's highest prevalence of HLA-linked classic congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency. The frequency was estimated to be between 1 in 282 and 1 in 490 liveborn infants. Studying 4 patients from 3 apparently unrelated Eskimo families residing in geographically distant villages, Speiser et al. (1992) found that all were homozygous for a substitution of G for A at base 656 in the second intron. They concluded that allele-specific hybridization should be an efficient means of prenatal diagnosis in this isolated population.
In the Spanish population, Ezquieta et al. (1995) found this splicing mutation in 30% of 41 mutant chromosomes, making it the most frequent cause of severe CAH in this population. They stated the mutation as an A-to-G change at nucleotide 655 of their clone. During the course of genetic analysis of CYP21 mutations in CAH families,
Day et al. (1996) noticed a number of relatives genotyped as nucleotide 656G homozygotes who showed no clinical signs of disease. They proposed that the putative asymptomatic nucleotide 656G/G individuals are incorrectly typed due to a dropout of 1 haplotype during PCR amplification of CYP21. They recommended that for prenatal diagnosis, microsatellite typing be used as a supplement to CYP21 genotyping in order to resolve ambiguities at nucleotide 656.
Lee et al. (2003) noted that approximately 75% of defective CYP21 genes that cause CAH are generated through intergenic recombination, termed apparent gene conversion, from the neighboring CYP21P pseudogene. Among them, the common intron 2 splice site mutation, which Lee et al. (2003) designated IVS2-12A/C-G, is believed to be derived from this mechanism and is the most prevalent case among all ethnic groups. However, mutation of 707-714delGAGACTAC (613815.0015) rarely exists alone, although this locus is 53 nucleotides away from IVS2-12A/C-G. From the molecular characterization of the mutation of IVS2-12A/C-G combined with 707-714delGAGACTAC in patients with congenital adrenal hyperplasia, Lee et al. (2003) found that it appeared to be in a 3.2- rather than a 3.7-kb fragment generated by Taq I digestion in a PCR product of the CYP21 gene. Interestingly, the 5-prime end region of such a CYP21 haplotype had CYP21P-specific sequences. The authors concluded that the coexistence of these 2 mutations is caused by deletion of the CYP21P, XA (TNXA; see 600985), RP2 (pseudogene of STK19, 604977), and C4B (120820) genes and intergenic recombination in the C4-CYP21 repeat module.
Among 370 unrelated alleles from patients in the Netherlands with 21-hydroxylase deficiency, Stikkelbroeck et al. (2003) found this to be the most common point mutation, occurring in 28.1% of alleles. They referred to the mutation as I2G (IVS2-13A/C-G; 656A/C-G).
Wedell et al. (1992) developed selective PCR amplification and direct sequencing of the full-length CYP21 gene and thereby identified 3 previously unknown mutations. One of them, in a patient with severe steroid 21-hydroxylase deficiency (201910), represented a substitution of serine for glycine-292 (G292S). The mutation was the result of a G-to-A transition at nucleotide 1718 in exon 7. The patient was 1 of 20 hemizygous patients, i.e., patients with only 1 copy of the functional CYP21 gene.
In a patient with severe steroid 21-hydroxylase deficiency (201910), Wedell et al. (1992) identified a change of a GG dinucleotide to a C in exon 10, resulting in a frameshift at arginine-484 and a predicted protein with 57 additional amino acids in the C-terminal end.
In 2 sibs with the late-onset form of 21-hydroxylase deficiency (201910) manifested by pseudoprecocious puberty, growth acceleration, and clitoral enlargement at ages 8 and 10 years, Wedell et al. (1992) identified hemizygosity (i.e., only 1 functional CYP21 gene was present) for 3 sequence changes: C to T at 4 bases upstream of translation initiation, pro106 to leu, and pro454 to ser. Since pro454 is conserved in 4 species, it is likely to be important for normal enzyme function. (White et al. (1994) later referred to the pro106-to-leu substitution as pro105 to leu, and Owerbach et al. (1992) referred to the pro454-to-ser substitution as pro453 to ser.)
Nikoshkov et al. (1997) tested the function of the -4, pro105-to-leu, and pro453-to-ser mutations by in vitro translation after expression of the mutant enzymes in cultured cells. While the -4 substitution had no measurable effect, the pro105-to-leu and pro453-to-ser mutations reduced enzyme activity to 62% and 68% for 17-hydroxyprogesterone and 64% and 46% for progesterone, respectively. When present in combination, these 2 mutations caused a reduction of enzyme activity to 10% for 17-hydroxyprogesterone and 7% for progesterone. These results indicated that pro105-to-leu and pro453-to-ser alleles should only cause very subtle disease when not in combination but may be considered when genotyping patients with the mildest forms of CAH1.
Using PCR in a study of the structure of the CYP21 gene in 13 unrelated nonclassic steroid 21-hydroxylase deficiency (201910) patients, 3 affected sibs, and 55 blood donors, Owerbach et al. (1992) found the val281-to-leu (613815.0002) and pro30-to-leu (613815.0004) mutations, as well as a pro453-to-ser (P453S) mutation in exon 10. The P453S mutation was identified in 46.2% of unrelated nonclassic CAH patients, but only 7.7% and 3.6% of salt-wasting CAH patients and blood donors, respectively. In contrast to the other 2 'nonclassic' mutations, pro453 to ser had not been detected in the CYP21 pseudogene and, therefore, probably had not arisen by gene conversion.
Soardi et al. (2008) found that P453S and another nonclassic mutation, H62L (613815.0034), had a synergistic interaction. When the mutant proteins were expressed together in COS cells, the activity of the enzyme was reduced to 4.1% and 2.3% toward 17OHP and progesterone, respectively. Two unrelated patients who both carried P453S+H62L on the paternal allele had a mild simple virilizing phenotype.
In 13 patients with congenital adrenal hyperplasia (201910), White et al. (1988) identified a deletion of approximately 30 kb, leaving behind the C4A gene (encoding the fourth component of complement; 120820) and a single CYP21P-like gene. The deletion prevents the synthesis of the protein and destroys all enzymatic activity. This mutation is very common and is found in 29% of all the salt-wasting cases.
Higashi et al. (1988) discovered that the CYP21P genes in 11 patients with congenital adrenal hyperplasia (201910) seemed to be replaced frequently in their 3-prime portions by the CYP21 gene sequences. All of these alterations occurred without changing the characteristic length (3.2 kb) of the TaqI fragment of the CYP21P gene, a result strongly suggesting that frequent gene conversions and/or intragenic recombinations have happened in the P-450 (C21) genes. This mutation results in a salt-wasting type and destroys all enzymatic activity. Gene conversions were observed in 8 normal individuals, suggesting that the resulting gene sequences do not always contain deleterious mutations from the CYP21 pseudogene.
Rodrigues et al. (1987) identified an insertion of CTG in exon 1 of the CYP21A2 gene at nucleotide position 28 coding for a leucine-10. This insertion has no effect on the enzymatic activity. This mutation is normally present in the CYP21 pseudogene.
Rodrigues et al. (1987) identified an A-to-G change at nucleotide 683 in exon 3 of the CYP21A2 gene, resulting in a substitution of arginine for tyrosine-102 (Y102R). There is normal enzymatic activity associated with this polymorphism.
By hybridization with specific oligonucleotide probes, White et al. (1988) showed an 8-bp deletion of nucleotides 707-714 in exon 3, typical of CYP21P, which prevents synthesis of the protein by a frameshift and causes the salt-wasting type of congenital adrenal hyperplasia. This mutation is present in about 8% of the salt-wasting CAH (201910) cases.
See 613815.0006 and Lee et al. (2003).
In a patient with the salt-wasting form of congenital adrenal hyperplasia caused by 21-hydroxylase deficiency (201910), Higashi et al. (1988) identified a cluster mutation in exon 6 of the CYP21A2 gene (ILE235ASN (I235N), VAL236GLU (V236E), and MET238LYS (M238K)). Each of these substitutions was caused by a T-to-A transversion at nucleotide position 1380, 1383, and 1389, respectively. This mutation was presumed to have arisen in a gene conversion event.
Tusie-Luna et al. (1990) expressed the exon 6 cluster mutation at high levels in cultured COS-1 cells using recombinant vaccinia virus to determine its functional effect. They found that this mutation had no detectable enzymatic activity.
Robins et al. (2005) excluded the M239K mutation as a disease-causing mutation in this cluster by demonstrating that it has no effect on enzyme activity. V237E abolished enzyme function and is thus a null mutation, whereas very low but measurable activity remained for I236N.
In a severely affected 21-hydroxylase deficiency (201910) patient, Wedell and Luthman (1993) identified a G-to-C substitution at nucleotide 177, the first nucleotide of the donor splice site of intron 7, resulting in abnormal splicing. This mutation was found in compound heterozygosity with a premature termination mutation (613815.0022).
Globerman et al. (1988) identified a T-to-C substitution at nucleotide 1994 in exon 8 of the CYP21A2 gene, resulting in a stop codon at position 318 (Q318X). Individuals homozygous for this mutation have the salt-wasting form of 21-hydroxylase deficiency (201910) and no enzymatic activity. This mutation is normally present in the CYP21 pseudogene.
In a Spanish population, Ezquieta et al. (2002) provided data on the contributions of gene conversion and founder effect to the distribution of the 2 most frequent severe point mutations of the CYP21A2 gene causing congenital adrenal hyperplasia: the 655G splicing mutation at intron 2 (613815.0006) and gln318-to-ter. Both mechanisms were found to contribute to the mutant alleles in different degrees. The 655G splicing mutation (accounting for 15.5% of alleles) seemed to be almost exclusively related to recent conversion events, whereas Q318X (accounting for 8.3% of alleles) was more likely to be due to the dissemination of remotely generated mutant alleles.
In a patient with a mild, nonclassic form of 21-hydroxylase deficiency (201910), Helmberg et al. (1992) 1 allele that carried 2 missense mutations in the CYP21A2 gene, R339H and P453S (see 613815.0010). The substitution of histidine for arginine-339 resulted from a G-to-C change at nucleotide 2058 in exon 8. The enzymatic activity associated with this mutation is lowered to 30 to 60% of normal.
In a patient with salt-wasting 21-hydroxylase deficiency (201910), Wedell and Luthman (1993) identified an A-to-G substitution at nucleotide 2339 in exon 9 of the CYP21A2 gene, causing a stop codon at position 406 (W406X).
In an HLA-homozygous patient with salt-losing congenital adrenal hyperplasia due to 21-hydroxylase deficiency (201910), Kirby-Keyser et al. (1997) demonstrated homozygosity for an E380D mutation in the CYP21 gene. Both parents and 1 sib were heterozygous for this mutation. E380D had not been identified in any pseudogenes, suggesting that the mutation had arisen through conventional means and not by gene conversion or similar mechanisms related to the neighboring pseudogene.
Billerbeck et al. (1999) sequenced the entire CYP21 gene of a Brazilian mulatto patient with the simple virilizing form of congenital adrenal hyperplasia (201910) who had the R356W mutation (613815.0003) in a heterozygous state. They identified a heterozygous G-to-A transition at nucleotide 2494, resulting in a gly424-to-ser (G424S) substitution in a region where glycine is conserved in at least 4 species. Overall, the gly424-to-ser mutation was found in a compound heterozygous state in 5 Brazilian families; 4 presented the simple virilizing form, and 1 presented the nonclassic form. Interestingly, 3 of the 5 families had a mulatto origin. All patients with the gly424-to-ser mutation had CYP21P and C4A (120810) gene deletions and human leukocyte antigen DR17 on the same haplotype, suggesting linkage disequilibrium and a probable founder effect.
In a female index patient and her 2 sisters presenting with classical congenital adrenal hyperplasia and severe genital virilization (201910), Baumgartner-Parzer et al. (2001) found hemizygosity for an arg426-to-his (R426H) mutation in the maternal CYP21B gene, resulting from a G-to-A transition in exon 10. The patients were compound heterozygous for a large gene deletion of the CYP21B (paternal) and CYP21A (maternal) genes. One of the 3 sisters had given birth to a daughter who was a clinically asymptomatic carrier of the R426H mutation. In vitro expression experiments showed the R426H mutant to exhibit only low enzyme activity toward the natural substrate 17-hydroxyprogesterone.
Genotyping of 41 Brazilian patients with the classical form of 21-hydroxylase deficiency revealed 64% microconversion, whereas deletions and large gene conversions accounted for up to 21% of the molecular defect (Araujo et al., 1996; Paulino et al., 1999). Lau et al. (2001) reported a novel mutation disclosed by sequencing the entire CYP21 gene of a patient in whom no pseudogene-originated mutation had been found. The patient, who had the classical form of 21-hydroxylase deficiency (201910), was the daughter of a consanguineous marriage; she was homozygous for a novel frameshift, an insertion of a cytosine between nucleotides 82 and 83, within exon 1. The mutation caused conversion of codon 28 from histidine to proline and premature termination at amino acid 78.
In 3 unrelated Brazilian patients with the classic form of the 21-hydroxylase deficiency (201910), Billerbeck et al. (2002) found 3 novel mutations after CYP21 gene sequencing. In 1 patient and her brother, both affected with the simple virilizing form, and in their aunt, with the nonclassic form, an AG-to-GG transition was found in the acceptor site of intron 2. In the sibs, this mutation was found in compound heterozygosity with the I172N mutation (613815.0001); in their aunt, it was found in compound heterozygosity with P30L (613815.0004), which confers more than 30% enzyme activity, explaining why she presented with the nonclassic form. In another patient with the salt-wasting form, they found an insertion of an adenine between nucleotides 1003 and 1004, in exon 4, that altered the reading frame and created a stop codon at codon 297 (613815.0029). In the third patient and his sister, they found a C-to-T transition in codon 408 predicted to encode an arg408-to-cys (R408C) substitution in a region where arginine is conserved in at least 4 different species. Microsatellite studies, using markers flanking CYP21 gene, revealed that each new mutation presents the same haplotype, suggesting a gene founder effect for each one.
For discussion of the 1-bp insertion (1003_1004insA) in exon 4 of the CYP21A2 gene that was found in compound heterozygous state in a patient with the salt-wasting form of 21-hydroxylase deficiency (201910) by Billerbeck et al. (2002), see 613815.0028.
For discussion of the arg408-to-cys (R408C) mutation in the CYP21A2 gene that was found in compound heterozygous state in 2 sibs with the salt-wasting form of 21-hydroxylase deficiency (201910) by Billerbeck et al. (2002), see 613815.0028.
In a woman with hyperandrogenism (201910), Lajic et al. (2002) identified a novel homozygous val304-to-met (V304M) mutation in the CYP21 gene. After expression in COS-1 cells, the mutated enzyme was found to have a residual activity of 46% for conversion of 17-hydroxyprogesterone and 26% for conversion of progesterone compared with the normal enzyme. A normal degradation pattern for this mutant enzyme indicated that the mutation is of functional, rather than structural, importance.
In a woman with signs of hyperandrogenism (201910), Lajic et al. (2002) identified a novel gly375-to-ser (G375S) mutation in the CYP21 gene in heterozygous state with a pro453-to-ser (P453S; 613815.0010) mutation, which is known to cause nonclassic CAH. The G375S variant almost completely abolished enzyme activity; conversion was 1.6% and 0.7% of normal for 17-hydroxyprogesterone and progesterone, respectively.
Stikkelbroeck et al. (2003) found a clustering of pseudogene-derived mutations in exons 7 and 8 of the CYP21A2 gene (val281 to leu, a 1-bp insertion after codon 306, gln318 to ter, and arg356 to trp) in 7 of 370 unrelated alleles (1.9%) from a population of Dutch patients with 21-hydroxylase deficiency (201910). This cluster had been reported by Koppens et al. (2000) in 2 Dutch patients (2 of 75 unrelated alleles) and by Wilson et al. (1995) in 2 patients (2 of 394 alleles). Stikkelbroeck et al. (2003) suggested that this cluster may be specific to the Dutch population and may be attributable to a common founder.
Of 60 novel mutations in CYP21 identified in a screen of 2,900 patients with steroid 21-hydroxylase deficiency (201910), Menassa et al. (2008) found that a his-to-leu substitution at codon 62 (H62L) was the most frequent. The H62L substitution, which arises from an A-to-T transversion at nucleotide 185 in exon 1 of the CYP21 gene, was found in 13 patients from 12 unrelated families, either isolated or associated on the same allele with a mild mutation. In isolation, or when associated with a partial conversion of the promoter, the H62L mutation was responsible for a nonclassic form; associated with the P453S (613815.0010) or P30L (613815.0004) mutations, H62L contributed to a simple virilizing phenotype more severe than that associated with P453S or P30L alone, but not as severe as the phenotype associated with I172N (613815.0001). Analysis of a 3-dimensional model structure of the CYP21 protein localized the H62L mutation to the beta-1-sheet region, in a large hydrophobic area considered important for membrane anchoring.
Soardi et al. (2008) found that the H62L mutant protein showed an activity compatible with a nonclassic mutation in functional assays. Determination of apparent kinetic constants revealed that the substrate binding capacity was in the same magnitude for mutant and normal enzyme. Soardi et al. (2008) found that the H62L mutation was associated with other mutations in both Brazilian and Scandinavian patients. In the Scandinavian patients H62L was associated on the paternal allele with the nonclassic P453S (613815.0010) mutation. In vitro activity data revealed a synergistic effect of the H62L+P453S mutation, which may explain the mild simple virilizing phenotype in these patients.
In a female patient with nonclassic 21-hydroxylase deficiency (201910), Riepe et al. (2008) detected heterozygosity for a novel mutation in the CYP21A2 gene, a 364A-C transversion in exon 3 resulting in a lys121-to-gln substitution (K121Q). This mutation was present on the maternal allele; the paternal allele carried a P453S mutation (613815.0010). In vitro expression analysis of the mutant K121Q enzyme in transiently transfected COS-7 cells revealed reduced CYP21 activity of approximately 14.0% for the conversion of 17-hydroxyprogesterone and 19.5% for the conversion of progesterone. K121 is located on helix C in the CYP21 protein, which is part of the heme coordinating system. In addition, helix C is involved in the interaction with the electron-providing enzyme P450 oxidoreductase (124015). Riepe et al. (2008) hypothesized that the K121Q mutation impairs electron flux between P450 oxidoreductase and CYP21 and alters substrate affinity by displacing the heme coordination site.
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