Gene conversion in steroid 21-hydroxylase genes.

The steroid 21-hydroxylase gene, CYP21B, encodes cytochrome P450c21, which mediates 21-hydroxylation. The gene is located about 30 kb downstream from pseudogene CYP21A. The CYP21A gene is homologous to the CYP21B gene but contains some mutations, including a C----T change which leads a termination codon, TAG, in the eighth exon. We found the same change in a mutant CYP21B gene isolated from a patient with 21-hydroxylase deficiency. Furthermore, a reciprocal change--i.e., a T----C change in the eighth exon of the CYP21A gene--was observed in the Japanese population and was associated with the two HLA haplotypes, HLA-B44-DRw13 and HLA-Bw46-DRw8. These changes may be considered the result of gene conversion-like events.     

Extended MHC haplotypes and CYP21/C4 gene organisation in Irish 21-hydroxylase deficiency families

Summary We have analysed fifteen classical 21-hydroxylase deficiency families from throughout Southern Ireland and report the serologically defined HLA-A, HLA-B, HLA-Cw, HLA-DR, C4A and C4B polymorphisms that characterize the inferred disease haplotypes. Additionally, we have used a combination of short and long range restriction mapping procedures in order to characterize the CYP21/C4 gene organization associated with individual serologically defined haplotypes. The results obtained indicate that disease haplotypes are characterized by a high frequency (33%) of CYP21B gene deletion and 8 out of 10 such deletion haplotypes are represented by the extended haplotype HLA-DR1, C4BQo, C4A3, HLA-B40(w60), HLA-Cw3, HLA-A3. Large scale length polymorphism in the CYP21/C4 gene cluster was found to conform strictly to a variable number of tandem repeats model with 4 alleles being detected. Disease haplotypes in which defective CYP21B gene expression is inferred to result from pathological point mutations show extensive diversity of associated HLA markers and include two examples of the extended HLA haplotype HLA-DR3, B8, Cw7, A1 haplotype, which has previously been reported to be negatively associated with 21-hydroxylase deficiency. One unusual disease haplotype has two CYP21 + C4 units, both of which appear to contain CYP21B-like genes.     

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.     

Direct analysis of CYP21B genes in 21-hydroxylase deficiency using polymerase chain reaction amplification

Steroid 21-hydroxylase deficiency is the leading cause of impaired cortisol synthesis in congenital adrenal hyperplasia (CAH). We have studied the structure of the CYP21B gene in 30 unrelated CAH patients using the polymerase chain reaction (PCR) to differentiate the active CYP21B gene from its highly related CYP21A pseudogene. The PCR approach obviates the need to distinguish the CYP21A and CYP21B genes by restriction endonuclease digestion and electrophoresis before analysis with labeled probes. Furthermore, direct nucleotide sequence analysis of CYP21B genes is demonstrated on the PCR-amplified DNA. Gene deletion of CYP21B, gene conversion of the entire CYP21B gene to CYP21A, frame shift mutations in exon 3, an intron 2 mutation that causes abnormal RNA splicing, and a mutation leading to a stop codon in exon 8 appear to be the major abnormalities of the CYP21B gene in our patients. These mutations appear to account for 21-hydroxylase deficiency in 22 of 26 of our salt-wasting CAH patients.     

Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants

Steroid 21-hydroxylase (cytochrome P450 21A2, CYP21A2) deficiency accounts for ∼95% of individuals with congenital adrenal hyperplasia, a common autosomal recessive metabolic disorder of adrenal steroidogenesis. The effects of amino acid mutations on CYP21A2 activity lead to impairment of the synthesis of cortisol and aldosterone and the excessive production of androgens. In order to understand the structural and molecular basis of this group of diseases, the bovine CYP21A2 crystal structure complexed with the substrate 17-hydroxyprogesterone (17OHP) was determined to 3.0 Å resolution. An intriguing result from this structure is that there are two molecules of 17OHP bound to the enzyme, the distal one being located at the entrance of the substrate access channel and the proximal one bound in the active site. The substrate binding features locate the key substrate recognition residues not only around the heme but also along the substrate access channel. In addition, orientation of the skeleton of the proximal molecule is toward the interior of the enzyme away from the substrate access channel. The 17OHP complex of CYP21A2 provides a good relationship between the crystal structure, clinical data, and genetic mutants documented in the literature, thereby enhancing our understanding of congenital adrenal hyperplasia. In addition, the location of certain CYP21A2 mutations provides general understanding of structure/function relationships in P450s.     

Susceptibility to IDDM Is marked by MHC supratypes rather than individual alleles

107 patients with insulin-dependent diabetes mellitus (IDDM) were typed for HLA A, B, C, and DR antigens, and for complement C4A, C4B, and Bf alleles, and the results were compared with those of a combined reference group of 332 appropriately matched healthy subjects. Supratypes (allelic combinations) were identified from the phenotype of each group, and it was shown that the frequency of several supratypes is increased in patients with IDDM, in particular supratypes (A1 Cw7) B8 C4AQ0 C4B1 BfS DR3 (P = 0.0001), (A30 Cw-) B18 C4A3 C4BQ0 BfF1 DR3 (P = 0.0003), (A2 Cw3) B62 C4AR C4B2.9 BfS DR4 (P = 0.0002), and three other supratypes including DR4. It was also shown that increases in the frequency of individual alleles are secondary to increases in supratype frequency. Moreover, supratypes appeared to interact; the presence of two relevant supratypes being particularly important. The absolute risk of IDDM was approximately 0.5 in subjects who were homozygous for B18 C4A3 C4BQ0 BfF1 DR3. We concluded that genetic susceptibility is best recognized by MHC supratypes rather than isolated alleles, and that supratype combinations make the identification of even greater disease risk possible.     

Analysis of steroid 21-hydroxylase gene in five unrelated Japanese patients with 21-hydroxylase deficiency

DNA samples from five unrelated Japanese patients with 21-hydroxylase (21-OHase) deficiency were studied by Southern analysis using human 21-OHase cDNA. Patterns seen after digestion with not only TaqI but also KpnI showed that two out of the five patients were homozygous for a deletion of the 21-OHase B gene. This result supports the report that the 21-OHase B gene is functional. In the other three, smaller mutations might be responsible for the disorder. The parents of one of the two patients with the deletion had a common ancestor. Hybridization patterns of DNA from members of the family of the patient were consistent with an autosomal recessive mode of inheritance of the deletion that correlates with the clinical phenotype. The deletion segregated with HLA-Aw 24; Bw 61; Cw 3. Heterozygous carriers of 21-OHase deficiency could be detected by comparing the patterns as well as the HLA haplotypes in this family. The application of the family study to the prenatal diagnosis is also discussed.     

Restriction maps and restriction fragment length polymorphisms of the human 21-hydroxylase genes

Restriction maps were constructed for the two human 21-hydroxylase genes (21-OHA and 21-OHB) by using DNA from subjects homozygous for a deletion of each gene. Comparing the patterns of these two genes, a KpnI restriction site occurred in the 21-OHA gene in place of a TaqI site in the 21-OHB gene about 1-kb from the 5' end of the gene, and an extra EcoRI site was located 500 bp 5' to the common EcoRI site. The DNA of fourteen unrelated normal subjects was digested with nine restriction endonucleases (AccI, BamHI, BgIII, EcoRI, HindIII, KpnI, MspI, SacI and TaqI). Restriction fragment length polymorphisms were found with EcoRI, HindIII and AccI that resulted from polymorphic endonuclease sites outside the genes.     

Sharing of MHC haplotypes among apparently unrelated patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

From the study of HLA, complement, and glyoxalase I alleles in 82 Venezuelan individuals belonging to 19 families of mixed ethnic origin having 20 affected newborns with salt-wasting congenital adrenal hyperplasia due to 21-hydroxylase (21-OH) deficiency, a total of 38 disease haplotypes and 53 nondisease haplotypes were found. Of the pathological haplotypes 47 % were found to share the HLA-B39 or -Bw62 specificities, 55 % of them in combination with the BFS, C2C, C4A4, C4B2 (SC42) complotype. The frequencies of HLA-B39 and -Bw62 among the affected haplotypes were 29 and 18% as compared with 6 and 0 % among the nondisease haplotypes of the same families. Statistical associations (P < 0.01) with salt-wasting adrenal hyperplasia were found with the SC42 complotype and with the combination SC42, HLA-B39. These results are markedly different from those reported in the literature which show an association at the population level among many Caucasoid samples of HLA-Bw47 and the extended haplotype (HLA-Bw47, DR7, FC91, 0) with the salt-wasting form of the disease. Furthermore, four of the unrelated patients reported here were homozygous for all the major histocompatibility complex loci tested, while three others were homozygous for at least two HLA loci. Analysis of the geographical origin of the grandparents indicated clustering of the deficiency carrier HLA haplotypes. This observation, together with the fact that there is an excess of homozygotes among the patients in Venezuela, strongly suggests that salt-wasting 21-OH deficiency congenital adrenal hyperplasia is mostly the result of a founder effect of relatively few independent mutations and, thus, of identity by descent of a few abnormal alleles at the 21-OHB locus in most cases. The mutation marked by HLA-Bw47 was not observed in this population.     

Two distinct areas of unequal crossingover within the steroid 21-hydroxylase genes produce absence of CYP21B

We mapped crossover sites in chimeric, recombinant CYP21 genes from six patients with salt-losing congenital adrenal hyperplasia (CAH). Nucleotide sequences unique to the CYP21A pseudogene or to the active CYP21B gene were mapped using gene-specific restriction sites and oligonucleotide hybridizations. Each chimeric CYP21 gene in the CYP21-deletion linked haplotypes contained sequences near the 5' end that were characteristic of CYP21A and only a single transition from sequences of CYP21A to those of CYP21B at the 3' end. The transitions all occurred within either of two discrete regions (+470 to +999 and +1375 to +1993). All eight chimeric CYP21 genes coupled with HLA-Bw47 in five unrelated patients had the CYP21A-CYP21B sequence transition within the same gene region (+1375 to +1993). One of the three other "CYP21B deletion" haplotypes (HLA-B7) had a sequence transition within this same region, while in the other two haplotypes (HLA-B61 and HLA-B18) the transition occurred between base pairs +470 and +999. By contrast, both CYP21 genes in a haplotype containing a gene conversion of CYP21B to CYP21A contained apparent transitions between sequences of CYP21A and CYP21B. We conclude that a single, unequal crossingover between the CYP21A and the CYP21B genes yields deletion of the active CYP21 gene and salt-losing CAH and that these crossingovers do not occur randomly within the CYP21 genes of our patients.     

Rearrangement of chromosome 21 in Alzheimer's disease

We have analyzed the DNA from four patients suffering from a dementia of the Alzheimer type and of four controls of identical age. Estimates of the copy numbers of two genes located on chromosome 21, SOD1 and ETS2, gave the following results: in all the cases there was duplication of ETS2 whereas SOD1 was normal. These preliminary results indicate that the segment of chromosome 21(q21----q22.1) is rearranged in all the four patients who were investigated.     

Deletion of complement C4 and steroid 21-hydroxylase genes in the HLA class III region

Molecular maps have been prepared of the HLA region on human chromosome 6 that includes the complement C4 and steroid 21-hydroxylase genes (21-OH), using DNA of individuals deficient (QO) in either of the two forms C4A or C4B. In all, 18 haplotypes with C4A QO were examined by Southern analysis and two had deletions of 28-30 kb that included both the C4A and 21-OHA genes. Of six C4B QO haplotypes, one had a deletion that included both the C4B and 21-OHA genes. Thus, some of the C4 null alleles are due to deletion of the gene but the majority in this sample are not. Deletion occurred in two common haplotypes suggesting that in the population as a whole, C4A deficiency is due to deletion in about one-half the C4A QO haplotypes. As duplication of C4A or C4B genes does occur, the possibility that unequal cross-over could explain the C4 deletion was examined by preparing cosmid clones from the DNA of an individual typed C4A QO. A cloned genomic fragment containing the single C4B gene was isolated and found to be similar to the homologous region of a cosmid from a normal individual carrying a C4A gene. This suggests that if a cross-over has occurred it is in a region where the two genes are identical. The biological significance of the rather frequent occurrence in the population of haplotypes with C4A or C4B deletion together with the accompanying deletion of the 21-OHA gene is discussed.     

CYP21 mutations in Brazilian patients with 21-hydroxylase deficiency

Congenital adrenal hyperplasia caused by 21-hydroxylase deficiency is a common autosomal recessive disorder resulting from mutations in the 21-hydroxylase (CYP21) gene. To develop a strategy to screen for the most commonly occurring CYP21 mutations in Brazil, we performed molecular genotype analysis on 73 children with CAH representing 71 unrelated families. The techniques used for CYP21 molecular genotype analysis were: restriction fragment length polymorphism, single-strand conformational polymorphism, allele-specific oligonucleotide hybridization, allele-specific polymerase chain reaction amplification, and heteroduplex analyses. Mutations were identified on all but eight affected alleles. The intron 2 splicing mutation was the most frequently identified mutation. Screening for the most common mutations detected at least one mutation on 132/142 (93%) alleles. Multiple CYP21 mutations were detected on 16.2% of alleles. The high frequency of multiple mutations on a single allele emphasizes the importance of thorough and accurate molecular genotype analysis of the complex CYP21 locus.     

Distribution of deletions and seven point mutations on CYP21B genes in three clinical forms of steroid 21-hydroxylase deficiency

To characterize mutations in the CYP21B gene that are responsible for congenital adrenal hyperplasia (CAH), DNA samples from 91 French patients have been studied by allelic-specific oligonucleotide hybridization and Southern blot analysis. Seven sites mostly found in the CYP21A pseudogene and deletions of the functional CYP21B gene have been screened. Gene conversions involving small DNA segments accounted for 57% of the tested mutations and probably cause 74% of the mutations responsible for the disease. Complete deletion of the CYP21B gene accounted for 18% of the CAH mutations in the whole sample and for 21% in the classical form of the disease. Three mutations were found associated with specific clinical forms of the disease: a G-C substitution in the seventh exon was associated with the late-onset form of the disease, and both an 8-bp depletion in the third exon and complete deletion of CYP21B were associated with the salt-wasting form.     

Radioimmunoassay of urinary 21-deoxytetrahydroaldosterone in primary aldosteronism and 21-hydroxylase deficiency

A radioimmunoassay of 21-deoxytetrahydroaldosterone was developed. Normal daily excretion of the unconjugated metabolite was 1.2 +/- 1.3 micrograms and of the glucuronized metabolite, 11.9 +/- 7 micrograms. The tetrahydroaldosterone/21-deoxytetrahydroaldosterone ratio varied more in patients with primary aldosteronism than in control subjects. Thus, measurements of the urinary excretion of the tetrahydroaldosterone or 21-deoxytetrahydroaldosterone alone did not provide an accurate expression for aldosterone production. Their sum correlated well with the clinical condition, i.e. clear-cut elevation in patients with primary aldosteronism. The diminished tetrahydroaldosterone/21-deoxytetrahydroaldosterone ratio found in patients with 21-hydroxylase deficiency may be attributed to increased bacterial conversion of tetrahydroaldosterone to 21-deoxytetrahydroaldosterone but could also stem from a deficiency implicating zona glomerulosa (aldosterone biosynthesis) regardless of the stage and clinical presentation of the disease.     

Molecular pathology of steroid 21-hydroxylase deficiency

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