CYP21/C4 gene organisation in Italian 21-hydroxylase deficiency families

Summary A total of 33 Italian 21-hydroxylase (21-OH) deficiency families were investigated using a combination of short and long range restriction mapping of the CYP21/C4 gene cluster. The analyses revealed that large-scale length polymorphism in this gene cluster strictly conformed to a compound variable number of tandem repeats (VNTR) plus insertion system with between one and four CYP21 + C4 units and seven BssHII restriction fragment length polymorphisms (RFLPs) (75kb, 80kb, 105kb, 110kb, 135kb, 140kb and 180kb). A total of 9/66 disease haplotypes, but only 1/61 nondisease haplotypes, showed evidence of gene addition by exhibiting three or more CYP21 + C4 repeat units. Of these, two were identified in one 21-OH deficiency patient who has a total of eight CYP21 + C4 units, being homozygous for the HLA haplotype DR2 DQ2 B5 A28. This haplotype carries four CYP21 + C4 units, three of which contain CYP21A-like genes and one of which contains a CYP21B-like gene that presumably carries a pathological point mutation. Of the other gene addition haplotypes associated with 21-OH deficiency, four show three CYP21 + C4 units flanked by HLA-DR1 and HLA-B14 markers. Although such haplotypes have commonly been associated with non-classical 21-OH deficiency, three examples in the present study are unexpectedly found in two salt-wasting patients, who are respectively homozygous or heterozygous for this haplotype. Only 7/66 disease haplotypes showed evidence of a CYP21B gene deletion.     

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.     

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.     

Determination of deletion sizes in the MHC-linked complement C4 and steroid 21-hydroxylase genes by pulsed-field gel electrophoresis

In man, the genes encoding the complement component C4 (C4A, C4B) of the immune system and the steroid 21-hydroxylase enzyme (CYP21A, CYP21B) of adrenal steroid biosynthesis are located in the major histocompatibility complex (MHC). Frequent gene deletions and duplications have been described in the C4 and CYP21 genes, particularly in patients with autoimmune diseases and congenital adrenal hyperplasia. Here we report the determination of deletion sizes in 11 chromosomes with six different deletions. The deletions spanned the C4A+CYP21A, C4B+CYP21A, and C4B+CYP21B gene pairs as determined by standard Southern blot analysis. The deletion size fell within the range of 30-38 kb in all the chromosomes, as determined by pulsed-field gel electrophoresis. Because the deletion sizes in most other gene clusters are more heterogeneous, the results suggest the involvement of a specific mechanism in the generation of C4+CYP21 deletions.     

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.     

Major-histocompatibility-complex gene markers and restriction-fragment analysis of steroid 21-hydroxylase (CYP21) and complement C4 genes in classical congenital adrenal hyperplasia patients in a single population

The gene CYP21B, encoding the steroid 21-hydroxylase enzyme of adrenal steroid biosynthesis, has been mapped to the human major histocompatibility complex (MHC). Deficiency of this enzyme leads to congenital adrenal hyperplasia (CAH). We report the phenotypes of the HLA and complement C4 and Bf genes, which are closely linked to the CYP21B gene, together with a detailed analysis of the CYP21 and C4 RFLP, in 17 Finnish families with CAH. The RFLP analysis with six restriction enzymes suggested that, altogether, 35% of the affected chromosomes had a CYP21B + C4B gene deletion, 9% an obvious gene conversion of the CYP21B gene to a CYP21A-like gene, and 3% a CYP21A + C4B duplication. The remaining 53% gave the RFLP patterns also found in nonaffected chromosomes. We also found that a 14.0-kb EcoRI RFLP marker of the CYP21 genes was strongly associated with the presence of a short C4B gene, suggesting that some of the RFLP markers found with the CYP21 probe may actually derive from C4B gene polymorphism. Three particular MHC haplotypes, each with a characteristic RFLP pattern, were found in many unrelated families. These three haplotypes accounted for 59% of the affected chromosomes in our study group, the rest (41%) of the affected chromosomes being distributed among various subtypes. The results suggest that, within a single, well-defined population such as in Finland, only a few CYP21B gene defects may constitute a substantial part of the affected chromosomes. This finding will help in genetic studies of CAH in such populations.     

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.     

Defective,deleted or converted CYP21B gene and negative association with a rare restriction fragment length polymorphism allele of the factor B gene in congenital adrenal hyperplasia

Summary Defects in the enzyme, steroid 21-hydroxylase, result in congenital adrenal hyperplasia (CAH), a common autosomal recessive disorder of cortisol biosynthesis. The gene encoding this protein (CYP21B) and a closely linked pseudogene (CYP21A) have been mapped in the HLA complex on chromosome 6p, adjacent to the complement genes C4B and C4A, about 80 kb from the factor B gene. Molecular analyses of patients with CAH have shown that the cause of the defect may be either a deletion, a point mutation or a conversion of the active gene. Linkage of the disease to HLA has previously been studied by several groups. We have analyzed DNAs from patients with classical and non-classical CAH and from their family members, by probing with CYP21, C4 and BF cDNAs. In 70% of the CAH haplotypes studied, the defective CYP21B gene was indistinguishable from its structurally intact corresponding gene in Southern blot analysis, and presumably bore point mutations. In the remaining chromosomes, evidence for gene conversions, deletions and various deleterious mutations of the CYP21B gene is given. Moreover, our linkage studies show that a polymorphic TaqI cleavage site in the factor B gene, recently described by us, may be a new and useful genetic marker, because we found this TaqI restriction site only in unaffected haplotypes carrying functional CYP21B genes and, therefore, in negative association with the defective CYP21B gene.     

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.     

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.     

Duplication of the CYP21A2 gene complicates mutation analysis of steroid 21-hydroxylase deficiency: characteristics of three unusual haplotypes

Steroid 21-hydroxylase deficiency, the primary cause of congenital adrenal hyperplasia, is caused by defects of the CYP21A2 gene. As a complement to hormonal measurements, mutation analysis of CYP21A2 is an important tool in the diagnosis of steroid 21-hydroxylase deficiency. Contemporary mutation-detection protocols based on the polymerase chain reaction often depend on the assumption that no more than one CYP21A2 gene is present on each chromosome 6. We describe three haplotypes with two CYP21A2 genes on the same chromosome, with defects typical of salt-losing steroid 21-hydroxylase deficiency in one of those genes, but not necessarily in the other. The frequency of these haplotypes in the general population is 6/365 (1.6%), so they are no less common than other haplotypes that indeed carry steroid 21-hydroxylase deficiency. Chromosomes that carry two CYP21A2 genes therefore represent a significant pitfall in the molecular diagnosis of steroid 21-hydroxylase deficiency. We recommend that, whenever CYP21A2 mutation analysis of an individual who is not a known carrier of steroid 21-hydroxylase deficiency is performed, the overall structure of the CYP21/ C4 region (the RCCX area) is determined by haplotyping to avoid erroneous assignment of carrier status.     

The implication of de novo 21-hydroxylase mutation in clinical and prenatal molecular diagnoses

We studied 37 unrelated families with a history of 21-hydroxylase deficiency (CYP21D) for eight common mutations and gene deletions in the 21-hydroxylase (CYP21) gene. We found de novo mutations in the CYP21 gene in two CYP21D patients. Analysis for eight common mutations in the 21-hydroxylase gene as well as large gene deletions was accomplished using polymerase chain reaction (PCR) followed by amplified created restriction site (ACRS) or restriction fragment length polymorphism (RFLP) and Southern blot followed by hybridization to a CYP21-specific probe. Linkage analysis was performed using microsatellite markers flanking the CYP21 gene. Ten short tandem repeat (STR) markers were used to confirm parentage in the two de novo mutation cases. In two prenatal diagnosis cases, an intron 2-13A/C>G mutation was identified in the proband, but not in the fetus, although the proband and fetus had identical linkage markers. Subsequently, the mutation was confirmed to be absent in the parents' genome and misparentage was ruled out. Our findings are consistent with previous studies showing a de novo mutation frequency of approximately 1.0-1.5% in the CYP21 gene. This new mutation rate is high relative to the rate of approximately one in one million for other autosomal recessive disorders. Thus, the de novo mutation rate in the CYP21 gene is not negligible. It must be considered and discussed in prenatal diagnosis and genetic counseling for this relatively common inherited disorder.     

Low frequency of CYP2B deletions in Brazilian patients with congenital adrenal hyperplasia due to 21-hydroxylas deficiency

The frequency of large mutations was determined in 131 Brazilian patients with different clinical forms of 21-hydroxylase deficiency, belonging to 116 families. DNA samples were examined by Southern blotting hybridization with genomic CYP21 and C4cDNA probes after Taql and Bg/II restriction. Large gene conversions were found in 6.6% and CYP21B deletions in 4.4% of the alleles. The breakpoint in these hybrid genes occurred after exon 3 in 92% of the alleles. All rearrangements involving CYP21B gene occurred in the heterozygous form, except in a patient with simple virilizing form who presented homozygous CYP21B deletion. Our data showed that in these Brazilian patients, CYP21B deletions were less frequent than in most of the large series previously reported.     

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.     

Prevalence of polymorphic 21-hydroxylase gene (CA21HB) mutations in salt-losing congenital adrenal hyperplasia

Using genomic restriction analysis of 14 unrelated patients with salt-losing congenital adrenal hyperplasia, we identified three different CA21HB mutation patterns: no detectable restriction fragment abnormalities (16/28 haplotypes), deletion of the active CA21HB gene (9/28), and apparent conversion of the active CA21HB gene to the pseudogene CA21HA (3/28). CA21HB gene deletion was associated with HLA-Bw47 in 6 haplotypes and with absent C4B expression in 7. A variety of HLA and C4 types was associated with the other mutations. Apparent conversion of CA21HB to CA21HA was identified by the disparity between the intensity ratios for the major TaqI and BglII hybridization fragments.     

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

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

Molecular genetics of 21-hydroxylase deficiency in congenital adrenal hyperplasia

21-hydroxylase gene analysis was performed on the genomic DNA from patients with congenital adrenal hyperplasia (CAH), their siblings, their parents as well as from a healthy individual serving as control. After digestion by the Taq I and Bgl II restriction enzymes, DNA was hybridized with specific nucleotidic probes: pC21a for the 21-hydroxylase genes, pAT-A for the C4 component Complement genes, closely linked to the 21-hydroxylase genes on the 6 chromosome. Likewise the pFB3B probe was used for the B factor gene located 80 kilobases upstream the 21-hydroxylase gene. From this molecular analysis on 11 families, we report here 4 investigations showing the most frequent genetic abnormalities we have encountered: gene deletions, gene conversions and point mutations. These data show that the molecular approach is a powerful tool for studying this endocrine disease at the clinical, genetic and fundamental point of view.     

Restriction fragment analysis of duplication of the fourth component of complement (C4A)

The two genes encoding the fourth component of complement (C4A and C4B) reside between HLA-B and HLA-DR on human chromosome 6. Two kilobases downstream from each C4 gene lies a 21-hydroxylase gene (CA21HA and CA21HB, respectively). Utilizing the method of Southern blotting and a 5'-end 2.4-kb BamHI/KpnI fragment of the C4 cDNA, we have analyzed TaqI-digested DNA from four pedigrees with one or more extended haplotypes containing a C4A duplication, as demonstrated by protein electrophoresis and segregation analysis. Two C4A protein duplications (C4A*2,A*3,C4B*QO and C4A*3,A*5,C4B*QO) segregated with two large TaqI DNA restriction fragments (7.0 and 6.0). In pedigree Fi, one individual homozygous for HLA-A3,B35,C4,DR1,DQ1,BFF,C2C,-C4A2,3,C4BQO had TaqI 7.0- and 6.0-kb restriction fragments with equal hybridization intensities as measured by two-dimensional densitometry (7.0/6.0 kb = 0.83, SD = 0.12, N = 7). A hybridization probe for the 21-hydroxylase gene also demonstrated equal gene dosage (CA21HA/CA21HB = 1.01). DNA from another individual (Ma I-2) with a different C4A gene duplication (C4A*3,A*5,C4B*QO) also had equal densitometry measurements (7.0/6.0 kb = 1.07). We conclude that two extended haplotypes from unrelated pedigrees have two C4 genes and both C4 genes encode separate C4A alleles. These findings are compatible with a gene conversion event of C4B to C4A.     

Low frequency of deletion alleles in patients with steroid 21-hydroxylase deficiency in a Mexican population

Steroid 21-hydroxylase deficiency is caused by mutations in the CYP21 gene. Approximately 95% of mutant alleles are generated by recombination events between the acitve gene CYP21 and its highly homologous pseudogene, CYP21P. Deletion alleles are generated by unequal crossing over, while point mutations are the result of gene conversion events. Deletions account for 20–25% of the 21-hydroxylase deficiency alleles in most populations studied. We have looked for deletions among 53 unrelated Mexican patients with steroid 21-hydroxylase deficiency and found that deletions represent less than 1% of the disease alleles. These findings suggest that nearly all mutant alleles in our patient population contain point mutations and that the low representation of deletion alleles among clinically diagnosed patients may be due to missing detection of salt wasters, mainly males, who may die during the neonatal period. Received: 17 November 1995 / Revised: 29 February 1996, 12 April 1996     

Modular variations of the human major histocompatibility complex class III genes for serine/threonine kinase RP, complement component C4, steroid 21-hydroxylase CYP21, and tenascin TNX (the RCCX module). A mechanism for gene deletions and disease associations

The frequent variations of human complement component C4 gene size and gene numbers, plus the extensive polymorphism of the proteins, render C4 an excellent marker for major histocompatibility complex disease associations. As shown by definitive RFLPs, the tandemly arranged genes RP, C4, CYP21, and TNX are duplicated together as a discrete genetic unit termed the RCCX module. Duplications of the RCCX modules occurred by the addition of genomic fragments containing a long (L) or a short (S) C4 gene, a CYP21A or a CYP21B gene, and the gene fragments TNXA and RP2. Four major RCCX structures with bimodular L-L, bimodular L-S, monomodular L, and monomodular S are present in the Caucasian population. These modules are readily detectable by TaqI RFLPs. The RCCX modular variations appear to be a root cause for the acquisition of deleterious mutations from pseudogenes or gene segments in the RCCX to their corresponding functional genes. In a patient with congenital adrenal hyperplasia, we discovered a TNXB-TNXA recombinant with the deletion of RP2-C4B-CYP21B. Elucidation of the DNA sequence for the recombination breakpoint region and sequence analyses yielded definitive proof for an unequal crossover between TNXA from a bimodular chromosome and TNXB from a monomodular chromosome.