Analysis of the duplicated human C4/P450c21/X gene cluster

Gene duplications, deletions and rearrangements occur with an unusually high frequency in the region of the P450c21 genes encoding 21-hydroxylase. In the human genome, the locus contains at least 6 genes, oriented 5′ C4A, P450c21A, XA, C4B, P450c21B, XB 3′. Sequence analysis of the XA gene, of the 5′ flanking DNA of the C4A gene, and of part of the XB gene revealed that this gene cluster was duplicated by nonhomologous recombination at a CAAG tetranucleotide. The location of this duplication suggests that it may have occurred after mammalian speciation. The XA gene is abundantly expressed in the human adrenal as a stable 2.6 kb RNA, but it is not known if that RNA serves a biological function. Knowledge of the anatomy of the XA gene facilitates genetic analysis of disease-causing lesions in the P450c21B gene. Southern blotting data show that about 76% of disordered P450c21B alleles bear gene microconversions that resemble point mutations; the remaining alleles are equally distributed between gene deletions and large gene conversions.     

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.     

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.     

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.     

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.     

Molecular characterization of the HLA-linked steroid 21-hydroxylase B gene from an individual with congenital adrenal hyperplasia

21-Hydroxylase deficiency which causes congenital adrenal hyperplasia is one of the most common defects of adrenal steroidogenesis. There are two 21-hydroxylase genes in man, A and B, and these have been mapped to the HLA class III region. Only the 21-hydroxylase B gene is thought to be active. To understand the molecular basis of congenital adrenal hyperplasia in a patient with the salt-wasting form of the disease, we cloned and characterized his single 21-hydroxylase B gene. The nucleotide sequence of this gene and a 21-hydroxylase B gene from a normal individual have been determined. Comparison of the two sequences has revealed 11 nucleotide alterations, of which two are in the 5' flanking region, four are in introns, one is in the 3' untranslated region and four are in exons. Two of the differences in exons cause codon changes, with Ser-269 and Asn-494 in the normal 21-hydroxylase B gene being converted to Thr and Ser, respectively. These amino acid substitutions may give an insight into those residues necessary for 21-hydroxylase enzymatic activity. We have also confirmed that the 21-hydroxylase A gene is a pseudogene due to three deleterious mutations in the exons. In addition, comparison of the 21-hydroxylase B gene sequence with other published sequences indicates that this microsomal cytochrome P-450 may be polymorphic.     

Pulsed field gel electrophoresis identifies a high degree of variability in the number of tandem 21-hydroxylase and complement C4 gene repeats in 21-hydroxylase deficiency haplotypes.

The human steroid 21-hydroxylase gene, CYP21B, and its closely homologous pseudogene, CYP21A, are each normally located centromeric to a complement C4 gene C4B and C4A respectively, in an organization suggesting tandem duplication of a CYP21 + C4 unit. Such an organization has been considered to facilitate gene deletion and addition events by unequal crossover between the tandem repeats. However, the large size (approximately 30 kb) of the individual CYP21 + C4 repeat units together with the difficulty in identifying reliable CYP21A- and CYP21B-specific markers has prevented direct monitoring of gene organization on individual haplotypes by conventional Southern analyses. In the present investigation we have sought to clarify the CYP21 and C4 gene organization in members of 32 British 21-hydroxylase deficiency families by employing additional experimental approaches, notably a long-range restriction mapping approach, which permits assessment through a VNTR type of analysis, of the number of CYP21 and C4 units on individual haplotypes. Our results show that there is a very high frequency (33%) of 21-hydroxylase deficiency haplotypes where functional CYP21B gene sequence has been removed as a consequence of CYP21 + C4 gene deletion while several haplotypes show evidence of gene addition. In each case that we have investigated the gene deletion and gene addition haplotypes differ in length from conventional haplotypes by integral multiples of approximately 30 kb, which strongly supports the involvement of unequal crossover mechanisms. Additionally, the comparatively frequent occurrence of CYP21 fusion genes which contain both CYP21A- and CYP21B-associated markers is suggested by the combined data from Southern analyses, long-range restriction mapping and characterization of selected regions of CYP21 genes which have been amplified in vitro.     

Decreased levels of steroid 21-hydroxylase [P450(c21)] and its mRNA in an adrenocortical adenoma associated with 21-hydroxylase deficiency

Adrenocortical adenoma incidentally found in a 37-yr-old female patient, with simple virilizing form of 21-hydroxylase deficiency, was studied. Cultured adenoma cells revealed excessive secretion of 17 alpha-hydroxyprogesterone in response to 10(-8) M ACTH, compared with those of 11-deoxycortisol and cortisol, which indicated impaired activity of the 21-hydroxylase. To elucidate the molecular mechanisms of this defective 21-hydroxylase in the adenoma, we analyzed the gene encoding specific cytochrome P450 (P450c21) for steroid 21-hydroxylation and its expression. DNA and RNA were extracted from the adrenal adenoma and were hybridized with a probe of human P450c21 gene, by Southern and Northern blot analysis. In Southern blot analysis with Taq I, Bgl II or Bam HI, there was no difference between the pattern of restriction fragments in DNA from the adenoma and normal peripheral leucocytes. Northern blot analysis of the adenoma showed the same size of P450c21 mRNA as in the normal adrenal gland, but the amount was low--about a half that of the normal adrenal. In Western blot analysis with polyclonal antibody to P450c21, only a small amount of P450c21 protein was detected in the adenoma, although it was found to be of the same molecular weight as that in the normal adrenal gland. In view of these findings it is conceivable as one of possibilities that a mild and small mutation in the structural or promotor region of the P450c21 gene may cause the decreased 21-hydroxylase activity in this adenoma.     

Heterogeneity in the structural basis of the human complement C4A null allele (C4A Q0) as revealed by HindIII restriction fragment length polymorphism analysis

The highly polymorphic fourth component of human complement (C4) is usually encoded by two genes. C4A and C4B, adjacent to the 21-hydroxylase (21-OH) genes, 21-OHA and 21-OHB, and is also remarkable in the high frequency of the 'null' alleles, C4A Q0 and C4B Q0. The molecular basis for the C4A Q0 allele was studied in 26 families through restriction fragment length polymorphism (RFLP) analysis with C4 and 21-OH cDNA probes after digestion of the DNA with the endonuclease HindIII. The individuals expressing the extended haplotype HLA-A1 (of A2) Cw7 B8 C2C BfS C4AQ0B1 DR3 have a large deletion taking off the C4A and 21-OHA genes.     

Substitution of Ile-172 to Asn in the steroid 21-hydroxylase B (P450c21B) gene in a Finnish patient with the simple virilizing form of congenital adrenal hyperplasia

Summary The steroid 21-hydroxylase enzyme (P450c21) is a member of the cytochrome P450 gene superfamily and is essential in the synthesis of cortisol and aldosterone. Defects in the P450c21B gene cause congenital adrenal hyperplasia (CAH), a common genetic disorder leading to virilization of newborn females. To avoid the standard cloning of mutant P450c21 genes from genomic libraries, we amplified the full-length genomic P450c21 genes by polymerase chain reaction (PCR). The amplification was followed by cloning and sequencing of a defective P450c21B gene. The strategy described here is generally applicable, thus making a simple characterization of the complete P450c21B gene possible. The method was tested in one patient suffering from the simple virilizing form of CAH. The sequence of three independent clones originating from the defective P450c21B showed that Ile at position 172 in exon 4 was substituted by Asn. The identical mutation also has been found in other patients with CAH.     

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.     

Prenatal diagnosis/treatment in families at risk for infants with steroid 21-hydroxylase deficiency (congenital adrenal hyperplasia)

The most common enzymatic defect of steroid synthesis is adrenal steroid 21-hydroxylase deficiency. Inhibited formation of cortisol causes increased pituitary release of ACTH, driving the adrenal cortex to overproduce androgens, whose synthesis does not involve the 21-hydroxylase enzyme. This hormonal setting is established in the embryonic period and affects development of genetic females, misdirecting differentiation of the external genitalia toward male type. At birth, the genitalia are visibly ambiguous (enlarged clitoris, fused labia) or in some cases even male in appearance {phallus with urethral opening, rugated scrotal sac), leading to wrong sex assignment. Adrenal steroid 21-hydroxylase deficiency is the most common basis of female pseudohermaphroditism. These females, however, have normal fertility and potential for gestation (gonads are functional and the internal duct-derived structures are well-formed), thus the sex of rearing should always be female. Management is by life-long hormonal (glucocorticoid) replacement, with surgical correction of the genital ambiguity. Prenatal diagnosis of 21-hydroxylase deficiency, first possible by steroid assay of the amniotic fluid, has utilized HLA typing for identification of loci (antigens B and DR) in close linkage with the 21-hydroxylase gene, and now increasingly relies on DNA analysis for linked HLA or C4 genes or for mutant 21-hydroxylase alleles directly by molecular genetic techniques. The most recent clinical advance is a program of combined prenatal diagnosis with karyotyping and suppression of fetal androgen production in genetic females by steroid administration to the mother. This is the first instance of an inborn metabolic error to be prenatally treated.

A series of 85 managed pregnancies is reported on, including accuracy of diagnosis, response of the mother to steroid treatment, and outcome for treated and untreated male and female fetuses (of 77 born by 6/91). Prenatal diagnosis by current techniques is accurate. Normal growth and development patterns postnatally suggest that dexamethasone treatment is safe.     

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.     

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.     

Molecular biology of disorders of sex differentiation.

Sexual ambiguity can be a difficult and sometimes confusing diagnostic problem in children. Recent developments in molecular biology have provided the opportunity to analyze the gene responsible for testicular determination, SRY, the androgen receptor gene and the gene encoding the cP450 enzyme specific for 21-hydroxylation, CYP21B, whose defects are responsible for congenital adrenal hyperplasia. Southern-blotting studies and PCR analyses of SRY, androgen receptor and CYP21B genes can be routinely used for the direct diagnosis of gonadal dysgenesis, androgen insensitivity syndromes and congenital adrenal hyperplasia, respectively. In sex-reversed XY females, several de novo mutations or deletions in the SRY gene have been reported. Defects in the human androgen receptor cause a spectrum of defects in male phenotypic sexual development associated with abnormalities in the receptor protein. Analyses of the androgen receptor gene structure have identified the causative mutation in some families: mutations that result in large-scale alterations of the structure of the androgen receptor, mRNA or gene mutations that alter the primary structure of the androgen receptor protein and mutations that alter the level of mRNA. The diversity of clinical phenotypes, apparent in 21-hydroxylase deficiency, is paralleled by a considerable degree of mutational heterogeneity in the CYP21 gene locus. Various changes causing severe 21-hydroxylase deficiency have been reported: point mutations, gene conversions and gene deletions. In conclusion, substantial progress has been made elucidating genetic defects causing sex reversal in XY females, the androgen insensitivity syndrome and congenital adrenal hyperplasia. Molecular genetics can also be applied for carrier identification and prenatal diagnosis.     

Basic and clinical aspects of congenital adrenal hyperplasia

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

Molecular analysis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency in Hong Kong Chinese patients

BackgroundCongenital adrenal hyperplasia (CAH) caused by 21-hydroxylase deficiency (21OHD) is an autosomal recessive disorder due to mutation in the CYP21A2 gene.ObjectiveTo elucidate the genetic basis of 21-hydroxylase-deficient CAH in Hong Kong Chinese patients.Patients and methodsMutational analysis of the CYP21A2 gene was performed on 35 Hong Kong Chinese patients with 21OHD using direct DNA sequencing and multiplex ligation-dependent probe amplification (MLPA).ResultsThe genetic findings of 21 male and 14 female patients are the following: c.293-13A/C>G (intron 2 splice site; 20 alleles), p.I172N (13), p.R356W (7), p.Q318X (4). A total of 20 mutant alleles contained gross deletion/conversion of all or part of the CYP21A2 gene. A novel mutation, c.1367delA (p.D456fs), was detected in one patient. One patient had only a heterozygous mutation detected. Out of 35 patients, 16 would have been incorrectly genotyped if either DNA sequencing or MLPA alone was used for molecular analysis.ConclusionsThe frequency of various mutations in the studied patients differs from those reported in other Asian populations. Gross deletion/conversion accounts for nearly one-third of the genetic defects. Therefore, laboratories must include methods for detecting point mutations as well as gross deletions/conversions to avoid misinterpretation of genotype. Genotyping has increasingly been proven to be a useful tool for supplementing, if not replacing, hormonal profiling for the diagnosis of 21OHD.     

Steroid 21-hydroxylase gene mutational spectrum in 50 Tunisian patients: Characterization of three novel polymorphisms

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease of steroid biosynthesis in humans. More than 90% of all CAH cases are caused by mutations of the 21-hydroxylase gene (CYP21A2), and approximately 75% of the defective CYP21A2 genes are generated through an intergenic recombination with the neighboring CYP21A1P pseudogene. In this study, the CYP21A2 gene was genotyped in 50 patients in Tunisia with the clinical diagnosis of 21-hydroxylase deficiency. CYP21A2 mutations were identified in 87% of the alleles. The most common point mutation in our population was the pseudogene specific variant p.Q318X (26%). Three novel single nucleotide polymorphism (SNP) loci were identified in the CYP21A2 gene which seems to be specific for the Tunisian population. The overall concordance between genotype and phenotype was 98%. With this study the molecular basis of CAH has been characterized, providing useful results for clinicians in terms of prediction of disease severity, genetic and prenatal counseling.     

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.