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.     

Identification of molecular defects causing congenital adrenal hyperplasia by cloning and differential hybridization of polymerase chain reaction-amplified 21-hydroxylase (CYP21) genes.

Congenital adrenal hyperplasia (CAH), one of the most common autosomal recessive disorders, is caused primarily by defects in the gene encoding steroid 21-hydroxylase, CYP21B. The molecular diagnosis of CAH, important for prenatal diagnosis, carrier detection, and a better understanding of the various clinical CAH forms, is complicated by the close proximity of a highly similar pseudogene, CYP21A, containing (and probably donating, by gene conversion-like events) most of the defects underlying CAH. In this study, we describe an efficient strategy to identify molecular defects causing CAH: polymerase chain reaction-amplified CYP21 loci are cloned and hybridized to a set of oligonucleotides, allowing rapid and allele-specific identification of all known CYP21B mutations relevant to 21-hydroxylase function. Possible new mutations can be identified by subsequent nucleic acid sequencing provided they reside within the cloned CYP21B fragment (from the TATA box to the 8th of the 10 CYP21B gene exons). Using this method, the CYP21B gene mutations of a heterozygous carrier and 25 CAH patients have been identified by oligonucleotide hybridization. All disease haplotypes seem to have been generated by recombinational events involving the CYP21A pseudogene. In 5 individuals, these data were subsequently verified by nucleic acid sequencing. The procedure can be used for diagnostic applications and may facilitate identification of new CYP21B defects.     

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.     

CYP21B gene conversion and complete CYP21A gene deletion in congenital adrenal hyperplasia

We studied a family in which one out of two children presented a non-salt wasting form of CAH. Genomic DNA of the patient, his brother, his parents and a normal control were digested by the Taq I and Bgl II restriction enzymes. The fragments were electrophoresed, transferred onto a nitrocellulose membrane and hybridized with two specific probes: pC21a for the CYP21 genes and pAT-A for the C4 genes. We performed simultaneous RFLP analyses of the CYP21 and C4 genes and determined the relative hybridization intensity of the genes using scanning densitometry of the X-ray films. The affected child had a CYP21B gene conversion in the CYP21A pseudogene on one chromosome inherited from his mother and a mutated CYP21B gene on the second chromosome inherited from his father. The second maternal chromosome, inherited by the unaffected brother, presented an unusual CYP21A gene deletion without a C4A or C4B gene deletion. Although CYP21A is a pseudogene, this type of complete CYP21A gene deletion associated with a CYP21B gene conversion has never been previously described.     

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.     

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.     

PCR-based detection of the CYP21 deletion and TNXA/TNXB hybrid in the RCCX module

Detection of the CYP21 deletion in congenital adrenal hyperplasia (CAH) in the RCCX module has been previously done by Southern blot analysis with multiple probes and separate digestions with the restriction endonucleases TaqI and BglII, which is laborious and indirect. Here, we describe an established PCR-based amplification method to analyze directly a CAH patient with a single CYP21 deletion, followed by RFLP analysis to characterize the interconversion region between tenascin A (TNXA) and tenascin B (TNXB). Data indicate that TaqI digestion of the defective CYP21 gene in the CAH patient produced 3.2-kb fragments. The CYP21 allele carried mutations in the CYP21P gene as determined by analysis with the amplification-created restriction site method. In addition, RFLP analysis indicated that the TNXB gene in the defective allele was replaced by TNXA to produce a TNXA/TNXB hybrid. We conclude that deletion of the RCCX module in this CAH patient included the RP2, C4B, and CYP21 genes and part of the TNXB gene. The junction of the recombination of the TNXA/TNXB hybrid may be located between IVS44 and exon 44 of the TNXB gene. This rapid, nonradioactive detection method will be beneficial for diagnostic purposes that are limited to the population originally studied.     

A missense mutation at Ile172----Asn or Arg356----Trp causes steroid 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is a common recessive genetic disease caused mainly by steroid 21-hydroxylase (P450c21) deficiency. Many forms of CAH exist resulting from various mutations of the CYP21B gene. We sequenced CYP21B cDNA from a normal person and its genes from a patient with simple virilizing CAH. When comparing several CYP21B sequences, we found it was polymorphic. In the patient, a single base substitution replaced Ile172 (ATC) with Asn (AAC) in one allele while Arg356 (CGG) was converted to Trp (TGG) in the other. A normal P450c21 cDNA clone was transfected into COS-1 cells to produce 21-hydroxylase activity toward its substrates, progesterone and 17-hydroxyprogesterone. Mutants corresponding to Asn172 or Trp356 mutation were constructed by site-directed mutagenesis of the normal c21 cDNA clone. They failed to produce active enzyme toward either substrate upon transfection into COS-1 cells, demonstrating that these mutations caused CAH. Aligning sequences with other P450s, Ile172 could be located in the membrane anchoring domain and Arg356 in the substrate-binding site of P450c21. Both mutations are present in the CYP21A1P pseudogene, suggesting that they may be transferred from CYP21A1P by gene conversion events.     

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.     

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.     

Molecular and endocrine characterization of a mutation involving a recombination between the steroid 21-hydroxylase functional gene and pseudogene

The gene encoding steroid 21-hydroxylase activity, P450c21B, is located in the major histocompatibility complex (MHC) class III region, in close proximity to a highly homologous pseudogene, P450c21A. Recombinations between P450c21B and P450c21A have been shown to result in deficiency of 21-hydroxylase activity, the usual cause of congenital adrenal hyperplasia (CAH). A mutant P450c21 gene from a patient with simple virilizing CAH was identified and shown to be consistent with a recombination between P450c21A and P450c21B. Sequence analysis of the mutant gene showed the recombination site to be located between the first exon and the second intron. The mutant gene encodes a leucine instead of the normal proline at codon 31. This mutation resides on a chromosome bearing the HLA-B44 serotype. A comparison of mutation associated with HLA-B44 and that normally found with the HLA-Bw47 serotype suggests that the HLA-B44 mutations are of more ancient origin. The patient's homologous chromosome has a deletion of P450c21B. Endocrinological testing therefore allows for testing of the mutant gene in genetic isolation. Such testing demonstrated that the patient was capable of producing aldosterone and retaining sodium in response to a low-sodium diet, indicating that the mutant gene encodes an enzyme with partial 21-hydroxylase activity.     

Identification of four novel mutations in the CYP21 gene in congenital adrenal hyperplasia in the Chinese

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. We have analyzed CYP21 gene sequences in 65 CAH families in Taiwan. All ten exons of the CYP21 gene were analyzed by differential polymerase chain reaction followed by single-strand conformation polymorphism electrophoresis and the amplification-created restriction site method. About 95% (123 chromosomes) contain mutations due to conversion of DNA sequences into its neighboring homologous pseudogene, CYP21P. Four novel mutations representing 5% of the total chromosomes have also been identified. The mutations were confirmed by sequencing an aberrant DNA fragment. These four mutations included a base change of the splicing donor site at intron 2 from GT to AT, a base substitution of C to T at codon 316, deletion of ten bases (TCCAGCTCCC) at codons 330–333 of exon 8, and duplication of 16 bases (CCTGGATGACACGGTC) at codons 393–397 of exon 9. The loss of the splicing donor site at intron 2 and the premature stop at codon 316 may result in aberrant splicing to reduce enzyme activity and a truncated protein with no enzyme activity, respectively. Likewise, both the duplication and the deletion forms create a frameshift and premature stop during translation. The resulting proteins lack the heme-binding domain and hence are expected to lose enzymatic activity. Since these mutations are not found in the neighboring CYP21P pseudogene, gene conversion should not be the cause of these novel mutations. Received: 20 April 1998 / Accepted: 30 May 1998     

Altered CYP21 genes in HLA-haplotypes associated with congenital adrenal hyperplasia (CAH): a family study

Disorders of the CYP21 gene, which is located within the major histocompatibility complex on the short arm of chromosome 6, are the leading causes of congenital adrenal hyperplasia (CAH). The coding gene and a highly homologous pseudogene are tandemly arranged with the two genes for the fourth component of complement (C4A and C4B). To analyse the prevalence rates of mutations of the CYP21 genes and the segregation of the CYP21 genes with their corresponding human leucocyte antigen (HLA)-haplotypes, 21 families with one or two children with the severe form of 21-hydroxylase deficiency were studied. Mutations of the CYP21 gene on their corresponding HLA-haplotype were detected by hybridisation of polymerase chain reaction (PCR)-amplified genomic DNA with sequence-specific oligonucleotides and solid phase direct sequencing. Our study has shown the following. (1) A single basepair mutation (AG or CG) within the second intron is the most frequent mutation leading to impaired 21-hydroxylase activity. This mutation is only detected in HLA-haplotypes associated with the salt-wasting form of CAH. (2) A large deletion of part or all of the CYP21 gene is associated with the HLA-haplotype A3, BW47, C6, DR7, DR53, DQ2 but is also observed in other HLA-haplotypes and can be detected by a simple rapid PCR restriction fragment length polymorphism method. (3) Two alleles of the coding CYP21 gene differing in a leucine codon within the first exon, (formerly described as a mutation associated with 21-hydroxylase deficiency) have been found with an equal distribution in patients with 21-hydroxylase deficiency, non-disease HLA-haplotypes and the local healthy controls.     

Diversity of the CYP21P-like gene in CYP21 deficiency

More than 90% of cases of congenital adrenal hyperplasia (CAH) are caused by mutations of the CYP21 gene. The occurrence of defective CYP21 genes, including 15 mutations, has been attributed to intergenic recombination of DNA sequences from CYP21P, and shows no influence on the RP1-C4A-CYP21P-XA-RP2-C4BCYP21- TNXB gene locus on chromosome 6p21.3. However, multiple gene deletions in this region produce at least three categories of gene arrangements: (a) C4A-CYP21P/CYP21-TNXB, in which there is a CYP21P/CYP21 fusion gene; (b) C4A-XCYP21-TNXB, where XCYP21 indicates that the CYP21 gene contains mutations of IVS2 (-12A/C>G and 707-714delGAGACTAC); and (c) C4A-CYP21P-TNXA/TNXB, in which the TNX A and B genes are fused. Among them, seven different structures of the CYP21 haplotype were found at these three loci. Formation of the C4A-CYP21P/CYP21-TNXB locus produced four distinct CYP21P/CYP21 chimeras. The C4A-XCYP21-TNXB locus contained the IVS2 mutation -12A/C>G and 707-714delGAGACTAC from the XCYP21 gene; and two kinds of TNXA/TNXB hybrids were found in the C4A-CYP21P-TNXA/TNXB locus. The seven different CYP21 alleles produced 3.2 kb Taq I fragments caused by deletion of the RP2-XA-C4B locus. Therefore, production of a 3.2-kb CYP21 allele shows diversity, but is not a unique feature of the CYP21P gene. Most of these gene arrangements probably exist in the C4A-XCYP21-TNXB and C4A-CYP21P/CYP21-TNXB gene loci. The existence of the C4A-CYP21P-TNXA/TNXB locus might not be common in CAH patients with 21-hydroxylase deficiency.     

Structural and functional analysis of a novel mutation of CYP21B in a heterozygote carrier of 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is one of the most common autosomal recessive disorders and occurs in its non-classical form in up to 6% of hirsute women. We report on a young woman with the clinical diagnosis of non-classical CAH and a novel, heterozygous missense mutation CTGGTG in exon 8, codon 317, of the steroid 21-hydroxylase CYP21B and complete loss of pseudogenes. Protein sequences of closely related P450 cytochromes and a homology-based 3D model of CYP21B were used for further functional analyses. We found that the mutated residue is part of a large cluster of hydrophobic residues. This cluster has three important features: (1) it is located directly next to the binding pocket, in close vicinity of the heme-cofactor, (2) all amino acids of the cluster are directly connected to two important binding regions, and (3) the packing within the cluster is very dense. Due to the tight packing in the cluster and its direct connection to the binding pocket region, any changes induced by the mutation of residue 317 can be expected to lead to structural shifts within the binding pocket and can explain the clinically observed impairment of 21-hydroxylase activity. In conclusion, the novel mutation L317V of the steroid 21-hydroxylase gene is associated with reduced steroid 21-hydroxylase activity probably due to structural shifts within the binding pocket and a mild phenotype of steroid 21-hydroxylase deficiency. In addition, the results support previous findings in which heterozygous CYP21 mutations are associated with symptoms of hyperandrogenism in susceptible individuals.     

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.     

Three novel mutations in Japanese patients with 21-hydroxylase deficiency

OBJECTIVE: This study analyzed the mutation of 21-hydroxylase deficiency (21-OHD) in 36 unrelated Japanese patients with congenital adrenal hyperplasia (CAH). METHODS: All the exons of the functional CYP21 gene (CYP21A2) were analyzed by polymerase chain reaction (PCR) and PCR direct sequencing. RESULTS: Apparent gene deletions and conversions were present in 23.6% of the 72 CAH alleles, in which the most frequent mutation was the IVS2-13 A/C>G (27.8%), followed by I172N (26.3%), consistent with the frequencies reported for other countries. Previously described mutations were not present in three unrelated cases. Sequence analysis of the complete functional CYP21A2 gene revealed three, not yet described mutations that represent a common pseudogene sequence. These three putative novel mutations are located in exon 1 (M1I), in exon 5 (1210-1211insT), and in exon 3 (R124H). CONCLUSIONS: In this study, we have identified three putative novel mutations. It remains to be determined whether these three mutations are responsible for the significant number of as yet uncharacterized CAH patients in Japan.     

Novel germline hMSH2 genomic deletion and somatic hMSH2 mutations in a hereditary nonpolyposis colorectal cancer family

Germline and somatic mutations of the hMSH2 gene were determined in a Japanese hereditary nonpolyposis colorectal cancer (HNPCC) family fulfilling the Amsterdam criteria. PCR-SSCP-sequencing of genomic DNA detected a somatic hMSH2 mutation of an A deletion at codon 227-229 in a duodenal carcinoma and a somatic hMSH2 mutation of an A insertion at codon 21 in a gastric carcinoma from affected family members, both carcinomas exhibiting high microsatellite instability. However, no germline hMSH2 mutation was detected by the PCR-SSCP-sequencing method. Genomic DNA was then analyzed by Southern blot hybridization using three hMSH2 cDNA probes (probe A involving exons 1-5, probe B involving exons 4-11 and probe C involving exons 9-16) after digestion by restriction enzymes, EcoRI, HindIII and NsiI. The NsiI digest of DNA from normal tissues of affected members exhibited an aberrant 8.6 kb restriction fragment, in addition to the normal 10.6 kb fragment, when hybridized to probes A and B. This suggested the presence of a heterozygous 2kb genomic deletion encompassing exon 4, 5 or 6. RT-PCR-sequencing revealed that the deleted region encompassed exon 5. This novel genomic deletion of the hMSH2 gene was confirmed to be pathogenic, and the Southern hybridization pattern was applied to the pre-symptomatic diagnosis.     

Analysis of the spectra of mutational damage of the 21-hydroxylase gene in patients with adreno-genital syndrome

The spectrum of mutations in the steroid 21-hydroxylase gene (CYP21B) and the frequency of 11 mutations among 66 patients with different forms of congenital adrenal hyperplasia (CAH) were analyzed by means of PCR amplification. Each of the CAH forms was characterized by specific spectrum of diagnostically important mutations. The salt-losing (SL) form of the disease was most frequently associated with gene deletion (39%) and the 668-13C-G mutation in the second intron (23.5%), whereas the majority of simple virilizing (SV) CAH cases were associated with the 1172N mutation in exon 4 (22%), gene deletion (16.5%), and the 668-13C-G mutation (16.5%). Mutations in the steroid 21-hydroxylase gene were detected in 70% of the chromosomes from the patients with the SL and SV forms of CAH, and only in 1.3% of the chromosomes from the patients with the nonclassic (NC) form. A total of 78 mutant chromosomes from the NC CAH patients were examined, and only one case of a gene deletion in the heterozygous state was revealed. In the individuals examined, the V281L and P30L mutations described in the NC CAH patients from other populations were not detected. This result can be explained either by the fact that NC CAH cases in Russia are associated with other major mutations, or by difficult clinical diagnosis questionable CAH cases.     

Evolutionary origin of mutations in the primate cytochrome P450c21 gene.

The CYP21 gene codes for the enzyme cytochrome P450c21 (21-hydroxylase), which is critically involved in the synthesis of glucocorticoids and mineralocorticoids. Standard human haplotypes contain two copies of CYP21--a functional gene and a pseudogene. Inactivation of the functional gene leads to congenital adrenal hyperplasia (CAH). The pseudogene has three main defects: an 8-bp deletion in exon 3, a T insertion in exon 7, and a stop codon in exon 8. To determine the origin of these defects and to shed light on the evolution of the CYP21 gene, we sequenced relevant segments of 10 primate CYP21 genes--three from a chimpanzee, another three from a gorilla, and four from an orangutan. We could show that the 8-bp deletion is present in the chimpanzee and humans, while the other two defects are restricted to humans only. In the gorilla and the 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 has 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.