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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Analysis of the association of HLA-DQ1 alleles with mutation of the 21-hydroxylase gene in patients with congenital adrenal hyperplasia

In 96 patients with congenital adrenal hyperplasia (CAH) and 50 healthy donors from northwestern Russia the distribution of the HLA-DQA1 alleles and the mutation spectrum and frequency at the CYP21B gene were examined. In the patients with nonclassical (NC) CAH, the distribution of the HLA-DQA1 polymorphic alleles was similar to that in the population sample. In the patients with the salt-wasting form of the disease a statistically significant decrease of the *0401 or *501 major allele frequency was observed. The prevalence of certain HLA-DQA1 genotypes, namely, HLA5, HLA3, and HLA4, was observed in the patients with the NC, salt-wasting (SW), and simple virilizing CAH, respectively. Each clinical group was characterized by a specific spectrum of clinically valuable mutations. An association between the CYP21B mutations most frequently found in case of SW and SV CAH (delB, I2splice, and I172N) and certain HLA-DQA1 alleles was demonstrated. The necessity of more precise clinical diagnostics of the NC CAH cases along with detailed examination of this group for determination of the major mutations typical of the NC CAH cases from northwestern Russia is discussed.     

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.     

Mutation-haplotype analysis of steroid 21-hydroxylase (CYP21) deficiency in Finland. Implications for the population history of defective alleles

Congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase deficiency is a common inherited defect of adrenal steroid hormone biosynthesis. Unusually for genetic disorders, the majority of mutations causing CAH apparently result from recombinations between the CYP21 gene encoding the 21-hydroxylase enzyme and the closely linked, highly homologous pseudogene CYP21P. The CYP21 and CYP21P genes are located in the major histocompatibility complex class III region on chromosome 6p21.3. We analyzed the mutations and recombination breakpoints in the CYP21 gene and determined the associated haplotypes in 51 unrelated Finnish families with CAH. They represent no less than half of all CYP21 deficiency patients in Finland. The results indicate the existence of multiple founder mutation-haplotype combinations in the population of Finnish CAH patients. The three most common haplotypes constituted half of all affected chromosomes; only one-sixth of the haplotypes represented single cases. Each of the common haplotypes was shown consistently to carry a typical CYP21 mutation and only in some cases was additional variation observed. Surprisingly, comparisons with previous published data revealed that several of the frequent mutation-haplotype combinations in Finland are in fact also found in many other populations of patients of European origin, thus suggesting that these haplotypes are of ancient origin. This is in clear contrast to many reports, including the present one, where a high frequency of de novo mutations in the CYP21 gene has been reported. In addition, two unique sequence aberrations in CYP21 (W302X and R356Q), not known to exist in the CYP21P pseudogene, were detected. Received: 5 September 1996 / Revised: 11 November 1996     

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.     

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.     

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.     

Exon 7 Ncol restriction site within CYP21B (steroid 21-hydroxylase) is a normal polymorphism

A point mutation within exon 7 producing an amino acid coding change and a recognition site for the endonuclease Ncol has been reported in the HLA-Bw47-linked CYP21A pseudogene and some mutant CYP21B (steroid 21-hydroxylase) genes of patients with congenital adrenal hyperplasia (CAH). Whether this mutation is deleterious was not demonstrated. We analyzed DNA from various subjects for the presence of the exon 7 Ncol site: group 1, 10 normal subjects; group 2, 11 patients with salt-losing CAH; and group 3, 18 members of an Amish pedigree in which 10 expressed HLA-Bw47 not linked to CAH. Southern blots of Ncol-digested genomic DNA which were hybridized with CYP21 cDNA showed that four subjects of group 1 had a heterozygous Ncol pattern. In group 2, seven patients had the Ncol site; two of them were homozygous for the site and had deletions of both CYP21B genes. The other five were heterozygous for the Ncol site, which was linked to a CYP21B deletion and a HLA-Bw47 haplotype. In group 3, no one exhibited the exon 7 Ncol site. To map the Ncol sites to CYP21A or CYP21B in the normal subjects, DNA from the four Ncol heterozygous subjects was double digested with Ncol and Mbol and hybridized with CYP21 cDNA. Ncol-Mbol fragments unique to CYP21A were identified in all four, but the smaller CYP21B-specific fragments were not detected. Their genomic DNA in the region of exon 7 (bases +1167 to +2058) was then amplified, cloned, and sequenced.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Major histocompatibility complex (MHC) class III genetics in two Amerindian tribes from Southern Brazil: the Kaingang and the Guarani

Population genetic studies of the major histocompatibility complex (MHC) class III region, comprising C2, BF and C4 phenotypes, and molecular genetic data are rarely available for populations other than Caucasoids. We have investigated three Amerindian populations from Southern Brazil: 131 Kaingang from Ivaí (KIV), 111 Kaingang (KRC) and 100 Guarani (GRC) from Rio das Cobras. Extended MHC haplotypes were derived after standard C2, BF, C4 phenotyping and restriction fragment length polymorphism (RFLP) analysis with TaqI, together with HLA data published previously by segregation analysis. C2 and BF frequencies corresponded to other Amerindian populations. C4B*Q0 frequency was high in the GRC (0.429) but low in the Kaingang. Unusual C4 alleles were found, viz. C4A*58, A*55 and C4B*22 (presumably non-Amerindian) and aberrant C4A*3 of Amerindian origin occurring with a frequency of 0.223 in the GRC. C4A*3 bands of homo- and heterozygous individuals carrying this variant were Rodgers 1 positive and Chido 1,3 positive, showed a C4A specific lysis type and a C4A like α-chain. Polymerase chain reaction studies and sequencing showed that this is based on a C4A*3 duplication with a regular C4A*3 and a partially converted C4A*0304 carrying the C4B specific epitopes Ch 6 and Ch 1,3. Associations of class III haplotypes with particular RFLP patterns were similar to those reported for Caucasoids. The previously described association between combined C4A and CYP21P deletions and the 6.4 kb TaqI fragment was not seen in these Amerindians. This fragment occurred within a regular two locus gene structure in the Kaingang, representing a “short” gene at C4 locus I. C4 and CYP21 duplications were frequently observed. The distribution of extended MHC haplotypes provides evidence for a close relationship between the KIV and KRC and a larger genetic distance between the two Kaingang groups and the GRC. Received: 6 March 1997 / Accepted: 13 May 1997     

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.     

Analysis of the Association between the HLA-DQA1 Alleles and the Steroid 21-Hydroxylase Gene Mutations in the Patients with Congenital Adrenal Hyperplasia

In 96 patients with congenital adrenal hyperplasia (CAH) and 50 healthy donors from northwestern Russia the distribution of the HLA-DQA1 alleles and the mutation spectrum and frequency at the CYP21B gene were examined. In the patients with nonclassical (NC) CAH, the distribution of the HLA-DQA1 polymorphic alleles was similar to that in the population sample. In the patients with the salt-wasting form of the disease a statistically significant decrease of the *0401 or *0501major allele frequency was observed. The prevalence of certain HLA-DQA1 genotypes, namely, HLA5, HLA3, and HLA4, was observed in the patients with the NC, salt-wasting (SW), and simple virilizing CAH, respectively. Each clinical group was characterized by a specific spectrum of clinically valuable mutations. An association between theCYP21B mutations most frequently found in case of SW and SV CAH (delB, I2splice, and I172N) and certain HLA-DQA1alleles was demonstrated. The necessity of more precise clinical diagnostics of the NC CAH cases along with detailed examination of this group for determination of the major mutations typical of the NC CAH cases from northwestern Russia is discussed.     

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.