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.     

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.     

Video enhanced imaging of the fluorescent Na+ probe SBFI indicates that colonic crypts absorb fluid by generating a hypertonic interstitial fluid

We report a rare ‘hypomorphic’ C4 allotype detected during routine screening in controls for the Rogers:1 epitope. C4B^*15 was distinguished by having only faint staining when using polyclonal anti-C4 antibody on agarose inimunoelectrophoresis (e.g. hypomorphic), having relatively weak hemolytic activity but being strongly reactive with monoclonal antibody to Rodgers 1. TaqI restriction fragment length polymorphism (RFLP) demonstrated that C4B^* 15 segregated with 7 kb and 5.4 kb C4 gene fragments and with the haplotype HLA-A2,C-, B50,BW6,DR7,DQ2,DR52,S07C2(1,15). The 5.4-kb fragment was more intense than the 7.0-kb fragment, suggesting duplication of the 5.4-kb fragment. This hypomorphic C4 allotype (genotype FREQUENCY = 0.0088) has diminished expression of C4 epitopes commonly recognized by polyclonal anti-C4 and may be missed by standard phenotyping methods.     

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.     

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.     

Evidence for frequent gene conversion in the steroid 21-hydroxylase P-450(C21) gene: implications for steroid 21-hydroxylase deficiency.

Oligonucleotide probes specific for the deleterious mutations harbored in the P-450(C21)A pseudogene and oligonucleotide probes specific for the corresponding sequences in the B gene were prepared to examine the molecular lesions in the P-450(C21) gene of P-450(C21)-deficient patients. Using these gene-specific probes, we performed Southern blot analyses of genomic DNAs from 11 patients and eight normal individuals. At least one allele of the B gene (the 3.7-kb TaqI fragment) in a patient was inactivated by mutations caused by recombination with the A gene. The A genes in normal individuals and patients seemed to be replaced frequently (i.e., 10/19 individuals) in their 3' portions by B gene sequences. All of these alterations occurred without changing the characteristic length (3.2 kb) of the TaqI fragment of the A gene, a result strongly suggesting that frequent gene conversions and/or intragenic recombinations have happened in the P-450(C21) genes. Densitometric analysis of the autoradiograms from hybridization experiments revealed extensive variation (from one to five copies) in the copy number of the A gene (the 3.2-kb TaqI fragment) whereas that of the B gene (the 3.7-kb TaqI fragment) was relatively constant at two or three copies.     

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.     

Marked genetic polymorphism of the swine steroid 21-hydroxylase gene, and its location between the SLA class I and class II regions

Restriction fragment length polymorphism (RFLP) analysis of the swine 21-hydroxylase (CYP21) region was conducted on 31 unrelated SLA class I typed pigs, mainly Large Whites, including 15 haplotypes. Ten haplotypes were from SLA genotypic homozygotes and five were from SLA class I phenotypic homozygotes. DNA digestion with Hin dIII, TaqI and PstI, and hybridization to a 4.5-kb swine CYP21 genomic probe yielded respectively two, four and three RFLP patterns. Six patterns were identified with combined RFLP. In addition, analysis of the CYP21 region in families comprising several SLA recombinants demonstrated that the CYP21 gene lies in the DNA segment between the SLA class I and class II regions. These overall results reinforce our previous conclusion about the existence in the pig of a single 21-hydroxylase gene. The characterization of at least six CYP21 allelic patterns provides a new tool for studying the associations between the SLA region and zootechnical traits.     

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.     

Polymorphism of the fourth component of complement in Turks

An analysis of polymorphism in the fourth component of human complement (C4) was performed on EDTA-plasma from 142 unrelated, randomly selected Turks without collagen-vascular disease or recurrent infections. Plasma samples treated with neuraminidase and carboxypeptidase-B were subjected to high-voltage agarose gel electrophoresis followed by immunofixation. C4B allotypes were further detected in some samples by Western blots with monoclonal antibody 1228 (anti-C4B/Ch1 reactivity). The frequencies of C4A and C4B alleles were determined. Allele C4B*5, which has been found to be relatively common in Asian (Oriental) populations, was not detected in this study. No specific predilection could be noted among the rare variants. C4A*3-C4B*1 was the most common haplotype (n = 40/142, or 28%) but was found less frequently than in Caucasian populations. This finding may be the result of the limited number of samples examined. C4A and/or C4B null allotypes were seen in 49 of 142 (34.6%) subjects. The most frequent C4 null allotype seen was C4B null (37/142, or 26%): 28 subjects had one C4B null allele; 1 had a homozygous deficiency of C4B (C4B*QO, *QO) and 7 had C4A*QO C4B*QO, a double heterozygous haplotype. Frequencies of homozygous haplotype C4A*Q0-C4B*Q0 in the population studied were found to be 0.007. The results of this study demonstrate that the genetic composition of the Turkish population exhibits both similarities and differences with the European population, and ranges between Caucasian and Mongoloid (Asian) populations.     

中国武汉地区汉族人补体第四成份(C4)的遗传多态现象

对神经氨酸酶处理去涎的血浆进行高压琼脂糖电泳后,分别应用免疫固定和溶血覆盖技术调查了我国武汉地区汉族180人的C4多态现象。在C4A座位发现5个变型:C4A4、3、2、1和91;在C4B座位发现6个变型:C4B 3、2、1、92、9W和96。两个座位均有静息等位基因(C4A~*QO和C4B~*QO)存在。它们相应的基因频率为:C4A~*4,0.014;3,0.633;2,0.192;1,0.011;91,0.003;QO,0.147;C4B~*3,0.006;2,0.127;1,0.751;92,0.041;9W,0.003;96,0.006;QO,0.0660单零C4A(c4A QO)表型个体占总体28.3％,单零C4B(C4B QO)占11.1％;纯合C4A缺乏(C4A QO,QO)和纯合C4B缺乏(C4BQO,QO)分别占0.56％。和1.11％卡方测验表明,C4A和C4B基因频率分别符合Hardy-Weinberg遗传平衡定律。     

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     

Genetic control of C6 polymorphism and C6 deficiency in rabbits

The genetic control of the sixth component of complement (C6) in rabbits has been studied by quantitation of C6 functional and antigenic levels and identification of polymorphism by isoelectric focusing (IEF) in gels. Patterns of inheritance of C6 variants in families carrying a silent gene for C6 were examined, and it was found that 3 common plasma phenotypic variants, C6 A, C6 B, and C6 QO were under the genetic control of allelic genes, C6*A, C6*B, and C6*QO. In IEF patterns, C6 A could be identified by its isoelectric point that was slightly more acidic than that of C6 B. C6 QO was undetectable because it lacked functional and antigenic activity. The C6*A/C6*B genotype displayed a mixed IEF pattern with bands characteristic of both C6 A and C6 B. Functional and antigenic levels of C6 that were found in heterozygous C6*A/C6*QO and C6*B/C6*QO rabbits were approximately one-half of the C6 levels found in the corresponding homozygous animals. The phenotypic variation closely resembles that previously observed in humans and rhesus monkeys, as well as preliminary data in rabbits. The patterns of inheritance indicated that the two common C6 structural genes and the deficiency gene were allelic variants at the same genetic locus.     

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

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

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.     

Partial C4 deficiency in subacute sclerosing panencephalitis

In an immunogenetic study, 23 subacute sclerosing panencephalitis (SSPE) patients and their families were studied for the HLA region markers HLA-A, B, C, DR, BF, C2, C4A, C4B, GLO I, and PGM3. In addition, C3, C4, and factor B serum levels were determined. A highly significant association of C4A^*QO with SSPE was found. Furthermore, two rare haplotypes, C4A*QOB*9QO, two C4ACh+ allotypes, and four Ch partial inhibitors were detected, which possibly impair the function of the C4 molecules. HLA-DR5 was increased. In addition, a number of rare HLA-A, C, B, DR haplotypes were observed. It is postulated that rare C4 molecular deficiency might be a predisposing factor in the pathogenesis of SSPE.     

A case of first trimester prenatal diagnosis of 21-hydroxylase deficiency with human complement C4 cDNA probe

Early prenatal diagnosis of 21-hydroxylase (21-OHase) deficiency would enable treatment to be done to protect the fetus from masculinization and/or life-threatening adrenal crisis at birth. We report here the prenatal diagnosis of 21-OHase deficiency with human complement component C4 cDNA to probe DNA from chorionic villi at 10 weeks of gestation. Southern analysis with human C4 cDNA identified TaqI restriction fragment length polymorphisms (RFLPs) in the family. Family analysis with these RELPs showed that the fetus was not affected at greater than 99% probability, because the frequency of recombination between the 21-OHase B gene and the C4 gene would be extremely low.     

Genetic mapping of the 21-hydroxylase locus: estimation of small recombination frequencies

The locus for 21-hydroxylase (CA21HB) has been mapped to the interval between the HLA-B and HLA-DR loci on chromosome 6. Several methods of estimating genetic distance were used to determine whether CA21HB is closer to HLA-B or HLA-DR based on data collected on 157 families ascertained through a proband with the classical form of 21-hydroxylase deficiency (CA12Hd). The results were inconclusive but serve to highlight the limitations of present methods of estimating genetic distance when recombination frequencies are of the order of .005.