Mapping of steroid 21-hydroxylase genes adjacent to complement component C4 genes in HLA, the major histocompatibility complex in man.

Abstract

The genes for four components (C) of complement in the human major histocompatibility complex (HLA) have been aligned previously in a series of overlapping cosmid cloned inserts. Those inserts, which contained the two C4 genes C4A and C4B, hybridized with human adrenal mRNA, indicating that they contain a gene expressed in the adrenal. The mRNA fraction of 2.4 kilobases (kb) hybridizes with genomic DNA of 4.5 kb, which is duplicated and lies about 1.5 kb 3' of both the C4A and the C4B complement genes. Sequencing of a 430-base section and comparison with the published cDNA sequence of bovine cytochrome P-450 21-hydroxylase, peptide sequences of porcine 21-hydroxylase, and a cDNA sequence of a rat liver cytochrome P-450 identified the gene as coding for human steroid 21-hydroxylase [steroid,hydrogen-donor:oxygen oxidoreductase (21-hydroxylating), EC 1.14.99.10]. Mapping of the gene was helped by use of a synthetic oligonudeotide based on the bovine cDNA sequence.The genes coding for the complement components C2, factor B, and two classes of C4 (C4A and C4B) lie between HLA-B and HLA-D in the major histocompatibility complex (HLA) in man (1). These genes have been mapped relative to each other, showing that those for C2 and factor B lie less than 1 kilobase (kb) apart and are separated from the C4A gene by about 30 kb (2). The C4B gene is about 10 kb from the C4A gene and, on a chromosome from one individual, a second C4B gene was found 10 kb further away (3).The most common form of congenital adrenal hyperplasia is due to a defect of steroidogenesis in the adrenal in which the enzyme steroid 21-hydroxylase [steroid,hydrogen-donor:oxygen oxidoreductase (21-hydroxylating), EC 1.14.99.10] is wholly or partly missing or inactive (4). This inherited defect is closely linked to the HLA complex (5). The 21-hydroxylase enzyme is a cytochrome P-450, and a cDNA clone coding for it has been obtained from bovine adrenal mRNA (6). The derived amino acid sequence showed strong homology with cystine-containing peptide sequences of porcine 21-hydroxylase (7), and there was limited homology between the cDNA sequence of the bovine 21-hydroxylase and of a rat liver cytochrome P-450 (8).To extend the characterization of the section of the HLA complex containing the complement genes, we tested the ability of cosmids (cos-containing plasmids) and their subcloned fragments to hybridize with human adrenal mRNA. This identified the position of the 21-hydroxylase genes in HLA, and use of a synthetic oligonucleotide based on the known cDNA and amino acid sequences helped to define the position of the genes. MATERIALS AND METHODSPreparation and Analysis of Genomic DNA. Genomic DNA was prepared from whole blood by the procedure of Bell et al. (9). For restriction enzyme analysis of DNA, 8-10 ,ug of genomic DNA was digested with one or two restriction endonucleases (5 units/,ug of genomic DNA) according to the manufacturer's instructions (New England Biolabs). DNA fragments were separated by electrophoresis in 0.7% agarose gels and t...

Uniparental isodisomy 6 associated with deficiency of the fourth component of complement.

“…DNA was isolated from peripheral mononuclear cells by phenol trophoresed in agarose, transferred to nylon membranes and hybridized with 32P-labeled probes, prior to autoradiography. The C4 probe used was a 500-bp fragment ofthe 5' end ofthe C4 cDNA, produced by Bam HI/Kpn I digest of pAT-A (12). The 21-hydroxylase (21-OH) probe was a 900 bp fragment, produced by a Bgl I digest ofthe genomic insert in p2 l-K4 (2).…”

Bases Moleculares da Hiperplasia Adrenal Congênita

“…A 21-hidroxilação é mediada por uma enzima mitocondrial específica denominada P450c21; seu gene codificador está localizado no locus do complexo principal de histocompatibilidade humana no braço curto do cromossomo 6, 6p21, especificamente dentro do locus do HLA classe III (5). Devido à íntima relação da 21OH com os genes do complexo HLA, observou-se que indivíduos afetados de uma mesma irmandade eram quase que invariavelmente HLA idênticos (6).…”

“…Os genes CYP21 e CYP21P se estendem sobre uma região de aproximadamente 30Kb, adjacentes e alternando com os genes C4A e C4B que codificam o quarto componente do complemento sérico (5,10). Recentemente foram descobertos outros pares de genes XA, XB, YA, YB, ZA e ZB (figura 1) (11).…”

A BF subtype “Fb1” is a marker gene of some mongoloid populations

“…The genetic locus of BF is structurally linked to the major histocompatibility complex (MHC) with other complement loci coding for C2, C4A, and C4B and two loci for 21-hydroxylase A (210HA) and B (21OHB) (Carroll et al, 1984(Carroll et al, , 1985White et al, 1985). Its genetic polymorphism has been extensively investigated on various populations since the first report of Alper et al (1972) by using immunofixation agarose gel electrophoresis.…”