Molecular cloning of steroid 21-hydroxylase

P. C. White, M. I. New, B. Dupont

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Congenital adrenal hyperplasia due to 21-hydroxylase (21-OH) deficiency affects approximately one in 5000 births and is, thus, one of the most common inborn errors of metabolism. It is inherited as a monogenic autosomal recessive trait closely linked to the HLA major histocompatibility complex. In this defect of cortisol biosynthesis, there is impaired conversion of 17-hydroxyprogesterone to 11-deoxycortisol. There are several forms of 21-OH deficiency that differ in severity and in HLA associations. An isolated enzyme defect in the zona fasciculata of the adrenal cortex, where cortisol is synthesized, is characteristically associated with HLA-Bw51(5). This is referred to as 'simple virilizing' disease because elevated levels of 17-hydroxyprogesterone result in excessive adrenal androgen synthesis. HLA-Bw47;DR7 and Bw60(40) are associated with severe 'salt-wasting' form of the disease where, in addition to the defect in the zona fasciculata, aldosterone synthesis in the zona glomerulosa (conversion of progesterone to 11-deoxycorticosterone) is impaired. If untreated, this can result in shock or death in the neonatal period from inability to conserve sodium. There are two related 'variant' forms associated with HLA-B14;DR1 in which patients have a relatively mild defect in cortisol synthesis, as measured by elevated baseline levels of 17-hydroxyprogesterone, and either remain asymptomatic ('cryptic' form) or develop symptoms of virilization in late childhood or at puberty ('late-onset' form). In addition to these HLA-B;DR associations, genetic linkage disequilibrium has been documented between complement allotypes and 21-OH deficiency. Of greatest interest are patients carrying the Bw47 antigen, who invariably have the unique haplotype HLA-Bw47;C4A*1;C4B*Q0 (null);Bf*F;C2*C. The Bw47(w4) antigen is serologically very similar to the more common antigen B13(w4) and these two antigens form identical banding patterns on isoelectric focusing. We, therefore, proposed that the HLA haplotype Cw6;Bw47; 21-OH*NULL; C4A*1; C4B*Q0;DR7 arose by a large genetic deletion or rearrangement of the original Cw6;B13;21-OH*NORMAL;C4A*3; C4B*1;DR7 haplotype. This implied that genomic DNA containing the 21-OH and C4B loci might be identified by comparing DNA from an individual with 21-OH deficiency who was homozygous for HLA-Bw47 and DNA from a normal individual homozygous for HLA-B13. The underlying biochemical defect in 21-OH deficiency has never been directly determined. In vitro 21-OH activity can be produced with two microsomal proteins from the adrenal cortex, an NADPH-dependent cytochrome reductase and a cytochrome P-450, termed P-T450(c21). P-450(c21) is one of a class of heme-containing monooxygenases with molecualr weights of about 50,000 which vary widely in substrate specificity and organ distribution. It is one of four cytochromes P-450 which are required to convert cholesterol to cortisol-the C22,27 side chain is cleaved by a P-450 to form pregnenolone; the 3β-hydroxyl is dehydrogenated, yielding progesterone, which is successively hydroxylated by three different cytochromes P-450 at the 17α, 21, and 11β positions to yield cortisol. As P-450(c21), but not cytochrome reductase, is substrate-specific, we hypothesized that the HLA-linked defect involved a structural gene for this protein. Therefore, we isolated a bovine adrenal cDNA clone encoding part of P-450(c21) in order to test this hypothesis. We found that a structural gene encoding P-450(c21) is indeed deleted when 21-OH deficiency is associated with HLA-Bw47.

Original languageEnglish (US)
Pages (from-to)277-287
Number of pages11
JournalAnnals of the New York Academy of Sciences
VolumeVOL. 458
StatePublished - 1985

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Steroid 21-Hydroxylase
Cloning
Molecular Cloning
Hydrocortisone
17-alpha-Hydroxyprogesterone
Defects
Zona Fasciculata
Cytochrome Reductases
Cytochrome P-450 Enzyme System
Haplotypes
Antigens
Adrenal Cortex
Progesterone
HLA-B14 Antigen
HLA-B13 Antigen
DNA
HLA-DR7 Antigen
HLA-DR1 Antigen
Cytochromes a
Cortodoxone

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Molecular cloning of steroid 21-hydroxylase. / White, P. C.; New, M. I.; Dupont, B.

In: Annals of the New York Academy of Sciences, Vol. VOL. 458, 1985, p. 277-287.

Research output: Contribution to journalArticle

White, P. C. ; New, M. I. ; Dupont, B. / Molecular cloning of steroid 21-hydroxylase. In: Annals of the New York Academy of Sciences. 1985 ; Vol. VOL. 458. pp. 277-287.
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T1 - Molecular cloning of steroid 21-hydroxylase

AU - White, P. C.

AU - New, M. I.

AU - Dupont, B.

PY - 1985

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N2 - Congenital adrenal hyperplasia due to 21-hydroxylase (21-OH) deficiency affects approximately one in 5000 births and is, thus, one of the most common inborn errors of metabolism. It is inherited as a monogenic autosomal recessive trait closely linked to the HLA major histocompatibility complex. In this defect of cortisol biosynthesis, there is impaired conversion of 17-hydroxyprogesterone to 11-deoxycortisol. There are several forms of 21-OH deficiency that differ in severity and in HLA associations. An isolated enzyme defect in the zona fasciculata of the adrenal cortex, where cortisol is synthesized, is characteristically associated with HLA-Bw51(5). This is referred to as 'simple virilizing' disease because elevated levels of 17-hydroxyprogesterone result in excessive adrenal androgen synthesis. HLA-Bw47;DR7 and Bw60(40) are associated with severe 'salt-wasting' form of the disease where, in addition to the defect in the zona fasciculata, aldosterone synthesis in the zona glomerulosa (conversion of progesterone to 11-deoxycorticosterone) is impaired. If untreated, this can result in shock or death in the neonatal period from inability to conserve sodium. There are two related 'variant' forms associated with HLA-B14;DR1 in which patients have a relatively mild defect in cortisol synthesis, as measured by elevated baseline levels of 17-hydroxyprogesterone, and either remain asymptomatic ('cryptic' form) or develop symptoms of virilization in late childhood or at puberty ('late-onset' form). In addition to these HLA-B;DR associations, genetic linkage disequilibrium has been documented between complement allotypes and 21-OH deficiency. Of greatest interest are patients carrying the Bw47 antigen, who invariably have the unique haplotype HLA-Bw47;C4A*1;C4B*Q0 (null);Bf*F;C2*C. The Bw47(w4) antigen is serologically very similar to the more common antigen B13(w4) and these two antigens form identical banding patterns on isoelectric focusing. We, therefore, proposed that the HLA haplotype Cw6;Bw47; 21-OH*NULL; C4A*1; C4B*Q0;DR7 arose by a large genetic deletion or rearrangement of the original Cw6;B13;21-OH*NORMAL;C4A*3; C4B*1;DR7 haplotype. This implied that genomic DNA containing the 21-OH and C4B loci might be identified by comparing DNA from an individual with 21-OH deficiency who was homozygous for HLA-Bw47 and DNA from a normal individual homozygous for HLA-B13. The underlying biochemical defect in 21-OH deficiency has never been directly determined. In vitro 21-OH activity can be produced with two microsomal proteins from the adrenal cortex, an NADPH-dependent cytochrome reductase and a cytochrome P-450, termed P-T450(c21). P-450(c21) is one of a class of heme-containing monooxygenases with molecualr weights of about 50,000 which vary widely in substrate specificity and organ distribution. It is one of four cytochromes P-450 which are required to convert cholesterol to cortisol-the C22,27 side chain is cleaved by a P-450 to form pregnenolone; the 3β-hydroxyl is dehydrogenated, yielding progesterone, which is successively hydroxylated by three different cytochromes P-450 at the 17α, 21, and 11β positions to yield cortisol. As P-450(c21), but not cytochrome reductase, is substrate-specific, we hypothesized that the HLA-linked defect involved a structural gene for this protein. Therefore, we isolated a bovine adrenal cDNA clone encoding part of P-450(c21) in order to test this hypothesis. We found that a structural gene encoding P-450(c21) is indeed deleted when 21-OH deficiency is associated with HLA-Bw47.

AB - Congenital adrenal hyperplasia due to 21-hydroxylase (21-OH) deficiency affects approximately one in 5000 births and is, thus, one of the most common inborn errors of metabolism. It is inherited as a monogenic autosomal recessive trait closely linked to the HLA major histocompatibility complex. In this defect of cortisol biosynthesis, there is impaired conversion of 17-hydroxyprogesterone to 11-deoxycortisol. There are several forms of 21-OH deficiency that differ in severity and in HLA associations. An isolated enzyme defect in the zona fasciculata of the adrenal cortex, where cortisol is synthesized, is characteristically associated with HLA-Bw51(5). This is referred to as 'simple virilizing' disease because elevated levels of 17-hydroxyprogesterone result in excessive adrenal androgen synthesis. HLA-Bw47;DR7 and Bw60(40) are associated with severe 'salt-wasting' form of the disease where, in addition to the defect in the zona fasciculata, aldosterone synthesis in the zona glomerulosa (conversion of progesterone to 11-deoxycorticosterone) is impaired. If untreated, this can result in shock or death in the neonatal period from inability to conserve sodium. There are two related 'variant' forms associated with HLA-B14;DR1 in which patients have a relatively mild defect in cortisol synthesis, as measured by elevated baseline levels of 17-hydroxyprogesterone, and either remain asymptomatic ('cryptic' form) or develop symptoms of virilization in late childhood or at puberty ('late-onset' form). In addition to these HLA-B;DR associations, genetic linkage disequilibrium has been documented between complement allotypes and 21-OH deficiency. Of greatest interest are patients carrying the Bw47 antigen, who invariably have the unique haplotype HLA-Bw47;C4A*1;C4B*Q0 (null);Bf*F;C2*C. The Bw47(w4) antigen is serologically very similar to the more common antigen B13(w4) and these two antigens form identical banding patterns on isoelectric focusing. We, therefore, proposed that the HLA haplotype Cw6;Bw47; 21-OH*NULL; C4A*1; C4B*Q0;DR7 arose by a large genetic deletion or rearrangement of the original Cw6;B13;21-OH*NORMAL;C4A*3; C4B*1;DR7 haplotype. This implied that genomic DNA containing the 21-OH and C4B loci might be identified by comparing DNA from an individual with 21-OH deficiency who was homozygous for HLA-Bw47 and DNA from a normal individual homozygous for HLA-B13. The underlying biochemical defect in 21-OH deficiency has never been directly determined. In vitro 21-OH activity can be produced with two microsomal proteins from the adrenal cortex, an NADPH-dependent cytochrome reductase and a cytochrome P-450, termed P-T450(c21). P-450(c21) is one of a class of heme-containing monooxygenases with molecualr weights of about 50,000 which vary widely in substrate specificity and organ distribution. It is one of four cytochromes P-450 which are required to convert cholesterol to cortisol-the C22,27 side chain is cleaved by a P-450 to form pregnenolone; the 3β-hydroxyl is dehydrogenated, yielding progesterone, which is successively hydroxylated by three different cytochromes P-450 at the 17α, 21, and 11β positions to yield cortisol. As P-450(c21), but not cytochrome reductase, is substrate-specific, we hypothesized that the HLA-linked defect involved a structural gene for this protein. Therefore, we isolated a bovine adrenal cDNA clone encoding part of P-450(c21) in order to test this hypothesis. We found that a structural gene encoding P-450(c21) is indeed deleted when 21-OH deficiency is associated with HLA-Bw47.

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