The steroid 11 beta-hydroxylase (P450c11) enzyme is responsible for the conversion of 11-deoxycortisol to cortisol in the zona fasciculata of the adrenal cortex. Animal studies have suggested that this enzyme or a closely related isozyme is also responsible for the successive 11 beta- and 18-hydroxylation and 18-oxidation of deoxycorticosterone required for aldosterone synthesis in the zona glomerulosa. There are two distinct 11 beta-hydroxylase genes in man, CYP11B1 and CYP11B2, which are predicted to encode proteins with 93% amino acid identity. We used a sensitive assay based on the polymerase chain reaction to analyze the expression of the CYP11B1 and B2 genes. Transcripts of CYP11B1 were detected at high levels in surgical specimens of normal adrenals and also in an aldosterone-secreting adrenal tumor. Transcripts of CYP11B2 were found at low levels in normal adrenals, but at a much higher level in the aldosterone-secreting tumor. CYP11B2 mRNA levels were increased in cultured zona glomerulosa cells by physiological levels of angiotensin-II. The entire coding regions of both CYP11B1 and B2 cDNAs were cloned from the tumor mRNA. Expression of these cDNAs in cultured COS-1 cells demonstrated that the CYP11B1 product could only 11 beta-hydroxylate 11-deoxycortisol or deoxycorticosterone, whereas the CYP11B2 product could also 18-hydroxylate cortisol or corticosterone. A small amount of aldosterone was synthesized from deoxycorticosterone only in cells expressing CYP11B2 cDNA. These data demonstrate that the product of CYP11B2 is required for the final steps in the synthesis of aldosterone.

Abstract-Anomalies in either of the tightly linked genes encoding the enzymes CYP11B1 (11␤-hydroxylase) or CYP11B2 (aldosterone synthase) can lead to important changes in arterial pressure and are responsible for several monogenically inherited forms of hypertension. Mutations in these genes or their regulatory regions could thus contribute to genetic variation in susceptibility to essential hypertension. To test this hypothesis, we performed 2 complementary studies of the CYP11B1/CYP11B2 locus in essential hypertension. After characterizing a DNA contig containing the CYP11B1 gene and mapping the gene in the Centre d'Etudes du Polymorphisme Humain reference panel of families, we performed a linkage study with 292 hypertensive sibling pairs and a highly informative microsatellite marker near CYP11B1. We also analyzed the association of 2 frequent biallelic polymorphisms of the CYP11B2 gene, 1 in the promoter at position Ϫ344 (Ϫ344C/T) and the other, a common gene conversion in intron 2, with hypertension in 380 hypertensive patients and 293 normotensive individuals. Statistical analyses did not show significant linkage of the CYP11B1 microsatellite marker to hypertension. No positive association with hypertension was found with the gene conversion in intron 2, but a positive association with hypertension was found with the Ϫ344T allele. The hypertensive and normotensive samples differed significantly in both genotype (Pϭ0.023) and allele frequencies (Pϭ0.010). Our data suggest a modest contribution of the CYP11B2 gene to essential hypertension. (Hypertension. 1998;32:198-204.)Key Words: aldosterone synthase Ⅲ steroid 11␤-hydroxylase Ⅲ biallelic polymorphism Ⅲ microsatellite marker Ⅲ association study Ⅲ linkage study T he cytochrome P450, CYP11B1, a steroid 11␤-hydroxylase, catalyzes the terminal step of cortisol biosynthesis.

Steroid 11,-hydroxylase deficiency (failure to convert 11-deoxycortisol to cortisol) is the second most common cause of congenital adrenal hyperplasia and results in a hypertensive form of the disease. The 11-hydroxylase enzyme is encoded by the CYPIIBI gene on chromosome 8q22. Two mutations in CYPIIBI have previously been reported in patients with 11(l-hydroxylase deficiency-Arg-448 -* His and a 2-bp insertion in codon 394. We now report eight previously uncharacterized mutations causing this disorder. Seven are point mutations (three nonsense and four missense) and one is a single base pair deletion causing a frameshift. We have used an in vitro transfection assay to show that all five known missense mutations causing 11l-hydroxylase deficiency abolish enzymatic activity. In principle, deletions of CYPIIBI could be generated by unequal crossing-over between CYPIIBI and the adjacent CYP11B2 gene, but no such deletions were found among the deficiency alleles in this study. Seven of the 10 known mutations are clustered in exons 6-8, a nonrandom distribution within the gene. This may reflect the location of functionally important amino acid residues within the enzyme or an increased tendency to develop mutations within this region of the gene.

Glucocorticoid-suppressible hyperaldosteronism (GSH) is an autosomal dint form of familial hypertension. The biochemical abnormalities seen in this disorder may be remedied by ami tion of dexamethse, implying that aldosterone synthesis is being abnormally regulated by corticotropin. The final three steps of aldosterone synthesis, 11(3 and 18-hydroxylation and 18-oxidation, are mediated by a cytochrome P450 in the zona glomerulo of the adrenal cortex termed CYP11B2. A related isozyme in the zona fascculata, CYPIlB1, is required for cortisol synthesis; this isozyme, which is normally expressed at much higher levels than CYP11B2, only has 1l-hydroxylase activity. These isozymes are encoded by genes on human chromosome 8q22. We have now studied four unrelated patients with GSH. We found that each patient has one chromosome that carries three CYPIIB genes instead of two. This has presumably been generated by unequal meiotic crossing-over. The extra gene is a hybrid with 5' regulatory and coding regions corresponding to CYPIIBi and 3' coding regions from CYPIIB2. The breakpoint is in intron 2 in two cases, intron 3 in one, and exon 4 in one. Cells transfected with hybrid cDNAs containing up to the first three exons of CYPIIBI synthesized aldosterone at levels near that of cells carrying normal CYPIIB2, but cells transfected with hybrids contining the first five or more exons of CYPIIBI could not synts detectable amounts of aldosterone. These data demonstrate that GSH is caused by expression of a gene that is regulated like CYPIIBI but that encodes a protein able to synthesize aldosterone.

Summary). In this study, we quantify the importance of the initially inherited telomere lengths within cells, in relation to other factors that influence telomere length during life. We have estimated the inheritance by measuring telomere length in monozygotic (MZ) twins using Q-FISH with a telomere specific peptide nucleic acid (PNA)-probe. Homologous chromosomes were identified using subtelomeric polymorphic markers. We found that identical homologous telomeres from two aged MZ twins show significantly less differences in relative telomere length than when comparing the two homologues within one individual. This result means that towards the end of life, individual telomeres retain the characteristic relative length they had at the outset of life and that any length alteration during the lifespan impacts equally on genetically identical homologues. As the result applies across independent individuals, we conclude that, at least in lymphocytes, epigenetic/environmental effects on relative telomere length are relatively minor during life.

Corticosterone methyloxidase H (CMO-II) deficiency is an autosomal recessive disorder of aldosterone biosynthesis, characterized by an elevated ratio of 18-hydroxycorticosterone to aldosterone in serum. It is genetically linked to the CYPiIB) and CYP11B2 genes that, respectively, encode two cytochrome P450 isozymes, P45OXIB1 and P450XIB2. Whereas P45OXIB1 only catalyzes hydroxylation at position 1113 of 11-deoxycorticosterone and 11-deoxycortisol, P450XIB2 catalyzes the synthesis of aldosterone from deoxycorticosterone, a process that successively requires hydroxylation at positions 1113 and 18 and oxidation at position 18. To determine the molecular genetic basis of CMO-II deficiency, seven kindreds of Iranian-Jewish origin were studied in which members suffered from CMO-Hl deficiency. No mutations were found in the CYP1IBl genes, but two candidate mutations, R181W and V386A, were found in the CYPJ1B2 genes. When these mutations were individually introduced into CYP11B2 cDNA and expressed in cultured cells, R181W reduced 18-hydroxylase and abolished 18-oxidase activities but left 1113-hydroxylase activity intact, whereas V386A caused a small but consistent reduction in the production of 18-hydroxycorticosterone. All individuals affected with CMO-ll deficiency were homozygous for both mutations, whereas eight asymptomatic subjects were homozygous for R181W alone and three were homozygous for V386A alone. These findings confirm that P450XIB2 is the major enzyme mediating oxidation at position 18 in the adrenal and suggest that a small amount of residual activity undetectable in in vitro assays is sufficient to synthesize normal amounts of aldosterone.

The most active corticosteroids are 11 beta-hydroxylated. Humans have two isozymes with 11 beta-hydroxylase activity that are respectively required for cortisol and aldosterone synthesis. CYP11B1 (11 beta-hydroxylase) is expressed at high levels and is regulated by ACTH, whereas CYP11B2 (aldosterone synthase) is normally expressed at low levels and is regulated by angiotensin II. In addition to 11 beta-hydroxylase activity, the latter enzyme has 18-hydroxylase and 18-oxidase activities and thus can synthesize aldosterone from deoxycorticosterone. Insights into the normal functioning of these enzymes are gained from studies of disorders involving them. Mutations in the CYP11B1 gene cause steroid 11 beta-hydroxylase deficiency, a form of congenital adrenal hyperplasia characterized by signs of androgen excess and by hypertension. Mutations in CYP11B2 result in aldosterone synthase (corticosterone methyloxidase) deficiency, an isolated defect in aldosterone biosynthesis that can cause hyponatremia, hyperkalemia, and hypovolemic shock in infancy and failure to thrive in childhood. These are both recessive disorders. Unequal crossing over between the CYP11B genes can generate a duplicated chimeric gene with the transcriptional regulatory region of CYP11B1 but sufficient coding sequences from CYP11B2 so that the encoded enzyme has aldosterone synthase (i.e. 18-oxidase) activity. This results in aldosterone biosynthesis being regulated by ACTH, a condition termed glucocorticoid-suppressible hyperaldosteronism. This form of genetic hypertension is inherited in an autosomal dominant manner.

Glucocorticoid-suppressible hyperaldosteronism is a dominantly inherited form of hypertension believed to be caused by the presence of a hybrid CYP1IB1/CYPJ1B2 gene which has arisen from an unequal crossing over between the two CYPJJB genes in a previous meiosis. We have studied a French pedigree with seven affected individuals in which two affected individuals also have adrenal tumors and two others have micronodular adrenal hyperplasia. One of the adrenal tumors and the surrounding adrenal tissue has been removed, giving a rare opportunity to study the regulation and action of the hybrid gene causing the disease. The hybrid CYPIIB gene was demonstrated to be expressed at higher levels than either CYPlJBI or CYPIJB2 in the cortex of the adrenal by RT-PCR and Northern blot analysis. In situ hybridization showed that both CYPIJBI and the hybrid gene were expressed in all three zones of the cortex. In cell culture experiments hybrid gene expression was stimulated by ACTH leading to increased production of aldosterone and the hybrid steroids characteristic of glucocorticoid-suppressible hyperaldosteronism. The genetic basis of the adrenal pathologies in this family is not known but may be related to the duplication causing the hyperaldosteronism. (J. Clin. Invest. 1995. 96:2236-2246