The molecular genetics of 21-hydroxylase deficiency.
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Adrenal Hyperplasia, Congenital↗
Biomedical subjects
W L Miller
Publications and source records attributed to W L Miller.
Integrated cardiac, renal, and endocrine actions of endothelin.
Endothelin, a newly discovered endothelial-derived peptide, has been demonstrated in vitro to have potent vasocontractile properties and has been speculated to play a role in vivo in arterial pressure-volume homeostasis. The present studies in anesthetized dogs were designed to determine the action of endothelin on cardiovascular-renal and endocrine function in vivo as in acute arterial pressure-volume regulation. Intravenous infusion of endothelin (50 ng/kg per min) increases arterial pressure by increasing peripheral vascular resistance but in association with an increase in coronary vascular resistance and decreases in cardiac output. Renal blood flow and glomerular filtration rate were markedly reduced in association with a sustained reduction in sodium excretion and an increase in plasma renin activity. Atrial natriuretic factor, vasopressin, and aldosterone were also elevated. These results indicate that endothelin is a potent vasoconstrictor that elevates systemic blood pressure in association with marked decreases in cardiovascular and renal function. This peptide may function as a counterregulatory hormone to the effects of endothelial-derived vasodilator agent(s).
Animals↗
Rearrangements and point mutations of P450c21 genes are distinguished by five restriction endonuclease haplotypes identified by a new probing strategy in 57 families with congenital adrenal hyperplasia.
Congenital adrenal hyperplasia (CAH) is caused by disorders of the P450c21B gene, which, with the P450c21A pseudogene, lies in the HLA locus on chromosome 6. The near identity of nucleotide sequences and endonuclease cleavage sites in these A and B loci makes genetic analysis of this disease difficult. We used a genomic DNA probe that detects the P450c21 genes (A pseudogene, 3.2 kb; B gene, 3.7 kb in Taq I digests) and the 3' flanking DNA not detected with cDNA probes (A pseudogene, 2.4 kb; B gene, 2.5 kb) to examine Southern blots of genomic DNA from 68 patients and 165 unaffected family members in 57 families with CAH. Of 116 CAH-bearing chromosomes, 114 could be sorted into five easily distinguished haplotypes based on blots of DNA digested with Taq I and Bgl II. Haplotype I (76 of 116, 65.6%) was indistinguishable from normal and therefore bore very small lesions, presumably point mutations. Haplotype II (4 of 116, 3.4%) and haplotype III (8 of 116, 6.9%) had deletions and duplications of the P450c21A pseudogene but had structurally intact P450c21B genes presumably bearing point mutations; point mutation thus was the genetic defect in 88 of 116 chromosomes (75.9%). Haplotypes IV and V lack the 3.7-kb Taq I band normally associated with the P450c21B gene. Haplotype IV (13 of 116, 11.2%) retains all other bands, indicating that the P450c21B gene has undergone a gene conversion event, so that it is now also associated with a 3.2-kb band. Haplotype V (13 of 116, 11.2%) lacks the 2.4-kb Taq I fragment and the 12-kb Bgl II fragments normally associated with the P450c21A pseudogene, as well as lacking the 3.7-kb Taq I fragment, indicating deletion of approximately 30 kb of DNA, resulting in a single hybrid P450c21A/B gene. Most (114 of 116, 98%) CAH alleles thus can easily be classified with this new probing strategy, eliminating many ambiguities resulting from probing with cDNA.
Adrenal Hyperplasia, Congenital↗
Extraadrenal steroid 21-hydroxylation is not mediated by P450c21.
The 21-hydroxylation of progesterone to deoxycorticosterone (DOC) and of 17-hydroxyprogesterone to 11-deoxycortisol in the human adrenal cortex is mediated by a single enzyme termed P450c21. Extraadrenal tissues can clear circulating progesterone and progesterone sulfate by 21-hydroxylation to DOC and DOC-sulfate. It has previously been established that such extraadrenal 21-hydroxylase activity is widely distributed in adult and fetal tissues, but it has not been known if extra-adrenal 21-hydroxylation is mediated by the same P450c21 enzyme found in the adrenal. We examined human RNA from fetal adrenal, liver, kidney, lung, brain, heart, skin, spleen, testis, and placenta by solution hybridization to human P450c21 probes transcribed from cloned human P450c21 cDNA, followed by nuclease protection and acrylamide gel electrophoresis. No P450c21 mRNA was detectable in any extraadrenal tissue. The sensitivity of the assay would have detected P450c21 mRNA at 0.01% of its abundance in the human fetal adrenal. Similar experiments in rats showed no P450c21 mRNA in brain, heart, kidney, liver, lung, testis, ovary, or uterus. These results clearly demonstrate that one or more enzymes other than the classical adrenal 21-hydroxylase are responsible for human and rat extraadrenal 21-hydroxylation.
17-alpha-Hydroxyprogesterone↗
Role of endogenous atrial natriuretic factor in acute congestive heart failure.
The current studies were designed to investigate the functional significance of elevated endogenous atrial natriuretic factor (ANF) in acute congestive heart failure (CHF). Integrated cardiorenal and endocrine function were measured in three models of acute low-output congestive heart failure with comparably reduced cardiac output (CO) and mean arterial pressure (MAP). Acute CHF was produced by rapid right ventricular pacing (group I, n = 5) which decreases CO and increases atrial pressures and plasma ANF. In group II, n = 5, thoracic inferior vena caval constriction (TIVCC) was produced to decrease venous return and CO but without increases in atrial pressure or plasma ANF. In group III, n = 5, TIVCC was performed and exogenous ANF infused to achieve plasma concentrations observed in acute CHF. In acute CHF with increases in endogenous ANF, sodium excretion (UNaV), renal blood flow (RBF), plasma renin activity (PRA), and plasma aldosterone (PA) were maintained despite decreases in CO and MAP. In contrast, TIVCC with similar reductions in CO and MAP but without increases in ANF resulted in decreases in UNaV and RBF and increases in PRA and PA. Exogenous administration of ANF in TIVCC to mimic levels in acute CHF prevented sodium retention, renal vasoconstriction, and activation of renin and aldosterone. These studies demonstrate that endogenous ANF serves as an important physiologic volume regulator in acute CHF to maintain sodium excretion and possibly participate in the suppression of activation of the renin-angiotensin-aldosterone system despite the stimulus of arterial hypotension.
Aldosterone↗
Regulation of growth hormone (GH) secretion by GH-releasing factor, somatostatin, and insulin-like growth factor I in ovine fetal and neonatal pituitary cells in vitro.
Serum GH concentrations in the ovine fetus are much higher than those in the neonate, and the maximal GH response induced by GRF is 5-fold greater in the fetus than in the neonate. To clarify these in vivo observations further, we studied the effects of GRF, somatostatin (SRIF), and insulin-like growth factor I (IGF-I) on primary cultures of fetal and neonatal ovine pituitary cells. GH secretion from fetal ovine pituitary cells increased from 148 +/- 34 to 950 +/- 130 ng/10(5) cells.3 h in response to 1 nM GRF, whereas GH secretion from neonatal pituitary cells rose from 113 +/- 26 to 1221 +/- 129 ng/10(5) cells.3 h, a significantly greater response (P less than 0.001). This greater GRF-induced GH response in neonatal than fetal cells differs from the response in vivo and suggests that the increased in vivo response in the fetus is not due to inherently increased sensitivity of pituitary cells to GRF. SRIF (10 nM) decreased maximal GRF-induced GH secretion by 37 +/- 3% in fetal cells compared with 59 +/- 8% in neonatal cells (P less than 0.01). This may explain in part the decreased in vivo sensitivity to SRIF in the ovine fetus compared to that in the neonatal lamb. In fetal pituitary cells, 10 nM GRF increased ovine (o) GH mRNA from 100 +/- 14% to 145 +/- 40%, SRIF decreased oGH mRNA to 84 +/- 3%, and GRF and SRIF in combination increased fetal oGH mRNA to 126 +/- 24%. Values in neonatal pituitary cell cultures were similar (control, 100 +/- 17%; GRF, 132 +/- 6%, SRIF, 85 +/- 15%; GRF plus SRIF, 105 +/- 26%). Pretreating fetal cells with 100 nM IGF-I for 3 days reduced GRF-stimulated GH secretion from 1049 +/- 38 to 232 +/- 8 ng/10(5) cells.3 h (P less than 0.001). Similarly, IGF-I pretreatment of neonatal cells reduced GRF-stimulated GH secretion from 810 +/- 18 to 419 +/- 16 ng/10(5) cells.3 h (P less than 0.001). The mean secreted IGF-I was 0.58 U/ml (36 nM) in culture medium from neonatal cells and was unchanged by incubation for 3 days with 5 micrograms/ml hGH. Secreted IGF-I in medium from fetal cells was 0.87 U/ml (54 nM) without GH and 0.81 U/ml (51 nM) after incubation with human GH. IGF-I mRNA was present in neonatal pituitary and brain.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Gonadotropin-releasing hormone-stimulated luteinizing hormone (LH) release from ovine gonadotrophs in culture is separate from phorbol ester-stimulated LH release.
The proposed involvement of protein kinase-C (PKC) as a mediator of GnRH action in pituitary cells was investigated. Treatment of dispersed ovine pituitary cells with the PKC activator phorbol 12-myristate-13-acetate (PMA) for 6 h at a dose of 10 nM stimulated the release of 5- to 10-fold more LH than did GnRH at the same dose. Analogs of PMA also stimulated LH release, but at levels parallel to their reported abilities to activate PKC. Treatment with GnRH plus PMA stimulated more LH release than either one alone in a strictly additive manner at all dose combinations from 10 pM to 100 nM. The PKC-activating diacylglycerol sn-1,2-dioctanoylglycerol also stimulated LH release that was additive with GnRH-stimulated LH release. Furthermore, retinal, an inhibitor of PKC, inhibited only PMA-stimulated LH release and not GnRH-stimulated LH release. These results do not support the theory that PKC activation is a necessary part of the mechanism of GnRH-stimulated LH release. The theory that calcium mobilization is also part of the mechanism of GnRH-stimulated LH release was, however, supported by the finding that GnRH did not stimulate LH release from ovine pituitary cells incubated in calcium-free medium. This demonstrates the absolute requirement for calcium in GnRH action, as proposed by others. In addition, the calcium ionophores A23187 and ionomycin were able to trigger LH release by themselves as well as augment GnRH- and PMA-stimulated LH release. This further strengthens the hypothesis that calcium mobilization is important in LH secretion.
Animals↗
Gene conversions and rearrangements cause discordance between inheritance of forms of 21-hydroxylase deficiency and HLA types.
Congenital adrenal hyperplasia (CAH) can be caused by a variety of defects in the functional gene encoding 21-hydroxylase (P450c21), which lies in the midst of the human leukocyte antigen (HLA) locus on chromosome 6. As a result, Mendelian genetics permit clinically distinct forms of CAH to be traced genetically by HLA and complement typing of family members. The recent cloning of probes for P450c21 now permits tracing of the affected gene directly. A consanguineous family had three members affected with three clinically distinct forms of CAH. Two of these individuals had identical extended haplotypes, including nine HLA and complement loci. Despite this extensive identity, the patterns of genomic DNA fragments digested with endonuclease EcoRI and detected by a P450c21 cDNA probe differed greatly in these two individuals. Thus, the DNA diagnosis of allelic variation was much more sensitive than the HLA diagnosis. Genomic DNA digested with endonuclease TaqI and probed with P450c21 cDNA revealed the 3.2-kilobase (kb) band, which is generally associated with the nonfunctional P450c21 A pseudogene, in all family members, and also revealed the 3.7-kb band associated with the functional P450c21 B gene in all family members except the severely affected index case. Probing of the same blots with a genomic probe also permitted examination of the adjacent downstream TaqI fragments, showing retention of both the 2.4-kb (A pseudogene) and 2.5-kb (B gene) fragments. Similarly, BglII-digested genomic DNA from all individuals contained both the 12-kb (A pseudogene) and 11-kb (B gene) bands. These data indicate that the basis of 21-hydroxylase deficiency in the index case was due to a homozygous gene conversion event and not to gene deletion. These results show that the DNA in and around the 21-hydroxylase gene is genetically very active, so that the usual generalization concerning linkage and inheritance may yield incorrect conclusions and diagnoses.
Adolescent↗
Molecular characterization of a Leydig cell tumor presenting as congenital adrenal hyperplasia.
We present an unusual patient with a Leydig cell tumor to show that greatly elevated serum concentrations of 17-hydroxyprogesterone (17OHP) may not be diagnostic of congenital adrenal hyperplasia (CAH). A 3.5-yr-old boy had a small testicular mass and plasma 17OHP concentrations of 147-333 nmol/L (4,850-11,000 ng/dL), suggesting CAH with adrenal rests. However, normal plasma cortisol values and the unresponsiveness of the 17OHP concentration to dexamethasone suppression or ACTH stimulation suggested a diagnosis of Leydig cell tumor. A 4-fold elevation in plasma 21-deoxycortisol compared with a 200-fold elevation in 17OHP suggested that the elevated 17OHP derived from the normal pathway of testosterone synthesis in the testis. This was proven by normalization of all hormonal values after tumor resection. Compared to the abundance of mRNA for P450c17, the tumor contained unusually large amounts of mRNA for P450scc, the cholesterol side-chain cleavage enzyme, which is the rate-limiting step in steroid hormone synthesis. Increased P450scc activity, which increased the conversion of cholesterol to pregnenolone, apparently permitted the 17,20-lyase activity of P450c17 to become rate limiting, thus accounting for the increased secretion of 17OHP. Thus, Leydig cell tumors can produce quantities of 17OHP previously reported only in CAH due to 21-hydroxylase deficiency. The molecular characterization of steroidogenic mRNAs in this tumor indicates an unusual ratio in the expression of the genes for the steroidogenic enzymes, probably accounting for the unusual pattern of serum steroids.
17-Ketosteroids↗
The transcriptional response of the human chorionic gonadotropin beta-subunit gene to cAMP is cycloheximide sensitive and is mediated by cis-acting sequences different from that found in the alpha-subunit gene.
Cyclic AMP stimulates transcription of the genes encoding the alpha- and beta-subunits of human CG (hCG). Although the cis-acting cAMP response element (CRE) of the alpha-subunit gene has been extensively characterized, relatively little is known about the putative response element of the hCG beta gene. Here, we demonstrate that cAMP stimulation of hCG beta gene transcription requires ongoing protein synthesis, whereas cAMP stimulation of alpha-subunit gene transcription does not. These results suggest that cAMP-stimulated transcription of the alpha and hCG beta genes may involve different cis-acting elements and trans-acting factors. Accordingly, we have constructed a series of deletion mutants consisting of fragments of the hCG beta promoter-regulatory region linked to the bacterial chloramphenicol acetyl transferase gene. To potentiate detection of a CRE, we constructed vectors containing the Rous sarcoma virus enhancer to augment transcriptional activity of the hCG beta-promoter. We also used vectors designed to determine whether regions containing a putative CG beta CRE could confer cAMP responsiveness to a heterologous promoter. These constructs were evaluated for activity by transfection into choriocarcinoma (BeWo) cells. Transient expression of these vectors identifies a broad region which renders transcription of the chloramphenicol acetyl transferase gene responsive to cAMP. Surprisingly, division of the hCG beta 5'-flanking and untranslated regions into smaller fragments led to loss of cAMP responsiveness, suggesting that the hCG beta CRE may be comprised of multiple domains dispersed across the proximal 650 base pairs of CG beta 5'-flanking and untranslated sequence.(ABSTRACT TRUNCATED AT 250 WORDS)
8-Bromo Cyclic Adenosine Monophosphate↗
Human adrenodoxin: cloning of three cDNAs and cycloheximide enhancement in JEG-3 cells.
Adrenodoxin is an iron-sulfur protein serving as an electron transport intermediate for two mitochondrial steroidogenic cytochromes P450. We have cloned and sequenced three human adrenal adrenodoxin cDNAs. The longest 5'-untranslated region was 131 bases long, and the coding sequences, identical in all three clones, predict a preprotein of 180 amino acids. The 3'-untranslated regions were 235, 596, and 776 bases long due to the presence of alternate polyadenylation sites. RNA transfer blots showed multiple size species of adrenodoxin mRNA consistent with finding multiple polyadenylation sites. Similar sized cross-hybridizing RNA species are found abundantly in the adrenal and testis and to a lesser degree in RNA from human fetal brain, spleen, placenta, kidney, liver, and intestine, as well as in cultured fibroblasts, suggesting the same or a very similar iron-sulfur protein is found in mitochondria of nonsteroidogenic tissues. JEG-3 cells, a transformed progesterone-producing line of trophoblastic origin, accumulate mRNAs for cytochrome P450scc (the mitochondrial cholesterol side-chain cleavage enzyme), adrenodoxin, and the fos oncogene when stimulated with 8-bromo-cyclic AMP. Addition of actinomycin D to such cultures blocked cAMP-induced accumulation of mRNAs for cytochrome P450scc and adrenodoxin. Addition of cycloheximide or puromycin to such cultures substantially reduced basal levels and markedly attenuated the cAMP-induced accumulation of cytochrome P450scc mRNA, but augmented the accumulation of adrenodoxin and fos mRNAs in additive and multiplicative fashions, respectively. These data indicate that the cAMP-induced synthesis of the steroidogenic machinery is not wholly dependent on cycloheximide-sensitive protein mediators.
8-Bromo Cyclic Adenosine Monophosphate↗
Nasal consonants in esophageal speech.
Nasalization of nasal consonants was measured using the ratio of a nasal accelerometer signal amplitude to air-borne microphone signal amplitude. Utterances produced by 3 esophageal speakers were studied. Word position (and time following air injection) of the nasal consonants was varied. Degree of nasal resonance was dependent on word position, with significantly higher values for word-final than for word-initial position.
Humans↗
Human adrenodoxin reductase: two mRNAs encoded by a single gene on chromosome 17cen----q25 are expressed in steroidogenic tissues.
Adrenodoxin reductase is a mitochondrial flavoprotein that receives electrons from NADPH, thus initiating the electron-transport chain serving mitochondrial cytochromes P450. We have cloned and sequenced two human adrenodoxin reductase cDNAs that differ by the presence of six additional codons in the middle of one clone. The sequence in this region indicates that these six extra codons arise by alternative splicing of the pre-mRNA. Southern blot hybridization patterns of human genomic DNA cut with four restriction enzymes indicate that the human genome has only one gene for adrenodoxin reductase. Analysis of a panel of mouse-human somatic cell hybrids localized this gene to chromosome 17cen----q25. The alternatively spliced mRNA containing the six extra codons represents 10-20% of all adrenodoxin reductase mRNA. The expression of the adrenodoxin reductase gene may be stimulated by pituitary tropic hormones acting through cAMP, but its response is quantitatively much less than the responses of P450scc and adrenodoxin.
Amino Acid Sequence↗
Synteny mapping of the genes for 21 steroid hydroxylase, alpha A crystallin, and class I bovine leukocyte antigen in cattle.
Synteny mapping of the genes for bovine alpha A crystallin (CRYA-1), steroid 21 hydroxylase (21OH), and a class I major histocompatibility locus bovine leukocyte antigen (BoLA) was accomplished by Southern blot analysis of DNA from bovine-hamster hybrid somatic cells. Comparison of the distribution patterns of CRYA-1, 21OH, and BoLA sequences among the hybrid cells revealed that the genes are located on two different chromosomes in the bovine genome. CRYA-1 mapped with superoxide dismutase (SOD1 in syntenic group U10, while 21OH and BoLA were concordant with glyoxylase (GLO1) in syntenic group U21. Therefore, the syntenic array of CRYA-1, 21OH, and BoLA in cattle is different from that observed in rodents, where the genes are closely linked to each other and to GLO1, and consistent with reported linkages in man.
Animals↗
Cloning and structure of the human adrenodoxin gene.
Adrenodoxin is an iron-sulfur protein that serves as an electron transport intermediate for all mitochondrial forms of cytochrome P450. To facilitate studying the regulation of adrenodoxin, we have cloned and determined the structure of the human adrenodoxin gene. It spans more than 20 kb, containing four exons and three introns. The first exon encodes the 60-amino-acid signal peptide, directing transport of the protein into the inner mitochondrial matrix. The mature peptide of 124 amino acids is encoded by the other three exons. The third exon encodes the portion of the protein containing the iron-sulfur center and a domain which binds other components of the electron transport chain. The transcriptional start sites were determined by primer extension and S1 nuclease mapping. The 5'-flanking region of this gene contains canonical promoters including a TATA box at nucleotide position -30 and two GC boxes at nucleotide positions -60 and -100. The sequence at nucleotides -234 to -252 is also highly homologous to the glucocorticoid-responsive element and the estrogen-responsive element.
Adrenodoxin↗
Differential H1- and H2-receptor-mediated histamine responses of canine epicardial conductance and distal resistance coronary vessels.
The contributions of histamine (H1 or H2) receptor-mediated responses and, therefore, the effects of histamine blocking agents are unclear with regard to regulation of proximal epicardial and distal resistance coronary arteries. This study was designed to evaluate the effects of selective H1- and H2-receptor antagonists on epicardial and resistance vessels in the closed chest dog model. Histamine, diphenhydramine (H1 blocker), and cimetidine (H2 blocker) were infused into the left anterior descending coronary artery (LAD), and responses were studied by quantitative coronary angiography and flow measurements (133Xe washout). Histamine infusion alone produced a significant dilation of the proximal LAD (13% above control) only at the highest dose (45 micrograms/min), while LAD flow was increased by 128%. In the presence of H1 blocker, histamine produced significantly greater epicardial dilation (55% above control). The flow response curve was shifted to the right in the presence of H1 blocker, but the flow attenuation was overcome by the highest histamine dose. In contrast, the H2 blocker attenuated both epicardial dilation (6% below control) and flow response (31% above control) to the highest histamine dose. Results support a differential regulation of proximal epicardial and distal resistance vessels to histamine with epicardial arteries demonstrating H1-mediated vasoconstriction and H2-mediated vasodilation and distal resistance vessels showing H1- and H2-mediated vasodilation. In addition, these findings suggest that H1 blockade may antagonize histamine-related vasoconstriction and vasospasm, while H2 blockers may permit unopposed H1-mediated vasoconstriction of epicardial arteries and also limit resistance vessel vasodilatory responsiveness in the presence of elevated tissue histamine, as may occur in atherosclerotic coronary artery disease.
Animals↗
Molecular biology of steroid hormone synthesis.
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Animals↗
Hormonal regulation of growth hormone secretion and messenger ribonucleic acid accumulation in cultured bovine pituitary cells.
In both primary cultures and transformed tumor cultures of rat pituitary cells, preincubation with T3 or dexamethasone increases GH secretion, mRNA accumulation, and gene transcription. GRF stimulates secretion and transcription in primary rat pituitary cultures. Sex steroids stimulate GH secretion in man, but contradictory findings have been reported in vitro. We studied the hormonal regulation of bovine GH (bGH) in primary monolayer cultures of adult bovine pituitaries. Neither T3 nor dexamethasone changed immunoreactive bGH secretion during a 3-h experimental incubation. After 72-h preincubation with T3 or dexamethasone, bGH secretion remained unchanged. T3 (10(-8) M) or dexamethasone (10(-8) M) did not alter the bGH secretory response to doses of GRF from 10(-12)-10(-8) M. However, T3 (P less than 0.001; r = 0.73) and dexamethasone (P less than 0.001; r = -0.71) decreased bGH mRNA content in dose-dependent fashions, as determined by Northern analysis and RNA dot blots probed with 32P-labeled bGH cDNA. T3 10(-7) M) decreased bGH mRNA content to 70% of the control value, 10(-7) M dexamethasone decreased bGH mRNA content to 77% of the control value, while GRF increased bGH mRNA content to 174% of the control value in a dose-dependent fashion (P less than 0.001; r = 0.72). Preincubation with testosterone or dihydrotestosterone did not change basal or GRF-stimulated GH secretion. Seventy-two-hour preincubation with 10(-8) M estradiol did not alter basal GH secretion, but increased the bGH secretory response to doses of GRF from 10(-11)-10(-8) M (P less than 0.001). Incubation with estradiol did not change bGH mRNA levels. These results demonstrate that, in contrast to rat GH mRNA, bGH mRNA accumulation is inhibited by T3 and dexamethasone. Estradiol augments the response to GRF, but this effect is not mediated by an increase in mRNA content. The hormonal responses of somatotropes vary significantly among mammalian species.
Animals↗