THE MOLECULAR defect underlying glucocorticoidsuppressible

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1 X/99/$03.00/0 Vol. 84, No. 11 The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A. Copyright 1999 by The Endocrine Society Biochemical Evidence of Aldosterone Overproduction and Abnormal Regulation in Normotensive Individuals with Familial Hyperaldosteronism Type I* MICHAEL STOWASSER, PHILLIP R. HUGGARD, TONY R. ROSSETTI, ANTHONY W. BACHMANN, AND RICHARD D. GORDON Hypertension Unit, University Department of Medicine, Greenslopes Hospital, Brisbane, 4120, Australia ABSTRACT We examined in detail biochemical characteristics of 10 normotensive individuals (6 females; age range, yr) with glucocorticoid-suppressible hyperaldosteronism (familial hyperaldosteronism type I) in an attempt to understand the development of hypertension in this disorder. All were normokalemic (median plasma potassium, mmol/l SD), and upright plasma aldosterone levels ( pmol/l) were within the normal range ( pmol/l) in nine subjects. However, upright PRA levels ( pmol/l min) were suppressed ( 13 pmol/l min), and the aldosterone to PRA ratio ( ) was elevated ( 65) in all but one subject. All subjects had elevated 24-h urinary levels of 18-oxo-cortisol ( nmol/ mmol creatinine; normal range, nmol/mmol creatinine). Plasma aldosterone failed to rise by at least 50% during 2 h ofupright THE MOLECULAR defect underlying glucocorticoidsuppressible hyperaldosteronism is a hybrid gene, the 5 sequences of which are derived from CYP11B1 and the 3 sequences are derived from CYP11B2 (1). The hybrid gene encodes an enzyme with aldosterone synthase activity, but its expression is regulated by ACTH by virtue of its CYP11B1 regulatory sequences, resulting in aldosterone production that is excessive and regulated by ACTH rather than by angiotensin II (AII) (1). We have designated this condition familial hyperaldosteronism type I (FH-I) (2) to distinguish it from a second familial variety of primary aldosteronism (FH-II) which, unlike FH-I, is not suppressible with glucocorticoids or associated with the hybrid gene mutation, but is frequently associated with the formation of aldosteroneproducing tumors. Hypertension in FH-I is often of early onset and may be of sufficient severity as to result in early death, commonly due to intracerebral hemorrhage (3 5). The development of genetic tests capable of reliably detecting the hybrid gene in DNA extracted from a peripheral blood sample (1, 6) collected anytime after birth (4, 7) has greatly facilitated the screening of families with FH-I and the detection of less Received March 3, Revision received June 4, Accepted August 3, Address correspondence and requests for reprints to: Dr Michael Stowasser, Hypertension Unit, University Department of Medicine, Greenslopes Hospital, Brisbane, Australia m.stowasser@ mailbox.uq.edu.au. * Supported by the Department of Veterans Affairs and the National Heart Foundation of Australia. posture in five of seven subjects, or during a 1-h infusion of angiotensin II (2 ng/kg min) in each of six subjects so studied. Serial, second-hourly (day-curve) aldosterone levels correlated tightly with cortisol (r , P 0.01 to 0.001), but not with PRA (r , not significant) levels in each of six subjects, and plasma aldosterone suppressed to less than 110 pmol/l during 4 days of dexamethasone administration (0.5 mg 6 hourly) in each of two studied, consistent with ACTH-regulated aldosterone production. In conclusion, biochemical evidence of excessive, abnormally regulated aldosterone production is present not only in hypertensive individuals with familial hyperaldosteronism type I, but also in those who are normotensive. The absence of hypertension in such individuals, therefore, cannot be attributed to lack of biochemical expression of the hybrid gene. (J Clin Endocrinol Metab 84: , 1999) floridly affected family members with only mildly elevated, or even normal blood pressure levels (2, 7 10). Normal blood pressure levels are encountered frequently among affected children, but in only a small proportion of adults found to have the condition by family screening (4, 9), suggesting that hypertension is likely to develop in most affected individuals following a variable normotensive phase. However, the presence and severity of hypertension varies considerably between affected individuals within any given age group, even among members of a single family sharing a common genetic abnormality (2, 4, 7 9), and some have remained normotensive until well into the 5th decade of life (11). In the current study, we examined in detail the biochemical characteristics of normotensive individuals with FH-I, to see if they were different from those described in hypertensive individuals with this disorder. Any such differences might expose factors important in the development of hypertension in this condition. Methods Diagnosis of FH-I and confirmation of normotensive state In all subjects studied, the diagnosis of FH-I was confirmed by demonstrating the presence of the hybrid gene in peripheral blood leucocyte DNA using a long-pcr -based method developed in this laboratory (4, 6) and validated against the Southern blot technique (1). Blood pressure was measured using a mercury sphygmomanometer after subjects had been quietly seated for at least 5 minutes. Three measurements, separated by at least 1 minute, were recorded. Subjects were regarded as normotensive if: 1) the mean of the last two systolic and diastolic measurements was within the normal range for age and sex (12); 2) they were not receiving antihypertensive medication; and 3) 4031

2 4032 STOWASSER ET AL. JCE&M 1999 Vol 84 No 11 there was no previous history of hypertension. Subjects were considered to be hypertensive if: 1) the mean of the last two systolic and diastolic measurements was above the normal range for age and sex; or 2) they were receiving medications as treatment for previously documented hypertension. Ten subjects (six females; median age when last assessed, yr sd; range, yr) of a series of 30 patients with FH-I in whom blood pressure status has been assessed according to these methods, were normotensive. Blood pressures remained normal in these individuals over yr (range, yr) of follow-up. All except one (patient 10, Table 1) of these 10 individuals belonged to a single large family (21 known affected members). At the time of assessment, 8 of the 10 normotensive subjects were not taking any regular medications. One female subject (patient 4) was receiving a combination oral contraceptive medication (cyproterone acetate 2000 g/ethinylestradiol 35 g, once daily), and another female (patient 10) was receiving hormone replacement therapy [twice weekly transdermal estradiol (2 mg), designed to release 25 g per 24 h]. During the biochemical studies, dietary salt intake was unrestricted. Midmorning upright plasma potassium, aldosterone and PRA, and urinary sodium and 18-oxo-cortisol levels Levels of plasma potassium, plasma aldosterone, PRA, and aldosterone to PRA ratios were measured in blood collected without stasis midmorning after at least 2 h of upright posture and compared with ranges established in our laboratory for normal subjects studied under similar conditions. Immediately after each collection, the blood was centrifuged and the plasma component was snap-frozen on dry ice and stored at 20 C pending assay. Levels of sodium and 18-oxo-cortisol (corrected for creatinine excretion) were measured in a 24-h urine collection. Aldosterone response to posture Plasma aldosterone was measured in blood collected at 0800 h following overnight recumbency and again at 1000 h following 2 h of upright posture (sitting, standing, or walking). Aldosterone was considered to be responsive to upright posture if the 1000-h upright levels were at least 50% higher than the 0800-h recumbent levels. Angiotensin II (AII) infusion studies AII infusion studies were performed during midmorning hours following at least 30 min of recumbency. AII was infused at a rate of 2 ng/kg min, and blood was collected basally and 60 min after commencement of the infusion for measurement of plasma aldosterone. Aldosterone was considered responsive to AII if levels rose by at least 50% during the infusion. Aldosterone, PRA, and cortisol day-curve studies For each day-curve study, a cannula was inserted into a forearm vein in the cubital fossa for blood sampling. Blood (15 ml) was collected every 2 h for 24 h from 1000 h to 1000 h for measurement of plasma aldosterone, PRA, potassium, and plasma cortisol. Posture was unrestricted until midnight, after which subjects remained recumbent until 0800 h and then assumed an upright posture until the completion of the day-curve at 1000 h. Dexamethasone suppression testing Plasma aldosterone, plasma cortisol, and PRA were measured in blood collected at 1000 h after 2 h ofupright posture following overnight recumbency, basally and also daily, during 4 days of dexamethasone administration (0.5 mg every 6 h). Assays Plasma aldosterone was measured by RIA (Coat-A-Count 125 I-Aldosterone RIA Kit; Diagnostic Products Corporation, Los Angeles, CA), in a modification of the method of Mayes et al. (13), with intra- and interassay coefficients of variation of 4.0% and 6.0%, respectively, and a lower detection limit of 69 pmol/l. PRA was measured by RIA (Gamma Coat [ 125 I] Plasma Renin Activity RIA Kit; INCSTAR Corp., Stillwater, MN) of generated angiotensin I in a modification of the method of Haber et al. (14), with intra- and interassay coefficients of variation of 4.3% and 7.2%, respectively, and a lower detection limit of 1.3 pmol/l/min. Plasma cortisol was measured by RIA (Quanticoat 125 I-Cortisol RIA Kit; Kallestad Diagnostics, Chaska, MN), with intra- and interassay coefficients of variation of 4.5% and 9.6%, respectively, and a lower detection limit of 28 nmol/l. Urinary 18-oxo-cortisol levels were determined by RIA using a method described previously (15). Data analysis Group data for the 10 normotensive subjects with FH-I are presented as median sd unless otherwise indicated. Results for hypertensive patients with FH-I have also been included, with the number value given in each case, for comparative purposes. For each day-curve study, Spearman rank correlation coefficients were determined for correlations between aldosterone and cortisol levels as a reflection of ACTH-dominated aldosterone regulation and between aldosterone and PRA levels as a reflection of AII-dominated regulation of aldosterone. Results Midmorning upright plasma potassium, aldosterone and PRA, and urinary 18-oxo-cortisol and sodium levels (Table 1) Plasma potassium levels were within the normal range ( mmol/l) for all 10 normotensive individuals, with a median sd for the group of mmol/l (compared with mmol/l in 19 hypertensive subjects with FH-I). TABLE 1. Basal clinical and biochemical characteristics of normotensive subjects with familial hyperaldosteronism type I Patient number Sex Age at diagnosis (yr) Age when last assessed (yr) Plasma potassium (mmol/l) Plasma aldosterone (pmol/l) Plasma renin activity (pmol/l.min) Aldosterone to PRA ratio Urinary 18-oxocortisol (nmol/mmol creatinine) ( ) a ( ) a (13 50) a (11 65) a ( ) a Urinary sodium (mmol/mmol creatinine) 1 F F M F F M M M F F Median SD a Normal ranges.

3 NORMOTENSIVE FH-I 4033 Upright plasma aldosterone levels were within the normal range ( pmol/l) in all except one (1412 pmol/l) of the normotensive subjects (patient 3, Table 1). The median level for the group was pmol/l (compared with pmol/l in 19 hypertensive subjects with FH-I). Upright PRA levels (median, pmol/l min compared with pmol/l min in 19 hypertensive subjects with FH-I) were suppressed below the lower limit of the normal range (13 pmol/l.min) and aldosterone to PRA ratios elevated (median, compared with in 19 hypertensive subjects with FH-I) above the upper limit of normal (65.0) in all but one subject (patient 4, Table 1). This 38-yr-old woman with normal plasma potassium (3.6 mmol/l) and aldosterone (646 pmol/l) levels, elevated PRA (99.0 pmol/l min), and low/normal aldosterone to PRA ratio (6.5) had never received diuretics, angiotensin-converting enzyme inhibitors, dihydropyridine calcium antagonists, or angiotensin type 1 receptor blockers, and did not demonstrate evidence of marked dietary salt restriction (24-h urinary sodium excretion 13.2 mmol/mmol creatinine) to explain the high PRA level, but was taking an estrogen and an androgen inhibitor. Repeat testing 3 months after changing her sole medication (a combination oral contraceptive medication containing 2000 g cyproterone acetate/35 g ethinylestradiol daily) to 150 g levonorgestrel/30 g ethinylestradiol daily gave similar results (blood pressure 110/ 80, plasma potassium 3.6 mmol/l, plasma aldosterone 755 pmol/l, PRA 74.6 pmol/l/min, and aldosterone to PRA ratio 10.1).With the exception of PRA and the aldosterone to PRA ratio, other biochemical findings in this patient (urinary 18-oxo-cortisol levels and results of posture studies, AII infusions, and day-curve studies) resembled those characteristic of FH-I. Urinary levels of 18-oxo-cortisol (median, nmol/mmol creatinine compared with nmol/ mmol creatinine in 15 hypertensive subjects with FH-I) were above the normal range ( nmol/mmol creatinine) in all subjects. Urinary sodium excretion ( mmol/mmol creatinine) was unremarkable in this group of normotensive patients with FH-I, and was not, specifically, consistent with marked dietary sodium restriction. Dynamic tests and day-curve studies Only two (patients 8 and 10, Table 2) of the seven subjects so studied demonstrated normal increases in plasma aldosterone of at least 50% above basal levels during assumption of upright posture (median response for the group, % compared with % in 19 hypertensive subjects with FH-I). However, in both of these responsive individuals, plasma cortisol also unexpectedly rose (by 130% and 59%, respectively), consistent with a concomitant rise in ACTH, and fell as expected (by 22%, 20%, 33%, and 8%, respectively) in the four unresponsive individuals in whom it was measured (patients 1, 4, 7, and 9). Although levels remained low, PRA rose during assumption of upright posture in five subjects (patients 1, 4, 5, 8, and 10; by 247%, 275%, 100%, 9%, and 97%, respectively), did not change in one (patient 9) and fell in one (patient 7, by 50%). None of the six subjects so studied demonstrated responsiveness of plasma aldosterone during an infusion of AII (Table 2; median response, % compared with % in 19 hypertensive patients with FH-I). Six subjects (patients 1, 4, and 7 10) underwent day-curve studies. In all six subjects, aldosterone correlated strongly with cortisol (r 0.79 to 0.97, median ; P 0.01 to 0.001), but not with PRA levels (r 0.13 to 0.40, median ; not significant). Two subjects (patients 5 and 10) underwent dexamethasone suppression testing (Fig. 1). Dexamethasone administration resulted in marked, persistent suppression of plasma cortisol, as expected. Within 24 h of dexamethasone administration, upright plasma aldosterone had fallen to less than 110 pmol/l (4 ng/dl), and PRA levels had begun to rise in both individuals. In both subjects, aldosterone to PRA ratios fell rapidly after commencement of dexamethasone and were still very low by day 5 (0 in patient 4, 1.9 in patient 10). One of the two individuals (patient 10) demonstrated evidence of early, partial recovery of AII-dependent aldosterone production, with plasma levels rising progressively from day 3 to day 5 of dexamethasone. This early recovery followed an earlier, marked rise in PRA levels, which by day 4 had reached pmol/l min (compared with corresponding levels ranging from pmol/l min in 15 other patients with FH-I studied within our unit). Patient 10 was a 40-yr-old normotensive female who was receiving treatment with 2 mg transdermal estradiol (designed to release 25 g per 24 h) twice weekly at the time of dexamethasone suppression testing. She demonstrated a sodium excretion rate of 9.4 mmol/ mmol creatinine (in the middle of the range of values for subjects in the current study) and gave no history of previous TABLE 2. Plasma aldosterone responses to upright posture and to angiotensin II infusion in normotensive patients with familial hyperaldosteronism type I Patient number Posture a Angiotensin II infusion (2 ng/kg min) Recumbent Upright % response Basal 60 min % response Not done Not done Median SD a For the posture studies, blood was collected at 0800 h after overnight recumbency and again at 1000 h after 2hofupright posture.

4 4034 STOWASSER ET AL. JCE&M 1999 Vol 84 No 11 FIG. 1. Changes in plasma aldosterone (F), plasma cortisol (Œ), and PRA (f) in response to dexamethasone administration (0.5 mg every 6 h) in two normotensive subjects with FH-I [patient 5 (a) and patient 10 (b)]. Levels were measured in blood collected at 1000 h after 2 h of upright posture. The shaded portion represents plasma aldosterone levels of 110 pmol/l (4 ng/dl) or less. treatment with diuretics to explain her brisk PRA response to dexamethasone. Discussion Among the current series of 30 individuals with FH-I proven by genetic testing and followed by us, 10 were normotensive at the time of diagnosis and have remained so during yr of follow-up. The fact that the median age of these 10 normotensive individuals when last examined approximates that of the 20 hypertensive patients with FH-I at the time that their hypertension was first detected suggests that at least some of these individuals, especially those who have already reached adulthood, have been relatively protected by some means from the hypertensive effects of the hybrid gene and that the development of hypertension in this disorder is not exclusively a function of age. Even in affected individuals who are hypertensive, similar protective mechanisms possibly operate to influence the severity of each patient s hypertension. In the current study, we examined the characteristics of normotensive individuals with FH-I to determine whether these subjects, in addition to having normal blood pressure, lacked any of the biochemical abnormalities that have been described for patients with complete expression of this disorder (2, 3, 5, 9 11, 16 20). These include hypokalemia, elevated plasma aldosterone, suppression of PRA, elevated aldosterone to PRA ratio, elevated urinary levels of the hybrid steroid 18-oxo-cortisol (17), unresponsiveness of aldosterone to upright posture or angiotensin II (AII) infusion (18), correlation of circadian aldosterone levels with those of cortisol (as a reflection of ACTH) rather than with those of PRA (as a reflection of AII) (11), and rapid, marked, and prolonged suppression of aldosterone during administration of dexamethasone (5, 19, 20). None of the normotensive patients with FH-I in this study were hypokalemic. However, several investigators have recently pointed out that hypokalemia is, in fact, uncommon in this condition, even when the patients are hypertensive, because it has been possible through genetic testing to identify all affected members of families, rather than identify only those with gross clinical or biochemical disturbances (2, 3, 8, 9). The same, more complete family studies have shown upright plasma aldosterone to be frequently within the normal range (2, 3, 9) in affected family members, even in hypertensive patients with FH-I. Only one of the normotensive patients in the present study demonstrated an elevated upright plasma aldosterone level. Patients with other forms of primary aldosteronism also frequently lack hypokalemia (2, 21 23) and demonstrate normal upright plasma aldosterone levels (2, 24 27). Excessive production of 18-hydroxy- and 18-oxo-cortisol (so-called hybrid steroids ) in FH-I is thought to result from aberrant expression of aldosterone synthase activity in zona fasciculata, where cortisol is available as a substrate (28, 29). Although an elevated urinary level of 18-oxo-cortisol is a sensitive biochemical marker of FH-I (3, 9), this steroid has only weak mineralocorticoid activity (30) and is believed not to play a major role in the development of hypertension in this condition (30). All normotensive subjects with FH-I showed elevated urinary levels of 18-oxo-cortisol. All but one subject (patient 4, who is discussed more fully below) in the current study demonstrated suppressed upright PRA levels and elevated aldosterone to PRA ratios, which are both biochemical indicators of hyperaldosteronism, evident in these individuals despite the absence of hypertension. Failure of plasma aldosterone to rise normally in response to upright posture or during AII infusion in FH-I is consistent with aldosterone production being predominantly regulated by ACTH (levels of which are falling during the morning hours when these tests are conducted) rather than by AII. In the current study, aldosterone responses to upright posture were seen in only two of seven subjects so studied and were attributable to concurrent unexpected rises in ACTH. Aldosterone failed to respond to AII infusion in these two patients, as well as in the other normotensive subjects with FH-I. Predominant regulation by ACTH also explains the strong tendency for circadian levels of aldosterone to tightly follow those of cortisol rather than PRA levels in this disorder (11 and the present study), and the ability of dexamethasone to rapidly induce marked suppression of plasma aldosterone, which usually lasts at least several days (9, 19, 20, and the present study). All the above features of FH-I were apparent in all of the subjects studied, despite the fact that they were normotensive.

5 NORMOTENSIVE FH-I 4035 Two female subjects (patients 4 and 10) demonstrated PRA levels and responses that differed from those of other normotensive individuals with FH-I, with one showing basal levels that were considerably higher and the other having levels that were suppressed basally, but showed a much brisker rise in response to dexamethasone administration. These two subjects were also the oldest among the current series of normotensive individuals with FH-I, suggesting that they might share factors rendering them particularly capable of resisting development of hypertension. At the time of assessment, both of these subjects were receiving treatment with preparations of estrogen. In normal females, the administration of estrogen preparations markedly increases hepatic synthesis and plasma levels of angiotensinogen (31). Reported effects of estrogen preparations on PRA levels have been variable, with earlier studies describing elevated levels (32, 33) and more recent studies (31, 34) reporting normal levels and reduced plasma renin concentrations, which presumably compensated for the increase in angiotensinogen production induced by these agents. It is possible, therefore, that the aberrant PRA findings in patients 4 and 10 can be explained solely on the basis of estrogen treatment. Cessation of the cyproterone acetate (an inhibitor of androgen action) component of patient 4 s combined oral contraceptive medication had no effect on her plasma aldosterone or PRA levels. Neither subject had ever received diuretic medications, and in neither subject were 24-h urinary sodium levels suggestive of marked dietary salt restriction to explain increased PRA levels or responsiveness. Reduced expression of the hybrid gene in certain patients with FH-I could result in higher PRA levels. However, urinary 18-oxo-cortisol levels were elevated in both subjects, and both demonstrated evidence of ACTH-regulated aldosterone production with plasma aldosterone levels falling during the early morning period (when ACTH falls as part of its normal diurnal rhythm) despite infusion of AII and tight correlation of circadian aldosterone levels with those of cortisol (but not PRA), suggesting functioning hybrid genes. An alternative explanation might be the coexistence in these subjects of a state of aldosterone resistance, perhaps genetically determined, which could protect them from the hypertensive and renin-suppressing effects of excessive aldosterone production. In conclusion, biochemical evidence of excessive, abnormally regulated aldosterone production characteristic of hypertensive individuals with FH-I was also present in normotensive individuals with FH-I. The absence of hypertension in such individuals cannot, therefore, be attributed to lack of expression of the hybrid gene. Given that urinary sodium excretion was unremarkable, the lack of hypertension in these individuals did not seem to be attributable to strict low dietary sodium intakes. Other investigators have reported more severe hypertension in this condition to be associated with lower urinary kallikrein levels (35) and maternal origin of the hybrid gene (8), but a lack of association of hypertension severity with urinary sodium excretion (35), urinary hybrid steroid levels (35), degree of hyperaldosteronism (8, 35), or position of the hybrid gene crossover point (8). The results of the current study exclude a complete lack of hybrid gene expression to explain the normal blood pressure levels. They do not exclude the possibility of a lower level of hybrid gene expression and of aldosterone overproduction contributing toward normality of blood pressure in normotensive individuals with FH-I. However, it is more likely that other genes involved in blood pressure regulation may determine whether an individual with this disorder develops hypertension. References 1. Lifton RP, Dluhy RG, Powers M, et al A chimeric 11 -hydroxylase/ aldosterone synthase gene causes glucocorticoid-remediable aldosteronism and human hypertension. Nature. 355: Gordon RD, Stowasser M Familial forms broaden the horizons for primary aldosteronism. Trends Endocrinol Metab. 9: Rich GM, Ulick S, Cook S, Wang JZ, Lifton RP, Dluhy RG Glucocorticoid-remediable aldosteronism in a large kindred: clinical spectrum and diagnosis using a characteristic biochemical phenotype. 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