Med Genet 1996;33:25-28 25 Variation of phenotype in patients with glucocorticoid remediable aldosteronism J Med Genet: first published as 10.1136/jmg.33.1.25 on 1 January 1996. Downloaded from

L J Gates, A A MacConnachie, R P Lifton, N E Haites, N Benjamin

Abstract gene would therefore have synthase Glucocorticoid remediable aldosteronism activity within the zona fasciculata because ofthe (GRA), an autosomal dominant cause of specificity ofits regulatory sequences and would , has been de- result in the synthesis of 18-oxocortisol, 18- scribed as resulting in severe hydroxycortisol, and aldosterone regulated in with premature death from . We des- response to adrenocorticotrophic cribe two new large pedigrees which in- (ACTH). Miyahara et all2 have shown that the clude subjects who have the abnormal protein encoded cDNA corresponding to the chimaeric gene strongly linked to GRA. chimaeric GRA gene exhibits aldosterone syn- The majority of affected members, who thase activity. In patients with GRA, treatment have only mild hypertension and normal with glucocorticoids causes feedback sup- biochemistry, are clinically indistin- pression of ACTH by its effect on the anterior guishable from patients with essential hy- pituitary, reducing aldosterone, 1 8-hydro- pertension. It is therefore likely that this xycortisol, and 18-oxocortisol synthesis.'3 condition is undercliagnosed. (J Med Genet 1996;33:25-28) Case reports FAMILY A Key words: hypertension; ; hypo- The proband (V-21, fig 4), a 55 year old farmer kalaemia. from Huntly, was first found to be hypertensive (blood pressure 220/150 mmHg) at the age of Glucocorticoid remediable aldosteronism 15 when he presented to his general practitioner (GRA) is a rare autosomal dominant' cause of with malaise. Evidence of sequelae of hyper- primary aldosteronism originally described in tension were apparent with ECG evidence ofleft 1966 by Sutherland et al' in a father and son ventricular hypertrophy with strain pattern, left with hypertension, low plasma con- ventricular enlargement on chest x ray, and centration, increased aldosterone secretion, in- grade 3 hypertensive retinopathy. Serum so- creased plasma volume, and suppressed plasma dium, potassium, and bicarbonate were initially activity, all of which were found to be within the normal range and there was no evid- reversed by low dose therapy. ence ofrenal disease by intravenous pyelography Further cases have been described of severe or renal biopsy. However, during the initial in- hypertension usually presenting at a young age patient assessment, there was a tendency to hy- http://jmg.bmj.com/ with signs ofprimary aldosteronism correctable pokalaemia with serum potassium recorded as by glucocorticoids and in association with 3 3, 3- 1, and 3 8 mmol/1 (NR 3 5-4 9 mmol/l). familial occurrence of hypertension6 and of A strong maternal family history ofhypertension cerebral haemorrhage.56 More recently the as- was also noted with his mother (IV- 17), grand- sociation of GRA with high levels of the ab- mother (III8), and two uncles (IV- 12 and Department of normal hybrid steroids 1 8-hydroxycortisol7 and IV- 18) being hypertensive and, in addition, on

Medicine and 18-oxocortisol8 has been reported. examination one of his sisters (V-22), aged 23, on October 1, 2021 by guest. Protected copyright. Therapeutics, University of We describe two pedigrees in which a total was also found to be hypertensive. There was Aberdeen, Polwarth of29 patients have been identified as possessing also a history ofcerebral haemorrhage in his ma- Building, Foresterhill, the abnormal 6-3 kb genomic DNA fragment, ternal grandmother. Aberdeen AB9 2ZD, following restriction analysis and detected by UK The proband had poor control of his blood Southern which is linked to L J Gates blotting, tightly pressure for many years while taking multiple A A MacConnachie GRA.9 In normal subjects, when genomic DNA antihypertensive medications and required sev- N Benjamin is digested with the restriction enzyme BamHI, eral admissions to hospital. He had previously Department of Southern blotted, and probed with a 11 3-hy- Molecular and Cell droxylase specific probe, there are two bands Biology, University of on the resultant autoradiograph of 8-5 and Fragment length Aberdeen, Polwarth 4 i5 kb, corresponding to 11 J-hydroxylase and Building, Foresterhill, Aberdeen AB9 2ZD, respectively. UK Aldosterone synthase and 11 P-hydroxylase are Aldosterone synthase 4.5 kb N E Haites >90% homologous at the nucleotide sequence level and both lie in tandem on chromosome 8q.'o Boyer Center for 1 1 f-hydroxylase Molecular Medicine, When genomic DNA from affected subjects is 8.5 kb Yale University School examined, the presence of a chimaeric gene of Medicine, results in a restriction fragment length poly- Connecticut, USA. Chimaeric gene 6.3 kb R P Lifton morphism (fig 1) and a third band on the auto- radiograph (fig 2). This new 6-3 kilobase frag- Correspondence to: regulatory sequence coding sequence| Dr Gates. ment results from the presence of a chimaeric E1 gene in which the sequences of 11 Received 21 April 1995 regulatory p- Figure 1 Schematic representation of BamHI restriction Revised version accepted for hydroxylase are fused to the coding sequences of sites to show the restriction fragment length polymorphism publication 30 August 1995 aldosterone synthase"1 (fig 3). The product ofthis present in DNA from patients with the chimaeric gene. 26 Gates, MacConnachie, Lifton, Haites, Benjamin

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Cco 01) a?sk 4 .--InL.-( °o =S 4 .R)S. -11a Figure 2 Autoradiograph of BamHI digested DNA with an ll/J-hydroxylase exon 3-5 .2)s. 0 00o probe. GRA mutation positive subjects have a third 6-3kb band corresponding to the Cl9\ presence of the chimaeric gene as well as the 8-5kb band and 4-5band corresponding to ;!,q r- )S. - CD Il/-hydroxylase and aldosterone synthase respectively. --E .2)S. 0 Lno .) 04 0co (.0 )S. CS O (I, All aldo synthase ACTH 11p-hydroxylase a) El I-1 EJ U) ao !EJ __ So _ crossing over -0 I a1) ;:,q 3 Coa1) .?,q -X o o Z ) ",)S. = -0 CD All aldo synthase ACTH aldo synthase ACTH 11f-hydroxylase 00*)S. g4- ~0 a) {: o° a1) http://jmg.bmj.com/ 0 °l c0 chimaeric genes D

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All= II ACTH=adrenocorticotrophic hormone aldo=aldosterone a1) on October 1, 2021 by guest. Protected copyright. regulatory sequence coding sequence E (Doo 0 CD- 0 CDo Co-tz 4 4- Figure 3 Schematic representation of crossing over during meiosis of ll/-hydroxylase .° and aldosterone synthase to produce the chimaenrc gene tightly linked to GRA. -00 0 xo ga) o 0 developed hypokalaemia (K + 2-8 mmol/l) while 0. on thiazide diuretics and required potassium sup- C was found to plementation. In October 1992, he - *-,k aldosteronism 2 have primary (renin pU/1 (NR -& 0Q0 ,0 5-50 ptU/1), aldosterone 24-9 ng/dl (NR<20)). z: ~ 0 U ECG again showed evidence of left ventricular _-0 hypertrophy. Abdominal CT scan showed no &.)q evidence of a Conn's adrenal and the raised aldosterone level was markedly suppressed from around 30 to 2 ng/dl two days after com- mencing 0 5 mg twice daily of dexamethasone -~~~~~~~~~~~~~~~~- (fig 5). DNA analysis was then carried out to confirm the clinical diagnosis of GRA. Since the diagnosis of GRA, the addition of dexa- methasone to his antihypertensive medication has improved the control of blood pressure and I'll he has been normokalaemic (October 1993, blood pressure 125/94 mmHg, serum potassium Variation ofphenotype in patients with glucocorticoid remediable aldosteronism 27

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~0'a J Med Genet: first published as 10.1136/jmg.33.1.25 on 1 January 1996. Downloaded from l 1 2 3 Day Figure 5 Aldosterone levels after starning oral dexamethasone 0-5mg twice daily in the proband offamilyA. -N O

4-1 mmol/l while taking dexamethasone -coo 0-25 mg twice daily, amiloride 10 mg bu- - o daily, l- metanide 1 mg daily, and quinapril 5 mg daily). Additional family members were traced from -LO 2o1 the family history and records of marriages, births, and deaths for individual parishes ofnorth ~ -v7 n0 east Scotland. Thirty-seven members offamilyA uno ~ ~ g c have been investigated to date and 15 (including c the proband) identified as possessing the I chimaeric gene strongly linked to GRA. As can a) be seen in the table, two family members (IV- 19 iIE0) and V 9) who have the abnormal chimaeric gene Q ° have no past medical history of hypertension, iI0 normal blood pressure recordings, and normal -~Io .s gC)o .1I plasma potassium concentrations. One of these, g-ogoC14N a) the 69 old maternal aunt (IV 4 proband's year 19), -aCDO _ O 0 had a bloodpressure of 152/88 mmHgandpotas- 10 sium 4-6 mmol/l at the time oftesting. S FAMILY B Ic The proband of family B (V 6, fig 6), a 25 year old man, had severe hypertension diagnosed at -a0 the of 15 a school medical. After 0 age following g. I admission for blood pressure assessment he was 0 a found to have a resting blood pressure of 150/ 0 00X 4- 114 mmHg, serum urea 4 0 mmol/l, and potas- . sium 3 9 mmol/l, and borderline criteria for left 'a

.0 http://jmg.bmj.com/ ventricular hypertrophy on ECG. There was a -aO uO CO i strong maternal family history of hypertension 0 o 0 in his mother (IV.2), grandmother (III4), and c uncle (IV-3). Despite antihypertensive med- ication there were problems with control. In 1993 he was noted to have hypokalaemic al- -CO-oo kalosis with serum potassium 3-3 mmol/l and co bicarbonate 34 mmol/l (NR 22-30 mmol/l). i on October 1, 2021 by guest. Protected copyright. On further investigation, he was found to have raised plasma aldosterone levels (970, -Uw 1 1350 pmol/l (NR 100-400 pmol/l)) which was also suppressed to 220 pmol/I following two C4 days of dexamethasone therapy at the dosage - 0 0~ 0 5 mg twice daily. His hypertension has con- -0O NO0 - K tinued to respond to low dose dexamethasone Ifl _Xsx H_~~.0 (0 75 mgdaily) and he is normokalaemic (serum 11 _ %m

Mutation positivefamily A members by Southern blotting

Age Sex BP* PMH ofHTt Biochemistry - ?~~~~~> tz4- IV- 17 82 F 190/116 HT aged 40 Episode of hypokalaemic alkalosis V-21 54 M 220/150 HT aged 15 Hypokalaemic alkalosis on thiazide V-22 50 F 180/104 HT aged 17 Hypokalaemic alkalosis IV-19 69 F 152/88 No PMH HT Normal range potassium 3-8 mmol/1). Of the 29 additional V 28 43 M 160/100 HT aged 27 Normal range family members investigated, we have identified VI 13 14 F No PMH HT IV- 18 68 M 170/120 HT aged 35 Hypokalaemia a further 14 possessing the abnormal chimaeric V25 31 M 170/114 HTaged 15 Normal range gene. Again, there is a spectrum of severity of V 24 30 M V10 35 M 170/110 HT aged 27 Hypokalaemia on thiazide diuretic clinical features; three mutation positive family Vi11 34 F 170/102 HTaged33 Normalrange members by DNA testing had no past medical V 9 28 F 146/97 No PMH HT Normal IV-2 33 F 160/100 Known HT Hypokalaemia on thiazide diuretic history ofhypertension with normal blood pres- IV-1 35 M 176/108 Known HT Hypokalaemia on thiazide diuretic sure and serum potassium levels while three * Blood pressure on no antihypertensive treatment. (JV 10, V- 17, V- 16) had hypertension diag- t Hypertension: sitting blood pressure> 160/95 mmHg. nosed in their teenage years. 28 Gates, MacConnachie, Lifton, Haites, Benjamin

The variation in phenotype of those having azide diuretics. The majority of positive family the mutation is illustrated by the immediate members are clinically indistinguishable from family members of the proband of family B. patients with essential hypertension and in His mother (IV-2) was diagnosed as having many cases were being treated as such before borderline blood pressures from the age of 19 the diagnosis. Few subjects are normotensive. J Med Genet: first published as 10.1136/jmg.33.1.25 on 1 January 1996. Downloaded from but was first treated for hypertension aged This form of hypertension is noteworthy be- 28 (BP 150/115 mmHg) and has had normal cause those with severe hypertension, such as serum biochemistry. She has been previously the probands ofthe two families described, may treated with a thiazide diuretic and is currently be unresponsive to standard antihypertensive treated with amiloride 5 mg daily with good medication but successfully managed by treat- blood pressure control. The proband has two ment with appropriate medical therapy such as sisters both possessing the chimaeric gene of dexamethasone, ,214 or amilor- GRA on DNA analysis, neither of whom had ide. 516 Expected benefit from identification and a past medical history of hypertension. The treatment of these cases is therefore high. It is oldest (V 5) is normotensive and has serum important to identify at risk subjects as they can potassium within normal limits; the younger be monitored and treated appropriately early in sister (VA4) had a borderline resting blood pres- life to prevent complications of severe hyper- sure but satisfactory daytime mean 24 hour tension. Each index case identified has had ambulatory blood pressure (136/80 mmHg) many affected relatives in whom the aetiology of and has not required therapy. At the time of their hypertension was previously undiagnosed diagnosis she had a potassium at the lower limit and in whom the blood pressure control may of normal (3 5 mmol/l). benefit from alternative or additional therapy to We are currently investigating a further three the standard antihypertensive medication, such families all with a proband found to possess as dexamethasone or anti-mineralocorticoid the chimaeric gene tightly linked to GRA. The medication. proband of the third family is a 30 year old We much appreciate the cooperation, help, and interest of the man from Peterhead, found on a screening families. We thank Dr N MacKay for his help and advice. medical to have a raised blood pressure. Initial 1 New MI, Oberfield SE, Levine LS, etal. Autosomal dominant investigations showed hypokalaemia (serum transmission and absence of HLA linkage in dexa- potassium 34mmol/1) and sequelae of hy- methasone suppressible hyperaldosteronism. Lancet 1980; i:550-1. pertension: left ventricular hypertrophy on 2 Sutherland DJA, Ruse JL, Laidlaw JC. Hypertension, in- ECG and grade I hypertensive fundal changes creased aldosterone secretion and low plasma renin activity relieved by dexamethasone. Can Med Assoc J 1966;95: with a strong maternal family history of hy- 1109-19. pertension (mother, aunt, and two uncles from 3 New MI, Peterson RE. A new form of congenital adrenal hyperplasia. JClin EndocrinolMetab 1967;27:300-5. the mother's four sibs), one ofthe uncles having 4 Miura K, Yoshinaga K, Goto K, etal. A case ofglucocorticoid- died from a cerebral haemorrhage aged 30. responsive aldosteronism. JClin EndocrinolMetab 1 968;28: 1807-15. The proband of the fourth family is a 56 year 5 Giebink GS, Gotlin RW, Biglieri EG, Katz FH. A kindred old woman from Huntly who has been treated with familial glucocorticoid-suppressible aldosteronism. J Clin EndocrinolMetab for hypertension since the age of 27, who has 1973;36:715-23. 6 Ganguly A, Grim CE, Bergstein J, et al. Genetic and patho- http://jmg.bmj.com/ been on dual therapy of beta blockade and physiologic studies of a new kindred with gluco- corticoid-suppressible hyperaldosteronism manifest in thiazide diuretic, and has had normal bio- three generations.J'CinEndocrinolMetab 1981 ;53: 1040-6. chemistry, normal fundoscopy, and no evidence 7 Chu MD, Ulick S. Isolation and identification of 1 8-hydroxy- from the of patients with primary hyper- of left ventricular hypertrophy on ECG, but aldosteronism. J Bio Chem 1982;257:2218-24. who had a family history of hypertension and 8 UlickS, Chu MD, Land M. Biosynthesis of 18-oxocortisol by aldosterone-producing adrenal tissue. JBiol Chem 1983; primary aldosteronism in her mother and of two 258:5498-502. maternal uncles with cerebral haemorrhage. 9 Lifton RP, Dluhy RG, Powers M, et al. A chimaeric11 I- hydroxylase/aldosterone synthase gene causes gluco- on October 1, 2021 by guest. Protected copyright. The proband of the fifth family has had high corticoid-remediable aldosteronism and human hyper- blood pressure since the age of 28, is currently tension. Nature 1992;355:262-5. 10 Mornet E, Dupont B, Vitek A, White PC. Characterisation treated with triple therapy of atenolol, enalapril, of two genes encoding human steroid11 I-hydroxylase (P- and the thiazide diuretic bendroflurazide, and 450iip).JjBiolChem 1989;264:20961-7. 11Lifton RP, Dluhy RG, Powers M, et al. Hereditary hy- has had a persistent mild hypokalaemic alkalosis pertension caused by chimaeric gene duplications and ec- (recent biochemistry potassium 3 6 mmol/l, bi- topic expression of aldosterone synthase. Nature Genet 1992;2:66-74. carbonate 32 mmol/l). His mother, aged 74, has 12 Miyahara K, Kawamoto T, Mitsuuchi Y,et al. The chimeric hypertension. gene linked to glucocorticoid suppressible hyperaldo- steronism encodes a fused P-450 protein possessing al- dosterone synthase activity. Biochem Biophys Res Commun. Discussion 1992;189:885-91. 13 Gomez-Sanchez CE, Gill JRJr, Ganguly A, Gordon RD. As can be seen from the clinical features of the Glucocorticoid-suppressible aldosteronism: a disorder of two families presented here, subjects affected the adrenal transitional zone. JClin EndocninolMetab 1988; 67:444-8. with GRA may present with a spectrum of 14 GillJR, BartterFC. Overproduction ofsodium-retainingster- disease with a few having severe hypertension oids by the zona glomerulosa is adrenocorticotrophin- dependent and mediates hypertension in dexamethasone- with attendant early morbidity and mortality, suppressible aldosteronism.ClinJ7 Endocrinol Metab 1981; primarily because of stroke. Although some 53:331-7. 15 Kremer D, Boddy K, BrownJJ, et al. Amiloride in the treat- of our family members described had evident ment of primary hyperaldosteronism and essential hy- hypokalaemic alkalosis, in most of the cases pertension.3rEiEndocnol 1977;7:151-7. 16 Stockigt JR, Scoggins BA. Long term evolution of gluco- hypokalaemia is only apparent when potassium corticoid-suppressible hyperaldosteronism. GlinJ En- homeostasis is stressed by treatment with thi- docninolMetab 1987;64:22-6.