Journal of Human (2004) 18, 47–51 & 2004 Nature Publishing Group All rights reserved 0950-9240/04 $25.00 www.nature.com/jhh ORIGINAL ARTICLE Coexistence of different phenotypes in a family with glucocorticoid-remediable aldosteronism

F Fallo1, C Pilon1, TA Williams2, N Sonino3, S Morra di Cella2, F Veglio2, R De Iasio4, P Montanari5 and P Mulatero2 1Department of Medical and Surgical Sciences, University of Padova, Italy; 2Department of Medicine and Experimental Oncology, University of Torino, Italy; 3Department of Statistical Sciences, University of Padova, Italy; 4CRBA, S Orsola Hospital, Bologna, Italy; 5City Hospital of Montecchio Emilia, Italy

In glucocorticoid-remediable aldosteronism (GRA), activity. Sibling 1 was normotensive, normokalaemic there is a large interfamily variation of phenotype. We and had normal PRA and . The father had report three subjects with GRA in a single family normal pressure and , low-normal PRA (parents, two brothers and two sisters), of whom only and normal aldosterone. All three subjects had elevated one (proband) displayed classical features of the levels of urinary 18-hydroxycortisol and 18-oxocortisol. excess. The proband was a man Baseline 11-deoxycorticosterone (DOC), corticosterone found to be hypertensive and hypokalaemic at the age (B) and aldosterone were high in the proband and of 24 years. Plasma activity was suppressed and normal in the father and sibling 1; 11-deoxycortisol (S) plasma aldosterone was repeatedly elevated. Blood and (F) were normal. ACTH induced a normal pressure and aldosterone levels normalized within 5 increase of B, DOC, S and F, and an excessive days of therapy. The presence of a aldosterone increase in all three patients. Abnormalities chimaeric CYP11B1/CYP11B2 gene was demonstrated in the chimaeric portions of CYB11B1 or CYP11B2 genes by long-PCR and Southern blotting (crossover site at the did not account for the phenotypic disparity of the end of intron 3) in the proband, in the younger sister different members in a single GRA family. Altered (sibling 1) and in the father. In these patients, sequen- regulation of the chimaeric gene may be responsible cing of the chimaeric portion of CYP11B1 did not reveal for differences in its activity. any mutation, while sequencing of the chimaeric portion Journal of Human Hypertension (2004) 18, 47–51. of CYP11B2 showed a V386A polymorphism in exon 7, doi:10.1038/sj.jhh.1001636 known to cause only a minimal impairment of enzymatic

Keywords: genotype; phenotype; glucocorticoid-remediable aldosteronism

Introduction coid therapy.2,3 The genetic abnormality results from the inheritance of a hybrid gene derived from the Glucocorticoid-remediable aldosteronism (GRA) is a crossover between the 50-regulatory ACTH-respon- rare autosomal dominant disorder of adrenocortical sive sequence of the CYP11B1 (11b-hydroxylase) steroid synthesis, in which aldosterone secretion is gene and the 30-coding region of the CYP11B2 solely regulated by adrenocorticotropin (ACTH) 4 1 () gene, and can be detected rather than II. In the classical form, by a reliable and simple long-PCR test.5 A consider- this syndrome is characterized by hypokalaemia, able interfamily variation has been described for and suppressed plasma renin many GRA phenotypic traits, including blood activity (PRA), presents at a young age in association pressure, aldosterone and PRA levels, hypokalaemia with familial occurrence of hypertension and cere- and occurrence of cerebrovascular accidents,2,3 and bral haemorrhage, and is correctable by glucocorti- no relationship between genotype and phenotype has been found.6,7 Scarce data are available on the intrafamily variation of phenotype in patients with Correspondence: Dr F Fallo, Department of Medical and Surgical genetically proved GRA. While in the family Sciences, University of Padova, Via Ospedale 105, 35128 Padova, previously reported by us8 and in that described Italy. 9 E-mail: [email protected] by Rich et al, the penetrance of hypertension and Received 12 May 2003; revised 5 August 2003; accepted 7 August aldosteronism was high in all affected pedigree 2003 members, in the family of Gates et al10 and in Different phenotypes in GRA F Fallo et al 48 another one more recently reported by us,11 the great 11-deoxycortisol (S) and cortisol (F) were normal. majority of affected members had mild hypertension ACTH administration induced a normal increase of and normal biochemistry, including serum potas- B, DOC, S and F, and an excessive increase of sium. Here we report a new family of six members in aldosterone. After 4 days of dexamethasone treat- which three subjects have been identified as ment (0.5 mg every 6 h) decreased, possessing the same type of CYP11B1/CYP11B2 aldosterone fell to undetectable levels and serum chimaeric fusion, but display diverse biochemical potassium increased slightly. On a long-term basis, and clinical phenotypes. dexamethasone 0.250 mg/day in combination with nifedipine 10 mg t.i.d. controlled blood pressure and serum potassium levels. The presence of the Case reports chimaeric CYP11B1/CYP11B2 gene was demon- strated by long PCR and Southern blotting of The Proband genomic DNA. Sequencing of the chimaeric PCR product demonstrated that the crossover site was A 34-year-old man was referred to our centre for located at the end of intron 3, just before the evaluation of refractory hypertension. He had been beginning of exon 4. initially diagnosed with hypertension and hypo- kalaemia at 24 years of age. was suspected on the basis of low PRA and elevated Family screening plasma and urinary aldosterone after repeated measurements. Abdominal CT scan and 75Se- All living first-degree family members were ana- methyl-nor-cholesterol adrenal scintiscan were in- lysed. The clinical, biochemical and genetic data are terpreted as showing adrenal bilateral , reported in Table 1. Body mass index (BMI) and and adrenal venous catheterization revealed sym- waist circumference were in the normal range for all metric, elevated secretion of aldosterone. The members, except the affected father and the un- patient had poorly controlled blood pressure for affected mother, who both showed mild overweight. many years while on multiple medical regimen, Blood pressure measurements and baseline data including , calcium-channel blockers were obtained in the two parents and the three and ACE inhibitors. He was evaluated as an out- siblings (one brother and two sisters), evaluated as patient on an unrestricted and potassium outpatients on an unrestricted sodium and potas- diet, and 3 weeks after withdrawal of antihyperten- sium diet. All siblings and the two parents were sive medications. There was no family history of found to be normotensive and normokalaemic, and hypertension or of early death from cerebrovascular had normal plasma and urinary aldosterone. The events. The proband’s blood pressure was 210/ father had low-normal PRA. No clinical conditions 110 mmHg, his serum potassium was 2.5 mmol/l, that could lower blood pressure, that is , upright PRA was suppressed and plasma and were present in the father. The father and the urinary aldosterone levels were elevated (Table 1). younger sister (ie, sibling 1) had high urinary 18- Routine biochemistry and renal scintigraphy were OHF and 18-oxoF. In these two subjects, an ACTH- normal. The EKG showed left ventricular hypertro- stimulation test was performed, following the same phy. Urinary levels of 18-hydroxycortisol (18-OHF) protocol described above. While baseline and and 18-oxocortisol (18-oxoF) were elevated. On a ACTH-stimulated plasma DOC, B, S and F were different day, ACTH 1-24 250 mg (Synacthen, Novar- normal, aldosterone was hyper-responsive to ACTH, tis Pharma) was injected as an i.v. bolus at 10.00 h as in the proband (Table 2). Sibling 1 and the father into the subject after a 30-min rest in a seated showed the same gene abnormality and the same position. Baseline 11-deoxycorticosterone (DOC), chimaeric gene crossover point of the proband. The corticosterone (B) and aldosterone were high, and crossover site was in the same region found in

Table 1 Clinical, biochemical and genetic data of the family members

Normal values Proband Sibling 1 Father Sibling 2 Sibling 3 Mother

Age (years)/sex (M/F) 34/M 21/F 65/M 36/F 29/M 61/F BMI (kg/m2) o25 22 19 27 22 24 24 Waist circumference (cm) o102 Mo88 F 82 67 99 79 86 84

CYP11B1/CYP11B2 chimaeric gene Positive Positive Positive Negative Negative Negative Blood pressure (mmHg) o140/o90 210/110 125/80 140/80 120/85 120/70 135/85 Serum K (mmol/l) 3.5–5.0 2.5 4.3 4.0 4.4 4.4 4.2 Upr. PRA (ng/ml.h) 1.3–5.2 0.1 2.6 1.0 4.8 3.9 3.5 Upr. Aldosterone (pmol/l) 140–830 1166 510 338 624 680 416 Urinary aldosterone (nmol/day) 8–83 138.1 48.8 40.8 50.2 42.8 32.9 Urinary 18-OHF (mmol/day) 0.05–0.29 6.61 1.83 5.81 0.15 0.24 0.29 Urinary 18-oxoF (nmol/day) 0.5–3.9 234.0 42.5 161.7 2.9 2.1 3.2

Journal of Human Hypertension Different phenotypes in GRA F Fallo et al 49 Table 2 Plasma steroid levels in the three patients with GRA and normal controls before and after ACTH administration

DOC (pmol/l) B (pmol/l) ALDO (pmol/l) S (nmol/l) F (nmol/l)

Proband Baseline 966 11 268 747 0.96 310 ACTH 2719 88 876 2916 3.64 625

Sibling 1 Baseline 466 3112 238 1.05 376 ACTH 1933 49 835 1105 4.51 774

Father Baseline 370 3910 397 0.57 292 ACTH 1537 47 291 2391 2.88 727

Normal subjects (n=10)a Baseline 449 7 88 4148 7 1155 318 7 117 0.82 7 0.35 358 7 116 ACTH 1330 7 508 62 058 7 9866 735 7 314 4.10 7 1.33 877 7 174

DOC, 11-deoxycorticosterone; B, corticosterone; ALDO, aldosterone; S, 11-deoxycortisol; F, cortisol. aMean 7 s.d. another Italian family from Sardinia, previously performed as described previously.5,15 In order to described by us.11 We analysed the sequences of sequence the entire chimaeric gene, a second long- the two hybrid genes to address the question of PCR was performed under similar conditions as whether the gene was the same, that is, the two described previously,15 but using primers comple- families had a common ancestor. In detail, we mentary to intron 3 of CYP11B1 and to 30UTR of compared two polymorphisms in intron 3, one in CYP11B2. The PCR products (3 kb) were purified exon 3 and one in intron 4. Both hybrid genes and sequenced as described previously.16 Primer carried a GAA at codon 173 codifying for lysine; sequences are available on request. however, the Sardinian family displayed a G in position +12 and a T in position +119 from the end of exon 3, and a C in position +140 from the end of Discussion exon 4. The family described here had three different nucleotides at the corresponding positions In our pedigree, we report a wide variation of (+12A/+119G/+140T), allowing us to exclude the phenotype between individuals affected by GRA. possibility of a common ancestor. Moreover, the two The proband had all classical features of primary Italian families were unrelated and lived in geogra- aldosteronism, that is, hypokalaemia, high aldoster- phical areas far from each other. one levels, suppressed PRA, in addition to the Since aldosterone levels were normal in sibling 1 presentation of hypertension at a young age. The and the father, we evaluated the possibility of a suppressibility of aldosterone by dexamethasone sequence alteration in the chimaeric portion of and the elevated levels of urinary 18-OHF and 18- CYP11B2 that would potentially lower aldosterone oxoF were also consistent with the clinical diag- synthase activity, thus explaining the occurrence of nosis, which was confirmed genetically by the normal aldosterone levels. Moreover, since these demonstration of the chimaeric CYP11B1/CYP11B2 GRA patients and the proband had no impairment of gene. Steroid precursors of aldosterone were ele- 11b-hydroxylase, in contrast to previous findings in vated in the proband, and DOC/B and S/F ratios GRA,18 the sequence of the chimaeric portion of were not different from normal either in baseline CYP11B1 was also examined. conditions (8 vs 10 and 0.2 vs 0.5, respectively, when expressed on an equimolar basis  100) or after ACTH stimulation (3 vs 2 and 0.2 vs 0.4, Laboratory evaluation respectively). The B/aldosterone ratio was also not different from normal in baseline conditions (15 vs Plasma and urinary aldosterone, PRA and F were 13, expressed on an equimolar basis), but was measured as described previously.5 Plasma DOC, greatly lower than normal after ACTH (30 vs 84), B and S were measured by an HPLC–RIA method reflecting the hyper-responsiveness of aldosterone as previously described.12 Urinary 18-OHF and to ACTH stimulation typical of this .8,17 18-oxoF were measured using an ELISA method.13,14 Moreover, a similar marked ACTH-induced decrease of B/aldosterone ratio due to aldosterone hyper- Molecular analysis response also occurred in the younger sister and in the father with GRA (45 and 20 vs 84, respectively). Long-PCR for the amplification of the chimaeric This steroid pattern is in disagreement gene and Southern blotting of genomic DNA were with findings in other studies of a deficient 11b-

Journal of Human Hypertension Different phenotypes in GRA F Fallo et al 50 hydroxylation in GRA,18 but is in accordance with 2 Dluhy RG, Lifton RF. Glucocorticoid-remediable aldos- an increased aldosterone synthase activity in this teronism. J Clin Endocrinol Metab 1999; 84: 4341– condition.19 The younger sister and the father were 4344. normotensive and normokalaemic. The father had 3 Stowasser M, Gordon RD. Primary aldosteronism: PRA in the lower limit of the normal range, learning from the study of familial varieties. J Hyper- tens 2000; 18: 1165–1176. consistent with the age. Lack of hypertension as 4 Lifton RB et al. A chimaeric 11b-hydroxylase/aldoster- well as of hypokalaemia was not surprising, because one synthase gene causes glucocorticoid remediable 2 this phenomenon has been frequently described. aldosteronism and human hypertension. Nature 1992; Differences in major common modifiers of blood 35: 262–265. pressure levels, for example, gender, age and BMI 5 Mulatero P et al. Diagnosis of glucocorticoid-remedi- (Table 1), did not appear to have an important role in able aldosteronism in primary aldosteronism: aldoster- inducing different phenotypes within the family. In one response to dexamethasone and long polymerase fact, the affected father, in spite of older age and chain reaction for chimeric gene. J Clin Endocrinol mild overweight, had normotension. All family Metab 1998; 83: 2573–2575. members lived in the same house, therefore suggest- 6 Pascoe L, Curnow KM, New MI, Corvol P. The relationship between phenotype and genotype ing that other factors potentially affecting pheno- on glucocorticoid-suppressible hyperaldosteronism type, such as dietary patterns and lifestyle, are (GSH). In: New MI (ed). Where Phenotype does not unlikely. Match Genotype, Ares-Serono Symposia SeriesFFron- All three subjects showed the same chimaeric tiers in , Vol. 16. Ares-Serono: Rome, gene crossover point. The possibility of an addi- 1996, pp 69–79. tional sequence alteration in the chimaeric portion 7 Dluhy RG, Lifton RP. Phenotypic variation in gluco- of CYP11B2, potentially lowering aldosterone corticoid-remediable aldosteronism (GRA). In: New MI (ed). Where phenotype does not Match Genotype, Ares- synthase activity and explaining the normal aldos- F terone levels in two of our GRA subjects, was Serono Symposia Series Frontiers in Endocrinology, excluded by our study. In particular, there was no Vol. 16. Ares-Serono: Rome, 1996, pp 81–90. 8 Fallo F et al. A new family with dexamethasone- mutation or gene conversion involving sequences of suppressible hyperaldosteronism: aldosterone unre- exons 5 and 6 responsible for 18-hydroxylase and sponsiveness to angiotensin II. Clin Endocrinol (Oxf) 15,20 18-oxidase activities. Moreover, in all three GRA 1985; 22: 777–785. subjects we detected the same V386A polymorph- 9 Rich GM et al. Glucocorticoid-remediable aldosteron- ism in the chimaeric portion of CYP11B2. This ism in a large kindred: clinical spectrum and diagnosis polymorphism is known to cause only minimal using a characteristic biochemical phenotype. Ann impairment of aldosterone synthase activity Intern Med 1992; 116: 813–820. in vitro,21 and has not been seen as an isolated 10 Gates LJ et al. Variation of phenotype in patients with homozygous change in patients with corticosterone glucocorticoid remediable aldosteronism. J Med Genet methyloxidase type I or II deficiency.22 It is rather 1996; 33: 25–28. 11 Mulatero P et al. Glucocorticoid remediable aldoster- possible that an altered regulation of the chimaeric onism: low morbidity and mortality in a four-genera- gene by specific inhibitors or repressors, or by other tion italian pedigree. J Clin Endocrinol Metab 2002; 87: genes lying outside the locus of the chimaeric gene 3187–3191. may be responsible for its differential expression 12 Boschi S et al. Measurement of steroid in and activity. Further, we cannot exclude the possi- plasma by isocratic high performance liquid chroma- bility that other genetic factors could interact with tography coupled by radioimmunoassay. Clin Chim the penetrance of the genetic alteration associated Acta 1994; 231: 107–113. with GRA. 13 Gomez-Sanchez CE, Leon LM, Gomez-Sanchez EP. Biotin-hydrazide derivatives for the development of steroid enzyme-linked immunoassay. J Steroid Bio- chem Mol Biol 1992; 43: 523–527. 14 Yamakita N et al. Simultaneous measurement of Acknowledgements plasma 18-oxocortisol and 18-hydroxycortisol We thank Professor Celso Gomez-Sanchez (Univer- levels in normal man. Eur J Endocrinol 1994; 131: 74–79. sity of Jackson, Mississippi, USA) for the kind gift of 15 Mulatero P et al. Recombinant CYP11B genes the antibodies used in the 18-OHF and 18-oxoF encode enzymes that can catalyze conversion of 11- assays. We are also indebted to Dr Leigh Pascoe deoxycortisol to cortisol, 18-hydroxycortisol, and (CEPH, Paris France) for helpful advice. 18-oxocortisol. J Clin Endocrinol Metab 1998; 83: 3996–4001. 16 Mulatero P et al. Blood pressure in patients with

primary aldosteronism is influenced by bradykinin B2 References receptor and a-adducin gene polymorphisms. J Clin Endocrinol Metab 2002; 87: 3337–3343. 1 Sutherland DJA, Ruse JL, Laidlaw JC. Hypertension, 17 Ganguly A, Weinberger MH, Guthrie GP, Fineberg NS. increased aldosterone secretion and low plasma renin Adrenal steroid response to ACTH in glucocorticoid- activity relieved by dexamethasone. Can Med Assoc J suppressible aldosteronism. Hypertension 1984; 6: 1966; 95: 1109–1119. 563–567.

Journal of Human Hypertension Different phenotypes in GRA F Fallo et al 51 18 Jamieson A et al. Altered 11b-hydroxylase activity cing enzyme, CYP11B1, into an aldosterone producing in glucocorticoid-suppressible hyperaldosteronism. enzyme. Nat Struct Biol 1997; 1: 32–35. J Clin Endocrinol Metab 1996; 81: 2298–2302. 21 Pascoe L et al. Mutations in the human CYP11B2 19 Fallo F, Kuhnle U, Boscaro Ma, Sonino N. Abnormality (aldosterone synthase) gene causing corticosterone of aldosterone and cortisol late pathways in glucocor- methyl oxidase II deficiency. Proc Natl Acad Sci ticoid-remediable aldosteronism. J Clin Endocrinol 1992; 89: 4996–5000. Metab 1994; 79: 772–774. 22 Wasniewska M et al. Aldosterone synthase deficiency 20 Curnow KM et al. The amino acid substitutions type 1 with no documented mutations in the CYP11B2 Ser288Gly and Val320Ala convert the cortisol produ- gene. Eur J Endocrinol 2001; 144: 59–62.

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