Proc. Natl. Acad. Sci. USA Vol. 95, pp. 10200 –10205, August 1998 Medical Sciences A genetic defect resulting in mild low-renin hypertension ROBERT C. WILSON*, SWATI DAVE-SHARMA*, JI-QING WEI*, VARUNI R. OBEYESEKERE†, KEVIN LI†, PAOLO FERRARI‡, ZYGMUNT S. KROZOWSKI†, CEDRIC H. L. SHACKLETON§, LEON BRADLOW*, TIMOTHY WIENS¶, AND MARIA I. NEW*i *Pediatric Endocrinology, The New York Hospital–Cornell Medical Center, 525 East 68th Street, New York, NY 10021; †Laboratory of Molecular Hypertension, Baker Medical Research Institute, P.O. Box 348 Prahran, Melbourne, Australia 3181; ‡Division of Nephrology, Inselspital 3010 Bern, Switzerland; §Mass Spectrometry Facility, Children’s Hospital Oakland, 747 52nd Street, Oakland, CA 94609-1809; and ¶Wichita Clinic, Bethel Office, 201 South Pine Street, Newton, KS 67114 Contributed by Maria I. New, June 24, 1998 ABSTRACT Severe low-renin hypertension has few known causes. Apparent mineralocorticoid excess (AME) is a genetic disorder that results in severe juvenile low-renin hypertension, hyporeninemia, hypoaldosteronemia, hypokalemic alkalosis, low birth weight, failure to thrive, poor growth, and in many cases nephrocalcinosis. In 1995, it was shown that mutations in the gene (HSD11B2) encoding the 11b-hydroxysteroid dehydroge- nase type 2 enzyme (11b-HSD2) cause AME. Typical patients with AME have defective 11b-HSD2 activity, as evidenced by an abnormal ratio of cortisol to cortisone metabolites and by an exceedingly diminished ability to convert [11-3H]cortisol to cortisone. Recently, we have studied an unusual patient with mild low-renin hypertension and a homozygous mutation in the HSD11B2 gene. The patient came from an inbred Mennonite family, and though the mutation identified her as a patient with AME, she did not demonstrate the typical features of AME. Biochemical analysis in this patient revealed a moderately ele- vated cortisol to cortisone metabolite ratio. The conversion of cortisol to cortisone was 58% compared with 0–6% in typical patients with AME whereas the normal conversion is 90–95%. Molecular analysis of the HSD11B2 gene of this patient showed a homozygous C3T transition in the second nucleotide of codon 227, resulting in a substitution of proline with leucine (P227L). The parents and sibs were heterozygous for this mutation. In vitro expression studies showed an increase in the Km (300 nM) over normal (54 nM). Because '40% of patients with essential hypertension demonstrate low renin, we suggest that such pa- tients should undergo genetic analysis of the HSD11B2 gene. A form of severe low-renin hypertension called apparent miner- alocorticoid excess (AME) first was described biochemically in 1977 (1). Similar patients subsequently were described, all of whom had prominent clinical signs and symptoms, including low birth weight, polyuria and polydipsia, failure to thrive, severe hypertension, hypokalemia, hypoaldosteronemia, nephrocalcino- sis, and suppressed plasma renin activity (PRA), and it has been associated with sudden fatality. The deficiency of 11b- hydroxysteroid dehydrogenase type 2 enzyme has been demon- strated in patients with AME and explains the pathogenesis of the disease, which results from excess cortisol binding to the miner- alocorticoid receptor (2). The cause of the disease was shown to be mutations in the HSD11B2 gene encoding the 11b-HSD2 enzyme (3). We report here on a form of low-renin hypertension in which a gene mutation produces a mild deficiency in the 11b-HSD2 enzyme. In contrast to previously described patients with AME, this new patient has low-renin hypertension and hypoaldosteronism but no other phenotypic features that would lead to the diagnosis of AME. Thus, the genetic mutation in the HSD11B2 gene, which results in a mild 11b-HSD2 enzyme deficiency, may be the cause of low-renin essential hypertension, the diagnostic basis of which is mostly unknown. Because 40% of patients with essential hypertension are associated with low renin, a number of these patients may have a mild form of AME. Further, as spironolactone causes ready remission, it is important to seek the diagnosis by genetic and clinical studies. Two isoforms of 11b-HSD have been identified and char- acterized, both of which originally were suspected of involve- ment in AME. The type 1 isoform (11b-HSD1) was cloned and characterized by Tannin et al. (4); it is NADP-dependent and is expressed in several human tissues (4, 5). This type was ruled out as the cause of AME (6). Later, a defect in the type 2 isoform was shown to be the cause of AME (2). The type 2 isoform of the 11b-HSD enzyme is NAD-dependent (7). It normally protects humans from cortisol intoxication by con- verting cortisol to cortisone, which does not bind to the type 1 mineralocorticoid receptor. Because aldosterone is not metabolized by the 11b-HSD enzyme, it normally has unim- peded access to the mineralocorticoid receptor, which has equal affinity to aldosterone and cortisol (8, 9). In patients with AME, however, because cortisol is secreted in milligram amounts whereas aldosterone is secreted in microgram amounts, cortisol saturates the mineralocorticoid receptor in patients with deficient 11b-HSD2 enzyme activity. The excess cortisol binding to the mineralocorticoid receptor produces a hypermineralocorticoid state, which results in hypokalemia, sodium retention, and volume expansion, thus suppressing plasma renin and aldosterone secretion. Signs of mineralocor- ticoid excess caused by cortisol binding to the mineralocorti- coid receptor in the absence of aldosterone is the hallmark of the disease, which has therefore been called ‘‘apparent min- eralocorticoid excess.’’ The cDNA for the enzyme 11b-HSD2 was cloned, and the gene (HSD11B2) encoding the enzyme was mapped to human chromosome 16q22 (10, 11). Immunohistological and activity studies localized the type 2 isoform to the distal nephron of the human kidney (12–16). Recently, researchers have identified mutations in the gene (HSD11B2) encoding 11b-hydroxysteroid dehydrogenase type 2. Thus far, 28 patients in 20 kindreds have had DNA analysis, revealing a total of 16 different mutations in the HSD11B2 gene (refs. 2, 3, 11, and 17–20; P. M. Stewart, personal communication) (Table 1). All of the patients had homozygous defects except three, who were compound heterozy- gotes (Table 1, Patients 3, 23, and 28). To date, there have been a total of '40 patients with AME and two 28-week fetuses with AME reported worldwide (1–3, 6, 11, 17, 21–36) (Table 1). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y9510200-6$2.00y0 PNAS is available online at www.pnas.org. Abbreviations: AME, apparent mineralocorticoid excess; PRA, plasma renin activity; THF, tetrahydrocortisol; THE, tetrahydrocortisone, CHOP, modified Chinese hamster ovary cell line. iTo whom reprint requests should be addressed at: Department of Pediatrics, Division of Pediatric Endocrinology, The New York Hospital–Cornell Medical Center, 525 East 68th Street, Box 103, New York, NY 10021. e-mail: minew@mail.med.cornell.edu. 10200 D o w n lo a d e d a t C a rn e g ie M e llo n U n iv e rs ity o n A p ri l 5 , 2 0 2 1 In a recent report of our extensive personal experience, geno- type, biochemical features, and phenotype of 14 patients with severe low-renin hypertension caused by AME (Patients 1–14, Table 1) were examined (2). All of the patients described had characteristic signs of a severe 11b-HSD2 defect. Birth weights were significantly lower than their unaffected sibs, and the patients were short, underweight, and hypertensive for age. Variable damage of one or more organs (kidneys, retina, heart, and central nervous system) was found in all of the patients except one. The follow-up studies of end organ damage after 2 to 13 years of treatment in six patients demonstrated significant im- provement in all patients. The urinary metabolites of cortisol demonstrated an abnormal ratio with predominance of cortisol metabolites (2); that is, Table 1. Review of AME patients worldwide: signs and biochemical features at presentation and subsequent biochemical and genetic evaluation Patient Kin- dred Ethnicity Age, years Sex Birth weight, kg Blood pressure, mmHg Blood pressure (90th centile for age) Serum K1, mmoly liter THF 1 5aTHFy THE F secretion rate, mgyd % Con- version, F 3 E HSD11B2 mutation Ref. 1* 1 American Indian 1.0 F 1.8 180y140 105y69 2.2 32.1 0.08 0 E356-1 Frameshift 3 2 2 Zoroastrian 9.0 M 2.0 250y180 110y71 3.5 9.1‡ 0.47 0 R337H, DY338 3 3 3 Italian-Moroccan 4.0 M 2.3 160y110 104y67 3.1 33.0 0.72 ND L250RyD244N 2 4 4 African American 9.3 F 2.1 130y90 109y71 2.7 8.9 0.12 2 R186C 3 5 4 African American 4.3 F 2.6 142y98 98y70 2.8 14.9 0.15 4.2 R186C 3 6 5 East Indian 0.8 M 2.0 150y100 105y67 2.4 20.1 0.05 6 R337H, DY338 3 7 6 East Indian 2.5 M 2.3 150y100 91y68 1.79 12.5 0.83 2 R337H, DY338 3 8 7 Middle Eastern 10.9 M 2.1 170y110 115y73 1.7 27.9 0.36 ND R208C 3 9 7 Middle Eastern 9.3 M 2.4 160y118 110y71 2.9 27.3 0.24 ND R208C 3 10 8 American Indian 3.3 M 2.0 205y130 99y60 0.9 26.8 0.51 1.5 L250P, L251S 3 11 9 Persian 14.0 F 2.2 220y160 116y79 2.8 8.91 ND ND R337C 17 12 9 Persian 11.6 M 2.1 170y110 110y72 2 6.85 ND ND R337C 17 13 9 Persian 4.0 F 2.4 160y100 98y70 3.1 6.7 ND ND R337C 17 14 10 Turkish 0.1 M 2.5 155y115 99y60 3.0 13.8 ND 4.5 N286-1 Frameshift 2 15 11 Mennonite 12.6 F 3.6 160y90 110y72 5.0 3.0 0.55 58.4 P227L This report 16 12 American Indian 9 M 170y100 110y71 14.4 R208C 11 17 13 CaucasianySouth American Indian 3 F 170y110 99y71 2.3 31.3 R213C 11, 23 18 13 CaucasianySouth American Indian 3.8 F 200y120 108y70 2 13.4 R213C 11, 23 19* 13 CaucasianySouth American Indian 6 M – ND 23 20 14 American Indian 1 F 142y92 105y69 – 73.8 L250P, L251S 11 21 15 European- American Indian 1.6 M 140y100 105y69 3.1 19.8 L250P, L251S 11, 35 22 16 Mexican American 26 F 180y120 123y88 – 7.9 Intron 3 (C to T) 11 23 17 Irish American 2.3 M 149y83 91y68 – 134 Codon 232, D9ntyCodon 305, D11nt 11 24 18 Asian-Pakistani 3.5 M 141y117 100y70 2.1 20 R374X 18, 31 25 18 Asian-Pakistani 1.4 M 144y91 105y69 3 50 R374X 18, 31 26*† 18 Asian-Pakistani – M – – – – – – – R374X 18, 31 27*† 18 Asian-Pakistani – M – – – – – – – R374X 18, 31 28 19 Japanese 2 M 2.5 160y90 91y68 2.7 43.7 R208Hy R337H, DY338 19 29 20 Brazilian 7 F 160y120 110y73 1.8 29.8 A328V 20, 25 30 21 German 3 F 175y110 99y71 2.8 ND 21 31 22 French 4 F 2.1 140y60 98y70 2 0.61‡ 1 ND 28 32* 23 American Indian 2.7 F 180y120 101y71 2.7 9.78 ND 35 33 24 Caucasian 2 M 110y65 91y68 2.2 15.87 ND 29 34 25 Northern European 1.6 M 2.17 150y110 105y69 2.6 45 ND 33 35* 26 Caucasian 0.4 M 2.36 200y100 105y69 1.8 68.8 ND 30 36 27 Caucasian 0.8 F 140y100 120y80 3.2 15‡ 8 R374X§ 34 37 28 Caucasian 21 M 200y145 121y81 1.7 13.6 0 ND 24 38* 18 Asian-Pakistani 3.5 M – low ND ND 18, 31 39 29 Turkish 1.3 M low 120y90 105y69 low 38.06 ND 32 40 30 French 2 M 3.1 140y80 91y68 2.5 22 ND 36 41 30 French 3.3 M 3.4 160y100 99y60 2.9 42 ND 36 42¶ 31 3.3 ND 27 43¶ 32 3.5 ND 27 44¶ 33 9 ND 27 Normal values .2.5 3.2–5.2 1.0 11.5 90 –95% *Died (Patient 19 died of stroke and may have been affected by AME. Patients 26 and 27 were still born. Patient 35 died after fever, persistently raised blood pressure, and hypokalemia. Patient 38, a twin of Patient 24, died of a diarrheal illness; AME is suspected). †Placental DNA. ‡THFyTHE. §Personal communication from P. M. Stewart. ¶Unreported potential AME Patients. ND, not done; F, cortisol; B, cortisone. Medical Sciences: Wilson et al. Proc. Natl. Acad. Sci. USA 95 (1998) 10201 D o w n lo a d e d a t C a rn e g ie M e llo n U n iv e rs ity o n A p ri l 5 , 2 0 2 1 [tetrahydrocortisol (THF) 1 5aTHF]ytetrahydrocortisone (THE) was 6.7 to 33 whereas the normal ratio is 1.0. Infusion of [11-3H]cortisol was used in patients to examine their ability to convert cortisol to cortisone by measuring the release of tritiated water. All of the previously reported patients with AME who were examined in this way exhibited little release of tritiated water compared with normal, indicating their failure to convert cortisol to cortisone. Because of the small number of patients with identical muta- tions, it was difficult to correlate genotype with phenotype. For example, Patient 1 had one of the most severe mutations, resulting in the truncation of the enzyme 11b-HSD2, and died at the age of 16 years while under treatment whereas Patient 14, with a similarly severe mutation, is thriving at age 3. Three patients (Patients 2, 6, and 7) with identical homozygous mutations from different families had varying degrees of severity in clinical and biochemical features. Herein we report studies of a patient with a mild form of low-renin hypertension. When the patient was referred with the diagnosis of AME but without hypokalemia and other classic signs and symptoms of the disease, 11b-HSD2 deficiency ap- peared unlikely. However, the diagnosis of AME proved correct on further examination. Though she demonstrated only mild hypertension and hypoaldosteronism and was otherwise asymp- tomatic, the patient clearly had AME based on biochemical evidence and later was proven to be homozygous for the P227L mutation of the HSD11B2 gene. CASE R EPORT This patient is a member of a highly inbred Mennonite population (Fig. 1). She was born by vaginal delivery with a normal birth weight (3.6 kg), similar to her unaffected her sibs (Table 1). She grew normally without typical symptoms of other patients with AME, such as polyuria and polydipsia, failure to thrive, or developmental delay. After the detection of asymptomatic mild low-renin hypertension during a routine sports physical at age 12.5, she was referred to a Kansas medical center for further evaluation. An extensive evaluation by her local Mennonite physician included normal serum electrolytes, urine analysis, urine culture, renal sonogram, renal scan, intravenous pyelogram, renal arteriogram, and urinary catecholamines. The patient was considered to be a potential AME patient by T.W., the local physician and co-author, and was referred to The New York Hospital–Cornell Medical Center Children’s Clinical Research Center for further evaluation. On examination at The New York Hospital–Cornell Medical Center at 13 years of age, her height was 146.4 cm (10th centile), her weight was 46.4 kg (50th centile), her body mass index was 21.8, and she was Tanner stage II for pubic hair and breast development. Her blood pressure was 148y92 (90th centile for age and sex, 124y78), she was normokale- mic (serum K1 concentration 4.1 mmolyliter), and did not have alkalosis (CO2 5 26 mmolyliter) (Table 2). Her endocrine evaluation revealed that her PRA was low (0.7 ngymlyhr) and that her hormone levels were unremarkable, with the exception of her serum aldosterone and urinary pH 1 aldosterone, which were both undetectable (see Tables 2, 3, and 4). MATERIALS AND METHODS This patient was studied under an institutionally approved pro- tocol on the Children’s Clinical Research Center of The New York Hospital–Cornell Medical Center. Blood pressures were measured every 2 hr with a mercury sphygmomanometer after the patient had been supine for at least 10 min throughout her hospitalization. The patient was given a diet calculated for calories, sodium, and potassium by the Children’s Clinical Re- search Center kitchen. Her 24-hr urines were collected and checked for urinary steroids, sodium, potassium, calcium, and creatinine. Blood samples for adrenal steroids, PRA, and elec- trolytes were collected daily at 8 a.m. Hormone Analysis. Hormone studies were performed after all antihypertensive medications had been discontinued for 10 days. Serum cortisol, aldosterone, deoxycortisone, corticosterone, tes- tosterone, dehydroepiandrosterone, D4-androstenedione, and es- tradiol were measured according to reported methods (37–41). PRA was measured by the method of Sealey et al. (42). Urinary steroid metabolites were measured by assays as described by Shackleton et al. (43). Corticotropin (ACTH) stimulation testing was performed by injecting 0.25 mg of Cortrosyn i.v. Serum hormones were assayed at 0 and 60 min after injection. Metabolic Studies. Cortisol secretion rate and cortisol half-life were determined as described (1, 2, 22, 44). To establish the FIG. 1. Pedigree of Mennonite family showing consanguinity (50). Table 2. Clinical and steroid hormone data Age, years Height, cm (centile) Weight, kg (centile) Blood pressure mmHg Blood pressure, mmHg (90th centile for age) K1, mmoly liter CO2, mmoly liter Serum F., mgydl Serum Aldo, ngydl Urine pH1 Aldo, mgy24 h PRA ngymlzh F secretion rate, mgym2yd Comments 12.6 147.5 (10) 45 (40) 160y90 123y79 5.0 ND ND ,1.2 ,1 UD At presentation in Kansas 13 149 (20) 47.4(50) 148y92 124y78 4.1 26 9.3 UD UD 0.7 ND Admission to New York Hospital 13.3 149 (20) 45.8 (50)* 141y81 124y78 4.5 28 12.9 14 ND 6.6 0.9 Spironolactone (25 mgyday) Chlorothiazide (125 mgyday) Normal 158 (50) 47 (50)† 110y72 3.2–5.2 22–32 5–15 4 –18 5–20 0.3–5.0 12.5 *Body mass index 5 20.8. †Body mass index 5 18.8. ND, Not done; UD, undetectable; Aldo, aldosterone; F, cortisol. 10202 Medical Sciences: Wilson et al. Proc. Natl. Acad. Sci. USA 95 (1998) D o w n lo a d e d a t C a rn e g ie M e llo n U n iv e rs ity o n A p ri l 5 , 2 0 2 1 dysfunction of the 11b-HSD2 enzyme, the metabolism of cortisol to cortisone was determined by measuring the release of tritiated water after [11-3H]cortisol infusion, as described by Hellman et al. (45). The tritiated water was recovered from the plasma by lyophilization. Molecular Analysis. In this patient, exons 2 through 5 of the HSD11B2 gene were sequenced, with the exception of the first 110 base pairs of exon 5. DNA sequencing was done after two rounds of PCR. In the first PCR, genomic DNA (100–500 ng obtained from peripheral blood leukocytes) as described in ref. 46 was denatured for 10 min at 98°C and was amplified using primers 54 GTGACTCTGGTTTTGGCAAGGA and 58 AAGTA- CAGTACATGCTTCCCTGTGG. The following reagents were added to the denatured DNA: 50 mM KCl, 10 mM TriszHCl (pH 8.3), 1.5 mM MgCl2, 0.01% gelatin, 0.75 units Taq polymerase (Life Technologies, Grand Island, NY), 200 mM dNTP, and 0.3 mM of each primer. The samples were subjected to denaturation at 94°C for 2 min. Five cycles consisting of 94°C for 1 min, 60°C for 1.5 min, and 72°C for 10 min were performed, followed by 30 cycles consisting of 94°C for 1 min, 60°C for 1.5 min, and 72°C for 4 min. A final cycle consisted of 94°C for 1 min, 60°C for 1.5 min, and 72°C for 10 min. The second PCR was performed in a 50-ml volume containing 2 ml from the first PCR with 15 pmols of forward primer and 5 pmols of reverse biotin primer, as described in ref. 2. The remaining reagents were identical to that of the first PCR. The samples were denatured for 1 min at 95°C and then for 4 cycles of 1 min at 95°C, 30 sec at 58°C, 10 min at 72°C, 30 cycles of 30 sec at 95°C, 30 sec at 58°C, 1 min at 72°C, 1 cycle of 1 min at 95°C, 1.5 min at 58°C, and 10 min at 72°C. The 59 end of exon 2 was PCR-amplified directly without the first round of PCR by using 100 –500 ng of genomic DNA, following the identical conditions of the second PCR by using primers F1 and B1 (2). DNA Sequencing. Sequencing of the HSD11B2 gene was performed using solid phase single-strand sequencing with the Sequenase Dye Primer Kit (Applied Biosystems) containing M13 primers. Single-stranded DNA from the PCR fragments was purified with streptavidin-bound magnetic beads (Dynal, Oslo), following the procedure in bulletin 21 from Applied Biosystems. After denaturation to remove the nonbiotinylated DNA strand, the bound DNA strand then was sequenced. Sequencing was done following the procedure described in the sequencing manual supplied with the sequencing kit with the following modifications: For the A and C reactions, 2 ml instead of 1 ml of the respective dye primers were used; and, instead 1 ml of the Sequenase enzyme, 2 ml were used. For the G and T reactions, 4 ml instead of 2 ml of the respective dye primers were used; and, instead of 2 ml of the Sequenase enzyme, 4 ml were used. The reactions were incubated at 37°C for 15 min, and then the four reactions were stopped by pooling them into 200 ml of TT buffer (10 mM TriszHCl, pH 8.0y0.1% Tween 20). The beads were concentrated with a magnet and were washed with an additional 200 ml of TT buffer. The sequencing products were electrophoresed and ana- lyzed with an Applied Biosystems Model 373A sequencer. In Vitro Expression Studies. Expression studies were per- formed as described (47, 48). The pALTER vector containing the insert from pHSD2 (10) was mutagenized with the following oligonucleotide: GACATGCCATATCTGTGCTTGGGGGCC. The mutated insert was subcloned in the mammalian expression vector pcDNAI (Invitrogen) to give the mutant plasmid pP227L. Chinese hamster ovary modified cell line (CHOP) cells were transfected, and homogenates were prepared and analyzed as described (10), with the exception of the presence or absence of 20% glycerol. The addition of glycerol had little or no effect on the percent conversion of cortisol to cortisone. The Km was determined in homogenates over the range of 25 nM to 400 nM cortisol. The Km in whole cells was determined over a range of 25nM to 800 nM cortisol. For Western blot analysis, 50 mg of transfected CHOP-cell homogenate protein was loaded per lane. Antibody HUH21 (12) was used at 1 mgyml. RESULTS Metabolic Studies. To study the patient’s cortisol secretion rate, she was infused with [1,2-3H]cortisol. Her secretion rate was 0.55 mgyday, which is extremely low (see Table 1). To study the patient’s ability to convert cortisol to cortisone, the patient was infused with [11-3H]cortisol. The release of tritiated water was used to measure the conversion of cortisol to cortisone. The patient converted 58.4% of [11-3H]cortisol to cortisone. Normal conversion is 90% to 95% whereas previously studied patients with AME converted only 0% to 6% (2, 3, 22, 26, 49). The patient’s 24-hr urinary cortisol metabolites are shown in Table 3. Her (THF 1 5aTHF)yTHE ratio was 3.0, whereas patients with FIG. 2. Mutations in the gene for 11b-hydroxysteroid dehydroge- nase type 2 in patients with AME. These patients were investigated by our group, and the phenotypes are well described. The HSD11B2 gene has five exons, is 6.2 kb long, and has been mapped to chromosome 16q22. The mutation in Patient 15 is reported here (identified by an arrow). All mutations found in affected patients are homozygous except for Patient 3, who is a compound heterozygote. Table 3. Urinary metabolites on admission to The New York Hospital–Cornell Medical Center Urinary metabolites Patient with mild AME Typical AME Normal controls THF (mgyd) 340 0.1– 479.5 1469 6 585 THE (mgyd) 320 UD–502.3 2589 6 1292 5aTHF (mgyd) 624 UD– 647.1 1373 6 701 THFyTHE 1.1 2.1–2.2 ,1 THF 1 5a THFyTHE 3.0 6.7–33 1 UD, undetectable. Table 4. ACTH stimulation test at admission to The New York Hospital–Cornell Medical Center Time 17-OHP, ngydl D5, ngydl Aldo, ngydl DOC, ngydl B, mgydl F, mgydl D4, ngydl T, ngydl DS, ngydl DHEA, ngydl E2, ngydl DHT, ngydl 0 19 40 B1 19 0.51 18.6 80 22 65 135 4.9 7 60 55 221 B1 53 2.05 35.5 91 26 86 240 3.7 7 17-OHP, 17-hydroxyprogesterone; D5, 17-hydroxypregnenolone; Aldo, aldosterone; DOC, deoxycorticosterone; B, corticosterone; F, cortisol; D4, D4-androstenedione; T, testosterone; DS, dehydroepiandrosterone sulfate; DHEA, dehydroepiandrosterone; Es, estradiol; DHT, dihydrotestos- terone. Medical Sciences: Wilson et al. Proc. Natl. Acad. Sci. USA 95 (1998) 10203 D o w n lo a d e d a t C a rn e g ie M e llo n U n iv e rs ity o n A p ri l 5 , 2 0 2 1 AME previously studied by us had (THF 1 5aTHF)yTHE ratios of 6.7 to 33 (2), and it has been reported to be as high as 134 (7). Adrenal Hormone Studies. The patient’s baseline and 60-min ACTH-stimulated adrenal hormones were all within the normal limits or close to normal, with the exception of aldosterone (see Table 4). As with most patients with AME, after ACTH stimu- lation, her aldosterone level remained undetectable. Gene Mutational Analysis. DNA analysis of the patient’s HSD11B2 gene revealed a homozygous C to T transition in the second nucleotide of codon 227 (CCG to CTG), resulting in a substitution of a proline for a leucine (P227L) (Fig. 2). The patient’s parents and her two siblings are heterozygous for this mutation. In Vitro Expression Studies. In vitro expression studies were used to determine the kinetics and activity of the 11b-HSD2 enzyme with a P227L mutation. Wild-type pHSD2 or mutant pP227L plasmids were transfected into CHOP cells. Kinetic analyses using cell homogenates revealed a Km of 54 nM (data not shown) from cells transfected with pHSD2 and a Km of 350 nM from cells transfected with pP227L (Fig. 3). Kinetic analyses also were done in whole cells and revealed similar Km [62 nM for cells transfected with the wild-type pHSD2 plasmid and 300 nM for cells transfected with the pP227L plasmid (data not shown)]. Whole cell expression studies resulted in similar conversion activities. The conversion of cortisol to cortisone using the wild-type pHSD2 plasmid was 15%, and the mutant pP227L plasmid resulted in 16% conversion (data not shown). However, expression studies using cell homogenates resulted in only 2% conversion of cortisol to cortisone with pP227L whereas pHSD2 resulted in 90% conversion (data not shown). The presence or absence of 20% glycerol made no difference in the percent conversion. Western analysis showed that comparable amounts of mutant and wild-type 11b-HSD2 were expressed (Fig. 4). DISCUSSION Essential hypertension has been estimated to occur in 15 million residents in the United States, and '40% are associated with low renin. Because mild low-renin hypertension can affect a young patient, it is imperative that it is detected as early as possible for prophylactic therapy and careful monitoring of the patient. Apparent mineralocorticoid excess is a severe form of low- renin hypertension that begins in childhood. Thus far, it has been rarely reported, with '40 patients identified worldwide. DNA analysis has now been performed in 29 patients with AME (Table 1; Fig. 2). We have examined the phenotype of 15 of these patients at The New York Hospital–Cornell Medical Center, including the mild case (Table 1, Patients 1–15). Before this report, all of the patients have displayed symptoms typical of AME, such as low birth weight, failure to thrive, poor growth, severe hypertension, hypokalemia, hyporeninemia, and hypoaldosteronemia. The pa- tient reported in this article did not report symptoms, though mild hypertension relative to her age and weight, low PRA, and low aldosterone were found on examination. She also had moderately abnormal findings on her electroencephalogram: Rare left front- to-central sharp waves occurred spontaneously accompanied by no observed behavioral change, which is suggestive of a seizure disorder of left front-to-central origin but is not diagnostic. From the data presented in Table 1, it is evident that the genetic defect in HSD11B2 is mild in this patient. She lacks (i) low birth weight, (ii) severe hypertension, (iii) failure to thrive, (iv) polyuria and polydipsia, (v) hypokalemia, and (vi) nephrocalcinosis and other significant end organ damage. Indeed, her major manifes- tations of AME are hyporeninemia, hypoaldosteronemia, and mild hypertension. In addition, the studies of conversion of cortisol to cortisone indicate that this patient has 11b-HSD2 activity intermediate between severely affected patients with AME and normals. The P227L construct gave a Km for cortisol of 350 nM as compared with a Km of 62 nM for the wild-type construct in whole cells. This is in contrast to the Km in an expression study of a severely affected patient with AME, which was totally devoid of activity. The Km of severely affected patients can be as high as 1,010 nM, as compared with the normal control of 110 nM (47). These studies indicate that the mutation P227L is less severe than the mutations reported in other patients with AME. Most reported patients with AME have homozygous muta- tions. Homozygosity for a rare mutation classically is explained by consanguinity, endogamy, or a founder effect. Historical evi- dence among reported patients with AME suggests that many are members of consanguineous populations (2, 3). The patient described here is from a consanguineous Mennonite family (of the Alexanderwohl Church) (see Pedigree, Fig. 1). It is possible that the mild form of AME seen in this patient may be prevalent in this inbred Mennonite population. A study is in place in which the Mennonite population will be analyzed to determine whether FIG. 3. Determination of Km value (Michaelis–Menten constant) for the metabolism of cortisol by 11b-HSD2 enzyme in CHOP cell homogenates transfected with pP227L in the presence of 20% glycerol (transfection with pHSD2 resulted in a Km of 54 nM). FIG. 4. Western blot analysis of homogenates from CHOP cells transfected with pP227L, wild type (pHSD2) or pcDNA1 vector. FIG. 5. Calculation of the frequency of a mild form of AME among an inbred Mennonite population of 2,000 members. This calculation would be a minimum frequency if no additional patients with AME are found. 10204 Medical Sciences: Wilson et al. Proc. Natl. Acad. Sci. USA 95 (1998) D o w n lo a d e d a t C a rn e g ie M e llo n U n iv e rs ity o n A p ri l 5 , 2 0 2 1 there are other cases of this mild form of AME. The gene frequency, heterozygote frequency, and disease frequency for AME in this population of 2,000 inbred Mennonites will be calculated according to the Hardy–Weinberg Equation (Fig. 5). 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