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Clinical Studies |
Endocrine Research Laboratory, Department of Medicine, St. Luke’s Medical Center, Medical College of Wisconsin (J.W.F., H.R.), Milwaukee, Wisconsin 53215; Howard Hughes Medical Institute, Departments of Medicine and Genetics, Boyer Center for Molecular Medicine, Yale University School of Medicine (J.H.H., R.P.L.), New Haven, Connecticut 06510
Address all correspondence and requests for reprints to: James W. Findling, M.D., Department of Medicine, St. Luke’s Health Science Office Building, 2901 West KK River Parkway, Suite 503, Milwaukee, Wisconsin 53215.
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Abstract |
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-subunits of the renal epithelial sodium channel. We
describe a kindred (K176) whose distinguishing clinical features were
mild hypertension and decreased aldosterone secretion. The index case
was a 16-yr-old girl with intermittent mild hypertension and
hypokalemia and subnormal PRA, aldosterone, 18-hydroxycorticosterone,
and deoxycortisol levels, but normal cortisol/cortisone metabolite
ratio and cortisol half-life. A frameshift mutation in the
carboxyl-terminus of the ß-subunit of the epithelial sodium channel
was identified in the index case, establishing the diagnosis of
Liddle’s syndrome. Sixteen at-risk relatives of the index case were
tested. Seven new subjects were heterozygous for the mutation found in
the index case, and two deceased obligate carriers were identified. All
genetically affected adult subjects had a history of mild hypertension,
and four had a history of hypokalemia. Basal and postcosyntropin plasma
aldosterone and urinary aldosterone levels were significantly
suppressed in those positive for the mutation. The family demonstrates
variability in the severity of hypertension and hypokalemia in this
disease, raising the possibility that this disease may be
underdiagnosed among patients with essential hypertension. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Recently, mutations in the carboxyl-terminus of the ß- or
-subunits of the renal epithelial sodium channel (ENaC) have been
shown to cause Liddle’s syndrome (3, 4, 5, 6). Expression of ENaC-containing
mutated subunits in Xenopus oocytes demonstrates a marked
increase in amiloride-sensitive sodium transport (7, 8, 9). These findings
demonstrate that Liddle’s syndrome arises from increased sodium
reabsorption in the distal nephron, leading to expanded plasma volume
and elevated blood pressure.
Only one large extended Liddle’s syndrome kindred has been reported to date, in which frank hypertension before the age of 20 yr segregated with the mutated gene as an autosomal dominant trait (3). It was consequently of interest to determine variability in the phenotype of different kindreds. We report the prospective diagnosis and clinical features of members of an extended kindred ascertained through an index case with Liddle’s syndrome.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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This was an asymptomatic 16-yr-old female in which hypertension (blood pressure, 136/114 to 142/100 mm Hg) was identified during a preschool examination. There was a family history of early onset of hypertension in her mother and two maternal aunts. She had menarche at age 12 yr and had normal menstrual cycles. Upon examination by us, she was a healthy-appearing pubertal female whose peak blood pressure was not elevated (118/70 mm Hg), pulse rate was 72 beats/min, height was 161 cm, and weight was 50 kg. Her physical examination was within normal limits.
Initial laboratory studies showed a serum sodium of 145 mmol/L,
potassium of 3.1 mmol/L, chloride of 102 mmol/L, CO2 of 32
mmol/L, urea nitrogen of 3.9 mmol/L (11 mg/dL), and creatinine of 71
µmol/L (0.8 mg/dL). PRA was less than 0.056 ng/L·s (<0.2
ng/mL·h). Upright plasma aldosterone was less than 28 pmol/L (<1
ng/dL), 18-hydroxycorticosterone was less than 138 pmol/L (<5 ng/dL),
and deoxycorticosterone was less than 30 pmol/L (<1 ng/dL). Urinary
tetrahydrocortisol, 5
-tetrahydrocortisol, and tetrahydrocortisone
were 3002, 3942, and 5331 nmol/day, respectively. The
cortisol/cortisone metabolite ratio (1.3), cortisol turnover (398), and
ring A reduction constant were within normal limits, excluding a
diagnosis of apparent mineralocorticoid excess syndrome.
The serum potassium concentration was measured again, and it ranged between 3.0–3.2 mmol/L. A 3-week trial of dexamethasone (0.5 mg/day) or spironolactone (100 mg/day) did not result in an increase in plasma potassium. In contrast, a 3-week trial of triamterene (100 mg/day) resulted in an increase in plasma potassium to 3.7 mmol/L. Subsequently, the patient was treated with trimethoprim-sulfamethoxazole for an upper respiratory infection, and her plasma potassium concentration increased further to 4.9 mmol/L. The finding of suppressed PRA, suppressed aldosterone levels, and a history of elevated blood pressure with hypokalemic alkalosis suggested a diagnosis of Liddle’s syndrome.
Subunits of the renal amiloride-sensitive epithelial sodium channel were screened for mutations, ultimately identifying a frameshift mutation in the cytoplasmic carboxyl-terminus of ßENaC in this patient (3). This mutation inserts an additional cytosine residue at codon 592, changing the encoded protein from amino acid 593 onward to a new termination at codon 605. This mutation thus removes the last 45 amino acids of the normal protein, including the proline-rich target. This mutation has not been found on over 1500 chromosomes from unrelated subjects, indicating that it is rare in the population.
Study of the family
Confidentiality of patient information was maintained in accordance with the Helsinki Declaration of 1975, as revised in 1983. All procedures were part of clinically indicated diagnostic testing requested by the families’ physicians.
Because of a strong family history, the mother (IV-2) of the
index case (V-2) was tested and found to carry the same frameshift
mutation, establishing that the mutation was transmitted from the
maternal lineage (Fig. 1
). A total of 22 available
at-risk subjects (excluding the index case) had genetic testing, and 16
of these subjects also had endocrine testing. Neither sodium nor
potassium intake was controlled during the studies. Medications were
discontinued for at least 24 h before endocrine testing (performed
between 0800–1000 h). Two subjects (IV-2 and III-3) were taking
captopril and hydrochlorothiazide, and one was taking nifedipine and
extended release potassium chloride (IV-10). No subject was taking
spironolactone or triamterine. For adult subjects, 24-h urine
collections were analyzed for creatinine, potassium, and aldosterone.
Basal venous blood samples were drawn between 0800–1000 h for DNA
analysis and determinations of PRA, plasma active renin, plasma
aldosterone, and plasma electrolytes. Then, cosyntropin [ACTH-(1–24);
250 µg Cortrosyn, Organon, Westbury, NY] was injected, and blood
samples for plasma aldosterone were drawn 30 min later. For children, a
single blood sample was drawn for genetic testing and determinations of
plasma potassium, PRA, active renin, and aldosterone. No children were
taking medication.
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DNA was extracted from whole blood leukocytes and was analyzed for the mutation identified in the index case of this kindred (K176) by single strand conformational polymorphism (SSCP) as described previously (3). DNA sequence analysis of samples from several patients confirmed that the SSCP variant encoded the frameshift mutation identified in the index case. Genetic diagnosis was performed by observers blinded to the clinical data.
Urinary potassium was measured by ion-specific electrodes (Nova 13, Waltham, MA). Urinary creatinine and plasma potassium were analyzed spectrophotometrically (Ectachem 700, Rochester, NY). PRA was measured by the RIA of angiotensin I generated in vitro (Incstar, Stillwater, MN). Plasma active renin was measured by direct two-site immunoradiometric assay (Nichols Institute, San Juan Capistrano, CA). Plasma aldosterone was measured directly by solid phase RIA (Diagnostic Product Corp., Los Angeles, CA). The same assay was used for urinary aldosterone after acidification with 3.2 N HCl and extraction with ethyl acetate.
Data analysis
Data were analyzed by unpaired t test or two-way ANOVA repeated on one dimension, followed by Duncan’s multiple range test. P < 0.05 was considered statistically significant. Data are presented as the mean ± SE.
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Results |
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shows the extended kindred (K176) of the index case
and the results of genetic screening for the Liddle’s frameshift
mutation identified in the index case (3). The mother (IV-2) of the
index case and two maternal aunts (IV-8 and IV-10) had a history of
hypertension and were positive for the mutation. One of these aunts
(IV-10) suffered a myocardial infarction in 1992 (2 yr before testing)
due to coronary spasm. Two maternal aunts (IV-5 and IV-12) had a
history of hypertension during pregnancy and for 1 yr postpartum and
were positive for the mutation. Two maternal aunts (IV-3 and IV-7) did
not have a history of hypertension and were negative for the mutation.
As the maternal grandmother (III-1) had no history of hypertension and
was negative for the mutation, we deduced that the maternal grandfather
(III-2) was the source of the mutation. III-2 died of complications of
hypertensive cardiovascular disease in the eighth decade of life. The
half-brother (III-3) of III-2 had a history of hypertension and was
positive for the mutation, supporting the inference that III-2 carried
the mutation. These half-siblings only shared genes by inheritance from
their mother (II-2), who, therefore, is also an obligate gene carrier.
II-2 had a long history of hypertension and died of a stroke at age 90
yr. The remaining mutation carrier (V-8) was 2 yr old and had a blood
pressure above the 90th percentile for age and gender (10) (see Table 2). DNA sequence analysis of samples from subjects III-3 and V-8
confirmed that the SSCP variant encoded the identical frameshift
mutation identified in the index case.
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). Four of the affected
subjects had previously documented findings of spontaneous hypokalemia
(Table 1).
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and 2).
There were no statistically significant differences in blood pressure
(Tables 1 and 2). However, systolic blood pressures of adult affected
subjects were, on the average, 20 mm Hg higher than those of unaffected
subjects, and diastolic blood pressure averaged 16 mm Hg higher,
despite the fact that three mutation carriers were taking
antihypertensive medication that was withdrawn only briefly before
testing. Similarly, there was a small but nonsignificant difference in
plasma potassium between these two groups. Interestingly, some affected
subjects, including one with normal blood pressure who was not
receiving antihypertensive therapy (IV-12), had normal renin levels,
and the mean PRA and PAR in affected and unaffected subjects were
similar.
There were dramatic differences in basal plasma aldosterone, plasma
aldosterone after cosyntropin administration, and 24-h urinary
aldosterone between these two groups, with all subjects carrying
mutations having lower aldosterone levels (Tables 1 and 2). This
difference persisted when urinary aldosterone was normalized to urinary
creatinine excretion in affected (0.21 ± 0.03 nmol/mmol) and
unaffected (2.60 ± 0.06 nmol/mmol) subjects (P <
0.001). Similar results were obtained in the pediatric subjects (Table 2), with the affected 2-yr-old child showing a PRA and aldosterone
concentration below the limits of detection.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunits
of ENaC. All disease mutations either remove or alter amino acids of
the target sequence TPPPXY and result in increased channel activity
(3, 4, 5, 6, 7, 8, 9). Recently, a protein that normally binds to this target sequence
has been identified and proposed as the negative regulator (12). This report describes the first kindred in which such prospective genetic diagnosis of Liddle’s syndrome has been performed, permitting unbiased assessment of the clinical manifestations resulting from the disease mutation. The kindred was of particular interest because, in contrast to most others, the hypertension in the index case was variable and mild. Genetic screening identified a total of 10 mutant gene and obligate carriers in this kindred. It is noteworthy that 2 of these subjects had hypertension only during and after pregnancy and have subsequently remained normotensive. Similarly, other affected subjects in this kindred have had either mild hypertension or hypertension that has been easily treated. Nonetheless, a history of hypertension was absent among family members who did not inherit the gene. The failure of the index case to demonstrate hypertension after initial evaluation seemed enigmatic. Recently, however, she has been diagnosed as having anorexia nervosa, raising the possibility that a reduction in sodium intake had reduced blood pressure from initial values. Hypokalemia has also been variable in affected subjects of this kindred, as has been reported in other kindreds (1, 2, 11). These findings demonstrate that sustained hypertension and hypokalemia are not obligatory among patients carrying mutations causing Liddle’s syndrome.
The typically mild hypertension seen in affected members of this kindred stands in contrast to the moderate to severe hypertension often encountered in other reported cases (1, 2, 3, 4, 11). There are several potential explanations for this apparently attenuated phenotype. One is an allele-specific effect, i.e. that this specific Liddle’s mutation causes less severe hypertension than other Liddle’s mutations. The demonstration that the major, if not sole, target of Liddle’s mutations is a proline-rich domain that is completely removed by the ßENaC mutation in the present kindred would make this explanation unlikely (3). The inheritance of modifying (i.e. blood pressure lowering) alleles and shared environmental factors may also account for the attenuated phenotype.
The features that completely distinguished gene carriers from noncarriers in this family were the aldosterone response to ACTH and the 24-h urinary aldosterone levels. Although this presumably reflects increased ENaC activity, leading to reduced renin secretion, PRA was highly variable among affected subjects and showed marked overlap with that observed in unaffected kindred members. Direct measurement of active renin, which obviates the interference of altered angiotensinogen (13), also did not distinguish between affected and unaffected subjects. The lack of a difference in renin between groups despite suppressed aldosterone may reflect the inability of a single basal PRA or PAR measurement to demonstrate a decrease in integrated angiotensin II levels during the entire day. This is analogous to ACTH or TSH concentrations being in the normal range in patients with hypoadrenalism or hypothyroidism secondary to hypopituitarism. This may be particularly true because aldosterone does not directly feedback on renin release, but suppresses it indirectly via volume expansion and changes in electrolyte balance.
The present studies demonstrated that low 24-h urinary aldosterone and/or a blunted response of plasma aldosterone to cosyntropin allow complete and impressive separation of affected and unaffected family members and consequently appear to be useful tests for excluding the diagnosis. The molecular data provide the opportunity for a rapid, sensitive, and specific genetic screening test for this disease.
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Acknowledgments |
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Footnotes |
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2 Investigator with the Howard Hughes Medical Institute.
Received September 26, 1996.
Revised November 18, 1996.
Accepted December 5, 1996.
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References |
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