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Division of Pediatric Endocrinology (K.M.K.-W., G.M.T., P.C.W.), University of Texas Southwestern Medical Center, Dallas, Texas 75390-9063; Section of Pediatric Endocrinology (D.S.), All Children’s Hospital, St. Petersburg, Florida 33701; (D.P.) Clearwater, Florida 33761; Department of Pediatrics (K.L.J.), University of California at San Diego, La Jolla, California 92093-6933; and Division of Endocrinology (L.K.), Texas Children’s Hospital, Houston, Texas 77030
Address all correspondence and requests for reprints to: Perrin C. White, M.D., Department of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9063. E-mail: perrin.white{at}utsouthwestern.edu
Abstract
Isolated hyperreninemic hypoaldosteronism presenting in infancy is usually caused by mutations in the CYP11B2 gene encoding aldosterone synthase. We studied five patients in four unrelated kindreds with hyperreninemic hypoaldosteronism, in whom we were unable to find such mutations. All presented in infancy with failure to thrive, hyponatremia, hyperkalemia, markedly elevated plasma renin activity, and low or inappropriately normal aldosterone levels. All had normal cortisol levels and no signs or symptoms of congenital adrenal hyperplasia. All responded to fludrocortisone treatment. There were no mutations detected in exons or splice junctions of CYP11B2. Linkage of the disorder to CYP11B2 was studied in two unrelated consanguineous patients and in an affected sib pair. The consanguineous patients were each heterozygous for at least one of three polymorphic microsatellite markers near CYP11B2, excluding linkage to CYP11B2. However, linkage of the disease to CYP11B2 could not be excluded in the affected sib pair. Genes involved in the regulation of aldosterone biosynthesis, including those encoding angiotensinogen, angiotensin-converting enzyme, and the AT1 angiotensin II receptor were similarly excluded from linkage. These results demonstrate the existence of an inherited form of hyperreninemic hypoaldosteronism distinct from aldosterone synthase deficiency. The affected gene(s) remain to be determined.
ALDOSTERONE REGULATES ELECTROLYTE excretion and intravascular volume mainly through effects on the distal nephron, where it increases sodium resorption from, and potassium excretion into, the urine by indirectly increasing activity of the epithelial sodium channel and the sodium-potassium ATPase. Aldosterone deficiency interferes with these processes and thus results in hyponatremia, hypovolemia, and hyperkalemia (1).
In primary aldosterone deficiency (i.e. inability of the adrenal gland to synthesize the hormone), plasma renin activity (PRA) is elevated, and so this condition is also referred to as hyperreninemic hypoaldosteronism. Renin is a proteolytic enzyme secreted by the nephron’s juxtaglomerular apparatus in response to low intravascular volume. It cleaves angiotensinogen to angiotensin I, which is converted to angiotensin II by angiotensin-converting enzyme (ACE). Angiotensin II binds to specific receptors on cells in the adrenal zona glomerulosa (the site of aldosterone synthesis), thus increasing aldosterone biosynthesis and secretion under normal circumstances (2).
Primary aldosterone deficiency can result from destruction of the entire adrenal cortex (Addison’s disease) by infection, injury, or autoimmune processes; from genetic disorders affecting the entire gland (such as lipoid hyperplasia, adrenoleukodystrophy, or adrenal hypoplasia congenita); or from genetic disorders affecting specific enzymatic conversions required for aldosterone biosynthesis. Two of these, the salt-wasting forms of 21-hydroxylase and 3ß-hydroxysteroid dehydrogenase deficiencies, also affect cortisol biosynthesis.
Rare patients have aldosterone deficiency with entirely normal cortisol and sex steroid synthesis. This is usually attributable to an inability to convert deoxycorticosterone to aldosterone, a three-step reaction normally mediated by the enzyme aldosterone synthase (3, 4). Two forms of aldosterone synthase (also termed: corticosterone methyloxidase) deficiency are recognized. These syndromes have identical clinical features but differ in profiles of secreted steroids. In particular, whereas excretion of 18-hydroxycorticosterone is mildly decreased in type I deficiency, urinary and serum levels of this steroid are markedly increased in patients with type II deficiency (5).
Aldosterone synthase is a mitochondrial cytochrome P450 enzyme, CYP11B2. It is encoded by the CYP11B2 gene located on chromosome 8, band q24.3, approximately 40 kb away from the 93%-identical CYP11B1 gene encoding the steroid 11ß-hydroxylase enzyme required for cortisol biosynthesis (6, 7). Most reported patients with presumed aldosterone synthase deficiency carry mutations in CYP11B2 (8, 9, 10, 11, 12, 13, 14, 15).
Here, we report five patients in four unrelated kindreds with hyperreninemic hypoaldosteronism presenting in early infancy in whom we were unable to detect mutations in CYP11B2. Two of these patients were of consanguineous parentage, strongly suggesting that the disease was inherited in an autosomal recessive manner. In these kindreds, the presence of consanguinity permitted the use of homozygosity mapping to exclude linkage of the disorder to CYP11B2, demonstrating the existence of an inherited form of hyperreninemic hypoaldosteronism distinct from aldosterone synthase deficiency.
Materials and Methods
Samples for DNA extraction were obtained from patients with
familial hyperreninemic hypoaldosteronism (Table 1
) after
obtaining appropriate informed
consent.
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.
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P]deoxy-ATP (>2500
Ci/mmol), and usually 1 µM of each primer, 125
µM deoxy-GTP, thymidine 5'-triphosphate, and deoxy-CTP, 1.25
µM unlabeled deoxy-ATP, 16 ng genomic DNA, and 0.2 U Taq
polymerase. Cycling conditions consisted of a 30-sec denaturation at 96
C, 30 sec annealing at 55 C, and 1 min extension at 72 C for 30 cycles.
A 5-min denaturation step (96 C) preceded cycling, and a 10-min primer
extension step and 4-C soak followed. A modified hot start was used as
previously described (16). Samples were subjected to electrophoresis in 6% denaturing polyacrylamide gels, fixed, dried, and autoradiographed for 1–18 h.
Results and Discussion
The clinical and biochemical characteristics of five patients in
four kindreds are summarized in Table 1
. All developed signs of
hypoaldosteronism within the first few weeks of life, including failure
to thrive, hyponatremia, and hyperkalemia. All had markedly elevated
PRA and low or inappropriately normal aldosterone levels. All responded
to treatment with fludrocortisone by normalizing serum electrolytes and
PRA.
Thus, these patients clearly had primary hypoaldosteronism with onset in early infancy. Two were of known consanguineous parentage consistent with an autosomal recessive disorder such as aldosterone synthase deficiency. All had normal 18-hydroxycorticosterone levels, suggesting that the patients did not have type II aldosterone synthase deficiency, but these results were entirely consistent with type I aldosterone synthase deficiency.
However, complete sequencing of all exons and introns 1, 4, 6, and 7,
plus partial sequencing of the remaining introns (including all splice
junctions) and 50 bp of the 5' flanking region failed to reveal
mutations in any of these patients. Patient 1 was heterozygous for a
V386A polymorphism; but this polymorphism, by itself, has a minimal
effect on enzymatic activity (8). Patient 2 was
heterozygous for the K173R polymorphism, which also has no effect on
enzyme activity (17). In any consanguineous kindred
carrying an autosomal recessive disease, the two mutant alleles in each
affected patient should be identical, and all polymorphic markers near
the affected gene should be homozygous. Thus, the fact that each of
these patients was heterozygous for a polymorphism within
CYP11B2 argues strongly against linkage of the disease to
this gene. However, both of these polymorphisms are normally present in
the closely linked CYP11B1 gene (6) and could
potentially represent recent gene conversions in these patients. To
definitively rule out linkage to CYP11B2, we examined three
highly polymorphic microsatellite markers near the gene in the two
consanguineous kindreds (Table 1, Fig. 1). In addition, these
microsatellite markers were examined in the two patients who were
siblings to see if they shared both haplotypes at this locus.
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We considered whether these patients might have had a known disorder other than aldosterone synthase deficiency. Although salt-wasting forms of congenital adrenal hyperplasia are relatively common causes of hyperreninemic hypoaldosteronism, all of our patients had normal cortisol levels, rendering this diagnosis unlikely. In particular, the normal 17-hydroxyprogesterone levels obtained in four patients rule out 21-hydroxylase deficiency, by far the most common salt-wasting form of congenital adrenal hyperplasia (18).
Normal cortisol biosynthesis was further demonstrated by cosyntropin stimulation testing and/or normal ACTH levels documented at 9 months of age or later (not shown). This suggests that none of these patients had an autosomal recessive form of adrenal hypoplasia congenita. This was important to rule out because aldosterone deficiency often presents before cortisol deficiency in this disorder (19).
Thus, it seems likely that these patients have a distinct autosomal recessive disorder that we term familial hyperreninemic hypoaldosteronism type 2, or FHHA2, to distinguish it from aldosterone synthase deficiency (FHHA1). This probably results from one or more defects in regulation of aldosterone biosynthesis. In principle, defects in genes encoding components of the renin-angiotensin system might be expected to interfere with aldosterone biosynthesis; such genes include those encoding angiotensinogen (AGT), angiotensin converting-enzyme (ACE, DCP1) and the AT1 type angiotensin II receptor. To evaluate this possibility, we typed the two consanguineous patients for two microsatellite polymorphisms near each of these genes. All patients were heterozygous for at least one marker at each locus, excluding these genes from linkage to the disease (not shown). In addition, we were able to exclude these genes from linkage to the disease in the affected sib pair, because the sibs carried different alleles at each of these loci.
Moreover, each of the corresponding genes has been knocked out in mice, and none of the knockouts have a phenotype of electrolyte abnormalities or hypoaldosteronism; instead, all are hypotensive (20, 21, 22). The lack of electrolyte abnormalities is consistent with the fact that aldosterone secretion is potently stimulated by hyperkalemia, which should compensate for the lack of stimulation by angiotensin II. As regards signaling steps downstream from the AT1 receptor, this receptor signals through G protein-coupled mechanisms that raise intracellular calcium levels (23, 24). These mechanisms are common to many cell types, and mutations affecting them would be expected to have much broader phenotypes than isolated aldosterone deficiency.
Possibly FHHA2 involves a trans acting factor affecting CYP11B2 expression and/or development of the adrenal zona glomerulosa. At present, these factors are still being defined, and none have been identified that are uniquely expressed in the zona glomerulosa.
FHHA2 may be more common than aldosterone synthase deficiency (FHHA1). Other than in Iranian Jewish kindreds, who all carry the same mutations (8), we failed to detect mutations in 4 of the 7 kindreds with hyperreninemic hypoaldosteronism we examined (unpublished observations). Similarly, no mutations affecting enzymatic activity could be detected in CYP11B2 in 4 of 11 published kindreds with apparent aldosterone synthase deficiency (9, 10, 11, 12, 13, 14, 15, 25, 26, 27). Considering the likelihood of bias against publishing negative results, this proportion of kindreds without CYP11B2 mutations is probably an underestimate. However, none of the published kindreds without detectable CYP11B2 mutations were consanguineous, and so linkage to CYP11B2 could not be formally excluded by the method used in the present study.
Elucidation of the cause(s) of FHHA2 awaits ascertainment of one (or more) consanguineous kindred(s) with enough affected individuals that homozygosity mapping can be used to determine the chromosomal location(s) of the affected gene(s).
Footnotes
This work was supported by Grant NIH R37-DK-37867 (to P.C.W.).
Abbreviations: ACE, Angiotensin-converting enzyme; AGT, angiotensinogen; PRA, plasma renin activity.
Received May 31, 2001.
Accepted July 26, 2001.
References
This article has been cited by other articles:
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  T. A. Williams, P. Mulatero, M. Bosio, S. Lewicka, M. Palermo, F. Veglio, and D. Armanini A Particular Phenotype in a Girl with Aldosterone Synthase Deficiency J. Clin. Endocrinol. Metab., July 1, 2004; 89(7): 3168 - 3172. [Abstract] [Full Text] [PDF]
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