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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 6 2518-2526
Copyright © 2003 by The Endocrine Society

A Compound Heterozygote Case of Type II Aldosterone Synthase Deficiency

Felicity M. Dunlop, Patricia A. Crock, Joseph Montalto, John W. Funder and Kathleen M. Curnow

Baker Medical Research Institute (F.M.D., J.W.F., K.M.C.), Melbourne 8008, Victoria, Australia; John Hunter Children’s Hospital, University of Newcastle (P.A.C.), Newcastle 2308, New South Wales, Australia; and Dorevitch Pathology (J.M.), Heidelberg 3084, Victoria, Australia

Address all correspondence and requests for reprints to: Prof. John W. Funder, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: john.funder{at}med.monash edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
An infant with failure to thrive, persistent hyponatremia and episodic vomiting and diarrhea was admitted to hospital at 9 months of age, and the diagnosis of type II aldosterone synthase deficiency was confirmed by plasma and urinary steroid determinations. The entire coding sequence of the aldosterone synthase gene (CYP11B2) was determined (both strands) in the affected infant, an unaffected sibling, and both parents. An exon 3 mutation (C554T, leading to amino acid T185I) was found in the father and both siblings, and an exon 9 mutation (A1492G, leading to T498A) was found in the affected infant and the mother. Expression of the mutant sequences in COS cells showed steroidogenic patterns typical of aldosterone synthase type II deficiency, including very low levels of aldosterone synthesis (<=0.5% of wild-type enzyme) consistent with the low aldosterone levels in the patient’s plasma. Both mutations in this compound heterozygote localize to the ß3-sheet in the cytochrome P450 enzyme structure, as does the previously characterized R181W mutation. This region of the enzyme is not part of the putative structural core, but mutations to this region suggest that it is important for conferring the unique ability of aldosterone synthase to catalyze efficient oxygenation of the C18 carbon of steroid substrates.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALDOSTERONE SYNTHASE DEFICIENCY usually presents in infancy as a life-threatening electrolyte imbalance. The major symptoms are failure to thrive, recurrent vomiting, and severe dehydration; a history of fever, diarrhea, lethargy, poor weight gain, and poor feeding since birth may also be observed (1, 2, 3). The key biochemical indicators are hyponatremia, hyperkalemia, natriuresis, metabolic acidosis, elevated plasma renin activity (PRA), and decreased plasma aldosterone. Renal function is normal, as indicated by normal serum creatinine levels; genitalia are similarly normal, in contradistinction with virilizing forms of congenital adrenal hyperplasia (1).

Plasma levels of adrenal steroids and their urinary metabolites are used in the diagnosis of aldosterone synthase deficiency. Very low or undetectable plasma aldosterone levels together with elevated corticosterone, 11-deoxycorticosterone, and 18-hydroxy-11-deoxycorticosterone are diagnostic of type II aldosterone synthase deficiency (1, 3). Aldosterone deficiency caused by other defects in adrenal steroid biosynthesis, including congenital adrenal hyperplasia due to 21-hydroxylase or 3ß-hydroxysteroid dehydrogenase deficiency, congenital adrenal hypoplasia due to a deficiency of the steroidogenic acute regulatory (StAR) protein and primary adrenocortical insufficiency can be excluded on the basis of steroid levels, both basal and in response to ACTH stimulation. Pseudohypoaldosteronism should also be considered, as its presentation may be similar to that of aldosterone deficiency but is characterized by elevated serum aldosterone levels (2).

Two types of aldosterone synthase deficiency [corticosterone methyl oxidase type I (CMOI) and type II (CMOII)] were previously thought to be the result of two separate enzyme deficiencies: CMOI, which was believed to 18-hydroxylate corticosterone, and CMOII, which was believed to catalyze a further 18-oxidation of 18-hydroxycorticosterone to produce aldosterone. As the identification of aldosterone synthase as the single enzyme responsible for the three terminal steps of aldosterone biosynthesis (11ß-hydroxylation, 18-hydroxylation, and 18-oxidation) from deoxycorticosterone (DOC), it appears that both types of aldosterone synthase deficiency are caused by mutations in the aldosterone synthase gene (4). Type I aldosterone synthase (CMOI) deficiency generally reflects mutations causing a complete loss or total inactivation of aldosterone synthase activity despite a clinical picture of blockade of 18-hydroxylation of corticosterone. In type II aldosterone synthase deficiency, the clinical picture suggests blockade of only the terminal 18-oxidation step, with some remaining aldosterone synthase activity.

In the present study the aldosterone synthase gene from an infant presenting with symptoms and signs consistent with type II aldosterone synthase deficiency was screened for mutations. Genomic DNA was isolated from blood samples taken from the infant, an unaffected sibling, and her parents, and the nine exons, including intron/exon boundaries, of the aldosterone synthase gene were amplified by PCR and sequenced. Two mutations were found, one of which was present in the mother’s DNA, and the other in the DNA of the father. The activity of the encoded enzymes was tested to confirm that the mutations were responsible for a deficiency in C18 activity of the enzyme and hence was responsible for the child’s phenotypic presentation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Clinical details

An apparently normal female infant was born at 41 wk to a 34-yr-old gravida 3 para 1 woman. Birth weight was 3720 g, length was 53.3 cm, and head circumference was 33.5 cm; her growth chart is shown in Fig. 1Go. The parents are Macedonian immigrants to Australia and nonconsanguineous. The infant was breast fed for the first 6 wk of life, but was noted to be weak and falling asleep during her feedings. She was changed to bottle feedings supplemented to increase caloric intake, but failed to thrive. At 4 months she was admitted for investigation of hyponatremia (serum sodium, 127 and 130 mmol/liter; normal, 134–142), dry peeling skin (Fig. 2AGo), and a sinus tachycardia of 198/min. Serum K+ was 5.9 mmol/liter (normal, 3.5–5.6), and she had mild hypercalcemia (Ca2+, 2.74 mmol/liter; normal, 2.10–2.64). A 24 h urinary sodium was low (2 mmol/d; normal, 60–200). There were no signs of salt-wasting nephropathy or enteropathy. A sweat test for cystic fibrosis was negative. At the age of 9 months, she was readmitted with growth failure (Fig. 1Go) and persistent hyponatremia (serum sodium, 124 mmol/liter) plus episodic diarrhea and vomiting. She was inactive and pale, with a prominent forehead and depressed nasal bridge. Marked fat dimpling over the abdomen (Fig. 2BGo) suggested GH deficiency. A spot urine again showed a low sodium level, and a urinary sample was sent to Dorevitch Pathology (Melbourne, Australia) for steroid analysis. Pituitary function was also assessed.



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  		Figure 1. Growth chart of the affected infant, demonstrating severe failure to thrive and dramatic catch-up growth after fludrocortisone and salt supplementation therapy.

 


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  		Figure 2. A, Lateral photograph of the chest and axilla of affected infant at age 4 months showing dry peeling skin. B, Photograph of the affected infant at age 9 months, showing central obesity with fat dimpling over the abdomen suggestive of GH deficiency.

 
DNA sequencing and segregation analysis

Informed consent was obtained from the parents on their behalf and on behalf of their children to study their family’s DNA to identify possible mutations causing deficient aldosterone synthesis. The Molecular Genetics Laboratory, Hunter Area Pathology Service, John Hunter Hospital, prepared genomic DNA from blood samples from the affected infant (100 µg/ml) and from the parents and sibling (500 µg/ml). The CYP11B2 gene was selectively amplified in five segments under the same PCR conditions and primers as those used by Portrat-Doyen et al. (5) with some modifications. Briefly, 50-µl reactions contained 375 ng genomic DNA, 50 pmol of each primer, 200 µM of each deoxy-NTP, 2.5 U Taq DNA polymerase (Promega Corp., Annandale, Australia; Perkin-Elmer Corp., Boston, MA), and 1x Taq reaction buffer with an optimum MgCl2 concentration for each reaction, as shown in Table 1Go. The PCR program on a GeneAmp 9700 thermocycler was 95 C for 5 min, followed by 30–35 cycles of 15 sec at 95 C, 15 sec at 56 C (50 C for exons 8–9), and 20 sec at 72 C, with a 72 C final extension for 10 min in the last cycle.


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  		Table 1. Primers (S sense, AS antisense) and MgCl2 concentration used for the selective amplification of aldosterone synthase

 
All primers were in the introns, except those used to amplify exons 8–9, with the exonic nucleotides in italics (Table 1Go). To sequence the remaining nucleotides not covered by the primers for exons 8–9, an additional set of primers was designed to amplify exons 7–9. Amplification of exons 7–9 was as described above, but with 200 ng genomic DNA and a 30-sec annealing step at 59 C. Some PCRs failed to produce sufficient product for sequencing (ostensibly due to contaminating salts in the DNA) and therefore required further optimization of the reaction conditions. This was particularly a problem with the affected infant’s DNA, which was at a lower concentration. The PCR parameters were therefore optimized, as outlined in Table 2Go. Additional PCR products, if required, were generated under the optimized reaction conditions from only 100 ng template DNA. The PCR products were purified after agarose gel electrophoresis (gel extraction kit; QIAGEN Clifton Hill, Australia), and the concentration was adjusted for sequencing after estimating the yield on an agarose gel by comparison with standards of known mass (Life Technologies, Inc., Mount Waverley, Australia). Where necessary PCR was repeated with gel extracts combined and then concentrated in a Speed-Vac to provide sufficient DNA for sequencing. In addition to the primers used for generation of the PCR products, additional primers were designed to obtain sequence for both strands (Table 3Go), and 30–90 ng PCR product were sequenced directly from both strands.


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  		Table 2. Optimized parameters for amplification of aldosterone synthase for specific DNA samples

 

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  		Table 3. Additional primers for sequencing aldosterone synthase PCR products

 
Recreation of the mutations found in the infant’s CYP11B2 gene into an aldosterone synthase cDNA expression plasmid for testing activity

The mammalian expression vector driven by a cytomegalovirus promoter (pCMV4) containing the coding sequence of CYP11B2 (pCMV4-CYP11B2), as described previously (6), was used as the template for introduction of the individual point mutations using Quik-Change mutagenesis according to the manufacturer’s instructions (Stratagene, Cedar Creek, TX).

Oligonucleotides for the introduction of the exon 3 mutation (underlined) were: sense, 5'-CGGGGGAGCCTGATCCTGGACGTCC-3'; and antisense, 5'-GGACGTCCAGGATCAGGCTCCCCCG-3'. Oligonucleotides for the introduction of the exon 9 mutation (underlined) were: sense, 5'-GTCCCCCCTCCTCGCTTTCAGAGCGATTAAC-3'; and antisense, 5'-GTTAATCGCTCTGAAAGCGAGGAGGGGGGAC-3'. The 50-µl reactions contained 50 ng pCMV4-CYP11B2, 1x Pfu reaction buffer, 125 ng of each complementary primer pair (with either exon 3 or exon 9 mutation), 200 µM of each deoxy-NTP, and 1.5 U Pfu and were overlaid with mineral oil. Reactions were carried out in an MJ minicycler (MJ Research, Inc., Cambridge, MA) at 95 C for 2 min, then for 12 cycles of 95 C for 30 sec, 55 C for 1 min, 68 C for 14 min, and then a 2-min additional extension 68 C. The PCR reactions were treated with DpnI to digest the template DNA for 3 h at 37 C, and then 1 µl was used to transform Epicurian XL-1 Blue supercompetent Escherichia coli according to the manufacturer’s protocol (Stratagene). Plasmids were purified from selected clones (QIAGEN), and the entire CYP11B2-coding region was sequenced to ensure that the desired mutations were present.

Expression of mutant coding sequence in COS-1 cells

COS-1 cells were maintained in DMEM with 10% fetal calf serum (FCS) and glutamine and passaged until subconfluent. For enzyme assays, cells were plated in 12-well plates (22-mm wells). After overnight recovery and anchorage, cells (~80% confluent) were transfected with 100 ng/well pCMV4 containing human adrenodoxin and either 500 ng/well pCMV4 alone or containing CYP11B1, CYP11B2, CYP11B2 with exon 3 mutation, CYP11B2 with exon 9 mutation, or CYP11B2 181W/386A mutations, in Opti-MEM with 4.8 µl/well Lipofectamine (Life Technologies, Inc., Mount Waverly, Victoria, Australia) according to the manufacturer’s instructions (6, 7). After a 5-h incubation, the medium was changed back to maintenance medium (DMEM/FCS/glutamine), and after a 23-h recovery the medium was replaced with 400 µl fresh DMEM/FCS/glutamine containing 0.4 nmol/well [14C]-11-DOC. After a 24-h incubation, 200 nmol nonradioactive 11-DOC was added to the medium, transferred into siliconized silica borate glass tubes for extraction with 2.5 vol ice-cold methylene chloride. The aqueous phase was removed, and the organic phase was dried completely by evaporation at 37 C. Extracted steroids were resuspended in 15 µl methylene chloride and spotted onto glass-backed, silica-coated, thin layer chromatography (TLC) plates. After chromatography in methylene chloride/methanol/water (300:20:1), 14C-labeled steroids were detected by phosphorimaging (6, 8). To demonstrate and quantitate the expression of the wild-type and mutated forms of aldosterone synthase in the transfected cells, the remaining cells were washed twice in 1 ml Hank’s Buffered Saline Solution and lysed in RIPA buffer [50 mM Tris-HCl (pH 7.5), 0.1% (wt/vol) sodium dodecyl sulfate, 0.5% (wt/vol) sodium deoxycholate, 1.0% (vol/vol) Triton X-100, 2 mM EDTA, 150 mM NaCl, 1 mM phenylmethylsulfonylfluoride, 5 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 µg/ml pepstatin] by rocking for 1 h at 4 C. Cell debris was removed by centrifugation at 12,000 x g for 20 min at 4 C, and the supernatant was retained for protein determination (Bio-Rad Laboratories, Inc., Regent Park, Australia). Equivalent amounts of protein were separated by 10% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes by Western blotting, and the blots were probed with an in-house antibody specific for CYP11B2.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Clinical

Initial urinary steroid profiles showed elevated urinary metabolites of corticosterone, 18-hydroxycorticosterone, and 18-hydroxy-11-dehydrocorticosterone, consistent with aldosterone synthase deficiency type II. Plasma aldosterone levels of 360 pmol/liter (normal range, 611-2491), associated with a high plasma renin level (30 ng/ml/h; normal range, 1.2–2.8), gave a very low plasma aldosterone/renin ratio of 11 (normal range, 100–200 for this age group). On Synacthen testing (synthetic corticotropin, 90 µg, im) serum cortisol rose from 266 to a peak of 550 nmol/liter at 30 min (normal, >600 nmol/liter). Baseline ACTH was undetectable. Thyroid function was normal (free T4, 18 pmol/liter; normal, 12–25).

The mother’s serum aldosterone level was 67 pmol/liter (normal, 80–1040); at a subsequent retest, aldosterone (160 pmol/liter) was within the normal range, but with an elevated PRA (5.1 ng/ml/h; normal, 1.2–2.8). The father’s serum aldosterone was 85 pmol/liter.

The diagnosis of aldosterone deficiency was made on the basis of the original urinary steroid profile. Therapy with fludrocortisone (Florinef, Bristol-Myers Squibb Pharmaceuticals, Noble Park, Victoria, Austrlia; 0.05 mg twice daily) and oral hypertonic saline was started, with a dramatic clinical response (Fig. 1Go). In view of the borderline response to Synacthen, we elected to treat her with hydrocortisone for several months, but eventually this was withdrawn. Intellectual development is normal.

Screening for mutations

Sequencing of PCR products amplified from the affected child’s aldosterone synthase gene revealed two separate heterozygous mutations (as shown in Fig. 3AGo), C554T in exon 3 and A1492G in exon 9, resulting in changes to amino acids T185I and T498A. Sequencing of the parents’ and unaffected sibling’s CYP11B2 gene revealed that the exon 3 mutation was inherited from the father and shared with the sibling, whereas the exon 9 mutation was inherited from the mother. All members of the family thus carry a single heterozygous mutation with the exception of the affected child, who inherited both mutated alleles (Fig. 3BGo).



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  		Figure 3. A, Results of automatic sequencing, showing the normal homozygous sequence and the heterozygous mutant sequence found in the affected child, with locations of the mutations depicted in exon-intron structure of the CYP11B2 gene. B, Family pedigree showing the inheritance of the two independently segregating heterozygous mutations. The father is represented by a square, and the mother and two daughters are represented by circles. The parents (F, father; M, mother) and the sister (S) are heterozygous and asymptomatic. The affected daughter (A) has two mutated alleles.

 
Expression and activity of mutant alleles in vitro

The T185I and T498A mutations were individually recreated in the CYP11B2 coding sequence contained in the pCMV4 mammalian expression vector and cotransfected with a construct expressing the electron shuttle protein adrenodoxin into COS-1 cells (6). Comparable expression levels were obtained for both wild-type CYP11B1 and CYP11B2 and for the CYP11B2 mutants, as determined by Western blot analysis shown in Fig. 4Go. A doublet at the expected size of approximately 50 kDa was detected, consistent with the precursor and mature (mitochondrially imported) forms of aldosterone synthase. A single nonspecific band at approximately 60 kDa shows that the levels of protein loaded were approximately equivalent for all lanes.



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  		Figure 4. Comparison of expression levels of aldosterone synthase from COS-1 cell lysates transiently transfected with the mammalian expression vector pCMV4 containing CYP11B coding sequences and cotransfected with adrenodoxin. The expression of wild-type aldosterone synthase and mutants containing mutations causing type II aldosterone synthase deficiency was detected by probing the Western blot with a rabbit antiserum generated against a peptide derived from aldosterone synthase.

 
Activity on TLC

Medium from transfected cells (containing [14C]-DOC as a substrate) was extracted after a 23-h incubation, and steroids were separated by TLC. The profile of steroid metabolites from both the T185I and T498A mutations found in the affected child’s CYP11B2 coding sequence and that of the classical 181W/386A mutant were virtually indistinguishable from the steroidogenic profile of CYP11B1 (Fig. 5AGo). Significant amounts of corticosterone and 18-hydroxycorticosterone were produced from DOC, but there was no discernible aldosterone. The production of 11-dehydrocorticosterone (Fig. 5AGo), presumably from corticosterone, was most likely due to the inherent hydroxysteroid dehydrogenase activity of the kidney-derived COS-1 cells. A range of inherent steroidogenic activities was apparent from the empty vector-transfected COS-1 cells, with a variety of different steroids produced from DOC; none of the steroids thus produced was a specific product of either CYP11B1 or CYP11B2.



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  		Figure 5. Aldosterone production from COS-1 cells transiently transfected with the mammalian expression vector pCMV4 containing coding sequences for mutant and wild-type aldosterone synthase. A, Steroids produced by these cells from [14C]-DOC were separated and identified by TLC; B, the amount of aldosterone produced in parallel transfection experiments incubated with unlabeled DOC substrate was determined by RIA. The percentage of aldosterone produced by mutants compared with wild-type enzyme is shown under the bars.

 
Activity by RIA

Quantification of aldosterone production by RIA in parallel experiments showed that all of the mutant forms of aldosterone synthase produced detectable levels of aldosterone, although only at a small fraction of wild-type levels (between 0.3–0.5%). Aldosterone production from the T185I and T498A mutants was comparable with that from the classical type II aldosterone synthase deficiency mutants (181W/386A) and thus most likely was causative for the current infant. Interestingly, in this assay aldosterone levels for CYP11B1 were consistently above those in the vector-only control, at a level comparable with the deficiency-causing mutants (Fig. 5BGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
On presentation the infant was profoundly affected, consistent with isolated congenital aldosterone synthase deficiency, despite measurable levels of endogenous aldosterone. The response to mineralocorticoid replacement and salt supplementation (dramatic catch-up growth, no further diarrhea or vomiting, increased energy, and normalized appetite) confirmed the diagnosis. The degree of growth failure can be explained by the severity of her salt-wasting state (9). Clinically this produced signs consistent with GH deficiency (frontal bossing and central obesity with fat dimpling), but other pituitary function was normal, and therefore, no formal GH testing was performed. The precise biochemical mechanism linking severe salt wasting and growth failure remains to be elucidated. The low urinary salt measurements on several occasions probably reflected the extent of total body salt depletion, but initially tended to cloud the diagnosis. In retrospect, the sinus tachycardia was presumably a response to volume depletion, as was the mild hypercalcemia. Both parents are clinically asymptomatic, presumably having 50% of the normal aldosterone synthetic capacity with one normal CYP11B2 allele each. The finding of low normal aldosterone levels in the mother and a single elevated PRA suggest that she may be biochemically affected by her heterozygous state; however, no formal sodium and fluid balance studies have been performed.

Of the two mutations found, the paternal T185I substitution has previously been reported as homozygous in association with type II aldosterone synthase deficiency (10). In that study no in vitro activity assay data were reported, but the parents were first degree cousins and also of middle eastern European origin (10, 11). Both parents of the infant examined here are of Macedonian origin, with no evidence for consanguinity. The in vitro activity assay results for both the T185I and T498A substitutions found in the current infant are consistent with significantly reduced C18 activity of aldosterone synthase, which coincides with the clinical diagnosis of type II aldosterone synthase deficiency.

In the majority of cases of clinically diagnosed congenital isolated aldosterone synthase deficiency mutations are subsequently found in the CYP11B2 gene, with in vitro activity consistent with the clinical picture. Theoretically, exceptions in which the in vitro activity of the mutants exceeds the expected level for a given clinical phenotype may be explained by additional mutations in the noncoding regions of the CYP11B2 gene. A range of completely inactivating mutations has been found to cause type I aldosterone synthase deficiency (Table 4Go), although the double homozygous mutation R181W/V386A until now is the only variant shown by in vitro activity assay to result in type II deficiency (Table 5Go). Of the mutations associated with type II deficiency only the homozygous deletion of R173, now in distinction from T185I, remains to be tested for in vitro activity (12).


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  		Table 4. Type I aldosterone synthase deficiency

 

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  		Table 5. Type II aldosterone synthase deficiency

 
The compound heterozygote reported by Zhang et al. (13) showed a clinical phenotype of type II deficiency, with both detectable serum aldosterone and elevated 18-hydroxycorticosterone, but in vitro no residual aldosterone synthase activity. The single T318M substitution as a component of the compound heterozygote was found to have in vitro activity comparable to the R181W/V386A mutant, and suggesting that it might alone cause type II deficiency. The activity test using steroidogenic MA-10 cells was sufficiently sensitive to detect activity for both the R181W/V386A mutant and CYP11B1, which had not previously been reported (13). In the present study aldosterone production from CYP11B1-transfected COS-1 cells was also consistently above vector-only controls, supporting the possibility that CYP11B1 may be capable of very low levels of aldosterone biosynthesis (14, 15). This may explain why some circulating aldosterone is still detectable, albeit at very low levels, in some patients with type I aldosterone synthase deficiency and no functional CYP11B2 gene.

The T498A substitution has not previously been described in association with type II aldosterone synthase deficiency; although only six residues from the carboxyl terminus, this threonine is conserved in both CYP11B1 and CYP11B2. No mutations causing either type of aldosterone synthase deficiency have previously been found in exon 9. The substitution C494F was reported as a possible cause of 11ß-hydroxylase (CYP11B1) deficiency, but was later found to be polymorphic (16, 17, 18). Residue swapping experiments of the diverging residues between CYP11B1 and CYP11B2 indicate that the carboxyl-terminal region is unimportant in determining the differing activities of the two enzymes (19, 20).

Members of the cytochrome P450 superfamily generally conform to a structure of four ß-sheets and approximately 13 {alpha}-helixes (21). The conserved P450 structural core consists of a four-helix bundle (helixes D, E, I, and L) and two structurally conserved ß-sheets (ß-sheet 1 containing five strands and ß-sheet 2 containing two strands), with the two ß-sheets forming part of the hydrophobic substrate access channel (21). Alignment of aldosterone synthase with members of the cytochrome P450 superfamily of known structure places residues 173 (helix D), 318 (helix I), and 386 (strand 4 of ß-sheet 1), known to be associated with type II deficiency, in the conserved structural core of the enzyme. The mutations in this study shown to be causative of type II deficiency (T185I and T498A) align to ß-sheet 3, strands 1 and 2, respectively. ß-Sheet 3 is associated with other mutations causing type II aldosterone synthase deficiency, as shown in Fig. 6Go (22, 23). Although this region is not considered part of the generally conserved structural core of cytochrome P450, it appears to play a role in determining the C18 activity of the enzyme.



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  		Figure 6. Ribbon drawing of cytochrome P450 BM-3, illustrating the structural fold conserved among members of the superfamily. The general fold consists of approximately 13 {alpha}-helixes and four ß-sheets, with helixes D, E, I, and L and ß-sheets 1 and 2 making up the conserved structural core. Shown as cross-section slab (A) or the complete molecule (B), the residues important for conferring aldosterone synthetic capacity on CYP11B1 (288 and 320) and those associated with type II aldosterone synthase deficiency (173, 181, 185, 318, 386, and 498) are labeled. Of particular note is ß-sheet 3 and the two mutations (T185I and T498A) found in the present study to cause type II aldosterone synthase deficiency.

 
In the present study two mutations were identified in the coding sequence of the aldosterone synthase gene from an infant diagnosed with type II aldosterone synthase deficiency. In vitro analysis of the enzyme activities of the novel T498A mutation and of the previously reported T185I allele showed each to have efficient 11ß-hydroxylase activity, but a loss of efficient C18 activity, resulting in poor aldosterone synthesis. This fits with both the clinical picture and the diagnosis. Both mutated residues align to the same region in the conserved cytochrome P450 structure fold (ß-sheet 3), as does R181W, highlighting a role for this region in conferring the efficient C18 activity of aldosterone synthase.


   Acknowledgments
 
We are grateful to the referring pediatrician, Dr. Lynn Banna, and to Dr. Stephen McInally for clinical photography. We thank Michelle Cinel for automated sequencing of amplified PCR products, and Kathy Myles for quantifying the aldosterone produced by transfected cells by RIA. We are indebted to Sue Smith for her expert secretarial assistance in the preparation of this manuscript.


   Footnotes
 
Present address for F.M.D.: Amrad Corporation Ltd., 576 Swan Street, Richmond, Victoria 3121, Australia.

Present address for J.W.F.: Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia.

Present address for K.M.C.: Pharmacia Australia, 59 Kirby Street, Rydalmere, New South Wales 2116, Australia.

Abbreviations: CMO, Corticosterone methyl oxidase; DOC, 11-deoxycorticosterone; FCS, fetal calf serum; pCMV4, mammalian expression vector driven by a cytomegalovirus promoter; PRA, plasma renin activity; TLC, thin layer chromatography.

Received February 28, 2003.

Accepted March 17, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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  6. Curnow KM, Tusie-Luna MT, Pascoe L, Natarajan R, Gu JL, Nadler JL, White PC 1991 The product of the CYP11B2 gene is required for aldosterone biosynthesis in the human adrenal cortex. Mol Endocrinol 5:1513–1522[Abstract]
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  9. Pareckh RS, Flynn JT, Smoyer WE, Milne JL, Kershaw DB, Bunchman TE, Sedman AB 2001 Improved growth in young children with severe chronic renal insufficiency who use specified nutritional therapy [Erratum: J Am Soc Nephrol 12:2418–26, 2002]. J Am Soc Nephrol 13:1421–1422[Abstract/Free Full Text]
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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.
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