Molecular Genetics and Metabolism 100 (2010) 357–364

Contents lists available at ScienceDirect

Molecular Genetics and Metabolism

journal homepage: www.elsevier .com/locate /ymgme

Five novel mutations in CYP11B2 gene detected in patients with aldosteronesynthase deficiency type I: Functional characterization and structural analyses

Huy-Hoang Nguyen a, Frank Hannemann a, Michaela F. Hartmann b, Ewa M. Malunowicz c,Stefan A. Wudy b, Rita Bernhardt a,*

a Department of Biochemistry, Saarland University, D-66041 Saarbrücken, Germanyb Steroid Research Unit, Division of Pediatric Endocrinology and Diabetology, Center of Child and Adolescent Medicine, Justus-Liebig-University, Giessen, Germanyc Department of Biochemistry and Experimental Medicine, The Children’s Memorial Health Institute, Warsaw, Poland

a r t i c l e i n f o a b s t r a c t

Article history:Received 25 March 2010Accepted 27 April 2010Available online 21 May 2010

Keywords:Aldosterone synthase deficiency (ASD)CYP11B2Steroid hydroxylation

1096-7192/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.ymgme.2010.04.016

* Corresponding author. Fax: +49 (0) 681 302 4739E-mail address: ritabern@mx.uni-saarland.de (R. B

Context: Aldosterone synthase deficiency (ASD) is an important differential diagnosis of diseases associ-ated with salt wasting in early infancy.Objective: The objective of this study was to investigate the molecular basis for the disorder by (1) molec-ular genetic analysis in the CYP11B2 from patients suffering from ASD type I. (2) Functional characteriza-tion of the missense mutant gene products. (3) Structural simulation of the missense mutations.Results: Patient 1 was a homozygous carrier of a novel mutation located in exon 4 causing a prematurestop codon (p.W260X). Patient 2 was analyzed to be compound heterozygous for two novel mutations:The first was an insertion mutation (p.G206WfsX51), and the second was a deletion mutation(p.L496SfsX169). Two siblings (patients 3 and 4) were compound heterozygous carriers of two novel mis-sense mutations (p.S315R, p.R374W). The expression studies of the mutant proteins in COS-1 cellsshowed a complete absence of CYP11B2 activity of p.S315R and p.R374W mutants for the conversionof 11-deoxycorticosterone to aldosterone. A 3-D model of CYP11B2 p.S315R and p.R374W indicated achange of the hydrogen bond network which might explain the cause of the dysfunction.Conclusion: We have identified the first CYP11B2 gene defects in two Polish families associated with phe-notypes of ASD type I. Analysis of the enzymatic function as a complementary procedure to genotypingrevealed data for understanding the clinical phenotype of ASD. Molecular modeling of the mutatedenzyme provided a rational basis for understanding the changed activities of the mutant proteins.

� 2010 Elsevier Inc. All rights reserved.

Introduction

Aldosterone is the most potent mineralocorticoid synthesizedin the zona glomerulosa of the adrenal cortex in all mammals [1].Aldosterone synthesis starting from 11-deoxycorticosterone isfacilitated by CYP11B2, which catalyzes three sequential reactions:the 11b-hydroxylation, the 18-hydroxylation, followed by 18-oxi-dation resulting in the formation of aldosterone [2,3].

Aldosterone synthase deficiency (ASD) is classified into twogroups, type I and type II CMO (corticosterone methyl oxidase)deficiencies. Patients with type I CMO deficiency (CMO I) havelow to normal levels of 18-OH-corticosterone and low levels ofplasma aldosterone or very low to undetectable levels of urinarytetrahydroaldosterone. On the contrary, type II CMO deficiency(CMO II) is related with high levels of 18-OH-corticosteroneand low to undetectable levels of plasma aldosterone or urinary

ll rights reserved.

.ernhardt).

tetrahydroaldosterone [3–7]. The two defects differ biochemicallyin that 18-hydroxycorticosterone is deficient in ASD type I, butoverproduced in ASD type II [6,7].

Both disorders are characterized by the same clinical signs andsymptoms: failure to thrive, vomiting, and severe dehydration; fur-ther concomitant biochemical features are hyperkalemia, hypona-tremia, and metabolic acidosis with elevated plasma renin activity[8]. CMO I and CMO II deficiencies are caused by mutations in thealdosterone synthase gene (CYP11B2) [3,9].

In this study, we performed molecular genetic analyses of theCYP11B2 gene from four patients suffering from aldosterone syn-thase deficiency. Sequence analysis of the CYP11B2 gene led tothe identification of 5 novel mutations. The functional analysis oftwo missense mutants in COS-1 cells indicated that mutantCYP11B2 proteins lost enzyme activity proving the pathogenicityof the mutations. The complete loss of mutant CYP11B2 enzymeactivities in the cell culture could be rationalized with the 3-Dmolecular model indicating a change of the hydrogen bond net-

358 H.-H. Nguyen et al. / Molecular Genetics and Metabolism 100 (2010) 357–364

work in the protein structure and providing further insights intothe function of cytochrome P450 enzymes.

Subjects and methods

Case reports

Patient 1The boy was born at term with a birth weight of 3450 g. It was

the first child of nonconsanguinous Polish parents from the Mazo-vian Region. The child with a history of recurrent vomiting, dehy-dration, poor weight gain and hypotonia since 3 weeks of life wasdiagnosed as salt water at the age of 2 months (plasma Na132 mol/l, plasma K 6.0 mol/l). Elevated plasma renin activity(PRA) of 50 ng/ml/h (normal range 2.2–10 ng/ml/h) and reducedplasma aldosterone of 84 pg/ml (normal: 140–1049 pg/ml) wereobserved. Plasma cortisol (9 lg/dL) and 17-hydroxyprogesterone(149 ng/dL) were normal.

Primary hypoaldosteronism was suspected and treatment onlywith fludrocortisone was instituted with good clinical response.At the age of 2 yrs fludrocortisone was stopped for 2 weeks toachieve a final diagnosis. Urinary steroid profiling by gas chroma-tography–mass spectrometry (GC–MS) showed the elevated excre-tion of metabolites of corticosterone, 11-dehydrocorticosterone,and diminished excretion of 18-hydroxycorticosterone and aldo-sterone metabolites, revealing ASD type I (see Table 1). Urinaryexcretion of cortisol metabolites was normal, markers of 21-hydroxylase deficiency were not elevated.

Patient 2The girl was born at term with a birth weight of 3820 g. She was

the first child of nonconsanguinous German parents. Recurrentvomiting, poor feeding and failure to thrive led to admission to apediatric hospital at the age of 6 weeks. Plasma Na was decreased(126 mmol/l), plasma K was elevated (6.26 mmol/l) and plasma re-nin was elevated (5214 ng/L; normal range 6.3–149 ng/L). Plasmacortisol (10.5 lg/dL) and 17-hydroxyprogesterone (1.45 ng/dL)were normal. A GC–MS urinary steroid profile showed elevatedmetabolites of corticosterone, 11-dehydrocorticosterone and de-creased aldosterone metabolites (THAldo) revealing ASD type I(see Table 1). Excretion of cortisol metabolites was normal, param-eters of 21-hydroxylase deficiency were not elevated. She re-sponded well to treatment with fludrocortisone, her growth andweight have normalized meanwhile.

Table 1Urinary steroid metabolite levels measured by gas chromatography-mass spectrometry (G

Patient Mutant on 2 alleles THDOC 6a-OH-THA

THA

(lg/24 h)

(lg/24 h) (lg/24 h)

Patient 12 yrs old

p.W260X/p.W260X 69 n.dt. 130

Patient 26 wks old

p.G206WfsX51/p.L496SfsX169

n.dt. 212 101

Patient 33 yrs old

p.S315R/p.R374 W 45 n.dt. 203

Patient 46 wks old

p.S315R/p.R374 W n.dt. 123 164

Reference range: 0–3 months 0–5 n.dt. 2–281–3 yrs 0–12 n.dt. 12–87

n.d., not determined; n.dt. no detectable, THDOC, tetrahydrodeoxycorticosterone; 6adehydrocorticosterone; aTHA, allo-tetrahydro-11-dehydrocorticosterone; THB, tetrahytetrahydro-11-dehydrocorticosterone; 18-OH-THB, 18-hydroxy-tetrahydrocorticosteron

Patient 3The girl was born at term with a birth weight of 2750 g. She was

the first child of nonconsanguinous Polish parents from the Mazo-vian Region. Since 2 months of age she presented with poorfeeding, episodic vomiting and diarrhea, failure to thrive, poorweight and growth failure. Since that age she was admitted afew times to the local pediatric hospital. She displayed hyponatre-mia and hyperkalemia, but the cause was undiagnosed. At the ageof 2 months serum cortisol (6 lg/dL) and plasma ACTH (22.5 pg/ml) were normal. The girl was treated with intravenous fluid infu-sion for 6–7 days to correct the abnormal levels of serum electro-lytes. Next she was examined in the outpatient department,showing persistent electrolyte disturbances. The parents stoppedthe child examination when she was about 1 year old. After diag-nosis of aldosterone synthase deficiency type 1 in her youngestbrother (patient No. 4) she was admitted to our hospital at theage of 3.2 years. She presented with growth retardation (height90 cm, 3rd percentile), no symptoms of dehydration and normalelectrolytes. Plasma aldosterone level was decreased with 30 pg/ml (normal range for age 3–5 years: 110–669 pg/ml) and plasmarenin activity (PRA) was increased with 15.6 ng/ml/h (normalrange for age 1–5 years: 1.5–5.7 ng/ml/h). An urinary steroid pro-file by GC–MS showed elevated metabolites of corticosterone,11-dehydrocorticosterone and reduced aldosterone metabolites(THAldo) consistent with ASD type I (see Table 1). The urinary ste-roid profile showed normal cortisol metabolites and no elevation ofmarkers of 21-hydroxylase deficiency.

Patient 4The boy was the youngest brother of patient No. 3. He was the

third child of the family, the second one was healthy. He was bornat term with a birth weight of 3900 g. At the age of 6 weeks he wasadmitted to our hospital for failure to thrive, dehydration symp-toms, dry peeling skin, presenting with salt wasting syndrome(plasma K 7.1 mol/l; Na 133 mol/l). Elevated plasma renin activityof 62.1 ng/ml/h (normal range for age 2–12 months: 2.2–10 ng/ml/h) and decreased plasma aldosterone of 41 pg/ml (normal range forage 2 weeks–3 months: 140–1409 pg/ml) were observed. Serumcortisol (13 lg/dL) and 17-hydroxyprogesterone (200 ng/dL) werenormal. At the age of 6 weeks a urinary steroid profile by GC–MSexcluded CAH due to 21-hydroxylase deficiency or 3b-hydroxy-steroid dehydrogenase deficiency but revealed increased cortico-sterone metabolite excretion with diminished excretion oftetrahydroaldosterone, revealing ASD type I (see Table 1).

C–MS).

aTHA THB aTHB 18-OH-THA

18-OH-THB

THAldo

(lg/24 h)

(lg/24 h)

(lg/24 h)

(lg/24 h) (lg/24 h) (lg/24 h)

280 60 148 n.dt. n.dt. 3

n.d. 9 24 n.dt. n.dt. n.dt.

49 49 301 n.dt. n.dt. 3

57 n.dt. n.dt. n.dt. n.dt. 3

3–42 1–10 6–24 3–12 n.dt. 4–1215–98 5–16 12–98 5–25 n.dt. 6–34

-OH-THA, 6a-hydroxy-tetrahydro-11-dehydrocorticosterone; THA, tetrahydro-11-drocorticosterone; aTHB, allo-tetrahydrocorticosterone; 18-OH-THA, 18-hydroxy-e; THAldo, tetrahydroaldosterone.

H.-H. Nguyen et al. / Molecular Genetics and Metabolism 100 (2010) 357–364 359

Gas chromatography–mass spectrometry (GC–MS)

Urinary steroids were profiled using GC–MS and selected ionmonitoring analysis was performed as described previously [10].Free and conjugated urinary steroids were extracted by solid phaseextraction (Sep-Pak C18 cartridge, Waters Associates, Milford, MA),and the conjugates were enzymatically hydrolyzed (type I pow-dered Helix pomatia, Sigma–Aldrich Corp., St. Louis, MO). Thehydrolyzed steroids were recovered by Sep-Pak extraction. Knownamounts of three internal standards (5a-androstane-3a,17a-diol,stigmasterol, and cholesteryl butyrate) were added to a portionof each extract before formation of methyloxime-trimethylsilylethers. GC was performed using an Optima-1 fused silica column(Macherey–Nagel, Dueren, Germany). Helium was used as carriergas at a flow rate of 1 ml/min. The gas chromatograph (Agilent6890 series GC, Agilent 7683 Series Injector, Agilent Technologies,Waldbronn, Germany) was directly interfaced to a mass selectivedetector (Agilent 5973 N MSD, Agilent Technologies) operated inthe selected ion monitoring mode. The injections took place withan 80 �C (2 min) GC oven; the temperature was then increasedby 20 �C/min to 190 �C (1 min). For separation of steroids, the tem-perature was increased by 2.5 �C/min to 272 �C. Values for theexcretion of individual steroids were determined by measuringthe selected ion peak areas against the internal standards.

Hormone assays

Plasma aldosterone and renin activity were assayed usingALDO-RIA-CT and REN-CT2 kits, respectively, from CIS bio interna-tional, Gif-sur-Yvette (Cedex, France).

PCR and sequencing

Genomic DNA was prepared from peripheral blood leukocytes,using a standard protocol. The CYP11B2 gene was selectivelyamplified by PCR and sequenced using oligonucleotides and condi-tions as described previously [5]. The sequences of DNA and pro-tein were analyzed with the program CLUSTALW 1.8.

Site-directed mutagenesis

The mutagenesis was performed with the vector pSVLh11B2 astemplate, using the Quik-Change Site-Directed Mutagenesis Kit(Stratagene Ltd., Cambridge, UK) according to manufacturer’sinstruction. The CYP11B2 sequence of the cell culture expressionconstruct pSVLh11B2 used as wild-type corresponds to that pub-lished by Kawamoto et al. [11] with one variation at position249, where we found Ser instead of Arg, as described by Mornetet al. [12]. Oligonucleotides corresponding to an amino acid ex-change of exon 5 were: sense, 50-CACTGCAGGGAGGGTGGACACGACAGC-30; and antisense, 50-GCTGTCGTGTCCACCCTCCCTGCAGTG-30. Oligonucleotides corresponding to an amino acid exchange ofexon 6 were: sense, 50-CAAGGAGACCTTGTGGCTCTACCC-30; andantisense, 50-GGGTAGAGCCACAAGGTCTCCTTG-30. PCR programfor site-directed mutagenesis was used to create the mutationsas described previously [5]. All changes were confirmed by auto-matic sequencing (MWG Eurofins, Ebersberg, Germany).

In vitro expression and assays of enzyme activity

COS-1 cells were grown following a standard protocol (Invitro-gen) using DMEM supplemented with 5% fetal bovine serum,0.1 mg/ml streptomycin, 100 U/ml penicillin, 1 mM pyruvate and4 mM L-glutamine. They were transiently cotransfected with1.5 lg of a pSVLh11B2 construct and 1.5 lg of a bovine adrenodoxinexpression vector (pbAdx) using the Effectene Transfection Reagent

Kit (Qiagen) after growing to a density of approximately 2 � 105 per6 cm dish cells. The pbAdx vector was kindly made available by Dr.M.R. Waterman (Vanderbilt University School of Medicine, Nash-ville, Tennessee, USA). After 6 h transfection, the cells were incu-bated for 72 h at 37 �C with 3 ml completed medium containing2 lM DOC and 5 nCi of 14C-labeled DOC. Steroids were extractedtwice from the cell culture supernatant (800 ll) with chloroformand the products were analyzed using high performance thin layerchromatography (HPTLC) as described previously [5].

Western blot analysis was performed to demonstrate theexpression of the wild-type and mutant of aldosterone synthasein the transfected cells. The antihuman-CYP11B rabbit antiserumwas kindly provided by Dr. H. Takemori (Department of MolecularPhysiological Chemistry, Osaka University Medical School, 2-2Yamadaoka, Suita, Osaka, Japan). Proteins were extracted fromthe COS-1 cells as described previously [5]. Total protein extracts(200 lg each lane) were subjected to 10% SDS–PAGE and thentransferred to nitrocellulose membrane. CYP11B2 was detectedwith an antihuman-CYP11B rabbit antiserum and visualized byusing the ECL Western analysis kit (Amersham Biosciences, UK)according to manufacturer’s instruction.

Molecular modeling

The changes of residues in CYP11B2 were modeled based on atemplate structure of CYP11B2 from our laboratory [13] by usingthe spdbv program (http://www.expasy.org/spdbv/) [14]. The ob-tained model structures were energy minimized using the steepestdescent algorithm implemented in the spdbv program. The struc-tural representations were generated by using the ViewerLiteprogram.

Results

Hormone assay

Urinary steroid metabolites from four patients were measuredby GC–MS (see Table 1). The profiles of the four patients were typ-ical for ASD type I, showing elevated excretion rates of metabolitesof corticosterone and 11-dehydrocorticosterone as well as dimin-ished excretion of 18-hydroxycorticosterone and aldosteronemetabolites.

PCR and sequencing

To analyze the CYP11B2 gene in the CMO I deficient patients,overlapping fragments of the CYP11B2 genes of the patients andtheir parents were amplified specifically from the genomic DNA,and all nine exons and the exon/intron boundaries were sequenced.

Patient 1 was detected to be homozygous for the as-yet-un-known A to G point mutation at bp 3722 (corresponding toc.780G > A) in exon 4 of the CYP11B2 gene (Genbank Number:D13752). This mutation results in a premature stop codon insteadof a tryptophan residue (p.W260X) (see Fig. 1A and B). The parentalsamples were unavailable for analysis.

Patient 2 carried a novel compound heterozygous mutation:insertion of T between position bp 3560 and 3561(g.3560_3561insT; c.618_619insT) and deletion of C at positionbp 6443 (g.6443delC; c.1489delC) of genomic DNA (Genbank Num-ber: D13752). The g.3560_3561insT mutation results in a frame-shift with predicted premature termination leading to atruncated protein at amino acid position 258 of the CYP11B2(p.G206WfsX51). The g.6443delC mutation leads to a frameshiftand additional 169 amino acids (p.L496SfsX169) (see Fig. 1A andC). The parental samples were unavailable for analysis.

Fig. 1. Mutant analysis by direct DNA sequencing. (A) Location of the novel mutations in the CYP11B2 gene. (B) The base changes from A to G point mutation at bp 3722(corresponding to c.780G > A) in exon 4, leads to a change of tryptophan 260 to a termination codon (p.W260X). The patient is homozygous for mutation p.W260X. (C) Thepatient is a compound heterozygous: insertion of T between position bp 3560 and 3561 (g.3560_3561insT; c.618_619insT) leads to a frameshift and stop codon at amino acidposition 258 in exon 4 (p.G206WfsX51), and deletion C at position bp 6443 (g.6443delC; c.1489delC) leads to a frameshift and additional 169 amino acids into CYP11B2protein (p.L496SfsX169). (D) The base changes from G to C at position bp 4173 (corresponding to c.945C > G) and T to C at position bp 5160 (corresponding to c.1120C > T)lead to the substitution of serine by arginine at amino acid position 315 and arginine by tryptophan at amino acid position 374, respectively. DNA sequencing of a healthycontrol shows the wild-type (WT) sequence, the father is heterozygous for p.S315R mutation, the mother is heterozygous for p.R374W mutation and the patients areheterozygous for p.S315R and p.R374W mutations.

360 H.-H. Nguyen et al. / Molecular Genetics and Metabolism 100 (2010) 357–364

Two siblings (patients 3 and 4) were found to be compound het-erozygous for the novel point mutations G to C at position bp 4173(corresponding to c.945C > G) in exon 5 (the paternal allele) and Tto C at position bp 5160 (corresponding to c.1120C > T) in exon 6(the maternal allele) of the CYP11B2 gene (Genbank Number:D13752). The g.4173C > G mutation leads to the substitution ofserine by arginine at amino acid position 315 of aldosterone syn-thase (p.S315R). The g.5160C > T mutation results in a change fromarginine by tryptophan at amino acid position 374 (p.R374W). Themutations were not found in two alleles of their healthy brother(see Fig. 1A and D).

In vitro expression and assays of enzyme activity

To analyse the enzymatic characteristics of the CYP11B2 mu-tants, as compared to the wild-type proteins, we cotransfectedthe resultant plasmids together with pBAdx4 into COS-1 cells.The co-expression of bAdx was demonstrated to be useful inincreasing the activity of human CYP11B2 in the COS-1 cell–cellsystem, as well as the sensitivity of the test system [5,15–18].

The expression of aldosterone synthase and the two single mu-tants was detected as a band of the correct size of approximately48.5 kDa [5,19]. The expression level of the two single mutants

Fig. 2. Western blot analysis of CYP11B2 expression in COS-1 cells. Transfectedcells were lysed after 48-h incubation with medium containing substrate (DOC).The proteins of CYP11B2-WT, p.S315R, p.R374W and Mock (pSVL without CYP11B2cDNA) were separated by SDS/PAGE. After transfer to a nitrocellulose membrane,the specific proteins were detected with an antihuman-CYP11B rabbit antiserumand visualized by ECL Western blot kit.

H.-H. Nguyen et al. / Molecular Genetics and Metabolism 100 (2010) 357–364 361

was similar to the CYP11B2-WT. Thus, Western blot analysis of theCYP11B2-WT and the two mutants (p.S315R, p.R374W) expressedin the cells demonstrated that none of the mutations significantlyaffected the translation efficiency (see Fig. 2).

After the expression of the proteins (p.S315R, p.R374W) wasconfirmed by Western blot analysis, the transfected COS-1 cellswere incubated with DOC as substrate to check the ability of theexpressed enzymes to convert the steroids. Steroids were extractedfrom medium after 72 h of incubation and separated by thin layerchromatography. The profiles of steroid metabolites from cellsexpressing the CYP11B2-WT enzyme contained all expected reac-tion products (corticosterone, 18-hydroxycorticosterone, and aldo-sterone). In contrast to this, the profiles of cells expressing thesingle CYP11B2 mutant p.R374W were similar to the profiles ofMock transfected cells (vector pSVL as a negative control), whichindicated that none of the steroid products was produced. In thesteroid pattern of cells expressing the single mutant p.S315R only9% of the substrate DOC was converted to the product corticoste-rone (B) but no further oxidation products were produced. Thissuggested that 11b-hydroxylation capacity of mutant p.S315Rwas dramatically impaired and 18-hydroxylation and 18-oxidationactivity was abolished. Thus, the expression studies showed thatthe two single mutants were not capable of converting DOC toaldosterone (see Fig. 3).

Cell culture experiments have not been performed for muta-tions p.W260X (truncated protein), p.G206WfsX51 (truncated

Fig. 3. Enzyme activities of aldosterone synthase in COS-1 cells. COS-1 cells weretransfected with 1.5 lg of pBAdx (bovine adrenodoxin) and 1.5 lg of pSVLcontaining the cDNA of CYP11B2-WT or the mutant constructs encoding thesubstitution p.S315R and p.R374 W or the vector pSVL without CYP11B2 cDNA as anegative control (Mock). The transfected cells were incubated with substrate DOC(2 lM DOC and 5 nCi of 11-[14C] deoxycorticosterone). Steroid patterns of DOCconversion are given as mean ± SEM of four similar independent experimentsperformed in duplicate. The amounts of the substrate, the intermediates cortico-sterone (B) and 18-hydroxycorticosterone (18-OH-B) and the final product aldo-sterone (Aldo) are presented as percentages of total radioactivity.

protein), and p.L496SfsX169 (additional 169 amino acids). Thesemutations lead in all probability to non-functional proteins.

Discussion

Clinically, all four patients exhibited salt wasting and growthfailure as a child during infancy. Biologically, high excretion ratesof corticosterone and 11-dehydrocorticosterone and decreasedmetabolites of 18-hydroxycorticosterone and aldosterone in uri-nary samples were observed by GC–MS steroid profiling. This isthe typical clinical and metabolic presentation of a ASD type I be-cause functionally impaired CYP11B2 does not convert corticoste-rone (B) efficiently to the 18-oxygenated metabolites and, as aconsequence, the B-derived metabolites, tetrahydro-11-dehydro-corticosterone (THA) and tetrahydrocorticosterone (THB) increasein the urine. B can, however, still be produced from 11-deoxycorti-costerone by the 93% identical mitochondrial isoenzyme CYP11B1[12]. However, CYP11B1 only catalyzes the 18-hydroxylation of Bto form 18-OH-B to a very small extent [20,21] and cannot furtheroxidize 18-OH-B to aldosterone. This leads to an accumulation ofderivatives of corticosterone.

In the present study, we describe five novel mutations:p.W260X, p.G206WfsX51, p.L496SfsX169, p.S315R, and p.R374Wwithin the CYP11B2 gene of patients suffering from ASD type Isymptoms. The mutants p.W260X from patient 1, p.G206WfsX51and p.L496SfsX169 from patient 2, were presumably non-func-tional proteins. Two mutants, p.W260X, p.G206WfsX51, are trun-cated and lack about half of the enzyme structure. Since theessential for the heme coordination cysteine of CYP11B2 is locatedat amino acid position 450, these proteins lack the heme and arethus functionally inactive. The mutant p.L496SfsX169 is a frame-shift, a non-stop mutation at the normal stop codon 504 and addi-tional 169 amino acids will be attached to the wild-type proteinstructure, which should lead to a non-functional enzyme. Together,these mutations could explain the clinical phenotype of patients 1and 2 causing ASD type I [22–26]. The missing or nearly missingenzyme activities of the two missense mutants (p.R374W andp.S315R) from two siblings (patients 3 and 4) analyzed in the cellculture are also consistent with the clinical phenotype of ASD typeI and comparable to data with other missense mutations causingASD type I [5,21,27,28].

The protein expression patterns of p.S315R and p.R374W mu-tants analyzed by Western blot (see Fig. 2) showed that the twomutants were expressed in COS-1 cells. When testing the CYP11B2activity using the substrate DOC in COS-1 cells, it was shown thatthe replacement p.R374W led to impaired activity of CYP11B2; andthe replacement p.S315R significantly reduced the production of Band abolished the formation of 18-OH-B and Aldo. This means thatthe patterns of activities of the mutant enzymes coincide with ASDtype I and, the molecular genetic results can explain clinical data,which also indicated no 18-oxygenated metabolites. The detectedB metabolites in urine samples were formed most probably dueto the activity of the isoenzyme CYP11B1.

To correlate the impaired activities of the amino acid replace-ments with structural data of CYP11B2, we performed alignmentswith other members of the P450 family and constructed com-puter models of the mutated CYP11B2 proteins. When consider-ing the alignment of amino acid sequences (see Fig. 4) it can beseen that the arginine 374 residue is highly conserved in CYP11B1and CYP11B2 enzymes of human, mouse, bovine, and pig, as wellas in the microbial enzymes CYP108 and CYP101, and humanCYP21A2. This indicates a functionally or structurally importantrole of R374 in cytochromes P450. R374 of CYP11B2 correspondto natural mutant R354H, R354C [29,30] of CYP21A2 whichresulted in a complete loss of enzyme activity consistent with

Fig. 4. Multiple sequence alignment of CYP11B1 and CYP11B2 of human (h), bovine (b), mouse (m), rat (r) and pig (p) with CYP108 and CYP101 of bacteria. The alignment wasdone using the program CLUSTALW 1.8. The underlined part in the CYP11B2 sequence corresponds to the helix. The white residues indicate mutations which were detected inpatients. The grey areas indicate SRS (substrate recognition site) in CYP2 family members identified by Gotoh [34].

362 H.-H. Nguyen et al. / Molecular Genetics and Metabolism 100 (2010) 357–364

the patients’ saltwasting phenotypes. In addition, residue R374 isinvariant in the K-helix Glu-X-X-Arg motif, which may be in-volved in stabilizing the core structure of the enzyme. The residueis located on the proximal side of the heme (the putative redoxpartner binding site) [31] and is also involved in forming the sub-strate recognition site 5 (SRS 5) (see Fig. 4). Hasemann [32] indi-cated that a variable-length of strand 1 of the b-sheet 6 segmentis anchored at one end by the K-helix Glu-X-X-Arg motif and atthe other end by the conserved arginine/histidine propionate li-gand in strand 4 of the b-sheet 1. Natural mutants occurring inthe Glu-X-X-Arg motif have been shown to have abolishedCYP11B1 activity [33].

Residue S315 in CYP11B2 has a non-polar character and plays arole in stabilizing the interaction of helix I with adjacent heliceswhich are in close relation to the heme cofactor [13,33,34]. TheI-helix of CYP11B1 and CYP11B2 also contains many hydrophobicamino acids which are the putative active site of CYP11B1 andCYP11B2 [13]. In addition, it has been demonstrated by changingCYP11B2 to CYP11B1 corresponding residues and vice versa[16,17] that the helix I of CYP11B1 and CYP11B2 is responsiblefor substrate specificity. The residue S315 is located within thecentral I-helix which is involved in heme binding and forms thesubstrate recognition site SRS4 (see Fig. 4) [13,33,34]. The residueS315 is highly conserved in human, mouse, bovine, and ratCYP11B1 and CYP11B2. This indicates a functionally or structurallyimportant role of S315 in cytochromes P450.

Hydrogen bonds are important landmarks in protein conforma-tion. They contribute to the stability of secondary structures and of

interactions between specific side chains and main chain polaratoms. Thus, the change of the hydrogen bond network of CYP11B2can influence substrate recognition and protein stability. To inves-tigate whether the two mutants (p.S315R, p.R374W) affect thestructure of CYP11B2, the changes of those residues in CYP11B2were modeled by using the spdbv program (http://www.exp-asy.org/spdbv/) [14]. When the arginine residue at position 374was substituted by tryptophan, twelve hydrogen bonds were chan-ged (see Fig. 5A). Also, two hydrogen bonds were changed whenserine 315 was substituted by arginine. Thus, the replacementp.R374W and p.S315R (see Fig. 5A and B) will result in a newhydrogen bond network of CYP11B2.

Thus, a point mutation (p.S315R) within the I-helix and theother point mutation (p.R374W) of the Glu-X-X-Arg motif and anew hydrogen bond network caused by these mutations ofCYP11B2 might generate deleterious conformational changes ofthe enzyme and of the the substrate recognition site leading toloose the enzymatic activity of CYP11B2.

In conclusion, mutations p.W260X, p.G206WfsX51,p.L496SfsX169, p.S315R, and p.R374W have been detected in theCYP11B2 genes of patients suffering from ASD type I symptoms.The expression of the two single mutant proteins in the COS-1 cellsindicated that mutation p.R374W abolished enzyme activity andmutation p.S315R decreased 11-hydroxylation activity and im-paired 18-hydroxylation and 18-oxidation activity. Moreover,alignment studies and our computer models indicated changes inthe protein structure of the mutants that could provide a rationalexplanation for the reduced or missing activities.

Fig. 5. Hydrogen bond network around residue 315 and 374 of different CYP11B2 species. The criteria for the hydrogen bonds were a minimum distance between 1.2 Å and amaximum distance of 2.8 Å between acceptor and donor as well as a minimum angle of 120� between acceptor and donor. Blue dashed lines: hydrogen bonds of CYP11B2. Thewhite arrows show that hydrogen bonds were changed. (A and B) Twelve white arrows show that hydrogen bonds were changed when arginine 374 was substituted bytrytophan. (A) Hydrogen bonds of WT (hydrogen bond around R374). (B) Hydrogen bonds of mutant (hydrogen bond around W374). (C and D) two white arrows show thathydrogen bonds were changed when serine 315 was substituted by arginine. C, hydrogen bonds of WT (hydrogen bond around S315). (B) Hydrogen bonds of mutant(hydrogen bond around R315).

H.-H. Nguyen et al. / Molecular Genetics and Metabolism 100 (2010) 357–364 363

Disclosure

The authors have nothing to disclose.

Acknowledgments

This work was supported by a grant of the Fonds der Chemis-chen Industrie to R.B. and a Ph.D fellowship of the VietnameseMinistry of Education and Training (MOET) to H.-H.N. The authorswould like to thank Dr. Norio Kagawa for proofreading of the man-uscript. The study was further supported by a grant from the Euro-pean Union Programme PERFECT-QLG1-CT-2002-90358 to E.M.M.,M.F.H., and S.A.W.

Reference

[1] K.M. Curnow, M.T. Tusie-Luna, L. Pascoe, R. Natarajan, J.L. Gu, J.L. Nadler, P.C.White, The product of the CYP11B2 gene is required for aldosteronebiosynthesis in the human adrenal cortex, Mol. Endocrinol. 5 (1991) 1513–1522.

[2] M. Bureik, M. Lisurek, R. Bernhardt, The human steroid hydroxylases CYP11B1and CYP11B2, Biol. Chem. 383 (2002) 1537–1551.

[3] M. Lisurek, R. Bernhardt, Modulation of aldosterone and cortisol synthesis onthe molecular level, Mol. Cell. Endocrinol. 215 (2004) 149–159.

[4] S. Ulick, J.Z. Wang, D.H. Morton, The biochemical phenotypes of two inbornerrors in the biosynthesis of aldosterone, J. Clin. Endocrinol. Metab. 74 (1992)1415–1420.

[5] H.H. Nguyen, F. Hannemann, M.F. Hartmann, S.A. Wudy, R. Bernhardt,Aldosterone synthase deficiency caused by a homozygous L451F mutation inthe CYP11B2 gene, Mol. Genet. Metab. 93 (2008) 458–467.

[6] G. Zhang, H. Rodriguez, C.E. Fardella, D.A. Harris, W.L. Miller, Mutation T318Min the CYP11B2 gene encoding P450c11AS (aldosterone synthase) causescorticosterone methyl oxidase II deficiency, Am. J. Hum. Genet. 57 (1995)1037–1043.

[7] M. Peter, J.M. Dubuis, W.G. Sippell, Disorders of the aldosterone synthase andsteroid 11beta-hydroxylase deficiencies, Horm. Res. 51 (1999) 211–222.

[8] M. Peter, C.J. Partsch, W.G. Sippell, Multisteroid analysis in children withterminal aldosterone biosynthesis defects, J. Clin. Endocrinol. Metab. 80 (1995)1622–1627.

[9] S. Ulick, Correction of the nomenclature and mechanism of thealdosterone biosynthetic defects, J. Clin. Endocrinol. Metab. 81 (1996)1299–1300.

[10] S.A. Wudy, M.F. Hartmann, Gas chromatography-mass spectrometry profilingof steroids in times of molecular biology, Horm. Metab. Res. 36 (2004) 415–422.

364 H.-H. Nguyen et al. / Molecular Genetics and Metabolism 100 (2010) 357–364

[11] T. Kawamoto, Y. Mitsuuchi, T. Ohnishi, Y. Ichikawa, Y. Yokoyama, H. Sumimoto,K. Toda, K. Miyahara, I. Kuribayashi, K. Nakao, And e. al., Cloning andexpression of a cDNA for human cytochrome P-450aldo as related to primaryaldosteronism, Biochem. Biophys. Res. Commun. 173 (1990) 309–316.

[12] E. Mornet, J. Dupont, A. Vitek, P.C. White, Characterization of two genesencoding human steroid 11 beta-hydroxylase (P-450(11) beta), J. Biol. Chem.264 (1989) 20961–20967.

[13] N.V. Belkina, M. Lisurek, A.S. Ivanov, R. Bernhardt, Modelling of three-dimensional structures of cytochromes P450 11B1 and 11B2, J. Inorg.Biochem. 87 (2001) 197–207.

[14] N. Guex, M.C. Peitsch, SWISS-MODEL and the Swiss-PdvViewer: anenvironment for comparative protein modeling, Electrophoresis 18 (1997)2714–2723.

[15] S. Bechtel, N. Belkina, R. Bernhardt, The effect of amino-acid substitutionsI112P, D147E and K152N in CYP11B2 on the catalytic activities of the enzyme,Eur. J. Biochem. 269 (2002) 1118–1127.

[16] B. Bottner, K. Denner, R. Bernhardt, Conferring aldosterone synthesis to humanCYP11B1 by replacing key amino acid residues with CYP11B2-specific ones,Eur. J. Biochem. 252 (1998) 458–466.

[17] B. Bottner, H. Schrauber, R. Bernhardt, Engineering a mineralocorticoid- to aglucocorticoid-synthesizing cytochrome P450, J. Biol. Chem. 271 (1996) 8028–8033.

[18] P.R. Cao, R. Bernhardt, Modulation of aldosterone biosynthesis by adrenodoxinmutants with different electron transport efficiencies, Eur. J. Biochem. 265(1999) 152–159.

[19] K. Lovas, I. McFarlane, H.H. Nguyen, S. Curran, J. Schwabe, D. Halsall, R.Bernhardt, A.M. Wallace, V.K. Chatterjee, A novel CYP11B2 gene mutation in anAsian family with aldosterone synthase deficiency, J. Clin. Endocrinol. Metab.94 (2009) 914–919.

[20] L. Pascoe, K.M. Curnow, L. Slutsker, A. Rosler, P.C. White, Mutations in thehuman CYP11B2 (aldosterone synthase) gene causing corticosteronemethyloxidase II deficiency, Proc. Natl. Acad. Sci. USA 89 (1992) 4996–5000.

[21] S. Portrat-Doyen, J. Tourniaire, O. Richard, P. Mulatero, B. Aupetit-Faisant, K.M.Curnow, L. Pascoe, Y. Morel, Isolated aldosterone synthase deficiency causedby simultaneous E198D and V386A mutations in the CYP11B2 gene, J. Clin.Endocrinol. Metab. 83 (1998) 4156–4161.

[22] T.A. Williams, P. Mulatero, M. Bosio, S. Lewicka, M. Palermo, F. Veglio, D.Armanini, A particular phenotype in a girl with aldosterone synthasedeficiency, J. Clin. Endocrinol. Metab. 89 (2004) 3168–3172.

[23] Y. Mitsuuchi, T. Kawamoto, K. Miyahara, S. Ulick, D.H. Morton, Y. Naiki, I.Kuribayashi, K. Toda, T. Hara, T. Orii, And et al., Congenitally defective

aldosterone biosynthesis in humans: inactivation of the P-450C18 gene(CYP11B2) due to nucleotide deletion in CMO I deficient patients, Biochem.Biophys. Res. Commun. 190 (1993) 864–869.

[24] M. Peter, W. Nikischin, P. Heinz-Erian, W. Fussenegger, K. Kapelari, W.G.Sippell, Homozygous deletion of arginine-173 in the CYP11B2 gene in a girlwith congenital hypoaldosteronism. Corticosterone methyloxidase deficiencytype II, Horm. Res. 50 (1998) 222–225.

[25] K.M. Kayes-Wandover, G.M. Tannin, D. Shulman, D. Peled, K.L. Jones, L.Karaviti, P.C. White, Congenital hyperreninemic hypoaldosteronism unlinkedto the aldosterone synthase (CYP11B2) gene, J. Clin. Endocrinol. Metab. 86(2001) 5379–5382.

[26] K.M. Kayes-Wandover, R.E. Schindler, H.C. Taylor, P.C. White, Type 1aldosterone synthase deficiency presenting in a middle-aged man, J. Clin.Endocrinol. Metab. 86 (2001) 1008–1012.

[27] S. Nomoto, G. Massa, F. Mitani, Y. Ishimura, K. Miyahara, K. Toda, I. Nagano, T.Yamashiro, S. Ogoshi, J. Fukata, S. Onishi, K. Hashimoto, Y. Doi, H. Imura, Y.Shizuta, CMO I deficiency caused by a point mutation in exon 8 of the humanCYP11B2 gene encoding steroid 18-hydroxylase (P450C18), Biochem. Biophys.Res. Commun. 234 (1997) 382–385.

[28] S. Geley, K. Johrer, M. Peter, K. Denner, R. Bernhardt, W.G. Sippell, R. Kofler,Amino acid substitution R384P in aldosterone synthase causes corticosteronemethyloxidase type I deficiency, J. Clin. Endocrinol. Metab. 80 (1995) 424–429.

[29] N. Krone, A. Braun, A.A. Roscher, D. Knorr, H.P. Schwarz, Predicting phenotypein steroid 21-hydroxylase deficiency? Comprehensive genotyping in 155unrelated, well defined patients from southern Germany, J. Clin. Endocrinol.Metab. 85 (2000) 1059–1065.

[30] B.S. Nunez, M.N. Lobato, P.C. White, A. Meseguer, Functional analysis of fourCYP21 mutations from Spanish patients with congenital adrenal hyperplasia,Biochem. Biophys. Res. Commun. 262 (1999) 635–637.

[31] J.A. Peterson, S.E. Graham, A close family resemblance: the importance ofstructure in understanding cytochromes P450, Structures 6 (1998) 1079–1085.

[32] C.A. Hasemann, R.G. Kurumbail, S.S. Boddupalli, J.A. Peterson, J. Deisenhofer,Structure and function of cytochromes P450: a comparative analysis of threecrystal structures, Structures 3 (1995) 41–62.

[33] J. Mestres, Structure conservation in cytochromes P450, Proteins 58 (2005)596–609.

[34] O. Gotoh, Substrate recognition sites in cytochrome P450 family 2 (CYP2)proteins inferred from comparative analyses of amino acid and codingnucleotide sequences, J. Biol. Chem. 267 (1992) 83–90.