Clinical, genetic, and structural basis of apparentmineralocorticoid excess due to 11β-hydroxysteroiddehydrogenase type 2 deficiencyMabel Yaua,1, Shozeb Haiderb,1, Ahmed Khattaba, Chen Lingb, Mehr Mathewc, Samir Zaidid, Madison Blochc,Monica Patelc, Sinead Ewertb, Wafa Abdullahe, Aysenur Toygarc, Vitalii Mudryib, Maryam Al Badie, Mouch Alzubdib,Robert C. Wilsonf, Hanan Said Al Azkawie, Hatice Nur Ozdemirc, Wahid Abu-Amerc, Jozef Hertecantg,Maryam Razzaghy-Azarh, John W. Funderi, Aisha Al Senanie, Li Sunc, Se-Min Kimc, Tony Yuenc,2, Mone Zaidic,2,3,and Maria I. Newa,2,3

aDepartment of Pediatrics, Icahn School of Medicine at Mount Sinai, New York, NY 10029; bDepartment of Pharmaceutical and Biological Chemistry,University College London School of Pharmacy, London WC1N 1AX, United Kingdom; cDepartment of Medicine, Icahn School of Medicine at Mount Sinai,New York, NY 10029; dDepartment of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114; eDepartment of Pediatrics,Royal Hospital, Muscat 111, Oman; fDepartment of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425;gDepartment of Pediatrics, Tawam Hospital, Abu Dhabi 15258, United Arab Emirates; hMetabolic Disorders Research Center, Tehran University of MedicalSciences, Tehran 1417613151, Iran; and iDepartment of Medicine, Monash University, Clayton, VIC 3800, Australia

Contributed by Maria I. New, November 14, 2017 (sent for review September 21, 2017; reviewed by Vidya Chandran Darbari, Lucia Ghizzoni, and Carlos Isales)

Mutations in 11β-hydroxysteroid dehydrogenase type 2 gene(HSD11B2) cause an extraordinarily rare autosomal recessive dis-order, apparent mineralocorticoid excess (AME). AME is a form oflow renin hypertension that is potentially fatal if untreated. Mu-tations in the HSD11B2 gene result either in severe AME or amilder phenotype (type 2 AME). To date, ∼40 causative mutationshave been identified. As part of the International Consortium forRare Steroid Disorders, we have diagnosed and followed the larg-est single worldwide cohort of 36 AME patients. Here, we presentthe genotype and clinical phenotype of these patients, promi-nently from consanguineous marriages in the Middle East, whodisplay profound hypertension and hypokalemic alkalosis. To cor-relate mutations with phenotypic severity, we constructed a com-putational model of the HSD11B2 protein. Having used a similarstrategy for the in silico evaluation of 150 mutations of CYP21A2,the disease-causing gene in congenital adrenal hyperplasia, we nowprovide a full structural explanation for the clinical severity of AMEresulting from each known HSD11B2 missense mutation. We findthat mutations that allow the formation of an inactive dimer, altersubstrate/coenzyme binding, or impair structural stability of HSD11B2yield severe AME. In contrast, mutations that cause an indirect dis-ruption of substrate binding or mildly alter intramolecular interac-tions result in type 2 AME. A simple in silico evaluation of novelmissense mutations could help predict the often-diverse phenotypesof an extremely rare monogenic disorder.

in silico molecular modeling | molecular dynamics | hypertension |congenital adrenal hyperplasia

Apparent mineralocorticoid excess (AME) is an ultrarareautosomal recessive disorder caused by a mutation in the

11β-hydroxysteroid dehydrogenase type 2 gene (HSD11B2), whichleads to a deficiency in the HSD11B2 enzyme. HSD11B2, amember of the short-chain alcohol dehydrogenase family, cat-alyzes the NAD+-dependent dehydrogenation of cortisol. In thekidney, HSD11B2 converts cortisol to inactive cortisone. Thismechanism protects the mineralocorticoid receptor from cor-tisol action, notwithstanding an equal in vitro affinity of themineralocorticoid receptor for cortisol and aldosterone, andthe 100- to 1,000-fold higher circulating levels of cortisol (1, 2).In 1977, we described the biochemical phenotype of a patient

with AME (3). We found that, in the absence of HSD11B2, themineralocorticoid receptor was occupied and stimulated by ex-cess cortisol, leading to hypertension without elevated aldoste-rone or renin levels. AME patients display low birth weight,failure to thrive, low-renin hypertension, and hypokalemia. De-

fective metabolism of cortisol to cortisone leads to the pathogno-monic elevation in the ratio of urinary tetrahydrocortisol (THF) plusallo-THF to tetrahydrocortisone (THE) [(THF+alloTHF)/THE].In a disorder in which one of the cardinal clinical features is hy-

pertension, insidious end-organ damage in renal, neurological,neuromuscular, cardiovascular, and ocular systems may arise at anearly age from chronic elevation of blood pressure or throughmetabolic alkalosis and severe hypokalemia, leading to renal dis-ease. Cardiac failure resulting from hypokalemia, hypertensive cri-ses, stroke, cardiomegaly, hypertensive retinopathy, and kidneyfailure are commonly reported complications of this disease. All ofthe AME patients reported until 1998 had the characteristic signs ofsevere AME. In 1998, the first patient with a mild form of AMEwho proved to have mutations that resulted in an HSD11B2 proteinwith attenuated activity was reported (4). Only three clinical featuresof the mild AME are the same as in severe AME: low renin, lowaldosterone, and elevated (THF+alloTHF)/THE ratio.Following our initial description of the disease-causing muta-

tion in the HSD11B2 gene (5, 6), around 40 additional mutations

Significance

Apparent mineralocorticoid excess, a rare autosomal recessivedisorder characterized by low renin hypertension, may displaya severe or mild phenotype in patients. The variability in clin-ical presentation stems from different extents of impairment ofthe 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) en-zyme arising from distinct mutations in the encoding gene. Thecomputational model of the HSD11B2 protein that we con-structed here will be useful in predicting disease severity fornewly reported missense mutations in this gene.

Author contributions: M.Y., S.H., T.Y., M.Z., and M.I.N. designed research; M.Y., S.H., A.K.,C.L., M.M., S.Z., S.E., W.A., V.M., M.A.B., M.A., R.C.W., H.S.A.A., and T.Y. performed re-search; M.Y., S.H., A.K., C.L., M.M., S.Z., M.B., M.P., S.E., W.A., A.T., V.M., M.A.B., M.A.,R.C.W., H.S.A.A., H.N.O., W.A.-A., J.H., M.R.-A., J.W.F., A.A.S., L.S., S.-M.K., T.Y., M.Z., andM.I.N. analyzed data; and M.Y., S.H., T.Y., M.Z., and M.I.N. wrote the paper.

Reviewers: V.C.D., QueenMary University of London; L.G., Universita degli Studi di Torino;and C.I., Augusta University.

The authors declare no conflict of interest.

Published under the PNAS license.1M.Y. and S.H. contributed equally to this work.2T.Y., M.Z., and M.I.N. contributed equally to this work.3To whom correspondence may be addressed. Email: mone.zaidi@mssm.edu or maria.new@mssm.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1716621115/-/DCSupplemental.

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have been described in fewer than 70 patients worldwide (Fig. 1A).Despite the small number of available patients, previous studieshave documented correlations between genotype and phenotype (7,8). For example, mutations that severely attenuate HSD11B2 ac-tivity, as assessed in vitro, cause severe disease, including early fa-tality, while mutations associated with mildly attenuated enzymeactivity give rise to type 2 AME (a milder phenotype of AME), oftenwith a late-onset presentation of mild hypertension.We recently published evidence for genotype–phenotype con-

cordance in 1,507 families with congenital adrenal hyperplasia(CAH) due to 21-hydroxylase deficiency and 220 CAH patientswith 11β-hydroxylase deficiency (9–11). In both studies, we used insilico dynamic modeling to define structural changes that eachCYP21A2 and CYP11B1 mutation induces in the respective en-zymes, namely 21-hydroxylase and 11β-hydroxylase. By correlatingthese changes with clinical phenotype, we defined structural de-rangements underpinning the clinical variants of CAH, namelysalt-wasting, simple virilizing, and nonclassic CAH (9). A previousattempt with AME mutations sought to correlate structure withfunction using a dimeric quaternary structure (12). This structure–function relationship is in contradiction with experimental evi-dence suggesting that the HSD11B2 monomer is, in fact, the ac-tive form of the enzyme (13).In this study, we provide extensive data on the demographics,

genotype, phenotype, and hormonal profile of 36 AME patientsfrom our International Consortium for Rare Steroid Disorders,together with long-term outcomes in a subset. To evaluate ge-notype–structure–phenotype correlations, we constructed a 3Dhomology model of HSD11B2, to which we mapped all of theknown HSD11B2 missense mutations, and additionally carriedout molecular dynamic (MD) simulations to understand alter-ations in structural flexibility. Thus, as with CAH, we present ourprediction of disease severity based on mutation-inducedchanges in HSD11B2 structure. This in silico strategy, we be-lieve, will facilitate the prediction of AME phenotype when newmutations are discovered.

ResultsWe report the demographics and clinical phenotype of 36 ge-netically confirmed AME patients from the International Con-sortium of Rare Steroid Disorders. The majority of patients wereof Omani (22 of 36) and Persian (6 of 36) descent, with 75%being from consanguineous marriages, mainly within Oman (Fig.1B and Table S1). The median age of AME diagnosis was 4.6 y(range: 0.1–15) and the cohort was distributed equally for eithersex. The majority of patients (86%) were born at term, with 76%being born small for gestational age. Fig. 1A shows that, in ourcohort, there were six missense, one insertion, two deletion, andthree frameshift mutations, and two indels. Expected from thepathophysiology of AME, the presenting mean arterial pressurewas elevated above the 90th percentile for all subjects (Fig. 1C).The average serum potassium level at diagnosis was low at2.72 mmol/L (range: 1.5–4.1 mmol/L), while serum bicarbonatewas high at 29.5 mmol/L (range: 20–38 mmol/L) (Fig. 1 D andE). Serum aldosterone levels were expectedly low (Fig. 1F), withtwo patients with elevated levels due to unclear reasons. Addi-tionally, renin levels were low, except for two patients (10.4 and14 ng/mL/h) that were on therapy (Table S1). In a subset of29 patients, the average (THF + alloTHF)/THE ratio, repre-senting the major urinary metabolites of cortisol and cortisone,was significantly elevated at 19 (range: 3–55, normal: 1.0) (Fig.1G). Of note is that 27 of 36 (75%) patients also presented withnephrocalcinosis. The nephrocalcinosis may arise from chroniclong-standing hypokalemia as noted, for example, in Barttersyndrome type III (14).To understand whether the clinical phenotype of HSD11B2

deficiency could be predicted by examining changes in enzymestructure induced by a given HSD11B2 mutation, we performed

in silico analytics using three estrogenic HSD17B1 [PDB IDcodes 1IOL (15), 1JTV (16), 1FDV (17)] that exhibited a 27%sequence similarity with HSD11B2 over 284 amino acids (Fig.S1A). The human HSD11B2 model exhibited a characteristicconserved NAD-binding Rossmann fold motif (Fig. S1B) with anadjacent substrate-binding site (Fig. S1 C and D). The 500-nsMD simulations of the monomer and dimeric forms of HSD11B2indicated a stable model with stably folded protein (Fig. S1 E–H).We used Internal Coordinate Modeling (ICM) software (www.molsoft.com) (18) to determine ΔΔG of missense mutationsfound in AME patients or experimentally engineered mutations.All mutations (Fig. 2A) showed an increase in ΔΔG values, sug-gesting the loss of protein stability and function (Fig. 2B).HSD11B2 exists as a homodimer in its inactive state (13). Four

residues—namely, R186, E190, A237, and R336—lie at the di-mer interface and could therefore interfere with dimer formation(Fig. 2C). Residues R186 and E190 are positioned at the dimerinterface on the α4 helix. An intersubunit ion-pair interaction(R186–E190) is formed as a result of the twofold inverted sym-metry of the adjacent subunit (Fig. 2D), akin to the R112–E120 ion pair in HSD17B1 (15–17). Importantly, this interactionwas found to be stable, and was maintained throughout thecourse of 500-ns MD simulations (Fig. 2 E–G). Furthermore, theinteraction was found to stabilize the α4 helix and maintainsflexibility around the coenzyme-binding site. Thus, the R186Cmutation results in the loss of intersubunit ion pair and pushesthe equilibrium toward formation of an active monomeric en-zyme (Fig. 2H). It also prevents the formation of the intrahelicalion-pair interaction and thus permits the destabilization ofstructural elements around the coenzyme-binding site.There are several other disruptive mutations at this dimer

interface involving residues A237, D244, L251, and D176. Theresidue A237 is surrounded by L241 and V239 from bothHSD11B2 subunits. Its mutation to Val enhances hydrophobicityof this cluster and strengthens the enzyme’s inactive dimeric state(Fig. 2I). The residue D244 not only creates an intrasubunit saltbridge with R336 that holds the α5 helix and β7 sheets together,but also hydrogen bond interactions with R360 from the adjacentsubunit (Fig. 2J). Its mutation to Asn decreases the strength ofthe R360 interaction, leading to the loss of the salt bridge withR366; this, in turn, impairs protein stability. Similarly, mutationof a hydrophobic L251 side chain to hydroxyl group of Ser in-creases polarity and forms a hydrogen bond with the positivelycharged side chain groups of R361; this enhances dimer binding(Fig. 2K). Finally, the mutation of D176 to Asn creates a hy-drogen bond between asparagine and T200, thus enhancing theinactive dimer state (Fig. 2L).Mutations of HSD11B2 residues which form the substrate or

coenzyme (NAD+) binding pocket have been shown in in vitrostudies to eliminate enzyme activity (19) and cause severe AME.For example, Y226 is located in the substrate-binding pocket(Fig. 3A). The aromatic phenol side chain forms hydrophobicinteractions with the aromatic ring of the substrate. The Y226Nmutation replaces this residue with a nonaromatic residue,resulting in the loss of hydrophobic stacking, which interfereswith substrate binding (Fig. 3A). Furthermore, this mutationchanges the side chain from being hydrophobic to hydrophilic,which is unfavorable for the binding of a hydrophobic substrate.The A221V mutation replaces the short side chain with a slightlybulkier side chain of Val, thus interfering with substrate bindingby reducing the volume of the pocket, while the A221G mutationdecreases hydrophobicity and introduces flexibility around therigid binding pocket (Fig. 3B).Our model shows that the polar 17-OH group in cortisol is

tucked in a hydrophobic region surrounded by Y226, P227, andL229. This hydroxyl group is absent in corticosterone, whichresults in enhanced hydrophobic interactions and ∼10-foldhigher binding affinity to HSD11B2 compared with cortisol

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(Fig. S2A). Besides the kidney, HSD11B2 is also expressed in theplacenta. Although the mineralocorticoid receptor is not expressedin this tissue, HSD11B2 inactivates cortisol to eliminate its growth-inhibiting and proapoptotic effects during embryonic development.During pregnancy, the high level of progesterone in the maternal

circulation can bind to and modulate HSD11B2 activity. This maytranslate into effects of any loss-of-function mutations that disruptprogesterone binding on birth weight. For example, the A221Vmutation severely affects the binding of progesterone more thancortisol because of the steric clashes between Val and the projecting

S26*R74Gfs*43

G89DE115_L116del

D144V

D176NL179RS180FF185SR186C

R208CR208HR213CA221GA221V

D223NY226NP227L

Y232CY232_T234delA237VD244N

F246+1ntL250R

L250_L251delinsPS

V254V (c.771C>G)V257Sfs*3T267A

R279CL287Cfs*36

Y299del

G305Δ11ntV321_V322insAPV

A328VR337C

R337_Y338delinsH

Y338HQ342Pfs*62E356Vfs*40

R359WL363PF367delR374*L376P

c.664+1G>Ac.664+14C>T

1 2 3 4 5

1 kb16q22

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/L25

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67A

/T26

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/L28

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*36

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299d

elV3

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US (7) - African American (2) - American Indian (1) - Chippewa (1) - Mennonite (1) - Pakistani (1) - Persian (1)

Oman (22) - Omani (22)

Iran (3) - Persian (3)

Australia (2) - Persian (1) - Turkish (1)

Italy (1) - Italian (1)

Canada (1) - Persian (1)

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Fig. 1. Clinical profile for patients with AME due to HSD11B2 deficiency from seven nations of the International Consortium for Rare Steroid Disorders. (A)Structure of the human HSD11B2 gene that contains five exons, showing known mutations and those harbored by 36 patients in this international cohort(bold). (B) Worldwide distribution of our cases of HSD11B2 deficiency. Mean arterial blood pressure (C), and serum potassium (D), bicarbonate (E), and al-dosterone (F) levels in our patient cohort shown as a function of age. Urinary (THF+alloTHF)/THE ratio (G) is shown for each genotype. Bold lines show the90th percentile (C), or upper (E) or lower (D and F) limits of normal ranges. Filled red and blue circles, respectively, represent females and males with overtAME; red open circle represents a female with AME type 2.

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NAD

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R186

R186

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E190

C186

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A237

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L241V239

A237V

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A BDisease Engineered

Mutant G (kcal/mol) Mutant G

(kcal/mol) Mutant G (kcal/mol) Mutant G

(kcal/mol)G89D 0.5 P227L 0.3 D91N 1.6 Y338H 1.8D144V 1.7 Y232C 3.8 E115G 3.3 Y338A 2.7D176N 2.1 A237V 0.8 E115K 2.6 Y338F 0.6L179R 0.5 D244N 1.1 Y232F 1.0 Y339A 3.3S180F 1.2 L250R 2.3 K236R 0.3 Y339H 1.8F185S 1.5 L250P 3.2 R335K 2.2 Y339F 0.8R186C 4.2 L251S 0.4 R335Q 1.6R208H 1.7 T267A 0.4 R335A 2.6R208C 4.8 R279C 4.1 R336K 1.2R213C 4.8 A328V 0.7 R336A 1.3A221V 0.4 R337C 2.8 R336Q 2.4A221G 1.3 Y338H 2.9 R337K 4.1D223N 1.0 R359W 4.0 R337A 2.8Y226N 2.0 R337Q 3.0

Not studied: L363P, L376P

Fig. 2. Disruptive mutations affecting the dimer interface of HSD11B2. (A) Human HSD11B2 dimer model constructed using existing HSD17B1 crystalstructures: 1IOL (cocrystallized with 17β-estradiol) that was the most complete structure, with no missing residues; 1JTV (1.5 Å) that was cocrystallized withtestosterone and exhibited higher resolution over 1IOL (2.3 Å); and 1FDV that was cocrystallized with the coenzyme NAD. The positions of all known mutatedresidues are indicated. (B) ΔΔG values for severe (red) or mild (black) HSD11B2 mutations. (C) At the dimer interface, two R186–E190 ion pairs are formedbetween the twofold inverted symmetry of the adjacent subunits. In case of the monomer (D), an intrahelical ion pair is formed, which is similar to theR112 and E120 salt bridge formed in the HSD17B1 templates. (E) Spatial positions of mutations (blue) mapped on one subunit. The two subunits are distinctlycolored (brown and blue). NAD+ (yellow) and cortisol (green) are illustrated as sticks. The ion-pair interaction at the dimer interface is stable throughout the500-ns simulation (F). However, some fluctuation is observed in the intrahelical interaction due to conformational changes in the monomer (G).(H) R186 makes ion pair interactions with E190 from adjacent subunit. The R186C mutation results in the loss of the ion pair interaction. An intrahelical ionpair formed between R186 and E190 in the monomer is also lost. (I) A237V mutation enhances hydrophobicity at the interface. (J) D244 forms an intrasubunition pair interaction with R336, as well as hydrogen bonds with R360 from the adjacent subunit. The polar uncharged side chain of asparagine in the mutant isunable to provide structural stability. (K) The OH group in the L251S mutation makes a hydrogen bond with R361 from the adjacent subunit, thus enhancingthe interaction at the dimer interface. (L) The hydrogen bond formed between D176N and T200 enhances the inactive dimer state.

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methyl groups in progesterone (Fig. S2B). Very interestingly, wefind that the two patients with the A221V mutations were small forgestational age (Table S1). Likewise, the A221G mutation affectsprogesterone binding indirectly by altering the structure of thebinding site. Similarly, the Y226N mutation is more detrimental toprogesterone binding as it reduces hydrophobic interactions be-tween the progesterone skeleton and the hydroxyphenyl ring oftyrosine (Fig. S2B). In contrast, P227 provides indirect structuralsupport to the binding site, and mutation to Leu has similar affectsfor both cortisol and progesterone (Fig. S2B).

There are four mutations that reside in and disrupt the co-enzyme binding site, thus severely impairing HSD11B2 activity.The side chain of residue L179 is normally positioned spatially ina short turn between the β4 sheet and α4 helix (Fig. 3C). Byallowing interactions with the hydrophobic side chains ofV174 and L282, these interactions make the coenzyme-bindingsite more flexible. Mutation to Arg introduces a guanidiniumside chain, which allows interactions with the carboxyl group sidechain of E172 and the hydroxyl group side chain of T184 (Fig.3C), thus introducing rigidity to the turn and decreasing theflexibility required for optimal enzyme function (7). Situated inthe coenzyme binding pocket, Y232 is a highly conserved residuein the Rossmann fold motif (20), suggesting that its mutation willbe detrimental to enzyme activity (21). The hydroxyl-phenyl sidechain of Y232 forms important hydrogen bonds with N171 andNAD+, holding the coenzyme in correct position for its catalyticfunction (Fig. 3D). This interaction is disrupted in the Y232Cmutation causing severe AME. The importance of this residue inenzyme activity has been independently confirmed by severalengineered mutations (21) (Fig. 3D). In the G89D mutation, thecarboxyl side chain of Asp displays severe steric clashes with theribose sugar moiety of adenosine, which affects the binding ofNAD+ (Fig. 3E). Another engineered mutation K236R leads tothe disruption of NAD binding (Fig. 3F). While retaining theability to interact with the NAD+ through hydrogen bonding, themutation abolishes enzyme activity (21).Impairments in structural stability of HSD11B2 disrupt its

tertiary structure, causing a marked attenuation of enzyme ac-tivity and severe AME. The residue L250 is positioned in theα5 helix, and its side chains sit in a tight hydrophobic pocketsurrounded by the side chains of L204, F246, W253, V255, andV257 (Fig. 4A). The hydrophobic pocket residues are locked byan ion-pair interaction between R208 and E249. Introduction ofa Pro side chain breaks the α5 helix by introducing rigidity. Thispocket is also too tight to accommodate the large, bulky guani-dinium side chain of Arg (Fig. 4A). A subsequent loss of hy-drophobicity in this region affects structural stability. Anotherstable intrahelical ion-pair interaction is formed betweenD144 and R147 (Fig. 4B). This interaction allows the α3 helix topack tightly with the adjacent β-sheets near the NAD+ bindingsite. The D144V mutation results in the loss of this interactionand destabilizes the packing arrangement (Fig. 4B). The hydro-phobic packing is also disrupted in F185S mutation, when thearomatic side chain of phenylalanine, surrounded by hydropho-bic residues of V182, C188, and M189, is replaced with a polarside chain of serine (Fig. 4C). Similarly, the hydrophobic sidechain of L363, surrounded by M347, I350, and F362, is posi-tioned in a helix-turn-helix (Fig. 4D). The L363P mutation dis-rupts the helix and the local structural environment.Furthermore, several mutations can disrupt packing of the

HSD11B2 protein to impair stability through the introduction ofa bulkier residue. Residue A328 forms hydrophobic interactionwith V215, I260, I325, and Y338 (Fig. 4E). By replacing the al-anine with a bulkier Val residue, the A328V mutation causessteric clashes with the side chain of I260 and Y338 from thesurrounding β-sheets (Fig. 4E). Residue S180 is positioned on aloop and its hydroxyl group side chain interacts with the sidechain and backbone nitrogen of T184 (Fig. 4F). This side chainlies within a very constricted space and restricts the accommo-dation of any bulkier residue, which causes steric clashes, in fact,without altering the protein structure. Thus, a Phe side chainwith a bulky aromatic ring, as in S180F mutation, is not toleratedat this position (Fig. 4F).Multiple hydrogen bonds and salt bridges between polar res-

idues stabilize the tertiary structure of the HSD11B2 enzyme.Certain mutations cause a loss of these interactions, resulting ininactive enzyme and severe AME. For example, the salt bridgebetween R208 and E249 is critical (Fig. 4G). Its loss, as in the

A

CortisolY226N

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Fig. 3. Interference with coenzyme or substrate binding to HSD11B2.(A) The tyrosine at position 226 forms the wall of the substrate-binding siteand imparts hydrophobicity. A mutation to N226 results in loss of hydro-phobic stacking rendering it unfavorable for substrate binding. (B) A221Vmutation reduces the volume of the substrate-binding pocket, whileA221G increased flexibility around the binding site. (C ) In the L179R mu-tation, the positively charged guanidinium side chain makes hydrogenbonds with the size chains of T184 and E172, thereby reducing the flexi-bility around the NAD+-binding site. (D) The hydroxy-phenyl side chain ofY232 makes hydrogen bonds with the hydroxyl group of ribose sugar andside chain of N171. These hydrogen bonds are essential, as mutations tophenylalanine, serine, or cysteine are not tolerated. The mutation tophenylalanine removes the hydroxyl group and leads to loss of the hydrogenbond, whereas mutation to serine with a shorter side chain results in increaseddistance of OH group from the NAD+, and again prevents effective hydrogenbonding; both mutations result in inactive enzyme. (E) The carboxyl side chainof aspartic acid in the G89D mutation causes severe steric clashes with the ri-bose sugar moiety of adenosine in NAD and affects coenzyme binding. (F) Thebulkier side chain in K236R mutation decreases the size of the coenzyme-binding pocket, making it unfavorable for optimal NAD+ binding.

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case of the R208C and R208H mutations, destabilizes the pro-tein and results in severe disease (Fig. 4G). Similarly, D223 formsa salt bridge with R337 (Fig. 4H). By replacing this salt bridge witha less-strong bond, a hydrogen bond, the D223N mutationchanges the negatively charged residue to an uncharged polarresidue resulting in a marked attenuation of in vitro activity (22).This also suggests that the salt bridge plays a crucial role inmaintaining HSD11B2 stability and activity. The side chains ofR213 form hydrogen bonds with the backbone atoms of residues

A331 and L329 (Fig. 4I). By diminishing these hydrogen bonds,which help maintain the tertiary structure of the protein, R213Cmutation impairs enzyme stability (Fig. 4I).The Arg/Tyr cluster from residues 335–339 has previously

been implicated in maintaining protein stability, although theprecise mechanism has remained unclear (23). Mutations in-volving these residues have been reported in AME patients, in-cluding R337C and Y338H. Simulations of the monomeric anddimeric HSD11B2 models suggest that residue R337 forms a salt

NAD

V215I260

Y338

A328VI325

L329

L330

M347

I350

L363P

F362

A B

F

V255

V257

L204

R208

W253 E249

F246

L250R/P

D223Y339

R337H/C/K/A

A331

L329R213C

D144VR147

D E

S180F

T184

R208H/C

E249

I

C

G H

V182

C188

M189F185S

J K

R337

Q261

D223N

A328

D327

Y338H/F

Fig. 4. Mutations affecting HSD11B2 stability. (A) L250 is positioned on α5 helix and the side chain is surrounded in a constricted hydrophobic pocket. Amutation to bulky arginine or proline that introduces helix distortion is not tolerated. (B) The D144V mutation results in the loss of the D144–R147 ion-pairinteraction and destabilizes the α3 helix packing arrangement near the NAD-binding site. (C) The polar side chain in F185S mutation disrupts the hydrophobicpacking formed by V182, C188, and M189. (D) The hydrophobic L363 residue is positioned in a helix-turn-helix, surrounded by M347, I350, and F362. Mutationto proline disrupts the helix and the local structural environment. (E) In the A328V mutant, the side chain of valine displays steric clashes within the hy-drophobic pocket. (F) The hydroxyl group side chain of S180 makes interactions with the backbone atoms and the side chain of T184. A phenylalanine sidechain abolishes these interactions and imparts flexibility to the loop. (G) The R208–E249 ion-pair interactions hold α4 and α5 helices together. R208H/Cmutants are unable to form this interaction. (H) The carboxyl side chain of D223 makes hydrogen bonds with the side chains of Q261 and R337. Mutation toAsn disrupts the hydrogen bond with R337. (I) The R213C mutation results in the loss of hydrogen bond interactions formed between the arginine side chainand the backbone atoms of A331 and L329. (J) R337 makes an ion pair interaction with D223 and also a hydrogen bond with Y339. While the lysine side chainretains decreased ability to form hydrogen bond with Y339, a mutation to histidine, cysteine, or alanine completely abolishes the R337–D223 ion-pair in-teraction. (K) The hydroxy-phenyl side chain of Y338 makes hydrogen bond with the backbone atoms of D327 and A328. These interactions maintain thespatial positions of α7 and β7. A mutation to histidine or phenylalanine results in the loss of these interactions and introduces flexibility.

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bridge interaction with D223 and hydrogen bonds with the OHgroup of Y339 (Fig. 4J). Their interactions appear to be importantin maintaining correct folding and stability of the protein. Thus,its mutation to Cys abolishes both the salt bridge and hydrogenbond to destabilize the enzyme and cause severe AME. Ge-netically engineered mutations of this residue further show thatR337K, which maintains polarity and positive charge, reducesenzyme activity by 70%, while R337A, which is similar toR337C in having uncharged and short side chains, inactivatesHSD11B2 completely (23) (Fig. 4J). In the same Arg/Tyrcluster, mutation of Y338 to His inactivates the enzyme tocause severe AME. Our model suggests that this Tyr residueforms hydrophobic interactions with A328 and hydrogen bondswith D327 (Fig. 4K). Both interactions help sustain proteinstability, so that the substitution of Tyr with His, for example,inactivates HSD11B2. This is because, although Y338H retains thehydrogen bonding ability, it has shorter side chains that destabilizethe enzyme. Similarly, the substitution of Tyr with Phe in anengineered Y338F construct is not tolerated. Here, hydropho-bic interactions are retained but hydrogen bonding is abolished(23). Finally, R337 and Y338 in the Arg/Tyr cluster are also highlyconserved across many species; thus, any changes to these res-idues are unlikely to be tolerated (23).Several mutations are reported to cause a milder form of

AME with less-severe clinical and biochemical manifestations.The relevant mutated constructs were shown to have main-tained HSD11B2 activity in vitro (4, 19, 24), consistent with theless-severe clinical phenotype. Structurally, these mutations caneither indirectly disrupt substrate binding (such as the P227Lmutation in one of our patient) or alter protein structure (suchas the R279C and R359W mutations) to attenuate, but notabolish enzyme activity (Fig. 5). Notably, while the P227 residuedoes not lie in a position that directly interacts with the sub-strate, its adjacent positioning to residue Y226 suggests that itmay have a role in substrate binding (Fig. 5A). In fact, this residueforms the “kink” in the loop region, maintaining the conformationof the active site and also holds Y226 in the correct position tooptimally interact with the substrate. Mutation of this residue toLeu (P227L) disrupts the kink and alters the positioning of Y226(Fig. 5A).Two nonconservative mutations thought to disrupt the tertiary

structure of HSD11B2 have been identified in patients withAME type 2. R279 forms a hydrogen bond with the backbone ofN171, which helps maintain and stabilize the local protein en-vironment (Fig. 5B). Replacement of R279 by a residue that doesnot form a hydrogen bond, as in the R279C mutation, leads tomild attenuation, but not total elimination of HSD11B2 activity(24), consistent with a mild clinical phenotype (Fig. 5B). Anothernonconservative mutation, R359W, changes the properties of theside chain from charged to hydrophobic. This alters the local

interaction within the protein structure, in which the positivelycharged side chain of R359 forms a hydrogen bond with thebackbone carbonyl oxygen of I350 (Fig. 5C). A loss of this in-teraction contributes toward the enhanced structural flexibility,causing attenuation of enzyme activity. Of note is that bothR279C and R359W cause minimal disruption to the intra-molecular interactions and occur near the surface of the protein.

DiscussionAME is an extremely rare disorder, with fewer than 100 casesreported to date (25). Most patients are found in populationswhere consanguinity and endogamy are common (26). The lowprevalence of AME and the relatively limited number of muta-tions in the HSD11B2 enzyme have together rendered it difficultto study genotype–phenotype associations. Our consortiumrepresents the largest AME cohort described phenotypically.Of the 16 patients we have reported preliminarily (6, 8, 27–

29), follow-up data were available on 8 males and 6 females (6–26 y of age). Like CAH, height of the patients improved, but fellbelow the target height (height SDS: −1.1 ± 0.6). Cardiovascularcomplications were prominent, with three patients dying fromcardiac arrest at 16, 16, and 17 y of age. Severe hypertension, leftventricular hypertrophy (12 of 16), and hypertensive retinopathy(10 of 16) were prominent clinical features. For the 13 survivingpatients with available follow-up echocardiograms, 12 had per-sistent left ventricular hypertrophy, 3 showed aortic root dilation,and 2 had aortic insufficiency. Another notable clinical featurewas nephrocalcinosis, which persisted in eight of nine patients at4.1–14.5 y follow-up, despite treatment of hypokalemia. Fourpatients developed renal insufficiency more than 10 y after di-agnosis, with two progressing to renal failure and received renaltransplantation (30). Interestingly, following transplant, thesepatients no longer showed clinical signs of AME, with a re-mission of the low-renin hypertension and hypokalemic alkalosiswhen medical treatment with spironolactone was discontinued.On follow-up, three females conceived spontaneously. One

patient had two miscarriages before a viable pregnancy that shecarried to term. Spironolactone was discontinued due to non-compliance, which resulted in severe hypertension and renalfailure. The second patient carried her pregnancy to term, withspironolactone being discontinued in the first trimester and en-suing blood pressure control with methyldopa. The third patientwas treated with eplerenone and hydrochlorothiazide duringpregnancy; she had severe hypertension (BP: 217/91) associatedwith preeclampsia at 24 wk of gestation and delivered a non-viable fetus. This long-range follow up of an ultrarare disorderhas profound implications on the future management of newlydiscovered AME patients.The role of the mineralocorticoid receptor and the HSD11B2

enzyme in essential hypertension is being increasingly recognized.

A B C

Cortisol

P227L

R279C

N171NAD

R359W

I350

Fig. 5. HSD11B2 mutations causing mild AME type 2. (A) P227 forms a kink in the loop and helps to correctly position Y226 to optimally interact with thesubstrate. An increase in flexibility of the loop in the P227L mutation results in misalignment of Y226. (B) The hydrogen bond between R279 and N171 is oneof many that hold β4 and β6 sheets together. It also orients the side chain of N171 to interact with NAD+. Mutation to cysteine results in the loss of theseinteractions. (C) R359–I350 interactions maintain the helix-turn-helix. The R359W mutation introduces flexibility to the region.

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The presentation of the mild form of AME mimics essential hy-pertension as there are no electrolyte abnormalities. Mild muta-tions were identified in patients with mild clinical markers ofAME, namely low aldosterone, hypertension, and abnormal ratioof cortisol to cortisone and in vitro studies demonstrated a verymild alteration in enzyme activity (6, 24). The mild form is bestidentified by mutations in the HSD11B2 gene as other hallmarks,such as low birth weight, poor growth, severe hypertension, andhypokalemia, are absent. Increased (THF+alloTHF)/THE ratioshave also been reported in 16–32% of essential hypertension,consistent with HSD11B2 dysfunction (31, 32). Furthermore,treatment with mineralocorticoid receptor antagonists has beenshown to lower blood pressure in patients with essential hyper-tension, heart failure, and hypertension resistant to conventionalantihypertensives (33–35). For a subset of patients with low renin–low aldosterone hypertension, mineralocorticoid receptor antag-onists may be the most effective therapy. HSD11B2 enzymedeficiency also accelerates atherogenesis and causes proinflam-matory changes in the endothelium of mouse models; this isreduced by eplerenone (36). Polymorphisms of the HSD11B2gene have been associated with hypertension (37). Despite theseoverlaps between clinical features of mild AME and essentialhypertension, the most definitive diagnosis of the former is bygenetic diagnosis.One of the main factors contributing to the development of hy-

pertension is the amount of salt intake. Nevertheless, not all indi-viduals respond to a salt load in the same way; notably, some develophypertension while others do not. AME is a form of salt-sensitivemonogenic hypertension. In addition to expression in the kidney,HSD11B2 is also expressed in the brain. Brain-specific Hsd11b2knockout mice show increased salt appetite and salt sensitivity (38).It has been suggested that minor abnormalities of this enzyme mayexplain salt-sensitivity in the pathogenesis in essential hypertension.Early reports suggested that changes in the structural integrity of

HSD11B2 can cause severe AME, whereas intronic mutations thatyield mildly impaired enzyme could result in type 2 disease (39). Tothis end, correlations between clinical phenotype and the magni-tude of reduction in enzymatic activity in vitro for certain mutationshave been described previously (7). Nonetheless, precise structuralchanges induced by specific mutations have hitherto remainedunsolved. A prior in silico model has used the dimeric quaternarystructure, but it was later shown experimentally that the active formof HSD11B2 was, in fact, the monomer (12, 13). Here, we reportthe successful identification, by computational modeling, of struc-tural changes in silico that are induced by HSD11B2 mutations. Wehave used this analysis to provide structural explanations for theeffect of mutations on HSD11B2 function. We find that mutationsthat cause severe AME are those that enhance dimerization, dis-rupt the substrate- or coenzyme-binding site, or severely impairstructural stability. We have also categorized mutations that resultin a mild AME (type 2) phenotype—namely P227L, R279C, andR359W—as those indirectly disrupting substrate binding or causingmild alterations in protein structure.Based on observations from the humanized model, we find that

most of the mutations occur in yet uncharacterized regions of theHSD11B2 protein. We thus employed MD simulations in explicitsolvent on both monomeric and dimeric models to understand thestructural flexibility of these residues. We conclude that HSD11B2mutations exert their effect by disrupting intramolecular interactions,which alter protein structure and impair structural stability. Of thesemutations, those that affect multiple salt-bridge interactions, hydro-gen bond networks, and hydrophobic patches have a greater impacton disrupting overall structural integrity. In contrast, mutations thatonly affect a single intramolecular interaction, such as R279C andR359W, cause relatively minor disruptions, and are, therefore, moretolerated. This is because loss of a single interaction is likely to becompensated by interactions formed between other residues.

The analysis presented here is based on a given missense mu-tation affecting the HSD11B2 protein. However, in the case ofcompound heterozygosity, we would expect the resulting clinicalphenotype to result from the combined effects of two mutations. Asan example, a patient presenting with mild AME (type 2) was acompound heterozygote having inherited two HSD11B2 mutations(19). As one of the mutations, Y232C, occurred at the coenzyme-binding site, we predict on basis of our in silico categorization, thatit would severely attenuate enzymatic activity. However, the othermutation, L376P, a residue that is C-terminal to residue 369 andwas not modeled, is expected to result in HSD11B2 with sufficientresidual activity. The resulting mild clinical phenotype is thereforeconsistent with only 50% of near-normal functioning enzyme. In yetanother example, despite inheriting two mutations expected to re-sult in severely attenuated HSD11B2, the patient did not presentwith AME until 21 y of age (19). There is no ready explanation forthis, but it is known that minimal residual enzyme activity might justbe sufficient for mineralocorticoid escape.Overall, therefore, we infer that there is close concordance

between the structural disruption of HSD11B2 caused by a givenmutation and the resulting clinical phenotype. We have recentlyperformed a similar exhaustive in silico analysis for the ∼150 knownmutations affecting the steroidogenic enzyme 21-hydroxylase (9).Almost all mutations were categorized in terms of specific patterns ofstructural changes that correlated tightly with the three known clinicalphenotypes of CAH, namely salt wasting, simple virilizing, and non-classic CAH. For example, if the mutation was located within 5 �Å ofthe substrate- or heme-binding site, the outcome invariably wassalt-wasting CAH, whereas if nonconserved hydrophobic patcheswere disrupted, the disease was a milder, simple virilizing variety.Our in silico categorization of clinical severity of mutations can

potentially be used to predict clinical severity or phenotype of newmutations very early during gestation, particularly with the avail-ability of prenatal diagnostic testing. To this end, using patientsfrom our CAH cohort, we can now establish, as early as 6 wk ofgestation, whether the fetus is affected or a carrier by next-generation sequencing of cell-free fetal DNA in the mother’splasma (40). Our in silico prediction tool, together with thistechnology, should change the paradigm for “early” prenatal diag-nosis of a variety of autosomal recessive disorders, including AME.

MethodsThe cohort of AME patients, whichwas a part of the International Consortiumof Rare Steroid Disorders (Principal Investigator: M.I.N.), was recruited in-dependently in seven countries by pediatric endocrinologists, with approvalfrom the Icahn School of Medicine at Mount Sinai Institutional Review Board.DNAwas extracted fromblood drawn frompatients after obtaining informedconsent. Genotyping was performed by PCR at Mount Sinai or at overseasinstitutions using appropriate primer sets, as described previously (6, 29).Serum potassium, bicarbonate, and aldosterone, and urinary THF and THElevels were measured locally at commercial laboratories.

The amino acid sequence for human HSD11B2 was obtained from UniProt(accessionno. P80365) andused to search for homologous proteins in the ProteinData Bank. The top searches identified three HSD17B1 templates [PDB ID codes1IOL, 1JTV, and 1FDV (15–17)]. Multiple sequence alignment was carried outusing ClustalX (41). Models were constructed using the Internal CoordinateModeling method implemented in the Molsoft ICM software (18). The spatialposition of coenzyme and ligand were retained in their respective binding sites.This was based on the assumption that the ligands and coenzyme interact inthe similar manner and replicate all conserved interactions from the templateinto the model. Several rounds of energy minimization yielded a final low-energy conformation with no steric clashes between side chains. The finalmodel, which accounts for residues 81–369, was chosen based on a low Cα rmsdoverlap between the HSD17B1 templates and the HSD11B2 model (0.11 Å, 0.15Å, 0.21 Å with 1IOL, 1JTV, and 1FDV, respectively). Stereochemical propertieswere evaluated using PROCHECK v3.5.4 (42).

HSD11B2 is amember of the short-chain dehydrogenase/reductase proteinfamily, which exhibit highly conserved coenzyme and ligand binding do-mains. High conservation in the overall fold between HSD17B1 and HSD11B2permitted the adoption of structural features of the template into the

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model. The 1FDV structure has been crystallized with NAD+ coenzyme. Alarge ligand-binding pocket located adjacent to the coenzyme-bindingsite accommodates estradiol (1IOL) and testosterone (1JTV) in two dif-ferent orientations. The natural substrate of HSD11B2, cortisol, wasdocked in the ligand-binding pocket using the docking suite of the ICMsoftware (18).

Models were initially generated as monomers. Dimers were created aftersuperimposition of themonomers on the twofold inverted-symmetry–relatedhalf of the monomer in the 1IOL template. Monomeric and the dimericforms of HSD11B2 were subjected to restraint-free atomistic molecular dy-namics simulations for 500 ns each to: (i) test the stability of the models and(ii) understand the structural role of mutations at the dimer interface.AMBER ff99SB-ILDN force field was used to describe the systems (43). Theforce-field parameters for NAD were obtained from the AMBER parameterdatabase (44). The coordinates for cortisol were sketched and minimized inthe ICM software (18) and parameterized using the Antechamber program(45). The systems were solvated using TIP3P water molecules in a box of size75 × 69 × 81 Å3 (monomer) and 104 × 69 × 81 Å3 (dimer), whose boundaries

extended at least 10 Å from the edge of the solute. Counter-ions wereadded to neutralize the charges. Equilibration was carried out in two phasesemploying 10 ns each of isothermal-isochoric (NVT) ensemble using a Lan-gevin thermostat set at 300 K and isothermal-isobaric (NPT) ensemble usinga Berendsen barostat set at 1 atm, hydrogen mass portioning, and a timestep of 4 fs. The final production run was carried out till 500 ns. ACEMD (46)was the preferred choice of MD engine, running on NVIDIA GTX780 GPUs.All analyses were carried out using the AMBER suite of programs (47).

Molecular graphics images were generated using the ICM browser (18). Alist of currently known missense mutations, their corresponding clinical fea-tures, and enzyme activity were extracted and cross-evaluated from variousoriginal publications.

ACKNOWLEDGMENTS. The authors acknowledged the summer studentshipfunding support from the Biochemical Society and the Nuffield Trust, andsupport from the National Institutes of Health: Grants AG40132, AG23176,AR65932, DK113627, and AR67066 (to M.Z.).

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