Research Article Functional and Structural Consequences of ...

Research ArticleFunctional and Structural Consequences of Nine CYP21A2Mutations Ranging from Very Mild to Severe Effects

Deacutebora de Paula Michelatto12 Leif Karlsson2 Ana Letiacutecia Gori Lusa1

Camila DrsquoAlmeida Mgnani Silva3 Linus Joakim Oumlstberg4 Bengt Persson45

Gil Guerra-Juacutenior3 Sofia Helena Valente de Lemos-Marini3 Michela Barbaro6

Maricilda Palandi de Mello1 and Svetlana Lajic2

1Laboratorio de Genetica Molecular Humana Centro de Biologia Molecular e Engenharia GeneticaUniversidade Estadual de Campinas Av Candido Rondon 400 13083-875 Campinas SP Brazil2Department of Womenrsquos and Childrenrsquos Health Karolinska Institutet Pediatric Endocrinology Unit (Q208)Karolinska University Hospital 171 76 Stockholm Sweden3Departamento de Pediatria Faculdade de Ciencias Medicas Universidade Estadual de CampinasRua Tessalia Vieira de Camargo 126 13083-887 Campinas SP Brazil4Science for Life Laboratory Department of Medical Biochemistry and Biophysics Karolinska Institutet 171 77 Stockholm Sweden5Science for Life Laboratory Department of Cell and Molecular Biology Uppsala University 751 24 Uppsala Sweden6Department of Molecular Medicine and Surgery Karolinska Institutet and Center for Inherited Metabolic Diseases (CMMS L705)Karolinska University Hospital 171 76 Stockholm Sweden

Correspondence should be addressed to Svetlana Lajic svetlanalajickise

Received 18 May 2016 Revised 12 August 2016 Accepted 18 August 2016

Academic Editor Maria L Dufau

Copyright copy 2016 Debora de Paula Michelatto et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

We present the functional and structural effects of seven novel (pLeu12Met pArg16Cys pSer101Asn pSer202Gly pPro267LeupGln389 Ala391del and pThr450Met) and two previously reported but not studied (pSer113Phe and pThr450Pro) CYP21A2mutations Functional analyses were complemented with in silico prediction of mutation pathogenicity based on the recentlycrystallized human CYP21A2 structure Mutated proteins were transiently expressed in COS-1 cells and enzyme activities towards17-hydroxyprogesterone and progesterone were determined Residual enzyme activities between 43 and 97 were obtainedfor pArg16Cys pSer101Asn pSer202Gly pPro267Leu and pThr450Met similar to the activities of the well-known nonclassicmutations pPro453Ser and pPro482Ser whereas the pLeu12Met variant showed an activity of 100 Conversely the novelpSer113Phe pGln389 Ala391del and pThr450Promutations drastically reduced the enzyme function below 4The119870

119898values for

all novel variants were in the same order of magnitude as for the wild-type protein except for pThe450MetThe maximum velocitywas decreased for all mutants except for pLeu12Met We conclude that pLeu12Met is a normal variant the mutations pArg16CyspSer101Asn pSer202Gly pPro267Leu and pThr450Met could be associated with very mild nonclassic CAH and the mutationspSer113Phe pGln389 Ala391del and pThr450Pro are associated with classic CAH The obtained residual activities indicated agood genotype-phenotype correlation

1 Introduction

Congenital adrenal hyperplasia (CAH) is caused by defectsin one of the steroidogenic enzymes involved in the adrenalsteroid biosynthesis from cholesterol to cortisol The mostcommon cause ofCAH is 21-hydroxylase deficiency (21OHD)

where patients with the classic form present with or withoutsalt loss (salt wasting or simple virilizing forms resp) duringthe neonatal period and affected females are born withvirilized external genitalia In the nonclassic form signsof androgen excess such as acne hirsutism and menstrualirregularities can be observed as late as during adolescence

Hindawi Publishing CorporationInternational Journal of EndocrinologyVolume 2016 Article ID 4209670 10 pageshttpdxdoiorg10115520164209670

2 International Journal of Endocrinology

and adulthood Children present with precocious pubarcheaccelerated growth velocity and advanced skeletal matu-ration [1] The worldwide incidence of classic 21OHD is1 10000 to 1 15000 live births while nonclassic 21OHDis much more prevalent occurring in 1 500 live births inCaucasian populations [2ndash5]

The CYP21A2 gene coding for the 21-hydroxylase enzymeis formed by 10 exons and 9 introns located on the shortarm of chromosome 6 [6 7] CYP21A2 is arranged in tandemwith a nonfunctional pseudogene (CYP21A1P) that shares98 sequence identity with the active gene [8 9] Thereare common pseudogene-derived mutations identified inCYP21A2 that are found in more than 95 of all CAH allelesIn general the less severe mutation present in the genotypedetermines the phenotype establishing a good genotype-phenotype correlation [10] Furthermore in vitro studiesindicate that mutated CYP21A2 residual enzyme activitiespresent a good correlation with in vivo disease severity [11ndash13] Therefore in vitro analysis for novel or rare mutationsis proposed as a complement for disease classification espe-cially where large groups of patients are not available forclinical investigation thereby improving genetic counsellingand clinical management [14ndash17]

In this report we describe a detailed evaluation of thefunctional role of seven novel (pLeu12Met pArg16CyspSer101Asn pSer202Gly pPro267Leu pGln389 Ala391deland pThr450Met) and two previously reported but not func-tionally studied (pSer113Phe and pThr450Pro) CYP21A2mutations [18 19] The aim of the study was to inves-tigate the pathogenicity of novelrare mutations using invitro assays and to establish a correlation between their invitro effect and a possible CAH phenotype In order toreach a reliable phenotype prediction we also expressedfour mutations (pIle172Asn pVal281Leu pPro453Ser andpPro482Ser) known to cause CAH of different severity aswell as a CYP21A2 normal variant (pAla15Thr) Further-more we complemented the functional analyses with in silicopredictions of mutation pathogenicity and the effects onprotein structure using the model of the recently crystallizedhuman CYP21A2 structure [20]

2 Material and Methods

21 Genotyping CYP21A2 genotypingwas performed at Uni-versidade Estadual de Campinas Brazil and at KarolinskaUniversity Hospital Sweden The study was approved by theEthics Committee of Universidade Estadual de Campinasand the Regional ethics committee of Karolinska InstitutetGenomic DNA was obtained from peripheral blood byphenolchloroformextractionTheCYP21A2 genewas specif-ically amplified in two or four fragments depending on thepresence or absence of the variant C at the intron 2 c290-13ACgtG position [21] Sequencing of the amplified productshas been performed using the BigDye Terminator v31 CycleSequencing kit (Applied Biosystems USA) according to themanufacturerrsquos instructions Fragments were separated on aGenetic Analyzer from Applied Biosystems (ABI PRISM3100 Genetic AnalyzerLife Technologies USA) Electro-pherograms were analyzed against the reference sequence

NM 0005006 Segregation analysis was performed in allsubjects by sequencing parental samples except for subject 3

22 Subjects Clinical and molecular data of the subjects aresummarized in Table 1

Subject number 1 a girl presented with premature pub-arche and accelerated growth velocity at the age of 4 yearsShe had clitoral enlargementwithout labioscrotal fusion sincebirth (Prader I) Due to advanced bone age (87 y at 64 ychronological age) she was subjected to 250120583g Synacthenstimulation test that exhibited an elevation in the 17OHPbasal level from 13 nM to gt154 nM at 60min The child wasdiagnosed with NC CAH and CYP21A2 genotyping revealeda complex genotype with a novel mutation pLeu12Mettogether with pGln318lowast on the maternal allele and the NCmutation pVal281Leu on the paternal allele

Subject number 2 came to medical attention when herdaughter was diagnosed with CAH and the family wasgenotyped for 21OHD She did not complain of any symptomsof androgen excess and stimulation with Synacthen raisedthe 17OHP basal level just above the cut-off at 60min (6to 31 nM) CYP21A2 genotyping revealed a novel mutationpSer101Asn in compound heterozygosity with pVal281LeuTherefore very mild NC CAH was suspected but was notclinically confirmed in this case

Subject number 3 a female presented with menstrualirregularities hirsutism and a clinical suspicion of NC CAHat the age of 26 yearsCYP21A2 genotyping revealed the com-mon pVal281Leu mutation and the novel pSer113Phe aminoacid change Although segregation analyses or biochemicalinvestigational data were not available we assumed that thegenotype could be responsible for her clinical presentationand NC CAH

Subject number 4 a girl was born appropriate forgestational age at gestational week (GW) 36 and detectedvia the neonatal screening program The 17OHP screeninglevel (106 nM) was slightly above the cut-off level (100 nM)The second tier remained elevated (66 nM cut-off 60 nMGW37+2) At birth she had no signs of virilization or salt lossAt 6 months of age a 250 120583g Synacthen stimulation test wasnormal Due to her borderline hormonal screening valuesCYP21A2 genotyping was performed The novel pSer202Glymutation was identified in trans with pGln318lowast Very mildNC CAH could at this stage not be ruled out

Heterozygosity for the pSer202Gly variant was also iden-tified in subjects number 5 and number 6 at the ages of 1month and 9 years respectively Both children were initiallyseen at a primary reference center and investigated for sus-pected signs of androgen excess However the analyses of17OHP genotyping and clinical examination could not con-firm the diagnosis of CAHThese subjects are thus heterozy-gous carriers for the novel pSer202Gly mutation

Subject number 7 a girl was virilized at birth (Prader IV)and presented with a salt-losing crisis at day 7 SW CAH wasconfirmed due to elevated levels of 17OHP and subsequentgenotyping identified that she was compound heterozygousfor the novel in-frame deletion pGln389 Ala319del inheritedfrom her father and a 30-kb deletion inherited from hermother

International Journal of Endocrinology 3

Table1Th

egenotypes

andsymptom

satd

iagn

osisof

theind

ividualscarrying

then

ovelmutations

(markedin

bold)

Subject

number

Sex

Age

atdiagno

sisSymptom

s17OHP(nmolliter)lowast

basalstim

ulated

Genotype

Phenotype

Cou

ntry

oforigin

Publication

1F

4years

Prader

Iprem

aturep

ubarche

acceleratedgrow

th13gt154

pLe

u12M

et+p

Gln318lowastpVal281Leu

NC

Brazil

Presentstudy

2F

Adult

Asym

ptom

atic

631

pSer101AsnpVal281Leu

mdashBrazil

Presentstudy

3F

Adult

Menstr

ualirregularities

and

hirsutism

NA

pSer113Ph

epV

al281Leu

NC

Brazil

Present

studylowastlowastlowast

4F

Atbirth

Positiven

eonatalscreening

17OHP106n

Mlowastlowast

521

Cortisol3601118

pSer202GlypGln318lowast

mdashSw

eden

Presentstudy

5F

1mon

thPrader

Iscreeningvalue17O

HP

23nM

NA

pSer202GlyW

THealth

ycarrier

Brazil

Presentstudy

6M

9years

Prem

aturep

ubarcheadvanced

BA(121y

at95

y)411

pSer202GlyW

THealth

ycarrier

Brazil

Presentstudy

7F

Atbirth

Prader

IVN

a(129m

M)

73N

Ap(G

ln38

9Ala39

1del)3

0-kb

deletio

nSW

Brazil

Presentstudy

8F

1mon

thPrader

VAd

renalcris

isday33

Na(

117mM)K(83mM)

hypo

glycem

ia(23mM)

247NA

pTh

r450

PropTh

r450

Pro

SWIran

[19]

9M

5years

Suspicionof

penile

grow

thpseud

oprecociou

spu

berty

15N

ApArg16Cy

sWT

Health

ycarrier

Brazil

Presentstudy

10F

4years

Prem

aturep

ubarcheBA

810yrs

at65y

rsof

age

27

pPro2

67Le

uWT

Health

ycarrier

Brazil

Presentstudy

11F

Adult

Acne

139

pTh

r450

MetW

THealth

ycarrier

Brazil

Presentstudy

lowast

Referencev

aluecut-offlevel17-OHP

6and30

nMbasaland60

min250120583gSynacthenstimulationtest

lowastlowast

Neonatalreference

valuecut-o

fflevel100

nM

lowastlowastlowast

Haidere

tal2013

[18]m

utationisrepo

rted

butn

opatie

ntph

enotyped

escriptio

nNAnot

availableSW

saltw

astin

gNC

nonclassicB

Abon

eageW

Twild

type


The detailed clinical presentation of subject number 8 hasbeen previously described [19] The girl was born virilized(Prader V) and presented with an adrenal crisis at day 33 Shewas found to be homozygous for the pThr450Pro mutation

Subjects numbers 9 10 and 11 presented with differentsigns of androgen excess and clinical suspicion of CAHfor summary see Table 1 However 17OHP levels remainedwithin the normal range even after Synacthen stimulationGenotyping revealed that these subjects were heterozygouscarriers for three novel mutations pArg16Cys pPro267Leuand pThr450Met respectively Since the functional con-sequences for these novel amino acid substitutions wereunknown they were included in the present investigation

23 Functional Studies The general description for the con-struction of vectors used in the COS-1 expression studies ofmutated CYP21A2 has been previously described [11 22]

Expression of wild-type and mutant CYP21A2 enzymesand assays of 21OH activity were performed as previouslydescribed [23 24] Enzyme activities were expressed as apercentage of conversion taking the apparent specific activityof the CYP21A2 wild type as 100 Assays were performedafter 40min of incubation time

Apparent kinetic constants were determined 24 h aftertransfection Intact cells were incubated as previouslydescribed [23] together with 05 10 20 30 or 60120583Mof unlabeled 17-hydroxyprogesterone (Sigma-Aldrich SaintLouis USA) as substrate After incubation at 37∘C for 20minsteroids were extracted and analyzed as previously described[23] Apparent kinetic constants were calculated after lin-ear regression of the data derived from determinations ofenzymatic activity at each of the five different substrateconcentrations

24 Western Blot To ascertain similar amount of CYP21A2expression in transfected cells Western blotting was per-formed using rabbit polyclonal antibodies against humanCYP21A2 as primary antibody (Sigma-Aldrich Saint LouisUSA) and anti-rabbit IgG (GE Healthcare Life SciencesFreiburg Germany) as the secondary antibody see Robinset al [23] for details

25 Structural Evaluation The resolved three-dimensionalstructure of human CYP21 pdb id 4y8w [18] was used asa starting point to evaluate the effect of the novel mutationson the protein structure The ICM molecular modellingsoftware (version 38-1 Molsoft LLC La Jolla CA) was usedto perform structural calculations of the structures of eachmutation expanding on the strategy previously described[25] First all missing intrastructural loops were added to the4y8w structure and the structure was optimized using energyminimization A model of each mutation was then generatedby modifying the corresponding amino acid residue fol-lowed by multiple steps of energy minimization The energyminimization was initially performed with strong backbonerestraints which were gradually relaxed and finally a globalminimization without backbone restraints was performedEach mutation was then evaluated based on energy and

distances to the steroid and heme as well as using theICM built-in function for evaluating stability changes frommutations by calculating the free energy changes

The BLAST web interface [26] was used to identify mam-malian proteins similar to the human CYP21A2 enzymeThesearch was performed on the Nov 2015 release of the Refseqdatabase and resulted in CYP21 protein sequences from 81mammals The sequences with the highest similarity to thehuman CYP21A2 protein one sequence per species wereretrieved from the database The retrieved sequences werealigned using the Linsi approach ofMAFFT [27] and for eachposition the sequence identity was calculated counting a gapas a mismatch

3 Results

We have determined the residual activities of seven novelCYP21A2 mutations (pLeu12Met pArg16Cys pSer101AsnpSer202Gly pPro267Leu pGln389 Ala391del andpThr450Met) and of two previously reported but not func-tionally studied alterations (pSer113Phe and pThr450Pro)Neither of the variants was present in the pseudogene Wecompared the data to the residual activities of fivewell-knownreference mutations (pAla15Thr pIle172Asn pVal281LeupPro453Ser and pPro482Ser) using in vitro functionalassays The different residual enzyme activities ranged fromnormal to completely abolished enzyme function and themutations could therefore be defined as being normal poly-morphisms or being able to cause CAH of various degrees ofseverity Figure 1 and Table 3 illustrate the results In orderto unravel the mechanism responsible for reduced enzymefunction and to better differentiate between a very mildmutation and a neutral amino acid change we determinedthe apparent kinetic constants for all mutants that exhibiteda residual enzyme activity above 80 compared to thewild-type protein (Table 2) 119870

119898(substrate-binding capacity)

for all variants was in the same order of magnitude as forthe wild-type protein except for the pThr450Met mutationThe maximum velocity (119881max) was decreased for all variantsexcept for pLeu12Met and for the reference mutationpArg15Thr and both are thus considered to be normalvariants (Table 2)

Equal protein expressions of mutant and wild-type pro-teins have been confirmed by Western blotting (data notshown)

Table 3 summarizes the biochemical results the putativeeffects on the protein structure for all mutants and thefinal prediction of the phenotype Structural calculations forpSer101Asn pSer113Phe Ser202Gly and pPro267Leu arealso illustrated in Figure 2The pPro267Leu mutation has noeffect on protein structure whereas pSer101Asn pSer202Glyand pThr450Met are predicted to have a minor effectConversely pSer113Phe produces a more severe effect onprotein structure since it interferes with the alfa helicescomprising the active siteThe pThr450Promutation and thepGln389 Ala391del in-frame deletion are predicted to havedeleterious effects on the CYP21A2 structure


NC 21OHDreferences

Classic 21OHDreference

NC 21OHDreferences Classic 21OHD

reference17OHP Progesterone

0102030405060708090

100

Enzy

me a

ctiv

ity (

)

0102030405060708090

100

Enzy

me a

ctiv

ity (

)

Wild

type

pA

la15

Thr

pPr

o482

Ser

pPr

o453

Ser

pVa

l281

Leu

pIle

172A

sn

pLe

u12M

etp

Pro2

67Le

up

Arg

16Cy

sp

Ser1

01A

snp

Ser2

02G

lyp

Thr4

50M

et

pSe

r113

Phe

pTh

r450

Pro

pG

ln38

9_A

la39

1del

Wild

type

pA

la15

Thr

pPr

o482

Ser

pPr

o453

Ser

pVa

l281

Leu

pIle

172A

sn

pLe

u12M

etp

Pro2

67Le

up

Arg

16Cy

sp

Ser1

01A

snp

Ser2

02G

lyp

Thr4

50M

et

pSe

r113

Phe

pTh

r450

Pro

pG

ln38

9_A

la39

1del

Figure 1 Enzymatic activities of CYP21A2 mutant proteins Activities are expressed as a percentage of wild-type activity which is arbitrarilydefined as 100 Conversion values are shown for the two natural substrates (17OHP and progesterone)

Table 2 Apparent kinetic constants using 17-OHP as substrate

119881max(pmolmgprotein per

min)

119870119898(120583M) 119881max119870119898

Wild type 519 (93) 125 (13) 42 (9)pLeu12Met 618 (207) 177 (52) 35 (4)pAla15Thr 671 (145) 169 (65) 42 (8)pArg16Cys 181 (31) 50 (11) 37 (3)pSer101Asn 326 (67) 97 (20) 34 (4)pSer202Gly 244 (47) 80 (15) 31 (5)pPro267Leu 320 (93) 84 (19) 38 (7)pThr450Met 43 (5) 12 (01) 35 (5)Values are present as the mean (1SD) of at least four experiments

4 Discussion

Nine novelrare mutations identified in individuals investi-gated and genotyped for CAH have been expressed in vitroand enzymatic activities were compared with the functionalactivity of the wild-type protein Five other well-knownreference mutations were included in the assayThe referencemutations present a gradient of increasing residual activitiesthat are associated with the CAH phenotype variability ThepIle172Asn mutation is a classic mutation in most casesleading to SV CAH [28] with a very low residual enzymeactivity towards both 17OHP and progesterone The muta-tions pVal281Leu pPro453Ser and pPro482Ser are threeNC CAHmutations that present different degrees of residualactivity pPro482Ser being the mildest variant [29] andpVal281Leu being the most frequent mutation causing NCCAH with a residual activity of approximately 50 [30] Atlast pAla15Thr is considered to be a neutral amino acidchange [29] Expressing these well-characterized mutationsat the same time as the novel mutations allowed a better

intercomparison among mutants and provided a genotype-phenotype prediction

Two of the analyzed mutations pLeu12Met andpArg16Cys are located in the first hydrophobic domain ofthe protein This region comprises the membrane targetinganchoring domain and is important for in vitro stability [31]It is unsuitable for computational protein structure analysisand is missing from the published crystal structures [20 32]Leu12 is highly conserved among mammalian species (88)Arg16 is also well conserved (69) and the correspondingamino acid residue in bovine is histidine indicating conser-vation of a positively charged polar residue Interestinglyanother mutation within this region pAla15Thr is a normalvariant [29] The mutation pLeu12Met was identified in ciswith the pGln318lowast mutation and consequently this is a nullallele whereas the pArg16Cys was identified in a heterozy-gous carrier Therefore it was impossible to evaluate theirpathogenicity based on the phenotype of the subjects(Table 1) In vitro functional studies showed that the mutantpLeu12Met did not demonstrate a significant difference inenzymatic activity or in kinetic constants when comparedwith the wild-type and pAla15Thr proteins It is thereforeconsidered to be a normal variant The pArg16Cys mutantdemonstrated a very mild reduction in enzyme activitysimilar to pPro482Ser (Table 3) in addition to a clear reduc-tion in apparent119881max (Table 2) Although we did not identifya compound heterozygous patient to confirm our hypothesisthis mutation may be associated with a very mild NC CAHphenotype

The pSer101Asn mutation was identified in an asymp-tomatic mother of a Brazilian CAH patient (Table 1) Shewas compound heterozygous for the pVal281Leu mutationthat was transmitted to her daughter Ser101 is conservedamongmammalian species (74) and is located between tworesidues that are part of the channel for product passage [32]However the structural calculations implied only a minoreffect on protein structure caused by the mutation In factin vitro studies confirmed a very mild effect with a reduction


Table3En

zymeactiv

itiesinsilico

predictio

nof

theeffecto

nproteinstr

uctureand

finalpredictio

nof

theph

enotypeforthe

novelm

utations

andforthe

common

mutations

used

asa

reference(in

bold)

Mutation(cDNA)

Proteinchange

Locatio

nin

the

protein

Insilico

predictio

nEn

zymea

ctivity

Phenotypep

rediction

(hem

izygou

sho

mozygou

sstate)

17OHP

Prog

c43GgtA

pAla15Th

rFirsth

ydroph

obic

domain

ND

100(0)

96(6)

Normalvarian

t(NV)

c34CgtA

pLeu12M

etFirsth

ydroph

obic

domain

ND

99(1)

100(1)

NV

c800Cgt

TpPro267Leu

Loop

between

120572-helixHand

120572-helixI

Noeffect

97(1)

87(7)

NVverymild

NC

c46CgtT

pArg16Cy

sFirsth

ydroph

obic

domain

ND

95(3)

81(3)

NVverymild

NC

c301

302T

CgtAA

pSer101As

nLo

opbetween

120572-helixB1015840

and

120572-helixC

Minor

effect

94(3)

74(2)

NVverymild

NC

c144

4CgtT

pPro4

82Ser

120573-she

et1205739

ND

91(6)

61(6)

Very

mild

NC

c604AgtG

pSer202Gly

Loop

between

120572-helixFand

120572-helixF1015840

Minor

effect

85(2)

81(3)

Very

mild

NC

c1349CgtT

pThr450Met

120573-sheet1205738

Minor

effect

78(6)

43(5)

Mild

NC

c1357CgtT

pPro4

53Ser

120573-she

et1205738

ND

73(8)

34(10)

NC

c841GgtT

pVa

l281Le

u120572-helix

IND

57(8)

29(5)

NC

c338Cgt

TpSer113Ph

eLo

opbetween

120572-helixB1015840

and

120572-helixC

Severe

effect

interfe

resw

ithactiv

esite

helices

4(1)

4(2)

CL

c1348AgtC

pThr450Pro

120573-sheet1205738

Very

severe

effect

lt1

lt1

CLc5

15Tgt

ApIle

172A

sn120572-helix

END

lt1

lt1

CLc1165117

3delCA

AGGCG

CCpGln389Ala391del

120572-helixL

Deleterious

0lt1

CLEn

zymea

ctivity

isexpressedin

of

wild

-type

activ

ityw

ithvalues

presentedas

mean(1SD

)ofatleastfour

independ

entexp

erim

ents

NDnot

determ

inedM

utations

areo

rdered

accordingto

ther

esidualenzym

eactiv

itywith

ther

eference

mutations

inbo

ld


(a)

(b)

(c)

(d)

Figure 2 Structural changes Visualization of the structural changes caused by fourmutations left wild type rightmutation (a) pSer101Asn(b) pSer113Phe (c) pSer202Gly and (d) pPro267Leu

of activity to 94 and 74 towards 17OHP and progesteronerespectively119881max value was also reduced as shown in Table 2Based on our results we may expect this mutation to resultin very mild NC CAH However we could not confirmour hypothesis because the individual did not present anyobvious symptoms and a neutral amino acid change could notcompletely be ruled out

The mutation pSer113Phe may disturb an adjacent 120572-helix based on the structural calculations for the model ofhumanCYP21A2 It is located in the vicinity of Ser109 Leu110and Trp117 which form part of the proximal substrate-binding pocket and the heme-binding region [32] Thereforethis amino acid substitution may interfere with the recogni-tion and binding of the substrate and consequently affect


enzyme activityThe in silico predictions were in line with thein vitro studies where a minimal activity of 4 towards bothsubstrates was obtained (Table 3) Consequently pSer113Phecan be associated with SV CAH if found in a homo- orhemizygous state Although pSer113Phe had been previouslyreported by Haider et al [18] as a NC mutation no patientdescription or in vitro studies were presented in the firstreport

The pSer202Gly mutation was identified in three inde-pendent subjects from two different continents The twoBrazilian carriers were genotyped because CAH was sus-pected but no othermutations were identified In the Swedishsubject however it was identified in compoundheterozygosiswith the null mutation pGln318lowast This girl has been followedduring her first year of life and so far no signs of virilizationhave been detected but a NC CAH phenotype cannot beruled out at this young age Functional studies showed areduction in enzyme activity to the level of the NC mutationpPro482Ser and a reduced 119881max (Tables 2 and 3) Thestructural calculations implied that this mutation causes aminor effect on protein structure since both serine andglycine are small amino acid residues and Ser202 is locatedin a loop close to the surface of the protein

The pPro267Leu mutation was identified in a girl thatwas investigated for premature pubarche at 4 years of ageCYP21A2 genotyping indicated that she was a heterozygouscarrier for this mutation Pro267 is weakly conserved amongmammalian species (51) and is located in a loop betweentwo 120572-helices The structural calculations did not show anynotable structural changes Functional studies showed aminor effect on enzyme activity similar if not milder than forpPro482Ser Kinetic studies showed a reduction in 119881max butto finally exclude pPro267Leu as a neutral variant additionaland symptomatic CAH cases with this mutation need to beidentified (Table 2)

The in-frame deletion of three amino acids (pGln389Ala391del) is the result of the nucleotide deletion c11651173delCAAGGCGCC in exon 9 Based on the phenotype ofthe patient who has SW CAH (Table 1) a drastic effect on theenzyme activity could be predictedThe patient is compoundheterozygous for this mutation inherited from her father anda 30-kb deletion inherited fromhermotherThe three deletedresidues (Glu Gly and Ala) are highly conserved amongmammalian species (83 94 and 94 resp) Based onthe structural analysis of human CYP21A2 these amino acidsare located inside an 120572-helix just before a His that has beenshown to interact with the EXXR structural motif importantfor the tertiary structure of the CYP21A2 enzyme [32] Thedeletion by itself may be deleterious to the structure or func-tion of the protein In vitro studies confirm the loss of enzymeactivity No reduction in protein level was seen by Westernblot analysis (data not shown) although specific stabilitystudies have not been performed

Finally the novel mutation pThr450Met has been iden-tified in a heterozygous carrier in the Brazilian populationPreviously the pThr450Promutationwas identified in an Ira-nian girl with SW CAH (Table 1) Thr450 is highly conserved(89) amongmammalian species and is located inside a short120573 sheet which is disturbed by the pThr450Pro mutation

The 120573 sheet is not central to the protein but the structuralcalculations imply that the mutation affects protein structuresignificantly The pThr450Met substitution however doesnot disturb the 120573 sheet to the same extent We expressedboth mutant proteins in vitro and obtained results that con-firmed the structural predictions (Table 3) The pThr450Promutation had a drastic effect on enzyme function similarto the classic reference mutation pIle172Asn whereas thepThr450Met mutant gives a milder reduction in enzymeactivity to the level of other NC mutations (Table 3) but witha more severe impact on apparent kinetic constants (Table 2)

5 Conclusions

We studied the functional effects of nine novelrare muta-tions in the CYP21A2 enzyme We conclude that pLeu12Metis a normal variant and the mutations pSer113Phe pGln389Ala391del and pThr450Pro are associated with classic CAHThe mutations pArg16Cys pSer101Asn pSer202GlypPro267Leu and pThr450Met could be associated withmilder forms of nonclassic CAH although the identificationof additional symptomatic cases will further define the clini-cal spectrum for these variants The study showed a goodcorrelation between genotype and phenotype for mutationsidentified in compound heterozygous individuals Further-more the putative effects on protein structure based onin silico predictions using the recently crystallized humanCYP21A2 structure confirmed the pathogenicity of themuta-tions and the results were in agreement with the functionalstudies

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

Acknowledgments

This work was supported by Sao Paulo Research Founda-tion (FAPESP) Grant no 201151808-2 to Maricilda Palandide Mello IFCAHESPE Marianne and Marcus WallenbergFoundation Stockholm County Council (ALF-SLL) Stif-telsen Frimurare Barnhuset Stiftelsen Samariten Jerring-fonden Stiftelsen Wera Ekstrom and Sallskapet BarnavardDebora de Paula Michelatto was supported by Sao PauloResearch Foundation (FAPESP) Grant no 201409844-0 andGrant no 201216815-0 The authors thank Cristiane dosSantos Cruz Piveta for technical support

References

[1] P W Speiser R Azziz L S Baskin et al ldquoCongenital adrenalhyperplasia due to steroid 21-hydroxylase deficiency an Endo-crine Society clinical practice guidelinerdquo Journal of ClinicalEndocrinology and Metabolism vol 95 no 9 pp 4133ndash41602010

[2] E Trakakis C Loghis and D Kassanos ldquoCongenital adrenalhyperplasia because of 21-hydroxylase deficiency a genetic


disorder of interest to obstetricians and gynecologistsrdquo Obstet-rical amp Gynecological Survey vol 64 no 3 pp 177ndash189 2009

[3] A Wedell ldquoMolecular genetics of 21-hydroxylase deficiencyrdquoPediatric Adrenal Diseases vol 20 pp 80ndash87 2010

[4] P W Speiser B Dupont P Rubinstein A Piazza A Kastelanand M I New ldquoHigh frequency of nonclassical steroid 21-hydroxylase deficiencyrdquo American Journal of Human Geneticsvol 37 no 4 pp 650ndash667 1985

[5] R C Wilson S Nimkarn M Dumic et al ldquoEthnic-specificdistribution ofmutations in 716 patientswith congenital adrenalhyperplasia owing to 21-hydroxylase deficiencyrdquo MolecularGenetics and Metabolism vol 90 no 4 pp 414ndash421 2007

[6] P C White D Grossberger B J Onufer et al ldquoTwo genesencoding steroid 21-hydroxylase are located near the genesencoding the fourth component of complement in manrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 82 no 4 pp 1089ndash1093 1985

[7] P C White and P W Speiser ldquoCongenital adrenal hyperplasiadue to 21-hydroxylase deficiencyrdquoEndocrine Reviews vol 21 no3 pp 245ndash291 2000

[8] Y Higashi H Yoshioka M Yamane O Gotoh and Y Fujii-Kuriyama ldquoComplete nucleotide sequence of two steroid 21-hydroxylase genes tandemly arranged in human chromosomea pseudogene and a genuine generdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 83 no9 pp 2841ndash2845 1986

[9] P C White M I New and B Dupont ldquoStructure of humansteroid 21-hydroxylase genesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 83 no14 pp 5111ndash5115 1986

[10] A Wedell B Stengler and H Luthman ldquoCharacterization ofmutations on the rare duplicatedC4CYP21 haplotype in steroid21-hydroxylase deficiencyrdquo Human Genetics vol 94 no 1 pp50ndash54 1994

[11] A Nikoshkov S Lajic M Holst A Wedell and H LuthmanldquoSynergistic effect of partially inactivating mutations in steroid21-hydroxylase deficiencyrdquoTheJournal of Clinical Endocrinologyamp Metabolism vol 82 no 1 pp 194ndash199 1997

[12] S Lajic T Robins N Krone H P Schwarz and A WedellldquoCYP21mutations in simple virilizing congenital adrenal hyper-plasiardquo Journal of Molecular Medicine vol 79 no 10 pp 581ndash586 2001

[13] V Tardy R Menassa V Sulmont et al ldquoPhenotype-genotypecorrelations of 13 rare CYP21A2 mutations detected in 46patients affected with 21-hydroxylase deficiency and in onecarrierrdquo Journal of Clinical Endocrinology and Metabolism vol95 no 3 pp 1288ndash1300 2010

[14] N Krone F G Riepe C-J Partsch W Vorhoff J Bramswigand W G Sippell ldquoThree novel point mutations of the CYP21gene detected in classical forms of congenital adrenal hyperpla-sia due to 21-hydroxylase deficiencyrdquo Experimental and ClinicalEndocrinology and Diabetes vol 114 no 3 pp 111ndash117 2006

[15] M Barbaro L Baldazzi A Balsamo et al ldquoFunctional studies oftwo novel and two rare mutations in the 21-hydroxylase generdquoJournal of Molecular Medicine vol 84 no 6 pp 521ndash528 2006

[16] M Taboas L Gomez Acuna M F Scaia et al ldquoFunctionalstudies of pR132C pR149C pM283V pE431K and a novelc652-2AgtG mutations of the CYP21A2 generdquo PLoS ONE vol9 no 3 Article ID e92181 2014

[17] A Massimi M Malaponti L Federici et al ldquoFunctional andstructural analysis of four novel mutations of CYP21A2 gene in

Italian patients with 21-hydroxylase deficiencyrdquo Hormone andMetabolic Research vol 46 no 7 pp 515ndash520 2014

[18] S Haider B Islam V DrsquoAtri et al ldquoStructure-phenotype cor-relations of human CYP21A2 mutations in congenital adrenalhyperplasiardquo Proceedings of the National Academy of Sciences ofthe United States of America vol 110 no 7 pp 2605ndash2610 2013

[19] A Baradaran-Heravi R Vakili T Robins et al ldquoThree novelCYP21A2 mutations and their protein modelling in patientswith classical 21-hydroxylase deficiency from northeasternIranrdquo Clinical Endocrinology vol 67 no 3 pp 335ndash341 2007

[20] P S Pallan C Wang L Lei et al ldquoHuman cytochrome P45021A2 the major steroid 21-hydroxylase structure of the enzymeprogesterone substrate complex and rate-limiting CndashH bondcleavagerdquo The Journal of Biological Chemistry vol 290 no 21pp 13128ndash13143 2015

[21] I F Lau F C Soardi S H V Lemos-Marini G Guerra Jr MT M Baptista and M P De Mello ldquoH28+C insertion in theCYP21 gene a novel frameshift mutation in a Brazilian patientwith the classical formof 21-hydroxylase deficiencyrdquoThe Journalof Clinical EndocrinologyampMetabolism vol 86 no 12 pp 5877ndash5880 2001

[22] S Lajic A Levo A Nikoshkov Y Lundberg J Partanen and AWedell ldquoA cluster of missense mutations at Arg356 of humansteroid 21-hydroxylase may impair redox partner interactionrdquoHuman Genetics vol 99 no 6 pp 704ndash709 1997

[23] T Robins M Barbaro S Lajic and A Wedell ldquoNot all aminoacid substitutions of the common cluster E6mutation in CYP21cause congenital adrenal hyperplasiardquo The Journal of ClinicalEndocrinologyampMetabolism vol 90 no 4 pp 2148ndash2153 2005

[24] F C Soardi M Barbaro I F Lau et al ldquoInhibition of CYP21A2enzyme activity caused by novel missense mutations identifiedin brazilian and scandinavian patientsrdquo Journal of ClinicalEndocrinology and Metabolism vol 93 no 6 pp 2416ndash24202008

[25] T Robins J Carlsson M Sunnerhagen A Wedell and B Pers-son ldquoMolecular model of human CYP21 based on mammalianCYP2C5 structural features correlate with clinical severity ofmutations causing congenital adrenal hyperplasiardquo MolecularEndocrinology vol 20 no 11 pp 2946ndash2964 2006

[26] NCBI Resource Coordinators ldquoDatabase resources of theNational Center for Biotechnology Informationrdquo Nucleic AcidsResearch vol 43 no 1 pp D6ndashD17 2015

[27] K Katoh and D M Standley ldquoMAFFT multiple sequencealignment software version 7 improvements in performanceand usabilityrdquo Molecular Biology and Evolution vol 30 no 4pp 772ndash780 2013

[28] M Amor K L Parker H Globerman M I New and P CWhite ldquoMutation in the CYP21B gene (Ile-172-gtAsn) causessteroid 21-hydroxylase deficiencyrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 85 no5 pp 1600ndash1604 1988

[29] M Barbaro S Lajic L Baldazzi et al ldquoFunctional analysisof two recurrent amino acid substitutions in the CYP21 genefrom Italian patients with congenital adrenal hyperplasiardquo TheJournal of Clinical Endocrinology amp Metabolism vol 89 no 5pp 2402ndash2407 2004

[30] M-T Tusie-Luna P Traktman andPCWhite ldquoDeterminationof functional effects of mutations in the steroid 21-hydroxylasegene (CYP21) using recombinant vaccinia virusrdquoThe Journal ofBiological Chemistry vol 265 no 34 pp 20916ndash20922 1990


[31] S Lajic A Nikoshkov M Holst and A Wedell ldquoEffects ofmissense mutations and deletions onmembrane anchoring andenzyme function of human steroid 21-hydroxylase (P450c21)rdquoBiochemical and Biophysical Research Communications vol 257no 2 pp 384ndash390 1999

[32] B Zhao L Lei N Kagawa et al ldquoThree-dimensional structureof steroid 21-hydroxylase (cytochrome P450 21A2) with twosubstrates reveals locations of disease-associated variantsrdquo TheJournal of Biological Chemistry vol 287 no 13 pp 10613ndash106222012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


and adulthood Children present with precocious pubarcheaccelerated growth velocity and advanced skeletal matu-ration [1] The worldwide incidence of classic 21OHD is1 10000 to 1 15000 live births while nonclassic 21OHDis much more prevalent occurring in 1 500 live births inCaucasian populations [2ndash5]

The CYP21A2 gene coding for the 21-hydroxylase enzymeis formed by 10 exons and 9 introns located on the shortarm of chromosome 6 [6 7] CYP21A2 is arranged in tandemwith a nonfunctional pseudogene (CYP21A1P) that shares98 sequence identity with the active gene [8 9] Thereare common pseudogene-derived mutations identified inCYP21A2 that are found in more than 95 of all CAH allelesIn general the less severe mutation present in the genotypedetermines the phenotype establishing a good genotype-phenotype correlation [10] Furthermore in vitro studiesindicate that mutated CYP21A2 residual enzyme activitiespresent a good correlation with in vivo disease severity [11ndash13] Therefore in vitro analysis for novel or rare mutationsis proposed as a complement for disease classification espe-cially where large groups of patients are not available forclinical investigation thereby improving genetic counsellingand clinical management [14ndash17]

In this report we describe a detailed evaluation of thefunctional role of seven novel (pLeu12Met pArg16CyspSer101Asn pSer202Gly pPro267Leu pGln389 Ala391deland pThr450Met) and two previously reported but not func-tionally studied (pSer113Phe and pThr450Pro) CYP21A2mutations [18 19] The aim of the study was to inves-tigate the pathogenicity of novelrare mutations using invitro assays and to establish a correlation between their invitro effect and a possible CAH phenotype In order toreach a reliable phenotype prediction we also expressedfour mutations (pIle172Asn pVal281Leu pPro453Ser andpPro482Ser) known to cause CAH of different severity aswell as a CYP21A2 normal variant (pAla15Thr) Further-more we complemented the functional analyses with in silicopredictions of mutation pathogenicity and the effects onprotein structure using the model of the recently crystallizedhuman CYP21A2 structure [20]

2 Material and Methods

21 Genotyping CYP21A2 genotypingwas performed at Uni-versidade Estadual de Campinas Brazil and at KarolinskaUniversity Hospital Sweden The study was approved by theEthics Committee of Universidade Estadual de Campinasand the Regional ethics committee of Karolinska InstitutetGenomic DNA was obtained from peripheral blood byphenolchloroformextractionTheCYP21A2 genewas specif-ically amplified in two or four fragments depending on thepresence or absence of the variant C at the intron 2 c290-13ACgtG position [21] Sequencing of the amplified productshas been performed using the BigDye Terminator v31 CycleSequencing kit (Applied Biosystems USA) according to themanufacturerrsquos instructions Fragments were separated on aGenetic Analyzer from Applied Biosystems (ABI PRISM3100 Genetic AnalyzerLife Technologies USA) Electro-pherograms were analyzed against the reference sequence

NM 0005006 Segregation analysis was performed in allsubjects by sequencing parental samples except for subject 3

22 Subjects Clinical and molecular data of the subjects aresummarized in Table 1

Subject number 1 a girl presented with premature pub-arche and accelerated growth velocity at the age of 4 yearsShe had clitoral enlargementwithout labioscrotal fusion sincebirth (Prader I) Due to advanced bone age (87 y at 64 ychronological age) she was subjected to 250120583g Synacthenstimulation test that exhibited an elevation in the 17OHPbasal level from 13 nM to gt154 nM at 60min The child wasdiagnosed with NC CAH and CYP21A2 genotyping revealeda complex genotype with a novel mutation pLeu12Mettogether with pGln318lowast on the maternal allele and the NCmutation pVal281Leu on the paternal allele

Subject number 2 came to medical attention when herdaughter was diagnosed with CAH and the family wasgenotyped for 21OHD She did not complain of any symptomsof androgen excess and stimulation with Synacthen raisedthe 17OHP basal level just above the cut-off at 60min (6to 31 nM) CYP21A2 genotyping revealed a novel mutationpSer101Asn in compound heterozygosity with pVal281LeuTherefore very mild NC CAH was suspected but was notclinically confirmed in this case

Subject number 3 a female presented with menstrualirregularities hirsutism and a clinical suspicion of NC CAHat the age of 26 yearsCYP21A2 genotyping revealed the com-mon pVal281Leu mutation and the novel pSer113Phe aminoacid change Although segregation analyses or biochemicalinvestigational data were not available we assumed that thegenotype could be responsible for her clinical presentationand NC CAH

Subject number 4 a girl was born appropriate forgestational age at gestational week (GW) 36 and detectedvia the neonatal screening program The 17OHP screeninglevel (106 nM) was slightly above the cut-off level (100 nM)The second tier remained elevated (66 nM cut-off 60 nMGW37+2) At birth she had no signs of virilization or salt lossAt 6 months of age a 250 120583g Synacthen stimulation test wasnormal Due to her borderline hormonal screening valuesCYP21A2 genotyping was performed The novel pSer202Glymutation was identified in trans with pGln318lowast Very mildNC CAH could at this stage not be ruled out

Heterozygosity for the pSer202Gly variant was also iden-tified in subjects number 5 and number 6 at the ages of 1month and 9 years respectively Both children were initiallyseen at a primary reference center and investigated for sus-pected signs of androgen excess However the analyses of17OHP genotyping and clinical examination could not con-firm the diagnosis of CAHThese subjects are thus heterozy-gous carriers for the novel pSer202Gly mutation

Subject number 7 a girl was virilized at birth (Prader IV)and presented with a salt-losing crisis at day 7 SW CAH wasconfirmed due to elevated levels of 17OHP and subsequentgenotyping identified that she was compound heterozygousfor the novel in-frame deletion pGln389 Ala319del inheritedfrom her father and a 30-kb deletion inherited from hermother


Table1Th

egenotypes

andsymptom

satd

iagn

osisof

theind

ividualscarrying

then

ovelmutations

(markedin

bold)

Subject

number

Sex

Age

atdiagno

sisSymptom


basalstim

ulated

Genotype

Phenotype

Cou

ntry

oforigin

Publication

1F

4years

Prader

Iprem

aturep

ubarche

acceleratedgrow

th13gt154

pLe

u12M

et+p


NC

Brazil

Presentstudy

2F

Adult

Asym

ptom

atic

631


mdashBrazil

Presentstudy

3F

Adult

Menstr

ualirregularities

and

hirsutism

NA

pSer113Ph

epV

al281Leu

NC

Brazil

Present


4F

Atbirth

Positiven

eonatalscreening

17OHP106n

Mlowastlowast

521

Cortisol3601118


mdashSw

eden

Presentstudy

5F

1mon

thPrader

Iscreeningvalue17O

HP

23nM

NA

pSer202GlyW

THealth

ycarrier

Brazil

Presentstudy

6M

9years

Prem

aturep

ubarcheadvanced

BA(121y

at95

y)411

pSer202GlyW

THealth

ycarrier

Brazil

Presentstudy

7F

Atbirth

Prader

IVN

a(129m

M)

73N

Ap(G

ln38

9Ala39

1del)3

0-kb

deletio

nSW

Brazil

Presentstudy

8F

1mon

thPrader

VAd

renalcris

isday33

Na(

117mM)K(83mM)

hypo

glycem

ia(23mM)

247NA

pTh

r450

PropTh

r450

Pro

SWIran

[19]

9M

5years

Suspicionof

penile

grow

thpseud

oprecociou

spu

berty

15N

ApArg16Cy

sWT

Health

ycarrier

Brazil

Presentstudy

10F

4years

Prem

aturep

ubarcheBA

810yrs

at65y

rsof

age

27

pPro2

67Le

uWT

Health

ycarrier

Brazil

Presentstudy

11F

Adult

Acne

139

pTh

r450

MetW

THealth

ycarrier

Brazil

Presentstudy

lowast

Referencev


6and30

nMbasaland60


lowastlowast

Neonatalreference

valuecut-o

fflevel100

nM

lowastlowastlowast

Haidere

tal2013

[18]m

utationisrepo

rted

butn

opatie

ntph

enotyped

escriptio

nNAnot

availableSW

saltw

astin

gNC

nonclassicB

Abon

eageW

Twild

type











3 Results







NC 21OHDreferences




0102030405060708090

100

Enzy

me a

ctiv

ity (

)

0102030405060708090

100

Enzy

me a

ctiv

ity (

)

Wild

type

pA

la15

Thr

pPr

o482

Ser

pPr

o453

Ser

pVa

l281

Leu

pIle

172A

sn

pLe

u12M

etp

Pro2

67Le

up

Arg

16Cy

sp

Ser1

01A

snp

Ser2

02G

lyp

Thr4

50M

et

pSe

r113

Phe

pTh

r450

Pro

pG

ln38

9_A

la39

1del

Wild

type

pA

la15

Thr

pPr

o482

Ser

pPr

o453

Ser

pVa

l281

Leu

pIle

172A

sn

pLe

u12M

etp

Pro2

67Le

up

Arg

16Cy

sp

Ser1

01A

snp

Ser2

02G

lyp

Thr4

50M

et

pSe

r113

Phe

pTh

r450

Pro

pG

ln38

9_A

la39

1del




min)

119870119898(120583M) 119881max119870119898


4 Discussion






Table3En

zymeactiv

itiesinsilico

predictio

nof

theeffecto

nproteinstr

uctureand

finalpredictio

nof

theph

enotypeforthe

novelm

utations

andforthe

common

mutations

used

asa

reference(in

bold)

Mutation(cDNA)

Proteinchange

Locatio

nin

the

protein

Insilico

predictio

nEn

zymea

ctivity

Phenotypep

rediction

(hem

izygou

sho

mozygou

sstate)

17OHP

Prog

c43GgtA

pAla15Th

rFirsth

ydroph

obic

domain

ND

100(0)

96(6)

Normalvarian

t(NV)

c34CgtA

pLeu12M

etFirsth

ydroph

obic

domain

ND

99(1)

100(1)

NV

c800Cgt

TpPro267Leu

Loop

between

120572-helixHand

120572-helixI

Noeffect

97(1)

87(7)

NVverymild

NC

c46CgtT

pArg16Cy

sFirsth

ydroph

obic

domain

ND

95(3)

81(3)

NVverymild

NC

c301

302T

CgtAA

pSer101As

nLo

opbetween

120572-helixB1015840

and

120572-helixC

Minor

effect

94(3)

74(2)

NVverymild

NC

c144

4CgtT

pPro4

82Ser

120573-she

et1205739

ND

91(6)

61(6)

Very

mild

NC

c604AgtG

pSer202Gly

Loop

between

120572-helixFand

120572-helixF1015840

Minor

effect

85(2)

81(3)

Very

mild

NC

c1349CgtT

pThr450Met

120573-sheet1205738

Minor

effect

78(6)

43(5)

Mild

NC

c1357CgtT

pPro4

53Ser

120573-she

et1205738

ND

73(8)

34(10)

NC

c841GgtT

pVa

l281Le

u120572-helix

IND

57(8)

29(5)

NC

c338Cgt

TpSer113Ph

eLo

opbetween

120572-helixB1015840

and

120572-helixC

Severe

effect

interfe

resw

ithactiv

esite

helices

4(1)

4(2)

CL

c1348AgtC

pThr450Pro

120573-sheet1205738

Very

severe

effect

lt1

lt1

CLc5

15Tgt

ApIle

172A

sn120572-helix

END

lt1

lt1

CLc1165117

3delCA

AGGCG

CCpGln389Ala391del

120572-helixL

Deleterious

0lt1

CLEn

zymea

ctivity

isexpressedin

of

wild

-type

activ

ityw

ithvalues

presentedas

mean(1SD

)ofatleastfour

independ

entexp

erim

ents

NDnot

determ

inedM

utations

areo

rdered

accordingto

ther

esidualenzym

eactiv

itywith

ther

eference

mutations

inbo

ld


(a)

(b)

(c)

(d)











5 Conclusions


Competing Interests


Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table1Th

egenotypes

andsymptom

satd

iagn

osisof

theind

ividualscarrying

then

ovelmutations

(markedin

bold)

Subject

number

Sex

Age

atdiagno

sisSymptom


basalstim

ulated

Genotype

Phenotype

Cou

ntry

oforigin

Publication

1F

4years

Prader

Iprem

aturep

ubarche

acceleratedgrow

th13gt154

pLe

u12M

et+p


NC

Brazil

Presentstudy

2F

Adult

Asym

ptom

atic

631


mdashBrazil

Presentstudy

3F

Adult

Menstr

ualirregularities

and

hirsutism

NA

pSer113Ph

epV

al281Leu

NC

Brazil

Present


4F

Atbirth

Positiven

eonatalscreening

17OHP106n

Mlowastlowast

521

Cortisol3601118


mdashSw

eden

Presentstudy

5F

1mon

thPrader

Iscreeningvalue17O

HP

23nM

NA

pSer202GlyW

THealth

ycarrier

Brazil

Presentstudy

6M

9years

Prem

aturep

ubarcheadvanced

BA(121y

at95

y)411

pSer202GlyW

THealth

ycarrier

Brazil

Presentstudy

7F

Atbirth

Prader

IVN

a(129m

M)

73N

Ap(G

ln38

9Ala39

1del)3

0-kb

deletio

nSW

Brazil

Presentstudy

8F

1mon

thPrader

VAd

renalcris

isday33

Na(

117mM)K(83mM)

hypo

glycem

ia(23mM)

247NA

pTh

r450

PropTh

r450

Pro

SWIran

[19]

9M

5years

Suspicionof

penile

grow

thpseud

oprecociou

spu

berty

15N

ApArg16Cy

sWT

Health

ycarrier

Brazil

Presentstudy

10F

4years

Prem

aturep

ubarcheBA

810yrs

at65y

rsof

age

27

pPro2

67Le

uWT

Health

ycarrier

Brazil

Presentstudy

11F

Adult

Acne

139

pTh

r450

MetW

THealth

ycarrier

Brazil

Presentstudy

lowast

Referencev


6and30

nMbasaland60


lowastlowast

Neonatalreference

valuecut-o

fflevel100

nM

lowastlowastlowast

Haidere

tal2013

[18]m

utationisrepo

rted

butn

opatie

ntph

enotyped

escriptio

nNAnot

availableSW

saltw

astin

gNC

nonclassicB

Abon

eageW

Twild

type











3 Results







NC 21OHDreferences




0102030405060708090

100

Enzy

me a

ctiv

ity (

)

0102030405060708090

100

Enzy

me a

ctiv

ity (

)

Wild

type

pA

la15

Thr

pPr

o482

Ser

pPr

o453

Ser

pVa

l281

Leu

pIle

172A

sn

pLe

u12M

etp

Pro2

67Le

up

Arg

16Cy

sp

Ser1

01A

snp

Ser2

02G

lyp

Thr4

50M

et

pSe

r113

Phe

pTh

r450

Pro

pG

ln38

9_A

la39

1del

Wild

type

pA

la15

Thr

pPr

o482

Ser

pPr

o453

Ser

pVa

l281

Leu

pIle

172A

sn

pLe

u12M

etp

Pro2

67Le

up

Arg

16Cy

sp

Ser1

01A

snp

Ser2

02G

lyp

Thr4

50M

et

pSe

r113

Phe

pTh

r450

Pro

pG

ln38

9_A

la39

1del




min)

119870119898(120583M) 119881max119870119898


4 Discussion






Table3En

zymeactiv

itiesinsilico

predictio

nof

theeffecto

nproteinstr

uctureand

finalpredictio

nof

theph

enotypeforthe

novelm

utations

andforthe

common

mutations

used

asa

reference(in

bold)

Mutation(cDNA)

Proteinchange

Locatio

nin

the

protein

Insilico

predictio

nEn

zymea

ctivity

Phenotypep

rediction

(hem

izygou

sho

mozygou

sstate)

17OHP

Prog

c43GgtA

pAla15Th

rFirsth

ydroph

obic

domain

ND

100(0)

96(6)

Normalvarian

t(NV)

c34CgtA

pLeu12M

etFirsth

ydroph

obic

domain

ND

99(1)

100(1)

NV

c800Cgt

TpPro267Leu

Loop

between

120572-helixHand

120572-helixI

Noeffect

97(1)

87(7)

NVverymild

NC

c46CgtT

pArg16Cy

sFirsth

ydroph

obic

domain

ND

95(3)

81(3)

NVverymild

NC

c301

302T

CgtAA

pSer101As

nLo

opbetween

120572-helixB1015840

and

120572-helixC

Minor

effect

94(3)

74(2)

NVverymild

NC

c144

4CgtT

pPro4

82Ser

120573-she

et1205739

ND

91(6)

61(6)

Very

mild

NC

c604AgtG

pSer202Gly

Loop

between

120572-helixFand

120572-helixF1015840

Minor

effect

85(2)

81(3)

Very

mild

NC

c1349CgtT

pThr450Met

120573-sheet1205738

Minor

effect

78(6)

43(5)

Mild

NC

c1357CgtT

pPro4

53Ser

120573-she

et1205738

ND

73(8)

34(10)

NC

c841GgtT

pVa

l281Le

u120572-helix

IND

57(8)

29(5)

NC

c338Cgt

TpSer113Ph

eLo

opbetween

120572-helixB1015840

and

120572-helixC

Severe

effect

interfe

resw

ithactiv

esite

helices

4(1)

4(2)

CL

c1348AgtC

pThr450Pro

120573-sheet1205738

Very

severe

effect

lt1

lt1

CLc5

15Tgt

ApIle

172A

sn120572-helix

END

lt1

lt1

CLc1165117

3delCA

AGGCG

CCpGln389Ala391del

120572-helixL

Deleterious

0lt1

CLEn

zymea

ctivity

isexpressedin

of

wild

-type

activ

ityw

ithvalues

presentedas

mean(1SD

)ofatleastfour

independ

entexp

erim

ents

NDnot

determ

inedM

utations

areo

rdered

accordingto

ther

esidualenzym

eactiv

itywith

ther

eference

mutations

inbo

ld


(a)

(b)

(c)

(d)











5 Conclusions


Competing Interests


Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























3 Results







NC 21OHDreferences




0102030405060708090

100

Enzy

me a

ctiv

ity (

)

0102030405060708090

100

Enzy

me a

ctiv

ity (

)

Wild

type

pA

la15

Thr

pPr

o482

Ser

pPr

o453

Ser

pVa

l281

Leu

pIle

172A

sn

pLe

u12M

etp

Pro2

67Le

up

Arg

16Cy

sp

Ser1

01A

snp

Ser2

02G

lyp

Thr4

50M

et

pSe

r113

Phe

pTh

r450

Pro

pG

ln38

9_A

la39

1del

Wild

type

pA

la15

Thr

pPr

o482

Ser

pPr

o453

Ser

pVa

l281

Leu

pIle

172A

sn

pLe

u12M

etp

Pro2

67Le

up

Arg

16Cy

sp

Ser1

01A

snp

Ser2

02G

lyp

Thr4

50M

et

pSe

r113

Phe

pTh

r450

Pro

pG

ln38

9_A

la39

1del




min)

119870119898(120583M) 119881max119870119898


4 Discussion






Table3En

zymeactiv

itiesinsilico

predictio

nof

theeffecto

nproteinstr

uctureand

finalpredictio

nof

theph

enotypeforthe

novelm

utations

andforthe

common

mutations

used

asa

reference(in

bold)

Mutation(cDNA)

Proteinchange

Locatio

nin

the

protein

Insilico

predictio

nEn

zymea

ctivity

Phenotypep

rediction

(hem

izygou

sho

mozygou

sstate)

17OHP

Prog

c43GgtA

pAla15Th

rFirsth

ydroph

obic

domain

ND

100(0)

96(6)

Normalvarian

t(NV)

c34CgtA

pLeu12M

etFirsth

ydroph

obic

domain

ND

99(1)

100(1)

NV

c800Cgt

TpPro267Leu

Loop

between

120572-helixHand

120572-helixI

Noeffect

97(1)

87(7)

NVverymild

NC

c46CgtT

pArg16Cy

sFirsth

ydroph

obic

domain

ND

95(3)

81(3)

NVverymild

NC

c301

302T

CgtAA

pSer101As

nLo

opbetween

120572-helixB1015840

and

120572-helixC

Minor

effect

94(3)

74(2)

NVverymild

NC

c144

4CgtT

pPro4

82Ser

120573-she

et1205739

ND

91(6)

61(6)

Very

mild

NC

c604AgtG

pSer202Gly

Loop

between

120572-helixFand

120572-helixF1015840

Minor

effect

85(2)

81(3)

Very

mild

NC

c1349CgtT

pThr450Met

120573-sheet1205738

Minor

effect

78(6)

43(5)

Mild

NC

c1357CgtT

pPro4

53Ser

120573-she

et1205738

ND

73(8)

34(10)

NC

c841GgtT

pVa

l281Le

u120572-helix

IND

57(8)

29(5)

NC

c338Cgt

TpSer113Ph

eLo

opbetween

120572-helixB1015840

and

120572-helixC

Severe

effect

interfe

resw

ithactiv

esite

helices

4(1)

4(2)

CL

c1348AgtC

pThr450Pro

120573-sheet1205738

Very

severe

effect

lt1

lt1

CLc5

15Tgt

ApIle

172A

sn120572-helix

END

lt1

lt1

CLc1165117

3delCA

AGGCG

CCpGln389Ala391del

120572-helixL

Deleterious

0lt1

CLEn

zymea

ctivity

isexpressedin

of

wild

-type

activ

ityw

ithvalues

presentedas

mean(1SD

)ofatleastfour

independ

entexp

erim

ents

NDnot

determ

inedM

utations

areo

rdered

accordingto

ther

esidualenzym

eactiv

itywith

ther

eference

mutations

inbo

ld


(a)

(b)

(c)

(d)











5 Conclusions


Competing Interests


Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NC 21OHDreferences




0102030405060708090

100

Enzy

me a

ctiv

ity (

)

0102030405060708090

100

Enzy

me a

ctiv

ity (

)

Wild

type

pA

la15

Thr

pPr

o482

Ser

pPr

o453

Ser

pVa

l281

Leu

pIle

172A

sn

pLe

u12M

etp

Pro2

67Le

up

Arg

16Cy

sp

Ser1

01A

snp

Ser2

02G

lyp

Thr4

50M

et

pSe

r113

Phe

pTh

r450

Pro

pG

ln38

9_A

la39

1del

Wild

type

pA

la15

Thr

pPr

o482

Ser

pPr

o453

Ser

pVa

l281

Leu

pIle

172A

sn

pLe

u12M

etp

Pro2

67Le

up

Arg

16Cy

sp

Ser1

01A

snp

Ser2

02G

lyp

Thr4

50M

et

pSe

r113

Phe

pTh

r450

Pro

pG

ln38

9_A

la39

1del




min)

119870119898(120583M) 119881max119870119898


4 Discussion






Table3En

zymeactiv

itiesinsilico

predictio

nof

theeffecto

nproteinstr

uctureand

finalpredictio

nof

theph

enotypeforthe

novelm

utations

andforthe

common

mutations

used

asa

reference(in

bold)

Mutation(cDNA)

Proteinchange

Locatio

nin

the

protein

Insilico

predictio

nEn

zymea

ctivity

Phenotypep

rediction

(hem

izygou

sho

mozygou

sstate)

17OHP

Prog

c43GgtA

pAla15Th

rFirsth

ydroph

obic

domain

ND

100(0)

96(6)

Normalvarian

t(NV)

c34CgtA

pLeu12M

etFirsth

ydroph

obic

domain

ND

99(1)

100(1)

NV

c800Cgt

TpPro267Leu

Loop

between

120572-helixHand

120572-helixI

Noeffect

97(1)

87(7)

NVverymild

NC

c46CgtT

pArg16Cy

sFirsth

ydroph

obic

domain

ND

95(3)

81(3)

NVverymild

NC

c301

302T

CgtAA

pSer101As

nLo

opbetween

120572-helixB1015840

and

120572-helixC

Minor

effect

94(3)

74(2)

NVverymild

NC

c144

4CgtT

pPro4

82Ser

120573-she

et1205739

ND

91(6)

61(6)

Very

mild

NC

c604AgtG

pSer202Gly

Loop

between

120572-helixFand

120572-helixF1015840

Minor

effect

85(2)

81(3)

Very

mild

NC

c1349CgtT

pThr450Met

120573-sheet1205738

Minor

effect

78(6)

43(5)

Mild

NC

c1357CgtT

pPro4

53Ser

120573-she

et1205738

ND

73(8)

34(10)

NC

c841GgtT

pVa

l281Le

u120572-helix

IND

57(8)

29(5)

NC

c338Cgt

TpSer113Ph

eLo

opbetween

120572-helixB1015840

and

120572-helixC

Severe

effect

interfe

resw

ithactiv

esite

helices

4(1)

4(2)

CL

c1348AgtC

pThr450Pro

120573-sheet1205738

Very

severe

effect

lt1

lt1

CLc5

15Tgt

ApIle

172A

sn120572-helix

END

lt1

lt1

CLc1165117

3delCA

AGGCG

CCpGln389Ala391del

120572-helixL

Deleterious

0lt1

CLEn

zymea

ctivity

isexpressedin

of

wild

-type

activ

ityw

ithvalues

presentedas

mean(1SD

)ofatleastfour

independ

entexp

erim

ents

NDnot

determ

inedM

utations

areo

rdered

accordingto

ther

esidualenzym

eactiv

itywith

ther

eference

mutations

inbo

ld


(a)

(b)

(c)

(d)











5 Conclusions


Competing Interests


Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table3En

zymeactiv

itiesinsilico

predictio

nof

theeffecto

nproteinstr

uctureand

finalpredictio

nof

theph

enotypeforthe

novelm

utations

andforthe

common

mutations

used

asa

reference(in

bold)

Mutation(cDNA)

Proteinchange

Locatio

nin

the

protein

Insilico

predictio

nEn

zymea

ctivity

Phenotypep

rediction

(hem

izygou

sho

mozygou

sstate)

17OHP

Prog

c43GgtA

pAla15Th

rFirsth

ydroph

obic

domain

ND

100(0)

96(6)

Normalvarian

t(NV)

c34CgtA

pLeu12M

etFirsth

ydroph

obic

domain

ND

99(1)

100(1)

NV

c800Cgt

TpPro267Leu

Loop

between

120572-helixHand

120572-helixI

Noeffect

97(1)

87(7)

NVverymild

NC

c46CgtT

pArg16Cy

sFirsth

ydroph

obic

domain

ND

95(3)

81(3)

NVverymild

NC

c301

302T

CgtAA

pSer101As

nLo

opbetween

120572-helixB1015840

and

120572-helixC

Minor

effect

94(3)

74(2)

NVverymild

NC

c144

4CgtT

pPro4

82Ser

120573-she

et1205739

ND

91(6)

61(6)

Very

mild

NC

c604AgtG

pSer202Gly

Loop

between

120572-helixFand

120572-helixF1015840

Minor

effect

85(2)

81(3)

Very

mild

NC

c1349CgtT

pThr450Met

120573-sheet1205738

Minor

effect

78(6)

43(5)

Mild

NC

c1357CgtT

pPro4

53Ser

120573-she

et1205738

ND

73(8)

34(10)

NC

c841GgtT

pVa

l281Le

u120572-helix

IND

57(8)

29(5)

NC

c338Cgt

TpSer113Ph

eLo

opbetween

120572-helixB1015840

and

120572-helixC

Severe

effect

interfe

resw

ithactiv

esite

helices

4(1)

4(2)

CL

c1348AgtC

pThr450Pro

120573-sheet1205738

Very

severe

effect

lt1

lt1

CLc5

15Tgt

ApIle

172A

sn120572-helix

END

lt1

lt1

CLc1165117

3delCA

AGGCG

CCpGln389Ala391del

120572-helixL

Deleterious

0lt1

CLEn

zymea

ctivity

isexpressedin

of

wild

-type

activ

ityw

ithvalues

presentedas

mean(1SD

)ofatleastfour

independ

entexp

erim

ents

NDnot

determ

inedM

utations

areo

rdered

accordingto

ther

esidualenzym

eactiv

itywith

ther

eference

mutations

inbo

ld


(a)

(b)

(c)

(d)











5 Conclusions


Competing Interests


Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

(b)

(c)

(d)











5 Conclusions


Competing Interests


Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























5 Conclusions


Competing Interests


Acknowledgments


References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Functional and Structural Consequences of ...

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