An insertion/deletion polymorphism in the
angiotensin I-converting enzyme gene
accounting for half the variance of serum
enzyme levels.
B Rigat, … , P Corvol, F Soubrier
J Clin Invest.
1990;
86(4)
:1343-1346.
https://doi.org/10.1172/JCI114844
.
A polymorphism consisting of the presence or absence of a 250-bp DNA fragment was
detected within the angiotensin I-converting enzyme gene (ACE) using the endothelial ACE
cDNA probe. This polymorphism was used as a marker genotype in a study involving 80
healthy subjects, whose serum ACE levels were concomitantly measured. Allele
frequencies were 0.6 for the shorter allele and 0.4 for the longer allele. A marked difference
in serum ACE levels was observed between subjects in each of the three ACE genotype
classes. Serum immunoreactive ACE concentrations were, respectively, 299.3 +/- 49, 392.6
+/- 66.8, and 494.1 +/- 88.3 micrograms/liter, for homozygotes with the longer allele (n = 14),
and heterozygotes (n = 37) and homozygotes (n = 29) with the shorter allele. The
insertion/deletion polymorphism accounted for 47% of the total phenotypic variance of
serum ACE, showing that the ACE gene locus is the major locus that determines serum
ACE concentration. Concomitant determination of the ACE genotype will improve
discrimination between normal and abnormal serum ACE values by allowing comparison
with a more appropriate reference interval.
Research Article
Rapid Publication
An Insertion/Deletion Polymorphism
in
the
Angiotensin I-converting Enzyme
Gene Accounting
for Half the Variance
of
Serum
Enzyme Levels
Brigitte Rigat, Christine Hubert, Francois Alhenc-Gelas, Franmois Cambien,* Pierre Corvol, and Florent Soubrier
Institut National de laSanteetRecherche Medicale U 36, College de France, 75005Paris;and*INSERJM U 258, 75014 Paris, France
Abstract
A polymorphism consisting of the presence or absence ofa
250-bp DNA fragment was detected within the angiotensin I-converting enzyme gene(ACE) using the endothelial ACE cDNA probe. This polymorphismwasusedas amarker geno-type in a study involving 80 healthy subjects, whose serum
ACE levels wereconcomitantly measured. Allele frequencies were0.6 for the shorter allele and0.4 for the longer allele.A
markeddifferenceinserumACE levelswasobserved between subjects in each of the three ACE genotype classes. Serum immunoreactive ACE concentrations were, respectively, 2993±49, 392.6±66.8,and494.1±883
,g/liter,
for homozy-gotes with the longer allele (n = 14), and heterozygotes (n=37)and homozygotes (n= 29)with the shorter allele. The insertion/deletion polymorphism accounted for 47% of the total phenotypic variance ofserum ACE, showing that the ACE gene locus is the major locus that determines serum ACE concentration. Concomitant determination of the ACE geno-type willimprove
discrimination
between normal and abnormalserumACE values byallowingcomparisonwitha more appro-priate reference interval. (J. Clin. Invest. 1990. 86:1343-1346.)Key words: genepolymorphism-genomicDNA
hybrid-ization
Introduction
Angiotensin I-converting enzyme
(ACE)'
(kininase II, EC 3.4.15.1) isazinc metallopeptidasewhose main known func-tionsare to convertangiotensinIintothevasoactiveand aldo-sterone-stimulating peptide angiotensin II, and to inactivate bradykinin (1). ACE is believed to have other physiological rolesbecauseof itswide enzymatic specificityandwide distri-bution(2).Thus, ACEis foundas amembrane-bound enzyme inendothelialcellsanddifferenttypesofepithelialandneuro-AddresscorrespondencetoFlorentSoubrier, INSERMU36,College
deFrance,3rued'Ulm,75005 Paris,France.
Receivedforpublication21May 1990 andinrevisedform28 June 1990.
1.Abbreviations usedin thispaper: ACE, angiotensinI-converting
enzyme;BMI, bodymassindex;DBP,diastolic bloodpressure; HMW,
high molecularweight; RFLP, restriction fragment length
polymor-phism; RIA, radioimmunoassay;SBP,systolicblood pressure.
epithelial cellsas well as in a circulating form in biological fluids, such as plasma and amniotic or seminal fluids. The mechanismsleadingtothebiosynthesis of thecirculatingform of ACEarestillunclear, but all the data available indicate that its structures is very similartothat of the cellular form (3).
Plasma ACE measurement iswidelyused for the diagnosis and follow-up of sarcoidosis because anelevation of the
en-zyme is often observed in this disease (4). Although plasma ACEconcentrationsareremarkablystable when measured
re-peatedlyinanormalsubject, largeinterindividual differences make it difficult to interpret plasma ACE levels in a given patient,asthe reference interval for normal values is large (5). The results ofastudyconducted inalarge sampleof healthy families showed an intrafamilial resemblance between ACE levels andalsosuggested thattheyaresubjecttotheeffect ofa
major gene (6).
Marker genotypes are useful tools to identify the alleles exerting agenetic effect on aquantitativetrait (7). Inan
at-tempttodefine the role of the ACE gene in the genetic control ofcirculating ACE we used the human endothelial ACE cDNAtodetectDNApolymorphismsatthe ACE gene locus.
An insertion/deletion polymorphism was identified at this locus and used as a marker genotype ina population study designed to explore the relationship between this polymor-phismand the serumACEconcentration.
Methods
Subjects
80healthy Caucasians (38males and 42females)wereselectedforthe
studyinacenterfor preventive medicine. Inclusionwasbasedbothon
clinical characteristicsand routine laboratorytestsperformedatthe
centre.Inclusion criteria were abodymassindex<28kg/m2,normal
bloodpressure on WHOcriteria (systolicblood pressure <160mmHg anddiastolicblood pressure<90mmHg measured in thesitting
posi-tionaftera5-minrest),glycemia<6mmol/liter,plasmagamma
glu-tamyltranspeptidase <30UI/liter, normalsedimentationrate, nor-mal chest x-ray, andabsence ofacute orchronic disease and drug
intake.
10 normalfamilies comprisingtwoparents and at least two
chil-drenwereselectedaccordingtothe samecriteria,to test themendelian inheritance oftheinsertion/deletion polymorphism foundat theACE gene locus(see Results).
Materials and experimental protocols
Serum ACE measurement. SerumACEconcentrationwasmeasured
in duplicate by direct radioimmunoassay(5).
DNA extraction.High-molecular weightDNA wasisolated from peripheralbloodleukocytesbystandardtechniques (8).DNA
concen-trationsweremeasuredby absorbanceat260nm.
HumanACEcDNAprobe. Thecomplete humanendothelial ACE
cDNA(clonespG19-22andpG21-11)wasusedinafirst approachto J.Clin. Invest.
© TheAmericanSocietyforClinical Investigation,Inc.
0021-9738/90/10/1343/04 $2.00
thedetection of restrictionfragmentlengthpolymorphism(RFLP)on
the human ACE gene(9). For subsequent detection of theinsertion/ deletion polymorphism, a 584-bp BanI restrictionfragment
corre-sponding to nucleotidepositions 2123to2707 of thepublishedcDNA
sequence (9) wasroutinely used. Inserts of plasmids pG19-22 and pG2 1-1 1, and theBan Ifragment of plasmid pG19-22,wereisolated from low-gelling temperature agarose gels (SeaPlaque, FMC Bio-products, Rockland, ME) and labeledathigh specificactivityby the random-primer labeling method (10) usingacommercial kit (Amer-shamInternational, Amersham, UK).
RFLP detection. Individual high-molecular weight DNAs were
digested by restriction enzymes (NewEnglandBiolabs, Beverly, MA)
undertheconditions advised by thesupplier,andweresubmittedto
electrophoresison0.7% agarosegels. DNAsweretransferred by
capil-lary blotting with alkalion nylon membranes (Hybond N+,
Amer-shamInternational)(1 1) andhybridizedtolabeledprobes,according
topreviously described protocols(8).Hybridizationandfilterwashing
were done underhigh stringency conditions (9).
Statisticalmethods. One-way analyses of variance were usedto compare groupmeansfor the different parameters studied.To account
for the association between meansand variancesof Ir-ACE levels in
thethree genotype groups,log-transformed valueswereusedfor the statisticaltest.Allelefrequencieswereestimated by the gene counting method andHardy-Weinberg'sequilibriumwaschecked by the chi-square test.Correction for biases of the estimated contribution of the locus to thephenotypic variance of ACE was computed according to Boerwinkle etal.(12). Pearson correlation coefficients were also com-puted when necessary.
Results
Clinicalparameters. The values for age, body massindex (BMI),systolicanddiastolicblood pressure (SBP and DBP) for menand women arelisted in Table I. BMI, which was slightly greater for menthan for women, was the only parameter that differedsignificantlybetween sexes(P<0.01).
Serum ACE concentrations. Serum ACE concentrations didnotdiffersignificantly betweenmenand women and were notcorrelated to age, SBP, DBP, or BMI (Table I). Conse-quently, these variableswere notadjusted forsubsequent anal-ysis (Table II).The mean value anddispersion of serum ACE levels in this population were similar to those observed in previous studies ofhealthysubjects (5, 6, 12a).
Identification ofaninsertion/deletion polymorphismatthe ACEgenelocus. Todetect DNA polymorphisms, gel-blot hy-bridization experiments were made on genomic DNA in 15
TableII.Clinical Parameters and Serum ACE Values in Groups
with
Diferent
ACEGenotypesII ID DD
Genotype n =14 n =37 n =29
Age 44.4±7.4 44.3±7.5 45.0±7.0
BMI
(Kg/m2)
22.9±3.1 23.2±2.6 22.0±2.3SBP(mmHg) 124.3±11.1 124.6±9.5 122.4± 10.5 DBP(mmHg) 77.1±8.2 76.9±6.7 77.1±7.3
Ir-ACE*
(Ag/liter)
299.3±49.0 392.6±66.8 494.1±88.3 Ir-ACE Ln(.ug/liter)
5.69±0.15* 5.96±0.17t 6.19±0.19tIcorresponds to the larger allele andD tothe shorterallele.Ir-ACE,
serumconcentrations obtained by directRIAof ACE. Ir-ACELn,
logarithm ofserumACEconcentrations. Resultsare means ±SD.
*For Ir-ACE comparisons, the testwasonly carriedout on log-trans-formed values.
tComparisonbetween genotype groups; P<0.001.
individuals, usingthe completeendothelialACE cDNA as a probe. A two-allele RFLP at the ACE locus was detected with thefollowing restriction enzymes: Xba I, Hind III, Bgl II, Kpn
I, and BamH I. Analysis of the fragment sizes obtained with these enzymesshowed that RFLPs resulted from a - 250-bp
insertion/deletion polymorphism. Hybridization of the filters with different parts of the cDNA showed that this polymor-phism could be detected by a 584-bpBan I restriction frag-ment,locatedbetweenpositions 2123and 2707ofthe cDNA sequence(9).For therestof thestudythisfragmentwas there-fore usedas aprobe, and the polymorphismwasdetected with the restriction enzymeHindIII.Theinseytion/larger alleleis designatedI, and thedeletion/shorterallele,D.
Genetic characterizationoftheinsertion/deletion polymor-phism. Mendelian inheritance ofthegenetic polymorphismat theACEgenelocus wasestablished in10normal nuclear fami-lies. Allele segregation in one nuclear family, analyzed by DNAgel-blothybridization, isshowninFig. 1. Inthis family, the fatherwashomozygote DD,themotherwashomozygote II, andbothchildren wereheterozygotes IDforthe polymor-phism.
ACEgenotypes weredetermined forallsubjects(TableII). Allelefrequencieswere0.406 forthe I alleleand 0.594forthe Dallele. The observed genotype distribution (II = 0.18, ID
TableI. Clinical Data and Serum ACE Concentrations of80Healthy Subjects
Men Women
n=38 n=42
Variable
Age 43.3±7.0 45.8±7.3
BMI
(Kg/IM2)
23.6±2.5 21.9±2.4*SBP(mmHg) 124.7±8.5 122.9±11.3
DBP(mmHg) 78.2±7.4 75.9±6.7 Ir-ACE
(ug/liter)
408.5±98.2 417.1±103.5Ir-ACE, serum ACE concentrations measured by direct RIA of ACE. Resultsaremeans ±SD.
*Comparisonbetween men and women:P <0.01.
1
2
3
4
a
-,r
9.5 0
>
9.25
Figure1. DNAgel-blot hybridization
experi-mentswith theBan I-Ban Irestriction
frag-mentof the ACE cDNA. GenomicDNA
digestionof members of
anuclearfamily bythe
restriction enzyme HindIII.DNAfrom thefather, mother,and
twochildrenareshown,
respectively, inlanes1, 2, 3, and4. DNA
frag-mentsizes,inkilobases,
areindicatedby
=0.46, and DD = 0.36) was in agreement with the Hardy-Weinberg proportion.
Relationship between the ACE genotype and serum ACE level. MeanserumACE levelswerethencomparedamong the three groups definedbythe ACEgene's polymorphism. The resultsof thiscomparison, giveninTableIIandFig. 2,showa
significant relationshipbetween the polymorphism and the levelofACE inthe serum ACElevel, withanadditive effectof the alleles. Thepolymorphismsaccounted for 47% of the vari-ance in serum ACE levels.
Discussion
Thestability ofthecirculatingACE level inagivenindividual allowsstudy ofthefactorsinfluencingitslong-term regulation. Alarge-scale studyofplasma ACE levels conducted in normal nuclear families has shown important interindividual varia-tion anddemonstrated the presence ofafamilialaggregation ofACE values dueto ageneticcontrol(6).Totestthe hypoth-esisthatanallelic variantof the ACE gene itselfwas responsi-ble for thiscontrol, we designed a studyto explore the rela-tionship between circulatingACE levels and ACE gene poly-morphisms.
Wedetected RFLPsattheACEgene locus usinganACE cDNAprobethatspanned complete endothelialACE mRNA sequence. With this probe,wepreviouslyshowed thatasingle gene codesfor ACEinman(9). Here,withmostof the restric-tion enzymes tested, we detected a two-allele RFLP
corre-sponding to an insertion/deletion polymorphism, withan al-lelic frequency of0.6forthe shorterallele and 0.4forthelarger allele. Analysis of polymorphic fragment segregation in ten
nuclear familiesdemonstrated the mendelian inheritance of thetwoidentified ACEgene alleles(Fig. 1).
Thepolymorphicinsertion (250 bp long) was located in-sidethe ACE geneasithybridized with internal fragments of the cDNA. Failure todetect the
insertion/deletion
polymor-phism withsomeoftherestrictionenzymesusingthe cDNAasprobe indicates thatthepolymorphic insertion isnotpresent in the cloned cDNA andisprobably flanked byintronic
se-quences. Genepolymorphisms caused bythe presenceor
ab-sence ofsequence insertions have been described in several mammaliangenes,for instancein the 5'flanking region ofthe humaninsulin-receptorgene and the 5'flanking region ofthe rat prolactin gene (13, 14). In both cases, sequence analysis revealed that DNAinsertions consisted of repetitiveelements. Allsubjects includedin the study were testedforACE gene polymorphisms by DNAgel-blot hybridization usingthe
re-strictionenzymeHindIII.When the levelofserumACE was comparedinsubjects ofthe three genotype classes (II, DD, and ID),amarkeddifferencewasfoundbetween the three groups. Subjects with the deletion polymorphism had higher ACE levels than thosewith theinsertion polymorphism. Interme-diatelevelswereobservedin heterozygotes(Table II andFig. 2). Thegenetic effectaccountedfor47% of the total variance ofserumACE. As the presenceofeach allele had an additive effect on serum ACE andtwo alleles had twice the effect of one, eachallelewascodominant.
Cambien etal. recently proposed a model forthegenetic controlofplasma ACE levels, based on the results of a family study(6).Inthisstudy, thegenetic analysis of familial pheno-typessuggested that these levels areaffected by a major gene, which was estimated to account for 29% of the total
pheno-730
700 600*
1-. 500
.Li 0 E c4
co
4001A
300- M±SD I
.
~~~~-200i n =14
Genotype 11
0
0 s
0~~~~o
"~~s
o~~~s
0 00 00
00~~~
00a
COO0 00
0 0
00
370 f
000 0
0
00
:37 n=29
ID DD
Figure 2.Serum immunoreactive ACE concentrations(ag/liter)for individual with theII,ID, andDD genotypes,respectively,shownin left, middle, and right panels. Solid vertical bars indicatemean
con-centration and standard deviationfor eachgroup.
typic variance of plasma ACE in adults. The present results confirm thismajor gene's effectas acleardifferenceinserum
ACE levelswas observed between the three genotype classes and the ACE genewasfoundtobe the
major
generesponsible
for thiseffect. Inadults,theinsertion/deletion
polymorphism
accountedfor about halfthe total
phenotypic variance,
a pro-portion somewhat larger than the 29% estimated from thefamily
study referredabove. Asonly47% oftheserum ACEvariance foundherewasduetothe alleleeffect, othergenetic
orenvironmentalfactorsmay beinvolvedin the interindivid-ualvariations in circulatingACE. However, in a studyof sev-eral candidate parameters (12a), noclear hormonal or envi-ronmental
influence
wasdetected.Gene alleleshave been showntoexertquantitative effects
on plasma concentrations ofother proteins. Thus, a Pvu II
polymorphism
ofthelipoprotein
lipasegenewasfoundtobeassociated with variationsin the concentrations ofserum tri-glycerides,butnosignificant relationshipwasfound withother polymorphisms ofthesamegene(15). Genetic variationatthe
fibrinogen
locus accountedforpartofthevariationinplasmafibrinogen
phenotypes (16). However, inboththesecases, thegenetic
effect appearedtobe weakerthan theserumACE-ge-notyperelationship, probablydueinpart tothe greater stabil-ity ofserumACE concentrations whicharelessinfluencedby environmentalfactorsthanfibrinogenandtriglyceridelevels.
CirculatingACEprobablyoriginates fromthe vascular
en-dothelial cells(1).ACEisa membrane-bound ectoenzymeof the cell
surface,
towhich it isanchoredbyacarboxyterminal hydrophobicdomain(9).Thebiochemical mechanismofcir-culating
ACEsecretion isnotclear,but severalhypothesescanbe made
including proteolytic
cleavage ofthe hydrophobic anchor andpassive leakage ofthe membrane-bound enzyme. Whatever the mechanism involved, it is likely that the ob-servedgenetic
control ofserum ACE level is exerted attheA
II
n=,,
14
transcriptionallevel. In that case, theinsertion/deletion itself maynotplay adirect part incontrollingACEtranscriptionbut is more likely to bein linkage disequilibrium withregulatory elementsof the ACE gene.
The present results of DNA gel-blothybridizationindicate thattheinsertion/deletion polymorphismis localized inside an intron. However, we cannotexclude the possibility that as a resultof differentACEpre-mRNA splicing, dependingonthe presence or absence of the insertion, part of this sequence mightbe present in the mature RNA, thusmodifyingits stabil-ityorgivingriseto thepresenceof an additional peptide inside theprotein, and consequently altering its secretionorstability. Suchpolymorphic insertionwas recentlydescribed inthe sig-nal peptideofthe apolipoprotein B gene (17). Lastly,the in-sertion itselfmight modify the splicing process ofthe ACE precursor mRNA by interfering with the lariat formation step (18).
Theobservation ofa geneticpolymorphismthatexplains much of theinterindividual variabilityin plasma ACE levels has clinical implications, particularly forthe diagnosis of granulomatous diseases,suchassarcoidosisinwhich circulat-ingACElevelsarehigh because of increasedACEsecretionby monocyte-derived cells (19). By determining thegenotype of patients,it may bepossibletoreducethesize of thereference interval to which agiven measurement ofthe plasma ACE level should be compared (Fig. 2). This would allow more accurate conclusions to be drawn about the
significance
of plasma ACElevelsinindividual patients (20). AsACE is im-plicated in vasoactive peptides metabolism, theACE geneisa candidate gene foressential hypertension. Consequently, the ACEgene polymorphismdescribed
here constitutes a poten-tial tool for genetic studies of hypertension.Acknowledgments
The authors wishtothank Drs.Champeau, Rene,andVilleneuvede Genty of the PreventionGenerale,Caisse Primaire d'Assurance Mala-die, Dr. Breda and Dr. Siest of the Center for PreventiveMedicine,
(Nancy), for selection of thesubjects,Mrs.D.Menard and C. Dollin fortechnical assistance,Mrs. M.Dreyfusforeditingthemanuscript,
Mrs. N. Braurefortypingthemanuscript,and Mrs.A.Depardieufor artwork.
Thiswork was supported by INSERM andbyagrantfrom Squibb LaboratoriesInc.
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