Expression of a missense mutation in the
messenger RNA for beta-myosin heavy chain in
myocardial tissue in hypertrophic
cardiomyopathy.
M B Perryman, … , R Hill, R Roberts
J Clin Invest.
1992;
90(1)
:271-277.
https://doi.org/10.1172/JCI115848
.
We have determined that a missense mutation in exon 13 of the beta-myosin heavy chain
(beta MHC) gene is expressed in the messenger RNA (mRNA) isolated from a right
ventricular endomyocardial biopsy obtained from the proband of a family with hypertrophic
cardiomyopathy. The mutation is the result of a substitution of an adenine for a guanine
residue in one allele of the beta MHC gene and creates a second recognition site for the
restriction endonuclease Ddel in exon 13. The mutation is inherited in a Mendelian fashion
and co-segregates with hypertrophic cardiomyopathy in this family. Complementary DNAs
synthesized from RNA isolated from the endomyocardial biopsy were cloned into a plasmid
vector and sequenced to confirm the expression of both the normal and mutant allele in
mRNA of myocardial tissue. This is the first report of the transcription of a mutant beta MHC
gene allele into mRNA of the myocardium.
Research Article
Find the latest version:
Rapid
Publication
Expression
of
a
Missense Mutation in the Messenger
RNA
for
,B-Myosin
Heavy
Chain in
Myocardial
Tissue in
Hypertrophic Cardiomyopathy
M. Benjamin Perryman, Qun-taoYu, Ali Jalilian Marian, Adolph Mares, Jr., GraceCzernuszewicz, Jonah lfegwu, RitaHill,
and Robert Roberts
Section of Cardiology, Baylor College ofMedicine, Houston, Texas, 77030
Abstract
Wehavedetermined thatamissense mutation inexon13 ofthe
#-myosin heavy
chain(fiMHC)
geneisexpressed
inthemessen-ger RNA
(mRNA)
isolated fromaright ventricular endomyo-cardialbiopsyobtained from the proband ofafamily with hy-pertrophic cardiomyopathy. The mutation is theresultofa sub-stitution ofanadenine foraguanine residuein onealleleof the/iMHC
gene and creates asecondrecognition site forthe restric-tionendonucleaseDdelin exon13.Themutationisinheritedin aMendelianfashionandco-segregates with hypertrophiccar-diomyopathy in this family. Complementary DNAs
synthe-sized fromRNAisolatedfrom the endomyocardial biopsywere
clonedintoaplasmid vectorandsequenced toconfirm the
ex-pression of both the normal and mutant allele in mRNA of myocardialtissue. This is the
first
reportof the transcription ofa mutant
#MHC
geneallele intomRNAof themyocardium.
(J. Clin. Invest. 1992. 90:271-277.) Keywords:hypertrophiccar-diomyopathy-moleculargenetics-mutation*
,-myosin
heavychain
Introduction
The first primary cardiomyopathy due to aninherited
defect
for which thegenehas been
identified
ishypertrophic
cardio-myopathy(HCM).'
Thedisease is inherited
inan autosomaldominant
mannerand theputative
disease geneis13-myosin
heavy
chain
(f3MHC)
locatedonchromosome14(1-4).
Several mutations have beenidentified
in the,3MHC
geneof affected
individuals
(5-9) and showntofollow Mendelian inheritance.f-myosin
heavy chain isexpressed in cardiac(10) and skeletal muscle(1 1) and is thepredominant
myosin
incardiac tissue. HCM is manifestedas amyriad
of clinicalfeatures including
sudden
death,
heartfailure,
syncope, andangina
with underly-ing pathology of left ventricularhypertrophy (12, 13). Thus,Addressreprint requests to Dr. Roberts, Section ofCardiology,Baylor College of Medicine, 6535 Fannin, MS F905, Houston,TX77030.
Receivedfor publication 13January 1992 and inrevisedform6
March 1992.
1. Abbreviations used in this paper: HCM,hypertrophic
cardiomyopa-thy;,BMHC,
f-myosin
heavychain; PCR, polymerasechainreaction;RT, reverse transcription. J. Clin. Invest.
©The American Society for Clinical Investigation, Inc.
0021-9738/92/07/0271/07 $2.00
Volume90,July 1992, 271-277
responsible for suchavaried phenotype particularly inasmuch
asearly studies on the 3MHC protein from patients with HCM showthe protein to be normal as determined by conventional techniques (14, 15). Although studies to date have shown the
f3-myosin
mutations to be present in genomic DNA (16), it remains to be determined whether the defect isexpressedin the tissue primarily responsible for the phenotype, namely, the car-diac myocyte. Documentation of the myosin mutation tran-scribed into the cardiac mRNA is an essential first step in deter-mining whether the defect is responsible for the disease. Accord-ingly,the present study was performed to determine whether a mutation in ,BMHC gene identified in afamily affected with HCM is transcribed into the mRNA of the cardiac myocyte. In afamily of 11 individuals(excludingspouses), six of which are affected withHCM, wehave shown that the DNA of the pro-band and affected individuals have a missense mutation in which adenine substitutes for guanine resulting in the substitu-tion of glutamine for arginine in 3MHC. The substitusubstitu-tionofadenine for guanine in exon 13 of the 3MHC gene creates a second recognition site in the exon for the restriction endonu-clease Ddel. Biopsy specimens of the heart were obtainedfrom
theproband and a portion of the 3MHC mRNAflankingthe exon13 mutation was converted to cDNA by reverse transcrip-tion and amplified using polymerase chain reaction (PCR). The amplifiedproduct wasdigestedwithDdel restriction en-zyme and both the normal and the mutant allele werefoundto be expressed.
Methods
Patientselection.Patients werediagnosedwith HCMbyusingthe echo-cardiographiccriteriadefined as thepresenceofseptalorventricular hypertrophywith a wall thickness of 13 mm or more in the absenceof other potentialcauses of hypertrophy such ashypertension (2). All procedures and protocols used in the evaluation of each subjectwere approved by the Institutional Review Boards of The Methodist Hospi-tal andBaylor College of Medicine. The pedigree referred to as number 155 wasestablished and eachfamilymemberwasevaluatedafter in-formedconsent wasobtained. In thecaseoftheproband,aright ventric-ularendomyocardial biopsywascollectedafterinformedconsent was
obtained.
Lymphocyte transformation and DNA isolation. Blood samples werecollected from all family membersof pedigree 155, DNA was
Amplification ofexon 13 ofi3MHC. The gene for
#MHC
was ampli-fied in theregionofexon13byPCR. Clonedwild-type genomicDNA in theregionsofJ3MHC exon 13 and the cloned mutant allele in the sameregioncontainingthe exon 13missense mutation from apatient with HCM werekindly providedby Dr. ChristineSeidman(Harvard Medical School, Boston) and were used as controls in all PCR and digestion reactions(6).Primer sequences weredesigned (18) byusing thepublishedflMHC
genesequence (19)and are asfollows: Sense-5'C-TCTTACCAACTTTGCTCTTGC3';and antisense-5'CTGCTGGA-CATTCTGCCCCTTGG3'. The sequence of the senseprimerwas de-signedtobecomplementaryto asequence inintron12located 35 bp upstreamfromexon 13 and theantisenseprimer complementaryto a sequence at the 3' endofexon13.Primeroligonucleotideswereused at afinalconcentration of0.2AM.
ThePCR reaction mixture contained 10mM Tris-HCl (pH8.3),50 mMKCI,0.001%(wt/vol)gelatin, 1.5 mMMgCI2, 200MM4dNTP,1 UThermus aquaticusDNA polymer-ase(5,000U/ml;Pharmacia,Inc.,Piscataway, NJ)and 250 ngof geno-micDNAper25 Mlofreactionvolume. DNAamplificationwas per-formed in a DNA thermalcycler(Perkin-Elmer Cetus, Norwalk, CT). The DNA wasinitiallydenatured at 94°C for 3 minfollowedby 30 cycles ofamplification accordingto the followingprotocol: denatur-ationat94°C for 1min, annealingat57°Cfor 30 s, and extension at 72°Cfor30 s.PCR products were separated by gelelectrophoresisin 2%NuSieve agarose(FMCBioproducts, Rockland, ME)inbuffer (50mMTris,50 mM boric acid, 1mMEDTA) at 100 Vfor2 h. The exon 13fragment wasisolated and extractedfromthe gelusingaSephaglas BandPrepkit (Pharmacia, Inc.). One-fourth of the volume containingthe eluted product was usedforPCRreamplificationusing conditionsand param-etersidenticaltothosedescribedabove. ThereamplifiedPCR product wasextracted andisolatedaccordingtothe procedure outlined above. ThePCR product (exon 13) was suspended in 20MLof Tris-EDTA bufferanddigested withtherestrictionendonuclease Ddel(48U)at 37°C for2 h. The productsofdigestionwereseparatedandidentified byethidiumbromidestaining after electrophoresisina3% agarosegel inTris-boric acid-EDTA bufferat100Vfor2 h.
Todeterminewhether infactthePCRprocedurewasspecifically amplifyingexon 13,theidentical protocolwasperformedonknown normal(individuals unaffected with HCM) DNA. Normalgenomic DNA wasamplifiedin theregion ofexon 13usingparametersidentical totheprocedureusedaboveforDNAamplification frommembersof pedigree 155. Theamplified product ofthe normal controls was iso-lated,extractedandsubjectedtodigestionwith the restriction endonu-cleases Rsal andDdel asdescribedabove.
Amplification of(3MHC
mRNAfromcardiac tissue.Aright ventric-ularendomyocardialbiopsywasobtained fromtheproband offamily 155.Theendomyocardial tissuewasfrozen rapidlyinliquidnitrogen forstorage at-135°C.Thetissue wasquicklythawed and homoge-nized bymeansofstandard methods at room temperature for total RNAisolation(RNAzolmethod,Cinna/Biotecx Laboratories, Hous-ton, TX) (20).Thehomogenate wasextracted withchloroformand total RNAwasprecipitatedinisopropanoland storedat-20°C over-night.Theprecipitatewasisolated bycentrifugationand washedtwice in75% ethanol. The pellet was gently Iyophilized and resuspended in 20Mlof sterilediethylpyrocarbonatedeionized water. Approximately 0.2Mgoftotal RNA was extractedfromasingle endomyocardial biopsy sampleand reverse transcription (RT) for the synthesis of the first strand cDNA wereperformed using50ng ofthe total RNA,utilizingan antisenseprimerto exon 16(5'ATCATCGATGTCCCTTTTGAGCT-CTGAGCACTCATCTTCC3'), by standard methodology (21). The conditions of theRTreaction were as follows: 50 ng of the total myo-cardialRNAwasadded to2Mlof reversetranscriptionbuffer(Sx HRT buffer[250mMTris-HCl,pH 8.3 at room temperature, 375mMKC1, 15mM
MgCI2J,
Gibco BRL,Gaithersburg,MD),and 1.5Mlof the 10 MMantisenseprimerto exon16 in a total volume of 10 Ml and dena-turedfor 5 min at85°C.RT wasaccomplishedbyadding 1MlofRTbuffer, 1.5
Mul
of0.1Mdithiothreitol, 1.5Mlof10mM 4dNTP, 0.5MlI RNasin (Promega Corp., Madison,WI) and 100 U Moloney murineleukemia virus RNase H- reverse transcriptase superscript (Gibco BRL)in a total volume of 15 Al. The final concentration ofantisense primer and 4dNTP in the mixture was 1 AM and 1 mM, respectively. The reaction was incubated at 420C for 2 h and was terminated by heat inactivation at 680C for 20 min.
The cDNA sequence was deduced from the genomic DNA se-quence forMHC (18). Primers were constructed to encompass the
regioncorrespondingtoregionswithinexons12-14of the cDNA. The sequence of the senseprimer wasdesignedtobecomplementaryto exon 12,(5'GAGGATCCCTCCATGTATAAGCTGACAGG3'), and that of the antisense primer (5TTAAGCTTTCTCCAGGGTGGCA-TTGATGCG3')to exon14;BamHl and Hindlilrestrictionsiteswere incorporated at the 5' end of the sense and antisenseprimers, respec-tively.
The RT product was diluted 1:4-fold with lx PCRbuffer[50 mM KCl, IO mM Tris-HCI (pH 9.0 at250C),1.5 mMMgCl2,0.01% gelatin (wt/vol) 0.1% Triton X-l00, PromegaCorp.] and wasamplifiedby PCR(RT-PCR).12.5,lofthedilutedRT product was added to 12.5 Ml of PCR mix which contained 0.2MM senseprimer,0.2 mMdNTP,1 U Taq DNA polymerase (Pharmacia, Inc.),resultingin a final concentra-tion of 0.1 MuM sense and antisenseprimersand 0.2mM dNTP. Cycling parameters for PCR were 950C for 3 min for one cycle followed by 35 cyclesof 940C for 1
min,
550C for 1min,
720C for1min,and afinal extension time of 4minat720C. The PCR product was separated by gelelectrophoresisona 2.5%lowmelting pointagarosegel.Theproduct was excised from thegelanddigested byDdel. The digestionwasperformedin a total volume of 60 Mlusingthe conditions outlinedpreviouslyfor thedigestion ofthe PCRproductofexon13.
Radiolabelingandelectrophoresis of the Ddel digestion products. TheamplifiedcDNAproduct wasdigestedby Ddel and labeled with 32Patthe 5' endusingstandardmethodology(22).Finalreaction condi-tions included 50 mM Tris-HCl (pH7.6), 10 mMMgCl2, 15 mM dithiothreitol and 10 UPolynucleotide Kinase (Pharmacia, Inc.)in a total volume of 30 MAl at 370C for 45 min. The reaction was terminated byheatinactivationat650C for 10 min. The products were precipi-tatedbyethanolusingstandardmethods andlyophilized.The pellet wasresuspended in 20MAl of sterile, deionizedwater and analiquotwas electrophoresed ona6% polyacrylamide gelfor 75 min. The DNA markerXOX174Hinflwaslabeled atthe 5' endwith 32Pand used as a size standard.
Cloningand sequencing.As aresult of incorporation of restriction sitesatthe 5' endoftheprimers,the321-bpRT-PCR product con-tainedBamHl andHindlll restriction sitesatends.The RT-PCR prod-uctwasdigestedwiththe above enzymes andpurified usinga
Centri-con100 ultrafiltration system(Amicon Corp.,Danvers, MA). The puri-fied productwascloned intoapGEMvectorand the recombinant DNA waspurifiedandsequenced bythedideoxy sequencingmethod usingaT7sequencingkit(Pharmacia, Inc.).
Results
Studieswere
performed
on afamily
(pedigree 155) consisting
of15
individuals including
spouses.Theproband had the clini-calfeatures of HCM and left ventricular hypertrophywascon-firmed
by two-dimensionalechocardiography
and shown tosatisfy
thediagnostic
criteriaaspreviously
described (2). Themutation,
a substitution of adenine for theguanine residue,
results in the creation ofa second recognition site for Ddel restriction endonuclease in themutantallele. TheDNA analy-siswas
performed using
PCRtoamplify
exon 13followed bydigestion
of theamplified
product
with the restrictionendonu-clease Ddel. Thesyntheticoligonucleotides used forPCR analy-siswereselected to
amplify
exon13 sequencesbetweennucleo-tides8744and 8897ofthe
fMHC
gene.After PCRamplifica-tionthe 154-bp DNA products were separated by agarose gel
Normal Mutant Dde I Dde I
site site
Pri
| l_Primer
--I
Primer
PCR
I
Product
Expected
Digestion
Pattern
Gel
Electrophoresis
xs)52
bp-32
bp--o~~~~~~~~~~~r
exon 13 4 PCR 154 bp
Dde I digestion
4
-Cas r *T.
I)Df11 ,a
_a
V
51)"C -~
i~m
Figure 1. Aschematic diagram of the relative locationsofthe normal and mutant Ddel re-striction sites inexon13 of the ,BMHCgeneis showninthe upperportion of the figure.The 154-bp PCRproduct andtherelative size of thenucleotidefragments produced after diges-tionof this product with Ddelarealso de-picted.The lowerportion ofthisfigureis a photograph ofanethidium bromide-stained agarosegel. It illustrates theelectrophoretic patternofthe PCR product (exon 13 of the
#MHC
geneamplified
fromDNAof members of Family 155)after digestionwith Ddel re-striction endonuclease. Lane 2 is wild-typeexon 13 DNAdigestedwith Ddel and shows twofragments of84 and 70 bp; lane 4 is exon
13 DNA from the mutant allele cloned from apatient withHCM and known missense mu-tationdigestedwith Ddel and shows three fragments of70, 52, and 32 bp. Exon 13 PCR productfroma normalindividual(containing
twonormalalleles) afterdigestionwith Ddel showstwofragmentsof 84 and 70 bp and exon
13 PCR productfromaffected individuals (containingonenormal and one mutant allele) showsfour fragmentsof84, 70,52, and 32bp. 151 hp The individuals from pedigree 155 are
indi-cated above theappropriatelanes.Size stan-82 bp dardsarein theextremerightlane and
undi-48 hp gested anddigestedPCR productsfroma nor-malindividualand the clonedmissense allele
arelabeledNormalandHCM, respectively.
Ddel endonuclease. DNA template
isolated
from normalindi-viduals
produces two DNAfragments of
84 and 70 bp whenanalyzed by
thisprocedure.
DNAisolated fromanindividual with the missense mutation, however, producesfour fragments of84, 70, 52,
and 32 bp in that both the normal andmutantallelearepresent
(Fig. 1).
GenomicDNAanalysis
showedthe probandtohaveamissense mutation inexon13ofthe ,BMHCgeneand all threechildren of the probandwere
positive for
the missense mutation while DNAfrom
theunaffected
husbandwasnormal (Fig. 1). Two of the childrenexhibited clinical
fea-tures
of HCM,
whereas thethird
child,
alsowith
themutation,
exhibits
noclinical features of
HCM. However, he isonly 11 yrof
age,and becausesymptomatology
isoften
notevident until
later in
life, it is
premature tomakeadefinitive diagnosis.
To determine if the
mutation is expressed
as mRNA in myocardial tissue, RNA wasisolated fromaright
ventricular endomyocardial biopsy obtained from the proband offamily
155 andwasusedas atemplate for RT-PCR. The cDNAwas
synthesized using
anoligonucleotide
primer
fromexon 16for
first strand transcription and nested oligonucleotide primers located in exons 12and 14forcDNA
amplification.
Theex-pected
321bp
RT-PCRproduct
wasproduced
fromRNAtem-plate isolated from
myocardium
ofan unaffected individualand that ofthe proband as shownin
Fig.
2.Genomic DNAtemplate
isolated fromtheproband
producedtheexpected872bp
(Fig.
2) product duetointronicsequences not presentin themRNA template. In lanes 3 and 4, there are low molecular weightnonspecific PCR products. To ensurethat the
nonspe-cific products donotinterfere with subsequent studies, the
321-bp productwasexcised from the gel and purifiedasdescribed
above and
digested
withDdelrestrictionenzyme.The restric-tionfragments
were5' end labeled with 32P and electrophoresedon acrylamidegels. The resulting autoradiogram anda sche-maticdrawing illustrating the resultsareshown inFig. 3. Diges-tion of cDNA from normal
myocardium
would be expectedtoproducetworestriction fragments of181 and 140 bp,whereas
digestion
of cDNA produced from RNAisolated
from themyocardial
biopsyof
the probandproducesfragments of 181,140, 149,
and32 bp in length duetothe expressionof
both the normal andmutantallele(Fig. 3). The lattercDNArestriction fragments fromtheprobandresultfromthetranscription ofasecond Ddel restriction site in mRNA correspondingto the
missensemutation inexon 13of the
3MHC
gene. Toconfirm thelocation of the mutation inthecDNAboththenormal and mutantRT-PCRproducts were cloned into a pGEM vectorfor nucleotidesequenceanalysis. Sequence analysis ofthe normalallele showsa
guanine
residue inposition
1208 whileanade-nine residue replacesguanine atthis position inthe mutant allele
creating
an additional Ddel restriction site (CTCAG)(Fig.
4).
*
-
x--Xo
z
c)
0
0
07
u
r-IT
=D
$-ct
a
-
-z
z
0
U-
4--A:
To
872
bp
--321
bp
Figure
2.Photograph
ofanethidium bromide agarosegel
of thecDNA
products produced by
RT-PCR of RNAisolated fromanen-domyocardial biopsy
from theproband
ofFamily
155.The cDNAproduct produced
from theproband
is labeled cDNA(HCM),
the cDNAproduct produced
fromanormal heart is labeled cDNA(nor-mal
heart-explanted
heartsofthepatients undergoing
cardiactransplantation
for ischemic heartdisease),
theRT-negative
control reaction is labeledControl(-RT),
the PCR control reactionproduct
is labeledcontrol(-DNA),
and the PCRproduct
obtainedusing
DNAasthe
template
is labeled Genomic DNA. In lanes 3 and 4 therearelow molecular
weight
PCRproducts.
Toensurethat thenonspecific
products
donotinterfere withsubsequent studies,
the 321-bp
product
wasexcised from the
gel
andpurified
asdescribed above anddigested
with Ddel restriction enzyme. The molecular size standardsarein thefarleftlane.Discussion
We havedetermined thatamissense mutation inexon 13of
the
,3MHC
geneisexpressed
in themRNAisolated fromaright
ventricular
endomyocardial biopsy
obtained from theproband
ofa
family (pedigree
155)
withhypertrophic cardiomyopathy.
Themutation,
whichwasfirst showntobeinherited
inaMen-delian fashion ina
large
French-Canadianfamily
(6)
andmorerecently
inothers(9),
istheresultofasubstitution ofanade-nine
for aguanine
residue in one allele of theflMHC
genewhichcreatesasecond
recognition
sitefor the restriction endo-nucleaseDdelinexon13ofthegene.Themutation isinherited in a Mendelian fashion infamily
155 and co-segregateswith theinheritance
ofhypertrophic cardiomyopathy.
Onesonwith the mutation(see
Fig.
1)
although
normalby
clinical andecho-cardiographic
criteria isonly
11 yrofage andthusisathigh
risk fordeveloping
symptomatology
laterin life sinceage-depen-dent penetrance is felt to be characteristic ofhypertrophic
car-diomyopathy(23).
The missense mutation in exon 13 presumablyresults in the substitution ofa glutamine residue for an arginine in
f3MHC
protein but to date transcription of a mutant,3-myosinallele hasnotbeen demonstratedto occurin the tissue affected
bythisdisease; namely,themyocardium.We used RT-PCRto examine the
f3MHC
gene mRNAtranscriptsfromaright ven-tricularendomyocardialbiopsyobtained from theprobandof Family 155.Our results show that both the normal andmutant j3MHC alleles are transcribed as mRNA in this patient. This is the first demonstration of thetranscription ofa mutantJ3MHCgene mutation in themyocardiumofapatientwith
hypertro-phic cardiomyopathy. Rosenzweig et al. (16) have recently
shown that a mutation in exon 9 of the
flMHC
gene is tran-scribed into mRNA inlymphocytes. However,because most, if not all, genes in the human genome are thought to be tran-scribed at very low levels inlymphocytes, these results donot address whether these mutations areexpressed in thepathologi-cally relevant organ. It is perhaps less likely that the mRNA is significantly translated into protein in thelymphocyte, at least thereis nopriori reason for cardiac ,3MHC to be abundantor evenpresent inlymphocytes. It may be argued that transcrip-tion of the mutant
f3MHC
allele in myocardium is also the result of low level transcription similar to that detected by Ro-senzweig et al. ( 16) in lymphocytes. However, in contrast to the lymphocyte, 3MHC isthe most abundantprotein inthe myo-cyte(24),thusthe presence of the transcribed mutation in the mRNAismostlikelytobe expressed asprotein.Inadditiontobeing transcribedinthe organresponsibleforthe
morphologi-caland clinical manifestation of the disease,it isalso of note that the amount of jMHC mutantmRNAin themyocardium
appears tobe ingreaterabundancethan inthe lymphocyte. We detected our,MHC mutant mRNA in the myocardium after only 35 cycles of amplification in contrast to the 80 cycles of
amplification requiredtodetectmutant mRNA inthe lympho-cyte(16).
Becauseofthesmallsizeof Family155,itisnotpossibleby
linkage studiestoverify thatthedisease in this family is
defini-tivelylinked tochromosome 14.However,thecompelling link-agedata reported byJarcho et al.(1)and Geisterfer-Lawrance etal.(6)forlinkagetochromosome 14and co-inheritanceof
the identicalmissense mutation in alargekindred make it
un-likelythatdiseaseinFamily155isnotproduced bythe muta-tion. However,until the entire fMHC gene,includingthe se-quences regulatinggene expressionare completely
character-ized, it is impossible to rule out the possibility of other
mutations in the gene. The ultimate goal of unravellingthe diversepathophysiologyof HCM now depends upon the
isola-tionandfunctional characterization ofthemutant,BMHC pro-tein and how it causes thedisease.
Documentation that a mutation in
3MHC
gene istran-scribed intocardiacmRNAinapatient affected with HCMis thefirst step indetermining whetherthedefect, in
f3MHC
gene,isresponsible for this disease. This is ofmore than usual impor-tance because, although the data linking the chromosomal locusof 14ql,thesite of
J3MHC
gene,isbeyondquestion inthe families showing such linkage, there are several reasons for concern overf3MHC
beingtheresponsiblegene.fMHC
is thelesion of HCM is essentially only present in the left ventricle and moreimportantly islocalized in themajorityof
individ-ualstotheseptum.Whyis itsolocalized, consideringthe myo-cardial abundance of distribution of ,3MHC? Furthermore, studies on a- and f3-myosin from patients affectedwith HCM
byconventionalbiochemicaltechniques performed before the
genetic studies have shown noabnormalities. Absolute
docu-mentation that the mutations inthe
flMHC
gene are responsi-ble forHCMmayrequiremanyyears asithasformostdiseases inwhichthe geneis identified through positional cloning (re-versegenetics) such as dystrophin for Duchenne musculardys-trophyorthegeneforcysticfibrosis. Toencourage suchstudies
and until theinformationisavailable, documentation that a mutant
f3MHC
gene, which co-segregates with the disease, isindeed transcribedin thecardiacmRNAprovides pivotal sup-portuntilfurtherenlightening scientific evidence is available. Cardiacgrowth and hypertrophy is the main adaptiveresponse
of the hearttoallinjuries and is universallypresentin cardiac failure. While it isexpected that elucidationofthemolecular
defect will lead to betterunderstanding, diagnosisand treat-mentofHCMit isalsolikelyto shedlight on cardiac growth andhypertrophyseeninresponse toinjury.It would also repre-sent the first defect for which the molecular basis has been
determined for sudden death.
Inthepresentstudy we made no attempt to relate our
mu-tationtothepathophysiologyor causeof this diseaseandthus,
atthisstage, one canonly postulatepotential
mechanisms.
Indeveloping
amodelto accountfor mutations inf3MHC
gene, Normal MutantDde I Dde I site site
asresponsible for this disease, one must first take into account
theknownobvious clinical findingsthatcharacterizethis
dis-ease. Findingsthat are critical todevelopment of any model
includelocalized septalhypertrophy,increased myocardial
con-tractility, increasedrateof cardiacejection,anddecreased car-diac relaxation. It is also well recognized that the human ven-tricleof the patient with HCM has an abnormal emptying pat-ternbeing more complete than normal as well as taking on an unusualshape referred to as the hour glass phenomenon. We believe one model that can explain these findings would be that the particular mutation in ,3MHC, incorporated into the myo-sin protein and thick filament of the sarcomere, imparts to the muscle theability to increase its force and velocity contraction,
which inturnwillultimately deform thegeometryofthe ven-tricle leading to an altered stress/strain relationship. In this mutation in which there is a substitution of glutamate for argi-nine there is anaccompanying changein the ionicchargeof ,BMHC from
positive
tonegative,
which may insomeway alterthe kinetics of its interaction with theactin-tropomyosin com-plex. It is well recognized that increased afterload leads to car-diachypertrophywhich isgenerallyconcentric in response to anhomogeneous stimulus such as hypertension. The increased contractility induced by the mutantmyosinisanalogoustothe stimulus ofincreasedafterload,however,the response is
asym-metrical sincethe alteredstress/strain relationship within the
ventricleimpactsonthe septum. Wewouldnot
anticipate,
nec-essarily, hypertrophyintherightventricle since it isalow pres-sure volume chamber and workload is less than one quarter
HCM
-Primer
exon 12 exon 13 exon 14
Primer | o
321 bp
-4 Dde I
digestion
Radiolabel
and
Gel Electrophoresis
.40I 32
bp-:*24
Figure3.Aschematicdiagram of the relative locations of the normal andmutantDdelrestriction sitesinexon13 of the ,BMHC cDNAisshown
intheleft portion of the figure. The 321 -bp PCR product and the relative size of the nucleotide fragments produced after digestion ofthisproduct with Ddel alsoaredepicted.Anautoradiogram ofanacrylamide gel of 32P-labeled Ddel endonuclease-digested cDNA fragments produced by RT-PCR of RNAisolated fromanendomyocardial biopsy from the proband of Family 155 is showninthe right portion of this figure. The
un-digestedPCRproductis labeledUndigested and the Ddel-digestedPCRproduct is labeled Digested. The undigested RT-PCR productis321bp
inlengthanddigestion with Ddel produces four fragments of181, 149, 140, and 32 bp. Themolecularsizemarkersarein the farrightlane.
MHCJS
cDNA
PCR
Product
v6
U0
321 bp-
-U
SiI bIp
Expected
Digestion
Pattern
181
bp-149bp L 140
bp-- 0o0
4.
a - 140
a
As o 2nd loiad
ACGT ACGT
ag.
NeW- C
aw M
"O "O_
_ _w~~4
. S.m
V_
T7
Ist load
ACGT
that observed in the left ventricle.The
subsequent
clinicalfind-ings of left ventricular obstruction
or decreasedcompliance
associated with manifestations such as
dyspnea, angina,
and failureareprobably secondary
tothehypertrophy
whichcon-tinuestoincreaseovertime. Inthefinalstagesof thedisease
one
often
sees cardiac failure with decreasedcontractility,
which
presumably
representsanendstage secondary
phenom-enon.
Themissense
mutation
inexon13 ofthe fMHCgeneis the first HCMmutation
tobe found inmorethanonefamily
with thedisease. Itwill beof
interesttodetermine if this mutationhasarisenmorethanonce
during
humanevolutionoriftwoseparate mutational eventshave
produced
theidenticalpoint
mutation. Studiesareunderwaytoexamine this
question.
Wegreatly appreciatetheexpertiseofTerry Tapscottinprovidingthe DNAfrom thetransformedlymphocytesand for thesecretarial
assis-tanceof Debora H. Weaver and Alexandra Pinckard.
:ad
,
-to
Figure4.Sequenceofthe cloned PCRproduct amplified
from cDNAsegmentencompassingexon12-14 in the
mutantallele ofproband 155.Substitutionof A for G in themutantallelecreatesanadditional Ddelrestriction site(CTCAG)indicatedbythe solidarrow(-~.The
normal Ddelrestrictionsite is indicatedbytheopenarrow.
This work issupportedinpartbygrantsfrom the NationalHeart,
Lung and Blood Institute, Specialized Centers of Research
(P50-HL42267-01),and the American HeartAssociation,Bugher Founda-tion Center for MolecularBiology (86-22 16) andagrantforfellowship
support(A.Mares, Jr.,M.D.)from the Robert Wood Johnson Founda-tion
(24-278223-974101).
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