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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:

(2)

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)

geneis

expressed

inthe

messen-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 hypertrophic

car-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 of

a mutant

#MHC

geneallele intomRNAof the

myocardium.

(J. Clin. Invest. 1992. 90:271-277.) Keywords:hypertrophic

car-diomyopathy-moleculargenetics-mutation*

,-myosin

heavy

chain

Introduction

The first primary cardiomyopathy due to aninherited

defect

for which thegenehas been

identified

is

hypertrophic

cardio-myopathy

(HCM).'

The

disease is inherited

inan autosomal

dominant

mannerand the

putative

disease geneis

13-myosin

heavy

chain

(f3MHC)

locatedonchromosome14

(1-4).

Several mutations have been

identified

in the

,3MHC

gene

of affected

individuals

(5-9) and showntofollow Mendelian inheritance.

f-myosin

heavy chain isexpressed in cardiac(10) and skeletal muscle(1 1) and is the

predominant

myosin

incardiac tissue. HCM is manifestedas a

myriad

of clinical

features including

sudden

death,

heart

failure,

syncope, and

angina

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-tionof

adenine 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

(3)

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 thepublished

flMHC

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 of0.2

AM.

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 1MlofRT

buffer, 1.5

Mul

of0.1Mdithiothreitol, 1.5Mlof10mM 4dNTP, 0.5MlI RNasin (Promega Corp., Madison,WI) and 100 U Moloney murine

leukemia 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 1

min,

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 a

family

(pedigree 155) consisting

of15

individuals including

spouses.Theproband had the clini-calfeatures of HCM and left ventricular hypertrophywas

con-firmed

by two-dimensional

echocardiography

and shown to

satisfy

the

diagnostic

criteriaas

previously

described (2). The

mutation,

a substitution of adenine for the

guanine residue,

results in the creation ofa second recognition site for Ddel restriction endonuclease in themutantallele. TheDNA analy-siswas

performed using

PCRto

amplify

exon 13followed by

digestion

of the

amplified

product

with the restriction

endonu-clease Ddel. Thesyntheticoligonucleotides used forPCR analy-siswereselected to

amplify

exon13 sequencesbetween

nucleo-tides8744and 8897ofthe

fMHC

gene.After PCR

amplifica-tionthe 154-bp DNA products were separated by agarose gel

(4)

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

gene

amplified

fromDNAof members of Family 155)after digestionwith Ddel re-striction endonuclease. Lane 2 is wild-type

exon 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 normal

indi-viduals

produces two DNA

fragments of

84 and 70 bp when

analyzed by

this

procedure.

DNAisolated fromanindividual with the missense mutation, however, producesfour fragments of

84, 70, 52,

and 32 bp in that both the normal andmutant

allelearepresent

(Fig. 1).

GenomicDNA

analysis

showedthe probandtohaveamissense mutation inexon13ofthe ,BMHC

geneand all threechildren of the probandwere

positive for

the missense mutation while DNA

from

the

unaffected

husband

wasnormal (Fig. 1). Two of the childrenexhibited clinical

fea-tures

of HCM,

whereas the

third

child,

also

with

the

mutation,

exhibits

no

clinical features of

HCM. However, he isonly 11 yr

of

age,and because

symptomatology

is

often

not

evident until

later in

life, it is

premature tomakea

definitive diagnosis.

To determine if the

mutation is expressed

as mRNA in myocardial tissue, RNA wasisolated froma

right

ventricular endomyocardial biopsy obtained from the proband of

family

155 andwasusedas atemplate for RT-PCR. The cDNAwas

synthesized using

an

oligonucleotide

primer

fromexon 16

for

first strand transcription and nested oligonucleotide primers located in exons 12and 14forcDNA

amplification.

The

ex-pected

321

bp

RT-PCR

product

was

produced

fromRNA

tem-plate isolated from

myocardium

ofan unaffected individual

and that ofthe proband as shownin

Fig.

2.Genomic DNA

template

isolated fromthe

proband

producedtheexpected872

bp

(Fig.

2) product duetointronicsequences not presentin the

mRNA 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-tion

fragments

were5' end labeled with 32P and electrophoresed

on acrylamidegels. The resulting autoradiogram anda sche-maticdrawing illustrating the resultsareshown inFig. 3. Diges-tion of cDNA from normal

myocardium

would be expectedto

producetworestriction fragments of181 and 140 bp,whereas

digestion

of cDNA produced from RNA

isolated

from the

myocardial

biopsy

of

the probandproducesfragments of 181,

140, 149,

and32 bp in length duetothe expression

of

both the normal andmutantallele(Fig. 3). The lattercDNArestriction fragments fromtheprobandresultfromthetranscription ofa

second 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 normal

allele showsa

guanine

residue in

position

1208 whilean

ade-nine residue replacesguanine atthis position inthe mutant allele

creating

an additional Ddel restriction site (CTCAG)

(Fig.

4).

(5)

*

-

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 agarose

gel

of the

cDNA

products produced by

RT-PCR of RNAisolated froman

en-domyocardial biopsy

from the

proband

of

Family

155.The cDNA

product produced

from the

proband

is labeled cDNA

(HCM),

the cDNA

product produced

fromanormal heart is labeled cDNA

(nor-mal

heart-explanted

heartsofthe

patients undergoing

cardiac

transplantation

for ischemic heart

disease),

the

RT-negative

control reaction is labeledControl

(-RT),

the PCR control reaction

product

is labeledcontrol

(-DNA),

and the PCR

product

obtained

using

DNA

asthe

template

is labeled Genomic DNA. In lanes 3 and 4 thereare

low molecular

weight

PCR

products.

Toensurethat the

nonspecific

products

donotinterfere with

subsequent studies,

the 32

1-bp

product

wasexcised from the

gel

and

purified

asdescribed above and

digested

with Ddel restriction enzyme. The molecular size standardsarein thefarleftlane.

Discussion

We havedetermined thatamissense mutation inexon 13of

the

,3MHC

geneis

expressed

in themRNAisolated froma

right

ventricular

endomyocardial biopsy

obtained from the

proband

ofa

family (pedigree

155)

with

hypertrophic cardiomyopathy.

The

mutation,

whichwasfirst showntobe

inherited

ina

Men-delian fashion ina

large

French-Canadian

family

(6)

andmore

recently

inothers

(9),

istheresultofasubstitution ofan

ade-nine

for a

guanine

residue in one allele of the

flMHC

gene

whichcreatesasecond

recognition

sitefor the restriction endo-nucleaseDdelinexon13ofthegene.Themutation isinherited in a Mendelian fashion in

family

155 and co-segregateswith the

inheritance

of

hypertrophic cardiomyopathy.

Onesonwith the mutation

(see

Fig.

1)

although

normal

by

clinical and

echo-cardiographic

criteria is

only

11 yrofage andthusisat

high

risk for

developing

symptomatology

laterin life since

age-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-myosin

allele 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 mutantJ3MHC

gene 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 the

pathologi-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.Inadditionto

being 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 is

tran-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 over

f3MHC

beingtheresponsiblegene.

fMHC

is the

(6)

lesion 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 muscular

dys-trophyorthegeneforcysticfibrosis. Toencourage suchstudies

and until theinformationisavailable, documentation that a mutant

f3MHC

gene, which co-segregates with the disease, is

indeed 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.

In

developing

amodelto accountfor mutations in

f3MHC

gene, Normal Mutant

Dde 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

to

negative,

which may insomeway alter

the 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

.40

I 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

(7)

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

clinical

find-ings of left ventricular obstruction

or decreased

compliance

associated with manifestations such as

dyspnea, angina,

and failureare

probably secondary

tothe

hypertrophy

which

con-tinuestoincreaseovertime. Inthefinalstagesof thedisease

one

often

sees cardiac failure with decreased

contractility,

which

presumably

representsan

endstage secondary

phenom-enon.

Themissense

mutation

inexon13 ofthe fMHCgeneis the first HCM

mutation

tobe found inmorethanone

family

with thedisease. Itwill be

of

interesttodetermine if this mutation

hasarisenmorethanonce

during

humanevolutionoriftwo

separate mutational eventshave

produced

theidentical

point

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).

References

1.Jarcho,J.A.,W. P.McKenna,J. A.Pare,S. D.Solomon,R.F.Holcombe,

S.Dickie,T.Levi,H.Donis-Keller,J.G. Seidman,and C. E. Seidman. 1989.

Mappingagene for familialhypertrophic cardiomyopathytochromosome14q1. N.Engi.J. Med. 321:1372-1378.

2.Hejtmancik,J.F.,P. A.Brink,J.Towbin,R.Hill,L.Brink,T.Tapscott,A.

Trakhtenbroit,and R. Roberts. 1991. Localization of gene for familial hypertro-phic cardiomyopathytochromosome

14q1I

inadiverse U.S.population. Circula-tion.83:1592-1597.

3.Solomon,S.D.,A. A. T.Geisterfer-Lowrance,H.-P.Vosberg,G.Hiller,

J. A.Jarcho,C. C.Morton,W.0.McBride,A. L.Mitchell,A.E.Bale,W. J.

McKenna, et al. 1990. A locus for familialhypertrophic cardiomyopathy is

closelylinkedtothe cardiacmyosinheavygenes,CRI-L436,andCRI-L329on

chromosome 14at

qlI

1-912. Am. J. Hum. Genet. 47:389-394.

(8)

-441--Takao.1989. Humancardiac myosin heavy chain gene mapped within chromo-someregion 14q1 1.2-q13. Am. J.Med. Genet. 32:279-284.

5.Epstein, N. D., L. Fananapazir, H. J. Lin, J. Mulvihill, R. White, J. M. LaLouel, R. P.Lipton, A. W. Nunhuis, and M. Leppert. 1992. Evidence of ge-neticheterogeneity in five kindreds with familial hypertrophic cardiomyopathy.

Circulation.85:635-647.

6. Geisterfer-Lawrance, A. A., S. Kass, G. Tanigawa, H.-P. Vosberg, W. McKenna,C. E.Seidman, and J. G. Seidman. 1990. A molecular basis for famil-ialhypertrophiccardiomyopathy: a beta-cardiac myosin heavy chain gene mis-sensemutation. Cell.62:999-1006.

7.Tanigawa,G., J. A.Jarcho, S. Kass, S. D. Solomon, and C. E. Seidman. 1990. A molecularbasis forfamilial hypertrophic cardiomyopathy: Analpha/

betacardiacmyosin heavy chain hybrid gene. Cell. 62:991-998.

8.Watkins, H., D.-S. Hwang, A. Rosenzweig, G. Tanigawa, J. Jarcho, W. McKenna, J. G.Seidman, and S. Seidman. 1991. Analysis of cardiac myosin heavychain genemutations that cause familial hypertrophic cardiomyopathy.

Circulation.84(Suppl.):II418.

9. Perryman, M. B., A. Mares, Jr., F. Hejtmancik, G. Gooch, and R. Roberts. 1991. The 3-myosinheavychainmissence mutation in exon 13, a putative defect

forHCMispresent inonlyoneof39families. Circulation84:11-418. (Abstr). 10. Emerson,C. P., and S. I.Bernstein. 1987. Molecular genetics of myosin. Annu.Rev.Biochem. 56:695-726.

11.Saez, L. J., K. M.Gionala, E. M. McNally, R. Fegally, R. Eddy, T. B.

Shows,and L.A.Lienward. 1987. Humancardiac myosinheavy chain genes and theirlinkagein the genome.Nucleic AcidsRes. 15:5443-5459.

12. Maron, B. J., S. E.Epstein, and W. C. Roberts. 1986. Causes of sudden death incompetitiveathletes. J.Am.Coll. Cardiol.7:204-214.

13. Maron, B. J., and W. C. Roberts. 1981.Hypertrophiccardiomyopathy

andcardiac muscle celldisorganization revisited: relation between the two and

significance.Am.Heart J. 102:95.

14.Schier,J.J., and R.S. Adelstein.1982.Structural andenzymatic

compari-sonofhuman cardiac muscle myosins from infants, adults and patients with hypertrophic cardiomyopathy. J. Clin. Invest. 69:816-825.

15.Vybiral, T., R. Roberts, H. F. Epstein. 1991. Myofibrillar structure is intact in interventricular septal biopsies of hypertrophic cardiomyopathy linked to chromosome 14q 1. Circulation. 84:II-419. (Abstr.)

16. Rosenzweig, A., H. Watkins, D. Hwang, M. Miri, W. McKenna, T. Traill, J. G.Seidman, and C. E. Seidman. 1991. Preclinical diagnosis of familial hyper-trophic cardiomyopathy by genetic analysis of blood lymphocytes. N. Engl. J. Med.325:1753-1760.

17. Neitzel, N. 1986. Aroutine method for the establishment of permanent growinglymphoblastoic cell lines. Hum. Genet. 73:320-326.

18. Lowe, T., J. Sharefkin, S. Q. Yang, and C. W. Dieffenbach. 1990. A computerprogramfor selection ofoligonucleotide primers for polymerase chain reactions.Nucleic Acids Res. 18:1757-1761.

19.Liew, C. C., M. J. Sole, K.Yamauchi-Takihara, B. Kellam, D. H. Ander-son, L. Lin, and J. C. Liew. 1990. Complete sequence and organization of the humancardiacbeta-myosin heavychain gene.NucleicAcidsRes.18:3647-365 1. 20.Chomczynski P: The RNAzol TM method. Cinna/Biotex Bull. No. 1, 1988.

21. Sambrook, J., E. F.Fritsch,and T. Maniatis. 1989. Construction and analysis ofcDNA libraries. In Molecular Cloning: A Laboratory Manual. J. Sam-brook, E. F.Fritsch, and T. Maniatis, editors. Cold Spring Harbor Press, Cold

SpringHarbor,NY. 8.60-8.63.

22.Sambrook, J., E. F.Fritsch,and T.Maniatis. 1989.Preparationof readio-labeled DNA and RNA probes. In: Molecular Cloning: A Laboratory Manual. J.

Sambrook,E. F.Fritsch,and T.Maniatis, editors. ColdSpringHarborPress,

ColdSpring Harbor, NY. 10.60-10.61.

23.Maron, B. J., P.Spirito, Y. Wesley, and J. Arce. 1986.Developmentand

progression of left ventricular hypertrophyinchildren withhypertrophic cardio-myopathy. N.Engl. J. Med. 3 15:610-614.

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