molecular cardiology and genetics in the 21st century—a primer

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Molecular Cardiology and Geneticsin the 21st Century—A Primer

Robert Roberts and Michael Gollob

Abstract: The terminology and technology of moleculargenetics and recombinant DNA have become an essentialpart of academic cardiology and will soon be applied atthe bedside. The treatise includes a brief summary of theessentials of the DNA molecule, the more common tech-niques, and their application to genetics and molecularcardiology. It is written to be understood by physicians,scientists, and paramedical personnel who would notnecessarily have a background in molecular biology.Inherent in the DNA molecule are three properties fun-damental to all of the diagnostic and therapeutic applica-tions, namely, the ability of DNA to separate into singlestrands, recombine (annealment or hybridization), andthe presence of the negative charge enables DNA frag-ments to be separated easily by electrophoresis. Geneticlinkage analysis of a family with an inherited diseaseenables one to identify the gene without knowing itsprotein product. Over 50 diseases in cardiology due tosingle-gene disorders have been identified and multiplemutations have been detected. The new therapeutic fron-tier will be stem cells and nuclear transfer. Identificationof genes responsible for coronary artery disease madepossible by genome-wide single nucleotide polymorphism(SNP) mapping techniques paves the way for personal-
ized medicine. (Curr Probl Cardiol 2006;31:637-701.)
M odern molecular biology has revolutionized research and ourunderstanding of the molecular pathogenesis of disease in variousbranches of medical disciplines. In the past, the nomenclature of

ecombinant DNA and other techniques of molecular biology remainedhe authors have no conflict of interest to disclose.urr Probl Cardiol 2006;31:637-701.
146-2806/$ – see front matteroi:10.1016/j.cpcardiol.2006.05.004
urr Probl Cardiol, October 2006 637

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omewhat foreign to the practicing cardiologist, in part because theechniques were only recently developed and their application to cardio-ascular diseases became prominent only over the past decade. In thisonograph, we will describe the historical development of the techniques

f recombinant DNA and of molecular biology. The unique features ofhese techniques over that of conventional scientific techniques will beiscussed together with how they solve cardiac problems in a way thatas previously not feasible. Inherited cardiac arrhythmias and cardiomy-pathies will be discussed as examples of the progress made in thepplication of molecular genetics to cardiac hereditary disorders. Theistorical perspective is intended to provide insight into why molecularechniques have blossomed and why they have an advantage over existingcientific techniques. It should be emphasized that the terminology andechniques are generic and are essentially the same regardless of thergan, organism, or field of research to which they are applied.1

he Evolution of Modern Molecular BiologyModern molecular biology is almost synonymous with the developmentf recombinant DNA technology. Despite the fundamental discoveries inhe 1950s and 1960s, application of these techniques did not emerge untilhe late 1970s and early 1980s.2 Miescher isolated DNA for the first timen 1869, and in 1944 Avery provided evidence beyond doubt that DNA,ather than protein, is responsible for transferring genetic informationuring bacterial transformation.3 In 1953, Watson and Crick4,5deducedhe double helix structure for DNA, which was based on the results of-ray diffraction by Franklin and Gosling6and Wilkins et al.7 The workf Watson, Crick, and Wilkins was rewarded with a Nobel Prize, the firstf three Nobel Prizes rewarded for advances in the understanding ofolecular genetics. Marmor, Lane,8 and Doty et al9 showed the double

elix of DNA could be separated into single strands by high temperaturesdenaturation) and reannealed (double stranded) with return to loweremperatures. This is a major property of DNA enabling many processesuch as DNA amplification by the polymerase chain reaction.

ichard A. Walsh: Miescher “discovered” DNA by noting there was aubstance derived from cell nuclei which differed from protein, and whichissolved in alkali but not water. He isolated the substance from salmonperm and bandages from surgical wounds and called it “nuclein.”

In 1964, Nirenberg and Matthaei10 and Nishimura et al11 elucidated the
enetic code by discovering the sequence of three DNA bases (the triplet
38 Curr Probl Cardiol, October 2006

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odon) code for each amino acid in a protein, a discovery that irrefutablyinked DNA as the molecule of life and resulted in another Nobel Prize inhe field of modern genetics. Olivera et al12 discovered DNA ligase, thenzyme used to join DNA fragments together, helping set the stage forecombinant DNA technology. However, the large size of the DNAolecule and its monotonous nature made it difficult to isolate andanipulate. The ability to isolate, manipulate, and clone DNA was, in

arge part, due to six further seminal contributions, as follows: (1) theiscovery of restriction endonucleases; (2) the discovery of reverseranscriptase; (3) the cloning of DNA; (4) the ability to sequence the basesomprising a DNA fragment; (5) the ability to mutate specific DNAesidues (altering specific amino acid codes) allowing for structure/unction studies of proteins; and (6) the advent of the polymerase chaineaction (PCR), which provided the tool to rapidly amplify exponentiallyelected fragments of DNA sequence.Restriction endonucleases are to the molecular biologist what the

calpel is to the surgeon. They cut double-stranded DNA within theolecule (hence, endonucleases) at basepair sequences that are specific

or each enzyme. The recognition sites for most enzymes are four to eightasepairs in length, with a few having recognition sites of only threeasepairs, and even fewer recognize eight basepairs. The restrictionndonucleases are isolated from bacteria where their normal function is asdefense mechanism, to digest foreign DNA, restricting it from being

ncorporated into the genome, and so are referred to as restrictionndonucleases. It is possible to cut DNA into fragments of a desired andonsistent size, knowing specifically where each cut is performed. Thebility to cut DNA into fragments of specific length was absolutelyssential to all of the recombinant techniques and more specifically for theevelopment of cloning. The existence of a DNA restriction endonucleaseas first discovered by Linn S in 196213; however, it was not until laterork,13-15 in 1970, that specific endonucleases were isolated and applied

o molecular genetic techniques.The second contribution was the independent discovery of reverse

ranscriptase in 1970 by two laboratories,16,17 which made it possible toenerate DNA complementary (cDNA) to messenger RNA (mRNA). Therst stage in generating a protein from a gene requires transcribinguclear DNA (transcription) into mRNA, which has the encodingequence for the amino acids of the protein. Thus, isolation of an mRNAnd conversion to cDNA provides one with a probe that will only bindith its complementary DNA (gene). The dogma for a long time was that
NA to RNA could not be reversed. The discovery of reverse transcrip-

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ion was worthy of more than a Nobel Prize for several reasons. RNAepresents the expressed form of a DNA gene. Only the gene in its DNAorm can be replicated such as with the cloning technique. The sequencef the DNA coding for protein is less than 1.5% of all DNA. Finding aene is like finding a needle in a haystack. Thus, being able to convert theNA into DNA sequences provided us with a new tool for identifyingenes. The observation, that cDNA contains only the coding sequence,as all that was needed to clone or express the gene was a major

evelation. The cDNA also provides a specific means to index its locationn the chromosome, as well as a more stable nucleic acid structuremRNA is easily degradable) for multiple applications, such as cloning.The third contribution was the birth of cloning. Cloning is a method tobtain multiple copies of a DNA fragment including a gene. In 1972,18

he first recombinant DNA molecule was generated at Stanford, and in973,19 the first foreign DNA fragment was inserted (recombined) into alasmid (DNA vector). Plasmids are autonomously replicating DNAolecules, commonly present in bacteria, and capable of replicating in

reat numbers within a bacterium using the molecular machinery of therganism. This first recombined plasmid was successfully reinserted (therocess of transformation) into a bacterium, which was grown in culturededia. The plasmid replicated providing millions of copies, and hence,

he first successful cloning of a foreign DNA fragment. Thus, it was nowossible to isolate mRNA known to code for a specific protein, and, witheverse transcriptase, convert it into a stable cDNA, which could then beecombined with a plasmid vector, transformed into a bacterium, andrown in culture, allowing for the cloning of large quantities of a specificNA sequence or gene.The fourth contribution was made in 1977, when Sanger et al20 atambridge and Maxam and Gilbert21 at Harvard independently devel-ped rapid nucleic acid sequencing techniques. These investigators wereubsequently awarded a Nobel Prize. Thus, DNA of unknown sequenceould now be cut into fragments of reasonable size, cloned into plasmidectors, replicated into large quantities, and the specific DNA sequenceetermined.In 1982,22 Smith et al described the technique of site-directed mutagen-

sis, a technique whereby a specific DNA fragment may be manipulatedr engineered, replacing a single basepair with another, resulting in anltered coding sequence, and the subsequent replacement of one aminocid for another. This powerful molecular technique enables the system-tic study of various regions of a gene (protein), to identify regions or
omains essential for specific functions, that is, structure–function anal-

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sis. Further, with the advances in human genetics leading to thedentification of disease-causing DNA mutations, site-directed mutagen-sis provided a tool to determine the physiological effect of a mutation.t also enabled the elucidation of the molecular pathogenesis of humanisorders. Site-directed mutagenesis in addition to in vitro systemstilizing recombinant DNA molecules can also be performed in vivo byirect injection of the gene into the germ line. Once incorporated into therganism’s genome, it can be transferred to succeeding generations. Thisrocess referred to a transgenesis is used to generate transgenic animalss models of human disease.

ichard A. Walsh: Genetically engineered mice have become a powerful toolo study specifically manufactured genetic changes and their resulted effectsn phenotypes in vivo. It is possible to overexpress a protein of interest orliminate a specific gene in an organ-, cell-, and time-dependent manner.hese have been referred to as transgenic or knockout animals, respectively.hile other mammalian species have been used for this purpose, mice have

redominated because of their short breeding time, relative cost, and ease ofanipulation of the germ line (see Cardiovascular Physiology in the Geneti-

ally Engineered Mouse, Second Edition, Hoit BD, Walsh RA, editors, Kluwercademic Publishers, 2002).

The development of the PCR to amplify selected DNA or RNAragments to several million copies instead of the need to clone providedhe final tool and provide the final tool for modern molecular biology.

hile cloning was a major breakthrough, PCR provides a more rapid andobust means to obtain millions of copies of a specific DNA moleculeithin hours. This discovery in 1985, also awarded with a Nobel Prize, ismajor discovery that has markedly facilitated advances over the last

ecade in the study of human genetic diseases. The technique of PCR ishe foundation of almost all investigations in modern molecular genetics.lthough many applications exist, as will be described later, PCR wasrst utilized to detect the genome of pathogens responsible for humanisease. For example, myocardial biopsies are obtained routinely inatients suspected of cardiomyopathy where the diagnosis is not evidentnd can be analyzed by PCR for the responsible pathogen.23,24 In essence,he presence of only one or two copies of RNA of DNA in a cell, whichannot be detected by conventional techniques, can be amplified by PCRo several million copies, putting particles such as viral RNA within thehreshold of detection for conventional techniques.In addition to the tremendous advances modern molecular genetic

echniques have provided in the study of human health and disease, such


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echniques have also proven invaluable in therapeutic drug development.or example, the first cardiac drug made by recombinant DNA techniquesas recombinant tissue plasminogen activator (rt-PA) in 1983, which

evolutionized the therapy of acute myocardial infarction. The develop-ent of this therapeutic agent serves to illustrate the value of the

echniques of recombinant DNA and molecular biology. A cDNAontaining all of the coding regions of the TPA gene was mass producedn bacterial and mammalian cell-culture systems. Site-directed mutagen-sis of the cDNA gene and its expression led to the identification of itsunctions, namely, lytic activity, fibrin affinity, and fibrin-dependentnhanced lytic activity.22 Five domains were recognized to have specificunctions that are coded by separate and autonomous portions (exons) ofhe gene: the finger domain and epidermal growth factor (EGF) domainsre responsible for fibrin binding; kringle1 and kringle2 are responsibleor enhancing lytic activity; and the light chain is responsible for theatalytic activity the binding site of the plasmin inhibitor. Similartructure–function analyses have been performed for urokinase, single-hain urokinase plasminogen activator (scu-PA), and, by splicing togetherarious portions of the genes, a variety of chimeric molecules have beenenerated. Site-directed mutagenesis is now a routine and invaluableechnique to determine molecular function. Hundreds of drugs have beenenerated by recombinant DNA technology.

nderstanding DNA: The Molecule of LifeThe DNA molecule is a linear polynucleotide consisting of repeatingnits of nucleotides. Each nucleotide consists of one base, a deoxyriboseugar, and a phosphate molecule. There are four bases: adenine, cytosine,uanine, and thymine. Adenine and guanine are purines and cytosine andhymine are pyrimidine bases. Each base is bound to one sugar and onehosphorous molecule. The ribose molecule lacks a hydroxyl group (OH)t the number 2 carbon on the ring and thus the designation deoxyribo-ucleic acid. The purine bases (adenine and quanine) are two-ringedtructures and the pyrimidine bases (thymidine and cytosine) are theingle-ringed structures (Fig 1). The double-helix structure arises from theomplementary pairing between the purine base of one strand and theyrimidine base of the second strand. The complementary pairing isptimally stabilized by hydrogen bonds formed between the purine andhe pyrimidine base, such that guanine (G) will always pair with cytosineC) via three hydrogen bonds and adenine (A) will always pair withhymidine (T) via two hydrogen bonds (Fig 2). Because of incompatible
ing conformations, cross pairing between the other purines and pyrimi-

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ines, ie, adenine with guanine and cytosine with thymine, do not occur.his complementary basepairing is the basis of molecular genetics. Theinding of a phosphorus molecule to each nucleoside gives rise to aucleotide. When each nucleotide strand is considered individually, theequence of nucleotide bases is always read from left to right from the 5=arbon to 3= carbon direction (Fig 3). In the double-helix configuration,he strand complementary to the 5= to 3= strand is oriented in a 3= to 5=irection relative to its complementary strand. This organization iseferred to as the antiparallel or antisense arrangement of the DNAtrands. For example, nucleotide pairing between a nucleotide strand with5= ATCCG 3= sequence has as its complementary strand 3= TAGGC 5=

n the double-helix configuration. Additionally, when describing seg-ents of DNA, it is common to refer to them by size. For example, a

egment of double-stranded DNA composed of 200 nucleotides is ofteneferred to as a 200-basepair (bp) segment. Similarly, 1000- and,000,000-nucleotide segments are 1-kb or 1000-kbp [1-megabase (Mb)]

IG 1. Building blocks of nucleic acids, DNA comprises a deoxyribose sugar backbone with theucleotide bases adenine, guanine, cytosine, and thymidine attached to the C1 position carbonn the sugar ring. Roberts R. Essentials of nucleic acids and proteins. In: Roberts R, editor. Arimer of Molecular Biology. New York, Elsevier Science Publishing, 1992. p. 19.

egments.


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The genome refers to the complete DNA sequence of an organism,hich is enclosed in the nucleus of a cell (Table 1). In the human, there

re 3 billion basepairs, which contain information that would more thanll a 500,000-page textbook. It is estimated that, in a single individual, ifll of the chromosomes were joined end to end, it would reach from thearth to the moon about 8000 times. The actual length of the DNA fromach cell is not apparent because the DNA helix of each chromosomexists as a compact, coiled structure stabilized by protein molecules,any of which are histones. The compact nature results in an increase in

iameter, thus allowing the DNA to be visible by electron microscopy.he coiled, compact character of DNA enables all of the genetic

nformation from a single cell to fit neatly into the cell’s nucleus, which

IG 2. Specificity of DNA basepairing. The two strands of DNA are bound together viaydrogen bonds between the nucleotide bases on each strand. The bonds are formed by strictairing between two complementary bases, AT or CG, such that each strand reflects the exactequence of the opposite strand (A, adenine; T, thymine; G, guanine; C, cytosine). The sugardart pentamer) and phosphate (dark circle) linkages form the backbone of the DNA strand.ource: Roberts R. Essentials of nucleic acids and proteins. In: Roberts R, editor. A Primer ofolecular Biology. New York: Elsevier Science Publishing, 1992. p. 22.

ccupies less than 10% of the total cell volume. The 25,000 genes


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IG 3. DNA replication conserves the nucleotide sequence. DNA is a double-stranded helicalolecule bound together by the nucleotide bases contained on each individual strand. Duringell division, two identical copies of the original parental strand are made by unwinding theNA and then synthesizing a complementary second strand to make two identical newaughter stands. Source: Roberts R. Essentials of nucleic acids and proteins. In: Roberts R,
ditor. A Primer of Molecular Biology. New York: Elsevier Science Publishing, 1992. p. 20.

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ncoding a human being accounts for about 1% of the DNA, thus most ofhe DNA is noncoding. There are 46 chromosomes, and each chromo-ome is a long continuous DNA molecule. The chromosomes vary in size,ut even chromosome 21, the smallest of them, contains more than0,000,000 basepairs.

he Definition of a GeneA gene is a distinct segment of the DNA forming a chromosome thatas the appropriate DNA sequences promoting transcription to mRNA,nd coding sequences to be transcribed and subsequently translated intosingle polypeptide (Fig 4). Typically, we think of a gene as having the= ends, which is not transcribed but is recognized by proteins that initiatehe transcription process, followed by the protein-coding sequence andhe 3= ends for stability. The coding sequence is referred to as the readingrame that starts with an ATG triplet, followed by various arrangementsf triplet bases (codons), which specify the amino acids to form aolypeptide. The 3= end of the gene is not translated into protein but isranscribed to impart stability to the messenger RNA. The codons TGA,r TAA or TAG (triplet stop codon), found at end of the reading frame,erminate the reading frame for amino acids. The protein coding se-uences (exons) for proteins are separated by noncoding sequencesintrons), with the latter spliced out during the transcription process andxcluded from the mRNA. The exon–intron boundaries have character-stic sequences, beginning with GT and ending with AG. Among theillion of bases of DNA, these sequence characteristics enable computerlgorithms to predict which segments of DNA contain genes coding forrotein. The gene requires many proteins to initiate and promote tran-cription (transcription factors) including enhancers and silencers (Fig 5).ranscription is usually initiated about 32 nucleotides upstream from thetarting codon of ATG at a sequence referred to as the TATA box.

ranscriptionThe central dogma of molecular biology is now well established,amely, DNA produces RNA, which in turn produces a polypeptide that

ABLE 1. The human genome (completed April 2003)

Number of bases 3.2 billionGenes estimated 30,000DNA for genes 1.5%

akes up the protein that provides the cell structure and performs the


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unctions of the cell (Fig 6). The genetic information inherited by eachndividual is encoded by the sequences of the bases of the DNA in theenome (the genotype), which is translated into proteins and provides theecognizable characteristics of the individual (the phenotype) such aseight and weight. For DNA to produce proteins, it must first go throughhe intermediary step of RNA. DNA, the double-stranded molecule,nwinds to give a single-stranded RNA molecule that serves as theemplate for protein (Fig 5). This process, since it goes from one nucleiccid to another nucleic acid, is referred to as transcription. This is a keyegulatory step in the old process of replicating and maintaining life.here are several aspects in the regulation of transcription, as indicated inable 2. The process of transcription is initiated by attachment of the

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IG 4. Regulation of gene activation. Schematic of the components of gene structure thatontributes to gene activation and protein synthesis. RNA polymerase binds to a site at theeginning of a gene0 (promoter region) that often contains a TATA box. Other gene elementsenhancers) may regulate this process. A heteronuclear RNA copy of the gene (hnRNA) isroduced that contains protein coding (exons) and noncoding (introns) sequences. Theoncoding introns are spliced out and the RNA is “capped” at the 5= end and polyadenylatedt the 3= end [poly(A) tail] to form a mature messenger RNA (mRNA). The mRNA is used toynthesize the protein encoded for by this gene in the cell cytoplasm. (Color version of figure isvailable online.)

nzyme RNA polymerase to specific recognition sites where the DNA is


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ouble-stranded but, upon activation by the enzyme, the strands selec-ively unwind and separate. The binding site for the RNA polymerase 2s always on the 5= end of the gene and travels on a single-stranded DNAowards the 3= end. Messenger RNA in addition to being single-strandedlso differs from DNA in that the deoxyribose sugar found in DNA iseplaced by ribose. Furthermore, uracil (U) replaces T and, like T, U pairsxclusively with A. The mRNA transcribed from DNA is usually referredo as the primary transcript and is a complementary copy of the DNA. TheRNA exits the nucleus but, prior to transport, undergoes extensive

osttranscriptional processing primarily through the three following mainvents: (1) the addition of the methylated guanosine (for methoguanosine

IG 5. Types of transcription factors which effect gene activation. Schematic representation ofhe shape of four types of protein transcription factors that bond to DNA and influence genectivation. Helix-turn-helix is a protein with two alpha-helices separated by a beta-turn. Leucineippers are protein dimers with interdigitating leucine amino acids. Zinc fingers have a peptideoop connected at the base by a zinc ion tetrahedron between cysteine and/or histidine inmino acids. The helix-loop-helix consists of beta-helix but utilizes leucine zippers and has a

oop between the beta-helices. The darkened areas are believed to be the regions of the proteinhat interact with the DNA to modulate transcription. Source: Roberts R. Essentials of nucleiccids and proteins. In: Roberts R, editor. A Primer of Molecular Biology, New York: Elseviercience Publishing, 1992. p. 34.

esidue) to the 5= end, referred to as a CAP, which is important for the


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nitiation of translation; (2) addition of a long tail of repeated adenineucleotides called the polyadenine tail to the 3= region of the mRNA,hich is essential for stability as it passes out into the cytoplasm to serve

s a template for protein synthesis; (3) the primary transcript, whichontains introns and exons, undergoes a specific splicing process wherebyhe introns are removed and exons are properly respliced together prior to

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Cell FunctionIG 6. DNA controls cell function; RNA is synthesized from a gene on the DNA template in theucleus. The protein is then synthesized from RNA to carry out cell function.

ABLE 2. Regulation of gene expression and protein synthesis

PretranscriptionTranscriptionRNA1PosttranscriptionTranslationPosttranslation

ource: Roberts R. Essentials of nucleic acids and proteins. In: Roberts R, editor. A Primer ofolecular Biology. New York: Elsevier Science Publishing, 1992. p. 30.

xit from the nucleus. It is then referred to as the mature mRNA. The


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xons of the 3= end do not code for proteins but for signals that terminateranslation and direct the addition of the polyadenine tail. The matureRNA exits the nucleus through nuclear pores and, upon entering the

ytoplasm, attaches to ribosomal RNA.

ranslationThe process whereby the mRNA encodes for a protein is called

ranslation (translates the nucleic acid code of DNA into the amino acidode of protein) (Fig 4). Protein translation occurs when a single mRNAoves along the ribosome and is read by tRNA molecules, which bring

he amino acid to the chain. Once a polypeptide is formed from thepecific mRNA, it may in itself form the protein or combine with otherolypeptides to form the mature protein. Once the protein is formed, it hasertain amino acid sequences which direct it to its specific compartmentn the cell. Secreted protein molecules, for example, contain a hydropho-ic tail sequence which directs it to the endoplasmic reticulum membrane.he proteins perform all of the work of the cell and are each synthesized

rom a unique mRNA. Gene expression refers to the whole process fromhe formation of the gene to a mature protein. Many proteins undergohemical modification posttranslation such as glycosylation.

ichard A. Walsh: This form of posttranslational molecular carpentry isarticularly common with cellular secretory or membrane proteins. It may

nvolve the development of disulfide bonds, proteolytic cleavage of the newlyynthesized protein, or the addition of carbohydrate moieties. These post-ranslational changes may importantly affect function and subcellular local-zation of proteins.

he Human Genome Project: Purpose and GoalThe Human Genome Project, the first large international effort in theistory of biological research, was initiated on October 1, 1990, to beompleted in the year 2005.25,26 However, with improvements in tech-ology and competition from the private sector, the timetable wasccelerated. A rough draft of 90% was completed in 2000, and theomplete sequence became available in 2003. The Human Genomeroject sequenced the DNA blueprint for the development of a singleertilized egg into a complex organism. This blueprint is written in theoded message given by the sequence of nucleotide bases—the A’s, C’s,’s, and T’s—that are strung together to make the DNA molecules in the
uman genome. However, while the overall objective was to sequence the

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uman genome, other goals were completed along the way that markedlyccelerated the efforts of all investigators involved in biological oredical research. The first goal was to develop a genetic map. This meant

eveloping markers (unique DNA sequences) along each chromosomehat would have a readily identifiable chromosomal position to provideighly informative signposts for the identification of nearby genes. Thisoal provided thousands of markers spaced 5 to 10 million basepairspart, spanning the entire human genome, leading to the creation of aenetic “roadmap” for each chromosome. As will become evident in auture section of this text, it is the use of this genetic map, with DNAequences (markers) of known positions (loci) along each chromosome,hat enables the mapping of a gene’s chromosomal location by geneticinkage analysis. The tool of genetic linkage analysis led to the acceler-tion of mapping the position of numerous genes responsible for diseasesf the cardiovascular system and other organs. Currently over 1500isease-causing genes are known, due to the more rapid identification ofenes facilitated by the Human Genome Project.The policy of the Human Genome Project is that the entire human DNA

equence, including all identified genes, will be available to the public.ach gene, as it is sequenced, is entered into a publicly accessibleatabase and available at no cost. In the United States, GenBank (atttp://www.ncvi.nlm.nih.gov) is run by the National Center for Biotech-ology Information and serves as the public repository of DNA sequencenformation. The results of the efforts of the publicly funded Humanenome Project consist of not only DNA sequences of the various genesut also the intervening sequences.Another goal was to develop a physical map of regions of the DNA that

re expressed as genes. These markers are referred to as expressedequence tags (ESTs) and contain short sequences of 200 to 300 bp.hese sequences are unique and represent a fragment of a yet to be fullyharacterized specific gene. ESTs are generated by extraction of all of theRNAs in a cell type, which represents all of the genes expressed at that

ime in that cell. The mRNA can be converted to cDNA with the enzymeeverse transcriptase and the sequences amplified by the polymerase chaineaction, from which unique sequences are selected and entered intoenBank as ESTs. The sequences of these ESTs are then matched to thelethora of sequences available in the DNA sequence repository. Thus,STs mapped to their chromosomal locations can be used as markers to

dentify novel genes responsible for disease. The development of thishysical map has tremendously accelerated the efforts of investigators to
dentify novel genes, relevant to normal physiology or disease. These

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STs serve as candidate genes if a locus harboring a disease gene isapped to a region; the ESTs in the region are potential candidate genes

nd greatly facilitate the identification of the gene of interest.

he HapMap ProjectWhile the Human Genome Sequencing project was completed in 2003,ther large-scale human genome projects continue. The sequence of theuman Genome differs by only 0.1% among human beings. Thisne-tenth of 1%, however, translates into 3 million bases. These 3 millionases are now considered to be responsible for essentially all of theuman variation including predisposition or resistance to diseases. Thus,t became evident that identifying the sequence responsible for humanariation would represent a major quest for the next decade.Essentially, all of human variation appears to be due to single-ucleotide polymorphisms referred to as single nucleotide polymorphismSNP)s, which are distributed throughout the human genome occurring at

frequency on average of about one SNP per 1000 basepairs. Whiledentifying the SNPs responsible for human variation and the mechanismhereby this sequence induces the change is of crucial importance, it iserhaps of even more immediate importance to identify those SNPs thatredispose to disease. Their potential to facilitate diagnosis, prevention,nd treatment could be enormous. The difficulty lies in how to identifyhose SNPs that predispose to disease. In searching for SNPs thatredispose to disease, it is quite a different task than identifying mutationsesponsible for single-gene disorders. A particular SNP is neither neces-ary nor required for a particular disease and thus contributes only a smallercentage of the predisposition to the disease. Inheriting several of theseNPs may give you an accumulative effect as expressed in the phenotypef a polygenetic disease. The diseases that ultimately must be understoodre those diseases due to multiple genes that interact significantly with thenvironment such as cardiac diseases, cancer, and mental illness. In anffort to facilitate future studies identifying SNPs and their relatedhenotype in polygenetic diseases, a consortium was formed consisting ofanada, Japan, United Kingdom, China, Nigeria, and United States to

equence and identify SNPs. The overriding question was to determinehether SNPs were coinherited in blocks and, hence, the term haplotype

nd the HapMap Project.27 The results were published and do indeedndicate that several of the SNPs are coinherited as blocks and exert aombined effect and thus one could select SNPs that are tagged to otherNPs, making it practical to scan the genome utilizing 300,000 to 500,000
NPs as opposed to several million. While each human being has only 3

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illion SNPs, in the general population it is estimated there are about 17illion. It would now appear that 500,000 SNP chips can be used for

enome-wide scans, which significantly decreases the cost compared toaving to utilize 2 or 3 million SNPs. One of the difficulties that continueso remain a challenge is the low frequency of occurrence of these SNPs.t would appear many of the SNPs occur at a frequency of less than 5%,hich makes detection by current technology very difficult (Table 4).ommon SNPs that occur with frequency of 5 or 10% can, however, beetected utilizing genome-wide scans with 500,000 SNPs as markers. Itppears that probably only 50,000 to 100,000 SNPs are responsible forroviding significant change in humans since most SNPs do not affectoding regions, although the percentage of SNPs present in noncodingromoter regions that may markedly influence transcription remains to beetermined.

hree Features Inherent in the DNA Moleculerovide the Foundation for Research and MedicalpplicationsThree features essential to all techniques of recombinant technology areorthy of note. The first is the ability of DNA, a double-strandedolecule, to denature and anneal or hybridize (Table 3). The double-

tranded DNA, held together by hydrogen bonding of the correspondingomplementary basis, will under exposure to high temperatures (95°C)eparate into two strands and, if the temperature is reduced, the comple-entary strands will again come together (anneal) and return to their

revious double-stranded state. The process of separating DNA intoeparate strands is referred to as denaturation and the recombiningrocess is known as annealing or hybridization with the latter termreferred if the two DNA fragments are from different sources. It isotable that the two strands of DNA join together identically to that of thearent molecule. This is because of complementary basepairing, whereby

must bind with T and C to G. The third feature utilized in the variousechniques is that of the high energy phosphorus, which is present in the

ABLE 3. Three unique features inherent in the DNA molecule

Ability of DNA molecule to separate into separate strandsAbility of DNA strands to reanneal or hybridizeThe inherent negative charge of phosphorous

NA molecule, providing it with a negative charge. These properties are


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ssential to all techniques as well diagnostic and therapeutic agents thatrise or are derived from recombinant techniques. One exploits theroperty of DNA to denature and recombine whereby a complementaryNA marker can be labeled with either a radionuclide or a color pigment

nd, if the complementary DNA is present in the sample, it willecombine (hybridize) with the indicator molecule to confirm the pres-nce of the complementary basepair. The probe is usually an oligonucle-tide of 20 or 30 bases, which is adequate to isolate DNA molecules. Onexample of this is confirming the diagnosis of a pathogen. The negativeharge imparted by the phosphorus is exploited to separate DNAolecules of different sizes through electrophoresis. By selecting the size

f the pores in a media, one can separate molecules of different sizesased on their charge as they move toward the positive electrode. Allechniques utilized in molecular biology utilize two or more of theserocedures.

echniques Used in Molecular Genetics

solation of DNADNA can be isolated from all human tissues with the exception ofature erythrocytes, which no longer contain a nucleus. Most procedures

sed in molecular biology require only nanogram (10�9 g) quantities ofNA, so enough tissue to yield these quantities may be obtained even

rom blood smears. In man, lymphocytes are a convenient and accessibleource of DNA, since they have the added advantage that they can beransfected by a virus (usually Epstein–Barr virus) to produce anmmortal cell line that can be propagated in the laboratory indefinitelynder appropriate cell-culture conditions. This process is called lympho-yte transformation and provides for a continuous renewable source of

ABLE 4. Discoveries seminal to modern molecular biology

1889 Isolation of DNA1944 DNA as hereditary material1953 DNA structure deduced1964 Genetic code deduced1970 Discovery of specific restriction endonuclease1970 Discovery of reverse transcriptase

1972/73 Development of the cloning technique1975/77 DNA sequencing

1980 Polymerase chain reaction1982 Site-directed mutagenesis

NA from an original specimen. However, today the technique of


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hole-genome amplification has all but replaced development of cellines as a means of having renewable DNA.Regardless of the source, the DNA present within the nucleus must be

xtracted and separated from the other cellular components. This iserformed by lysis of the cell and nuclear membrane, removal of allroteins, and isolation of the DNA. Classically, this procedure involvesysis of the cell using a lytic enzyme and a nonionic detergent, depro-einization of the lysed products using the organic solvents phenol andhloroform, and precipitation of the DNA by ethanol from a solution ofigh salt concentration. If performed appropriately, this procedure yieldsfairly pure product visible to the naked eye. This process is universallysed and has led to the production of automated instruments thatffectively and efficiently extract the DNA such that 10 to 15 ml of wholelood will typically yield approximately 50 to 100 �g of genomic DNA.

eparation of DNA Fragments by Gel ElectrophoresisAfter DNA is isolated, it is digested with a restriction endonuclease and

oaded into a well of agarose or polyacrylamide gel and subjected to anlectric current (Fig 7). Each individual nucleotide because of itshosphorus has a net negative charge that forms the basis of separation ofNA fragments in an electrical current. The DNA fragments migrate

oward the anode according to their size with the larger fragmentsigrating slowest. Following separation of the fragments, the gel is

tained with ethidium bromide. To determine the size of the fragments ofhe unknown DNA, standard DNA fragments of known size are concom-tantly electrophoresed for comparison. Electrophoresis through agaroseill separate double-stranded DNA fragments varying from 1000 to00,000 bp and polyacrylamide is used to separate DNA fragments fromto 1000 bp. Theoretically, polyacrylamide gels should separate frag-ents differing in size by only 1 bp. Utilizing these gels, one can detect

ands as little as 1 ng and a difference between fragments of 0.5% of theirize.

outhern, Northern, and Western BlottingThe ability to identify specific fragments of DNA after electrophoresisecame routine after the development of the Southern transfer techniqueFig 8) by E. M. Southern in 1975,28 now referred to as Southern blotting.e developed a procedure whereby DNA fragments separated by gel

lectrophoresis are transferred by capillary action to a filter and theattern of the bands are permanently fixed identical to the pattern
btained on the gel. Briefly, the electrophoresed double-stranded DNA is

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hemically denatured into single strands while in the gel and passivelyransferred onto a nitrocellulose filter or a nylon membrane. The trans-erred DNA is then irreversibly bound to the filter by baking the filter athigh temperature. DNA can be effectively transferred by this procedure

fter several hours with the resultant blot being the exact duplicate of theel containing the electrophoresed DNA.The blotted membrane can then be used to identify particular segmentsf DNA by reacting (hybridizing) the single-stranded DNA on theembrane with a solution containing a probe such as a 32P radioactively

IG 7. A typical Southern blot with distinct bands. Each vertical lane consists of DNA from aeparate individual. Source: Roberts R. Techniques of molecular biology. In: Roberts R, editor.

Primer of Molecular Biology. New York: Elsevier Science Publishing, 1992. p. 57.

agged single-stranded segment of DNA, which is complementary to a


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NA region within the targeted segment. The conditions of the hybrid-zation process are empirically established such that the probe attachesnly to it complementary segment of DNA, and the resultant double-tranded product is analogous to the native double-stranded DNA. Therobe may be a segment of DNA defined by its sequence (oligonucleo-ide) or merely a small fragment of DNA from a much larger segment ofnterest. X-ray film is then exposed to the tagged membrane at �70°C in

process called autoradiography. The resultant pattern shown on the-ray film reflects complementary fragments of DNA on the membrane

hat hybridized to the probe. Analysis of RNA by a similar technique isermed Northern blotting and analysis of proteins is termed Westernlotting.In human disease caused by large DNA deletions rather than singleasepair changes, Southern blotting is the method of choice for detection.he use of a DNA probe covering the known sequence of the deletion willot be visible after hybridization of the probe if the deletion is present inhe DNA segment, but will be visualized in a normal DNA fragment.

IG 8. Southern transfer apparatus. Source: Roberts R. Techniques of molecular biology. In:oberts R, editor. A Primer of Molecular Biology. New York: Elsevier Science Publishing, 1992.. 56.


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IG 9. The cloning of a DNA fragment utilizing a plasmid as the vector. Source: Roberts R.echniques of molecular biology. In: Roberts R, editor. A Primer of Molecular Biology. New
ork: Elsevier Science Publishing, 1992. p. 63.

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NA CloningThe prerequisites for cloning are depicted in Fig 9 and consist of the

ollowing: (1) an isolated DNA fragment to serve as an insert; (2) a vectorplasmid); (3) a restriction endonuclease site common to both the insertnd the vector so that the DNA ends will be compatible, allowing theragment to be ligated or inserted into the vector; (4) a DNA ligase toigate the insert into the vector; and (5) a means to differentiate the hostells that have incorporated the vector with the insert from those that haveot.Fundamentally, DNA cloning is a technique that replicates a specific

ragment of DNA in a replicating organism. The foreign DNA fragmentinsert) is ligated (inserted) into a larger segment of DNA (vector). Theector (containing insert) is then placed in its host cell, where it replicatesnd amplifies. There are several means of detecting those bacteria whichave incorporated the vector (with insert). One approach is to modify theector to include a gene that expresses a protein resistant to ampicillin.nother is to express a gene which emits a color. The bacteria are grown

n media containing ampicillin and only those that have the resistanceene (and therefore the vector) will survive. Thus after the bacteria arerown and plated out on agar, monoclonal bacterial colonies (clones) areelected on the basis of ampicillin resistance. Growth in this mediandicates that the vector was successfully incorporated into the bacterialolony and the vector with insert was successfully cloned. A singleolony of bacteria is selected and further amplified in culture media. DNAragments from any source can be amplified at least a million-fold.Following amplification, the vector containing the insert of interest isurified, and the cloned insert isolated in large quantities. Typically,ectors used are plasmids, circular DNA molecules that naturally repli-ate in various strains of bacteria. Inserts may represent any DNAragment of interest, typically a gene coding for a protein of interest thatay then be made in large quantities for biochemical studies. The route

ield from cloning is about 1 million copies of the desired DNA fragment.Cloning is used by industry to produce for pharmaceutical purposes vast

mounts of purified substances that normally are found in limiteduantities. The practice of cardiology was significantly impacted by theroduction of rt-PA for treatment of acute myocardial infarctions.imilarly, endocrinologists treat patients with diabetes mellitus usingecombinant human insulin.


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olymerase Chain ReactionThe development of the PCR (Fig 10) revolutionized the approach toolecular biology and molecular genetics.23,24 PCR is an automated in

IG 10. Polymerase chain reaction. Source: Roberts R. Techniques of molecular biology. In:oberts R, editor. A Primer of Molecular Biology. New York: Elsevier Science Publishing, 1992.. 70.

itro repetitive reaction that uses a heat-stable DNA polymerase to


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mplify a specified segment of DNA. The elegance of PCR lies in itsimplicity and the property to amplify a single copy of the target fragmentf DNA up to 1 million-fold in a matter of hours without the use ofedious classic cloning techniques, thus enabling easy access to largeuantities of DNA. An essential property of DNA making PCR possibles the property of DNA to denature and separate into its two complemen-ary strands and, under appropriate conditions, reanneal and again becomeouble-stranded (hybridization). The complementary basepairing and thebility to hybridize underlies all the essential techniques of molecularenetics.Practically the only limitation to PCR is the need to know a short stretchf base sequences flanking both the 5= and the 3= ends of the desiredegment of DNA. A sequence of approximately 20 bp on both ends of theesired fragment is all that is necessary. These oligonucleotide sequences,alled primers, are complementary to small regions of both strands ofarget DNA (template) located at both ends (5= and the 3= end). A senserimer is designed with a sequence identical to the 5= end of the templatend an antisense primer is designed with the sequence of the strand thats the complement to the 3= end of the template. Under the usual reactionarameters used, primers of 18 to 24 bp have a significant affinity for onlyhe target template and, therefore, the vast majority of the reactionroduct is specific. The strategic location of the primers and the 5= to 3=ction of DNA polymerase dictate that the amplified region will beverything located between the primers. A reaction cycle consists ofenaturation of a single double-stranded DNA template; annealing of therimers to the specified regions on the template; and extension from bothrimer sites by DNA polymerase in the 5= to 3= direction, to yield twoouble-stranded products. This reaction is cyclically repeated to increasehe number of products exponentially until a plateau is reached afterpproximately 25 to 30 cycles. It is routinely possible to amplifyragments up to 10,000 bp (10 kb).The applications of PCR are many. The PCR product, which may

epresent a portion of a gene, may be screened for a genetic mutation byne of numerous mutation detection systems. In principle, mutationetection systems are designed to detect a change in DNA sequence bybserving shifts in gel mobility or melting properties of a mutant DNAragment, as compared to a fragment with normal sequence. Mutationetection systems are sensitive to the level of detecting a single basepairlteration. A commonly used technique in human genetics is single-tranded conformation polymorphism (SSCP) analysis, whereby a single
asepair change from normal sequence may show a differing migration

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attern on a non-denaturing gel, caused by a change in secondarytructure of the mutated fragment. Similarly, a mutated DNA sequenceay alter the melting temperature of DNA, resulting in differential

etention of the fragment (compared to normal sequence) on a chroma-ography column (denaturing high-performance liquid chromatography,HPLC). Alternatively, a researcher may choose to directly sequence theucleotide DNA sequence and compare the sequence to normal sequence.The process of PCR may also be used to derive full-length cDNA (gene

equence without intervening introns) from single-copy mRNA of ex-ressed genes by a method called rapid amplification of cDNA ends. Thisethod uses multiple primer pairs located within the region of interest asell as at the end of the mRNA sequence, including the poly(A) tail.mplification using multiple primer sets generates multiple overlappingroducts; the overlapping regions can be identified and the remainingequences can be combined to define the entire cDNA. The diagnostictility of PCR has been well established in infectious disease, includingiral myocarditis. Conventionally, detection of viral DNA required ateast 50,000 copies of viral nucleic acid per cell, making the diagnosis ofiral myocarditis virtually impossible using myocardial biopsy samples.resently, single-copy viral genes can be amplified for detection by PCR.hese are just a few of the potential applications of PCR and it is most

ikely that PCR will continue to have its impact on medicine for years toome.

NA SequencingDNA sequencing is the determination of the precise sequence ofucleotides in a sample of DNA. The most commonly used method isalled the dideoxy method. DNA sequencing reactions all use a primer tonitiate DNA synthesis. This primer will determine the starting point ofhe sequence being read, and the direction of the sequencing reaction. Theuccess of this technique is based on the use of fluorescently labeledideoxy nucleotides for each of the bases comprising DNA (ddATP,dCTP, ddGTP, ddTTP). As opposed to deoxynucleotides, dideoxynucle-tides (ddNTPs) lack of a free 3= OH group. Although ddNTPs may bedded to a DNA template being elongated by DNA polymerase, thesedNTPs prevent the next nucleotide from being added, and the chainould terminate. In a sequencing PCR reaction, both normal andideoxynucleotides are present and the incorporation of a ddNTP willccur randomly. Through repeated cycles of denaturing, primer anneal-ng, and extension/termination, new strands of DNA may be synthesized
hat will all vary in where in the fragment termination occurred due to the

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ncorporation of a fluorescently labeled ddNTP. Thus, at the conclusion ofDNA sequencing reaction, a pool of DNA exists ranging from the

mallest possible fragment size of 1 bp to the maximize size of theragment. In automated DNA sequencers, the different fluorescent labelsttached to each of the four dideoxynucleotides (ddA, ddC, ddG, and ddT)ill be detected from base 1 to the final base, providing a color-codedNA sequence.DNA sequencing has become commonplace and new technologies have

nhanced the rate and precision for sequence determination. It is presentlyossible to sequence up to millions of basepairs per day using automatedechnology.

ite-Directed MutagenesisThe technique of site-directed mutagenesis provided researchers theltimate tool for studying the pathophysiological basis of disease causedy genetic mutations. This technique was made possible by previousnnovations in molecular biology, including PCR, insertion of a DNAragment into a vector, and cloning. A circular plasmid moleculeontaining a gene of interest may serve as a template for PCR. Primers areesigned to purposely mismatch a single nucleotide in the gene, engi-eering an altered codon, replacing one amino acid for another. FollowingCR, the amplified plasmids will then contain the insert with the mutatedodon, and the mutated gene may be cloned for further studies.The advances in molecular biology leading to the identification ofisease-causing DNA mutations required a technique to generate mutantroteins and to analyze their function in vitro to elucidate the abnormalhysiology leading to disease. Site-directed mutagenesis enables thetudy of mutant proteins in in vitro systems and recombinant DNAolecules can also be injected into the germ line of mice and successfully

xpressed in succeeding generations. These transgenic animals thereafterecome a model for in vivo analysis of the pathogenesis of the disease.

olecular Genetics: Genetic TransmissionAll hereditary information is transmitted from one generation to thether through DNA. The basic hereditary unit is referred to as a gene andonsists of a distinct fragment of DNA which encodes a specificolypeptide (protein). Each individual has two copies of each gene, calledlleles, one from the mother and one from the father. It is estimated theuman genome has 25,000 genes localized in a linear sequence along 23airs of chromosomes, including 22 pairs of autosomes (chromosomes 1
o 22) and one pair of sex chromosomes, X and Y. Females have two X

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hromosomes, while males carry one X and one Y chromosome. Eacharent must contribute one of each chromosomal pair and thus one copyf each gene. The gene is located at a particular site on the chromosomend is referred to as the chromosomal locus or genetic locus. A given genelways resides at the same genetic locus on a particular chromosome sohe loci on homologous chromosomes are identical. However, allelesesiding at these loci may be homozygous (identical alleles) or heterozy-ous (two different alleles).

lassification of Inherited DisordersThe DNA molecule is notable for its stability and seldom changes fromne generation to the other. Nevertheless, occasional base sequencehanges do occur, referred to as mutations. Mutations are defined astable sequence changes in DNA that are inherited. Mutations occur at arequency of approximately one every 200 years. Mutations may involveportion of the chromosome, a single nucleotide as either a substitution,deletion, or an insertion, or multiple nucleotides.It is thus convenient to classify heredity diseases into three broad

ategories, as follows: (1) chromosomal abnormalities; (2) single-gene oronogenic disorders; (3) polygenic disorders or complex traits which are

ue to interactions of multiple genes and nongenetic factors.

hromosomal AbnormalitiesHuman cells each have two copies of each chromosome (diploids) and

ach chromosome has two arms referred to as the long (Q) or the short (P)rms (Fig 11). The arms of the chromosome meet at primary constrictioneferred to as the centromere. Chromosomal abnormalities are commonnd are the most common cause for spontaneous abortions. Chromosomalbnormalities are much more a concern of pediatrics than of adult disease.he chromosomal abnormalities are usually large and can be detectedost of the time by doing karyotyping or simply microscopic analysis of

he chromosomes. They will not be discussed except to state that the twoost common adult cardiovascular chromosomal diseases are Down

yndrome (Trisomy 21) and Turner syndrome (xo), both due to non-isjunctions of the chromosomes.

ichard A. Walsh: Non-disjunction refers to the failure of a homologous pairf chromosomes to separate during meiosis. When an additional copy of ahromosome is added during fertilization, three copies of the same chromo-ome (Down syndrome) or only one copy (Turner syndrome) is found in theygote rather than a chromosome pair.


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ingle-Gene DisordersA single-gene disorder is an inherited disease cause by mutations in a

ingle gene that are necessary and sufficient for the development of thehenotype. They show a Mendelian pattern of inheritance classified asutosomal-dominant, autosomal-recessive, or X-linked (dominant or re-essive). Mitochondria has its own DNA which encodes for 37 genes.iseases due to mitochondrial DNA mutations are only transmitted from

he mother (no male-to-male transmission), since only ovum has mito-hondria. It is important to realize that only a very small fraction ofardiovascular disorders are single-gene disorders and in the wholeopulation the prevalence of single-gene disorders is rare, varying from 1n 1000 to 1 in 10,000 and essentially never exceeds 1 in 500 (0.5%). Its estimated there are about 14,000 single-gene disorders, of which over500 of the genes have been identified. The genotype refers to the geneticasis, while phenotype refers to the observable features such as height,eight, or clinical features of a disease. One may possess the gene but not

xpress the phenotype. The percentage of individuals with a gene that isxpressed as a phenotype is referred to as penetrance and the variabilityn the clinical features of a particular expressed phenotype is termed

Chromosome classification

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xpressivity. On average, a mutation occurs every 106 cell divisions or


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nce every 200,000 years. Only mutations occurring in the gametes areransmitted. The patterns of inheritance are shown in the diagram in Fig2. In autosomal-dominant disorders males and females are equallyffected; an offspring of an affected parent will have a 50% chance ofnheriting the mutant allele. In sporadic cases, the mutations occur deovo in one of the germ lines of the parents but by definition is absent inhe somatic cells of parents. Autosomal-dominant inheritance usually hasariable expressivity. The following features are characteristic of auto-omal-dominant inheritances (Fig 12): (1) each affected individual has anffected parent unless the disease occurs due to a new mutation or theres low penetrance; (2) there is usually an equal split (50/50) of normal andffected offspring born to an affected individual; (3) normal children ofn affected individual will have only normal offspring; (4) equal portionsf males and females are affected; (5) both sexes are equally likely toransmit the abnormal allele to male and female offspring and male-to-ale transmission occurs; and (6) vertical transmissions through succes-

IG 12. Pedigrees outlining the usual inheritance patterns for autosomal-dominant and recessiveraits, X-linked inheritance, and mitochondrial inheritance. Squares signify males and circlesignify females. Filled circles and squares are affected females and males, respectively. Source:arian AJ, Bruqada R, Roberts R, Hurst The Heart, ed. 11, New York, McGraw-Hill, p. 750,004.

ive generations occur. Two other characteristic features that help


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ifferentiate this type of inheritance for autosomal-recessive disorders areelayed age of onset and variable clinical expression. In autosomal-ominant, the phenotype is observed despite only one of the gene orlleles being affected. In autosomal-recessive inheritance, both alleles areffected, otherwise there is no phenotype. Males and females are equallyffected. Clinical uniformity is more typical and disease onset generallyccurs much earlier in life than in autosomal-dominant. Recessiveisorders are more commonly diagnosed in childhood and, on average,nly one in four children or 25% will be affected. The following areharacteristic of autosomal-recessive disorders: (1) parents are clinicallyormal in alternate generations (genetically are heterozygous); (2) alter-ate generations are affected, with no vertical transmission; (3) bothexes are affected with equal frequency; (4) each offspring of hetero-ygous carriers has a 25% chance of being affected, a 50% chance ofeing an unaffected carrier, and a 25% chance of inheriting only normallleles.X-linked inherited disorders are caused by defects in genes located on

he X chromosome. Females have two X chromosomes and thus, if onlyne mutant allele, may seldom develop the phenotype. On the other hand,ales have a single X chromosome and are more likely to display the full

yndrome whenever an abnormal gene is inherited from the mother. Theharacteristic features of X-linked inheritance include (1) no male-to-ale transmission; (2) all daughters of affected males are carriers; (3)

ons of carrier females have a 50% risk of being affected and daughtersave a 50% chance of being carriers; (4) affected homozygous femalesccur only when an affected male and carrier female have children; and5) the pedigree pattern in X-linked recessive traits tends to be obliqueecause of the occurrence of the trait of the sons of normal carriers but notn sisters of affected males. Examples of X-linked disorders of the heartnclude X-linked cardiomyopathy, Bart syndrome, a Duchenne/Becker,nd Emery–Dreifuss muscular dystrophy.Since mitochondria DNA is transmitted to the next generation onlyy the female, DNA mutations can only be inherited by the mother.he characteristic features of mitochondria inheritance include the

ollowing: (1) equal frequency and severity of disease for each sex;2) transmission through females only, with offspring of affectedales being unaffected; (3) all offspring of affected females may be

ffected; (4) extreme variability of expression of disease within aamily; (5) phenotype might be age-dependent; (6) organ mosaicism is
ommon.

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apping the Chromosomal Location (Locus) of Genes byinkage AnalysisChromosomal DNA Markers. To understand chromosomal mapping ofenes, one must first understand chromosomal markers, chromosomalrossovers (recombination), and the concept of genetic linkage.29,30 Forost inherited diseases, we do not know the responsible gene or protein.or some time, it has been possible to map the location of genes withoutnowledge of the causative gene or protein. This was initially referred tos reverse genetics and now more appropriately named positional map-ing and cloning since one clone’s the gene knowing only its chromo-omal position relative to another chromosomal marker. A chromosomes a linear molecule of DNA, varying in length from 50 million bpchromosome 21, smallest) to 263 million bp (chromosome 1, largest). Ahromosomal marker is a polymorphic sequence of DNA (referred to asenotyping) with known chromosomal position which can be detected bynalyzing (genotyping) an individual’s DNA. DNA markers are nowvailable to span each chromosome at intervals of 3 to 5 million bp. Theoutine is to screen with a set of 300 to 800 markers selected to span theuman genome. DNA markers, like genes, have two alleles per individ-al, one from each parent, and are transmitted to offspring according toendel’s law with the individual being heterozygous or homozygous for

hat marker. For a marker to be informative it must be heterozygous.hen all of the markers are placed together on each chromosome and the

enetic distance estimated, a genetic map is produced. Genetic distance iseasured in terms of centamorgans (cM), named after the geneticist,. H. Morgan. One centamorgan approximates 1 million basepairs (mbp).he availability of the human genome DNA sequence now makes itossible to estimate the precise physical distance in basepairs rather thanelying on a genetic estimate. Over 5000 highly informative chromosomalarkers spanning the entire genome are now available. Identification ofparticular locus housing a gene of interest is made possible by showing

hat the causal gene of interest is in close proximity to one of the DNAarkers of known chromosomal location, a method referred to as genetic

inkage analysis. Once a disease is linked to a marker of knownhromosomal locus, it means the disease locus and the marker are on theame chromosome and in close physical proximity. One then attempts todentify other DNA markers to flank the disease locus to reduce theistance between it and the markers.Chromosomal Crossover (Recombination). Since humans inherit two

ets of autosomal chromosomes (diploid), one from each parent, all of the


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enes carried by the autosomal chromosomes have two forms, referred tos alleles, one on each chromosome. The two alleles occupy the samehromosomal locus on different chromosomes (homologous), which giveise to the terminology of homologous loci on homologous chromosomes.

hich of the parents two chromosomes is inherited by the offspring isandom, meaning there is only a 50% chance as to which of the parents’wo chromosomes will be inherited by the offspring. In addition which ofhe parents’ two alleles is transmitted to the offspring depends on anotherrocess referred to as chromosomal crossover, which occurs betweenairs of homologous chromosomes. Prior to meiosis, homologous chro-osomes and only homologous chromosomes come together and form

ridges (chiasmata, usually two per pair), between them such thategments of equal proportions are exchanged between them, giving rise torossover of the accompanying genes (Fig 13). In genetic parlancehromosomal crossover is referred to as recombination since a segment ofne chromosome has broken away and replaces the same segment of thether pair such there is simply an exchange of equal proportions betweenhe two pairs. Thus there is no loss or gain of chromosome or genes. This

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ffspring will have the same genes. However, it is important to point outhat this process of recombination means the allele crossing over to itsomologous chromosome l occupies the same location (locus) as on itsrevious chromosome. Thus, the actual position of the gene or marker onach chromosome referred to as the locus remains the same for anyarticular allele or marker. Whether genes are separated by recombinationepends on the distance between them on the chromosome and theumber of meiosis that occur. The further apart the genes are, the moreikely they are to be separated (recombination) and the chances increaseith every meiosis. While genes are independent units and are passed on

n random fashion if two or more genes are close together on the samehromosome and no chiasma is formed between them, they will beoinherited in the offspring. In genetic parlance the two genes areenetically linked. In chromosomal mapping to identify the location of annknown gene responsible for disease in a family we take advantage ofhis principal. We analyze (genotype) the DNA of all of the familyembers, normals and affecteds, for DNA markers spanning each

hromosome. If we observe a particular marker or set of markers inheritedy the affecteds but not by the normals, it means that marker is in suchlose physical proximity to the gene that causes the disease that everyime the gene causing the disease is inherited so is the DNA marker. Thearker and the disease gene are linked and we now know which

hromosome and the approximate location (locus) of the gene. It turns outhat any two markers, two genes, or a marker and a gene will separaterecombine) at a frequency of 1% per 1,000,000 bp of distance betweenhem. Thus, recombination is very much related to the physical distanceetween the marker and the gene. The recombination frequency isalculated by dividing the number of crossover events or recombinationsy the total number of meioses. Once the locus of a gene is mapped, onean further saturate that region with additional chromosomal markers toinimize the distance between the flanking markers. One would prefer to

arrow the region to about 1 million bp, although this is not alwaysossible.The basis for chromosomal crossover is illustrated in Fig 14. The locusesignated with “A” carries the allele responsible for the disease. Theorresponding locus “a” on the homologous chromosome has the allelehat codes for the same protein but has not undergone a mutation and ishus the normal allele. The loci designated “B” “b” represent alleles of aNA marker of known chromosomal location that has nothing to do with

he disease. In the right-hand panel the disease and marker loci are so
lose that they tend to be coinherited in the subsequent offspring, whereas

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n the left-hand panel the DNA marker of known location is so far fromhe locus carrying the disease of the allele that it is far less likely to beoinherited in the offspring.Genetic Linkage Analysis. Genetic linkage analysis is only appropriate

f one has a family of two or three generations in which a particularisease is segregating across the generations and exhibits a Mendelianattern of inheritance. The family is phenotyped, meaning each familyember is assessed clinically for the disease and phenotyped as affected,

naffected, or indeterminant (diagnosis cannot be ascertained). A pedi-ree is then constructed of the families showing affected, unaffected, andndeterminant, as indicated in Fig 15. DNA is analyzed for the wholeamily of both affected, normal, and indeterminant individuals, whichonsists of genotyping for all of the DNA markers selected to span theuman genome, initially a set of 300 to 800 markers. Followingenotyping to exclude or prove linkage to a DNA marker, it is necessaryo perform this analysis utilizing computerized techniques. Frequently,

IG 14. Genetic linkage.

he marker and the gene are coinherited only in affected individuals but


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IG 15. A pedigree of three generations having individuals affected with hypertrophicardiomyopathy (HCM). The open circles indicate unaffected females; the open squaresndicate unaffected males; the solid symbols indicate affected individuals (both male andemale); the slash through a symbol indicated the patient is dead; and a circle or squareithin the circle or square indicated the diagnosis is uncertain. DNA was analyzed for

estriction fragment length polymorphisms (RFLPs) by Southern blotting, and the results arehown on this autoradiograph for 11 of the individuals in the pedigree. Each vertical laneepresents the DNA of the individual indicated by the number above, which corresponds tohe same number on the pedigree. The DNA was digested with the restriction endonucleaseaq1 and separated on agarose gel electrophoresis. It was then denatured into its twoeparate strands, transferred to a nylon membrane by the Southern transfer technique, androbed with a 32P-labeled probe. The probe, referred to as P436, was derived from part of

he beta-myosin gene, which is known to be located on the long arm of the chromosome 14.his probe recognizes two alleles, one at 4.2 kb and the other at 1.8 kb. The largerragment at the top is consistently present in all of the individuals, so we will be examininghe polymorphic alleles of 4.2 kb (A1) and 1.8 kb (A2).

Individual 51 is an affected female who is heterozygous, having received the A1 allele fromne of her parents and the A2 allele from the other parent. Individual 49, in contrast, is


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his may not always be 100%, keeping in mind anything over 50%oinheritance could reflect genetic linkage. Several methods have beensed all based on computer analysis with the most common beingaximal Likelihood Estimate.31 One estimates the probability of a

articular inheritance pattern indicating linkage. This probability can thene compared to the probability of that particular inheritance pattern noteing linked. The ratio of these two probabilities (that is, of linkage at aiven recombination fraction versus nonlinkage) is called the Odds Ratioor Linkage. This ratio is usually expressed as a log rhythm to base 10.he value is called the log rhythm of the odds or LOD score. Thus, a LODcore of one represents 101 odds that a marker is genetically linked to theene. If the odds are 1000:1 or 103, the log rhythm of these odds woulde 3 and is referred to as a LOD score of 3. The minimum LOD score forenetic linkage is 3 in the case of autosomal-dominant disease. In the casef X-linked disease a LOD score of 2 is accepted for linkage. To excludeenetic linkage simply requires a LOD score of �2 or less. In biostatis-ical terms a LOD score of 3 represent 95% likelihood of linkage, whereasLOD score of 4 represents 99% chance of linkage.

omozygous, having inherited the identical A2 allele from both the mother and the father.ndividual 53, a normal female, is also homozygous for the A2 allele. Individual 57, a normalemale, is heterozygous at this locus, having both the A1 and the A2 alleles. Individual 59, anffected male with HCM, is also heterozygous. Individual 64, an affected male, is homozygous,ith both alleles being A2. Individual 66, an affected female, is heterozygous, having both the1 and the A2 alleles. Individuals 67 (normal male), 72 (normal female), and 78 (affected

emale) are all homozygous for the A2 allele. Individual 79 is a normal male and iseterozygous, having both A1 and A2 alleles. Computer analysis of the beta-myosin gene inhis family together with that in other families showed linkage between this marker and theisease for hypertrophic cardiomyopathy. A LOD score was obtained of greater than 4,

ndicating the odds for linkage are more than 99%.The analysis of this Southern blot illustrates several of the key features of linkage analysis

xplained in the text: (1) the same polymorphic pattern at the marker locus can be seen in bothnormal and an affected individual within the same family; and (2) some affected individualsre homozygous at the marker locus while others are heterozygous, and, as indicated in the

ext, only those individuals who are heterozygous for the two alleles will provide information forinkage analysis with this particular marker locus. Thus, which allele is inherited by the siblingrom the parents at the marker locus is completely random and independent of which allele isnherited at the disease gene locus, despite the two loci being linked. The analysis in this familylso shows how, because of the lack of information, one may require a larger number of

ndividuals than initially expected to ascertain whether linkage is present between the markerocus and that of the disease. In several of the individuals shown here the marker locus isomozygous and therefore will contribute almost no information to the linkage analysis. For arobe to be informative, it must be heterozygous, which is frequently not the case, as illustrated

n this pedigree analysis. Source: Roberts R. Essentials of molecular genetics. In: Roberts R,
ditor. A Primer of Molecular Biology. New York: Elsevier Science Publishing, 1992. p. 114.

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To map a chromosomal locus by linkage analysis usually requires aedigree of at least two generations and preferably three generations havingt least 10 affected individuals. A major problem is always the certaintyhereby the phenotype can be determined, which is very much the respon-

ibility of the physician. It is hoped that future efforts to more preciselyhenotype will be developed to facilitate our search for disease-related genes.

Summary of Axioms of Genetic Linkage Analysis.

. In reference to genes on autosomal chromosomes, every individual hastwo forms of the gene, referred to as alleles, one being inherited fromthe mother and the other inherited from the father. In individuals withdominant disease, one allele is defective and the other is normal.

. The DNA marker of known chromosomal location to which a diseasegene is linked also has two alleles, one from the father and one fromthe mother.

. When a DNA marker and disease-related gene are said to begenetically linked, it means that the two loci are linked, not theiralleles.

. Since the two loci are linked, and not the alleles, which allele aparticular offspring gets is total chance since the inheritance of eitheror both alleles at a particular locus is independent of the other.

. Neither of the alleles at the maker locus has anything to do withcausing the disease. Both alleles of the marker locus occur in thegeneral population and do not themselves cause disease. They simplyreside at a locus that is in close enough physical proximity to the locusthat contains the disease-producing gene to be coinherited more oftenthan by chance.

. To be informative for linkage analysis, the alleles of the marker locusmust be heterozygous. This means that the two alleles at the markerlocus must not have a nucleotide sequence identical to that of the probebeing utilized for their detection but must be polymorphic.

. Linkage of a marker locus and a disease-related locus implies that thetwo are coinherited more often than by chance alone, which meansmore often than 50% of the time. It does not mean, however, that thetwo loci are always coinherited; in fact, only if they are extremelyclose would this be true.

. It follows from previous axioms that, in analyzing the DNA of themarker loci of individuals within families affected with the disease, thesame pattern may be seen in an individual without the disease as in
those individuals with the disease. This is why computer analysis is

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necessary to ascertain whether the disease allele is more commonlyinherited with one or more of the alleles at the marker loci than wouldbe expected by chance.

. Crossover or recombination occurs between, and only between,homologous chromosomes, so the alleles that cross over or recombineoccupy the same locus on their new chromosomes as they did on theprevious ones.

solation and Identification of the GeneOnce the locus of the gene has been determined, one attempts to narrow

he region between the flanking markers before proceeding to identify theene. Today with the sequence of the genome known and many genesaving already been mapped to their chromosomal locus, the firstpproach is to sequence genes in the mapped region as potential candidateenes. If the candidate genes in the region after being sequenced do notontain the responsible mutation, it may be necessary to clone the regionnd identify novel genes to be sequenced as candidates for the mutation.nce the mutation is identified, one then determines if it is indeed the

ausative mutation. The minimum requirement is the mutation be foundn affecteds and not in normal family members and be absent in at least00 normal individuals representative of the population from which theamily with the disease was selected (eg, Caucasian, African, or Chinese).

verview of Phenotyping, Genotyping, Mapping,nd Identification of the GeneThe overall approach to chromosomal mapping of heredity diseases by

inkage analysis and subsequent isolation of the gene may be summarizedategorically as follows: (1) collection of data from families havingndividuals affected by this specific disease through two or three gener-tions; (2) the disease segregates in a Mendelian pattern; (3) clinicalssessment to provide an accurate diagnosis of the disease using consis-ent and objective criteria to separate normal individuals from thoseffected and those who are indeterminate or unknown; (4) collection oflood samples for extraction of DNA for immediate analysis andubsequent whole-genome amplification should be stored in small ali-uots to avoid repeated freezing and thawing; (5) development of aedigree for analysis of the families; (6) DNA genotyping with a largeumber of DNA markers of known chromosomal loci that span theuman genome; (7) linkage analysis is performed on the genotypes to
ap the chromosomal locus; (8) development of flanking markers around

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he region containing the disease locus; (9) isolation and cloning of theegion of DNA containing the gene; (10) sequence analysis of the gene todentify the precise mutation causing the disease; (11) demonstration ofhe causal relationship between the defective gene and the disease byhowing segregation of the mutation in affected individuals only andbsence in an independent, unrelated normal population.

nherited Cardiovascular Diseases

ardiomyopathiesHypertrophic Cardiomyopathy. Hypertrophic cardiomyopathy (HCM)

s the most common cardiovascular disease inherited as an autosomal-ominant single-gene disorder (Fig 16). Although recognized as a familialisease for many decades, it was not until the landmark genetic discoveryf Christine and Jonathan Seidman in 1990 that the molecular basis of thisisorder was elucidated.32 Their finding that a single-basepair mutation inhe beta-myosin heavy chain (MYH7), resulting in an arginine-to-lutamine amino acid change, indicated that this disease is primarily aisease of contractile sarcomeric proteins. Since this initial discovery,ver 400 different mutations in 12 genes have been identified (Table 5),

GENES RESPONSIBLE FORCARDIOVASCULAR DISEASE

Aortic Aneurysm – 1 gene

Marfan Syndrome – 1 gene

Lev’s Disorder – 1 gene

ASD – 2 genes

Holt-Oram Syndrome – 2 genesSinus Node Dysfunction – 1 locus

Supra-Ventricular Aortic Stenosis – 1 geneHeart Block – 1 locus

Bicuspid Aortic Valve – 1 geneWPW – 1 gene

Mitral Valve Prolapse – 4 lociShort QT Syndrome – 1 gene

ARVD - 7 loci (4 genes)Brugada Syndrome - 2 genes

DCM - 23 loci (5 genes)Long QT Syndrome - 3 genes

HCM – 12 genesAtrial Fibrillation - 6 loci (4 genes)

Structural Heart Disease

Conduction & Arrhythmia

Abnormalities

IG 16. Genes responsible for cardiovascular disease.

ndicating HCM is a genetically heterogeneous disease.33


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Genetic studies of HCM families from various regions of the worldndicate that mutations in MYHC and myosin binding protein CMYBPC) are the most common causes of human HCM, responsible forpproximately half of all cases.33 Troponin T (TNNT2) and Troponin ITNNTI3) are also relatively common, each accounting for 5 to 10% ofases. Thus, mutations in MYHC, MYBPC, TNNT2, and TNNI3 are theisease-causing genes in up to two-thirds of all HCM index cases.Modifier Genes and Phenotypic Variability. A common feature ofCM, and many other single-gene disorders, is the presence of significantariability in the phenotypic expression of affected patients. This vari-bility is seen between families and even within affected members of theame family and causative mutation. A significant factor for this geneticackground is the presence of genomic DNA polymorphisms or SNPs inenes other than the disease-causing gene. SNPs are located in coding oregulatory regions of genes and can affect the gene expression andunction. SNPs imposing functional differences for proteins involved inathways of cardiac hypertrophy phenotype will alter the end phenotypen single-gene disorders, and thus, are referred to as “modifier genes.”

odifier genes are neither necessary nor sufficient to cause HCM but maynfluence the severity of cardiac hypertrophy or risk of sudden cardiaceath (SCD). Angiotensin I converting enzyme (ACE-I) was the first genessociated with modifying disease phenotype in HCM.34 ACE-I catalyzes

ABLE 5. Causal genes for hypertrophic cardiomyopathy (sarcomeric genes)

Gene Symbol Locus Frequency Predominant mutations

eta-Myosin heavychain

MYH7 14q12 �35% Missense

yosin binding protein MYBPC3 11p11.2 �20% Splice-junction and insertion/deletion

ardiac troponin T TNNT2 1q32 �20% Missenseardiac troponin I TNNI3 19q13.2 �5% Missense and deletionlpha-Tropomyosin TPM1 15q22.1 �5% Missensessential myosin lightchain

MYL3 3q21.3 �5% Missense

egulatory myosin lightchain

MYL2 12q23-24.3 �5% Missense and one truncation

ardiac alpha-actin ACTC 15q11 �5% Missense mutationsitin TTN 2q24.1 �5% Missense mutationlpha-Myosin heavychain

MYH6 14q1 Rare Missense and rearrangementmutations

ardiac troponin C TNNC1 3q21.3-3p14.3

Rare Missense mutation

onversion of angiotensin I to angiotensin II and inactivates bradykinin,


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modulator of cardiac growth and cellular proliferation.35 A commonCE-I polymorphism is an insertion (I) or deletion (D) of a 287-bp Alu

epeat in intron 16. Although the direct functional effect of the I/Dolymorphisms is unknown, the genotype of these alleles in affectedCM patients have been associated with the severity of cardiac hyper-

rophy and risk of SCD in most but not all studies.34,36-41 Specifically, theD genotype is more common in families with severe hypertrophy and

ncreased incidence of SCD. Variants of endothelin-1, tumor necrosisactor, angiotensinogen, and angiotensin II receptor I have also beenssociated with the degree of cardiac hypertrophy.42-44 The identity ofost modifier genes for HCM remains largely unknown. The identifica-

ion of these modifiers will provide additional substrates for potentialherapeutic intervention.Although phenotypic variability may exist in patients with HCM causedy the same gene, due to genetic modifiers, correlation between the causalene and the degree of HCM severity and risk of SCD exists. Mutationsn MYH7 are associated with an early onset of disease, extensiveypertrophy, and an increased vulnerability for SCD (Fig 17). SomeYH7 mutations are considered prognosticators in HCM. However, due

IG 17. Pathogenesis of FHCM. Source: Marian AJ, Bruqada R, Roberts R, Hurst The Hear, ed.1, New York, McGraw-Hill, p. 750, 2004.

o the low frequency of most mutations consistent correlation to outcome


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an be made for only a few mutations. In contrast to MYH7 mutations,YPBC3 mutations are associated with late-onset, mild hypertrophy, and

ow incidence of SCD.45,46 Importantly, it is necessary to recognize theimitations of these generalizations. Many confounding variables, such asmall number of families with identical mutations, the influence ofodifier genes, and coexisting morbidities make strict genotype–pheno-

ype correlations presumptive at best.Pathogenesis of HCM. Cardiac hypertrophy resulting from sarcomericene mutations is considered to be a compensatory phenotype resultingrom inefficient myocyte contractility. Research on mutant sarcomericroteins has indicated potential mechanisms leading to this hypertrophicompensatory response (Fig 18). Mutant sarcomeric proteins have beenemonstrated to have reduced myofibrillar Ca� sensitivity and decreasedTPase activity.47 Trafficking defects leading to impaired sarcomeric

ssembly and protein localization have been demonstrated.47 The netffect in all cases is inefficient sarcomeric contractility inducing theelease of signaling molecules and growth factors that stimulate inductionf compensatory cardiac hypertrophy and fibrosis. Evidence suggestinghe compensatory nature of cardiac hypertrophy comes from animal

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IG 18. Pathogenesis of FHCM.

odels and includes the observed upregulation and expression of mole-


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ules involved in cellular growth and proliferation. Such moleculesnclude endothelin-1, transforming growth factor beta-1, and insulin-likerowth factor 1.48,49 Development of genetic animal models expressinghe human mutations,50,51 and their utilization to seek new treatments, hashown reversal of the phenotype with Losartan,52 Simvastatin,53 andpironolactone.54 A clinical trial is now ongoing to evaluate these

herapies.Dilated Cardiomyopathy. As in the case for HCM, dilated cardiomy-pathy (DCM) is a genetically heterogeneous disease. In contrast toCM, autosomal-recessive and X-linked inherited DCM also exists.

nterestingly, the vast majority of genes responsible for DCM encodearcomeric proteins, identical to the causal genes for HCM.55 Thus,espite the contrasting phenotypes of HCM and DCM, mutations inarcomeric genes may result in either disease. Many other causal genesor DCM involve the myocyte cytoskeleton, leading to the description ofenetic DCM as a disease of cytoskeletal proteins. The first genedentified for autosomal-dominant DCM was alpha-actin.56 Subsequently,utations in genes encoding additional components of the sarcomere,

ncluding MYH7, TNNT2, and TTN, were found in patients with DCM.55

ince mutations in ACTA, MYH, and TNNT2 are also known to causeCM, these findings point to the commonality of the genetic basis ofCM and HCM. This observation also suggests that, depending on whichomain or region a mutation resides in these genes, a phenotype of HCMr DCM might arise, presumably reflecting the interaction of mutatedomain with other proteins or molecules, ie, protein structure-function.or example, a mutation in TNNT2 required for adequate Ca� bindingight reduce contractility and lead to compensatory hypertrophy,hereas a mutation in a domain interacting with a cytoskeletal proteinay induce cardiac dilatation.Indeed, cytoskeletal proteins are also important causes of DCM.utations in a variety of muscle-specific genes give rise to a combined

henotype of DCM and skeletal muscle myopathy. For example, muta-ions in alpha-sarcoglycan (adhalin) cause an autosomal-recessive form ofCM that occurs in association with limb-girdle muscular dystrophy.57

utations in the intermediary filament desmin and its associated proteinlpha-B-crystallin have been identified in patients with DCM and skeletalyopathy, leading the classification of this disease phenotype as desmin-

elated myopathy.57,58 The gene responsible for Duchenne/Becker mus-ular dystrophy is dystrophin, located on Xp21, which encodes a largeytoskeletal protein.58 Mutations in this gene, commonly insertions or
eletions rather than point mutations, may also lead to both DCM and

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keletal myopathy. Reflecting the relevance of protein structure-function,utations in the five regions of the dystrophin gene often cause DCMithout skeletal involvement.59

Perhaps the most intriguing causal gene for familial DCM is the lamin/C gene,60,61 which encodes a nuclear envelop protein. The observedhenotype resulting from mutations in the rod domain of lamin A/C isrogressive conduction disease, atrial arrhythmias, heart failure, andCD. Mutations in this gene may also give rise to over 10 distincthenotypes involving a variety of tissues, including adipose tissuelipodystrophy), peripheral nervous tissue (Charcot-Marie-Tooth syn-rome), and bone tissue (mandibuloacral dysplasia), and most recentlyas been associated with premature aging (progeria). Again, tissue orrgan systems involved may be predicted by domain-specific mutations inhe gene/protein.Pathogenesis of DCM. Since DCM may be caused by mutations inenes encoding various classifications of proteins, various mechanisms ofathogenesis are likely. For example, mutations in the sarcomericroteins, cardiac alpha-actin, beta-myosin heavy chain, cardiac troponin, impart a dominant-negative effect on transmission of the contractile

orce to the extracellular matrix proteins.55,62 Cytoskeletal proteins areritical to intracellular organization, force transduction, and membranetability. Experimental evidence indicates that mutations in proteins suchs dystrophin and muscle LIM protein impair the integrity of the myocytedisk and disrupt sarcolemmal folding, leading to deficient mechanical

oupling and myocyte shortening.63,64 Pathogenesis of DCM resultingrom mutations in desmin and alpha-B-crystallin involves deposition ofesmin and alpha-B-crystallin aggregates in the myocardium.65 Theolecular pathogenesis of DCM caused by mutations in lamin A/C are

argely unknown but are likely to involve disruption of integrity of theytoskeleton.

ichard A. Walsh: Generally mutations in sarcomeric proteins are associatedith hypertrophic cardiomyopathy, while mutations of cytoskeletal proteinsre often associated with dilated cardiomyopathy. There are many excep-ions, however, which include variable penetration of the phenotype for aiven mutation in humans and divergence of phenotype of the sameutations between species. For example, mutations in the gene-encodingyosin binding protein C, which are among the most commonly associatedith hypertrophic cardiomyopathy, produce a dilated cardiomyopathic phe-otype in mice. The precise mechanisms by which mutations in genesncoding cardiomyocyte sarcomeric or cytoskeletal proteins produce alteredardiac size, geometry, and function are incompletely understood.


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Arrhythmogenic Right Ventricular Cardiomyopathy. Arrhythmogenicight ventricular cardiomyopathy (ARVC) is a primary disorder of theyocardium characterized by progressive loss of myocytes, fatty infil-

ration, and replacement fibrosis.66 The predominant site of involvements the right ventricle; however, the left ventricle and the interventriculareptum may be involved in some cases. In contrast to HCM and DCM,ffected patients with ARVC often have only subtle clinical symptomsince left ventricular ejection fraction is usually preserved. More com-only, this condition presents with ventricular arrhythmias originating

rom the right ventricle and the first clinical manifestation of disease maye sudden cardiac death.The most common mode of inheritance is autosomal-dominant, but an

utosomal-recessive form in conjunction with keratoderma and woolyair (Naxos disease) has been reported.67 At least eight loci for autoso-al-dominant ARVC have been mapped and three genes have been

dentified. (Table 6) Four known causal genes are RyR2,68 which encodeshe cardiac ryanodine receptor; JUP, encoding junction Plakoglobin,68

SP,69 which codes for desmoplakin; and PKP2, encoding plakophilin-2.utations in RYR2 are also known to cause catecholaminergic polymor-

hic ventricular tachycardia (CPVT), which is not associated with cardiactructural abnormalities. As ARVC may be a challenging disease toiagnose, the possibility exists that some families believed to have ARVCay indeed suffer with CPVT instead. The most recently identified gene

ausative for ARVC is PKP2 and is proposed to be responsible for up to5% of cases.70

Pathogenesis of ARVC. Although the pathogenesis of ARVC remains

ABLE 6. Chromosomal loci and causal genes for ARVD

Chromosome Symbol Protein Function

RVC1 14q24.3 RYR2 Ryanodine receptor 2 Calcium channelRVC2 1q42RVC3 14q11-q12RVC4 2q32RVC5 3p23RVC6 10p12-p14RVC7 10q22RVC8 6p28 DSP Desmoplakin Cell–cell adhesionaxos disease 17q21 JUP Plakoglobin Cell–cell adhesionporadic/familial ARVC 12p11 PKP2 Plakophilin-2 Cell–cell adhesion

ource: Fuster V, et al. Cardiovascular Diseased due to Genetic Abnormalities. In: Hurst Theeart, 11th ed. McGraw-Hill, 2004. Vol. 72. p. 1750.

argely unknown, considerable insight is gleaned from the identification


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f the disease-causing genes. Plakoglobin, desmoplakin, and plakophi-in-2 are all components of desmosomes. Desmosomes are complexultiprotein structures anchored to the cell membrane and promote

ell-to-cell adhesion. Thus, it is likely that defective cell-to-cell adhesiont adherens junctions is the basis of the pathology for ARVC. Mutationsn these genes may result in defective heart morphogenesis and myocar-ial architecture leading to replacement fibrosis. Fibrosis, in turn, inddition to poor myocyte coupling is likely the key factor in theulnerability to ventricular arrhythmias from this condition. The predi-ection for right ventricular involvement remains unknown. It has beenypothesized that the right ventricular outflow tract and inferobasalegion of the right ventricle, areas with frequent involvement, may bender increased stress and stretch in early development, promoting areater degree of pathology in these regions.70

ardiac ArrhythmiasIon channels provide the molecular basis for cardiac excitability and are

nchored in the cell or luminal membranes of cardiac myocytes (and otherxcitable tissues). These channels have specific ion selectivity and allowhe passage of charged ions, such as Na or K, when in their active or opentate. The precision and timeliness of the open and closed state of cardiacon channels are the basis for a normal cardiac action potential inyocytes. A mutation in an ion channel may perturb this precise

egulation in the kinetics of the channel, leading to alterations in cardiacction potential duration or vulnerability of the cell to abnormal “after-epolarizations,” triggering cardiac conduction abnormalities or danger-us ventricular arrhythmias, respectively. In the last decade, numerousardiac ion channels have been implicated as the molecular basis forarious cardiac arrhythmogenic syndromes, providing the molecularargets for understanding the basis of arrhythmogenesis in humans.Long QT Syndrome. Long-QT syndrome (LQTS) is a disease ofentricular repolarization identified by prolongation of the QT interval onhe ECG.71 It is characterized by recurrent syncopal episodes and

alignant ventricular arrhythmia, typically Torsades de pointes. Al-hough most patients are asymptomatic, sudden cardiac death may be therst clinical manifestation.Two patterns of inheritance have been described in familial LQTS: (1)

utosomal-recessive disease, described by Jervell and Lang-Nielsen in957, which is associated with deafness; and (2) autosomal-dominant
isease, described by Romano and Ward, which is not associated with

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eafness and is more common than the recessive form. Since the seminalnding in the laboratory of Keating identifying the first gene causative foramilial LQTS,72 six additional genes have been identified (Fig 17). Theenetic studies of LQTS led to the term “channelopathies,” as thedentified genes were shown to be ion channels, with one exception. Mostamilial cases of LQTS (40 to 70%) are due to mutations in genesesponsible for LQTS types 1, 2, and 3. The consistent electrophysiologicbnormality caused by mutations in these genes is prolongation of theardiac action potential (repolarization phase), translating into prolongedT interval on the 12-lead ECG.LQT1 (Long QT Type 1)The causal gene for LQT1 is KVLQT1 (or KCNQ1), which encodes aoltage-gated potassium channel alpha-subunit.73 Co-assembly with aeta-subunit, MinK (KCNE1), is required to form the slow component ofhe delayed rectifier current, Iks. Mutations in this gene lead to LQT byeduction of this potassium current. In normal physiology, Iks is mostensitive to beta-adrenergic stimulation. This may explain the observationhat patients with mutations in the KCNQ1 gene are more likely to haveardiac events triggered by adrenergic stress. Event rates are noted toecline appreciably with beta-blocker therapy. Homozygous mutations inCNQ1 give rise to the Jervell and Lange–Nielsen syndrome, character-

zed by sensineural deafness and features of LQTS.LQT2The LQT2 gene is HERG (KCNH2), which was isolated in 1994 from

he hippocampus and named human ether-a-go-go-related gene due to itsomology to the ether-a-go-go gene in Drosophila. The reference togo-go” reflects the evident dancing movement in Drosophila in responseo ether, which requires HERG channel activity. HERG encodes theotassium channel alpha-subunit for the rapid component of the delayedectifier potassium current, IKr, after co-assembly with MIRP1 (KCNE2).s in the case of LQT1, mutations in HERG lead to reduced potassium

urrent and increased action potential duration.LQT3The causal gene is SCN5A, encoding for the alpha-subunit of the

ardiac voltage-dependent sodium channel.74 In contrast to the loss ofurrent produced by mutations in potassium currents, mutations inCN5A cause LQTS by augmenting sodium current during the initialepolarizing phase of the action potential (phase 0), which also increasesction potential duration. Mutations in SCN5A also cause Brugadayndrome, progressive conduction system disease, and idiopathic ventric-
lar fibrillation.

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LQT4This genetic form of LQTS is the exception to the “channelopathy”asis for LQTS, although mutations in the causative gene likely result inbnormal ion channel function. In a large French family with 65 affectedembers having LQT and sinus node dysfunction, the causal gene was

dentified as ANKB (also known as ANK2), which encodes ankyrin B.75

utations in ankyrin B disrupt cellular localization of the sodium pump,he sodium/calcium exchanger, and the inositol-1,4,5-triphosphate recep-ors, reducing their expression levels and affecting Ca�2 signaling indult cardiac myocytes.75 This finding suggests that mutations in proteinsecessary for the appropriate localization and electrical signaling cas-ades in the myocyte may also alter cardiac electrophysiology and inducerolonged cardiac repolarization.LQT5As noted above, MinK (minimal potassium ion channel) co-assemblesith KCNQ1 to form the slow component of the delayed rectifier current,

ks. Mutations in this gene have been identified as causing both autoso-al-dominant and autosomal-recessive LQTS.LQT6LQT6 is caused by mutations in MirP1 (MinK-related peptide)

KCNE2). MirP1 assembles with HERG to form the rapid component ofhe delayed rectifier potassium current, IKr. As with HERG, mutations in

irP1 lead to reduced potassium current and increased action potentialuration.LQT7Andersen’s syndrome is a rare autosomal-dominant inherited disorder

haracterized by the constellation of periodic paralysis, cardiac arrhyth-ias, LQT syndrome, and dysmorphic features such as short stature,

coliosis, clinodactyly, hypertelorism, low-set or slanted ears, microgna-hia, and broad forehead.76 The causal gene is KCNJ2, located onhromosome 17q23; it encodes for the channel responsible for the inwardectifier potassium current, Kir2. Electrophysiologic studies indicate thathe mutations in KCNJ2 lead to a loss-of-function on Kir2.1 current,rolonging action potential duration.

ichard A. Walsh: In summary six of the seven mutations causing geneticallyetermined long QT intervals are “channelopathies.” In one case, sodiumhannel inflow kinetics are increased leading to prolonged repolarization

LQT3). In five cases, potassium channel outflow kinetics are decreased withhe same effect. Before the availability of automatic implantable defibrillators
or symptomatic patients, Type 1 antiarrhythmic agents and beta-blockade,

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espectively, produced salutary effects on ion channel kinetics in patientsith sodium and potassium channelopathies.

Drug-Induced LQTS. A long list of medications may cause long QTrolongation and susceptibility to Torsades de pointes. Although most ofhese drugs are utilized for noncardiac illness, they commonly have thebility to block potassium channels, leading to a reduction in function ofhe delayed rectifier potassium current, IK. Interestingly, individualsemonstrating drug-induced QT prolongation often have a genetic pre-isposition, harboring polymorphisms in genes known to cause LQTS.napp et al have demonstrated that, in up to 15% of patients with

acquired” LQT secondary to medication, genetic polymorphisms inCNQ1 and HERG exist.77 Presumably, in the absence of the drug-

nduced risk factor, these patients would never have developed LQT orxperienced a sudden arrhythmic event. Similarly, Splawski et al haveeported the presence of a sodium channel genetic variant (SCN5Aer1102Tyr) known to occur in over 10% of healthy, African-Americanontrol patients. However, these authors have reported that amiodaronenduces long QT and polymorphic ventricular tachycardia in the presencef this polymorphism.78

Brugada Syndrome. Brugada syndrome is identified by a characteristicCG pattern consisting of ST-segment elevation in leads V1 to V3 andseudo-right-bundle branch block. Brugada syndrome frequently mani-ests itself in the third and fourth decades of life with unexplainedyncope or ventricular fibrillation causing cardiac arrest, although eventsave been reported in all age groups. The characteristic ECG sign is oftenetected in asymptomatic individuals. Stratification of asymptomaticatients for risk of cardiac events remains controversial. As no effectiveedical therapy is available, it is generally accepted that patients with

nexplained syncope and Brugada ECG sign should receive an implant-ble cardioverter-defibrillator.The alpha-subunit of the cardiac sodium channel, SCN5A, was identi-ed as a cause for Brugada syndrome in 1998.79 Over 70 differentutations in SCN5A have been identified that collectively account for

pproximately 20% of all cases with Brugada syndrome. As in manyther genetic disorders, Brugada syndrome also exhibits locus heteroge-eity, and a second locus on chromosome 3 has been mapped.80 SCN5Autations induce a large spectrum of phenotypes, including Brugada

yndrome, long-QT syndrome (LQT3), and progressive cardiac conduc-


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ion defect. Occasionally, families with mixed phenotypes due to theame SCN5A mutation are identified.81,82

Collectively, these data suggest that mutations in SCN5A cause variablehenotypic manifestations that span Brugada syndrome, LQT3, androgressive conduction defects. This spectrum of disease reflects thepecific mechanistic defect in the sodium channel caused by a mutation.iophysical characterization of mutations in SCN5A giving rise torugada syndrome suggests that mutations decrease the availability ofa1� current by two main mechanisms: decreased expression of theutant channel or acceleration of inactivation of the channel. Compared

o LQT3, the pathogenesis of the Brugada syndrome could be consideredirectly opposing. Biophysical data indicate that LQT3 mutations cause aelayed inactivation of the channel,71 which is exactly the opposite inrugada syndrome, where there is an accelerated inactivation.83 Thebserved loss of sodium current caused by mutations in SCN5A hasroven useful in clinical practice to unmask the ECG characteristics ofhis condition in suspected cases. Specifically, anti-arrhythmic medica-ions with sodium channel-blocking properties such as ajmaline, procain-mide, flecainide, and pilsicainide have been shown to induce STlevation in leads V1 and V2, characteristic of the Brugada ECG sign, inarriers of SCN5A mutations.Catecholaminergic Polymorphic Ventricular Tachycardia. CPVT is aeritable disorder that presents as exercise- or stress-induced syncope,entricular arrhythmias, or sudden death. This condition is most com-only inherited as an autosomal-dominant disease, although rare auto-

omal-recessive inheritance is also known. Clinically, the diagnosishould be suspected in individuals experiencing syncope or sudden deathuring exercise and may be confirmed by the observation of characteristicolymorphic ventricular tachycardia induced on exercise treadmill. Aommon observation in affected patients is the development of charac-eristic bidirectional VT, akin to that observed in digitalis toxicity.Autosomal-dominant CPVT is caused by mutations in the cardiac

yanodine receptor (RyR2),84 an ion channel localized to the luminalembrane of sarcoplasmic reticulum (SR), the cellular organelle respon-

ible for Ca storage and release during excitation-contraction coupling.he rare form of autosomal-recessive CPVT is caused by mutations inalsequestrin 2 (CASQ2), a protein also closely linked to intracellularalcium handling.85

RyR2 and CSQ are molecules critical to intracellular Ca2� homeostasis.unctional studies on RyR2 mutations indicate a “gain-of-function”

nRyR2 channel activity, leading inappropriate Ca “leak” or release from


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he SR. Inappropriate Ca release may lead to the phenomenon ofafterdepolarizations” on the cardiac action potential, triggering polymor-hic ventricular arrhythmia. Given the propensity for arrhythmia devel-pment during exercise, beta-blocker therapy has proven useful iniminishing arrhythmia burden in some patients.Familial Cardiac Conduction System Disease (CCD). Inherited cardiac

onduction disease presents with variable phenotypic expressions anday be associated with other genetic syndromes. Progressive familial

eart block (PFHB) has been described in a large, South African kindred.inkage analysis indicates the disease-causing gene resides chromosome9 (19q13.2-q13.3); however, it has not yet been identified.86 Lenegre–ev disease, also a progressive conduction system disease, is now known

o be caused by mutations in SCN5A.87 Thus, SCN5A mutations areesponsible for three distinct phenotypes: LQT3, Brugada syndrome, andonduction system disease. The mechanism(s) by which mutations in thisene produce conduction disease remains somewhat unclear, althoughCN5A mutations giving rise to conduction disease demonstrate “loss-f-function” of channel activity in cellular models.Conduction system disease or atrioventricular block (AV block) is also

ound in association with genetic disease, giving rise to structural heartefects. The homeobox transcription factor, NKX2.5, causes familialtrial septal defect with multiple affected individuals also demonstratingV block. Similarly, Holt–Oram syndrome, which may result in atrial orentricular septal defects, often is associated with conduction disease.resumably, in the context of these structural heart diseases AV block isecondary to anatomic abnormalities in the AV conduction axis, aspposed to ion channel dysfunction.Familial Wolff–Parkinson–White (WPW) Syndrome. In 2001, the firstene for a familial form of WPW was reported.88 The phenotypic featuresf kindreds with this genetic syndrome were diverse, unlike the moreommonly observed cases of sporadic WPW.88 Affected patients typi-ally demonstrate evidence of ventricular preexcitation on 12-lead ECG,ommonly with extraordinary large QRS amplitudes. Supraventricularachycardias are common in youth. Electrophysiologic studies haveemonstrated tachycardias are mediated by accessory bypass tracts, anduccessful catheter ablation can be performed, as is seen in typical WPWatients. However, the clinical course of patients and associated cardiacbnormalities of patients with this genetic syndrome differ significantlyrom sporadic WPW. At least one-third of patients show evidence ofardiac hypertrophy, often severe. Progression to dilated cardiomyopathy
ay occur. As patients enter their fourth decade, progressive conduction

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ystem disease is common. Over 50% of patients develop paroxysmalollowed by chronic atrial fibrillation.The disease-causing gene was identified to be the gamma-2 regulatory

ubunit (PRKAG2) of AMP-activated protein kinase (AMPK). Thisrotein plays a key role in regulating cardiac glucose metabolism and mayegulate ion channel activity by phosphorylation. Given the role ofMPK in cardiac metabolism, the finding that cardiac hypertrophy in

hese patients is due to glycogen storage disease was not surprising. Theole of this protein in regulating cardiac development or normal cellularlectrophysiology remains to be determined and is a continued focus ofurrent research. Controversy exists as to whether or not mutations inRKAG2 result in a loss or gain of function in AMPK activity.89 Geneticnimal models expressing the PRKAG2 mutation exhibit a phenotypedentical to that observed in humans.89

Familial Atrial Fibrillation. Atrial fibrillation is the most commonustained cardiac arrhythmia. It is estimated there are over 3 millioneople with atrial fibrillation in the United States, which will increase to.6 million by 2050.90-92 Atrial fibrillation may cause significant morbid-ty, accounting for up to one-third of all strokes over the age of 60 years.93

he first locus responsible for atrial fibrillation was mapped to 10q22-4.94 Six loci have since been mapped but only four genes identified, allrom within families of mainland China.92 The identified genes encodeotassium channels also known to cause LQTS. Whether these geneticndings reflect the cause for atrial fibrillation from other geographicegions or in the more common idiopathic cases of atrial fibrillationemains to be determined.KCNQ1, the cause of LQT1, was the first gene identified in familial

trial fibrillation. This was identified in a single, four-generation Chineseamily showing autosomal-dominant inheritance.89,92 Expression of thedentified mutant KCNQ1 gene in COS cells showed increased potassiumurrent, thereby shortening phase 3 of the cardiac action potential, inirect contrast to LQT causing mutations in KCNQ1. Thus, the mutationnduced a gain-of-function, which shortens the atrial action potentialuration and the effective atrial refractory period. Several investigatorsith multiple families had shown KCNQ1 is not responsible for atrialbrillation in their families,92,95 suggesting this is an uncommon causeor atrial fibrillation.The same Chinese investigators identified a mutation in KCNE2, known

o cause LQTS6, in another single Chinese family with autosomal-ominant atrial fibrillation.92,96 Functional studies again showed that the
utant shortened the atrial action potential duration and the atrial

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ffective refractory period. The third gene was identified to be KCNJ2,he cause for LQTS7. The identified mutation confers a gain-of-functionn channel activity, producing the same effects on action potentialuration as described above.92,97 Thus, the common functional abnormal-ty caused by mutations in these potassium channels results in shorteningf the cardiac action potential and predicted shortening of the atrialefractory period, a known phenomenon in atrial fibrillation. Although its premature to extrapolate these findings in rare families to all-comersith atrial fibrillation, these findings provide insight for the developmentf novel anti-arrhythmic therapies.

nimal Models of Cardiovascular Genetic DiseaseAs described in an earlier section, the advances in recombinant DNA

echnology has enabled the genetic engineering of animals for the purposef creating models of human disease. Typically mice are used, althoughat and rabbit models have been developed (Fig 19). These animals maye engineered to contain a human disease-causing gene in their genome,r alternatively have their native gene (homologous to the human gene)utated to harbor a human disease-causing mutation. Thereafter, theseice can be bred to transmit the gene of interest, with expression of theutant protein in succeeding generations. These transgenic animals

hereafter become a model for in vivo analysis of the pathogenesis of theisease and to seek novel therapies.Numerous animal models of human disease have been created as a result of

he identification of disease-causing genes. In cardiovascular disease, condi-ions such as HCM, DCM, LQTS, WPW, CPVT,50,51,88,98-100 and manythers may now be studied in animal models. All of these conditions haveeen well known in the field of cardiology for decades, but remained annsolved mystery in regards to pathogenesis. The development of animalodels provides the investigator an opportunity to determine the molecular

athways and evaluate novel therapeutic strategies. To illustrate this concept,uture therapeutic treatments for HCM as gleaned from research in animalodels will be briefly discussed.Pathologically, HCM may lead to severe myocardial wall thickening,

nducing significant hemodynamic impairment. Current pharmacologicherapy in HCM is empiric and has not been shown to induce regressionf hypertrophy. In conjunction with severe hypertrophy, significantyocardial fibrosis develops, a key determinant leading to the vulnera-

ility of ventricular arrhythmias and sudden cardiac death. Studies in
nimal models now suggest potential clinical utility of angiotensin II

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eceptor blockers and HMG-CoA reductase inhibitors in the attenuationnd reversal of cardiac hypertrophy and fibrosis in animal models ofCM.52,53

In a cardiac troponin T-Q92 transgenic mouse model producing thehenotype of HCM, blockade of angiotensin II receptor 1 reducednterstitial collagen volume, expression levels of collagen alpha1 (I)RNA, and TGF-�1 protein, the latter a known mediator of profibrotic

ffects of angiotensin II, by approximately 50% to the normal levels.52

Equally encouraging has been the observation that HMG-CoA reduc-ase inhibitors may prevent or attenuate cardiac hypertrophy and fibro-is.101-103 Simvastatin, a pleiotropic HMG-CoA reductase inhibitor,

IG 19. Gene transplantation into germ cells. Source: Roberts R, Towbin J. Principles andechniques of molecular biology. In: Roberts R, editor. Molecular Basis of Cardiology.ambridge, MA: Blackwell Scientific Publications, 1993. Vol. 8, p. 78.

educed left ventricular mass by 37%, wall thickness by 20%, and


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ollagen volume fraction by 50% in the �-MyHC-Q403 transgenic rabbitodel of human HCM.53 In addition, indices of left ventricular filling

ressure were improved significantly. These results in genetically engi-eered animal models provide the impetus to pursue clinical trials inumans to determine whether treatment with HMG-CoA reductasenhibitors or angiotensin II blockade induces regression of hypertrophy oriminished risk of sudden cardiac death in patients with HCM.

uturistic Therapy Utilizing Stem Cells and NuclearransferThere is tremendous interest in stem cells as therapy for cardiovasculariseases and in particular for regenerating myocardium following myo-ardium infarction. The hope is to stimulate stem cells to differentiate intoparticular desired cell such as a cardiomyocyte with all of its contractileroperties. Another hope is to stimulate such progenitor cells to grow newlood vessels in the hope of preventing myocardial ischemia andnfarction. Stem cells are thus being explored as a major research interestoth in vivo and in vitro together with clinical studies in human beings.hile this field of research is still in its infancy, it is very exciting and the

otential is perhaps much greater than for gene therapy and likely to yieldherapeutic benefit within the next 5 to 10 years.There are two types of stem cells: embryonic and adult. Embryonic stem

ells (ES) are totipotent cells derived from the inner cell mass of thelastocyst that can be replicated indefinitely in an undifferentiated state inell culture. These ES cells can in vivo be stimulated to differentiate intoany cell types. The human being, while thought to have a million trillion

ells, has only about 200 different cells as defined by a specific function.t is well recognized that all of these cells come from a single stem cell,amely, the egg and the sperm, and from that probably develops about 20mbryonic undifferentiated stem cells from which are derived the 200ifferentiated cells. The use of embryonic stem cells is very much mirednto ethical issues that have prohibited this research from proceeding asapidly as perhaps possible. This has also spurred great interest ino-called adult stems cells. It has become evident from several sourceshat adult stems cells exist in most organs. The heart is considered to be

terminally differentiated organ and as such does not have stem cells;owever, there is increasing evidence to suggest the occasional stem cellay be present in the heart, although data is still not yet convincing.here are considerable data to indicate stem cells exist in the bonearrow, the brain, the liver, and other organs. There are numerous studies

o show that, when one has a myocardial infarction, certain progenitor


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one marrow cells are attracted to the area and appear to have someherapeutic effect whether it is through release of paracrine factors orther means. Embryonic stem cells could, of course, lead to rejection andn a very small percentage may proliferate into undifferentiated tumorsuch as teratomas. In contrast, adult stems when utilized from thendividual’s bone marrow for example would not be rejected and there areonsiderable data to show that they are less likely to develop intoeratomas. There are many studies ongoing in animals and to a lesserxtent in humans utilizing bone marrow adult stem cells with the hopehey will regenerate myocardium or induce the formation of bloodessels.The limitation of adult stem cells is their lack of plasticity since they are

lready to some extent committed to differentiate into their own cellineage, in contrast to embryonic stem cells, which are capable ofifferentiating into any form of cells. Adult stem cells are a fruitful areaf research and will, of course, be pursued with great interest over theext decade. The other approach that is receiving some interest but withess success is attempts to reverse the commitment or differentiation andorce the cell back into a replicating cell cycle such that it can betimulated to metamorphosis into the cell of choice. The embryonic stemell has the most potential with respect to differentiating into the desiredells such as that of the cardiomyocyte or that of the endothelial cell.egardless of the potential applications for either the ES or the adult stemells, research currently must be directed to understanding the mecha-isms involved in cell differentiation. Even if ES cells were available,here remains to be determined which factors are required to stimulateheir differentiation into a cardiomyocyte or an endothelial cell. It is likelyhat extensive fundamental research is required to answer these questionsefore one is likely to achieve therapeutic success in any of these avenues.tudies would suggest that adult stem cells such as those from bonearrow have a homing instinct to be taken up by the heart at times of

amage such as with myocardial infarction and they appear to have aignificant effect on function. This effect could be from paracrine growthactors, and another possibility is fusion of these cells with myocytes,hich in turn induce cardiac regeneration. It remains to be determined as

o whether there are progenitor stem cells present in the myocardium andf so can they be triggered to proliferate and differentiate into cardiomy-cytes or blood vessels.The other area of research that is also being pursued but less intensely

s that of nuclear transfer. Nuclear transfer, whereby a nucleus of one’s
wn cells is inserted into a prior enucleated stem cell, also has great

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romise. This would avoid the problem of rejection as well as that oferatomas associated with ES cells and would indeed regenerate a cellith a genotype identical to that of the individual. This technique haseen off limits from an ethical point of view since theoretically one couldlone human beings utilizing this technique. However, nuclear transfer iseing pursued in countries such as Britain, Russia, and Sweden with theppropriate prohibition not to clone human embryos. These studies areven more primitive than those of stem cells but can be expected to enternto the mainstream as we unravel the fundamental mechanisms andppreciate their true potential as therapeutic weapons.

dentification of Genes Causing Polygeniciseases—The New FrontierGreat progress has been made in single-gene disorders. Since a singleene is in large part responsible for the phenotype, it is possible throughenetic linkage analysis to map the chromosomal location of the geneesponsible for a disease segregating in a two- to three-generation family.his is not possible in a polygenic disease such as atherosclerosis orypertension. Atherosclerosis is due to multiple genes each contributingsmall percentage to the phenotype. Thus, no one gene is responsible for

he phenotype. The two approaches to atherosclerosis have been caseontrol association studies of either the direct or the indirect method.ost studies to date have been indirect assessing the frequency of a

olymorphism in individuals with the disease versus its frequency inontrols. Studies that have been performed generally involved sampleizes that were inadequate. The indirect approach consisting of genome-ide scans has not been feasible due to inadequate number of markers

nd inadequate sample size. Studies performed to date have included0,000 to 100,000 markers but it requires hundreds of thousands ofarkers. Second, the population should be analyzed in at least two

ndependent populations utilizing 400,000 to 500,000 SNPs as markers inn initial population of at least 2000 (1000 affected, 1000 controls)ollowed by a population of at least 12,000. In 2005,104 Hinds et alnalyzed 1.5 million SNPs selected to span the human genome andhowed a minimum of 275,000 SNPs are required to obtain the samenformation. A workshop on association studies for polygenic diseaseslso recommends 500,000 SNP scans and the use of at least twondependent populations. Based on our calculations, to detect genes fororonary artery disease, we recommend the use of the 500,000 SNParker chip in an initial population of 1000 affecteds and 1000 controls

o detect a size difference between the two populations of at least 0.2.


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hose loci showing an association with a selected P value such as 0.001ould be further assessed for more stringent association (eg, P value.000001) in a second population of 8000 affecteds and 4000 controls. Inhe second population, one can map with customized chips only to thoseegions showing an initial association in the first study. Approaches suchs this should identify genes responsible for diseases such as coronaryrtery disease, hypertension, and the metabolic syndrome.

ichard A. Walsh: Molecular Cardiology and Genetics in the 21st Century byoberts and Gollob is a clearly written and richly illustrated introduction to a

apidly evolving area of biomedical research. It is particularly useful for clinicalardiologists who desire a state-of-the-art overview of the field as a basis fornderstanding the current literature and thinking about potential futureardiovascular clinical applications.It is easy to imagine possible applications of the current and evolving statef genomics and genetics to the diagnosis, prognosis, and treatment ofardiovascular disease. Indeed, there is a growing sense that identification ofhe monogenic and more common polygenic factors for acute and chronicisease will lead to a refined and more effective personalized medicine. Thisnderstandable enthusiasm should be tempered by the real challenges to theoncept that genotype exclusively determines phenotype. Obviously, envi-onmental influences play powerful and modifiable roles in the determinationf disease phenotypes. Differences in genetic background and epigeneticactors can profoundly modify the phenotypic expression of disease due to

onogenic or polygenic mutations or polymorphisms (J Mol Cell Cardiol006;40:201-4). The complex interaction among genetic, genetic back-round, epigenetic, and disease progression is an area of current intense

nterest and research. Future progress in this area requires interdisciplinaryesearch among geneticists, cell biologists, computational biologists, physi-logists, and clinicians. As always, the future is bright but increasinglyomplicated.For a more extensive examination of the molecular and genetic basis for

ardiovascular disease, the reader is referred to “Molecular Management ofardiac Hypertrophy and Failure” edited by Richard A. Walsh, Abingdon, UK,aylor and Francis Publishers, 2005.

AcknowledgmentsThis article was supported by a grant from Canadian Institutes of Healthesearch: Treatment and Mechanisms of Sudden Death in Heart Failure; Heartnd Stroke Foundation of Canada: Genetic Influence on Arrhythmias in Heartailure and Genetic Analysis in Survivors of Unexplained Cardiac Arrest. Wecknowledge Fran Baas and Sue Slater in the completion of this article.

REFERENCES1. Roberts R. Integrated program for the training of cardiovascular fellows in

molecular biology. In: Albertini A, Lenfant C, Paoletti R, editors. Biotechnology in
Clinical Medicine. New York: Raven Press, 1987. p. 99-104.

6

2. Roberts R. Impact for molecular biology in cardiology. Am J Physiol1991;261:8-14.

3. Avery O, MacLeod DM, MacCarty M. Studies on the chemical nature of thesubstance inducing transformation of pneumococcal types. J Exp Med1944;79:137-58.

4. Watson JD, Crick FHC. Molecular structure of nucleic acids: a structure fordeoxyribose nucleic acid. Nature 1953;171:737-8.

5. Watson JD, Crick FHC. Genetical implications of the structure of deoxyribonucleicacid. Nature 1953;171:964-7.

6. Franklin RE, Gosling RG. Molecular configuration in sodium thymonucleate.Nature 1953;171:740-1.

7. Wilkins MHF, Stokes AR, Wilson HR. Molecular structure of deoxypentosenucleic acids. Nature 1953;171:738-40.

8. Marmor J, Lane L. Strand separation and specific recombination of deoxyribonu-cleic acids: biological studies. Proc Natl Acad Sci USA 1960;46:453-61.

9. Doty P, Marmor J, Eigner J, et al. Strand separation and specific recombination indeoxyribonucleic acids: physical chemical studies. Proc Natl Acad Sci USA1960;46:461-76.

10. Nirenberg M, Matthaei JH. The dependence of cell-free protein synthesis in E. coliupon naturally occurring or synthetic polyribonucleotides. Proc Natl Acad Sci USA1961;47:1588-602.

11. Nishimura S, Jones DS, Khorana HG. The in vitro synthesis of a co-polypeptidecontaining two amino acids in alternative sequence dependent upon a DNA-likepolymer containing two nucleotides in alternating sequence. J Mol Biol1981;146:1-21.

12. Olivera BM, Hall ZW, Lehman IR. Enzymatic joining of polynucleotides. V. ADNA adenylate intermediate in the polynucleotide joining reaction Proc Natl AcadSci USA 1968;61:237-44.

13. Linn S, Arbor W. Host specialty of DNA produced by Escherichia coli. X: In vitrorestriction of phage fd replicative form. Proc Natl Acad Sci USA 1968;59:1300-6.

14. Smith HO, Wilcox KW. A restriction enzyme from Hemophilias influenzae. I.Purification and general properties. J Mol Biol 1970;51:379-91.

15. Kelly TJ Jr, Smith HO. A restriction enzyme from Hemophilias influenzae. II. Basesequence of the recognition site. J Mol Biol 1970;51:393-409.

16. Baltimore D. Viral RNA-dependent DNA polymerase. Nature 1970;226:1209-11.17. Termin HM, Mizutani S. RNA-dependent DNA polymerase in virions of Rous

sarcoma virus. Nature 1970;226:1211-3.18. Cohen S, Chang A, Boyer H, et al. Construction of biological functional bacterial

plasmids in vitro. Proc Natl Acad Sci USA 1973;70:3240-4.19. Danna KJ, Nathans D. Specific cleavage of simian virus 40 DNA by restriction

endonuclease of Hemophilus influenzae. Proc Natl Acad Sci USA 1971;68:2913-7.20. Sanger F, Air M, Barrel BG, et al. Nucleotide sequence of bacteriophage phi X174

DNA. Nature 1977;265:687-95.21. Maxam AM, Gilbert W. A new method of sequencing DNA. Proc Natl Acad Sci

USA 1977;74:560-4.22. Gething MJ, Adler B, Boose JA, et al. Variants of human tissue-type plasminogen

activator that lack specific structural domains of the heavy chain. EMBO J
1988;7:2731-40.

C

23. Saiki RK, Scharf S, Faloona F, et al. Enzymatic amplification of beta-globingenomic sequences and restriction site analysis for diagnosis of sickle cell anemia.Science 1985;230:1350-4.

24. Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic amplification ofDNA with a thermostable DNA polymerase. Science 1988;239:487-91.

25. The Chromosome 21 Mapping and Sequencing Consortium. Nature 2000;405:311-9.

26. Katsanis N, Worley KC, Lupski JR. An evaluation of the draft human genomesequence. Nat Genet 2001;29(1):88-91.

27. Altshuler D, Brooks LD, Chakravarti A, et al. A haplotype map of the humangenome—The International HapMap Consortium. Nature 2005;427:1299-320.

28. Southern EM. Detection of specific sequences among DNA fragments separated bygel electrophoresis. J Mol Biol 1975;98:503-17.

29. Roberts R, Towbin J, Parker TG, et al. A Primer of Molecular Biology. New York:Elsevier Science Publishing Co., Inc., 1992.

30. Marian AJ, Brugada R, Roberts R. Hurst’s the Heart—Chapter 72—CardiovascularDiseases due to Genetic Abnormalities, 11th ed. 2004. p. 1747-83.

31. Ott J. Analysis of human genetic linkage. Baltimore, MD: The Johns HopkinsUniversity Press, 1991.

32. Geisterfer-Lowrance AA, Kass S, Tanigawa G, et al. A molecular basis for familialhypertrophic cardiomyopathy: a beta-cardiac myosin heavy chain gene missensemutation. Cell 1990;62:999-1006.

33. Marian AJ, Roberts R. The molecular genetic basis for hypertrophic cardiomyop-athy. J Mol Cell Cardiol 2001;33(4):655-70.

34. Marian AJ, Yu QT, Workman R, et al. Angiotensin converting enzyme polymor-phism in hypertrophic cardiomyopathy and sudden cardiac death. Lancet1993;342:1085-6.

35. Yamazaki T, Komuro I, Yazaki Y. Role of the renin-angiotensin system in cardiachypertrophy. Am J Cardiol 1999;83(12A):53H-7H.

36. Lechin M, Quinones MA, Omran A, et al. Angiotensin I converting enzymegenotypes and left ventricular hypertrophy in patients with hypertrophic cardiomy-opathy. Circulation 1995;92:1808-12.

37. Tesson F, Dufour C, Moolman JC, et al. The influence of the angiotensin Iconverting enzyme genotype in familial hypertrophic cardiomyopathy varies withthe disease gene mutation. J Mol Cell Cardiol 1997;29:831-8.

38. Pfeufer A, Osterziel KJ, Urata H, et al. Angiotensin-converting enzyme and heartchymase gene polymorphisms in hyptertrophic cardiomyopathy. Am J Cardiol1995;78:362-4.

39. Yoneya K, Okamoto H, Machida M, et al. Angiotensin-converting enzyme genepolymorphism in Japanese patients with hypertrophic cardiomyopathy. Am Heart J1995;130:1089-93.

40. Yamada Y, Ichihara S, Fujimura T, et al. Lack of association of polymorphisms ofthe angiotensin converting enzyme and angiotensionogen genes with nonfamilialhypetrophic or dilated cardiomyopathy. Am J Hypertens 1997;10:921-8.

41. Osterop AP, Kofflard MI, Sandkuiji LA, et al. AT1 receptor A/C1166 polymor-phism contributes to cardiac hypertrophy in subjects with hypertrophic cardiomy-
opathy. Hypertension 1998;32:825-30.

6

42. Brugada R, Kelsey W, Lechin M, et al. Role of candidate modifier genes on thephenotypic expression of hypertrophy in patients with hypertrophic cardiomyopa-thy. J Investig Med 1997;45:542-51.

43. Patel R, Lim DS, Reddy D, et al. Variants of trophic factors and expression ofcardiac hypertrophy in patients with hypertrophic cardiomyopathy. J Mol CellCardiol 2000 Dec;32(12):2369-77.

44. Yamada Y, Ichihara S, Izawa H, et al. Association of a G994-T(Val279-Phe)polymorphism of the plasma platelet-activating factor acetylhydrolase gene withmyocardial damage in Japanese patients with nonfamilial hypertrophic cardiomy-opathy. J Hum Genet 2001;46:436-41.

45. Niimura H, Bachinski LL, Sangwatanaroj S, et al. Mutations in the gene for cardiacmyosin-binding protein C and late-onset familial hypertrophic cardiomyopathy.N Engl J Med 1998;338(1248):1257.

46. Erdmann J, Raible J, Maki-Abadi J, et al. Spectrum of clinical phenotypes and genevariants in cardiac myosin-binding protein C mutation carriers with hypertrophiccardiomyopathy. J Am Coll Cardiol 2001;38:322-30.

47. Fatkin D, McConnell BK, Mudd JO, et al. An abnormal Ca(2�) response in mutantsarcomere protein-mediated familial hypertrophic cardiomyopathy. J Clin Invest2000 Dec;106(11):1351-9.

48. Hasegawa K, Fujiware H, Koshiji M, et al. Endothelin-1 and its receptor inhypertrophic cardiomyopathy. Hypertension 1996 Feb 27;27(2):259-64.

49. Li RK, Li G, Mickle DA, et al. Overexpression of transforming growth factor-beta1and insulin-like growth factor-I in patients with idiopathic hypertrophic cardiomy-opathy. Circulation 1997;96:874-81.

50. Oberst L, Park J-T, Wu Y, et al. Decreased left ventricular ejection fraction,detected by 178TA radionuclide angiography in transgenic mice expressing amutant cardiac troponin T-Gln92: altered function precedes structural abnormalities.Circ Res 1999 (in press).

51. Marian AJ, Wu Y, Lim D-S, et al. A transgenic rabbit model for humanhypertrophic cardiomyopathy. J Clin Invest 1999;104:1683-92.

52. Lim DS, Lutucuta S, Bachireddy P, et al. Angiotensin II blockade reversesmyocardial fibrosis in a transgenic mouse model of human hypertrophic cardio-myopathy. Circulation 2001 Feb 13;103(6):789-91.

53. Patel R, Nagueh SF, Tsybouleva N, et al. Simvastatin induces regression of cardiachypertrophy and fibrosis and improves cardiac function in a transgenic rabbit modelof human hypertrophic cardiomyopathy. Circulation 2001;104:r27-r34.

54. Tsybouleva N, Zhang L, Chen S, et al. Aldosterone, through novel signalingproteins, is a fundamental molecular bridge between the genetic defect and thecardiac phenotype of hypertrophic cardiomyopathy. Circulation 2004;109:r59-r66.

55. Kamisago M, Sharma SD, DePalma SR, et al. Mutations in sarcomere protein genesas a cause of dilated cardiomyopathy. New Engl J Med 2000;343:1688-96.

56. Olson TM, Michels VV, Thibodeau SN, et al. Actin mutations in dilatedcardiomyopathy, a heritable form of heart failure. Science 1998;280:750-2.

57. Coral-Vazquez R, Cohn RD, Moore SA, et al. Disruption of the sarcoglycan-sarcospan complex in vascular smooth muscle: a novel mechanism for cardiomy-opathy and muscular dystrophy. Cell 1999;98:465-74.

58. Malhotra SB, Hart KA, Klamut HJ, et al. Frame-shift deletions in patients with
Duchenne and Becker muscular dystrophy. Science 1988;242:755-9.

C

59. Muntoni F, Wilson L, Marrosu G, et al. A mutation in the dystrophin geneselectively affecting dystrophin expression in the heart. J Clin Invest1995;96:693-9.

60. Fatkin D, MacRae C, Sasaki T, et al. Missense Mutations in the rod domain of thelamin A/C gene as causes of dilated cardiomyopathy and conduction-systemdisease. N Engl J Med 1999;341:1715-24.

61. Rosengart TK, Patel SR, Crystal RG. Therapeutic angiogenesis: protein and genetherapy delivery strategies. J Cardiovasc Risk 1999;6:29-40.

62. Thiene G, Basso C, Danieli G, et al. Arrhythmogenic right ventricular cardiomy-opathy. Trends Cardiovasc Med 1997;7:84-90.

63. Tsubata S, Bowles KR, Vatta M, et al. Mutations in the human delta-sarcoglycangene in familial and sporadic dilated cardiomyopathy. J Clin Invest 2000Sep;106(5):655-62.

64. Bonne G, Di Barletta MR, Varnous S, et al. Mutations in the gene encoding laminA/C cause autosomal dominant Emery-Dreifuss muscular dystrophy. Nat Genet1999;21:285-8.

65. Bova MP, Yaron O, Huang Q, et al. Mutation R120G in alphaB-crystallin, whichis linked to a desmin-related myopathy, results in an irregular structure anddefective chaperone-like function. Proc Natl Acad Sci USA 1999;96:6137-42.

66. Corrado D, Fontaine G, Marcus FI, et al. Arrhythmogenic right ventriculardysplasia/cardiomyopathy: need for an international registry. Study Group onArrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy of the WorkingGroups on Myocardial and Pericardial Disease and Arrhythmias of the EuropeanSociety of Cardiology and of the Scientific Council on Cardiomyopathies of theWorld Heart Federation. Circulation 2000 Mar 21;101(11):E101-6.

67. McKoy G, Protonotarios N, Crosby A, et al. Identification of a deletion inplakoglobin in arrhythmogenic right ventricular cardiomyopathy with palmoplantarkeratoderma and wooly hair (Naxos disease). Lancet 2000;355:2119-24.

68. Tiso N, Stephan DA, Nava A, et al. Identification of mutations in the cardiacryanodine receptor gene in families affected with arrhythmogenic right ventricularcardiomyopathy type 2 (ARVD2). Hum Mol Genet 2001;10:189-94.

69. Rampazzo A, Nava A, Malacrida S, et al. Mutation in human desmoplakin domainbinding to plakoglobin causes a dominant forum of arrhythmogenic right ventric-ular cardiomyopathy. Am Hum Genet 2002;71:1200-6.

70. Gerull B, Heuser A, Wichter T, et al. Mutations in the desmosomal proteinplakophilin-2 are common in arrhythmogenic right ventricular cardiomyopathy.Nat Genet 2005;36(11):1162-4.

71. Roden DM, Lazzara R, Rosen M, et al. Multiple mechanisms in the Long-QTSyndrome: current knowledge, gaps, and future direction. Circulation1996;94(8):1996-2012.

72. Sanguinetti MC, Jiang C, Curran ME, et al. A mechanistic link between aninherited and an acquired cardiac arrhythmia: HERG encodes the Ik potassiumchannelr. Cell 1995;81:299-307.

73. Barhanin J, Lesage F, Guillemare E, et al. K(V)LQT1 and lsK (minK) proteinsassociate to form the I(Ks) cardiac potassium current [see comments]. Nature 1996Nov 7;384(6604):78-80.

74. Wang Q, Shen J, Splawski I, et al. SCN5A mutations associated with an inherited
cardiac arrhythmia, long QT syndrome. Cell 1995;80:805-11.

7

75. Mohler PJ, Schott JJ, Gramolini AO, et al. Ankyrin-B mutation causes type 4long-QT cardiac arrhythmia and sudden cardiac death. Nature 2003 Feb6;421(6923):634-9.

76. Plaster NM, Tawil R, Tristani-Firouzi M, et al. Mutations in Kir2.1 cause thedevelopmental and episodic electrical phenotypes of Andersen’s syndrome. Cell2001;105:511-9.

77. Knapp M, Seuchter SA, Baur MP. Two-locus disease models with two marker loci:the power of affected-sib-pair tests. Am J Hum Genet 1994 Nov;55(5):1030-41.

78. Splawski I, Timothy KW, Tateyama M, et al. Variant of SCN5A sodium channelimplicated in risk of cardiac arrhythmia. Science 2002;297:1333-6.

79. Chen Q, Kirsch GE, Zhang D, et al. Genetic basis and molecular mechanism foridiopathic ventricular fibrillation. Nature 1998 Mar 19;392(6673):293-6.

80. Weiss RM, Barmada MM, Nguyen T, et al. Clinical and molecular heterogeneity inthe Brugaga syndrome: a novel gene locus on chromosome 3. Circulation2002;105:707-13.

81. Bezzina C, Veldkamp MW, van Den Berg MP, et al. A single Na(�) channelmutation causing both long-QT and Brugada syndromes. Circ Res 1999 Dec3;85(12):1206-13.

82. Kyndt F, Probst V, Potet F, et al. Novel SCN5A mutation leading either to isolatedcardiac conduction defect or Brugada syndrome in a large French family.Circulation 2001 Dec 18;104(25):3081-6.

83. Dumaine R, Towbin JA, Brugada P, et al. Ionic mechanisms responsible for theelectrocardiographic phenotype of the Brugada syndrome are temperature depen-dent [see comments]. Circ Res 1999 Oct 29;85(9):803-9.

84. Priori SG, Napolitano C, Tiso N, et al. Mutations in the cardiac ryanodine receptorgene (hRyR2) underlie catecholaminergic polymorphic ventricular tachycardia.Circulation 2001 Jan 16;103(2):196-200.

85. Lahat H, Pras E, Olender T, et al. A missense mutation in a highly conserved regionof CASQ2 is associated with autosomal recessive catecholamine-induced polymor-phic ventricular tachycardia in Bedouin families from Israel. Am J Hum Genet2001;69(6):1378.

86. Brink PA, Ferreira A, Moolman JC, et al. Gene for progressive familial heart blocktype I maps to chromosome 19q13. Circulation 1995;91:1633-40.

87. Schott JJ, Alshinawi C, Kyndt F, et al. Cardiac conduction defects associate withmutations in SCN5A. Nat Genet 1999 Sep;23(1):20-1.

88. Gollob MH, Green MS, Tang A, et al. Identification of a gene responsible forfamilial Wolff-Parkinson-White syndrome. New Engl J Med 2001;344(24):1823-64.

89. Sidhu J, Yadavendra S, Rajawat YS, et al. Transgenic mouse model of ventricularpreecitation and atrioventricular reentrant tachycardia induced by an AMP-activated protein kinase loss-of-function mutation responsible for Wolff-Parkinson-White Syndrome. Circulation 2005;111:21-9.

90. Dobrev D, Graf E, Wettwer E, et al. Molecular basis of downregulation ofG-protein-coupled inward rectifying K(�) current I(K,ACh) in chronic humanatrial fibrillation: decrease in GIRK4 mRNA correlates with reduced I(K,ACh) andmuscarinic receptor-mediated shortening of action potentials. Circulation
2001;104:2551-7.

1

1

1

1

1

C

91. Chen YH, Xu SJ, Bendahhou S, et al. KCNQ1 gain-of-function mutation in familialatrial fibrillation. N Engl J Med 1997;299:251-4.

92. Roberts R. Genetic mechanisms of atrial fibrillation. Nature 2005 (in press).93. Darbar D, Herron K, Ballew JD, et al. Familial atrial fibrillation is a genetically

heterogeneous disorder. J Am Cardiol 2003;41(12):2185-92.94. Brugada R, Tapscott T, Czernuszewicz GZ, et al. Identification of a genetic locus

for familial atrial fibrillation. N Engl J Med 1997;336:905-11.95. Blair E, Redwood CS, Ashrafian H, et al. Mutations in the gamma(2) subunit of

AMP-activated protein kinase cause hypertrophic cardiomyopathy: evidence for thecentral role of energy comp disease pathogenesis. Hum Mol Genet 2001;10(11):1215-20.

96. Gollob MH, Seger JJ, Gollob TN, et al. Novel PRKAG2 mutation in the geneticsyndrome of ventricular preexcitation and conduction defects with childhood onsetand absence of cardiac hypertrophy. Circulation 2001;104:3030-3.

97. Roberts R, Brugada J. Genetic aspects of arrhythmias. Am J Med Genet2000;97(4):310.

98. Sidhu J, duanxiang L, Wang A, et al. Two transgenic animal models expressinghuman troponin T gene mutations: one exhibiting dilated cardiomyopathy (W141)and the other exhibiting hypertrophic cardiomyopathy (Q92). Journal of AmericanCollege of Cardiology 3-3-2004;43:216A.

99. Nuyens D, Stengl M, Dugarmaa S, et al. Abrupt rate accelerations or prematurebeats cause life-threatening arrhythmias in mice with long-QT3 syndrome. Nat Med2001;7(9):1021-7.

00. Cerrone M, Colombi B, Santoro M, et al. Bidirectional ventricular tachycardia andfibrillation elicited in a knock-in mouse model carrier of a mutation in the cardiacryanodine receptor. Circ Res 2005;96(10):77-82.

01. Oi S, Haneda T, Osaki J, et al. Lovastatin prevents angiotensin II-induced cardiachypertrophy in cultured neonatal rat heart cells. Eur J Pharmacol 1999;376:139-48.

02. Park HJ, Galper JB. 3-Hydroxy-3-methylglutaryl CoA reductase inhibitors up-regulate transforming growth factor-beta signaling in cultured heart cells viainhibition of geranylgeranylation of RhoA GTPase. Proc Natl Acad Sci USA1999;96:11525-30.

03. Su SF, Hsiao CL, Chu CC, et al. Effects of pravastatin on left ventricular mass inpatients with hyperlipidemia and essential hypertension. Am J Cardiol2000;86:514-8.

04. Hinds DA, Stuve LL, Nilsen GB, et al. Whole-genome patterns of common DNAvariation in three human populations. Science 2005;307:1072-9.


molecular cardiology and genetics in the 21st century—a primer

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