Artículo 4. How to use an article about genetic association JAMA 2009

8/2/2019 Artculo 4. How to use an article about genetic association JAMA 2009

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. 2009;301(1):74-81 (doi:10.1001/jama.2008.901)JAMA

John Attia; John P. A. Ioannidis; Ammarin Thakkinstian; et al.

Background ConceptsHow to Use an Article About Genetic Association: A:

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USERS GUIDES TO THE MEDICAL LITERATURE

How to Use an ArticleAbout Genetic AssociationA: Background ConceptsJohn Attia, MD, PhD

John P. A. Ioannidis, MD, PhD

Ammarin Thakkinstian, PhD

Mark McEvoy, MMedSc

Rodney J. Scott, PhD

Cosetta Minelli, PhD

John Thompson, PhD

Claire Infante-Rivard, MD, PhD

Gordon Guyatt, MD, MSc

CLINICAL SCENARIO

A 55-year-old man consults you, wor-ried about his risk of developing Alz-heimer disease. His grandfather hadde-mentia in his 70s, and his own fatherwas diagnosed with dementia at 65years. He has been a smoker since hewas 20 years of age, works as an elec-

trician, and has been taking antihyper-tensive medication (thiazide and -blocker) for the last 5 years. He hasnever hadhischolesterol level checked.He has recently read a news story aboutgenetic tests and asks you whether heshould have any genetic test for de-mentia risk, in particular for a genecalled APOE.

THE SEARCH

Consultingyourelectronicmedicaltexts,

youfindasectionaddressinggeneticriskfactors for dementia. You come acrosswordssuchasallele, genotype,and geneticassociation andencountermanyabbrevia-tions, includingAPOE, e3/e4, and e4/e4(BOX). A colleague with an interest ingenetics steers you toward a helpfulWebsite, the HuGE Navigator,1 andyoufind that there areapparently more than

1000 publications on genetic associa-tions of Alzheimer disease that dis-

cuss several hundred genes; the wholeendeavor seems daunting. A quick In-ternet search reveals that many com-panies offer genetic tests for Alzhei-mer disease, often as part of testinghundreds of thousandsof genetic varia-tions. You realize that you need to re-view yourbasic geneticsknowledge anddiscover more about how to read ge-netic association studies.

The Human Genome Project hasgreatly stimulated interestin genetic de-

terminants of disease. The determi-nants of common mendelian diseasesthat involve a single gene (eg, cystic fi-brosis, Huntington disease) are well es-tablished. Current research addressesthe role of genetics in the chronic dis-eases composing the major causes ofhuman morbidity and mortality, dis-eases that result from the concomitanteffect of environmental, behavioral, and

This is the first in a series of 3 articles serving as an introduction to clinicians

wishing to read and critically appraise genetic association studies. We sum-

marize the key concepts in genetics that clinicians must understand to review

these studies, including the structure of DNA, transcription and translation, pat-

terns of inheritance, Hardy-Weinberg equilibrium, and linkage disequilib-

rium. We review the types of DNA variation, including single-nucleotide poly-

morphisms (SNPs), insertions, and deletions, and how these can affect protein

function. We introduce the idea of genetic association for both single-

candidate gene and genome-wide association studies, in which thousands of

genetic variants are tested for association with disease. We usetheAPOEpoly-

morphism and its association with dementia as a case study to demonstrate

the concepts and introduce the terminology used in this field. The second and

third articles will focus on issues of validity and applicability.

JAMA. 2009;301(1):74-81 www.jama.com

AuthorAffiliations: Centre forClinicalEpidemiology andBiostatistics, University of Newcastle, Hunter MedicalResearch Institute, and Department of General Medi-cine,John Hunter Hospital, Newcastle,Australia (Dr At-

tia and Mr McEvoy); Department of Hygiene and Epi-demiology, University of Ioannina, School of Medicine,Ioannina, Greece, and Center for Genetic Epidemiol-ogy and Modeling, Tufts Medical Center, Departmentof Medicine, Tufts University School of Medicine, Bos-ton, Massachusetts (Dr Ioannidis); Clinical Epidemiol-ogy Unit, Faculty of Medicine, Ramathibodi Hospital,Mahidol University, Bangkok, Thailand (Dr Thakkin-stian); Division of Genetics, Hunter Area PathologySer-vice, JohnHunter Hospital,New Lambton, Australia, andCentre for Information Based Medicine, Faculty ofHealth, University of Newcastle, Hunter Medical

Research Institute, Newcastle, Australia (Dr Scott); Res-piratory Epidemiologyand Public Health, NationalHeartand Lung Institute, Imperial College, London, England(Dr Minelli); Department of Health Sciences, Univer-

sity of Leicester, Leicester,England (Dr Thompson);De-partment of Epidemiology, Biostatistics and Occupa-tional Health, Faculty of Medicine, McGill University,Montreal, Canada (Dr Infante-Rivard); and Depart-mentof Clinical Epidemiologyand Biostatistics,McMasterUniversity, Hamilton, Canada (Dr Guyatt).Corresponding Author: John Attia, MD, PhD, Centrefor ClinicalEpidemiology and Biostatistics,University ofNewcastle, Level 3, David Maddison Bldg, Newcastle2300, Australia ([email protected]).Users Guides to the Medical Literature Section Edi-tor: Drummond Rennie, MD, Deputy Editor, JAMA.

74 JAMA, January 7, 2009Vol 301, No. 1 (Reprinted) 2009 American Medical Association. All rights reserved.

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Box. Glossary

Additive

Describes any trait that increases proportionately in expres-sion when comparing those with no copy, 1 copy, or 2 cop-ies of that allele, ie, those with 1 copy ofthe alleleshowmore

of the trait than those without, and in turn, those with 2copies show more of the trait than those with 1 copy

Allele

One of several variants of a gene, usually referring to a spe-cific site within the gene

Candidate gene study

A study that evaluates association of specific genetic vari-ants with outcomes or traits of interest, selecting the vari-ants to be tested according to explicit considerations (knownor postulated biology or function, previous studies, etc)

Chromosome

Self-replicating structures in the nucleus of a cell that carry

the genetic informationDominant

Describes any trait that is expressed in a heterozygote, ie,one copy of that allele is sufficient to manifest its effect

Genome

Theentire collection of genetic information (orgenes) thatan organism possesses

Genome-wide association study

A study that evaluates association of genetic variation withoutcomes ortraits ofinterest by using 100 000 to 1 000 000or more markers across the genome

Genotype

The genetic constitution of an individual, either overall orat a specific gene

Haplotype

Alleles that tend to occur together on the same chromo-some because of SNPs being in proximity and therefore in-herited together

Heterozygous

An individual is heterozygousat a gene locationif he or shehas2 differentalleles (one on the maternal chromosome andone on the paternal) at that location

Homozygous

An individual is homozygous at a gene location if he or shehas 2 identical alleles at that location

Isoform

Variant in the amino acid sequence of a protein

Linkage

The tendency of genes or other DNA sequences at specificloci to be inherited together as a consequence of their physi-cal proximity on a single chromosome

Linkage disequilibrium

A measure of association between alleles at different loci

Locus/loci

Thesite(s) ona chromosome atwhich thegene fora particu-lar trait is located or on a gene at which a particular SNP islocated

Messenger RNA

A ribonucleic acidcontaining single-strand copy of a genethat migrates outof thecell nucleus to the ribosome, whereit is translated into a protein

Mutation

A rare variant in a gene, occurring in 1% of a population;cfpolymorphism

Pedigree

A diagram depicting heritable traits across 2 or moregenerations of a family

Phenotype

The observable characteristics of a cell or organism, usu-ally being the result of the product coded by a gene (geno-type)

Polymorphism

The existence of 2 or more variants of a gene, occurring ina population, with at least 1% frequency of the less com-mon variant (cfmutation)

Recessive

Describes any trait that is expressed in a homozygote butnot a heterozygote, ie, 2 copies of that allele are necessaryto manifest its effect

Ribosome

The protein synthesis machinery of a cell where messengerRNA translation occurs

SNP

Abbreviation for single-nucleotide polymorphism, asingle base pair change in the DNA sequence at a particu-lar point compared with the common or wild-typesequence

Synonymous SNP

A SNP that does not lead to a change in the amino acidsequence compared with the common or wild-typesequence; cf nonsynonymous , in which there is a changein the amino acid sequence as a result of the SNP

Variant allele

The allele at a particular SNP that is the least frequent in apopulation

Wild-type allele

The allele ata particular SNP that is most frequent ina popu-lation, also called common allele


2009 American Medical Association. All rights reserved. (Reprinted) JAMA, January 7, 2009Vol 301, No. 1 75

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genetic factors. In the last year alone,major studies have tested hundreds ofthousands of genetic variations (poly-morphisms),trying to establishthe ge-netic determinants of coronary arterydisease,2 type 2 diabetes,3 stroke,4 mul-

tiple sclerosis,

5

breast cancer,

6

bipolar

disorder,7 rheumatoid arthritis,8 Crohndisease,7 and Alzheimer disease.9-11

There are many reasons why a clini-cian may wish to understand the re-sults of genetic association studies. Thisgenetic information may shed light on

pathways involved in disease and iden-

tify new targetsfor therapeuticinterven-tions, advancesthat areof interestin ba-sicandapplied research. Genetics mightbe able to improve diagnosis or im-prove theabilityto usetherapeuticagentsmore efficiently andwithless risk, an area

termedpharmacogenomics. Althoughfewgenomic applications have reached thislevel ofutility, despite theclaimsof com-panies marketing genetictests, thismightchange in the future. The most likelyshort-term clinical effect of this geneticinformation is to enhance risk stratifi-cation and provide patients with infor-mation about their prognosis.

This series of 3 articles aims to pro-vide clinicians with an introduction toreading andunderstandinggenetic stud-ies in medicine. This first article sum-marizes the key genetic concepts clini-

cians must understand to read articlesoffering genetic associations for pos-sibleclinicaluse; those familiar withbasicgeneticsmaywishto movestraighttothesecond article. That article, along withthe third, will focus on issuesof validityand applicability fundamental to criti-cal appraisal of such studies. Through-out the series, our presentation will besimplified but sufficiently detailed toallow readers to judge therelevantissues.

THE GENETIC BLUEPRINT

In1953,James Watsonand FrancisCrickproposeda windingstaircase (doublehe-lix) structure of DNA (FIGURE 1). Thesides of the staircase or ladder, calledstrands, are formedby alternating sugar(deoxyribose) and phosphate mol-ecules; the rungs of the ladder areformed by 4 nitrogen-containing ringcompounds called bases: adenine (A),thymine (T), guanine (G), and cyto-sine (C). A pair of these bases formseach rung of the ladder; adenine al-ways binds to thymine and cytosine al-

ways binds to guanine to form the fullrung. Thus, each rung of the helix lad-der is called a base pair. A single baseplus its associated sugar and phos-phate groups is called a nucleotide.

One long stretch of double-strandedDNA, forming the spiral staircase, con-stitutes 1 chromosome, on which therearemanygenes,agenebeingastretchof

Figure 1. Components and Structure of DNA

C

G

G

C

T

A

G

C

C

G

C

G

A

T

G

C

T

A

DNA double helix

Sugar

Adenine (A)

Guanine (G)

Thymine (T)

Cytosine (C)

BasesSugar-phosphate

group

Nucleotide Linked nucleotides

DNA building blocks

Phosphate

Single DNA strand

Double-stranded DNA

Sugar-phosphate backbone

A double helix

GC

AC

Hydrogen bonds

Base

pairs

5

3

5

5

Base

3

Single DNA strand

Sugar-phosphate backbone

3

3

3

5

5

A

G

T

C

CA G

TG

C CA G

T

1

4 3

25

3

5

3

5

Base

pairs

C T

The building block of DNA is the nucleotidea sugar (deoxyribose) with a phosphate group at the 5 carbonand a base (adenine, thymine, guanine, or cytosine) at the 1 carbon. Nucleotides link together by a bondbetween thephosphate group of onenucleotide andthe 3 carbonof thepreviousnucleotide,to form a singleDNA strandwith a resultingdirectionality of 5 to 3. Twostrands with opposite directionality combine to forma doublehelix that is held together by hydrogen bonds acrossthe bases.Adenine always binds to thymine andguanine always binds to cytosine. The sequence of base pairs encodes the genetic information.



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DNA that typically codes for 1 protein.Twenty-threechromosomesinthespermandthe corresponding23 chromosomesin the egg come together at fertilizationtoformtheentireDNAsetofahuman,calledthegenome.Eachpersontherefore

has 23 pairs of chromosomes, of which1pairisthesexchromosomesthatdeter-minesex.Theother22pairsarenumbered1to22,providingeachpersonwith2ver-sionsofeachgene,oneonthematernallyinheritedandoneonthepaternallyinher-ited chromosome (FIGURE 2).

DNAis the blueprint for making pro-teins that build cells and tissues and en-zymes that catalyze biochemical reac-tions within a cell. The information onthe DNA guides the production of pro-teins through a 2-step process of tran-scription and translation.

The first step involves transcribingDNA into messenger RNA (mRNA)(FIGURE 3). The mRNA molecule mi-gratesoutofthenucleustothecytoplasmto reach the protein-building machin-ery of the cell, known as the ribosome.Here,duringthesecondstep,themRNAmoleculeistranslatedintoprotein,usingthecodetolinkaminoacidsoneatatime.These processes of transcription andtranslation convert thegenetic informa-tionatonegene,calledthegenotype,intothe protein that, in concert with other

genes, theirproteins, andenvironmen-tal exposures, determines the final at-tribute, the phenotype (eg, hair color,height, thrombophilia).

HUMAN VARIATION

Sequencing the human genomeidentifying the entire sequence of basepairs in the 25 000 genes that consti-tutehuman DNAhas revealed thatthesequence is more than 99% identicalacross different people.12 However, thehuman genomeincludes 3.3billion base

pairs; thus, even with this high level ofsimilarity, there are still more than 12million potential variations between 2peoples genomes.13-15 Differences ingene structure that occur infrequentlyin populations, ie, less than 1%, arecalled mutations, whereas differencesthat occur more frequently, ie, greaterthan or equal to 1%, are called poly-

morphisms (FIGURE4). These polymor-phisms may take a number of differ-ent forms:

1. the presenceorabsence ofan entirestretchofDNA(insertion/deletionpoly-morphisms),avariationofwhichinvolves

DNA duplication, called copy numbervariation, or CNV;2. repeating patterns of DNA that

vary in the number of repeats; each re-peating unit may vary from 2 to 3 tohundreds of base pairs long and mayrepeat a few times to hundreds of times;and

3. a singlebase pair change, calleda single-nucleotide polymorphism,orSNP(pronounced snip). This is by far themost common polymorphism; scien-tists have cataloged more than 12 mil-lion SNPs to date.13,15 Some SNPs are

in parts of the gene that are translated,ie, code for protein: among them, non-synonymous SNPs lead to a change inamino acid sequence of the resultantprotein, whereas synonymous SNPs donot result in amino acid change. OtherSNPs are in areas of the chromosomethat do not directly code for protein butmaystill influence cell functionthroughother means, such as controlling theamount of the protein that the cellbuilds. Given the very large number ofSNPs, their nomenclature can be con-

fusing, but the most common systemuses a number with the prefix rs (forreference SNP), eg, rs1228756.

The different forms or variants that aparticular polymorphism may take arecalledalleles.Forexample,theAPOEgeneintheclinicalscenariohas3alleles,namede2, e3, ande4 (e for epsilon; for histori-calreasons,thereis no e1). ThelocationalongtheDNAstrandatwhichaparticu-lar allele is present is called a locus. I n aferalmoment,geneticistsdecidedto calltheformofthegenethatismostcommon

inthepopulationthewildtype;wewillusethe less colorful term common allele;and the less common allele(s), variantallele(s).Thedifferentallelesinthecaseof SNPs, only the nonsynonymousSNPsresult in production of differentforms of the protein for which the geneisresponsible.Thesedifferentproteinsarecalled isoforms.

Of theAPOE alleles, allele e3 is mostcommon in white populations (78%)and thus represents the common al-lele; e2 and e4 are variant alleles (witha frequency of 6% and 16%, respec-tively, among white individuals). Physi-

ologically, the apoE protein carries aform ofcholesterolandbinds to theapoEreceptor on the surface of cells for thecholesterol to be metabolized. Of the 3protein isoforms that result from the 3correspondingAPOE alleles, the e2 iso-form has decreased strength of bind-ing,or affinity, to theapoEreceptor. Theproteins resulting from the e3 and e4alleles, the e3 and e4 isoforms, havehigher affinity compared with e2. (Forthe sake of simplicity, we have pre-sented the 3 alleles of the APOE gene,leading to the 3 isoforms, as if they are

changes at one point in the DNA. In fact,these isoforms are defined by changesat 2 points in the DNA, leading tochanges in 2 amino acids (positions 112and 158) that make up the apoE pro-tein: alleles e2, e3, and e4 at the DNAlevel lead to amino acids cysteine/cysteine,cysteine/arginine, and arginine/arginine at these 2 sites, respectively).

Because each person has 2 versionsofeach chromosome, onefromthe motherandone from thefather, individualshave2 APOE genes. The 2 relevant chromo-

somes (in the case of apoE, the 19thchromosome) may carry the e3 allele(denoted e3/e3) or 1 each of e3 and e2alleles (denoted e2/e3), or any othercombination, eg, e2/e4, e3/e3. Thisshorthand denotes thegenotype of theindividual. Individuals with 2 copies ofthe same allele are said to be homozy-gous, or a homozygote; ie, an individualwith e3/e3 is homozygous for e3, or ane3 homozygote. Conversely, an indi-vidual with 2 different alleles, eg, e2/e3, is a heterozygote, or heterozygous.

Individuals with e2/e3 produce somee2 protein and some e3 protein. Thequestion then follows: which proteinfunction wins? If the variant allele isdominant, it need be present in only 1of the 2 genes to carry all the biologi-cal activity. In such cases, the allele onthe other chromosome will remain bio-logically silent. Conversely, an al-



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lele that is recessive will need to be pre-sent on both genes, ie, homozygous, toaffect function; it will otherwise re-

main silent. If 2 differing protein iso-forms resulting from 2 different alleles(eg, e2, e3) share function, the model

of protein function is known as an ad-ditive, orper-allele, model. These domi-nant, recessive, and additive/per-

Figure 2. Human Male Karyotype, Chromosome Structure and Mapping, and Location of APOE

1 2 3 4 5

6 7 8 9 10 11 12

16 17 1813 14 15

19 20 21 22 X YSex chromosomes

Molecular location of APOE

(base pairs 50100879 to 50104490)

A Normal karyotype B Chromosome structure

C Chromosome 19 mapping (ideogram) and location of APOE

Intron 2DNA

Intron 3

Exon 1 Exon 3 Exon 4

Coding regionUntranslated region

5 3

Telomere

Telomere

Centromere

30-nm fiber

Nucleosome

Histones

DNA double helix

Loops

19q13.2

p

Centromere

q

Telomere

Centromere

Telomere

Intron 1

Exon 2

13.3

13.3113.32

13.33

13.4113.4213.43

13.2

13.2

13.13

13.13

13.12

13.12

13.11

12

12

1111

13.11

Telomere

50103500 bp 50104500 bp50102500 bp50101500 bp50100500 bp

A, Typically, an individual has 23 pairs of chromosomes. One member of each pair is inherited from the mother and one from the father. Chromosomes shown in thekaryotype were obtained when the cell was not dividing, stained using Giemsa, and ordered by size. B, The DNA double helix is wound around proteins called histonesto form small packages called nucleosomes. The nucleosomes in turn are wound around themselves to form loops that make up the chromosome. The region of thechromosome near the center is called the centromere, and each end is called a telomere. C, The centromere is not exactly at the center of the chromosome, resultingin a shorter arm, named p forpetit(French for small) and a longer arm, named q. Chromosome 19 is the site of the APOEgene, which is composed of sequences withregulatory functions (untranslated regions) and sequences with coding functions. Regions of the gene that are spliced out during transcription to messenger RNA arecalled introns. The remaining regions, exons, contain the sequences that code for the final protein product.



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allele models are called models ofinheritance, or genetic models.

As it turns out, the APOE alleles actpretty much in an additive manner;thus, e2/e3 individuals have overallapoE function that is partway be-

tween the reduced affinity of an e2 ho-mozygote and the higher affinity of ane3 homozygote. With most genetic as-sociation studies for complex dis-eases, the inheritance model remainsunknown.

EXAMINING GENESAT THE POPULATION LEVELIn genetics, it is usual to describe thedistribution of the alleles of interest inthe population. In the same way thatmost continuous variables in medi-

cine observe a normaldistribution,mostallele distributions observe what iscalled Hardy-Weinberg equilibrium(HWE).TheHardy-Weinberg lawstatesthat if there are 2 alleles at a particularlocus, named A and a, with frequency

p and q, respectively, then after 1 gen-eration of random mating the geno-type frequencies of the AA, Aa, and aagroupsin thepopulation will be p2, 2pq,and q2, respectively. Given that thereare only 2 alleles possible, A or a, then

p

q=1,andp

22pq

q

2

=1.It isgen-eral practice in a genetic associationstudy to check whether the allele fre-quencies observe HWE proportions.Deviations from HWE in the popula-tion may be due to the following:

Figure 3. Transcription and Translation

Mature mRNA transcript

DNA

5

N U C L E U S

mRNA

Posttranscriptional modification of mRNAand transportation to cytoplasm

Ribosome

5

3

RNA polymerase

Primary

mRNA transcript

5

3 5

3

5

3

Direction of

transcription

Transcription

Nucleus

Cytoplasm

Cell

AREA OF

DETAIL

Exon 1 Exon 2Intron 1 Intron 2

CodingregionPromoter UTR UTR

DNA

Gene

5

5

3

3

Deletion of introns (splicing)

Transcription

5 3

CYTOP L A SM

Translation

Direction of

translation

Primary

mRNA

transcript

Mature

mRNA transcript

mRNA Synthesis

5 3

Protein Amino acid

Transfer RNA (tRNA)

During transcription, the DNA double helix is split apart, and RNA polymerase synthesizes messenger RNA (mRNA) using one DNA strand as a template. Sections ofthe primary mRNA transcript, called introns, are spliced out to form the mature mRNA, which moves into the cytoplasm. The ribosome uses the mRNA sequence tobuild the protein. A specific sequence of 3 bases codes for each amino acid, which is delivered to the ribosome by transfer RNA. UTR indicates untranslated region.



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Inbreeding, ie, marryingclose rela-tives, because HWE depends on ran-dom (with respect to the relevant gene)mating

Genetic drift, a process in whicha population is isolated, with a limitednumber of possible matings

Migration

New mutations; only very newmutations upset HWE because equi-librium is usually reached within1 gen-eration in a sufficiently large popula-tion (this is where the equilibrium inHWE comes from)

Selection, eg, a selective disadvan-tage of a particular allele that leads tofetal death.

Deviations from HWE may also sig-nal methodological problems with thegeneticstudy (eg,error in genotyping orpopulation stratification), a possibilitydiscussedin thenext articlein this series.

CANDIDATE GENE VSGENOME-WIDE APPROACHES

Investigators conducting genetic asso-ciationstudiesmaytargetgenesforinves-tigation according to the known or pos-

tulated biology and previous results, anapproach knownascandidate gene asso-ciation.Alternatively,they mayscreentheentire genome for associations, anapproach thathas,in recent years, trans-formed the field of genetic associationstudies. Researchersnowinvestigatehun-dreds of thousands of SNPs across theentire genome, without any previoushypotheses about potential mecha-nismsor candidates.This type ofagnos-tic study, genome-wide association,or GWA, hasgreatly accelerated thepace

of discovery of genetic associations.16,17

As discussedin thenext article in thisseries, testing so many potentialgenessi-multaneously carries the risk of findingmanyspuriousassociations.For thisrea-son, SNPs that seem to have strong orsuggestive statistical signals in an initialGWA study are then tested for replica-tion in other large data sets or studies.Toensurethatthediscoveriesarenot juststatistical flukes, replication studies areoften published along with the initialdata.7 The boundaries between candi-

date gene studies and agnostic GWAstudies can become blurred, and the 2types of studies are not mutually exclu-sive: GWA studies propose new candi-dates for replication but may also inter-rogate traditional candidates.

Whether hypothesis-driven or GWAstudies, genetic association studies usu-allyrepresentpopulation-basedinvesti-

Figure 4. Common (Wild-Type) Allele and 4 Types of Genetic Polymorphisms

C

T

G C

A Common (wild-type) allele

B Polymorphisms

Repeated segments

Nucleotide variation

Deletion

Insertion

Tandem repeat

Single-nucleotide polymorphism (SNP)

Inserted segment

Deletedsegment

T TG

T TG

CA G

T

CA G

TG

C CA G

T

CA A T G C CA G T

CA G

TG

C

A GC

A GT

T TG

C

DNA polymorphisms include deletions, in which a DNA sequence is missing compared with the common al-lele, and insertions, in which a DNA sequence is added compared with the common allele. Repeats may alsooccur,in which thesame sequence repeats multiple times.Depending on thesize of therepeating unit andthenumber of repeats, these variants may have different names, such as satellites, microsatellites, minisatellites,or copy number variants. Single-nucleotide polymorphisms (SNPs), variations at a single base-pair location,are the most common type of polymorphism in the human genome.



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gationsinwhichdiseasedandnondiseasedindividuals are unrelated. Genetic stud-ies(eithercandidateorgenome-wide)mayalso be carried out among family mem-bers in large pedigrees affected by a rarediseasecausedbyararemutation.Insuch

studies,thepresenceofcertainstretchesofDNAorgenesinfamilymemberswithdisease and their absence in those with-outdiseaseiscalled linkageanalysis.Themethodsandinterpretationoffamilystud-ies differ radically from those of pop-ulation-based studies,18,19 andthisseriesis restricted to discussion of the latter.

BecausedetectingSNPsisrelativelyeasyand they are responsible for most of thegenetic variation in humans, they havebeenthefocusofmostoftheresearchex-ploring gene-diseaseassociation.Theba-sicideaofagene-diseaseassociationstudy

isrelatively simple. Inthesameway thatvariation in an exposure is linked to anoutcome(eg,cholesterol andmyocardialinfarction)ina traditionalepidemiologicstudy, variation in a gene is linked to anoutcome(eg,aSNPinthecholesteryles-tertransferasegeneandmyocardialinfarc-tion) in a genetic association study.

LINKAGE DISEQUILIBRIUM

One goal of traditional epidemiology,elucidating causation, can be frus-trated by noncausal associations. Tra-

ditional epidemiology tries to deal withthis problem by adjusted or multivar-iate analysis. For example, when ex-aminingthe associationof hypertensionwith stroke, investigators will simulta-neously adjust for other variables, in-cluding age, sex, smoking, and obesity.

In genetic association studies, onegoal may be to establish whether a SNPis causally associated with the out-come (eg, presence or absence of dis-ease). This requires isolating the func-tion of a particular SNP from the otherSNPs that may be nearby in the gene.In practice, because stretches of the ge-nome tend to be inherited together asa unit (a situation known as linkage dis-equilibrium), this may be difficult. Thus,the association of a SNP with an out-come, no matter how strong, may benoncausal. It is possible that some otherSNP in linkage disequilibrium with the

one under study is the true causal vari-able. Although this is an important dis-tinction if the aim is to understand theunderlying biology to develop a newtherapeutic agent, it is not critical if theaim isto use the SNP simply asa marker

of risk.The linkage disequilibrium phenom-enon results in haplotype blocks,stretchesofDNAdefinedbythepresenceofhighlinkage disequilibriumamongtheSNPspresent.TwoormoreSNPsthatareinlinkagedisequilibriuminthesamehap-lotype block candefine haplotypes, spe-cificcombinationsofvariantsacrosstheseSNPs. Thecombinations ofvariantsthatcanarisearedictatedbytheextentoflink-age disequilibrium.

As an example, consider SNP A withacommonallelefrequency(A)of80%and

SNP B with a common allele frequency(B) of 60%. If there is no linkage, alleleA at SNP A and allele B at SNP B willbe found together in the same person0.800.60=36%ofthetime,ie,consis-tentwithchance.Withperfectlinkagedis-equilibrium,eg,wheretheSNPsareveryclosetogether, it may happen that alleleA is always found with allele B. The ex-tentofthislinkagedisequilibriummaybeexpressedanumberofdifferentways,withmetricssuchasr2 (aplaincorrelationco-efficient)or D.Describingtheproperties

of each metric goes beyond the scope ofthis article16; however, in general, thegreaterthesemeasures,thegreaterthede-greeoflinkagebetweenthevariants,witha D of 1 indicating that 2 alleles are al-ways found together.

Armed with this background knowl-edgeofgeneticconceptsandterminology,youarenowreadytoembarkonanevalu-ation of validity, results, and applica-bility in genetic association studies. Theremaining 2 articles in this series willaddress these issues.

Author Contributions: Drs Attia, Ioannidis, and Guy-att had full access to all of the data in the study andtake responsibility forthe integrityof thedataand theaccuracy of the data analysis.Financial Disclosures: Dr Guyatt reports that his in-stitution receives royalties from publication of theUsers Guides to the Medical Literaturebook. No otherauthors reported disclosures.Additional Contributions: We wish to thank JulianHiggins, PhD (Cambridge University), and JohnDanesh, MBBS,DPhil (Cambridge University),for help-ful comments on early drafts of this series. We also

wish to thank Jane McDonald, BA PGCertEdit (Uni-versityof Newcastle, Australia), forinvaluable help increating the original figures.

REFERENCES

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Artículo 4. How to use an article about genetic association JAMA 2009

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Transcript of Artículo 4. How to use an article about genetic association JAMA 2009