1、Genetics of complex human disease traits.Daniel T.OC onnor,M.D.Department of Medicine.Nopm-252.First year curriculum in human genetics.Wed Apr 1,2009.CMME-2047.PURPOSE.In the next two hours,we plan to cover the role of heredity and genes invery common,non-Mendelian traits that are frequently seen by
2、 primary carephysicians.We will illustrate how we establish the role of heredity on any trait,and then methods to position the particular genes that influence such a trait.WHAT IS A COMPLEX TRAIT?Trait=phenotype.Disease causation/etiology/origin:The old conundrum of:Nature(heredity)versus nurture(en
3、vironment).How to solve this riddle:Family/pedigree or twin studies(see below).Frequency.Most(95%)of the disease encountered in internal medicine,familymedicine,pediatrics,neurology,or psychiatry is complex,and its origin is not wellunderstood.Not clearly completely hereditary(Mendelian)or environme
4、ntal.Read:Hypertension,coronary artery disease,arrhythmia,stroke,aneurysm,asthma,COPD,diabetes,obesity,schizophrenia,bipolar disorder.Multifactorial:Genes,environment,gene-by-environment interactions.Non-Mendelian.Mendelian:Gene Bimodality:Hallmark of a major gene effect on a quantitative trait.Bimo
5、dality:Hallmark of a major gene effect on a quantitative trait.Polygenic Traits01231 Gene 3 Genotypes 3 Phenotypes01232 Genes 9 Genotypes 5 Phenotypes012345673 Genes 27 Genotypes 7 Phenotypes051015204 Genes 81 Genotypes 9 PhenotypesComplex Trait ModelDisease PhenotypeCommonenvironmentMarkerGene1Indi
6、vidualenvironmentPolygenicbackgroundGene2Gene3Linkage Linkagedisequilibrium Mode ofinheritance LinkageAssociation APPROACHES TO COMPLEX TRAITS.(Genetic)epidemiology.Demographics(age,sex,ethnicity,geography,fam hx).Risk(susceptibility)factors(see above).Relative risk(RR):Given a risk factor,what is t
7、he increase in traitprevalence?Estimator of RR:Odds ratio(OR).Given a risk factor,OR=(have trait/donot have trait).OR+/-confidence interval(+/-95%CI)versus reference(no risk)=1.No risk:RR or OR=1.OR+/-CI 1 CHGA genetic variation:Risk factor for hypertensive ESRD in blacksJ Am Soc Nephrol.2008 Mar;19
8、(3):600-14.Chromogranin A polymorphisms are associated with hypertensive renal disease.Salem RM,Cadman PE,Chen Y,Rao F,Wen G,Hamilton BA,Rana BK,Smith DW,Stridsberg M,Ward HJ,Mahata M,Mahata SK,Bowden DW,Hicks PJ,Freedman BI,Schork NJ,OConnor DT.Estimator of RR:Odds ratio(OR).Given a risk factor,OR=
9、(have trait/do not have trait).Intermediate(risk)traits(phenotypes).Intermediate in time and mechanism.Bridging genotype and ultimate,late disease trait.Ideally:Greater h2,earlier penetrance.If biochemical assays:Biomarkers.Intermediate phenotypeGene “Intermediate”intime and causality MechanismTwins
10、Cardiorenal disease trait(later life)Time(decades.)Gene(fixed at conception)Twins:window into heritability(h2)of any phenotypeMonozygotic(MZ,identical)twins:Billy and Benny.VP=VG+VEh2=VG/VP=2(RMZ-RDZ)Source:Guinness Book of World Records.Total mole count for MZ and DZ twins01002003004000100200300400
11、Twin 2Twin 101002003004000100200300400Twin 2Twin 1MZ twins-153 pairs,r=0.94DZ twins-199 pairs,r=0.60020406080100HeightWeightSBPDBPCardiac outputSVRBaroreflex upBaroreflex downPlasma norepiPlasma epiHeritability(h2)of traits in human twin pairsHeritability(h2),mean+/-SEMTrait87+/-291+/-147+/-750+/-67
12、1+/-459+/-633+/-943+/-770+/-467+/-4PhysicalPhysiologicalBiochemicalFamily studies(twin pairs,pedigrees).Fam hx as a risk factor.In 1st degree relatives:Parents,siblings.Heritability(h2):Fraction of trait variance accounted for by genetic variance.VP=VG+VE h2=VG/VP Estimate h2 from twin pair(or pedig
13、ree)studies:Type of twin pairAllele sharing across the genome.MZ=monozygotic=identical.100%DZ=dizygotic=fraternal50%(on average),like any sib pair Quick-and-dirty algorithm:h2=VG/VP=2(RMZ RDZ)West J Med.1984 Dec;141(6):799-806.Understanding genetic and environmental risk factors in susceptible perso
14、ns.Williams RR.Family history as a risk factor for complex traits.Familyhistory(Genetic)linkage:Co-segregation of marker and trait.Versus independent segregation:Different chromosome,or far apart on samechromosome.Thus,marker and trait loci are within 50 cM of genetic distance.cM:Just count the meio
15、tic recombinants versus non-recombinantscM=(Recombinant meioses/Total meioses)*(100)Lower cM means closer(marker and trait loci)Calibration.cM(genetic/meiotic distance)vs Mb(physical distance):1 cM=1 Mb(actually 0.5-2.0).Markers to span the genome for linkage:3000 cM/50 cM=60 fully informative(heter
16、ozygosity)markers in theoryIn practice:400-800 highly informative markers(multiallelic microsatellites)2000-10,000 less informative markers(biallelic SNPs)Linkage=Meiotic co-segregationA2A4A3A4A1A3A1A2A2A3A1A2A1A4A3A4A3A2Marker allele A1cosegregates withdominant disease Linkage MarkersThomas Hunt Mo
17、rgan discoverer of linkageIdiosyncratic features of genetic linkage(=meiotic co-segregation).Units.Metric=meiotic recombination(50 meioses/generation).Units of genetic distance=recombination during meiosis(cM).cM=(recombinant meioses/total meioses)*100 E.g.:8/(8+86)*100=(8/94)*100 =8.5 cM1 cM 1 Mb R
18、ange 0.5-2.0 Varies by species,sex,chromosomal region(meiotic“hot spots”)Significance.“LOD”scores.LOD=Log10 of the odds ratio for linkageOdds ratio:Co-segregation(marker and trait)NotSignificant:LOD 3.0(i.e.,odds ratio 1000/1)Why 3.0?50“linkage groups”(meiotic breaks/generation),target=0.05.1/50*1/2
19、0=1/1000.Genetic linkage:Meiotic recombination distance in cMcM=(recombinant meioses/total meioses)*1008/(8+86)*100=(8/94)*100 =8.5 cMMahata SK,Kozak CA,Szpirer J,Szpirer C,Modi WS,Gerdes HH,Huttner WB,OConnor DT.Dispersion of chromogranin/secretogranin secretory protein family loci in mammalian gen
20、omes.Genomics.1996 Apr 1;33(1):135-9.1 cM 1 MbRange 0.5-2.0Varies by species,sex,chromosomal region(meiotic“hot spots”)Mouse SBP crosses:“Genome scan”linkage.Wright FA,OConnor DT,Roberts E,Kutey G,Berry CC,Yoneda LU,Timberlake D,Schlager G.Genome scan for blood pressure loci in mice.Hypertension.199
21、9 Oct;34(4 Pt 1):625-30.LOD score:Log10 of the OR for linkage(marker,trait).Log10(non-recombinants/total meioses).Significant LO D 3(i.e.,OR 1000).Why?50 linkage groups(meiotic breaks/generation),target J Clin Invest.1996 May 1;97(9):2111-8.Quantitative trait locus mapping of human blood pressure to
22、 a genetic region at or near the lipoprotein lipase gene locus on chromosome 8p22.Wu DA,Bu X,Warden CH,Shen DD,Jeng CY,Sheu WH,Fuh MM,Katsuya T,Dzau VJ,Reaven GM,Lusis AJ,Rotter JI,Chen YD.Genetic linkage:What the data(marker,trait)look like.J Clin Invest.1996 May 1;97(9):2111-8.Quantitative trait l
23、ocus mapping of human blood pressure to a genetic region at or near the lipoprotein lipase gene locus on chromosome 8p22.Wu DA,Bu X,Warden CH,Shen DD,Jeng CY,Sheu WH,Fuh MM,Katsuya T,Dzau VJ,Reaven GM,Lusis AJ,Rotter JI,Chen YD.Genetic linkage:What the data(marker,trait)look like.(Allelic)associatio
24、n:Marker The catecholamine biosynthetic pathway.T Flatmark.Regulation of catecholamine biosynthesis.Acta Physiol Scand 168:1-17,2000.Figure 8:TH haplotypes in vivo6810121416182.4 1042.6 1042.8 1043 1043.2 1043.4 1043.6 104Tyrosine hydroxylase promoter haplotype 2:Pleiotropy.Coordinate effects on bot
25、h catecholamine excretionand stress blood pressure response in twinsChange in DBP during cold stress,mmHgNorpinephrine excretion,ng/gmHaplotype 2n=2 copies(n=32 individuals)Haplotype 2n=1 copy(n=164 individuals)Haplotype 2n=0 copies(n=131 individuals)Norepinephrine h2=49.6+/-6.7%,p=0.0001*Haplotype
26、2 on norepinephrine:p=0.0125*,4.06%variation explained 6065707580859095100C/CC/TT/TTyrosine hydroxylase regulatory polymorphism:Interaction of genotype and sex to affect DBPMale DBPFemale DBPDBP,mmHg(mean+/-SEM)Tyrosine hydroxylase(TH)promoter C-824T diploid genotype78.3+/-1.5(n=234)2-way ANOVA:(Cov
27、ariates:age,BMI)Overall F=12.4,p0.001*Genotype F=1.30,p=0.273Sex F=31.6,p0.001*Genotype*Sex F=3.14,p=0.044*C-824T explains 3.4%of DBP varianceAlleles:C=61%,T=39%HWE:Figure 7:Intermediate phenotypesMechanismTimeGene Biochemical trait Physiological traitDisease traitHypertension:“Intermediate”phenotyp
28、es and candidate genes.Tyrosine hydroxylase C-824T Catecholamines Baroreceptor function Stress blood pressure Hypertension GWAS(Genome Wide Association Study).Hypothesis:Commondisease/Common Variant.Markers to span the genome for association:3.3 Gbp(1 Gbp=109 bp)genome/500K SNPs=6000 bp(6 kbp).The o
29、nly variants spaced this closely(i.e.,the common)are SNPs.Spacing based on HapMap and LD(linkagedisequilibrium)blocks.HapMap:270 people world-wide typed at 4 million SNPs across thegenome.Within an LD block,SNPs are highly correlated(r2 0.6-1.0).Try to tag each LD block across the genome.Advantage:U
30、nrelated individuals(do not need families).Problems:Population stratification(artifactual association as a result of allelefrequency differences across populations).Statistical challenges to GWAS:Many LD blocks tested,modifiedtarget p=5x10-7.Solution:Replication in an independent sample,for joint(mu
31、ltiplicative,)probability.pter qter5 3ChromosomePaternalMaternalT C A G C T G A Haplotype:Ordered array of alleles along a single chromosome.Biallelic SNPs(single nucleotide polymorphisms).Typically“transitions”:Purine Purine(G A)Pyrimidine Pyrimidine(C T)5 3PaternalMaternalT A C G T A C GC G T C C
32、G T A“Linkage disequilibrium”(LD):Local,marker-on-marker locus Equilibrium=randomness(no correlation,r2=0)Disequilibrium=non-random(correlated,r20)Marker-on-trait locus:Mapping tool0.9 0.9 0.9 0.0 0.9 0.9 0.9 0.5 0.50.0Ancestral(shared)Meiotic recombinationr2BiallelicSNPs In population genetics,link
33、age disequilibrium is the non-non-randomrandom association of alleles at two or more loci.Linkage disequilibrium describes a situation in which some combinations of alleles or genetic markers occur more or less frequently in a population than would be expected from a randomrandom formation of haplot
34、ypes from alleles based on their frequencies.Non-randomNon-random associations between polymorphisms at different loci are measured by the degree of linkage disequilibrium(LD).The level of linkage disequilibrium is influenced by a number of factors including the rate of meiotic recombination meiotic
35、 recombination(crossovers(crossovers)and the rate of mutation.Linkage disequilibrium(LD).Marker trait Marker markerHapMap:View variation patternsTriangle plot shows LD values using r2 or D/LOD scores in one or more HapMap populationThe International HapMap Project(Identification of SNPs that tag hap
36、lotypes within blocks)Daly,M.J.,Rioux,J.D.,Schaffner,S.F.,Hudson,T.J.and Lander,E.S.(2001).High-resolution haplotype structure in the human genome.Nature Genet.29:229-232.Linkage disequilibrium(LD)“blocks”on human chromosome 14q32 100 kbp displayed,from 3 short-range(30 kbp)LD blocksNo long-range(10
37、0 kbp)LDQ ui ckTi m e?and aTI FF(LZW)decom pressorare needed to see thi s pi cture.Region of the genome around around SNP rs9941339 in CDH13(T-cadherin=novel adiponectin receptor)on 16q24 associated with intra MZ pair differences in HDL cholesterol(GWAS in n=1662 MZ pairs).Black points represent SNP
38、s genotyped in the study and gray points represent SNPs whose genotypes were imputed.In middle panel,red line shows the fine-scale recombination rate(centimorgans per Mb)estimated from Phase II HapMap and the black line shows the cumulative genetic distance(in cM).Association p=8.5x10-8.Gene-by-Envi
39、ronment(GxE)interaction probed by MZ twin intra-pair trait differences:HDL-cholesterol effect of T-cadherin(CDH13,novel adiponectin receptor)genetic variation revealed by dense,genome-wide profiling in 1662 MZ pairsp=8.5x10-8 100 kbp Common disease/Rare variant hypothesis.Or,accumulation of excess r
40、are variants:Non-synonymous(amino acidreplacement)cSNPs.Technology:Extensive re-sequencing in large numbers of cases vscontrols.Typically resequence a p athway in population trait extreme individuals(boost statistical power).Analyses:Summed c2,cases versus controls.Computational assessmentof amino a
41、cid change functionality(SIFT,PolyPhen).Ultimately functionalstudies.Accumulation of deleterious rare amino acid substitution variants at extremes of human body mass index(BMI)Ahituv N,Kavaslar N,Schackwitz W,Ustaszewska A,Martin J,Hebert S,Doelle H,Ersoy B,Kryukov G,Schmidt S,Yosef N,Ruppin E,Shara
42、n R,Vaisse C,Sunyaev S,Dent R,Cohen J,McPherson R,Pennacchio LA.Medical sequencing at the extremes of human body mass.Am J Hum Genet.2007 Apr;80(4):779-91.Dr illing down to the QTN(Quantitative Trait Nucleotide).Problem:Even after successful allelic association,the lower limit ofresolution is the LD
43、 block(3-50 kbp).So where,within that LD block,is thecausative variant?Solution:Studies of the putative responsible variant in a system outside ofthe human organism:in vitro(test-tube enzymology),in cella(transfection intocultured cells),or in vivo(transgenic mice).Drilling down to the“QTN”(“Quantit
44、ative Trait Nucleotide”)Haplotype“block”is the lower limit of resolution of marker-on-trait mapping.Switch to studies of associated variants:In cella.E.g.,transfected/expressed variants.In vitro.E.g.,kinetic properties of variants.In vivo:transgenic mice(BAC haplotype variant expression on knockout
45、background).Positional candidate genetic loci.“Positional candidate”locusWong C,Mahapatra NR,Chitbangonsyn S,Mahboubi P,Mahata M,Mahata SK,OConnor DT.The angiotensin II receptor(Agtr1a):functional regulatory polymorphisms in a locus genetically linked to blood pressure variation in the mouse.Physiol
46、 Genomics.2003 Jun 24;14(1):83-93.“Positional candidate”locusWong C,Mahapatra NR,Chitbangonsyn S,Mahboubi P,Mahata M,Mahata SK,OConnor DT.The angiotensin II receptor(Agtr1a):functional regulatory polymorphisms in a locus genetically linked to blood pressure variation in the mouse.Physiol Genomics.20
47、03 Jun 24;14(1):83-93.“Positional candidate”locusWong C,Mahapatra NR,Chitbangonsyn S,Mahboubi P,Mahata M,Mahata SK,OConnor DT.The angiotensin II receptor(Agtr1a):functional regulatory polymorphisms in a locus genetically linked to blood pressure variation in the mouse.Physiol Genomics.2003 Jun 24;14
48、(1):83-93.Wild-typeAgtr1a promoterLuciferasereporterTranscriptionPromoter/reporterplasmid(pGL3-Basic)VariantAgtr1a promoterLuciferasereporterTranscriptionPromoter/reporterplasmid(pGL3-Basic)LuciferasetranscriptionNucleusLuciferasetranslationCytosolChromaffin cellFirefly luciferaseenzymatic activity
49、assayTransfectionCelllysisPromoter variant characterizationWong C,Mahapatra NR,Chitbangonsyn S,Mahboubi P,Mahata M,Mahata SK,OConnor DT.The angiotensin II receptor(Agtr1a):functional regulatory polymorphisms in a locus genetically linked to blood pressure variation in the mouse.Physiol Genomics.2003
50、 Jun 24;14(1):83-93.“Positional candidate”locus R isk allele(gene)versus modifier a llele(gene).Risk allele:Allele that increases risk/susceptibility for disease,inlongitudinal studies.Example:CFTR(Cl-channel)DF508 ExposureDzStableRapid declineInitiation(case/control study)Outcome(longitudinal study
