GGAW - Oct, 2001M-W LIN Study Design for Linkage, Association and TDT Studies 林明薇 Ming-Wei Lin, PhD 陽明大學醫學系家庭醫學科 台北榮民總醫院教學研究部

GGAW - Oct, 2001M-W LIN Collins FS. (1992) Nature genetics 1:3-6

GGAW - Oct, 2001M-W LIN Collins FS. (1992) Nature genetics 1:3-6

GGAW - Oct, 2001M-W LIN Linkage Mapping for Disease Genes Linkage analysis (Lod score method) Allele-sharing methods

GGAW - Oct, 2001M-W LIN Gregor Mendel The principle of segregation of alleles. The principle of independent assortment.

GGAW - Oct, 2001M-W LIN Linkage Linkage describes the phenomenon whereby allele at neighbouring loci are close to one another on the same chromosome, they will be transmitted together more frequently than chance.

GGAW - Oct, 2001M-W LIN Linkage Family

GGAW - Oct, 2001M-W LIN Linkage Analysis Family

GGAW - Oct, 2001M-W LIN Recombinant Gametes Crossing over between two neighbouring loci will produce recombinant gametes.

GGAW - Oct, 2001M-W LIN Recombination Fraction Recombination fraction (θ) = number of recombinant gametes total gametes

GGAW - Oct, 2001M-W LIN Estimation of Recombination Fraction Direct Method: count recombinants. Maximum Likelihood Method: Unknown phases Incomplete penetrance Heterogeneity

GGAW - Oct, 2001M-W LIN

GGAW - Oct, 2001M-W LIN

GGAW - Oct, 2001M-W LIN Recombination Fraction Recombination fraction is a measure of genetic distance. 1cM= 1% chance of recombination between two loci.

GGAW - Oct, 2001M-W LIN Likelihood Odds Likelihood of data if loci linked at θ Likelihood odds = Likelihood of data if loci unlinked L(θ< 0.5) = L(θ= 0.5)

GGAW - Oct, 2001M-W LIN Lod Score L(θ< 0.5) Lod score (θ) = log 10 L(θ = 0.5)

GGAW - Oct, 2001M-W LIN Linkage Analysis Methods Direct counting recombinants and non-recombinants Maximum Likelihood Estimate

GGAW - Oct, 2001M-W LIN Phase Known Family

GGAW - Oct, 2001M-W LIN Phase Known L(θ) = (θ/2) r ((1-θ)/2) n-r r:No. of recombinants n:All meiosis

GGAW - Oct, 2001M-W LIN Lod Score Phase Known L(θ) LOD = log L(θ= 0.5) (θ/2) r [(1-θ) / 2] n-r = log { } (0.25) n = log 2 n θ r (1-θ) n-r

GGAW - Oct, 2001M-W LIN Phase Unknown Family

GGAW - Oct, 2001M-W LIN Phase Unknown L(θ) = 1/2 (θ/2) r [(1-θ)/2] n-r +1/2 (θ/2) n-r [(1-θ)/2] r r:No. of recombinants n:All meiosis

GGAW - Oct, 2001M-W LIN Lod Score Phase Unknown L(θ) LOD = log L(θ= 0.5) 1/2 [(θ/2) r [(1-θ)/2] n-r +(θ/2) n-r [(1-θ)/2] r ] =log { } (0.25) n = log {2 n-1 [θ r (1-θ) n-r +θ n-r (1-θ) r ]}

GGAW - Oct, 2001M-W LIN Lod Score - Maximum Likelihood Estimate (Z) Can be calculated at any values of  between 0 and 0.5, but are conventionally reported at  =0, 0.01, 0.05, 0.1, 0.2, 0.3, and 0.4. Z max is the maximum likelihood estimate (MLE) of . Lod score can be converted to a chi- square statistic by 2(loge10)  4.6.

GGAW - Oct, 2001M-W LIN Total Lod Score Lod score obtained from individual families can be added together to calculate the total lod score.

GGAW - Oct, 2001M-W LIN Statistical Significance of the Lod Score lod score > 3: evidence of linkage 2 < lod score < 3: suggestive evidence of linkage -2 < lod score < 2: uninformative of linkage lod score < -2: exclusion of linkage

GGAW - Oct, 2001M-W LIN Is a Pedigree Useful for linkage Analysis? Are critical individuals in the pedigrees doubly heterozygous at the loci? (Informative) Can the offsprings be scored as recombinants or nonrecombinants? (Phase)

GGAW - Oct, 2001M-W LIN Parameters Assumed in Lod Score Analysis Transmission mode of disease Recombination fraction Trait allele frequencies Penetrance values for each possible disease phenotypes Marker allele frequencies.

GGAW - Oct, 2001M-W LIN Advantages of Lod Score Analysis Statistically, it is more powerful approach than any nonparametric method. Utilizes every family member’s phenotypic and genotypic information. Provides an estimate of the recombination fraction. Provides a statistical test for linkage and for genetic (locus) heterogeneity.

GGAW - Oct, 2001M-W LIN Limitations of Lod Score Method assumes single locus inheritance requires specification of disease gene frequency and penetrance has reduced power when disease model is grossly misspecified

GGAW - Oct, 2001M-W LIN Complex Diseases No clear pattern of Mendelian inheritance A mix of genetic and environmental factors Incomplete penetrance Phenocopies Oligogenic or polygenic Heterogeneity High frequency of disease-causing allele

GGAW - Oct, 2001M-W LIN Recurrence Risk (λ) Frequency in relatives of affected person λ r = Population frequency r denotes the degree of relationship

GGAW - Oct, 2001M-W LIN Recurrence Risk Genetic mapping is much easier for traits with high λ s (λ s > 10) than for those with low λ s (λ s < 2).

GGAW - Oct, 2001M-W LIN Recurrence Risk of Different Diseases

GGAW - Oct, 2001M-W LIN Allele-sharing Methods Identical by state (I.B.S.) Two alleles of the same form. Identical by descent (I.B.D.) Two alleles are descended from the same ancestral allele.

GGAW - Oct, 2001M-W LIN Allele-sharing Methods Testing whether affected relatives inherited a region IBD (or IBS) more often than expected under random Mendelian segregation.

GGAW - Oct, 2001M-W LIN IBD = 2IBD = 1 IBD = 0 ACAB BC ACBC ACAB CD ADBC

GGAW - Oct, 2001M-W LIN IBS = 2IBS = 1 IBS = 0 BC AC ABADBC

GGAW - Oct, 2001M-W LIN Affected Sib-pair Methods An affected sib-pair may share 0,1, 2 alleles identical by descent (IBD) with probabilities of 0.25, 0.5, 0.25, respectively, at any marker locus.

GGAW - Oct, 2001M-W LIN IBD = 2 ACAB BC AB AC BCAA IBD = 1 IBD = 0 25% 50% 25%

GGAW - Oct, 2001M-W LIN Affected Sib-pair Methods If the marker locus is independent of the trait locus, the probabilities of the affected sib-pairs share 0,1, 2 alleles ibd will remain as 0.25, 0.50, 0.25.

GGAW - Oct, 2001M-W LIN Affected Sib-pair Methods If the marker locus is linked to the trait locus, an excess of affected sib-pair sharing two alleles ibd will be expected.

GGAW - Oct, 2001M-W LIN Allele-sharing Methods Affected Sib-pairs Affected Pedigree Member

GGAW - Oct, 2001M-W LIN Pearson  2 statistics Comparing observed numbers of sib-pairs sharing 0, 1, 2 alleles IBD with their expectations under the null hypothesis.

GGAW - Oct, 2001M-W LIN Pearson  2 statistics Alternative hypothesis: IBD sharing012 observedn 0 n 1 n 2 N = n 0 + n 1 + n 2 Null hypothesis: IBD sharing:012 expected N/4N/2 N/4

GGAW - Oct, 2001M-W LIN Comments on Allele-Sharing Method  There is no need to specify any genetic parameters of the transmission model.  Less powerful to detect linkage compared with the lod score method if the genetic transmission model can be specified correctly.  It is poor at providing a precise location of the disease gene.

GGAW - Oct, 2001M-W LIN Thresholds for Mapping Complex Traits

GGAW - Oct, 2001M-W LIN Association Study Case-Control study Transmission disequilibrium test (TDT)

GGAW - Oct, 2001M-W LIN ○□ ○□ □ ○ ○ □ ○ □ ADAD ACAC BCACAC AB BCCDA ADAD ACAC ■●■ ●●■ ● ■ ■ ● DDACAC BDCDCD CDCD BCABADAD BDAD Case-Control study

GGAW - Oct, 2001M-W LIN Linkage Disequilibrium Linkage disequilibrium is the non-random association in a population of alleles at closely linked loci.

GGAW - Oct, 2001M-W LIN Linkage Disequilibrium A2---B1-----C2---X----D3-----E4----F2  A2---B1-----C2---X----D3-----E4  A2---B1-----C2---X----D3  B1-----C2---X----D3  C2---X----D3  C2---X N generations

GGAW - Oct, 2001M-W LIN TDT Study To examine the transmission of a particular allele at a locus from heterozygous parents to their affected offspring.

GGAW - Oct, 2001M-W LIN □○ ● □○ ■ □○ ■ □○ ● BCABBCBB ABAC ACAC BCACAC BBBC AB “Trios” for TDT study “transmitted allele“  “case” “Non-transmitted allele”  “control”

GGAW - Oct, 2001M-W LIN What does a positive association imply? Direct causal effect Linkage disequilibrium Population stratification

GGAW - Oct, 2001M-W LIN When to Use Association Study Candidate gene Positive evidence of linkage Candidate region allelic associations

GGAW - Oct, 2001M-W LIN Suitable Sample for Linkage Disequilibrium Mapping Genetically isolated populations Younger populations

GGAW - Oct, 2001M-W LIN Successful Examples of Mapping Genes by Association Studies Autoimmune diseases associated with HLA  IDDM  multiple sclerosis  ankylosing spondylitis  rheumatoid arthritis Angiotensin-converting enzyme and heart disease low-density lipoprotein receptor and heart disease insulin locus and IDDM

GGAW - Oct, 2001M-W LIN Sample Size Required Linkage for Monogenic Traits One large family at least 40 informative meioses 20 cM marker density Expected lod score > 3

GGAW - Oct, 2001M-W LIN Sample Size Required Allele-Sharing λ s = 2 at least 600 affected sib pairs narrow down the region to 1 cM

GGAW - Oct, 2001M-W LIN Sample Size Required Linkage for Complex Traits Sham, Lin et al (2000) Am J Human Genetics 66,

GGAW - Oct, 2001M-W LIN Genetic Markers A complete informative marker locus at 0 recombination fraction to the disease locus.

GGAW - Oct, 2001M-W LIN Genetic Models Kp: population risk, q: disease allele frequency f 0 : penetrance for the genotype AA; f 1 : penetrance for the genotype Aa f 2 : penetrance for the genotype aa

GGAW - Oct, 2001M-W LIN Pedigree Types

GGAW - Oct, 2001M-W LIN Number of Pedigrees Required  = , Power = 90%, Homogeneity

GGAW - Oct, 2001M-W LIN Number of Pedigrees Required  = , Power = 90%, Heterogeneity (  = 0.5)

GGAW - Oct, 2001M-W LIN Sample Size Required Case-Control Study (  = 0.05, Power = 90%)

GGAW - Oct, 2001M-W LIN Sample Size Required Case-Control Study (  = 0.05, Power = 90%)

GGAW - Oct, 2001M-W LIN Sample Size Required TDT Study (  = 0.001, Power = 80%)

GGAW - Oct, 2001M-W LIN Define phenotype Identify evidence of genetic component Extended families Define study design Sib pairsSingle affected member Family, clinical information and DNA collection Genotyping Data analysis Identify regions of interest Physical Mapping / Gene Identification

GGAW - Oct, 2001M-W LIN Successful Examples Cystic fibrosis Huntington disease Early onset breast cancer (BRCA1, BRCA2) Alzheimer disease (chr14, chr1) Maturity-onset diabetes of the young (MODY) (chr12)...