1. Single Nucleotide Polymorphism And Association Studies
Stat 115/215

2. International HapMap Project
The International HapMap project is a recent, large-scale effort to facilitate GWAS studies:
Phase 1: 269 samples, 1.1 M SNPs
Phase 2: 270 samples, 3.9 M SNPs
Phase 3: 1115 samples, 1.6 M SNPs
Phase 3 platforms:
Illumina Human1M (by Wellcome Trust Sanger Institute)
Affymetrix SNP 6.0 (by Broad Institute)

3. Phase 1 & 2
90 Yoruba individuals (30 parent-parent-offspring trios) from Ibadan, Nigeria (YRI)
90 individuals (30 trios) of European descent from Utah (CEU)
• 45 Han Chinese individuals from Beijing (CHB)
• 45 Japanese individuals from Tokyo (JPT)

4. Phase III samples Population descriptors:
ASW (A): African ancestry in Southwest USA
CEU (C): Utah residents with Northern and Western European ancestry from the CEPH collection
CHB (H): Han Chinese in Beijing, China
CHD (D): Chinese in Metropolitan Denver, Colorado
GIH (G): Gujarati Indians in Houston, Texas
JPT (J): Japanese in Tokyo, Japan
LWK (L): Luhya in Webuye, Kenya
MEX (M): Mexican ancestry in Los Angeles, California
MKK (K): Maasai in Kinyawa, Kenya
TSI (T): Toscans in Italy
YRI (Y): Yoruba in Ibadan, Nigeria

5. Using 4242 independent SNPs and applying STRUCTURE
ASW
CEU
CHB
CHD ç
JPT
LWK
MEX
MKK
TSI
YRI

6. PCA plot

7. Non-African Populations
GIH
MEX

8. Haplotype Maps
Although there are around 10,000,000 SNPs, they group into a small number of groups of SNPs that are correlated with each other.
So, there are around around 300,000 unique arrangements of the SNPS
This is not that big of a number!
CS people can imagine an exhaustive search

9. SNP Characteristics: Linkage Disequilibrium
Hardy-Weinberg equilibrium
In a population with genotypes AA, aa, and Aa, if p = freq(A), q =freq(a), the frequency of AA, aa and Aa will be p2, q2, and 2pq, respectively at equilibrium.
Similarly with two loci, each two alleles Aa, Bb

10. SNP Characteristics: Linkage Disequilibrium
Equilibrium Disequilibrium
LD: If Alleles occur together more often than can be accounted for by chance, then indicate two alleles are physically close on the DNA
LD expected to decay monotonically on either side of each SNP
In mammals, LD is often lost at ~100 KB
In fly, LD often decays within a few hundred bases

11. Low LD -> Recombination
Basic Concepts
Parent 1
Parent 2
A B
a b
A B
a b X
A B
a b
A b
A B
A B
a b OR
a B
A B
A b
a b
A B
a B
A b
A B
a b
A B
etc…
High LD -> No Recombination
(r2 = 1) SNP1 “tags” SNP2
Low LD -> Recombination
Many possibilities

12. SNP Characteristics: Linkage Disequilibrium
Three ways to calculate LD
Observed
Expected

13. SNP Characteristics: Linkage Disequilibrium
D’ = D / Dmax (Lewontin 1964)
D = 0.1, Dmax = 0.24, D’ = 0.1/0.24 = 0.427
p1 = 0.6, q1 = 0.6

14. SNP Characteristics: Linkage Disequilibrium
Statistical Significance of LD
Chi-square test with 1 df
General chi-square tests
Permutation tests B1 B2
Total A1
n11
n12
n1. A2
n21
n22
n2.
n.1
n.2 nT

15. SNP Characteristics: Linkage Disequilibrium
Can see haplotype block: a cluster of linked SNPs

16. Haplotype: an illustration
A1A1, A2B2, A3A3
A1B1, B2B2, B3B3
A1B1, B2B2, A3B3 A1 B2 A3 B1 B3 A1 B2 A3 B1 B3
A1A1, B2B2, A3B3
B1B1, B2B2, A3B3
A1B1, B2B2, A3B3 or

17. Haplotype
Definition: an ordered list of alleles of multiple linked loci on a single chromosome
A1 A2 A A4 A5 A6 A7 A8
Marker loci
chromosome
Status C C C C N N N N
Haplotypes

18. SNP Characteristics: Linkage Disequilibrium
Haplotype boundary: blocks of sequence with strong LD within blocks and no LD between blocks
Haplotype size distribution

19. SNP Characteristics: Linkage Disequilibrium
[C/T] A T X C [A/C] [T/A]
Possible haplotype: 23
In reality, a few common haplotypes explain 90% variations
Tagging SNPs:
SNPs that capture
most variations
in haplotypes
removes
redundancy
Redundant

20. SNP Characteristics: Population Stratification
Population Stratification: individuals selected from two genetically different populations in different proportions
Stratification may be environmental, cultural, or genetic
Could give spurious results in case control association studies (later this lecture)

21. SNP Discovery Methods Where are the SNPs in human genome?
Sequence many individuals, find mismatches in alignments, too costly to sequence all
Computational:
Align genome assembly to EST (mRNA) for SNPs in the coding regions
Need to differentiate between SNP and sequencing error
Resequence to verify
dbSNP: 6 M SNPs

22. SNP Discovery Methods Sequence-free SNP detection
First check whether big regions have SNPs
Basic idea: denature and re-anneal two samples, detect heterduplex
Can pool samples (e.g. 10 African with 10 Caucasians) to speed screening
Then sequence smaller regions to verify

23. SNP Genotyping
For a known locus TT C/A AG, does this individual have CC, AA or AC?
Use PCR to amply enough of the bigger region
Primer before SNP, then ddCTP and ddATP
Sequence a few bp: add A,C,G,T in turn, right nt incorporated to give light proportional # of incorporated nt
Use florescent probes (CTGAA): give out light if hybridized ’- GACTT -5’
SNP chip (simultaneously genotype thousands of SNPs) CC AA CA

24. Association Studies Association between genetic markers and phenotype
Especially, find disease genes, SNP / haplotype markers, for susceptibility prediction and diagnosis
Two strategies:
Population-based case-control association studies
Family-based association studies

25. Case-Control Association Studies
SNP/haplotype marker frequency in sample of affected cases compared to that in age /sex /population-matched sample of unaffected controls
Expected:
( ) * ( ) / ( ) = 49
( ) * (86+296) / ( ) = 321
2 = 27.5, 1df, p < 0.001

26. Pitfalls of Association Studies
Association  causal
Difficult when several genes affecting a quantitative trait
Penetrance (fraction of people with the marker who show the trait) and expressivity (severity of the effect)
Population stratification
e.g. some SNP unique to ethnic group
Need to make sure sample groups match
Hidden environmental structure
Not very reproducible

27. Family-based Association Studies (TDT)
Look at allele transmission in unrelated families and one affected child in each
Could also compare
allele frequency
between affected vs
unaffected children
in the same family
Like coin toss

28. Reproducibility of Association Studies
Most reported associations have not been consistently reproduced
Hirschhorn et al, Genetics in Medicine, 2002, review of association studies
603 associations of polymorphisms and disease
166 studied in at least three populations
Only 6 seen in > 75% studies

29. Cause for Inconsistency
What explains the lack of reproducibility?
False positives
Multiple hypothesis testing
Ethnic admixture/Stratification
False negatives
Lack of power for weak effects
Population differences
Variable LD with causal SNP
Population-specific modifiers

30. Causes for Inconsistency
A sizable fraction (but less than half) of reported associations are likely correct
Genetic effects are generally modest
Beware the winner’s curse (auction theory)
In association studies, first positive report is equivalent to the winning bid
Large study sizes are
needed to detect these
reliably

31. Should we Believe Association Study Results?
Initial skepticism is warranted
Replication, especially with low p values, is encouraging
Large sample sizes are crucial
E.g. PPARg
Pro12Ala &
Diabetes

32. Acknowledgement Tim Niu Kenneth Kidd, Judith Kidd and Glenys Thomson
Joel Hirschhorn
Greg Gibson & Spencer Muse