1. Case Study #2 Session 1, Day 3, Liu
PLOS Genetics | DOI: /journal.pgen

2. Enantiomer
Mirror image
Methadone

3. Methadone
Kreek, 1973, 1976

4. Methadone Maintenance Therapy
Long-term use of Methadone
“Plasma concentration of methadone is a primary index for quantifying and determining therapy responses to MMT”
What kind of biomarker is this?
Invasive, cost, slow, non-predictive
During treatment
Other
Markers?
Before treatment
Plasma
concentration
Clinical
response

5. Metabolism Pathways Partially known
Journal of Analytical Toxicology 2013;37:476 –485

6. Previous Candidate Gene-based Studies
Anesthesiology 2015, Vol.123,

7. Previous Candidate Gene-based Studies
Crettols et al., Clin Pharmacol Ther. 2006;80(6):668-81

8. Study Design QC QC QC QC Discovery set N=360 Validation set N=78
Phenotype
Genotype
Phenotype
Genotype QC QC QC QC
Candidate SNPs
Candidate Haplotypes
Confirmed SNPs
Confirmed Haplotypes
Evaluation

9. Why?

10. Covariates and Phenotypes
Age, gender, BMI
Race?
The plasma concentrations of the methadone R- and S-enantiomers and their metabolites

11. Phenotypic data QC
Top 5 percentile
Bottom 5 percentile

14. Single Locus Association
Multivariate linear regression
Genotype 1 2
Phenotype
-1.2
0.8
1.5
0.03
1.2
1.3 …
Age 32 45 31 39 65 72
Gender
BMI
22.1
23.2 25 33 29
22.5
Ancestry
0.5
0.7
0.98
0.93
0.45
0.39
covariates X

15. Haplotype-based Analysis
Biological relevant: a haplotype is more likely to alter the overall function than a single allele
Combining SNPs into haplotypes will reduce the data
Increase the genotypic variance to increase power
22, 23, 24
Average length of a LD block in Asian/Cau~5 SNPs

16. Single SNP Results R-Methadone: S-Methadone, R-/S-EDDP
Rs on chromosome 9intergenic region
S-Methadone, R-/S-EDDP
no single SNP was significant after a multiple-test correction of the false discovery rate.
AX (raw p = 4.83 × 10−7), SPON1, Chr 11
rs (raw p = 8.18 × 10−7), intergenic
and AX (raw p = 2.08 × 10−6), intergenic

18. Haplotype analysis Why haplotype analysis 5-SNP window
Among the 25 most significant windows in each of the analyses of different window sizes, higher than 92.5% of the significant windows were overlapped. Second, in the study population, a window size of 5 provided a distribution of window width nearest to the distribution of the block size of the LD of the Asian population in the International HapMap II Project

19. Results R-Methadone: S-Methadone: R-/S-EDDP 4 windows: all intergenic
4th window overlaps with rs
S-Methadone:
23 windows: 17 haplotypes
Chr 11: overlap with SPON1
Chr 16: GSG1L
Chr 19: CYP450: CYP2B6
R-/S-EDDP
None

20. SNP vs Phenotypic Variation
Similar to a predictive model in biomarker discovery
R-Methadone:
Rs accounted for 9.541%: tagSNP of 4 in the LD block
4 haplotypes: 11.35%
S-Methadone
17 significant haplotypes accounted for 23.96%

21. Validation and Replication
78 samples, same criteria, same array
urine morphine test
5/17 haplotypes replicated at p<0.05
Together account for 17.95% and 19.51% of S-Methadone
Same direction
CYP2B6, SPON1, and GSG1L
Rs : none

22. Mechanistic
Constitutive androstane receptor (CAR)-activated coexpression of CYP2B6, SPON1, andGSG1L

23. rs
Rs and heterochromatic histone H3 lysine 9 trimethylation (H3K9me3) in the primary T regulatory cells from peripheral blood (p-value signal score was ). 
Imputed information of 2-Mb flanking region of rs on chromosome 9
The imputed SNP rs at 82,938,858 bp (Raw p = 6.80 ×10−9) was even more significant than rs at 82,944,201 bp (Raw p = 2.24 ×10−8). All of the significant SNPs were located in intergenic regions.

24. Power and sample size
Under powered?

25. Winner’s Curse Replication issue Disease Is it really worth the value?
Phenotype Winner
Does it really confer that high risk?
Disease
Replication issue

26. Homework Projects
Nonalcoholic Fatty liver disease (NAFLD)

27. Background NAFLD is present in 30% US adults and 10-15% children
By MRI in general population 35% - far higher in select population such as type 2 diabetics
By CT and US – about 15-17%
By AST/ALT – 7%
Most common cause of elevated liver enzymes in both adults and children
Up to 5% US adults may have NASH

28. Homework Projects Two set of questions: Overall phenotypes: NAFLD
Two specific phenotypes:
NAFLD vs Normal: binary
Hepatic Fat Content (HFC): continuous
Molecular data:
Gene expression: 1000 genes
SNPs: Chr1, Chr 2, Chr 3

29. Projects Project 1: Biomarker discovery for FLD
Project 2: Biomarker discovery for HFC
Project 3: GWAS for FLD (Choose from 1 Chr data)
Project 4: GWAS for HFC (Choose from 1 Chr data)

30. Deliverables Biomarker study: GWAS study:
A smallest number of genes as a signature.
Explain your design strategy
Explain your result
Explain your future direction
GWAS study:
Top SNPs and the associated genes

31. Operation and Presentation
Group discussion
Team work
TA guided
15-20min / group
5 min discussion