Session 1, Day 3, Liu Case Study #2 PLOS Genetics DOI:10.1371/journal.pgen.1005910
Enantiomer Mirror image Methadone
Methadone Kreek, 1973, 1976
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 Other Markers? Plasma concentration During treatment Clinical response Before treatment
Metabolism Pathways Partially known Journal of Analytical Toxicology 2013;37:476 485
Previous Candidate Gene-based Studies Anesthesiology 2015, Vol.123, 1142-1153
Previous Candidate Gene-based Studies Crettols et al., Clin Pharmacol Ther. 2006;80(6):668-81
Study Design 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
Why?
Covariates and Phenotypes Age, gender, BMI Race? The plasma concentrations of the methadone R- and S-enantiomers and their metabolites
Phenotypic data QC 1 5 0 0 Top 5 percentile 1 0 0 0 5 0 0 0 Bottom 5 percentile - 5 0 0 O r ig in a l W in s o r iz e d
Single Locus Association Multivariate linear regression covariates Genotype Phenotype Age Gender BMI Ancestry 0 1 2 X -1.2 0.8 1.5 0.03 1.2 1.3 32 45 31 39 65 72 1 2 2 1 1 2 22.1 23.2 25 33 29 22.5 0.5 0.7 0.98 0.93 0.45 0.39
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 2 2, 2 3, 2 4 Average length of a LD block in Asian/Cau~5 SNPs
R-Methadone: Single SNP Results Rs17180299 on chromosome 9àintergenic region S-Methadone, R-/S-EDDP no single SNP was significant after a multiple-test correction of the false discovery rate. AX-16534452 (raw p = 4.83 10 7), SPON1, Chr 11 rs1448332 (raw p = 8.18 10 7), intergenic and AX-13599782 (raw p = 2.08 10 6), intergenic
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
Results R-Methadone: 4 windows: all intergenic 4 th window overlaps with rs17180299 S-Methadone: 23 windows: 17 haplotypes Chr 11: overlap with SPON1 Chr 16: GSG1L Chr 19: CYP450: CYP2B6 R-/S-EDDP None
SNP vs Phenotypic Variation Similar to a predictive model in biomarker discovery R-Methadone: Rs17180299 accounted for 9.541%: tagsnp of 4 in the LD block 4 haplotypes: 11.35% S-Methadone 17 significant haplotypes accounted for 23.96%
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 Rs17180299: none
Mechanistic Constitutive androstane receptor (CAR)-activated coexpression of CYP2B6, SPON1, andgsg1l
rs17180299 Rs17180299 and heterochromatic histone H3 lysine 9 trimethylation (H3K9me3) in the primary T regulatory cells from peripheral blood (p-value signal score was 2.47354). Imputed information of 2-Mb flanking region of rs17180299 on chromosome 9 The imputed SNP rs17083093 at 82,938,858 bp (Raw p = 6.80 10 9 ) was even more significant than rs17180299 at 82,944,201 bp (Raw p = 2.24 10 8 ). All of the significant SNPs were located in intergenic regions.
Power and sample size Under powered?
Winner s Curse Is it really worth the value? Phenotype Winner Disease Does it really confer that high risk? Replication issue
Homework Projects Nonalcoholic Fatty liver disease (NAFLD) Normal Steatosis (NAFL) Steatohepatitis (NASH) Fibrosis/ cirrhosis
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
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
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)
Biomarker study: Deliverables 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 Explain your design strategy Explain your result Explain your future direction
Operation and Presentation Group discussion Team work TA guided 15-20min / group 5 min discussion