Polysubstance Abuse–Vulnerability Genes: Genome Scans for Association,

Using 1,004 Subjects and 1,494 Single-Nucleotide Polymorphisms

Uhl, Liu, Walther, Hess, NaimanUhl, Liu, Walther, Hess, Naiman

Presented by Brian O’LearyPresented by Brian O’Leary

Why This Article?Why This Article?

Talkin’ about substance abuseTalkin’ about substance abuse The goal is similar to TrAPSSThe goal is similar to TrAPSS UI is interested in using this UI is interested in using this

methodmethod Cheep: 11,500 SNP Chip for Cheep: 11,500 SNP Chip for

$500$500 Possible computing projects for Possible computing projects for

sophomores who need sophomores who need something better to do than something better to do than drink and play UT.drink and play UT.

Key DefinitionsKey Definitions

SNPSNP MicroarrayMicroarray Association genome scanningAssociation genome scanning Pooled analysisPooled analysis

SNPSNP

Stands for: Stands for: Single Nucleotide PolymorphismSingle Nucleotide Polymorphism

Which means:Which means:Single Nucleotide PolymorphismSingle Nucleotide Polymorphism

For example: For example: AAAAGGCTAA GGCTAA A ATTGGCTAA.GGCTAA.

““For a variation to be considered a SNP, it For a variation to be considered a SNP, it must occur in at least 1% of the population. must occur in at least 1% of the population. SNPs, which make up about 90% of all SNPs, which make up about 90% of all human genetic variation, occur every 100 human genetic variation, occur every 100 to 300 bases along the 3-billion-base to 300 bases along the 3-billion-base human genome. “human genome. “http://www.ornl.gov/TechResources/Human_Genome/faq/snps.htmlhttp://www.ornl.gov/TechResources/Human_Genome/faq/snps.html

Microarray TechnologyMicroarray Technology

A technique that utilizes A technique that utilizes hybridization to evaluate hybridization to evaluate expression levels of thousands expression levels of thousands of genes in a single experiment of genes in a single experiment

A microarray consists of a chip A microarray consists of a chip with oligonucleotides designed with oligonucleotides designed to hybridize to different parts of to hybridize to different parts of the genomethe genome

Association Genome Association Genome ScanningScanning Technique that uses co-segregation Technique that uses co-segregation

to determine a marker/locus to determine a marker/locus associated with a specific phenotypeassociated with a specific phenotype

Makes use of higher genomic marker Makes use of higher genomic marker densitiesdensities

Does not require the participation of Does not require the participation of family members which increases the family members which increases the size of the samplesize of the sample

Downside:Downside: Founder effect – multiple founders of Founder effect – multiple founders of

same phenotypesame phenotype

Pooled AnalysisPooled Analysis

Performing an analysis on one Performing an analysis on one set of people vs. another set (as set of people vs. another set (as opposed to one individual vs. opposed to one individual vs. another)another)

This analysis technique loses This analysis technique loses individuality but gains a large individuality but gains a large amount of data at reduced cost. amount of data at reduced cost.

Snapshot of ArticleSnapshot of Article

Few chromosomal locations Few chromosomal locations confirmed for drug-abuse confirmed for drug-abuse vulnerability allelesvulnerability alleles

Used pooled-sample SNP Used pooled-sample SNP microarray analysis to define microarray analysis to define markers and identify markers and identify chromosomal regions containing chromosomal regions containing allelic variants related to drug-allelic variants related to drug-abuse vulnerabilityabuse vulnerability

MotivationMotivation

Improve Improve understanding of understanding of human addictionshuman addictions

Assist efforts to Assist efforts to prevent and/or treat prevent and/or treat individuals with individuals with vulnerability to vulnerability to addictionsaddictions

Methods – whoMethods – who

Research volunteers provided Research volunteers provided ethnicity and drug historyethnicity and drug history

European American pools:European American pools: 239 control individuals239 control individuals 415 abusers415 abusers

African American pools:African American pools: 99 control individuals99 control individuals 252 abusers252 abusers

Methods – data techniqueMethods – data technique

Washed pooled DNA over an Washed pooled DNA over an Affymetrix HuSNP GeneChip Affymetrix HuSNP GeneChip microarraymicroarray Assessed total of 1,494 SNP Assessed total of 1,494 SNP

markersmarkers Genotype calls were made Genotype calls were made

using Affymetrix GeneChip using Affymetrix GeneChip softwaresoftware

A/B SNP ratios were computedA/B SNP ratios were computed Experiment replicated twiceExperiment replicated twice

Methods – analysisMethods – analysis

Top and bottom 5% of the Top and bottom 5% of the distributions for European distributions for European American abuser/control American abuser/control hybridization ratios considered hybridization ratios considered “candidate positive” SNPs“candidate positive” SNPs

Reproducibility sought by finding Reproducibility sought by finding SNPs in top and bottom 7.5% of SNPs in top and bottom 7.5% of the African American the African American abuser/control hybridization abuser/control hybridization ratiosratios

Methods – analysisMethods – analysis

Mapviewer used to determine Mapviewer used to determine SNPs close to markers linked to SNPs close to markers linked to alcohol or nicotine dependencealcohol or nicotine dependence

Pooled results compared to Pooled results compared to individual DNA samplesindividual DNA samples

Monte Carlo simulations used to Monte Carlo simulations used to compare results with results compare results with results expected by chanceexpected by chance

ResultsResults

““Pooled SNP genotyping was performed with acceptable reproducibility and evidence for validity.”

Of 150 candidate positive SNPs in European Americans, 42 were also in the candidate list for African Americans.

By chance, 22 markers would have appeared in both sets

ResultsResults

Three pairs of markers close to each Three pairs of markers close to each other in genomeother in genome

Monte Carlo simulation indicated a Monte Carlo simulation indicated a 0.042 probability that this would 0.042 probability that this would occur by chanceoccur by chance

We Know What We’re Doing We Know What We’re Doing So There.So There. ““Features of the SNP-analysis

method used here also indicate sufficient reliability and validity to test our working hypotheses.”

1. Test-retest, sense/antisense, and pooled and individual genotypes correlations

2. Reproducible positive results

3. Location of markers not likely due to chance

That’s ItThat’s It