Doctoral Thesis Presentation

Title: Roles of CYP2A6 Gene Polymorphism in Treatment of Nicotine

Dependence.

Yawo Mawuli Akrodou

October 2014

Walden University

College of Health Sciences

Presentation Overview

• Problem statement.

• Study background.

• Data collection, processing and analyses results.

• Research questions and hypotheses.

• Presentation of findings and their implications.

• Study limitations, recommendations, and social implications.

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Background: Problem Statement

• General Problem: Lack of systematic nicotine dependence treatment. Classic nicotine therapies provide only temporary abstinence. More than 80% nicotine dependent treated relapse within a year (Foulds, 2006, p. 1).

• Specific Problem: Difficulty to translate CYP2A6 gene variants nicotine metabolizers capability of metabolism into nicotine dependence treatment. Inconsistent information, interindividual variability, different genes

frequencies, and study design issues (Kortmann et al., 2010).

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Background: Tobacco Harm and Related Diseases

• Association of tobacco’s chemicals with pulmonary cancers. 1950s Doll and Bradford study in United Kingdom (Medical Research Council [MRC], 2013).1950s Hammond and Horn study in United States (Schneider, 2006).

• Current high prevalence of nicotine and nicotine related diseases.The prevalence of nicotine is stalled at 20% 400 thousand deaths from nicotine-related diseases in US (CDC, 2011).6 million people deaths from tobacco smoking (WHO, 2012). More than 26% of heart attacks, 12% to 19% of strokes attributable to smoking.

http://galleryhip.com/nicotine-gum-cancer.html

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Difference X-ray of smokers organs and Nonsmokers ones.

• http://addictionblog.org/

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Background: Nicotine Metabolism Pathways

• Nicotine: Powerful psychoactive chemicals or mood-altering drug.• Approximately 1.0-1.5mg of nicotine is absorbed in the lung with pKa = 0.8.• Transits by the blood-stream to the brain in 10 seconds. (Remington et al.,

2010).• Biologically, 70%-80% of nicotine is catalyzed by the enzyme cytochrome

P450 2A6 (CYP2A6).• Cotinine is completely transformed by CYP2A6 into trans-3-hydroxycotinine

(Hukkanen et al, 2005).

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Background: Nicotine Dependence

• Nicotine Dependence: State of switching from regular smoking to permanent nicotine user.

NB: Nicotine dependence is assessed using nicotine withdrawal syndrome scale nicotine dependence Syndrome scale.

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Background: Nicotine Dependence Assessment Instruments

• Fagerström Test of Nicotine Dependence (FTND; Piper et al., 2008).

• Diagnostic and Statistical Manual [DSM-IV]) criteria (Mezzich, 2002).

• Nicotine Dependence Syndrome Scale (NDSS; Shiffman, Waters, & Hickcox, 2004).

• Wisconsin Inventory of Smoking Dependence Motives (WISDM; Piper et al., 2004).

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Background: Nicotine Dependence Treatment

Nicotine Dependence Pharmacotherapies

• Nicotine replacement therapy (NRT).Patch, spray, gum, chewing gum, transdermal patches, nasal sprays, vapor

inhalers, and sublingual tablets and lozenges.• Pharmacology (FDA approved drugs)

Bupropion (Zyban®), and Varenicline (Champix ®)

Cognitive behavioral therapy• Advising and motivational techniques.

Hukkanen et al. (2005).

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Background: Treatment Limitation Facts

• More than 80% of nicotine dependents treated relapse within 6 months or 1 year

• Stall of the decrease of the prevalence of nicotine dependence at 20% in USA.

• 400 thousand nicotine dependence-related deaths a year.• More than $176 billion yearly nicotine-related disease treatments’

expenses labor and work loss hours.

Center of Diseases Control and Protection (2011).

10 Oral Defense

Background: Lack of Personalized Treatment

• Lack of effective and personalized treatment. • One size fits all paradigm.• Difficulty using genes information in treatment.

Example of Nicotine Genes. DRD4: Reinforce nicotine dependenceMA-O: Predispose to nicotine dependenceCYP2A6: Dependence and Cessation CYP2B6: Cessation

Ho et al.(2010).

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Example of Nicotine Genes

Table Example of Nicotine Genes

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Background : Cytochrome P450 CYP 2A6 (CYP2A6)• CYP2A6 Polymorphisms

Influences on nicotine metabolism Decrease nicotine metabolism. CY2A6*4A : Deleted , no function

decrease. CYP2A*1H, *9A, &*12A: At least one loss-of-function allele.

Normal Metabolizers CYP2A6*1A full function alleles. Increase nicotine metabolism.

CPY2B6*6 : Nicotine dependence cessation.

Mroziewicz & Tyndale (2010).

Figure. CYP2A6 Molecular Location on chromosome 19: base pairs 40,843,537 to 40,850,446

http://ghr.nlm.nih.gov/gene/CYP2A6

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Example of CYP2A6 Variants

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Table Example of CYP2A6 Variants

Cytochrome P450 CYP 2B6 (CYP2B6)

• Cytochrome P450 CYP2B6 (CYP2B6) CYP2B6 is known as a nicotine gene candidate that interacts with CYP2A6 and

is responsible for bupropion metabolism and influence nicotine cessation. (National Center of Biotechnology Information [NCBI], 2012).

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Background: Study Purpose Statement

Quantitative Cross-sectional study design to evaluate variables correlation.

Assess CYP2A6 variants impact on nicotine treatment. Assess the interaction of CYP2A6 gene variants,

and CYP2B6*6 variant impact in nicotine treatment.

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Study Research Questions 1 and 2

•Research Question 1:How do the associations between CYP2A6 slow nicotine metabolizer gene variants, nicotine dependence syndrome or nicotine withdrawal syndrome, and therapy type (bupropion or NRT) affect nicotine dependence treatment outcome?

•Research Question 2: How do the associations between CYP2A6 normal nicotine metabolizer gene variants, nicotine dependence syndrome or nicotine withdrawal syndrome, and therapy type (bupropion or NRT) affect nicotine dependence treatment outcome?

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Study Research Questions 2 and 3

• Research Question 3: How does an interaction between CYP2A6 slow nicotine metabolizer gene variants, gene variant CYP2B6*6, and therapy type affect nicotine dependence treatment outcome?

• Research Question 4: How does an interaction between CYP2A6 normal nicotine metabolizer gene variants, gene variant CYP2B6*6, and therapy type (bupropion or NRT) affect nicotine dependence treatment outcome?

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Study Hypotheses 1 and 2

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Hypothesis 1 Ho: The associations between CYP2A6 slow nicotine metabolizer gene variants, nicotine dependence syndrome or nicotine withdrawal syndrome, and therapy type (bupropion, or NRT) do not have a significant impact on nicotine dependence treatment outcome.

Ha: The associations between CYP2A6 slow nicotine metabolizer gene variants, nicotine dependence nicotine or nicotine withdrawal syndrome, and therapy type (bupropion or NRT) have a significant impact on nicotine dependence treatment outcome.

Hypothesis 2 Ho: The associations between CYP2A6 normal nicotine metabolizer gene variants, nicotine

dependence syndrome, or nicotine withdrawal syndrome and therapy type do not have a significant impact on nicotine dependence treatment outcome.

Ha: The associations between CYP2A6 normal nicotine metabolizer gene variants, nicotine dependence syndrome or nicotine withdrawal syndrome and therapy type (bupropion or NRT) have a significant impact on nicotine dependence treatment outcome.

Hypotheses 3 and 4

Hypothesis 3 Ho: CYP2A6 slow metabolizer gene variants of nicotine interaction with gene variant CYP2B6*6 and therapy type do not have a significant impact on nicotine dependence treatment outcome. Ha: CYP2A6 slow metabolizer of nicotine gene variant interaction with gene variant CYP2B6*6 and therapy type have a significant impact on nicotine dependence treatment outcome.

Hypothesis 4 Ho: CYP2A6 normal metabolizer of nicotine gene variants’ interaction with gene variant CYP2B6*6 and therapy type do not have a significant impact on nicotine dependence treatment outcome. Ha: CYP2A6 normal metabolizer of nicotine gene variants’ interaction with gene variant CYP2B6*6 and therapy type have a significant impact on nicotine dependence treatment outcome.

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Theoretical Foundation

Theoretical Foundation: Behavioral genetic theory developed in 1869 by Galton in United Kingdom. Theory Principle

• Causal relationships between genes and behavior.

• Gene-genes, and gene-environmental interactions influences.

• Genes use in diseases treatments.

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Factors Influencing Nicotine Behavior and Metabolism Rate

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Note. Environmental and biological factors that influence nicotine behavior and modulate nicotine metabolism.

Figure 1. Factors influencing nicotine behavior and metabolism rate

Theoretical Framework

Theoretical Framework: Personalized treatment concept.• Genetic makeup of individuals contributes to the liability to nicotine, or

drug addictions (Hall et al. 2002).

• Two individuals are 99.9% identical.

• Zero one point % difference in DNA constitutes the origin of profound variation,

diverse behavior, and visible traits.• Treatment tailoring to patient genotype.

• Medication selection to maximize successful treatment odds.

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Schema illustrating personalized medicine concept and its difficulties

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Note. Dash lines indicate difficulties achieving personalized medicine. Figure 2. Schema illustrating personalized medicine concept and its difficulties

Study Method/Design

• The study used cross-sectional quantitative method research study.

• Allow to exanimate the correlation of independent and dependent variables in a

clinical setting (Checkwoay, Pearce, & Kriebel 2004)

• Increase the study power to detect association and to find interactions between

gene variants (Cordell, 2009).

• Aim to characterize and substantiate gene-gene and gene-environmental

interactions (Cordell, 2009).

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Study Participants and Sample size

Population: Participants were drawn from nicotine-dependent individuals seeking cessation treatment in Madison and Milwaukee, WI, USA. More than 9,000 adult smokers between the ages of 18 and 80 were sampled (NCBI, 2011) as participants for randomized clinical trials organized by UW-TTURC from 2001–2009. Approximately 2,575 individuals were enrolled in randomized clinical trials involving different medication treatments and have provided blood samples.

The sample size consists of to 1,862 participants. This number is based on calculation using Quanto Version 1.2 genetic software for genes-genes and genes-environmental effect determination in clinical treatment.

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Study Variables

Dependent variable: Quitting Status (QS)

Independent variables SM: Slow major nicotine metabolizer genes NM: Normal major nicotine metabolizers genes WS: Withdrawal syndrome Score low score 0-4 and high score is 5-10 on scales

ND: Nicotine dependence score the same

Th: Treatment type (NRT, Bupropion, and Placebo)

• Covariate: Age, Education Attainment, Ethnicity/Race and Gender

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Data Sources

• Clinical Studies of Wisconsin Transdisciplinary Tobacco Use Research Center (UW-TTURC) from 2001–2009 in Madison and Milwaukee, WI, USA (NCBI, 2011).

Approval and Permission

• Walden University Internal Review Board approval. • National Health Institute permission.

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Data Sources: Original Clinical Trial Studies Description

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Table 7  Original Clinical Trial Studies Description

Data Collection Methods

• Survey questionnaires • Alveolar carbon monoxide (CO) level testing to confirm level of CO in the

blood.• Genotyping using 96 well plates using the Illumine HumanOmni2.5-4v1 D

array. • Gene sampling and clustering using GenomeStudio version 2010.2 Genotyping

Module version 1.7.4 and GenTrain version 1.0. Genotyping call rates for all SNPs were >> 98.71% Mean = 0.99, SD = 0.0071, max = 1.00, min = 0.85.

National Center of Bioinformation (2011).

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Data Analysis Methods

• Descriptive statistic was used to process and to determine: sample composition, Percentage of dependence and withdrawal syndrome scores.

• Beagle and Plink software were used to predict participants genotype.

• Chi-square (χ2) test was used to verify genotype normal distribution in the population.

• Logistic regression model was used to test hypotheses.

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Data Processing Results

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Original sample N = 1, 972 Missing dependence score = 6 Missing Withdrawal score = 28 Mismatched and genotypes = 51 Unknown = 55 Other races = 4

FTND: Dependence scores Mean = 4.92, SD = 1.31WS: Withdrawal score Mean = 5.41, SD = 2.15

Final Sample N = 1862

Data Processing Results

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Table Data Processing Results

Data Analysis Results: Sample Demographics

The study Sample is composed by the most

1099(59.0%) participants of 31-50 of age.

262 (14.1%) African Americans.

1600 (85.59%) Caucasians.

802 (43.1%) Graduates.

772 (41.4%) Females.

1090 (58.6%) Males.

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Study Sample Demographics

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Table. Sample Demographics

Data Analysis Results : FTND and WS Scores

FTND: Nicotine Dependence

• Thirty-two point one percent (32.1%) of

participants reported a low level of nicotine

dependence, and 67.9% reported a high level

of nicotine dependence.

WS: Withdrawal Syndrome

• Forty point eight percent (40.8%) of all

participants reported experiencing nicotine

withdrawal syndrome during treatment,

and 59.2% reported craving.

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Low and High Dependence Subgroups

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Data Analysis Results : Genotype Distribution Frequency, Treatment Group, Quiting Status.

Nicotine Genes Variant Carriers • Normal Metabolizer (NM):

CYP2A6*1A: 69.1% • Slow Nicotine Metabolizers

Carrier CYP2A6*4A: 56.5% CYP2A6*1H: 46.9% CYP2A*9: 38.2% CYP2A6*12A: 58.6% CYP2B6*6: 48.1%

Treatment Group

• Receive Nicotine replacement therapies (NRT): 45.1%

Received bupropion: 35% Received placebo: 10.5% placebo. Quitting Status within 6 months

Reported quitting: 29.4% Did not quit : 70.6%

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Genotype distribution frequency in the whole population

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Figure 4. Genotype distribution frequency in the whole population

Hypothesis 1: Test Result for Nicotine Syndrome Association Slow Metabolizers

•Research Question 1: How do the associations between CYP2A6 slow nicotine metabolizer gene variants, with nicotine withdrawal syndrome or nicotine dependence syndrome, and therapy type (bupropion or NRT) affect nicotine dependence treatment outcome?

Result: Significant impact with CYP2A6*1H (OR = 1.55, 95% CI [1.12-1.90] ; p < 0.001). CYP2A6*4A (OR = 1.60, 95% CI [ 1.13-1.95] ; p < 0.001). CYP2A6*9A (OR = 1.35, 95% CI [1.10-1.65] ; p < 0.001). CYP2A6*12A ( OR = 1.46, 95% CI[1.18-1.80] ; p < 0.001).

Conclusion: Participants carrying one of these slow nicotine metabolizer gene variants were 1.35, 1.46, 1.55, or 1.60 times more likely to maintain abstinence 6 month posttreatment.

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Hypothesis 1: Test Result for Withdrawal Syndrome Association with Slow Metabolizer

Result : Significant impact with:

CYP2A6*1H (OR = 1.54, 95% CI [1.26-1.89] ; p < 0.001).

CYP2A6*4A, (OR = 1.59, CI [1.30-1.95] ; p < 0.001).

CYP2A6*9A (OR = 1.45, CI[1.18-1.80]; p < 0.001).

CYP2A6*12A( OR = 1.46, CI[0.10-1.65] ; p = 0.001). Conclusion: Participants carrying one of these slow nicotine metabolizer gene

variants were 1.35, 1.45, 1.54, or 1.59 more likely to maintain abstinence 6 month posttreatment.

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Hypothesis 2: Test Result for Nicotine and withdrawal Syndromes Association with Normal Metabolizers

• Research Question 2: How do the associations between CYP2A6 normal nicotine metabolizer gene variants, nicotine dependence syndrome, or withdrawal dependence syndrome, and therapy type (bupropion or NRT) affect nicotine dependence treatment outcome?

Result: Significant impact on nicotine dependence treatment outcome with CYP2A6*1A (OR = 1.35, 95% CI [1.11-1.70]; p < 0.003) for nicotine dependence

syndrome. CYP2A6*1A (OR = 1.40, 95% CI [1.11-1.75]; p < 0.004). nicotine withdrawal

syndrome. Conclusion: Participants carrying CYP2A6*1A were 1.35 times more likely to maintain

abstinence 6 month posttreatment period, and were 1.40 times more likely to maintain abstinence 6 month posttreatment period for those who had experienced nicotine withdrawal syndrome during treatment.

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Hypothesis 3: Test Result for Nicotine Syndrome Association with Slow Metabolizer • Research Question 3: How does an interaction between CYP2A6 slow nicotine

metabolizer gene variants, gene variant CYP2B6*6, and therapy type affect nicotine dependence treatment outcome?

Result : Significant impact on treatment outcomes with:

CYP2A6*1H (OR = 1.56, 95% CI: [1.26-1.90] ; p < 0.001).

CYP2A6*4A (OR = 1.61, 95% CI [ 1.31-1.96] ; p < 0.001).

CYP2A6*9A (OR = 1.47, CI [1.18-1.88] ; p < 0.001).

CYP2A6*12A (OR = 1.35, CI[ 1.11-6.7] ; p=0.004). Conclusion: Participant having one of these specific gene variants were 1.35, 1.47, 1.56 or

1.61 times more likely to maintain abstinence for s 6 month posttreatment period.

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Hypothesis 3: Test Result for Withdrawal Syndrome Association with Slow Metabolizer

Result : Significant impact for each slow nicotine metabolizer gene variant:

CYP2A6*1H (OR = 1.57, 95% CI:[1.13-1.89] ; p < 0.001).

CYP2A6*4A (OR = 1.70, 95% CI [01.15-1.95] ; p < 0.001).

CYP2A6*9A (OR = 1.44, 95% CI [1.17-1.95] ; p < 0.001).

CYP2A6*12A (OR = 1.32, 95% CI[1.10-1.64] ; p < 0.003).

Conclusion: Participant having one of these specific slow nicotine metabolizer gene

variants and gene variant CYP2B6*6 were 1.32, 1.44, 1.57, or 1.70 times more likely to

maintain abstinence for six month posttreatment period.

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Hypothesis 4: Test Result for Nicotine or Withdrawal Syndromes Association with NM

• Research Question 4: How does an interaction between CYP2A6 normal nicotine metabolizer gene variants, gene variant CYP2B6*6, and therapy type (bupropion or NRT) affect nicotine dependence treatment outcome?

Result 1: Significant impact on nicotine treatment outcome with: CYP2A6*1A (OR = 1.37, 95% CI [1.10-1.69]; p < 0.003) for nicotine dependence syndrome. Conclusion: Normal nicotine metabolizer CYP2A6*1A gene variant and CYP2B6*6 gene variant carriers were 1.37 times more likely to maintain abstinence for 6 month posttreatment period.

Result 2: Significant impact on nicotine treatment outcome with: CYP2A6*1A (OR = 1.39, 95% CI [1.07-1.72] ; p < 0.004) nicotine withdrawal syndrome. Conclusion: Patients carrying normal nicotine metabolizer CYP2A6*1A gene variant and CYP2B6*6 gene variant were 1.39 times more likely to maintain abstinence for 6 month posttreatment period.

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ANOVA (3x2) Genes Interaction Main Effect Testing for Hypotheses 3 and 4

• For hypothesis 3 slow nicotine metabolizers CYP2A6*4A and CYP2A6*12A interactions’ main effects with CYP2B6*6 were significant CYP2A6*4A, F (1, 50.2) = 134.49, partial η² = 0.068, p < 0.001, and CYP2A6*12, F (1, 34.9) = 132.38, partial η² = 0.059, p < 0.001. CYP2A6*4A and CYP2A6*12 tended to have a greater main effect with CYP2A6*6

in nicotine dependence treatment.

• For Hypothesis 4, ANOVA result indicated a significant interaction main effect between normal nicotine metabolizer CYP26*1A and CYP2B6*6 gene variants, F (1, 38.2) = 127.72 partial η² = 0.065, p < 0.001 in nicotine dependence treatment. Both gene variants tended to generate a greater main effect on nicotine dependence

treatment.

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Findings and Previous Studies

• How do findings related to the literature ? Nakajima et al. (2006) and Ho et al. (2010) case-control studies finding. Slow nicotine metabolizers carriers were 1.7 times more likely to quit smoking than noncarriers.

• Epistasis consists of the interaction of many genes to express a specific phenotype or to play a specific function” (Russell, 2002).

• Ring and Valdez (2007) finding.CYP2B6 interacted with CYP2A6 (p < 0.002 and 0.003) to clear nicotine from the organism.

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Findings Related to Theoretical and Conceptual Framework.

• How do findings relate to conceptual/theoretical framework?

Gene-genes interaction impacts on nicotine treatment outcome.

CYP2A6 and CYP2B6*6 interact to increase successful nicotine treatment.

outcome as suggested by Ring and Valdez (2007) .

Gene-gene environmental interaction .

Education level, nicotine exposure to nicotine association impact nicotine

treatments.

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Example of Testing Among Different Subgroups

• Example of Logistic regression analysis of the association of slow nicotine metabolizer gene variant CYP2A6*4A with nicotine dependence syndrome

High odd ratios-61 to 80 years old (OR= 1.83, 95% CI[1.12-2.78], p = 0.002).

Lower odd ratios 31-50 years old (OR = 1.57, 95% CI [1.20-2.02] ; p < 0.001).

Lower female subjects ( OR = 1.27, 95% CI[ 0.93-1.75], p < 0.001) than for male subjects ( OR = 1.86, 95% CI[ 1.40-2.42] ; p < 0.001).

Higher odds ratio for college graduates (OR = 1.30, 95% CI [0.10-1.63] ; p = 0.001) than for other

educational background groups.

Higher odds ratio for Caucasian descendants (OR = 1.63, 95% CI[ 0.93-2.0] ; p < 0.085) than for African American descendants (OR = 1.59, 95% CI [1.28-1.98] ; p = 0.001) .

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Limitations

• Correlational designed limitation.

• Use of secondary datasets.

• Environmental factor influence on genes.

• Extreme variability of CYP2A6 and CYP2B6 gene variants and variability of

genes frequencies (Khoury et al., 2010).

• Limited subsets of ethnicities used (Caucasian and African American

descendants).

• Study design issues.

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Recommendations for action

• Replication of the current study in other populations with more diverse ethnicities.

• Bigger sample sizes use.• Use of wide range of sets of alleles.• Pharmacogenomics: Assess the strength of medication used.• Use of cognitive variables.• Incorporate gene therapies in treatments.

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Social Change Implications

• Improve genetic profiling, gene screening, and gene testing results

interpretations.

• Better assessment of nicotine gene risk factors.

• Advance individualized disease treatment.

• Motivate population and parents gene screening.

• Strengthen cognitive behavioral therapy.

• Promote healthcare policy making.

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Abstract – see notes section on components for abstract• Statement: Existing nicotine dependence therapies have decreased smoking prevalence in the United States, but

the decline in the number of adult smokers is stalling, due, in part, to the limited efficacy of current therapies that lack treatment personalization. Cytochrome P450 2A6 (CYP2A6) gene variants are known to metabolize nicotine and possibly influence nicotine dependence treatment. These genes’ inconsistent information, interindividual variability, interactions with other genes, and environmental factors have made it difficult to use their information to improve nicotine dependence therapy.

• Method and Design: This cross-sectional study based on behavioral genetic theory stating that environmental and genetic factors cause behavioral disorders, assessed the impact of slow nicotine metabolizers (CYP2A6*1H, CYP2A6*4A, CYP2A6*9, and CYP2A6*12A) and normal (fast) nicotine metabolizers (CYP2A6*1A) gene variants and their interactions with CYP2B*6 associated with nicotine therapy type and nicotine dependence and withdrawal syndromes on nicotine dependence outcome.

• Results: CYP2A6*4A (OR = 1.60, CI [1.13-1.95]; p < 0.001) and CYP2A6*9A (OR = 1.47, CI[1.18-1.88] ; p < 0.001) were the most linked to the highest odds of successful treatment outcome, indicating that carriers of slow nicotine metabolizers were more likely to maintain abstinence 6 months post period treatment than normal (fast) metabolizer CYP2A6*1A (OR = 1.35, 95% CI[ 1.11-1.70] ; p < 0.003) carriers.

• Social Implications: Study findings may be useful in gene counseling and nicotine gene therapy to tailor individualized nicotine clinical treatments, to increase smoking quit rates, and to induce positive social change by improving the lives of smokers and their families.

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Thank you and questions

• Chair: Dr. Peter Anderson • 2nd committee member: Dr. Sandra• URR: Dr. Roland Thorpe

Questions?

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