Genetics and Pharmacogenetics in Addictions Wade Berrettini, MD, PhD Karl E. Rickels Professor of Psychiatry Director, Center for Neurobiology and Behavior, Department of Psychiatry, School of Medicine University of Pennsylvania (wadeb@mail.med.upenn.edu)
Definitions: Addiction & Dependence Dependence: is defined by withdrawal and tolerance Tolerance: higher dose needed to achieve same effect; 2 types: Pharmacokinetic tolerance: Change in drug metabolism Pharmacodynamic tolerance: Change in cellular response to the drug. Withdrawal: characteristic syndrome which appears shortly after abrupt cessation of daily drug intake. Addiction: chronic, compulsive, maladaptive pattern of drug use, despite adverse consequences due to drug use: loss of job, family, development of medical problems, legal difficulties, etc. Addiction implies dependence.
Two Topics to be Covered Mu opioid receptor variant predicts therapeutic response to naltrexone in alcohol addiction (or what I learned at my visit to the FDA) Alpha3/5 nicotinic receptor subunit genes in nicotine addiction
Naltexone Treatment of Alcohol Addiction Naltrexone (NTX), a mu opioid receptor antagonist, is efficacious (at 50-100 mg daily) in the treatment of ethanol addiction. It reduces risk for relapse to heavy drinking (> 4 drinks daily for a woman, > 5 drinks daily for a man), but does not influence abstinence. It reduces the euphoria from ethanol, and thereby reduces the drive to drink excessively. Because alcohol addiction is so common in our country, widespread use of naltrexone could improve public health considerably.
Randomized Placebo Controlled NTX Trials Studies supporting efficacy Studies not supporting efficacy Study # Ss Notes Study # Ss Notes Volpicelli et al 1992 70 None Oslin et al 1997 44 Older O Malley et al 1992 97 None Kranzler et al 2000 183 None Volpicelli et al 1997 97 None Krystal et al 2001 627 VA only Kranzler et al 1998 20 Depot Lee et al 2001 (Singapore) 53 None Anton et al 1999 131 None Gastpar et al 2002 (Germ.) 171 None Chick et al 2000 (UK) 169 Adherence Kranzler et al 2004 315 Depot Monterosso et al 2001 183 None Killeen et al 2004 145 None Morris et al 2001 (Australia) 111 None Oslin et al in press 240 None Heinala et al 2001 (Finland) 121 Nonabst. Latt et al 2002 (Australia) 107 None Ahmadi and Ahmadi 2002 (Iran) 116 None Guardia et al 2002 (Spain) 202 None Balldin 2003 118 None Kiefer et al 2003 (Germany) 160 None Kranzler et al 2003 153 None Kranzler et al 2004 315 For drinking not relapse Anton et al 2004 270 None
Mu Opioid Receptor Amino Acid Sequence Extracellular Space N40D (A118G) SNP; N is a glycosylation site cytoplasm
Dopamine neurons, with cell bodies in the VTA project to the n. Acc. & the medial prefrontal cortex. (n. Acc.) (ventral Activation of these neurons is tegmental area) a key brain signal of reward. A MAJOR CNS REWARD SYSTEM
Self (1997) Neurobiological Adaptations to Drug Use, Hospital Practice.
OPRM1 A118G EFFECT ON TRANSCRIPTION Zhang et al, JBC, 2005
OPRM1 A118G EFFECT ON TRANSLATION Zhang et al, JBC, 2005
G Allele Effects Miotic Response to a mu Opioid Receptor Agonist in vivo 45 40 35 30 25 20 15 10 5 0 (n) 45 +/- 8 miosis 33 +/- 6* 24 +/- 7* AA AG GG (40) OPRM1 GENOTYPE (8) (3) p<0.001 AA vs AG/GG Lotsch et al, 2006 Are G allele carriers hyporesponsive to mu receptor agonists?
G Allele Carriers are Hyporesponsive to mu Opioid Receptor Agonists Romberg et al. Polymorphism of mu-opioid receptor gene (OPRM1:c.118AG) does not protect against opioid-induced respiratory depression despite reduced analgesic response. Anesthesiology 2005;102:522-30. Klepstad et al. The 118 A G polymorphism in the human mu-opioid receptor gene may increase morphine requirements in patients with pain caused by malignant disease. Acta Anaesthesiol Scand 2004;48:1232-9
G Allele Carriers Hyper-responsive to Antagonists Wand et al, Neuropsychopharm 26:106 114, 2002
Alcohol effects by genotype Self-reported Stimulation (SHAS) 50 45 40 35 30 25 20 15 10 5 0 AA allele AG allele 0.02 0.04 0.06 Breath Alcohol Concentration Ray and Hutchinson, 2004
NTX Blunts Alcohol-Induced High in OPRM1 118G Carriers P<0.01, genotype x medication x BAC But not in OPRM1 AA Homozygotes (Ray & Hutchinson, 2004)
Ethnicity & A118G Allele Frequency Based on multiple studies, allele frequencies differ markedly across ethnicities for the A118G SNP in the mu opioid receptor gene. It arose after the out-of of-africa migration. Crowley et al, 2003 Gelernter et al, 1999 Tan et al, 2003 Bart et al, 2004 ETHNICITY f(g) ETHNICITY f(g) African 1% Koreans 31% African- American 3% Chinese 35% Swedish 17% Malaysian 45% European- origin US 15% Indian 47%
Human migration Adapted from Cavalli-Sforza et al, 2003
SUMMARY The A118G mis-sense sense SNP changes asparagine to aspartate, with resultant loss of an N-glycosylation N site, and with functional consequences. The G allele results in a marked decrease in OPRM1 transcription and translation ex vivo and in vitro. G allele carriers are hypo-responsive to mu opioid receptor agonists and hyper-responsive responsive to antagonists in vivo. The A118G SNP shows marked ethnicity-related variations in allele frequency.
Does A118G genotype influence treatment response to naltrexone among alcohol addicted patients?
Naltrexone (NTX) in Clinical Trials for Alcoholism Patients were selected from 3 NTX multiple double-blind, placebocontrolled trials. These trials were 12 weeks, and include selfreport measures of drinking along with biochemical verification (liver enzymes and carbohydrate deficient transferrin). Primary endpoint was relapse to heavy drinking. Medication was given in blister packs and compliance was determined by pill counts. Participants had twice weekly abstinence counseling sessions. Only those persons completing the trial, with outcome data, were contacted later to provide a blood sample for DNA studies.
OPRM1 VARIANTS IN NALTREXONE Rx FOR ALCOHOL DEPENDENCE Cumulative Survival (time to relapse) *p = 0.04 vs NTX Asn40 odds ratio = 3.4 Naltrexone / Asp40 Allele (A/G, G/G), N = 23* Naltrexone Asn40 Allele (A/A), N = 48 Placebo / Asp40 Allele (A/G, G/G), N = 41 Placebo / Asn40 Allele (A/A), N = 18 Days Oslin et al 2003
Fig 6: 118G Allele Predicts Good NTX Response % G O O D O U T C O M E P = 0.005, genotype x medication interaction odds ratio = 5.8, A/A vs. G/* PLA A/A NTX PLA NTX G/A, G/G The COMBINE study was a multi-site trial of NTX, acamprosate or both drugs, +/- addiction counseling, for alcohol addiction. The NTX & placebo groups were compared by A118G genotype for outcome after 14 weeks of treatment.
Questions for the FDA What types of trials (retrospective and prospective, double-blind, placebo-controlled, etc.) would be required for the FDA to modify the current naltrexone indication to a genotype-specific indication for alcoholism? ANS: 2 prospective placebo-controlled trials in which persons with the response allele, treated with naltrexone, have outcomes statistically superior to persons without the response allele, treated with naltrexone. Further, in both trials, placebo-treated groups do not differentiate from the naltrexone-treated group without the response allele. After one such trial, given the two retrospective studies, labeling could be changed to indicate the advantage of naltrexone pharmacotherapy in persons with the response allele. Would such an indication lead to the genetic test itself being FDA approved as a diagnostic test? ANS: Yes Are there separate requirements for approval of different formulations of the medication - depot vs oral naltrexone? ANS: The change in labeling would be specific to the formulation used.
Planned Study Design Features for an FDA Pharmacogenetic Registration Trial of Naltrexone in Alcohol Addiction 150 G allele subjects (190 A/A) Placebo controlled Randomization stratified by gender and genotype (compare A/A vs A/G + G/G) Measures of stimulation / high and craving 12 weeks, relapse to heavy drinking is the primary endpoint Medication Management as psychosocial platform Use depot NTX to minimize compliance problems.
SUMMARY For a genotype-specific indication, the FDA may require two independent studies, both of which are placebo-controlled, double-blind, with randomization stratified by genotype. Both studies must show: 1) that outcome for persons with the favorable allele, randomized to the drug, differentiates from persons with the favorable allele randomized to placebo; and 2) outcomes for persons without the favorable allele, randomized to the drug, do not differentiate from persons without the favorable allele randomized to placebo.
Two Topics to be Covered Mu opioid receptor variant predicts therapeutic response to naltrexone in alcohol addiction Alpha3/5 nicotinic receptor subunit genes in nicotine addiction
Public Health Perspectives If current trends continue, the annual number of deaths (world-wide) from tobacco-related diseases will double from 5 million in the year 2000 to 10 million in 2020 (WHO, http://www.who.int/whosis ). In the US, ~ 13% of adults are ND. Smoking is the largest preventable cause of morbidity and mortality in the US, with obesity being the second largest.
Patch Efficacy Effectiveness of Patch therapy (vs. Placebo) No. of studies No. of participants Odds Ratio ( 95% CI) 37 16228 1.84 [1.65, 2.06] Fiore et al., 2000; Silagy et al., 2004
Bupropion 300mg used for 9 weeks Mechanism may involve blockade of DA/NE reuptake Effective for only a subset of smokers Effectiveness of Bupropion (vs. Placebo) No. of studies No. of participants Odds Ratio (95% CI) 21 7171 1.99 [1.73, 2.30] Fiore et al., 2000; Hughes et al., 2005
Varenicline nicotinic receptor partial agonist Eliminates reward from smoking and craving and withdrawal AEs are comparable to placebo (nausea is biggest concern) 60 50 Onckene et al., 2005 48 4-week Continuous Abstinence % 40 30 20 17.1 33 28.6 37.3 10 0 Placebo Bupropion 0.3mg Once/day 1mg Once/day 1mg Twice/day
Drug Dependence* is > 3 of These 1. Tolerance (e.g., the need to take more drug to achieve the desired effect) 2. Withdrawal: Daily use for at least several weeks; for nicotine, following abrupt cessation, four or more of the following signs: (A) dysphoric or depressed mood (B) insomnia (C) irritability, difficulty managing anger (D) anxiety (E) difficulty in concentration (F) restlessness (G) decreased heart rate (H) increased appetite or weight gain 3. Repeated unsuccessful attempts to quit or reduce using 4. Continued drug use despite medical or psychological harm 5. Drug use is often greater than intended or longer than intended. 6. Reduction or elimination of social, recreational or occupational activities because of drug use 7. Much time is spent obtaining and using the drug, & recovering from use. For nicotine dependence (ND) #6 & #7 are not very relevant. Thus, DSM criteria for drug dependence are not ideal for ND. *From the Diagnostic and Statistical Manual of Mental Disorders, 4 th edition, 1994
Hall et al, Tob Control 11:119, 2002
NachRs Influence Transmitter Release dopamine molecules NachR NachRs modulate neurotransmitter release via pre-synaptic receptors. Nicotine s ability to modulate release of dopamine may be key to its addictive quality. dopamine receptors Post-synaptic NachRs modulate the response of the post-synaptic neuron.
Electrophysiology of Nicotine Reward Nicotine increases release of dopamine in the n. Accumbens and medial prefrontal cortex. Dopamine is released at low levels tonically in the n. Accumbens, but at high levels during phasic (burst) firing. The difference (delta) of dopamine released between tonic and phasic modes is thought to be a signature of reward. Electrophysiologic studies show that nicotine increases dopamine release during phasic, as opposed to tonic, firing. This may underlie the rewarding effect of nicotine. Rice & Cragg, Nature Neurosci 7:583, 2004 Zhang & Sulzer ibid 7:581, 2004
Nicotine Increases Dopamine Release During Phasic Firing in n. Accumbens D O P A M I N E At low frequency stimulation of VTA dopaminergic neurons (eg, tonic activity), nicotine decreases the amount of dopamine released. At high frequency stimulation (eg, phasic activity), nicotine increases the amount of dopamine released. Rice & Cragg, Nature Neurosci 7:583, 2004
Nicotine Increases Dopamine Release During Phasic Firing in n. Accumbens Rice & Cragg, Nature Neurosci 7:583, 2004 Nicotine increases the phasic (burst firing) dopamine release from accumbal slices and inhibits the tonic firing dopamine release, thereby creating a larger difference (delta) between basal synaptic dopamine levels and phasic firing synaptic dopamine levels. The magnitude of this delta may be an electrophysiologic measure of the rewarding valence of a drug.
Single Nucleotide Polymorphisms (SNPs) The most common variation in DNA is the SNP. An example of a SNP is shown below. At a hypothetical point in the human genome (the fifth base pair in the sequence), humans can have a T:A base pair or a C:G base pair. ALLELE 1 ALLELE 2 CGATTGCACC CGATCGCACC GCTAACGTGG GCTAGCGTGG SNPs are the variants which increase risk for common diseases. A common SNP occurs every 1000 base pairs across the 3 billion bases of DNA in the human genome.
The Human Genome Project Among the ~3 million common SNPs, which are functional?
ADVANCES in SNP TECHNOLOGY ADVANCES in SNP TECHNOLOGY These 1,000,000 SNP CHIPs provide on average 1 SNP every ~ 4000 base pairs across the genome, allowing study of most chromosomal regions, with assessment of copy number variations (insertions, deletions, duplications) and mitochondrial DNA.
Genome-Wide Association Study (GWAS) for Cigarettes per Day GWAS of common diseases (eg, asthma, hypertension) have been done (or are in process) at GSK to find new targets for medication development. Some of these studies collected crude smoking data, including the answer to the question: If you ever smoked daily, what was the greatest number of cigarettes per day (CPD) you smoked for at least a month? This CPD variable is related to a DSM-IV concept of nicotine dependence (ND) in a complex manner, but virtually everyone who ever smoked > 25 CPD would satisfy criteria for ND.
NESARC: National Epidemiologic Survey on Alcohol and Related Conditions Survey methods Interviews conducted 2001-02 Conducted by National Institute on Alcohol Abuse and Alcoholism (NIAAA) Data were collected in personal interviews conducted in respondents homes One sample adult 18 or over was selected randomly for interview in each household Population Represents the civilian, non-institutionalized adult population of US Including DC, Alaska and Hawaii African Americans, Hispanics and adults aged 18-24 years over-sampled Overall response rate 81% (n=43,093) Smoking measures Nicotine Dependence using DSM-IV criteria Number of Cigarettes smoked usual quantity currently smoked or when you did smoke
Sensitivity and Specificity for Usual Quantity Smoked by Lifetime History of Nicotine Dependence among Smokers (n=18,930) Cutoff Sensitivity Specificity 1 0.97 0.0809 5 0.8855 0.308 10 0.7877 0.4307 15 0.603 0.3936 20 0.5373 0.6499 25 0.1813 0.8933 30 0.1676 0.899 35 0.1013 0.9367 40 0.0984 0.9378 45 0.0282 0.98 50 0.0276 0.9804 55 0.0197 0.9846 60 0.0194 0.9847 70 0.0046 0.9963 ~ 65% specificity for persons habitually smoking >20 CPD for criteria for DSM-IV ND. This suggests that the concept of DSM-IV ND might require revision, so that a greater fraction of people who habitually smoke > 20 CPD would meet criteria. When the CPD value is > 25, specificity improves to 90% for DSM-IV criteria for ND.
GEMS and Lausanne Genes Correlated with CPD SNP ChromoStartPos Gene GEMS p Laus p pooled p RS6495308 15 76694711 CHRNA3 0.008723 0.000601 6.90E-05 RS7804771 7 1.37E+08 DGKI 0.007254 0.001059 9.81E-05 RS2645339 5 1.78E+08 GRM6 0.000916 0.02548 0.000272 RS5522 4 1.5E+08 NR3C2 0.000589 0.001751 1.52E-05 RS5525 4 1.5E+08 NR3C2 0.01019 0.000269 3.78E-05 RS10869409 9 76313209 RORB 0.01129 0.000365 5.53E-05 RS7846903 9 76304931 RORB 0.009396 0.001039 0.000122 RS13293006 9 76326716 RORB 0.02351 0.002327 0.000592 RS7873840 9 76340109 RORB 0.01027 0.01714 0.001698 RS4932598 15 90338849 SLCO3A1 0.0249 0.000639 0.000192 RS4932597 15 90338621 SLCO3A1 0.03262 0.000637 0.000245 RS12439738 15 90336555 SLCO3A1 0.00379 0.01282 0.000531 RS12439765 15 90336606 SLCO3A1 0.004227 0.01374 0.000625 GEMS (2000 people) and Lausanne (6000 people) are two studies of cardiovascular disease risk, in which CPD was treated as a quantitative variable. CHRNA3 is the alpha 3 subunit of the nicotinic receptor, a biologically plausible candidate gene. Berrettini et al, 2008
Comparison of GEMS/Lausanne (Using CPD as a Quantitative Trait) and HITDIP Analysis in a Case (> 25 CPD) Control (< 5 CPD) Mode GENE CHRNA3 (alpha 3 subunit of nicotinic receptor) GEMS/Lausanne* combined p Value 0.000069 (rs6495308) ehitdip^ p Value 0.0000026 (rs1317286) GSK3B (glycogen synthase kinase 3, beta subunit): P=0.008 p = 0.004 ADAMTS1: ADAM metallopeptidase with thrombospondin type 1 motif, 1 3 SNPs 0.0005<p<0.005 3 SNPs** 0.006<p<0.034 ABCC11: ATP-binding cassette, subfamily C (CFTR/MRP), member 11 p = 0.012 P = 0.008 CHRNA3 significant in a candidate gene study (Saccone et al, HMG 16:34, 2007) of ~1000 ND cases and ~ 1000 controls (p = 0.0003, same SNP not tested) *GEMS: ~1000 dyslipidemia cases + ~ 1000 controls; Lausanne: ~5600 persons ^HITDIP: ~1740 cases and ~6200 controls **No overlapping SNPs
CHRNA3 Gene Structure & SNPs in LD with CPD Rs578776: 76,675,454 Risk allele = C F(G) = 0.72 Saccone et al, 2007 P = 0.0003 Rs1051730: 76,681,392 Risk allele = T F(A) = 0.32 Saccone et al, 2007 P = 0.001 RS1317286: 76683184 ehitdip Risk allele = G F(C) = 0.33 P = 0.0000026 RS6495308: 76694711 GEMS/Lausanne Risk allele = T F(T) = 0.77 P = 0.000069 Berrettini et al, 2008
CHRNA5 CHRNB4 CHRNA3 Bierut et al, 2008, P = 0.007; Chen et al, in press P=0.003 and 0.007; Weiss et al, 2008 p = 0.0009 Saccone et al, 2007 P=0.003 Saccone, et al, 2007, P=0.0003 Thorgeirsson et al, 2008, P=10-20 Berrettini et al, 2008 (P=0.000003 Berrettini et al, 2008 (p = 0.00007) Risk alleles are boxed and all are found on a common haplotype (frequency = 38%)
Prominent Neuronal NachR Subtypes Alpha5 subunits cannot form functioning receptors alone or in combination with only one other subtype. Alpha 5 subunits do not participate in forming the acetylcholine binding site. (Courtesy of Jon Lindstrom, PhD)
5 mrna in Human Brain (GeneLogic) RED = ABSENT, BLUE = PRESENT, GREEN = MARGINAL
3 mrna in Human Brain (GeneLogic) RED = ABSENT, BLUE = PRESENT, GREEN = MARGINAL
CHRNA5 GENE STRUCTURE: Mis-sense SNP in Exon 5 A/G (N/D) SNP with A as the minor allele (~35% frequency in individuals of European origin). This SNP occurs in a region of high conservation, and may be functional. The A allele increases risk for ND.
Mol Psychiatry, 2008 Biol Psychiatry, 2008
CHRNA5 SNP rs588765 Affects mrna Transcription *P<0.05 Wang et al, Mol Psychiatry, 2008
CHRNA3/5 Alleles Increase Lung CA Risk (Hung et al, Nature, 2008) This was found even though cases and controls were closely matched for smoking history!
CHRNA3/5 Alleles Increase Risk for Lung CA # 2 This was found even though cases and controls were closely matched for smoking history! Amos et al, Nat Genetics, 2008 CHRNA5 CHRNA3
PNAS, 2006 Lung CA cell lines
Future Directions The functional significance of SNPs in the implicated haplotype block must be established through molecular and biochemical studies. Use the results above to guide new medication development for ND. Conduct pharmacogenetic studies to determine whether the risk haplotype predicts response to nicotine replacement, varenicline or bupropion therapies. Study other populations in which ND is common (alcoholism and schizophrenia)