1. Statistical Genetics of Substance Use (SU) and Substance Use Disorders Kenneth S. Kendler, MD Virginia Institute of Psychiatric and Behavioral Genetics Virginia Commonwealth University Advanced Genetic Epidemiology Statistical Workshop Oct 26, 2015

3. Paradigm 1- Basic Genetic Epidemiology - What Have We Learned? Genetic factors play a substantial role in the etiology of Substance Use Disorders (AD). Heritability – the proportion of individual differences in a particular disorder or trait in a particular population that results from genetic differences between individuals. Heritability estimates typically in the range of 50-60% How does this compare to other psychiatric and biomedical disorders?

4. HeritabilityPsychiatric DisordersOther Important Familial Traits ~zeroLanguage Religion 20-40%Anxiety disorders, Depression, Bulimia, Personality Disorders Myocardial Infarction, Normative Personality, Breast Cancer, Hip Fracture 40-60%Alcohol Dependence Drug Dependence Blood Pressure, Asthma Plasma cholesterol, Prostate Cancer, Adult-onset diabetes 60-80%Schizophrenia Bipolar Illness Weight, Bone Mineral Density 80-100%AutismHeight, Total Brain Volume Heritability Of Psychiatric Disorders

5. How Consistent are the Estimates of Heritability of AD Across Space? Heritability is not a characteristic of a disorder – rather it is a feature of a disorder in a specific population at a specific time. We will look quickly at twin studies of AD and other SUDs.

8. Interactions between gender, culture and genes – the role of social factors to constrain behavior. Tobacco consumption and year of birth in Swedish twins. Study done with Nancy Pedersen on the SATSA sample. Study males and females separately

9. Prevalence And Heritability Of Regular Tobacco Use Three Birth Cohorts Of Men And Women In Sweden Heritability

10. How Consistent are the Estimates of Heritability of SUDs Across Space and Time? So, to the best of our knowledge, the heritability of AD is relatively robust – across multiple European populations living in Australia, North American and Europe and across a half century of Swedish history that saw dramatic changes in that country. But a quite different picture is seen for regular smoking behavior with a large gene x cohort interaction in women. Know much less about results for other psychoactive substance abuse and dependence.

11. Paradigm 2- Advance Genetic Epidemiology Many questions relevant to SUDs Begin with question of multivariate models – What is the relationship between the genetic and environmental risk factors for SUDs and for psychiatric disorders?

12. Paradigm 2- Advance Genetic Epidemiology – Multivariate Models Examine this question in 2,111 personally interviewed young adult members of the Norwegian Institute of Public Health Twin Panel. Statistical analyses were performed with the Mx and Mplus programs.

14. Paradigm 2- Advanced Genetic Epidemiology – Multivariate Models Replicating earlier results from our Virginia twin analyses and from the Minnesota group, SUDs are genetically part of the externalizing group of disorders.

15. Paradigm 2- Advanced Genetic Epidemiology – Multivariate Models Let’s drill down deeper into the relationship between AD and SUD to directly address the question of the specificity or non-specificity of genetic risk factors for AD.

16. Illicit Substance Genetic Factor Alcohol Dependence Cannabis Dependence Cocaine Dependence Nicotine Dependence AAAA EEEE E1.68.82.35.20.31.68.37.27.48.18.53.47.28.48 Caffeine Dependence A.56 E.14.80 Licit Substance Genetic Factor.77.52.15.82 Alcohol Dependence Cannabis Dependence Cocaine Dependence Nicotine Dependence Caffeine Dependence

17. Paradigm 2- Advance Genetic Epidemiology – Multivariate Models Similar to prior analyses from this sample, these results suggest that ~ 70% of heritabiity for AD is shared (this time with other drugs of abuse) and 30% unique to AD. For cocaine dependence, for example, 85% of total heritability is shared with other drugs and 15% is unique. In general, pretty clear that non-specific genetic effects outweigh specific effects.

18. Paradigm 2- Advance Genetic Epidemiology – Development Genes and environment act through time. Focus on alcohol intake in 1796 members of male-male pairs from the Virginia Adult Twin Study of Psychiatric and Substance Use Disorders. Assessed retrospectively using a life-history calendar.

19. NICOTINE

22. Paradigm 2- Advanced Genetic Epidemiology – Development One more developmental question – Do we see differential developmental changes in the impact of specific genetic risk factors for alcohol use over time? Again ~ 1700 males from VATSPSUD

23. Dynamic Genetic Influences on Alcohol Consumption  The heritability of many alcohol-related phenotypes changes over time. Are there also qualitative changes in genetic influences?  VATSPSUD: adult twins recruited in Virginia and assessed for psychiatric and substance use disorders; only males were included in this analysis  Average alcohol consumption between age 12- 33 was assessed using a life history calendar vipbg Edwards & Kendler, 2013 Psychol Med

24. Multiple (2 genetic, 1 shared env) factors for alcohol consumption vipbg  The relative influence of A1 vs. A2 changed from adolescence into middle adulthood

25. Does A1 reflect specific risk factors for AUD and A2 more adolescent limited externalizing vulnerability?  The existence of qualitative changes in genetic factors influencing alcohol consumption has implications for gene identification efforts vipbg

26. Paradigm 2- Advanced Genetic Epidemiology Twin-family designs – ask a new set of questions.

31. Paradigm 2- Advanced Genetic Epidemiology How to capture the conditionality of genetic influences on SUDs. No initiation, no chance to express genetic risk. How to model? CCC model – causal, contingent, common pathway.

34. Paradigm 2- Advanced Genetic Epidemiology – Gene x Environment Interaction Definition – the impact of genetic risk factors on disease risk is dependent on the history of environmental exposures. OR – the impact of environment risk factors on disease risk is dependent on genotype. Probably no area of psychiatric genetics research that is more controversial and artifact prone. A range of conceptual and statistical issues - Buyer beware!

35. Gene x Environment Interaction Just show one classical example – in type I (adult non-ASPD) alcoholism from Cloninger’s Swedish adoption studies. Risk only high in subjects at high genetic risk and exposed to high risk environment.

37. Paradigm 2- Advanced Genetic Epidemiology – Gene x Environment Interaction Again ~ 1700 males from VATSPSUD Asked – would the heritability of alcohol consumption in adolescence be modified by key environmental risk factors –Alcohol Availability –Peer Deviance –Prosocial Behaviors

41. Paradigm 2- Advance Genetic Epidemiology – Gene x Environment Interaction Many other interesting G x E findings for alcohol use. A few other examples many from the work of Danielle Dick. One general theme – Genetic effects on alcohol use are more pronounced when social constraints are minimized and/or when the environment permits easy access to alcohol and/or encourage its use.

42. Gene-Environment Interaction Alcohol Use Marital Status (Heath et al., 1989) Religiosity (Koopmans et al., 1999) Urban/rural residency (Rose et al., 2001) Neighborhood characteristics (Dick et al., 2001) Parenting/Peers (Dick et al., 2006, 2007)

43. Adoption Studies Let’s take a short detour in to adoption studies. Obvious point about scientific inference More secure about any finding when you can reach it by multiple routes, esp when they have different potential biases.

45. Sample Follow-up in 9 public data bases (1961-2009) in Sweden of adoptees and their biological and adoptive relatives. Identified 18,115 adoptees born 1950-1993; 78,079 biological parents and siblings; 51,208 adoptive parents and siblings. DA recorded in medical, legal or pharmacy registry records.

46. Sources of Data 1. The Swedish Hospital Discharge Register included all hospitalizations (including for DA) for all people in Sweden from 1964-2009. Every record has the main discharge diagnosis and eight secondary diagnoses. 2. The Swedish Prescribed Drug Register included all prescriptions in Sweden picked up by patients from May 1 st 2005 through 2009. It is complete, as all prescriptions are registered at the National Board of Health and Welfare. 3. The Swedish mortality register contained all causes of death and time of death from 1961-2009.

47. Sources of Data 4. The National Censuses provided information on education and marital status in 1960, 1970, 1980, and 1990. 5. The Total Population Registry included annual data on education and marital status from 1990-2009. 6. The Multi-Generation Register provided information on family relationships from 1932 to 2009 including all adoptions and adoptive and biological parents and siblings. Biological siblings reared with the adoptee were excluded. 7. The Outpatient Care Register included information from all outpatient clinics in Sweden from 2001-2009.

48. Sources of Data 8. The Primary Health Care Register included outpatient care data on diagnoses and time for diagnoses 2001- 2007 for 1 million patients from Stockholm and middle Sweden. 9 The Swedish Crime Register included national complete data on all convictions, including those for DA, from 1973-2007.

49. Definition of Drug Abuse We identified DA in the Swedish hospital discharge, mortality, primary care, and outpatient care registers by the following ICD codes: ICD8: Drug dependence (304); ICD9: Drug psychoses (292), and Drug dependence (304); ICD10: Mental and behavioral disorders due to psychoactive substance use (F10-F19), except those due to alcohol (F10) or tobacco (F17).

50. Definition of Drug Abuse DA was also identified in the Crime Register by codes 5011, 5012 which reflect crimes related to DA. Crimes related only to alcohol abuse or to trafficking in or possession of drugs of abuse were excluded. DA was identified in individuals in the Prescribed Drug Register who had retrieved (on average) more than 4 daily doses a day for 12 months from either of Hypnotics and Sedatives (Anatomical Therapeutic Chemical (ATC) Classification System N05C and N05BA) or Opioids (ATC: N02A). Cancer patients were excluded.

51. The 820 unique cases of DA in our cohort came from the following registries: Discharge– 527, Crime – 313, Outpatient – 264, Prescribed Drug – 118, and Primary Health Care – 8. No unique cases of DA were identified through the mortality register.

52. Odds Ratios and 95% Confidence Intervals for the Registration of Drug Abuse Between the Five Registers Used in this Study Hospital Discharge Outpatient Primary Health Care Drug Prescription Crime 32.9 (32.2- 33.4) 65.2 (63.9-66.5)47.4 (41.8-53.7)5.6 (5.3-5.9) Hospital Discharge 118.0 (115.7- 120.4) 69.8 (61.8-78.7)20.9 (20.2-21.7) Outpatient 94.4 (83.5- 106.8) 29.6 (28.5- 30.8) Primary Health Care 37.9 (30.9-46.4)

54. In this study, we could perform both adoption designs –Affected parent → adopted away offspring –Affected adoptee → biological and adoptive relatives

55. Design # 1 – –Risk for DA was significantly elevated in adopted away offspring of biological parents with DA (OR=2.09, 95% CIs 1.66-2.62). Design # 2 –Risk for DA was significantly elevated in biological full and half-siblings of adoptees with DA (OR=1.84, 1.28-2.64 and OR=1.41, 1.19-1.67, respectively). –Risk for DA was significantly elevated in adoptive siblings of adoptees with DA (OR=1.95, 1.43-2.65).

56. Next, sought to create empirical indices of genetic and environmental risk for DA in the adoptees. Used a multiple regression approach from which we derived an index.

57. Genetic risk for DA in the adoptee was indexed by a range of features including – biological parental low educational attainment and divorce –a parental or sibling history of DA, criminal activity treatment for psychiatric or alcohol problems.

58. Environmental risk for DA was predicted by a diverse set of characteristics including –adoptive parental history of divorce, premature death, criminal activity, hospitalization for medical or alcohol problems, –adoptive sibling history of DA hospitalization for psychiatric, alcohol or medical problems.

59. Examined individually in logistic regression, where the genetic and environmental risk indices were divided into ten deciles, both risk scores were strongly predictive of DA. OR per decile = 1.13 for genetic risk (1.13 10 =3.39) OR per decile = 1.10 for environmental risk (1.10 10 =2.59,) The correlation between risk scores (a measure of assortative placement) was small (+0.11) but significant (p<0.001).

61. Our key dependent variable, DA, is dichotomous. We initially used logistic regression and modelled DA as a function of the genetic risk score, the environmental risk score, sex of the adoptee and AFCAP. However a key a priori goal of these analyses was to determine if genetic and environmental risk factors interacted in the etiology of DA. We have previously argued that the scale of raw probabilities, rather than the logistic scale, is more appropriate for such analyses 16. Therefore, for our analyses of gene x environment interaction, we used PROC GENMOD in SAS 17 with the identity link and specified the variance to be binomial. We specified the effects of the explanatory variables (and the interaction term) to be additive on the scale of probabilities. All p values are reported two-tailed

63. DA is an etiologically complex syndrome strongly influenced by a diverse set of genetic risk factors reflecting a specific liability to DA and a vulnerability to other externalizing disorders and by a range of environmental factors reflecting marital instability, and psychopathology and criminal behavior in the adoptive home. Adverse environmental effects on DA are more pathogenic in individuals with high levels of genetic risk.

64. The Sources of Parent-Child Transmission of Drug Abuse: Path Analyses of Not-Lived-With Parental, Step-Parental, Triparental and Adoptive Families To clarify the origins of parent-child resemblance for drug abuse (DA), using national Swedish data, we fit path models to information on DA in parents and children from six informative family types: i) not-lived- with father, ii) not-lived-with mother, iii) step-father, iv) step-mother, v) triparental, and vi) adoptive. From these families, we estimated parent-offspring resemblance reflecting the effects of genes + rearing, genes only and rearing only.

65. Biological Mother Cohabited With Offspring For All of First 15 Years Biological Father Never Resided With or Near Offspring Unrelated Stepfather Cohabited With Offspring ≥ 10 of First 15 Years Offspring Risk G + EG OnlyE Only

68. Gene- Environment Correlation Better termed genetic control of sensitivity to the environment. Time is too limited to give details. My sense is that this process is of substantial importance in mediating the impact of genetic risk factors on SUDs. That is, in part, genes impact on risk for SUDs by increasing the chances that individuals seek out high risk environments which expose them to substances of abuse and encourage them in their use and misuse.

69. Paradigm 2- Advance Genetic Epidemiology Integrated etiologic models. To just get a start looking at causal pathways.

73. Paradigm 2- Advance Genetic Epidemiology – Integrative Developmental Model Evidence for two etiologic pathways characterized by genetic and temperamental factors and by psychosocial adversity.

74. Paradigm 2- Advance Genetic Epidemiology – Multivariate Model for DSM-IV Criteria for AD Attempted to distinguish two hypotheses. 1. Each of the seven AD criteria index the same set of risk genes so that the diagnosis of AD is genetically homogeneous. 2. The DSM-IV syndrome of AD is genetically heterogeneous, arising from multiple sets of risk genes that are each reflected by a distinct set of diagnostic criteria. –Rodent studies suggest relatively distinct set of risk genes for different alcohol- related traits.

75. Paradigm 2- Advance Genetic Epidemiology – Multivariate Model for DSM-IV Criteria for AD Long arduous task of complex model fitting. 7,548 personally interviewed male and female twins from the Virginia Adult Twin Study of Psychiatric and Substance Use Disorders Had to take account of the fact that lots of people did not meet our screening criteria and skipped out of the alcohol section. This is the best fit model --

76. Excessive Quantity / Frequency Perception of Alcohol Problem ToleranceWithdrawal Loss of Control Desire to Quit Preoccu- pation Activities Given Up Continued Use Despite Problems A1A1 A2A2 A3A3.29.22.67.26.51.42.24.30.54.30.33.43.30.27.50.19.28.51.50.44.48.33.47.38.29 E1E1 E2E2.17.36.51.22.67.01.58.19.43.45.33.45.63.28.39.58.28.47.41.34.36.30.45.42.34.29.59 E S1 E S2 E S3 E S4 E S5 E S6 E S7 E S8 E S9

77. Paradigm 2- Advance Genetic Epidemiology – Multivariate Model for DSM-IV Criteria for AD This is the best fit model – Robustly supported second hypothesis – evidence for three genetic factors, which we tentatively called: –heavy use and tolerance –loss of control with alcohol associated social dysfunction –withdrawal and continued use despite known problems.

78. 8 Major Conclusions 1. SUDs are substantially heritable and heritability estimates for AUD appear to be relatively stable across studied populations. For smoking, we have evidence of potentially strong gene x cohort effects. 2. Roughly 2/3rds of genetic risk factors for AUD and other SUDs are not-disorder specific but are shared with other forms of substance abuse and with other externalizing disorders generally.

79. 3. In early adolescence, siblings resemblance for alcohol, nicotine and cannabis consumption is entirely due to environmental factors. With increasing age, we see an increasing degree of genetic influence. 4. For at least AUD and SUDs, we have from adoption and adoption-like designs strong evidence for parent-offspring environmental transmission (but not for AUD from twin family designs). 5. Genes for SUDs appear to be rather substantially moderated by environmental exposures, especially those which either relax social constraints and/or permit easy access but also those that reflect adverse family environments.

80. Conclusions 6. G-E correlation is probably an important etiologic factor in SUDs. Genes can impact on SUDs via outside the skin pathways. 7. I presented one very rough integrated etiologic model for AUD – showing how genetic/termpermental and environmental adversity pathways might inter-relate in the etiology. 8. DSM-IV criteria for AD appear, from a genetic perspective, to be etiologically complex reflecting multiple dimensions of genetic risk. Would we see the same for other SUDs?

81. Key Collaborators Mike Neale PhD Alexis Edwards PhD Carol Prescott PhD Hermine Maes PhD Lindon Eaves PhD Charles Gardner PhD Steve Aggen PhD John Myers MA Ted Reichborn- Kjennerud MD Nathan Gillespie Jan Sundquist Kristina Sundquist

82. Support NIAAA including our Alcohol Research Center at VCU NIDA NIMH Virginia Commonwealth University’s generous support for the Virginia Institute for Psychiatric and Behavioral Genetics No conflicts of interest