Inter- and transgenerational heritability of preconception chronic stress or alcohol exposure: Translational outcomes in brain and behavior.

Chronic stress and alcohol (ethanol) use are highly interrelated and can change an individual's behavior through molecular adaptations that do not change the DNA sequence, but instead change gene expression. A recent wealth of research has found that these nongenomic changes can be transmitted across generations, which could partially account for the "missing heritability" observed in genome-wide association studies of alcohol use disorder and other stress-related neuropsychiatric disorders. In this review, we summarize the molecular and behavioral outcomes of nongenomic inheritance of chronic stress and ethanol exposure and the germline mechanisms that could give rise to this heritability. In doing so, we outline the need for further research to: (1) Investigate individual germline mechanisms of paternal, maternal, and biparental nongenomic chronic stress- and ethanol-related inheritance; (2) Synthesize and dissect cross-generational chronic stress and ethanol exposure; (3) Determine cross-generational molecular outcomes of preconception ethanol exposure that contribute to alcohol-related disease risk, using cancer as an example. A detailed understanding of the cross-generational nongenomic effects of stress and/or ethanol will yield novel insight into the impact of ancestral perturbations on disease risk across generations and uncover actionable targets to improve human health.

A whole exome sequencing study to identify rare variants in multiplex families with alcohol use disorder.

Background: Alcohol use disorder (AUD) runs in families and is accompanied by genetic variation. Some families exhibit an extreme susceptibility in which multiple cases are found and often with an early onset of the disorder. Large scale genome-wide association studies have identified several genes with impressive statistical probabilities. Most of these genes are common variants. Our goal was to perform exome sequencing in families characterized by multiple cases (multiplex families) to determine if rare variants might be segregating with disease status. Methods: A case-control approach was used to leverage the power of a large control sample of unrelated individuals (N = 8,983) with exome sequencing [Institute for Genomic Medicine (IGM)], for comparison with probands with AUD (N = 53) from families selected for AUD multiplex status. The probands were sequenced at IGM using similar protocols to those used for the archival controls. Specifically, the presence of a same-sex pair of adult siblings with AUD was the minimal criteria for inclusion. Using a gene-based collapsing analysis strategy, a search for qualifying variants within the sequence data was undertaken to identify ultra-rare non-synonymous variants. Results: We searched 18,666 protein coding genes to identify an excess of rare deleterious genetic variation using whole exome sequence data in the 53 AUD individuals from a total of 282 family members. To complete a case/control analysis of unrelated individuals, probands were compared to unrelated controls. Case enrichment for 16 genes with significance at 10-4 and one at 10-5 are plausible candidates for follow-up studies. Six genes were ultra rare [minor allele frequency (MAF) of 0.0005]: CDSN, CHRNA9, IFT43, TLR6, SELENBP1, and GMPPB. Eight genes with MAF of 0.001: ZNF514, OXGR1, DIEXF, TMX4, MTBP, PON2, CRHBP, and ANKRD46 were identified along with three protein-truncating variants associated with loss-of-function: AGTRAP, ANKRD46, and PPA1. Using an ancestry filtered control group (N = 2,814), nine genes were found; three were also significant in the comparison to the larger control group including CHRNA9 previously implicated in alcohol and nicotine dependence. Conclusion: This study implicates ultra-rare loss-of-function genes in AUD cases. Among the genes identified include those previously reported for nicotine and alcohol dependence (CHRNA9 and CRHBP).

Epigenetic Effects in HPA Axis Genes Associated with Cortical Thickness, ERP Components and SUD Outcome.

Association between familial loading for alcohol use disorders (AUD) and event-related potentials (ERPs) suggests a genetic basis for these oscillations though much less is known about epigenetic pathways influenced by environmental variation. Early life adversity (ELA) influences negative outcomes much later in life. The stress-activated neuropeptide corticotropin-releasing hormone (CRH) contributes to the deleterious effects of ELA on brain structure and function in animals. Accordingly, we hypothesized that ELA would be related to cortical thickness and electrophysiological characteristics through an epigenetic effect on CRH receptor type-1 (CRHR1) methylation. A total of 217 adolescent and young adult participants from either multiplex alcohol dependence or control families were scanned using magnetic resonance imaging (MRI) at 3T and cortical thickness was determined. Longitudinal follow-up across childhood, adolescence, and young adulthood provided developmental ERP data and measures of adversity. Blood samples for genetic and epigenetic analyses were obtained in childhood. Cortical thickness and visual ERP components were analyzed for their association and tested for familial risk group differences. Visual P300 amplitude at Pz and cortical thickness of the left lateral orbitofrontal region (LOFC), were significantly related to risk group status. LOFC cortical thickness showed a negative correlation with CRHR1 methylation status and with childhood total stress scores from the Life Stressors and Social Resources Inventory (LISRES). Stress scores were also significantly related to P300 amplitude recorded in childhood. The present results suggest that early life adversity reflected in greater total LISRES stress scores in childhood can impact the methylation of the CRHR1 gene with implications for brain development as seen in cortical thickness and electrophysiological signals emanating from particular brain regions.

Structural Brain Correlates of Childhood Inhibited Temperament: An ENIGMA-Anxiety Mega-analysis.

Temperament involves stable behavioral and emotional tendencies that differ between individuals, which can be first observed in infancy or early childhood and relate to behavior in many contexts and over many years.1 One of the most rigorously characterized temperament classifications relates to the tendency of individuals to avoid the unfamiliar and to withdraw from unfamiliar people, objects, and unexpected events. This temperament is referred to as behavioral inhibition or inhibited temperament (IT).2 IT is a moderately heritable trait1 that can be measured in multiple species.3 In humans, levels of IT can be quantified from the first year of life through direct behavioral observations or reports by caregivers or teachers. Similar approaches as well as self-report questionnaires on current and/or retrospective levels of IT1 can be used later in life.

Factors influencing COVID-19 Infection in older individuals: History of Alcohol Use Disorder, Major Depressive illness, genetic variation and current use of alcohol.

Introduction: The Coronavirus Disease 2019 (COVID-19) pandemic continues to be a major public health problem. Vulnerable populations include older individuals with presumed weakening of the immune response. Identification of factors influencing COVID-19 infection could provide an additional means for protecting such individuals. Methods: Members of a family study previously interviewed as middle aged individuals were re-contacted and asked to participate in extended phone interview (2-3 hours) covering past and current mental health issues, physical health diagnoses, use of alcohol and drugs, and exposure to anyone with COVID-19. The average follow-up period was 32 years. Detailed medication use was collected to confirm medical diagnoses and to reveal possible protective effects of particular drug classes currently prescribed for the participant by their physician. Serology was available for red cell antigens (ABO, Kell, Duffy, Kidd, Rhesus) and HLA subtypes. Analyses were conducted to contrast COVID-19 + and COVID-19 - individuals for physical and mental health diagnoses, use of alcohol and drugs, and red cell and HLA serology. Additionally, analyses were conducted to contrast these groups with a group reporting known exposure but absence of COVID-19 symptoms or diagnosis by a health professional. Results: Interviews were completed between September 2020 and November 2021. A total of 42 of the 90 individuals interviewed had been vaccinated at the time of interview. At the time of interview, 11.1% reported having developed COVID-19.Using quantity per occasion (QPO) and quantity by frequency (QXF) totals in the past month by type of alcohol consumed, we found a significant association between QPO for liquor (p=0.017) and marginal effects for QXF for liquor consumption (p=0.06). Exposed individuals who were COVID-19 negative tended to drink more liquor than those who were positive, an average of about one drink per day. Beer and wine consumption were not statistically significant. A diagnosis of alcohol use disorder at baseline evaluation was not a significant predictor of being COVID positive or negative.Self-reported current depression or depression in the past only was not a predictor of COVID-19 status based on a single question "Are you depressed currently or only in the past?". In contrast, completion of a clinical interview designed to elicit depressed mood and concurrent symptoms for determination of the lifetime presence or absence of a depressive episode did reveal a significant effect. Comparison of responses at baseline to follow-up showed those most resilient to developing COVID-19 were those without evidence of a depressive episode by lifetime history at both points in time.Physical health issues were analyzed for those that were frequently occurring in our sample such as hypertension but not found to be significant. BMI was analyzed and found to be statistically non-significant.Analysis of HLA variation across the whole sample did not reveal a significant association but among males two variants, A1 and B8, did show significant variation associated with COVID-19+ and COVID-19- status. Analyses of the red cell antigens revealed one significant red cell effect; Kidd genotypic variation was associated with COVID-19 status. Interpretation: We tentatively conclude that use of specific types of alcohol, namely liquor, is associated with reduced frequency of COVID-19. However, the amount is low, averaging about 1 drink per day. Enlarged samples are needed to confirm these results. The finding that past history of alcohol use disorder does not increase likelihood of developing COVID-19 is important. It should be noted that the 34 individuals diagnosed with AUD at baseline had survived an average of 32 years in order to participate in the current interview suggesting they may be especially resilient to adverse health conditions. The finding that a single question designed to elicit the presence or absence of depressed mood either currently or in the past was not a risk factor for COVID-19 in contrast to report of a clinically significant past history of a depressive episode based on more extensive examination using DSM criteria is important. Results for the KIDD blood group are novel and warrant further investigation.

Suicidal ideation and aggression in childhood, genetic variation and young adult depression.

BACKGROUND: Suicidal thoughts and behaviors have been studied in association with a variety of risk factors. The aim of the present study was to determine if levels of child/adolescent aggression and/or variation in candidate genes previously associated with suicidal behaviors in adults would influence the presence of suicidal ideation in childhood/adolescence, and to determine if ideation was associated with young adult depression. METHODS: A longitudinal study of children, adolescents and young adults who were at high or low risk for alcohol and other substance use disorders by familial background were assessed. The Child Behavior Checklist (CBCL) aggression scale scores with derived subtypes (physical and relational) and genetic variation (ANKK1, DRD2, COMT, SLC6A4, HTR2C) were used as predictors of the presence and onset of suicidal ideation in childhood using survival analysis. Structural equation models (SEM) were fit to determine the relative importance of the predictors controlling for background variables. RESULTS: CBCL aggression was significantly associated with child/adolescent suicidal ideation. One SNP in the ANKK1 gene (rs1800497), one in the HTR2C gene (rs6318), and two haplotypes, AAAC in the ANKK1-DRD2 complex and the CCC haplotype of the HTR2C gene, were significantly associated with the presence and onset of child/adolescent suicidal ideation. Follow up in young adulthood showed a significant relationship between suicidal ideation in childhood/adolescence and young adult depression. CONCLUSIONS: Genetic variation and presence of elevated aggression scores from the childhood CBCL are significant predictors of childhood suicidal ideation. Suicidal ideation in childhood and being female are predictors of young adult depression.

Accuracy of self-reported hypertension: Effect of age, gender, and history of alcohol dependence.

Patient awareness of medical conditions may influence treatment seeking and monitoring of these conditions. Accurate awareness of hypertension reported to clinicians evaluating patients for whom clinical history is limited, such as in emergency care, can aid in diagnosis by revealing whether measured hypertension is typical or atypical. Measurement of blood pressure in a laboratory study was assessed at rest, immediately before phlebotomy, and within 10 minutes after. The resting measure was used to determine the accuracy of self-reported hypertension in 283 adults. Parametric analyses were conducted to identify potential variables influencing accuracy of self-reported hypertension. Sensitivity, specificity, and the kappa coefficient of agreement were calculated to determine the influence of alcohol dependence (AD), sex, age, and cigarette smoking on hypertension awareness. Self-report was mildly sensitive, correctly identifying individuals with hypertension in approximately 37% of the cases, but was highly specific (95%) in identifying individuals without hypertension. Similar sensitivities were found in analyses separated by sex. Sensitivity was greater in those over age 55 (53%) in comparison with those <54, as well as in those who were not smoking. Comparison of those with and without a history of AD revealed that both groups show similar accuracy in reporting hypertension. Absence of hypertension can be accurately determined with self-report data in those without hypertension. A significant proportion of those with measured hypertension report an absence of hypertension.

DRD2 methylation and regional grey matter volumes in young adult offspring from families at ultra-high risk for alcohol dependence.

Dopaminergic alteration is a prominent feature in those with AD and may influence brain development in those with a family history of AD. MRI scans (3T) from 43 HR offspring (27.4 ±â€¯3.6 years) and 45 controls (24.5 ±â€¯4.1 years) provided whole brain (WB) and region of interest (ROI) analyses. The VBM8 toolbox was used for WB analysis (threshold p < 0.005; cluster = 100 voxels); the MarsBaR ROI toolbox provided region of interest data. Pyrosequencing of CpG sites within the DRD2 gene was performed. DRD2 methylation was significantly increased in association with familial high-risk status. Significant familial risk group differences were seen with HR individuals showing reduced volume of the Left Inferior Temporal, Left Fusiform and Left Insula regions relative to LR controls. These regions have previously been linked to social cognition. DRD2 methylation was negatively related to grey matter volumes in these regions. Because these regions, have been previously linked to facial affect perception and social cognition, lesser grey matter volumes in individuals at high-risk for developing AD suggests that neural underpinnings of social cognitive impairment may be a premorbid risk factors for AD.

Cross-generational effects of alcohol dependence in humans on HRAS and TP53 methylation in offspring.

AIM: We hypothesized that cross-generational effects of alcohol exposure could alter DNA methylation and expression of the HRAS oncogene and TP53 tumor suppressor gene that drive cancer development. METHODS: DNA methylation of the HRAS and TP53 genes was tested in samples from young participants (Mean age of 13.4 years). RESULTS: Controlling for both personal use and maternal use of substances during pregnancy, familial alcohol dependence was associated with hypomethylation of CpG sites in the HRAS promoter region and hypermethylation of the TP53 gene. CONCLUSION: The results suggest that ancestral exposure to alcohol can have enduring effects that impact epigenetic processes such as DNA methylation that controls expression of genes that drive cancer development such as HRAS and TP53.

Neural predictors of substance use disorders in Young adulthood.

Offspring from multiplex, alcohol-dependent families are at heightened risk for substance use disorders (SUDs) in adolescence and young adulthood. These high-risk offspring have also been shown to have atypical structure and function of brain regions implicated in emotion regulation, social cognition, and reward processing. This study assessed the relationship between amygdala and orbitofrontal cortex (OFC) volumes obtained in adolescence and SUD outcomes in young adulthood among high-risk offspring and low-risk controls. A total of 78 participants (40 high-risk; 38 low-risk) from a longitudinal family study, ages 8-19, underwent magnetic resonance imaging; volumes of the amygdala and OFC were obtained with manual tracing. SUD outcomes were assessed at approximately yearly intervals. Cox regression survival analyses were used to assess the effect of regional brain volumes on SUD outcomes. The ratio of OFC to amygdala volume significantly predicted SUD survival time across the sample; reduction in survival time was seen in those with smaller ratios for both high-risk and low-risk groups. Morphology of prefrontal relative to limbic regions in adolescence prospectively predicts age of onset for substance use disorders.

Differentiating the Effects of Familial Risk for Alcohol Dependence and Prenatal Exposure to Alcohol on Offspring Brain Morphology.

BACKGROUND: Offspring with a family history of alcohol dependence (AD) have been shown to have altered structural and functional integrity of corticolimbic brain structures. Similarly, prenatal exposure to alcohol is associated with a variety of structural and functional brain changes. The goal of this study was to differentiate the brain gray matter volumetric differences associated with familial risk and prenatal exposure to alcohol among offspring while controlling for lifetime personal exposures to alcohol and drugs. METHODS: A total of 52 high-risk (HR) offspring from maternal multiplex families with a high proportion of AD were studied along with 55 low-risk (LR) offspring. Voxel-based morphometric analysis was performed using statistical parametric mapping (SPM8) software using 3T structural images from these offspring to identify gray matter volume differences associated with familial risk and prenatal exposure. RESULTS: Significant familial risk group differences were seen with HR males showing reduced volume of the left inferior temporal, left fusiform, and left and right insula regions relative to LR males, controlling for prenatal exposure to alcohol drugs and cigarettes. HR females showed a reduction in the right fusiform but also showed a reduction in volume in portions of the cerebellum (left crus I and left lobe 8). Prenatal alcohol exposure effects, assessed within the familial HR group, was associated with reduced right middle cingulum and left middle temporal volume. Even low exposure resulting from mothers drinking in amounts less than the median of those who drank (53 drinks or less over the course of the pregnancy) showed a reduction in volume in the right anterior cingulum and in the left cerebellum (lobes 4 and 5). CONCLUSIONS: Familial risk for AD and prenatal exposure to alcohol and other drugs show independent effects on brain morphology.

Lifetime use of cannabis from longitudinal assessments, cannabinoid receptor (CNR1) variation, and reduced volume of the right anterior cingulate.

Lifetime measures of cannabis use and co-occurring exposures were obtained from a longitudinal cohort followed an average of 13 years at the time they received a structural MRI scan. MRI scans were analyzed for 88 participants (mean age=25.9 years), 34 of whom were regular users of cannabis. Whole brain voxel based morphometry analyses (SPM8) were conducted using 50 voxel clusters at p=0.005. Controlling for age, familial risk, and gender, we found reduced volume in Regular Users compared to Non-Users, in the lingual gyrus, anterior cingulum (right and left), and the rolandic operculum (right). The right anterior cingulum reached family-wise error statistical significance at p=0.001, controlling for personal lifetime use of alcohol and cigarettes and any prenatal exposures. CNR1 haplotypes were formed from four CNR1 SNPs (rs806368, rs1049353, rs2023239, and rs6454674) and tested with level of cannabis exposure to assess their interactive effects on the lingual gyrus, cingulum (right and left) and rolandic operculum, regions showing cannabis exposure effects in the SPM8 analyses. These analyses used mixed model analyses (SPSS) to control for multiple potentially confounding variables. Level of cannabis exposure was associated with decreased volume of the right anterior cingulum and showed interaction effects with haplotype variation.

Longitudinal predictors of cannabis use and dependence in offspring from families at ultra high risk for alcohol dependence and in control families.

Cannabis use is common among adolescents. Identification of the factors associated with continued heavy use into young adulthood and development of cannabis abuse and dependence is of considerable importance. The role of familial risk for addiction and an associated endophenotype, P300 amplitude, has not previously been related to cannabis use and dependence. A prospective longitudinal study spanning childhood and young adulthood provided the opportunity for exploring these factors, along with genetic variation, in the cannabis use behaviors of 338 young adult offspring from high and low familial risk for alcohol dependence families (ages 19-30). P300 data were collected multiple times in childhood. The association between young adult patterns of cannabis use or cannabis abuse/dependence was tested with genetic variation in the cannabinoid gene, CNR1, the ANKK1-DRD2 gene, and childhood developmental trajectories of P300. Young adult patterns of cannabis use was characterized by three patterns: (i) no use throughout; (ii) declining use from adolescence through young adulthood; and (iii) frequent use throughout. Following the low P300 trajectory in childhood predicted cannabis abuse and dependence by young adulthood. A four SNP ANKK1-DRD2 haplotype (G-G-G-C) was found to be significantly associated with the frequency of use patterns (P = 0.0008). Although CNR1 variation overall was not significantly associated with these patterns, among individuals with cannabis abuse/dependence the presence of one or both copies of the rs806368 A > G minor allele conferred a 5.4-fold increase (P = 0.003) in the likelihood that they would be in the frequent and persistent use group rather than the declining use group.

Volumetric Differences in Cerebellar Lobes in Individuals from Multiplex Alcohol Dependence Families and Controls: Their Relationship to Externalizing and Internalizing Disorders and Working Memory.

Offspring from families with multiple cases of alcohol dependence have a greater likelihood of developing alcohol dependence and related substance use disorders. Greater susceptibility for these disorders may be related to cerebellar morphology. Because posterior regions of the cerebellum are associated with cognitive abilities, we investigated whether high-risk offspring would display regionally specific differences in cerebellar morphology and whether these would be related to working memory performance. The relationship to externalizing and internalizing psychopathology was of interest because cerebellar morphology has previously been associated with a cognitive affective syndrome. A total of 131 participants underwent magnetic resonance imaging (MRI) with volumes of the cerebellar lobes obtained with manual tracing. These individuals were from high-risk (HR) for alcohol dependence families (N = 72) or from low-risk (LR) control families (N = 59). All were enrolled in a longitudinal follow-up that included repeated clinical assessments during childhood and young-adulthood prior to the scan that provided information on Axis I psychopathology. The Working Memory Index of the Wechsler Memory Scale was given at the time of the scan. Larger volumes of the corpus medullare and inferior posterior lobes and poorer working memory performance were found for the HR offspring relative to LR controls. Across all subjects, a significant positive association between working memory and total volume of corpus of the cerebellum was seen, controlling for familial risk. Presence of an internalizing or externalizing disorder interacting with familial risk was also associated with volume of the corpus medullare.

Psychological and Neurobiological Precursors of Alcohol Use Disorders in High Risk Youth.

It has long been known that Alcohol Use Disorders (AUDs) run in families with substantial heritability. Determining the specific genetic underpinnings of these disorders has been challenging because of the clinical heterogeneity and variable expression across the lifespan. The search for endophenotypic biological variation associated with the AUD and related substance use disorder (SUD) phenotypes is based on the belief that an endophenotype is more proximal to the causative gene. Identification of genes conferring increased susceptibility has important implications for treatment through the potential development of medications that target specific genetic pathways. High risk family designs that contrast offspring with and without a familial/genetic background have provided valuable insights into the psychological characteristics (executive control, affective regulation, decision making and social cognition) that differentiate such individuals. The current chapter will review these with a focus on brain morphology of specific regions, the coordinated activity of neural networks, and developmental trajectories of electrophysiological activity.

ACN9 and alcohol dependence: family-based association analysis in multiplex alcohol dependence families.

A previous genome-wide linkage study of alcohol dependence (AD) in the Pittsburgh-based multiplex family study found suggestive evidence for linkage on Chromosome 7q, a region in which the ACN9 gene is located. Using the same two generation Pittsburgh family data in which linkage was found, data for a third generation was added. The expanded sample included 133 pedigrees with 995 individuals. Finer mapping was undertaken using six SNPs extending from rs1917939 to rs13475 with minor allele frequency (MAF) ≥0.15 and pair-wise linkage disequilibrium (LD) of r(2) <0.8 using the HapMap CEU population. Binary affection status, visual, and auditory P300 data were tested for family-based association. Family-based analyses found all six SNPs associated with affected status. Three SNPs are located upstream of the gene, two SNPs are within intron 1 and one is in Exon 4. FBAT P-values for the six SNPs ranged between 0.05 and 0.0005. Haplotype analysis revealed one four-SNP block formed by rs10499934, rs7794886, rs12056091, and rs13475 with an overall significant association at P = 0.0008. Analysis of visual P300 amplitude data, a known endophenotype of alcohol dependence risk, revealed a significant association for SNPs within intron 1 and exon 4 under a dominant model of transmission. Family-based association analysis shows the ACN9 gene significantly associated with alcohol dependence and P300 amplitude variation. The potential importance of the ACN9 gene for AD risk may be related to its role in gluconeogenesis which may be involved in the regulation of alcohol metabolism.

Familial risk for alcohol dependence and developmental changes in BMI: the moderating influence of addiction and obesity genes.

AIM: Familial loading for alcohol dependence (AD) and variation in genes reported to be associated with AD or BMI were tested in a longitudinal study. MATERIALS & METHODS: Growth curve analyses of BMI data collected at approximately yearly intervals and obesity status (BMI > 30) were examined. RESULTS: High-risk males were found to have higher BMI than low-risk males, beginning at age 15 years (2.0 kg/m(2) difference; p = 0.046), persisting through age 19 years (3.3 kg/m(2) difference; p = 0.005). CHRM2 genotypic variance predicted longitudinal BMI and obesity status. Interactions with risk status and sex were also observed for DRD2 and FTO gene variation. CONCLUSION: Variation at loci implicated in addiction may be influential in determining susceptibility to increased BMI in childhood and adolescence.