Gene-Environment Interactions and the Regulation of Behavior Workshop 

June 2–3, 2004
Holiday Inn Select
Bethesda, MD

This meeting was held in conjunction with the National Children’s Study, which is led by a consortium of federal agency partners: the U.S. Department of Health and Human Services (including the National Institute of Child Health and Human Development [NICHD] and the National Institute of Environmental Health Sciences [NIEHS], two parts of the National Institutes of Health, and the Centers for Disease Control and Prevention [CDC]) and the U.S. Environmental Protection Agency (EPA).

Meeting chaired and report written by Sarah S. Knox, Ph.D., NICHD, NIH, DHHS

Purpose

The purpose of this workshop was to inform the protocol planning process of the National Children’s Study concerning psychosocial and environmental factors that influence gene expression related to complex behaviors. The goal was to incorporate lessons learned from research on animals and humans into recommendations for future epidemiologic research on gene/environment interactions in the regulation of behavior. The focus of the meeting was on interactions with the social environment because a separate meeting on interactions with chemical exposures is being planned for September.

Background

Animal research on genes associated with behavior and on the modification of gene expression by psychosocial factors has been accumulating for many years. This research covers a fairly broad range of factors from physiological risk to behavioral traits, and has emphasized the extent to which the functional importance of a gene is determined by factors that influence expression (1). Intra- and extracellular environments play an important role not only in the magnitude of expression but also in the direction of expression, i.e., whether it is up- or downregulated. The intracellular environment is a function not only of its genetic components, but also of influences from extracellular factors such as hormones, neurotransmitters, cytokines, and nutrients (1).Variation in these constituents can determine how and when a gene is functionally expressed. Psychosocial factors are involved in these interactions through their influence on hormones and neurotransmitters. This can be illustrated by the influence of stress on transcription factors. Glucocorticoids such as cortisol are part of the hormonal response to stress. The transcription factors cJun and cFos in the cell are sequence-specific DNA-binding proteins that bind to DNA in a multi-protein complex that controls cell proliferation and growth. These transcription factors are extremely responsive to extracellular stimuli (2) and it has been shown that they influence glucocorticoid receptor-induced transcription of proliferin, causing it to increase or decrease according to their presence together or alone in the surrounding environment (3). Research in rats has demonstrated that immobilization stress (physically restraining the animal) upregulates both of these transcription factors in endothelial, myocardial, and smooth muscle cells of coronary vessels (4). Similarly, it has been demonstrated that immobilization stress influences gene expression in the hippocampus in rats, causing increased expression of corticotropin-releasing hormone mRNA (5) and a decrease in 5-HT1 _a mRNA levels in the dentate gyrus (6-7). These latter data complement other research which has demonstrated that serotonin polymorphisms differentially influence heart rate and blood pressure responsivity to stress (8), by showing that stress also influences the functional expression of serotonin polymorphisms.Similar interactions have been demonstrated for the 2-adrenoceptor variants, which influence the magnitude of stress responsivity. Animal research shows that the functional expression of this gene is itself influenced by stress (9). In addition, stress induced expression of corticosteroid receptors has been demonstrated to vary by gender (10). Together, these data illustrate the complexity and ongoing nature of gene/environment interactions, emphasizing that "genes or environment?" is not a meaningful question. The presence of a linked allele at a locus is an indication of risk but it is not enough to predict functional expression. Understanding function requires knowledge of the factors that influence when and in what manner (e.g., up or down regulation) it is expressed.

What is interesting about the influence of psychosocial factors on gene expression is that the changes in expression are sometimes permanent. This has been illustrated in a strain of mice (BALB cByJ), that is more reactive to stressors than other (e.g., C57BL 6ByJ) mice, as defined by hypersecretion of hypothalamic-pituitary-adrenal (HPA) hormones and more behavioral disturbances. In these genetically hyper-reactive mice, it has been demonstrated that early life handling can prevent performance deficits and attenuate HPA hypersecretion to stress in adulthood (11). Also, it has been demonstrated that when the hyper-reactive mice are raised by normal (C57BL 6ByJ) mothers, the excessive stress-elicited HPA activity is reduced, as are other behavioral impairments (11), indicating an effect of early rearing on gene expression. Offspring of mothers who exhibited more licking and grooming of pups during the first 10 days of life have been shown to have reduced plasma adrenocorticotropic hormone and corticosterone responses to acute stress, increased hippocampal glucocorticoid receptor messenger RNA expression, enhanced glucocorticoid feedback sensitivity, and decreased levels of hypothalamic corticotropin-releasing hormone messenger RNA. Each of these measures was significantly correlated with the frequency of maternal licking and grooming (12-13). Although the mechanism responsible for these changes has not been conclusively established, it may involve oxytocin release. Oxytocin, which is secreted from the hypothalamus, has antistress effects (14), can be elicited by stroking or licking, and has been hypothesized as the mechanism for blood pressure reduction in massaged rats (15).

Research on non-human primates has also demonstrated the influence of mothering on gene expression. Research in Rhesus monkeys has demonstrated that a short form of the serotonin transporter (rh5- 5-HTTLPR) gene is associated with drinking alcohol to excess in monkeys reared in an environment with same age peers and no mother, indicating a genetic link to alcohol abuse. However, monkeys with this same genotype reared together with their mothers, actually consume less alcohol (16), indicating that this same polymorphism under different circumstances, confers a protective effect. These data further illustrate that knowing the genotype is not sufficient for predicting phenotypic expression without additional knowledge of the psychosocial environment.

The goal of this workshop was to attempt to identify candidate genes that would be important to behavioral development, specify environmental factors that may influence their expression, and discuss mechanisms of influence as well as relevant methodology for a longitudinal birth cohort study.

Candidate Genes

There is an accumulating body of research on animals and humans that supports the influence of the social environment on gene expression, as well as the moderation of environmental influences by specific allelic variations. Based on group discussion, participants developed the following list of genes warranting further consideration:

A number of genes that have been associated with behavior and that may be susceptible to or moderated by environmental factors were discussed. In addition to stress and depression associated with the serotonin transporter 5-HTTLPR gene mentioned above, variation in the serotonin transporter gene has also been associated with alcohol sensitivity in Rhesus macaques exposed to early life stress (17).

The monoamine oxidase A gene, MAOA, located on the X- chromosome, encodes the MAO enzyme, which metabolizes neurotransmitters such as norepinephrine and serotonin (18). In the Dunedin birth cohort study, which had well characterized environmental adversity histories, it was found to moderate the effects of child maltreatment. Although there was no main effect of a well characterized tandem repeat (VNTR) polymorphism in the promoter region of the MAOA on antisocial behavior, and there was a main effect for child maltreatment, the interaction showed that antisocial behavior was significantly weaker among those with high MAOA activity than those with low activity (18). In this study, maltreatment groups did not vary in MAOA activity, indicating that the genotype did not influence exposure to maltreatment. There is also evidence that this genotype may be associated with higher tobacco consumption (19).

Hox genes modulate other genes in early embryogenesis and HOXA1 and HOXB1 loci are candidates for markers of genetic susceptibility to autism spectrum disorders (20). However, the literature on the association of autism to these HOX genes is not consistent (21-22). Whether there are environmental factors that can influence the variation in these results, has not yet been investigated.

Several polymorphic sites, including Val158Met of the catechol-O-methyltransferase (COMT) gene, which is involved in the degradation of catecholamine neurotransmitters, have long been implicated in schizophrenia. However, the data are inconsistent, perhaps due partly to having cohorts from different populations. One study found three polymorphic sites (including Val158Met) that were significantly associated with schizophrenia in Ashkenazi Jews (23). However, these authors also reported that the schizophrenia-associated haplotype is significantly heterogeneous in populations worldwide. A study in Chinese schizophrenics could not find a significant association between the Val158 Met polymorphism and scores on the Brief Psychiatric Rating Scale or Mini-Mental State Examination, nor did it seem to significantly affect susceptibility, symptomatology, global cognitive function, or prognosis in Chinese patients with schizophrenia (24). Although the data are inconsistent (25-26), COMT seems to be a strong candidate for schizophrenia.

Dopamine, a precursor of norepinephrine, has been associated with multiple disorders. Dopamine D2 receptor-binding density has been reported as being increased in the brains of schizophrenics and a meta-analysis has shown a highly significant association with the Cys allele (27). Long forms of the DRD4 gene VNTR have been reported to be more prevalent in substance abusers (28) and the same gene has been associated with attention deficit/hyperactivity disorder, specifically the 7-repeat allele of the dopamine receptor D4 gene (29). It has also been demonstrated in animal research that repeated exposure to social stress has long-term effects on indirect markers of dopaminergic activity in brain regions associated with motivated behavior (30).

Definition of Behavior

It was emphasized during the meeting that although psychiatric diagnoses are important, the definition of behavior should be broader. The developmental domains of language, cognition, and emotion are all thought to be influenced by gene/environment interactions. A broader context of behavioral issues thought to be particularly relevant were enumerated:

Early relational aggression
Refractory physical aggression
Reaction to novelty
Delayed fine and gross motor development
Sensory processing
Behavioral reactivity to novelty - characterized from animal and human research
Inappropriate emotional expression
Reactivity to stress
Resilience
Self-esteem
Sociability
Pro-social behavior
Suicidal tendencies
Early relational depression
Shyness
Maternal depression
Extreme irritability (from infancy on)
Impulsivity
Working memory
Executive functions
Information processing efficiency
Fears
Sleep patterns
Stereotypic behaviors
Relations with peers
Anxiety
ADHD
Autism

The issue of child abuse was also discussed, not just with respect to its influence as an environmental factor, but also in the context of whether it elicits specific behaviors that should be measured. Participants noted that some exposures, such as trauma, have variable or delayed expression, presenting methodological challenges with respect to capturing information.

With respect to psychiatric diagnoses, it was noted that although the diagnosis of schizophrenia is usually not made until after age 20, psychotic symptoms may be reported before that time and that focus in a longitudinal study of a birth cohort should be on premorbid signs. It was noted that all measures of normal behavioral development, including motor development, are skewed to the delayed side in children who subsequently become schizophrenic. One issue that may be important to take into consideration in this context is population migration. Second-generation immigrant populations seem to have a greater prevalence of schizophrenia than the first generation, the explanation being that social cohesion in the first generation of immigrants functions as a significant buffer factor. This would also indicate a gene/environment interaction in the manifestation of full-blown disease.

Temperament as a developmental process was also emphasized. Animal models show that temperament traits, such as a shy/anxious personality can be bred but that environment has a major influence on phenotypic expression. High-reactive monkeys that are cross fostered to nurturant females display less behavioral disturbance during weaning than other high-reactive monkeys and even less than control infants. In contrast to normally reactive monkeys and high-reactive monkeys who are not cross fostered to nurturing mothers, they also develop in a behaviorally precocious manner (31). Similar results have been demonstrated in human adoption studies. Children at risk for schizophrenia based on parental history, show a protective effect against developing it when placed in psychologically healthy families (32). Thus, the meeting participants emphasized the dynamic process involved and the need for multiple measurements at different time points.

It was agreed that autism, which affects about 3% of the population, would be a good candidate for investigation in a study of this size. There is a consensus in the scientific community that autism is a genetic disorder but the extent to which it is influenced by the environment is still unclear. One third of the children exposed to thalidomide in utero are eventually diagnosed with autism. Other factors that have been reported in association with autism are exposure to valproic acid, phosphoric acid, rubella (now controlled by vaccinations), and prostaglandin. However, one participant expressed the opinion that environmental influences on gene expression related to autism would be limited to the time period during which the neural tube is formed, because this is the only time during which the genes are active. Whether or not these are the only genes involved has not been conclusively established. Gene/environment interactions on chromosome 2 have been pinpointed, but up to 15 loci may be involved. Identifying environmental influences will be difficult because phenotypic effects are not seen until long after exposure.

Environmental Factors That Influence Gene Expression

The interplay between genes and the environment in the regulation of complex behaviors evolves as part of a dynamic process that changes as the child is exposed to multiple chemical, biological, social, and psychological factors during the course of continuing growth and development. Exposures such as parenting or family environment change over time and the child’s vulnerability varies by developmental stage, necessitating measurements at multiple time points. Understanding the primacy of this dynamic process is paramount to properly designing a study in which interactions will be investigated. The implications are that good environments can help to protect against "bad genes" and "good genes" can help to protect against bad environments. The goal of research is to understand the mechanisms involved so that it is possible to facilitate optimal outcomes.

In deciding which environmental factors to prioritize in a birth cohort study of gene x environment interactions, the specificity of knowledge gained from animal data was seen as an important starting point. Other important sources are environmental factors that have been pinpointed in twin and adoption studies, as well as environmental factors that are known to influence human development in general and may be candidates for influences on gene expression. Integrating results from multiple methods helps to define the relevant domains and the vulnerable time points for different types of exposures in children. It was also noted that characteristics of environments that predispose to adverse outcomes tend to cluster (e.g., low socioeconomic status, poor housing, and lead exposure) and that this clustering should be taken into account when analyzing the data. There may be synergistic effects of multiple exposures.

Beginning with the intra-uterine environment, factors considered to be important included: maternal smoking, maternal substance abuse, psychosocial stress (including the measurement of biological markers such as catecholamines and cortisol), infectious exposure, physical trauma, and thyroid hormone levels. It has been shown in monkeys that prenatal stress, both early and late in pregnancy, results in reduced hippocampal volume and inhibition of neurogenesis in the dentate gyrus (33). With respect to the perinatal environment, lack of oxygen was listed as a key risk factor.

In early childhood, factors such as nurturance, breast vs. bottle-feeding, discipline techniques, parental emotional expressiveness and maternal responsiveness, cognitive stimulation, media exposure, environmental stability, and paternal involvement were considered important candidates for influences on gene expression. As demonstrated in the examples given on cross-fostering in monkeys and humans, parenting is of primary importance to early gene x environment interactions. During the meeting, parenting behaviors were summarized under four headings:

• Parental control of the macro-environment: childcare, health care, where the family lives, friends, economic situation
• Parental stimulation of cognitive development: didactic behavior on the part of the parents
• Parent as protector and regulator of child’s emotional state
• Parent as protector and regulator of child’s physical state

The importance of including fathers in the study was also emphasized.

The Canadian National Longitudinal Survey of Children and Youth, in which parents respond to an 11-point, interviewer-administered questionnaire, assesses parents on several additional characteristics including whether they are: authoritative vs. authoritarian, engaged vs. disengaged, consistent vs. inconsistent, and how secure they feel that people in the neighborhood look out for their children (neighborhood social cohesion). They combine this with a visual observation of the physical characteristics of the neighborhood that quantifies specific characteristics such as graffiti. In this study, whether or not the parents feel that their children are safe in the neighborhood discriminates cognitive and behavioral development at ages 3 and 4, after controlling for SES, parenting, and other covariates. The implications of these results are that it is important to capture not only the family environment, but also the macro-level environments.

Environmental influences are thought to have more impact during specific developmental stages and transition periods. Critical periods suggested were intrauterine, birth/perinatal, 8-10 months, 18-24 months, and thereafter, 2 years, 4 years, 7 years, and adolescence. The period from the middle of the first year through the pre-school years was seen as an especially vulnerable period. This is the period, during which an increase in synaptic density occurs and regulation of the stress system in the frontal-cortical and cortical-limbic systems is being formed. During this time, domestic violence is also an important issue. Children under two years of age respond with withdrawal but later, with acting out.

Since the majority of children spend many hours in some type of day care, characteristics of childcare were also considered to be highly important. At a minimum, type of day care, number of changes in day care provider, the ratio of children to the number of caretakers, whether day care was with a family member or outside source, and the number of child care arrangements each day (e.g., before and after school) were seen as important, as well as any additional measures of quality of care that could be obtained.

Other variables of primary interest that extend across childhood stages are socioeconomic status; the interaction between the macro-environment (neighborhood cohesiveness and safety) and micro-environment (family relations, nurturance, discipline); quality of parental marital relations; parental health behaviors such as smoking, diet, and alcohol consumption; social support; intrusiveness of parents; and school experiences. Again, the dynamics of change over time were emphasized. Socioeconomic status of individuals and communities can change and migration influences social cohesion. This may be especially important in rural areas where stability of the environment is related to economy.

It was emphasized several times during the workshop that it is as important to be as exacting and explicit about measurements of the environment as it is to be explicit about the specificity of the alleles. Analyses and interpretations of outcomes are only reliable and valid if the initial measurements are accurate.

Gene/Environment Interactions: Mechanisms

Development is not dominated by either genes or the environment but by a continual interaction of both, stemming from the influence of intra- and extra-cellular factors on gene expression (34). Animal studies have provided insight not only into environmental risk factors related to gene expression, but also into possible mechanistic pathways. Particularly vulnerable periods occur during development of the nervous system relevant to proliferation, migration, differentiation, myelination, synaptogenesis, and apotosis (35). Exposure to toxicants such as lead, methyl mercury, and ethanol during these critical processes can lead to developmental neurotoxicity (35). However, also neurotransmitters such as serotonin, acetylcholine, and catecholaminergic agonists and antagonists, which are responsive to psychosocial stress, function differently in the developing nervous system than they do in adults. They can react with receptors over a greater distance, forming a morphogenetic gradient that contributes to pattern formation of different regions of the nervous system (35).

Glucocorticoids are not only steroid hormones, but also important gene transcription factors (38\6) and part of the HPA-axis response to stress. Ligand-activated, these receptors, along with mineralcorticoid receptors, can contribute to the regulation of gene transcription (37). There is also abundant evidence that chronic elevation of corticosteroid levels in the brain can lead to neurodegeneration or suppressed neurogenesis in the hippocampus and that early life experience can cause changes in stress responsivity that persist into adulthood (37,38). It has been shown in several studies that when a toddler is separated from its mother but is in the presence of a babysitter to whom it has a secure attachment, cortisol levels do not rise. However, children left with an insecure attachment figure are much more likely to show increases (39). Not only glucocorticoids but also the secretion of growth hormone is reduced in rat pups after maternal separation and appears to be due to the lack of tactile stimulation by the mother (40), again, a mechanism that may possibly be related to oxytocin secretion. Social processes, particularly between caregivers and infants, play critical roles in regulating the activity of the HPA axis during development in the first years of life.

Animal research investigating the mechanisms through which maternal behavior can influence gene expression has established a strong link with methylation in the promoter region of the glucocorticoid receptor gene promoter region in the hippocampus (41). Investigators mapped differences in the methylation status of individual cytosines and found a specific dinucleotide of the exon17 promoter region that was always methylated in low licking and grooming and arched back nursing (LG-ABN) mothers but rarely methylated in high-LG-ABN dams. When they cross-fostered pups from low-LG-ABN mothers to high-LG-ABN dams, the methylation status of this same dinucleotide was indistinguishable from that of biological offspring of the high-LG-ABN dams, illustrating that it was changed through maternal behavior.

Methodology Relevant to Epidemiologic Studies

As the above literature suggests, a great deal of the research on mechanisms involving influences of the social environment on gene expression has been done in animals, where it is possible to take tissue samples from the brain and other areas of the body to measure expression in conjunction with specific environmental exposures. These types of gene expression experiments are not possible in humans, due to the ethical issues involved. Measuring gene x environment interactions in the regulation of behavior in humans is further complicated by the fact that behaviors are complex, multiple loci and epistasis are involved, and by variation in the dominance and penetrance of different alleles in the population under study. A probability-based sample of 100,000 children and their families would help control for some of the bias related to dominance and penetrance.

Despite the problems, large longitudinal cohort studies provide a powerful way to examine these interactions in humans. Using genome scans, the level of association between specific alleles and specific diagnoses or phenotypes can be ascertained. Then polymorphisms of these candidate genes can be more closely examined to see if the association varies by environmental conditions. An example of how fruitful this research can be is the Dunedin Multidisciplinary Health and Development Study, which has followed a representative birth cohort of children for 26 years (42). Based on data that the short allele in the 5-HTTLPR is associated with lower transcriptional efficiency of the promoter compared with the long, and on human and animal research showing that the stress response is mediated by variations in the 5-HTTLPR, the authors performed a hierarchical regression of DSMIV diagnosed depression on the 5-HTTLPR polymorphism, stressful life events and their interaction, with gender as a covariate. There was a main effect for stressful life events, no main effect for 5-HTTLPR, but a significant interaction effect for the gene x stress interaction, such that individuals who had at least one short version of this allele had increasing depression with increasing life events. The National Children’s Study will provide an unprecedented opportunity to investigate gene x environment interactions due to the broad range of physical, chemical, and psychosocial exposures that will be measured at multiple time points and the number of families that will be involved.

It was also pointed out that analysis of quantitative data on several thousand markers would likely reveal patterns of association in addition to associations with individual alleles. Several members of the group emphasized that it would be important to identify twin births, which constitute about 1% of all live births. In a cohort of 100,000, that means about 1000 pairs, which would be sufficient for a subset of analyses based on twins.

A broader methodological problem inherent in human research, which was beyond the scope of this workshop but which nevertheless remains to be solved, is the problem with linear, additive models. Twin, linkage, and association equations all assume separate genetic and environmental components that are linear and additive. The specificity obtainable from animal models has made it abundantly clear that there is an ongoing dynamic interaction between genes and their surrounding environments that cannot be captured by linear, additive models. Intra- and extra-cellular environmental factors influence gene expression in such a way that, "phenotype emerges as a function of this constant dialog, and any effort to ascribe percentage values to isolated variables is likely to be biologically meaningless" (22). Although this problem is acknowledged and periodically broached at genetics meetings, solving it will be a long and arduous process. For the present, we must simply be aware of the pitfalls it presents in designing human studies.

It was also emphasized during the workshop that it is important to keep in mind that risk factors tend to cluster, and that such interactions must be taken into account. Urban areas with low socioeconomic status are often polluted and have multiple social problems.Methodologically, this might be addressed by using information from other data banks, for instance an EPA data bank that could identify relatively unpolluted areas with low socioeconomic status for comparison, or by using census data and General Social Survey data to obtain information on social qualities of different areas.

Ethical Considerations

There are important ethical issues connected with genetic studies, not the least of which is confidentiality. There was consensus that confidentiality was of the utmost importance and should be a high priority. However, several other important issues related to ethical considerations were also broached. The wording of consent forms will need to convey the way in which samples will be utilized and must also inform the participant about whether the samples will be stored for future use. The issue of the point at which consent is required from the child as well as the parent was also discussed. Still another issue concerned whether participants should be informed about specific results from genetic testing. Some workshop participants thought that it should not be done and others felt that subject buy-in and attrition risks were important factors to be considered in making a decision. The ambivalence stemmed from the fact that although many participants may want to know about any heritable health risks, interpretation of genetic testing is always complicated. There is also the risk that if insurance companies find out about participation in the study and the release of genetic information, they will require participants to reveal the information as a prerequisite for coverage, creating the risk that they may denied if the information is detrimental. Although there are legal precedents for patient confidentiality that prevent misuse of the data, it is important to thoroughly examine the ethical issues from multiple perspectives before proceeding.

Participants

Marion J. Balsam, M.D., NICHD, NIH, DHHS
Adelaide Barnes, B.A., NICHD, NIH, DHHS
Stanley Barone, Ph.D., Office of Research and Development, EPA
W. Thomas Boyce, M.D., University of California, Berkeley
Stephen L. Buka, Sc.D., Harvard University
Dennis S. Charney, M.D., NIMH, NIH, DHHS
Terri Damstra, NIEHS, NIH, DHHS
Terence Dwyer, M.D., M.P.H., NICHD, NIH, DHHS
M. Daniele Fallin, Ph.D., Johns Hopkins University
Alan R. Fleischman, M.D., New York Academy of Medicine
Nathan Fox, Ph.D., University of Maryland
Megan B. Gunnar, Ph.D., University of Minnesota
Clyde Hertzman, M.D., M.Sc., FRCPC, University of British Columbia
Sarah S. Knox, Ph.D., NICHD, NIH, DHHS
Cynthia A. Moore, M.D., Ph.D., CDC, DHHS
Daniel Pérusse, Ph.D., University of Montreal
Paul M. Plotsky, Ph.D., Emory University
David Reiss, M.D., George Washington University
Patricia M. Rodier, Ph.D., University of Rochester
Peter C. Scheidt, M.D., M.P.H., NICHD, NIH, DHHS
M. Anne Spence, Ph.D., University of California, Irvine
Stephen J. Suomi, Ph.D., NICHD, NIH, DHHS
Diane K. Wagener, Ph.D., RTI International
Barry S. Zuckerman, M.D., Boston University

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