Genetic sensitivity to the caregiving context: The influence of 5httlpr and BDNF val66met on indiscriminate social behavior

1. INTRODUCTION

Gene × environment studies have been a source of both tremendous enthusiasm as well as frustration. Although the initial demonstration of gene x environment interactions were promising[1], failed replications of both candidate gene and genome wide association studies (GWAS) have dampened the enthusiasm for genetic studies in psychiatry[2, 3]. The lack of consistent findings appears to challenge the utility of genetic studies for complex phenotypes, as well as the standard genetic risk and resilience model. An alternative conceptualization, differential susceptibility, may offer significant insight into these discrepant findings and enhance the understanding of the influence genetic variation has on complex psychological and behavioral phenotypes. In this model, specific polymorphisms confer a differential responsiveness to the environment, instead of risk or resilience per se [4]. These “plasticity alleles” are predicted to enhance outcomes in positive environments yet elevate vulnerability in adverse environments [5, 6]. Individuals with the non-plastic, “fixed” alleles, are expected to demonstrate few differences in behavior or outcomes between positive or negative environments. Randomized controlled trials provide a unique opportunity to directly test this model as there is a specific manipulation of the environmental context and established outcome measures by which responsiveness to the environmental change can be measured. [7] Randomized controlled trials of alterations of the environment, particularly the caregiving environment, though common in animal studies are rare in human research and thus represent an important source for hypothesis testing related to differential susceptibility.

Extremes of early caregiving adversity, including severe social deprivation as a result of institutional rearing, are associated with a range of clinical and behavioral problems that in normative social conditions often result in significant impairment. One behavioral construct which has been demonstrated across studies of children reared in institutions and proposed to be part of a deprivation specific pattern (as reviewed in[8]) is indiscriminate social behavior. Indiscriminate social behavior is also elevated in children exposed to early maltreatment. [9] The core features of indiscriminate behavior include lack of reticence with unfamiliar adults, inappropriate social boundaries and affection with strangers, willingness to accompany strangers, and failure to check back with a familiar caregiver when in an unfamiliar setting. While these behaviors may have an unidentified adaptive purpose in inconsistent or inadequate caregiving environments, these same behaviors in normative environments are impairing across multiple domains in part consistent with a mismatch theory of early behavior. Recently, construct and criterion validity as well as stability of indiscriminate behaviors have been reported in a longitudinal study of institutionalized children [10-12]. Despite its etiologic association with adverse caregiving(13) indiscriminate behavior persists in a significant proportion of children years after restoration of adequate caregiving environment [11, 13-15] indicating that other factors contribute to both the development and the persistence of indiscriminate social behavior.

A model of genetically driven differential susceptibility in this context would predict that indiscriminate behavior in some children, “sensitive” individuals, would be quite responsive to changes in the caregiving environment whereas others, “fixed” individuals, would demonstrate little change when moved to an improved environment. This model predicts that children who carry differentially susceptible genetic alleles would demonstrate both the greatest amount of symptoms in the adverse environment and the least amount of symptoms in the positive environment. To test this theory directly we explored the contribution of genotype to change in levels of indiscriminate behavior in the setting of a randomized controlled trial of foster care compared to institutional rearing, the Bucharest Early Intervention Project [16-19].

The biological substrate of indiscriminate behavior is unknown. However, given the association of indiscriminate behavior with a range of negative behavioral and psychological outcomes we selected functional polymorphisms in two genes, the 5httplr in the serotonin transporter gene and the met66val polymorphism in BDNF. These genes were selected a priori because they have been associated with differential susceptibility in both preclinical animal studies and human research, have established roles in social behavior, and have been associated with a range of psychopathology. [3, 20-31] There exists significant evidence, particularly for the 5httlpr, that individuals with the short (“s”) allele, particularly those with the s/s genotye, are not only at increased risk for psychopathology with exposure to high levels of stress or adversity, but these same individuals also appear to benefit disproportionately from supportive environments.[32-34] BDNF is critically involved in neuroplasticity and neurodevelopment and BDNF levels have been found to moderate the association between early adversity and anxiety. [35] The functional val66met polymorphism has been studied across psychopathology. Although a number of studies have demonstrated that the val allele is the protective allele a recent meta-analysis revealed that the met allele was protective for neuroticism.[36] The differential impact of the met allele was also demonstrated in association with high levels of exercise and depression as well as protective relative to psychological disorders when associated with elevated fear processing. [37, 38] Because variation in gene expression levels have been demonstrated during early development for both genes we further evaluated whether these genotypes exhibited any differential timing effects.

The differential susceptibility model further predicts a multiplicative genetic effect where the responsiveness to the environment may be greater in individuals with more than one plasticity allele. Given that both gene-gene and epistatic interactions have been demonstrated repeatedly with these specific polymorphisms [20, 39-43] we examined both their independent and combined impact on indiscriminate social behavior. We hypothesized that cumulative genetic plasticity would be associated with the greatest sensitivity to the caregiving environment. We predicted that children with both plasticity genotypes would exhibit the greatest number of symptoms in the negative caregiving environment (institutional) but the lowest amount of symptoms in the positive caregiving environment (foster care). We further predicted that children with no plasticity alleles would exhibit little difference in indiscriminate behavior between the extreme caregiving environments. [25, 44, 45].

2. METHODS

3. RESULTS

98 individuals had complete data sets that included DAI scores at all time points and valid genotype for both the BDNF and 5htt polymorphisms. No differences in DAI scores were found in those children from whom genotype was available and those who did not have genotype data and DAI scores were not associated with gender or ethnicity. Genotype was not associated with gender, randomization group, or ethnic background (Table 1). Frequency of non-collapsed genotypes were s/s=22, s/l=54, and l/l=24 for the 5httlpr and met/met=1, met/val=24 and val/val=73. Genotype categorized in this manner was not significantly associated with gender, group or ethnic background.

4 DISCUSSION

These results support our hypothesis that differential susceptibility to the caregiving context, determined by the s/s genotype of the 5httlpr and the met allele of BDNF, contributes significantly to indiscriminate behavior in children exposed to early severe social deprivation and adds to the growing body of literature supporting a differential susceptibility model in developmental psychopathology. [7] Our findings demonstrate, in the setting of a longitudinal randomized controlled trial, that individual differences in responsiveness to changes in the caregiving environment are influenced by genetic variation. Children with “sensitive” or plastic genotypes (s/s 5httlpr or met allele carriers of BDNF) had the most signs of indiscriminate behavior when randomized to CAUG. However children with this same “sensitive” genotype, when placed into the enhanced caregiving environment of high quality foster care, had the least signs of indiscriminate behavior at 54 months of age and the greatest decrease in indiscriminate behavior. As expected children with the alternative “fixed” genotypes (5httlpr l/s and l/l or BDNF val/val) demonstrated little change in indiscriminate behavior with alterations in the caregiving environment at any time point.

Interestingly, we also noted that the influence of genotype appears to follow a different time course for each genotype in this study. The 5httlpr genotype appears to have an earlier impact, before the 42 month assessment, while the impact of the BDNF genotype happened slightly later in development, between 30 months and 54 months. This may reflect developmental differences in the impact of these genes or alternatively differences in the underlying mechanism through which they influence changes in indiscriminate behavior in response to caregiving changes. Our findings also indicate that genetic sensitivity to change in the caregiving context did not occur immediately. Rather the decline in indiscriminate behavior occurred over time as their experiences in a different caregiving environment accumulated. The fact that a period of time in the new caregiving context is required for the expression of this differential sensitivity is consistent with a model that includes neuronal plasticity or alteration in neurodevelopmental trajectories occurring prior to detectable changes in a behavioral phenotype. This hypothesis should be tested in existing animal models of early adversity and maternal separation paradigms.

Consistent with other studies our results demonstrate a cumulative effect of plasticity genotype. Children who were carriers of both the BDNF met allele and the s/s 5httplr genotype, two plastic genotypes, had the greatest decline in indiscriminate behavior by 54 months- if they were placed in foster care. However if they remained in the institutional care they had the highest level of indiscriminate symptoms at 54 months. Children who had neither sensitive genotype had little change in symptoms.

The 5httlpr and BDNF val66met polymorphisms have been studied across a range of disorders. Although some have suggested that this raises the question of false positive findings, others propose this reflects a more widespread involvement in neurodevelopment consistent with their broad expression in the central nervous system. The BDNF val66met polymorphism has been associated with anxiety and mood disorders, neuroticism and HPA axis reactivity [26, 59, 60]. Additionally BDNF is known to modulate neuronal plasticity, increase hippocampal dependent memory and learning, is up-regulated in response to stress exposure, and implicated in serotonergic neuronal development and function [61, 62]. Consistent with our results, previous studies have demonstrated that the s/s genotype of the 5httlpr is protective within supportive early or current environments but risk amplifying with exposure to adversity, as noted in one previous study of adolescents with a history of institutionalization[24, 63, 64]. An additional study demonstrated the lowest level of plasticity in response to early childhood adversity in children with the val/val BDNF genotype and long allele of the 5httlpr in effortful control [25] similar to our results which demonstrated little change in levels of indiscriminate behavior with this genotype combination.

The finding of an additive impact of these two polymorphisms is consistent with the evidence of their interaction at the biological and cellular level [43, 65, 66]. Additionally, a significant number of studies have demonstrated both epistatic and gene x gene interactions with 5httlpr and BDNF, particularly in the presence of environmental adversity [20, 25, 39, 67-71]. In a study of HPA axis reactivity in preschool children, Dougherty and colleagues demonstrated an interaction between the s/s genotype and BDNF met carriers on cortisol levels and stress reactivity in preschool children consistent with their hypothesis that children with this genetic combination maybe less reactive under low stress conditions and more reactive under high stress conditions. These findings of genetic plasticity with the s/s and met allele are consistent with our own findings in which children with this combination of polymorphisms demonstrated significant improvement in indiscriminate social behavior when placed in foster care but elevated indiscriminate behavior with prolonged institutional rearing[39].

There are several limitations to this study. First, we did not correct for genetic admixture and possible population stratification. However, for population stratification to present a statistical problem, two conditions need to be met [72]. First, there must be significant differences in allele frequencies by ethnicity. Second, baseline differences in indiscriminate behavior between ethnic groups must be present. As neither of these conditions was present, we did not correct for genetic admixture. Second, we examined only the s and l alleles of the 5httlpr. Other studies have considered an additional polymorphism in the 5httlpr (rs 25531), but we did not further subdivide the genotypes due to concerns about sample size and uncertain functional significance of additional SNPs located within this polymorphism. Although there is some evidence that this additional SNP has functional significance more detailed studies have revealed up to 10 allelic versions in this same polymorphism thus questioning whether other variants could be functionally significant [73]. Additionally we characterized the plasticity genotype for the 5httlpr alleles as s/s compared to s/l and l/l genotypes together. This was done apriori as in previous studies the s/s genotype has demonstrated the highest level of plasticity compared to s/l or l/l genotypes. [27, 74, 75] A third limitation is sample size and the resulting limited statistical power, particularly when considering stratification by group and combined genotype, increasing the risk of a Type II error. The number of children with both plasticity alleles was particularly small and while this is the largest randomized controlled trial of foster care compared to institutional care the issue of sample size needs to be considered. Lastly, we performed analysis with two genes thereby increasing the potential for type I errors. To address this limitation we only presented findings that remained significant after Bonferroni correction had been applied. Our analyses were driven by clearly delineated a priori hypotheses based on previous studies that have demonstrated plasticity with these genotypes, the known interactions between these genes at the molecular and cellular level, and previous evidence of gene x gene x environment interactions. [40, 68, 76, 77].

5. CONCLUSIONS

These results replicate and extend previous findings related to differential susceptibility in general and these polymorphisms in particular. The demonstration of this effect in a randomized controlled trial utilizing the same outcome measure over time represents one of the first studies to demonstrate differential susceptibility in the same individuals over time and offers significant support for the model. [78] The development of disinhibited social behavior in children exposed to extremes of early adverse caregiving which, in the majority of individuals appears to be resistant to later alterations of the caregiving environment, is also consistent with the mismatch hypothesis in that this type of behavior, in the context of unpredictable or limited social stimulation may be adaptive and developmentally set a behavioral repertoire of seeking social interaction from any individual [79]. For the majority of children who experience adverse early caregiving resistance to relying on a specific caregiver, if there is little future expectation that a reliable caregiver would be identified, may be an appropriate evolutionarily adaptation. However by having a limited number of individuals within a population able benefit from novel positive environmental exposures, such as supportive caregivers, at the risk of increased vulnerability to negative environments, could offer evolutionary advantage to the group as a whole by permitting a subset to have significant benefit should rare changes in circumstances occur.

Conceptualizing gene x environment interactions in terms of differential susceptibility represents a potential important paradigm shift for genetic studies of developmental psychology and psychopathology that often treat genetic influences as fixed effects rather than fluid interactions. The redefining of genetic variants, particularly ones with a high prevalence in a population, as neither “good” nor “bad” but rather in terms of responsiveness to environmental changes is more consistent with the complexities of biological systems. Thoughtful testing of hypotheses derived from previous research, combined with evidence found in preclinical and translational studies, may decrease the number of failed replications and enhance our understanding of the genetic influence on developmental psychopathology.