Summary of aims and objectives: This study will capitalise on environmental, biological and

behavioural data that, together with sequential DNA collections over four time points, from infant

and child saliva, are already available from the Wirral Child Health and Development Study

(WCHADS). This is an MRC funded cohort recruited in pregnancy and followed, so far, to age 5

years, established to identify prenatal, infancy and early childhood risks for child psychopathology.

There is a strong focus on early processes underpinning risks for the conduct disorders, but with

emerging findings also in internalising symptoms. The study has limited funding for genotyping but

none for epigenetics. It is distinctive among cohort studies in using a two stage design that enables

detailed observational and experimental data obtained during pregnancy, infancy and childhood to

be conducted on an epidemiological sample. We are not aware of any other studies that combine

such intensive measurement with sequential DNA collection early in life. We will carry out

comprehensive analyses of DNA methylation allowing us to develop a detailed picture of the

epigenome and gene regulation over early development and in relation to continuities and

discontinuities in early experiences, and stable and changing behavioural phenotypes. The case for

the application is made on the basis of the distinctiveness of the data available from this sample,

and also the degree of overlap and complementarity with collaborating studies, which will allow for

data sharing and replication of novel findings. So far collaborations on methylation analyses have

been established with Barker & Mills (NIH funded study) who have a study of methylation pathways

to conduct disorder using a subsample of ALSPAC participants; Generation R Holland, Teiemier;

McGill University, Canada, Meaney, O'Donnell. We also anticipate collaborative data sharing with a

much wider range of groups, especially those that have identified methylation over regions of

interest later in life, and wish to examine the contributions of early experiences in those regions.

Specific aims include a) to provide evidence on methylation patterns mediating links between

prenatal and early postnatal environmental exposures and later biological and behavioural

outcomes, b) examine the role of differential methylation in explaining gene by environment

interactions in early development, c) use environmental predictors to identify novel environment

sensitive regions of the genome for further study in relation to behavioural phenotypes, d) make use

of our repeated DNA and observational measures to extend findings in other studies, e.g. examine

earlier methylation patterns through infancy in pathways to conduct disorders identified in a

subgroup of the ALSPAC cohort (NIH; Barker & Mills) on the basis of methylation estimated at birth

and later childhood at ages 7 and 9 years.

Background.

The intestinal microbiota has coevolved with the human genome, and numerous studies suggest that the gut microbiome and human physiology and metabolism are integrated. The composition of the gut microbiota has been associated with immune development and regulation (1), lipid deposition (2), and plasma glucose levels (3). In humans, treatment with vancomycin is associated with development of adiposity (4) and exposure to antibiotics in early life was recently linked to increased body mass index in childhood (5).

Potential effects of the gut microbiota on bone metabolism have only been investigated in a limited number of studies. In mice, absence of gut microbiota leads to increased bone mass and fewer osteoclasts in the trabecular bone as well as a lower number of CD4 positive cells and osteoclast precursor cells in the bone marrow. Colonisation of germ free mice normalised bone mass and the number of CD4 positive cells in the bone marrow (6). Furthermore, treatment of mice with penicillin, vancomycin or the combination of vancomycin and ampicillin is associated with increased bone mass and size (7).

Effects on bone health of increasing activity of selective gut microbes, through the use of prebiotics, have been assessed in a number of small laboratory and clinical studies. Compared to placebo, intake of a prebiotic (non-digestible oligosaccharides derived from lactose) increased trabecular bone mass in rats, possibly due to improved utilization of calcium and magnesium (8). In humans, treatment with prebiotics (short- and long-chain inulin-type fructans) has been shown to increase calcium absorption and bone mineralization during pubertal growth, and the effects appears to be modulated by common genetic variations in the vitamin D receptor (9). In addition, prebiotic and antibiotic treatments in humans are associated with changes in the secretion of glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (10, 11) and these gut hormones may play a role in the regulation of bone formation and resorption (12, 13).

The modulation of gut microbiota by antibiotics could potentially affect clinically relevant measures of bone mass and quality in humans, in particular attainment of peak bone mass, and, consequently, the risk of fragility fractures in adulthood.

Aims.

* To investigate if exposure to antibiotics in early life is associated with changes in total body bone mass in childhood.

* To determine if associations between early antibiotic use and subsequent total body bone mass persist after adjustment for potential confounding factors such as altered body composition.

* To examine if exposure to antibiotics in early life is likely to affect the risk of clinical events related to osteoporosis in later life, such as hip fracture, based on associations with hip BMD.

* To study the relative contribution of alterations in cortical bone size, thickness, density and turnover to relationships between early life exposure to antibiotics and bone mass which we observe, based on tibial pQCT scan measurements and CTX results.

Hypothesis.

Early life exposure to antibiotics influences gut microbiota, which changes bone metabolism. These alterations cause increases in bone mass in late childhood.

Exposure variables.

Antibiotics in the first 24 months of life.

Outcome variables.

* Indices of total body bone mass as measured by dual-energy x-ray absorptiometry at ages 9.9, 15.5 and 17.8 years.

* Hip BMD as measured at age 13.5 and 15.5

* Cortical bone indices as measured by tibial pQCT at age 15.5 years and 17.8 years

* CTX at age 15.5 years

Confounding variables.

Sex, height, weight, fat mass, lean mass, smoking in first trimester, breast feeding, Tanner stage, physical activity, vitamin D status, socio-economic status/maternal education, insulin, glucose, lipids, leptin, adiponectin, CRP

References.

1. Sommer F, Backhed F. The gut microbiota--masters of host development and physiology. Nature reviews Microbiology 2013; 11(4): 227-38.

2. Backhed F, Ding H, Wang T, et al. The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci U S A 2004; 101(44): 15718-23.

3. Karlsson FH, Tremaroli V, Nookaew I, et al. Gut metagenome in European women with normal, impaired and diabetic glucose control. Nature 2013; 498(7452): 99-103.

4. Thuny F, Richet H, Casalta JP, Angelakis E, Habib G, Raoult D. Vancomycin treatment of infective endocarditis is linked with recently acquired obesity. PLoS One 2010; 5(2): e9074.

5. Blustein J, Attina T, Liu M, et al. Association of caesarean delivery with child adiposity from age 6 weeks to 15 years. Int J Obes (Lond) 2013.

6. Sjogren K, Engdahl C, Henning P, et al. The gut microbiota regulates bone mass in mice. J Bone Miner Res 2012; 27(6): 1357-67.

7. Cho I, Yamanishi S, Cox L, et al. Antibiotics in early life alter the murine colonic microbiome and adiposity. Nature 2012; 488(7413): 621-6.

8. Weaver CM, Martin BR, Nakatsu CH, et al. Galactooligosaccharides improve mineral absorption and bone properties in growing rats through gut fermentation. Journal of agricultural and food chemistry 2011; 59(12): 6501-10.

9. Abrams SA, Griffin IJ, Hawthorne KM, et al. A combination of prebiotic short- and long-chain inulin-type fructans enhances calcium absorption and bone mineralization in young adolescents. Am J Clin Nutr 2005; 82(2): 471-6.

10. Cani PD, Lecourt E, Dewulf EM, et al. Gut microbiota fermentation of prebiotics increases satietogenic and incretin gut peptide production with consequences for appetite sensation and glucose response after a meal. Am J Clin Nutr 2009; 90(5): 1236-43.

11. Francois F, Roper J, Joseph N, et al. The effect of H. pylori eradication on meal-associated changes in plasma ghrelin and leptin. BMC gastroenterology 2011; 11: 37.

12. Tsukiyama K, Yamada Y, Yamada C, et al. Gastric inhibitory polypeptide as an endogenous factor promoting new bone formation after food ingestion. Mol Endocrinol 2006; 20(7): 1644-51.

13. Yamada C, Yamada Y, Tsukiyama K, et al. The murine glucagon-like peptide-1 receptor is essential for control of bone resorption. Endocrinology 2008; 149(2): 574-9.

The societal impact of conduct problems (e.g., fighting, stealing) is profound, but the degree of impact is related to when conduct problems start (1). Compared with their later onset counterparts, individuals with an onset of conduct problems in early childhood are at high risk for persistent criminal behaviors and the development of antisocial personality disorder (2); these youth are often also impaired in multiple settings (social, educational and interpersonal) and are at increased risk for developing mental and physical health problems in adulthood (3). Research has highlighted that childhood onset individuals can present impaired cognitive (e.g., IQ, attention, inhibition) and emotional (e.g., callousness, anxiety, depression) function (4-6), and the role of neurobiological factors in explaining persistent offending in the early onset group has attracted attention. However, only a handful of neuroimaging studies to date have focused on children with conduct problems and these have often identified structural and functional atypicalities in affect processing and affect regulation areas of the brain (e.g. amygdala, orbitofrontal cortex and anterior cingulate cortex) (7). Most brain imaging studies, however, have either not assessed the timing of onset of conduct problems (8, 9), or are based on cross-sectional designs with retrospective reporting used to establish childhood onset (10). Moreover, few published neuroimaging studies have taken into account the risk environments (e.g., poverty, stressful life events, poor parenting) that associate with abnormal brain structure and/or function, and potential biological mechanisms (e.g., DNA methylation) that could at least partially explain brain differences for those exposed to chronic risk. These two limitations are important to redress, for at least three reasons. First, childhood onset children are shown to be exposed to a high incidence of prenatal and early postnatal risks that also associate with impaired (long-term) brain function and structure (11). Second, in certain studies, risk exposures explain differences in the cognitive functioning of delinquents (12). Third, a potential biological mechanism that can affect both the structure and function of the brain, on the experience of chronic risk, is epigenetics (13, 14). The potentially modifiable nature of environmental and epigenetic risk holds particular relevance for clinical and public health policies aimed at reducing the prevalence of conduct problems.

This application proposes the first systematic cross-national examination of potential neurobiological differences in different onsets of conduct problems in two comparable longitudinal epidemiological birth cohorts: The Avon Longitudinal Study of Parents and Children (ALSPAC; UK) and Generation R (GenR; The Netherlands). The proposal includes extensive measures of environmental stress exposures beginning prenatally and extending to late-childhood; DNA sampling from the same individuals at birth, age 7, age 9 (ALSPAC) and age 10 (GenR); brain imaging at ages 10 (GenR) and age 18 (ALSPAC); and the construction of developmental phenotypes of conduct problem trajectories between ages 4 and 13 (ALSPAC), and age 3 and 10 (GenR). This novel and innovative application has five specific aims:

Aim 1. Does structural and functional brain imaging differ between early childhood onset and later onset conduct problem trajectories?

Aim 2. Do any identified differences in brain structure and/or function associate with environmental risk exposure (prenatal and/or postnatal)?

Aim 3. Are risk exposure-to-brain associations at least partially explained by DNA methylation differences?

Aim 4. Is the impact of risk on brain structure/function and DNA methylation greater the earlier and more chronic the experience? (e.g., prenatal vs. postnatal).

Aim 5. Are the above Aims replicable in ALSPAC and GenR?

REFERENCES

1. Moffitt TE, Arseneault L, Jaffee S, Kim-Cohen J, Koenen KC, Odgers CL, et al. Research Review: DSM-V conduct disorder: research needs for an evidence base. J Child Psychol Psychiatry. 2008;49:3-33.

2. Moffitt TE. Life-course persistent versus adolescence-limited antisocial behavior. In: Cicchetti D, Cohen DJ, editors. Developmental Psychopathology. 2nd ed. NY: Wiley; 2006. p. 570-98.

3. Odgers CL, Caspi A, Broadbent JM, Dickson N, Hancox R, Harringthon H, et al. Conduct problem subtypes in males predict differential adult health burden. Arch Gen Psychiatry. 2007;64:476-84.

4. Fairchild G, van Goozen SHM, Stollery SJ, Aitken MRF, Savage J, Moore SC, et al. Decision Making and Executive Function in Male Adolescents with Early-Onset or Adolescence-Onset Conduct Disorder and Control Subjects. Biological Psychiatry. 2009;66(2):162-8.

5. Barker ED, Seguin JR, White HR, Bates ME, Lacourse E, Carbonneau R, et al. Developmental trajectories of male physical violence and theft: relations to neurocognitive performance. Arch Gen Psychiatry. 2007;64(5):592-9.

6. Barker ED, Oliver BR, Maughan B. Co-occurring problems of early onset persistent, childhood limited, and adolescent onset conduct problem youth. J Child Psychol Psychiatry. 2010;51:1217-26.

7. Blair RJR. Neuroimaging of Psychopathy and Antisocial Behavior: A Targeted Review. Current Psychiatry Reports. 2010;12(1):76-82.

8. Sterzer P, Stadler C, Krebs A, Kleinschmidt A, Poustka F. Abnormal neural responses to emotional visual stimuli in adolescents with conduct disorder. Biological Psychiatry. 2005;57:7-15.

9. De Brito SA, Mechelli A, Wilke M, Laurens KR, Jones AP, Barker GJ, et al. Size matters: Increased grey matter in boys with conduct problems and callousunemotional traits. Brain. 2009;132:843-52.

10. Fairchild G, Passamonti L, Hurford G, Hagan CC, von dem Hagen EA, van Goozen SH, et al. Brain structure abnormalities in early-onset and adolescent-onset conduct disorder. American Journal of Psychiatry. 2011;168:624-33.

11. Barker ED, Maughan B. Differentiating Early-Onset Persistent Versus Childhood-Limited Conduct Problem Youth. American Journal of Psychiatry. 2009;166:900-8.

12. Lynam D, Moffitt TE, Stouthamer-Loeber M. Explaining the relation between IQ and delinquency: Class, race, test motivation, school failure, or self-control ? J Abnorm Psychol. 1993;102(2):187-96.

13. Labonte B, Yerko V, Gross J, Mechawar N, Meaney MJ, Szyf M, et al. Differential Glucocorticoid Receptor Exon 1B, 1C, and 1H Expression and Methylation in Suicide Completers with a History of Childhood Abuse. Biological Psychiatry. 2012;72(1):41-8.

14. Jensen Pena C, Monk C, Champagne FA. Epigenetic Effects of Prenatal Stress on 11beta-Hydroxysteroid Dehydrogenase-2 in the Placenta and Fetal Brain. PLoS ONE. 2012;7:e39791.

Although individual genetic markers from genome-wide association studies (GWAS) usually explain limited heritability especially in psychiatric traits, polygenic scores can be a better way to summarise genetic effects of a large number of markers that do not individually achieve geneome-wide significance. (Dudbridge, 2013). This approach has been applied succesfully to schizophrenia (Purcell et al, 2009) and bipolar disorder (Hamshere et al, 2011).

Attention-deficit hyperactivity disorder (ADHD) is a highly heritable neurodevelopmental disorder. Traits of ADHD are present in family members of those with the conditions, as well as in the general population. Learning disabilities are common in ADHD with up to 40% of children with a diagnosis of ADHD presenting with them (Willcutt et al, 2000). In addition, neurocognitive deficits are seen in ADHD with executive function being the primary deficit (Willcutt et al, 2000). Given the attention problems that children with ADHD face, it is expected that their school performance will be affected.

Our hypothesis is that polygenic risk scores based on a clinical sample of ADHD will predict educational attainment and IQ in children from the general polulation.

Polygenic risk scores were calcualted on ALSPAC children using a GWAS of 727 ADHD patients and 5,081 controls (Stergiakouli et al, 2012) as a discovery sample (project B1342) and were provided by colleagues in Cardiff.

Aims of proposal:

To test if polygenic risk scores predict school performance in ALSPAC children using SATS school scores. We are expecting that higher polygenic scores for ADHD will be associated with worse performance in school.

To test if polygenic risk scores predict IQ in ALSPAC children using the WASI IQ score from Focus@3 clinic. We are expecting that higher polygenic scores for ADHD will be associated with lower IQ scores at age 15.

The training sample GWAS from Cardiff did not identify any genome-wide significant hits but it did find that 13 biological pathways enriched for SNP association significantly overlapped with those enriched for rare CNVs. These included cholesterol-related pathways. At the level of individual genes, CHRNA7, which encodes a nicotinic receptor subunit previously implicated in neuropsychiatric disorders, was affected by six large duplications in case subjects (none in comparison subjects) (Stergiakouli et al, 2012). Our hypothesis is that polygenic risk scores based on genetic variants from the GWAS mentioned above could predict obesity and smoking behaviour in the general population.

Aims of proposal:

To test if polygenic risk scores predict BMI at ages 7, 9 and 15 and lipid levels at age 9.

To test if polygenic risk scores predict smoking behaviour at age 15.

Aims

The aims of this project are to assess the long-term effects of adolescent cannabis use on cognitive functioning. Previous research has suggested heavy cannabis use in the teenage years may lead to persistent neuropsychological deficits; however much of the research to date has been cross-sectional and therefore unable to assess whether pre-exposure group differences are driving the association. A recent longitudinal study found chronic heavy cannabis use was associated with a decline in IQ in those with adolescent-onset cannabis use, but not in those with adult-onset use (Meier et al, 2012). However this study had a number of important limitations, including small group sizes and failure to account for a number of potential confounding variables adequately. The present study will address these issues with an independent, larger sample, and assess other factors that may contribute to the relationship.

Research Questions

1. Is heavy cannabis use in adolescence associated with a decline in IQ?

2. Does the neuropsychological functioning of different cannabis-exposure groups differ in childhood, i.e. before onset of drug use? Do the exposure groups differ in other ways (e.g. early environment, socioeconomic status) that might influence neuropsychological functioning?

3. Does adolescent cannabis use affect cognitive function once pre-exposure functioning and other potential confounders (e.g. mental illness or socioeconomic status) have been accounted for? If so what is the size of this effect, and is this also reflected in academic acheivement?

4. Are any deficits seen globally across all neuropsychological functioning, or are they specific to certain domains (e.g. attention or decision making)?

Exposure variables:

1. Cannabis use. This measure will be defined according to lifetime reported usage between ages 13-18 years.

2. Cannabis dependence. This measure will be defined according to criteria for cannabis dependence between ages 16-18 years.

Outcome variables:

1. IQ change from childhood to adolescence. This outcome will be defined based on performance on IQ assessments completed at ages 8 and 16.

2. Neuropsychological functioning. This outcome will be defined by performance on cognitive tasks completed between ages 16-18 years.

3. Academic acheivement. This outcome will be defined by performance on academic assessments completed at school year 11 (Key Stage 4).

Confounding variables:

1. Pre-exposure neuropsychological functioning. Pre-existing differences or deficits in cognitive and academic ability are likely to be related to post-exposure functioning

2. Pre-exposure academic performance. Problems at school in childhood may influence cognitive development and may also be related to the initiation of cannabis use

3. Childhood temperament and behaviour. Childhood traits may influence cognitive development and may also be related to initiation of cannabis use

4. Mental illness (including depression, psychosis and conduct disorder) will likely influence neuropsychological functioning and task performance

5. Sex, socioeconomic status (e.g. parental years of education) and early environment/ parental factors (e.g. parental IQ or drug use) will likely influence neuropsychological functioning

6. Other substance use and dependence (including alcohol and tobacco) may influence neuropsychological functioning

7. Recent cannabis exposure at testing will likely affect performance on neuropsychological assessment due to acute (use in the past 24 hours) and residual (use in the past month) effects of the drug

Reference

Meier, M.H., Caspi, A., Ambler, A., Harrington, H., Houts, R., Keefe, R. S. E., McDonald, K., Ward, A., Poulton, R., & Moffitt, T. E. (2012) Persistent cannabis users show neuropsychological decline from childhood to midlife. PNAS, 109, 1-8.

This proposal will address the need to better understand the interplay between behavioural and biological mechanisms in human development. It will make substantial contributions to our theoretical understanding of social and cognitive development, social behaviour, and their impact on health and behaviour in childhood, adolescence and early adulthood, integrating recent developments in epigenetics. It will develop the empirical knowledge base in these areas, in order to support the development of novel interventions, and create an international hub linked to existing major research groupings.

Intrapersonal, interpersonal and environmental influences (such as family environment, early adversity, and socioeconomic position) at different stages of the life course are critical to the development of behaviour. These influences need to be understood in order to develop and optimise interventions to improve health and wellbeing. However, knowledge of the behavioural and psychological mechanisms of behavioural control, their development through infancy, adolescence and early adulthood, and their relationship with social behaviour, health and wellbeing, remain poorly integrated, and are rarely (if ever) investigated within a single data set. We propose a cohesive programme of research intended to provide a comprehensive understanding of the relationship between cognition, social behaviour, and impulsivity and risk taking. This will be allied to a life course perspective, to understand the role of social and environmental influences at every stage of development into early adulthood.

The Avon Longitudinal Study of Parents and Children (ALSPAC) forms the core resource on which this proposal is structured, utilising the wide range of phenotypic and environmental measures available, in addition to biological samples, genetic and epigenetic information and linkage to health and administrative records. This proposal is a collaborative project across the Universities of Bath, Bristol, Cardiff and Exeter, and will be supported by a wider network of national and international collaborators. It will be structured into three main workstreams, corresponding to our three main aims.

Aim 1: Understand the cognitive precursors (e.g., attentional and perceptual processes) of social behaviour, and health and wellbeing.

Aim 2: Understand the developmental pathways, trajectories and mechanisms associated with social behaviour (in particular sexual behaviour and aggressive behaviour).

Aim 3: Understand social, cognitive and environmental influences on impulsivity and high-risk behaviours, and subsequent health and wellbeing.

Workstream 1 (Social and Cognitive Development): This work will focus on the cognitive precursors of social behaviour, and health and wellbeing. There is growing interest in the extent to which domain-general processes, particularly attentional and perceptual processes, play a role in the development of social cognition and theory of mind. In addition, social compliance, particularly in children, may depend on individuals' ability to adopt, maintain, and execute appropriate goals. As a consequence, executive control functions, including working memory, constrain the manifestation of social behaviour, including the impact of social context, parenting and imitation. However, a central problem that has limited progress in this area is the poor specification of executive control, particularly in children. Until recently, working definitions of the construct have relied on a loose collection of putative executive functions. Structural equation modelling has since provided frameworks for organizing these functions, with a focus on updating, inhibition, and shifting, but these still lack a clear theoretical basis. We will therefore examine the development of executive control in children using a principled approach that sets these candidate functions in a theoretical context. This follows directly from two claims: 1) executive function represents the individual's ability to maintain task goals (updating) and to use these to prevent them from acting inappropriately in response to external stimuli (inhibition), and 2) executive control has a temporal aspect that is not independent of previous experience (shifting). We have the expertise to employ precise measures of these functions and, crucially, manipulate them orthogonally to properly test their interplay. Armed with this suite of novel but theoretically-motivated tasks, the relationship between executive control, measures of attention/perception, measures of social context and parenting, and social behaviour will be investigated. We will explore the relationship of executive function in early life and later trajectories of social and cognitive development, social behaviour, and impulsivity and high-risk behaviour. This will be done in a new longitudinal study across three time points over three years. We will also explore atypical groups based on measures of autism spectrum disorder and attention deficit hyperactivity disorder, the developmental trajectories of these traits over time in ALSPAC, and use targeted recall studies of ALSPAC participants who score at the extreme of measures of executive function to investigate cognitive mechanisms in more detail, using both behavioural and fMRI methods.

Workstream 2 (Social Behaviour): This work will examine the developmental pathways, trajectories and psychological mechanisms associated with outcomes in two aspects of social behaviour that have significant societal impacts: sexual behaviour and aggressive behaviour. Life history theory provides a meta-theory for the coherent study of sociosexual and aggressive behaviour at functional, ontogenetic and mechanistic levels. It proposes that timing of important events across the lifespan, and the strategies employed to achieve such goals, are determined by flexible mechanisms that function to increase reproductive success in response to environmental conditions. Human behavioural ecologists have successfully applied this theoretical approach to societally-important issues such age of first reproduction, family size, and parental investment as a function of factors such as socio-economic status and childhood social experiences, and researchers in other disciplines have investigated the neural, endocrinological and psychological mechanisms underlying such behaviour. We will integrate work across these levels of explanation (function, ontogeny, and mechanism), with a particular focus on the development of emotional processing strategies, which play an important role in sexual and aggressive behaviour. Our underlying theory is that biases in emotional processing often reflect adaptive calibration of socio-cognitive mechanisms to the current environment. For example, links between hostile childhood environments and later aggressive behaviour are well established, and may be functional: when exposed to chronically hostile environments in development, aggression may be an adaptive, rather than a maladaptive, strategy in many cases. We will use the ALSPAC cohort to identify emotion processing strategies that mediate the relationships between childhood environments and social outcomes in adolescence and young adulthood. This will allow us to examine trajectory and development of social behaviours, investigating the role of genetic, epigenetic and social/environmental factors (including physical trauma such as head injury) on development. We will use these analyses to design targeted recall studies in the ALSPAC cohort, to investigate underlying psychological mechanisms using both behavioural and fMRI methods.

Workstream 3 (Health and Wellbeing): This work will examine the relationship between social and cognitive development, social behaviour and health behaviour in adolescence and early adulthood. Psychological models of health behaviour have traditionally focused on explicit decisions arising from cognitive appraisal processes. More recently, dual-process models have emerged which integrate evidence that automatic, impulsive processes also play an important (and perhaps dominant) role. Critically, a constellation of high-risk behaviours (e.g., aggressive behaviour, sexual behaviour, substance use) frequently co-occur, and many adolescents engage in these behaviours at levels harmful to health and wellbeing. It is therefore logical to focus on determinants of these multiple risk behaviours, as this may provide single targets for intervention with the potential for broad impact. Current dual-process models argue that interacting, neural systems control decision making, with an impulsive system focused on immediate consequences and a reflective system focused on future prospects. The reflective system governs the impulsive system; however, there are individual differences in the extent to which this is the case, and this is related to executive function. Therefore, factors which influence cognitive and social development in childhood may in turn influence impulsive and high-risk behaviours in adolescence and early adulthood, both directly (via individual differences in executive function) and indirectly (via exposure to peer groups and other environmental risks). We will explore the relationship between cognitive and social development in early life, and subsequent impulsive and high-risk behaviour in adolescence and early adulthood. We will identify the impact of environmental exposures at multiple time points (e.g., tobacco exposure in utero, in childhood in the home, in adolescence in the home and via peer groups) on subsequent impulsive and high-risk behaviours, and explore the mechanistic relationships between these variables using targeted recall methods to enable more intensive characterisation of relevant constructs, epigenetic markers of exposure, and Mendelian randomisation where possible.

AIMS:

Is the presence of parent-reported "choosy" or "difficult to feed" behavior in children associated with GI symptoms (gastroenteritis, diarrhoea, vomiting, stomach ache and stool type)?

Is the presence of parent-reported "choosy" or "difficult to feed" behavior in children associated with alterations to either parent or child quality of life?

Is the presence of parent-reported "choosy" or "difficult to feed" behaviour in children associated with differences in the child's dietary intake at 1.5, 3.5 and 10 years or later food preference (e.g. large intakes of milk/dairy foods or sweet foods)?

Is early life dietary intake/ timing of weaning or complementary foods (from Infant FFQs at 6 & 15 months of age) associated with eating behaviors (choosiness or difficulty feeding)?

Is early life dietary intake/ timing of weaning or complementary foods (from Infant FFQs at 6 & 15 months of age) associated with reports of child GI symptoms (gastroenteritis, diarrhoea, vomiting, stomach ache and stool type)?

Is early life dietary intake/ timing of weaning or complementary foods (from Infant FFQs at 6 & 15 months of age) associated with differences in the child's dietary intake at 1.5, 3.5 and 10 years or later food preference (e.g. large intakes of milk/dairy foods or sweet foods)?

Is maternal dietary intake during pregnancy associated with eating behaviors (choosiness or difficulty feeding)?

Is maternal dietary intake during pregnancy associated with reports of child GI symptoms (gastroenteritis, diarrhoea, vomiting, stomach ache and stool type)?

Is maternal dietary intake during pregnancy associated with differences in the child's dietary intake at 1.5, 3.5 and 10 years or later food preference (large intakes of milk/dairy foods or sweet foods)?

There is evidence that both particular genes (heritability as high as 60%) and particular environments contribute to vulnerability to subsequent alcohol abuse and dependence [Mcgue, 1999; Enoch and Goldman, 2001; Lesch 2005]. However, there is relatively little data on gene-environment interactions in the aetiology of alcohol use disorders (AUDs) [van der Zwaluw and Engels, 2009]. Further, little is known about the role of internalising behaviour in mediating the relationship between genes, environments and subsequent AUDs.

The Avon Longitudinal Study of Parents and Children (ALSPAC) provide a unique opportunity to try and determine the relationship between genes and environments in AUDs, through the mediation of internalising behaviour. First, ALSPAC includes data on genetic variants and on adverse environments (e.g. stressful life events (SLEs)). Second, ALSPAC includes comprehensive data on internalising behaviour experienced during childhood.

Heavy alcohol use is positively associated with the experience of environmental stressors such as job, health-related, social and legal stress [Dawson et al., 2005]. In turn, the behavioural response to stress is mediated by corticotrophin-releasing factor (CRF) [Arborelius et al., 1999; Binder and Nemeroff, 2010]. Studies involving animal models have shown that polymorphisms within the CRF receptor gene (CRHR1), together with the experience of environmental stress factors, result in greater alcohol consumption [Sillaber et al., 2001; Hansson et al., 2006]. Similarly, in humans, polymorphisms in the CRHR1 gene interact with negative life events to predict heavy alcohol use [Treutlein et al., 2006; Blomeyer et al., 2008]. Besides the CRHR1 gene, the CRF pathway consists of the following genes: POMC, UCN, CRH, CRHR2, UCN3 and UCN2 [PharmGkB. Date accessed: 13 March 2013]. These genes are relatively unexplored in terms of vulnerability to alcohol dependence.

It is often argued that AUDs are characterised by externalising behaviour [Kumpulainen, 2000; Englund et al., 2008]. Findings regarding the link between behavioural inhibition/internalizing symptoms and AUDs are less emphasised in the literature, although a few studies have shown that internalising symptoms, such as anxiety and depression, are associated with alcohol use [Loeber et al., 1999; Fite et al., 2006]. CRF has also been shown to play a role in internalising behaviours; indeed polymorphisms within CRHR1 have shown to have a significant association with anxious temperament and have an influence on the metabolic activity of local brain regions in rhesus macaques [Rogers et al., 2012]. There has been little work, however, on the relationship between variants in the CRF pathway and internalising behaviour in humans.

Methodology

1) Select variants from the genes involved in the CRF pathway based on a systematic review of the literature.

2) Include genotype in the models as a (a) quasi-continuous, (b) time-invariant predictor of latent continuous and/or (c) categorical variable

i. Test for association with the extended alcohol use phenotype measured by the 10-item Alcohol Use Disorders Identification Test (AUDIT) administered at 16 years of age or later [Heron et al., 2012].

3) Identify mediating pathways by which genes confer susceptibility

i. Hypothesized that CRF genes will be likely to confer risk via internalising behaviour evident in early childhood. Internalising behaviour will be measured by the parent-rated, emotional subscale of the Strength and Difficulties Questionnaire (SDQ), administered at 7 years of age or older as described by Araya et al. (2008) and Evans et al. (2008).

ii. Hypothesized that CRF genes will be related directly to alcohol use and risk pathways in adolescence that emerge concomitantly with AU and not involved in pathways with origins in childhood

4) Test GxE with environments previously shown to moderate genetic risk. Environmental factors such as SLEs will be measured using Section D of the parent-rated questionnaire "My Son's/Daughter's Health and Behaviour-42 months". SLEs will also be assessed by the "Life Events" questionnaire completed by the Mother of the "child" at 18, 30, 42 and 57 months as well as 5, 6 and 8 years and completed by the "child" at 16 years of age.

i. Regressing outcome variables onto (a) genotype (b) an environment, and (3) and interaction term reflecting the product of genotype and environment.

We hypothesize that the enhanced asthma risk following infant RSV exposure is due to two non-mutually exclusive factors: (1) a genetic predisposition common to both diseases and (2) infant RSV infection acting as a causal agent in asthma development. We will use genetic epidemiological methods to determine the extent to which infant RSV infection plays a causal role in the development of asthma or if it merely serves as a marker of shared genetic risk with asthma. This study leverages multiple existing US based cohorts, including the Infant Susceptibility to Pulmonary Infections and Asthma Following RSV Exposure (INSPIRE; Hartert, PI), the Tennessee Children's Respiratory Initiative (TCRI; Hartert, PI), RSV Bronchiolitis in Early Life (RBEL I and II; Castro, PI), and Childhood Origins of Asthma (COAST; Lemanske, PI). This revised grant improves power for genetic studies and builds infrastructure to understand infant RSV infection and its respiratory sequelae by collaborating with longitudinal cohorts that have well-characterized respiratory infections in infants during the first year of life followed through to early childhood wheezing/asthma outcomes. We will first identify the genes associated with response to RSV infection (Aim 1) and then determine the genetic bases of shared RSV infection and recurrent wheezing/ early asthma susceptibility (Aim 2). This would be the first GWAS of RSV infection in infancy and its respiratory sequelae of recurrent wheezing/asthma.

We would like to use existing GWAS data from ALSPAC for replication of our primary findings.

Specific Aim 1a:

1a) We will identify the genetic determinants of the severity of RSV infection in the first year of life among ~2000 infants from multiple US cohorts with confirmed RSV exposure or infection. For GWAS genotyping, we will use the Illumina HumanCore + Exome BeadChip in European-Americans (EAs) and the Illumina Omni 2.5M + Exome BeadChip in African-Americans (AAs) and Hispanic-Americans (HAs).

Specific Aim 1b) We will perform a pathway analysis of genome-wide data that addresses limitations of SNP analyses to identify genes or pathways that are associated with severity of RSV infection during infancy.

Specific Aim 2: We will identify the specific genetic variants that lead to the shared genetic risk of severity of RSV infection and early childhood recurrent wheezing/asthma.

This proposed collaboration will address an important gap in understanding how severe infant RSV infection increases the risk of recurrent wheezing/asthma. The results will provide novel insights into both the timing and mechanism for developing primary prevention strategies.

Based on two papers from the ALSPAC cohort, both involving collaborator John Henderson, there are 284 infants hospitalized with infant bronchiolitis. The ALSPAC cohort has well characterized wheezing and asthma outcomes. Using this existing data, we would like to replicate our SNPs predicting severe infant bronchiolitis, measured as hospitalization (y/n). We would also like to replicate SNPs that examine risk of wheezing/asthma after hospitalization for bronchiolitis. We propose in Specific Aim 1b and in exploratory analyses for asthma outcomes to conduct pathway analyses. We would like to use existing ALSPAC data to conduct a replication of our pathway analysis. This will entail the analysis of existing GWAS data.

If the sample size for hospitalization with bronchiolitis followed to to wheezing / asthma outcomes includes enough children with DNA available, we would like to consider using your genetics lab for replication of 480 SNPS from Aim 1 and 480 SNPs from Aim 2 using cases a set of matched controls. At this point we do not know whether our primary SNPs will exist in your data set. We also do not know whether there will be enough children from the case group with DNA.