Our group has already completed a three-stage genomewide association study of mathematical ability in 10 year-olds drawn from the Twins Early Development Study (TEDS) (under review). 43 SNPs were nominated from the first two stages which involved assaying pooled DNA on Affymetrix DNA microarrays to compare individuals of high mathematical ability to those of low mathematical ability. In the third stage, these 43 SNPs were individually genotyped in a sample which spanned the entire spectrum of ability. 10 SNP associations remained significant. Though effect sizes were extremely small, when a composite SNP-score was created for subjects in the final sample by summing the number of 'mathemtics score increasing alleles', these 10 SNPs accounted for 2.9% of the phenotypic variation in the sample. Interestingly, when the remaining 33 SNPs were added to this score, they did not mask this effect on mathematical performance, but rather increased it. This suggests to us that many of these remaining 33 SNPs may show true influences over mathematical ability which when analysed alone, our sample was underpowered to detect.

As the true test of association is replication, we are currently seeking out a comparable sample with mathematics ability data. With access to 1) existing ALSPAC mathematics scores, and 2) existing genotype data - collected on Illumina microarrays - we would be able to simply and easily test for the association of the 43 implicated SNPs (or where necessary, their proxies on the Illumina platform) with mathematical ability in the ALSPAC sample. As quantitative genetic results indicate that some of the genetic action over mathematics is general across ages, whilst some is not, we propose to investigate the action of the 43 SNPs over mathematical ability at all ages for which there is data available (we have been informed that this is 4-5, 11 and 15 years). Further to this, we are especially interested in investigating the combined influence of 1) the top 10 SNPs and 2) all 43 SNPs over mathematical ability. As mentioned above, this may be achieved by simply summing the math-increasing alleles possesed by each subject, and investigating the relationship of this SNP-set score with mathematical ability. If linear models are used to investigate SNP associations, then a simple alternative might be to add all 10/43 SNPs into the model. Whilst we would greatly prefer to conduct these analyses ourselves - we do understand that there may be restrictions on the release of some or all of the data we have requested, and so would also be very happy to work closely with ALSPAC researchers on this project.

As much of the work our group conducts is funded by the Wellcome Trust, we follow the same open access publishing policy as ALSPAC. We will therefore ensure that any publications resulting from this collaboration will be freely available to all researchers via PubMed Central.

ALSPAC is an important resource in the study of child development. Researchers using the cohort have shown an association between factors such as antenatal diet, maternal and paternal anxiety and depression and later child development.(1-5) There is strong evidence that child development and school performance are also influenced by socioeconomic factors including: parental occupation, marital status, income and parental education. (6-10) Maternal smoking and social adversity, specifically during pregnancy, were independently associated with an increase in risk of child symptoms of inattention and hyperactivity in a large multi-centre study of environmental risk factors for ADHD.(11) However, the causal mechanism of how childhood adversity affects development is unclear.

Early parent-child interaction is the basis of the attachment relationship which in turn allows the child to develop a template for social and emotional relationships.(12) Although there has been work looking at the effect of parenting and childhood attachment difficulties on general health, behaviour and self regulation, potentially mediated through the hypothalamic-pituitary-adrenal axis, the evidence base for the effect of parenting on other aspects of childhood development and later scholastic competence is less clear.(13, 14) The NICHD Study of Early Child Care and Youth Development, looking at 1000 US families, showed that early attachment predicted emotional development and language skills in younger children but not cognitive functioning; however the effect of attachment was dependent on socioeconomic background.(15, 16)

Dr Waylen and Professor Stewart-Brown have worked extensively with ALSPAC and have developed and validated a parenting measure which incorporates parental reports of attitude, behaviours and feelings towards the child. This measure of parenting has been shown to be independently associated with maternal reported child health.(17) In addition, ALSPAC has repeated measures of self-reported parenting activity scores between 6 months and 38 months. Professor Emond, Dr Deave and Dr Lingam have experience in the analysis of the modified Denver Developmental Screening Test (modified DDST) measured at 6, 18, 30 and 42 months along with measures of later development including motor coordination, speech and language skills, non verbal skills, social communication, and academic ability (reading and spelling).(2, 18)

Research questions

1. What is the effect of parenting on child development measured using standardised tests, controlling for potential confounding and modifying factors?
2. To what extent does parenting mediate the effect of known risk factors for child development such as socioeconomic status and parental education?

Methods

The primary exposure variable will be the parenting measure developed by Dr Waylen and Professor Stewart-Brown. A secondary exposure variable Parenting Activity will be developed using the ALSPAC parenting activity scores.

Outcome variables will be the modified Denver Developmental Screening Test (modified DDST) measured at 6, 18 and 30 months and later developmental measures including inattention and hyperactivity using the Development and Well-Being Assessment- DAWBA; difficulties in social cognition (The Social and Communication Disorders Checklist- SCDC; poor reading skills (using the Weschler Objective Reading Dimensions- WORD and NARA II); expressive language and comprehension using the WOLD, nonverbal impairment using the DANVA and Cognitive ability using the abbreviated WISK III measured between 7 and 9 years. (19-25)

A series of multivariable linear regression and logistic regression models (for continuous and binary outcomes respectively) will be created to look at the associations between parenting measures and child development accounting for potential confounding and mediating factors identified from the literature. Adjustment will be made for the bias introduced by missing data using multiple imputation by chained equation.(26)

Variable needed

Dr Lingam already has a dataset containing the developmental variables and some of the parenting activity scores needed for the analysis as part of his PhD looking at the causes and consequences of developmental coordination disorder. Use of the derived parenting measure has been granted by Dr Waylen and Professor Stewart Brown. Full details of all the variables in the analysis are attached in table 1.

Power calculation

The prevalence of suboptimal parenting (increased hostility, resentment and hitting/shouting) has been reported in between 10 and 30% of the ALSPAC cohort at different time points.(17) Taking 7000 to be the study population (approximate children that attended focus at 7) and our outcome variable to be the lowest 10% of a given developmental trait, the relative risk that it will possible to be 80% sure of detecting at the 5% level of significance is between 1.27 and 1.42.

References

1. Hibbeln JR, Davis JM, Steer C, Emmett P, Rogers I, Williams C, et al. Maternal seafood consumption in pregnancy and neurodevelopmental outcomes in childhood (ALSPAC study): an observational cohort study. Lancet. 2007 Feb 17;369(9561):578-85.

2. Deave T, Heron J, Evans J, Emond A. The impact of maternal depression in pregnancy on early child development. BJOG. 2008 Jul;115(8):1043-51.

3. Ramchandani P, Stein A, Evans J, O'Connor TG. Paternal depression in the postnatal period and child development: a prospective population study. Lancet. 2005 Jun 25-Jul 1;365(9478):2201-5.

4. Ramchandani PG, Stein A, O'Connor TG, Heron J, Murray L, Evans J. Depression in men in the postnatal period and later child psychopathology: a population cohort study. J Am Acad Child Adolesc Psychiatry. 2008 Apr;47(4):390-8.

5. Talge NM, Neal C, Glover V. Antenatal maternal stress and long-term effects on child neurodevelopment: how and why? J Child Psychol Psychiatry. 2007 Mar-Apr;48(3-4):245-61.

6. Brooks-Gunn J, Duncan GJ. The effects of poverty on children. Future Child. 1997 Summer-Fall;7(2):55-71.

7. Duncan GJ, Brooks-Gunn J. Family poverty, welfare reform, and child development. Child Dev. 2000 Jan-Feb;71(1):188-96.

8. Votruba-Drzal E. Economic disparities in middle childhood development: does income matter? Dev Psychol. 2006 Nov;42(6):1154-67.

9. Prathanee B, Thinkhamrop B, Dechongkit S. Factors associated with specific language impairment and later language development during early life: a literature review. Clin Pediatr (Phila). 2007 Jan;46(1):22-9.

10. Stanton-Chapman TL, Chapman DA, Bainbridge NL, Scott KG. Identification of early risk factors for language impairment. Res Dev Disabil. 2002 Nov-Dec;23(6):390-405.

11. Rodriguez A, Olsen J, Kotimaa AJ, Kaakinen M, Moilanen I, Henriksen TB, et al. Is prenatal alcohol exposure related to inattention and hyperactivity symptoms in children? Disentangling the effects of social adversity. J Child Psychol Psychiatry. 2009 Feb 27.

12. Bowlby J. Attachment and loss: Attachment Vol1 London: Hogarth Press; 1969.

13. Rees CA. Thinking about children's attachments. Arch Dis Child. 2005 Oct;90(10):1058-65.

14. Stewart-Brown SL, Fletcher L, Wadsworth ME. Parent-child relationships and health problems in adulthood in three UK national birth cohort studies. Eur J Public Health. 2005 Dec;15(6):640-6.

15. Belsky J, Fearon RM. Infant-mother attachment security, contextual risk, and early development: a moderational analysis. Dev Psychopathol. 2002 Spring;14(2):293-310.

16. Friedman SL, Boyle DE. Attachment in US children experiencing nonmaternal care in the early 1990s. Attach Hum Dev. 2008 Sep;10(3):225-61.

17. Waylen A, Stallard N, Stewart-Brown S. Parenting and health in mid-childhood: a longitudinal study. Eur J Public Health. 2008 Jun;18(3):300-5.

18. Lingam R, Hunt L, Golding J, Jongmans M, Emond A. Prevalence of developmental coordination disorder using the DSM-IV at 7 years of age: a UK population-based study. Pediatrics. 2009 Apr;123(4):e693-700.

19. Frankenburg WK, Dodds J, Archer P. The Denver II: a major revision and restandardization of the Denver Developmental Screening Test Pediatrics. 1992;89 (1):91-7.

20. Goodman R, Ford T, Richards H, Gatward R, Meltzer H. The Development and Well-Being Assessment: description and initial validation of an integrated assessment of child and adolescent psychopathology. Journal of Child Psychology and Psychiatry. 2000 Jul;41(5):645-55.

21. Skuse DH, James RS, Bishop DV, Coppin B, Dalton P, Aamodt-Leeper G, et al. Evidence from Turner's syndrome of an imprinted X-linked locus affecting cognitive function. Nature. 1997 Jun 12;387(6634):705-8.

22. Rust J, Golombok S, Trickey G. WORD Wechsler Objective Reading Dimensions Manual. Sidcup, UK: The Psychological Corporation; 1993.

23. Rust J. WOLD Wechsler Objective Language Dimensions Manual. London, UK: The Psychological Corporation; 1996.

24. Nowicki S, Duke MP. Individual differences in the nonverbal communication of affect: the Diagnostic Analysis of NonVerbal Accuracy scale. Journal of Nonverbal Behaviour. 1994;18:9-35.

25. Weschsler D, Golombok S, Rust J. WISC-IIIUK Wechsler Intelligence Scale for Children -Third Edition UK Manual. 3rd ed. Sidcup, UK: The Psychological Corporation; 1992.

26. Stuart EA, Azur M, Frangakis C, Leaf P. Multiple imputation with large data sets: a case study of the Children's Mental Health Initiative. Am J Epidemiol. 2009 May 1;169(9):1133-9.

ALSPAC provides an ideal opportunity for a pilot study to explore the extent to which the newly-designed "contour card" test indicates the integrity of any visuoperceptual functions in children aged 11-13 years. ALSPAC is an ongoing birth cohort study with a large amount of prospectively-collected data on visual functions and other abilities, in a contemporary sample of people who are now in their late teens. The contour cards were used in one of the ALSPAC clinics when the children were aged 11 years. An estimate of visuoperceptual skills in the children has been obtained by asking their mothers a battery of questions about their child's visuoperceptual abilities (based on Houliston et al., 1999). Both the raw visuoperceptual question responses, and the results of a factor analysis of these data, are being prepared for publication. We propose to compare the ALSPAC cohort members' performance in the contour card test at 11 with their visuoperceptual skills (as elicited in these questions), and their reading abilities, as theory and previous research predict that these might be linked with performance in the contour test. We would also include basic demographic data such as age and sex, and relevant eye problems such as strabismus and amblyopia. Outputs would be an MRes thesis to University of Bristol and if appropriate, a paper in a peer-reviewed journal.

Background

One of the oldest unsolved problems in vision science is how the visual system groups spatially separate elements of a display to form a coherent whole (May and Hess 2007). In vision science this is termed perceptual grouping, which refers to our ability to extract visual information from lower-level features to obtain perception of a higher-level structure. Psychophysical research has attempted to delineate principles by which local orientations and motions are combined across space to facilitate the detection of simple spatial contours. The perceived global structure of a scene depends heavily on the configuration and properties of its constituent local visual features (Ledgeway et al., 2005). Kovacs et al., 1999 developed a test of perceptual grouping, using "contour integration", that is suitable for use with paediatric patients in a clinical setting. This was motivated by research (1996) which found the detectability of a closed path (contour) defined by Gabor elements is significantly degraded in amblyopic eyes. These studies found that performance was degraded in strabismic, but not anisometropic amblyopia. Cells in the early parts of visual pathway have small receptive fields that are well tuned for orientation, direction of motion, and disparity (Norcia et al., 2005). Contours defined by Gabor patches cannot be detected by large filter mechanisms operating on the scale of the contour itself. Such elements force the observer to detect the contour on the basis of long-range comparisons between local orientation measurements at the defined spatial scale. The contour test has been devised to test perceptual grouping and has been shown to detect differences in people without ocular problems vs. those with amblyopia, as the above references mention. However, it has not been demonstrated whether the contour test can be useful as a measure of higher visual functions in children. We are interested to test children with problems in integrating different aspects of visual information (ie visuoperceptual problems), specifically those with "simultanagnosia". "Simultanagnosia" is a visuo-perceptual difficultly linked to object recognition in a crowded scene. This is thought to involve difficulties in using perceptual grouping to see the bigger picture made up from a number of component elements, thus "simultanagnosia" may be associated with poor performance in the contour test.

Details of the Contour detection card test

The cards consist of a closed chain of Gabor signals that roughly model the receptive field properties of orientation-selective simple cells in V1 (Kovacs et al., 1999). Contours defined by Gabor patches force the observer to detect the contour on the basis of long-range comparisons (Pennefather et al., 1998) for orientation differences of up to +/- 60(masculine ordinal indicator) amongst elements, which implies an association field and the path is reliably identified (Field et al., 1993; Loffer, 2008). Normal observers' performance on contour task starts to degrade at contrasts below 7-10% whilst amblyopes had thresholds outside of the 95% confidence interval range of normal observers (Kovacs et al., 2000). Contour integration deficits thus constitute an aspect of visual function that is disrupted by abnormal visual experience (Pennefather et al., 1999). (See below for an example of contour detection card).

The parameter (delta) is the ratio of background element spacing to contour element spacing. Variation of the parameter allows one to isolate first-order and second-order integration mechanisms, since the detection of the contour at values of (delta) less than 1 is impossible using first-order cues alone. The advantage of using (delta) parameter is that one can directly study the efficiency of long-range interactions that contribute to the integration of spatially distributed objects. By changing (delta), only the signal-to-noise ratio changes, while the shape of the contour, the global and local curvatures, the number of contour elements, the length of the contour, the spacing along the contour and the eccentricity of the elements are all kept constant. Contour integration deficits were found predominantly when the task required second-order information (ratio drops below 1) (Kovacs et al., 1999).

Few studies have been conducted to explore to what extent the contour test reflects day-to-day performance in particular tasks. However, adults with reading difficulties (diagnosed with dyslexia) have been reported to perform less well in the contour test than age-matched adults without reading problems (Simmers et al) .

Simultanagnosia

Simultanagnosia is a visual deficit described as a relative inability to comprehend more than one visual element in a scene and hence picture the whole. It has been described after prenatal damage to the brain, or after later-acquired parietal lobe injuries. We would like to test for covariation between results in the test of 'perceptual grouping' (the contour test) in children aged 11 and the reports of childrens' mothers on their abilities in the questions designed to elicit symptoms of visuoperceptual difficulties, including simultanagnosia. The questions asked in the ALSPAC questionnaire were originally devised to aid history-taking in patients with hydrocephalus, who often have vision and visuoperceptual problems. Questions about visuoperceptual problems have not been widely used in samples of normally developing children. The ALSPAC mothers' responses to the questions have been analysed looking for common underlying themes, using principal components analysis (PCA) and the data suggest that the mothers' responses cluster around 3 "factors": Factor 1 correlates with questions about seeing objects surrounded by clutter (similar to simultanagnosia as described above); Factor 2 correlates with visuomotor problems (difficulties making judgements about positions of self or objects within 3D space), and Factor 3 with questions about recognition of faces. The first two are thought to be mediated primarily by a part of the brain known as "the dorsal pathway" and the latter by another area called "the ventral pathway". These hypothesized functional sub-divisions within the brain are based on experimental evidence and on observations in clinical literature: the dorsal pathway is thought to carry information about where objects are in space, whilst the ventral pathway is more responsible for fine detail and recognition.

Objective

We would like to test the childrens' raw visuoperceptual scores as reported by the mothers, and 3 PCA-derived factors of visuoperceptual skills, in comparison with the contour test results. We will take account of other potentially important variables such as visual acuity (at appropriate age) and factors known to impair performance in the contour test eg strabismus and amblyopia. We would also like to look at a measure of reading ability with respect to the contour results, to estimate whether the contour test will usefully predict reading difficulties in this cohort. We hypothesize that the contour test results will show stronger associations with Factor 1 than with Factors 2 or 3. We also hypothesize that some children with reading difficulties (ie reading worse than that predicted by their IQ) will perform less well in the contour test than will children with reading skills commensurate with their IQ.

We therefore request access to these specific variables:

* Child's sex

* Mother's highest educational attainment

* Parent's socio-economic status (SES from pregnancy questionnaires)

* Child's reading ability at 13 (TOWRE test at tf2)

* Childs IQ (WISC at F8 clinic)

* Child's visual status (strabismus and/or amblyopia assessed at F7 clinic)

* Child's contour test results (F11 clinic)

* Child's visuoperceptual skills (questions asked of mum in 13-yr child-based questionnaire).

The analytic strategy will start with tests of correlation, either parametric or non-parametric depending on the distribution of the data. We will then use Logistic regression and/or Generalised Linear Modelling (GLM) to obtain estimates for the associations between the contour test results and either reading or visuoperceptual skills, adjusted for relevant potential confounders.

References

Field, D.J., Hayes, A., Hess, R.F. (1993) Contour integration by human visual system: evidence for a "local association field" Vision Res, 33, 173-93

Houliston, M.J., Taguri, A.H., Dutton, G.N., Hajivssiliou, C., Young, D.G. (1999) Evidence of cognitive visual problems in children with hydrocephalus: a structured clinical history-taking strategy. Dev Med Neurol, 41, 298-306

Kovacs, I., Kozma, P., Feher, A., Benedek, G. (1999) Late maturation of visual spatial integration in humans. Proc Natl Acad Sci USA, 96, 12204-9

Ledgeway, T., Hess, R.F., and Geisler, W.S. (2005). Grouping local orientation and direction signals to extract spatial contours. Empirical tests of "association field" models of contour integration. Vision Research, 45, 2511-2522

Loffler, G. (2008) 'Perception of contours and shapes: Low and intermediate stage mechanisms', Vision Research, 48, 2106-27

May, K.A. And Hess, R.F. (2007). Dynamics of snakes and ladders. Journal of Vision, 7, 1-9

Norcia, A.M., Sampath, V., Hou, C. (2005). Experience-expectant development of contour integration mechanisms in human visual cortex. Journal of Vision. 5, 116-130

Pennefather, P.M., Chandna, A., Kovacs, I., Polat, U., and Norcia, A.M., (1999) Contour detection threshold: repeatability and learning with 'contour cards'. Spatial Vision 12, 257-266

Simmer and Bex. Deficits in Visual Contour Integration in Dyslexia. IOVS 2001 42: 2737 - 2742.

Smoking has been found to be associated with an increased risk of depression, although the direction of causality is unclear. Munafo et al (2008) found that in ALSPAC smoking cessation was associated with a reduction in depression symptom score. However, this finding could be explained by confounding by other factors, or reverse causation where women who are feeling more positive are more likely to stop smoking. Freathy et al (2009) have identified a genetic variant in the CHRNA5-CHRNA3-CHRNB4) gene cluster which is strongly associated with quitting smoking in pregnancy in ALSPAC, but which is not associated with typical confounding factors.

We would like to use this variant to carry-out a Mendelian randomization analysis to determine whether a causal association between quitting smoking during pregnancy and a reduction in depressive symptom score exists.

We already have data on depression scores and potential confounders which has been used for another analysis of folate and depression so we would only require the genotype data in order to carry-out this analysis.

References:

Freathy RM, Ring SM, Shields B, Galobardes B, Knight B, Weedon MN, Smith GD, Frayling TM, Hattersley AT.A common genetic variant in the 15q24 nicotinic acetylcholine receptor gene cluster (CHRNA5-CHRNA3-CHRNB4) is associated with a reduced ability of women to quit smoking in pregnancy. Hum Mol Genet. 2009 May 9. [Epub ahead of print]

Munafo MR, Heron J, Araya R. Smoking patterns during pregnancy and postnatal period and depressive symptoms. Nicotine Tob Res. 2008 Nov;10(11):1609-20.

Using an existing IQ GWAS dataset, we have conducted a targeted candidate gene and candidate region analysis for intelligence. We identified several new genes in previously identified candidate regions. We currently have three other replication samples, and would also like to add the ALSPAC study as an independent relication.

We will need to have access (through an ALSPAC collaborator) to the (results of association analysis of) IQ scores of all individuals and to the genotypes of SNPs in three genes that were obtained using the Illumina 317k platform.

23andMe, Inc. (the "Company") is excited to support the Avon Longitudinal Study of Parents and Children ("ALSPAC") by providing the genotyping support described in this letter agreement. With the goal of genotyping 11,000 children already enrolled in the ALSPAC study, the Company will provide Illumina Human550+ Quad DNA Analysis BeadChips (each of which can process four samples and analyses approximately 610,000 loci and each of which contain the custom content generated and designed by 23andMe) (the "BeadChips") to process a maximum of 11,000 samples, plus another 1,000 for failures in processing.

The Company will ship that number of BeadChips to process approximately 1,000 samples to the Wellcome Trust Sanger Institute laboratory ("Sanger") and the remainder of the BeadChips to the National Genetics Institute laboratory ("NGI") (collectively the "Laboratories"), at which Laboratories the samples will be genotyped. Sanger's processing fees per sample shall be as set forth on Exhibit A, which cost shall not exceed the actual cost of processing by Sanger as represented by the Sanger. The Company will pay these processing costs as the genotyping occurs and is confirmed to the Company on written invoice. The Company will also pay the processing costs for samples processed by NGI pursuant to the terms of a separate agreement. The University of Bristol represents that only samples of children already enrolled in the ALSPAC study will be shipped to the Laboratories to be genotyped with the BeadChips and that the 11,000 number of samples processed shall only be exceeded to re-process samples that fail processing up to the maximum of 12,000. The Company is aware that genotyping data produced by the Laboratories of the ALSPAC participants under this letter agreement shall be the property of University of Bristol and reintegrated into the ALSPAC dataset.

As consideration for the support described above, each of Bristol University and Sanger, to the extent applicable, agrees that: 1) it shall not utilize the BeadChips except for the purposes set forth in this letter agreement, 2) it shall not utilize any information provided by 23andMe, Inc. to process the BeadChips, including but not limited to decoding files or other services provided to aid in the decoding of the DNA samples using the BeadChips, other than in order to process the BeadChips for the purposes set forth in this letter agreement (the BeadChips and information provided therewith the "Proprietary Information"), 3) shall maintain as confidential the Proprietary Information, applying the same measures to protect such Proprietary Information as it does its own confidential information, and 4) that 23andMe shall be permitted to discuss and announce that it is funding the genotyping of the ALSPAC children and that it may send a Research Fellow to analyze the ALSPAC dataset.

The Company agrees that the Laboratories shall provide the resulting data from the genotyping directly to the University of Bristol and that the Company shall not receive such data from either laboratory. However, if either laboratory requests aid from the Company in processing the data under this letter agreement, the Company shall be happy to provide reasonable assistance.

The University of Bristol agrees that a Research Fellow can be attached to the MRC Centre for Causal Analyses and Translational Epidemiology at the Department of Social Medicine, University of Bristol, where they will be able to analyze the data in the same manner as the other analysts, for a period of up to six (6) months subject to the terms of a secondment agreement to be agreed between University of Bristol and 23andMe.

Although religious practice is well recognised in the American literature as related to positive health, very little research has been carried out in the UK.

I deliberately asked Ursula King to develop questions on religious belief and practice to be administered to the parents in pregnancy (D and PB files) with the aim of assessing how well religious belief related to the ability to look at resilence when major life events had occurred.

This project therefore will compare those with and without religious beliefs in regard to their response to major life events that have occurred subsequent to the religious belief questions being administered. The current suggestion is that this should be the 8 months major life events scale, which considers life events after the birth of the child.

The outcomes to be considered will be those measured between 8 months and 5 years and will include:

1. Maternal depression and anxiety subsequent to 8 months (allowing for prenatal depression and anxiety).

2. Parenting activities and strategies (allowing for prenatal attitudes to parenting).

3. Behaviour of the parent with the child (e.g. the frequency with which the mother is irritated and or loses their temper).

4. Relationship between the parents (allowing for prenatal relationships)

5. Use of alcohol and illicit drugs (compared with prepregnancy and prenatal assessments).

6. Later religious belief (as measured at 61 months).

There are a variety of factors that will be taken into account in the logistic and multiple regression analyses: these will include parental ages, number of other children in the household, social network and social support, parental personality, parental education levels, childhood life events,life events prior to the birth of the child, and other factors that distinguish those who have a belief system from those who do not at the time of pregnancy.

We wish to examine the dietary patterns in the ALSPAC partners (when children are 47 months of age) which will be obtained using Principal Components Analaysis. This work will feed into project B 716, which examines the association between maternal and child dietary patterns. This work was recently well received at the ICDAM7 conference in Washington and we feel it is important to also take into account any effects of partner's diet.

Before we add partner's patterns into our models forming project B716, we plan to publish a paper first, describing partner's dietary patterns; examining social and demographic associations with them and also examining associations with maternal dietary patterns assessed at the same time.

We request permission to apply for a research grant to examine cord blood samples in the ALSPAC offspring. This proposal will determine whether offspring metabolic, vascular and inflammatory phenotypes are predetermined by the time of birth. Specifically we wish to measure IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests in cord blood. We are aware that some analysis of cord blood has already been performed in small subsets, specifically leptin in 197 infants1, and IGF-II, soluble IGF2R, insulin, IGF-I, IGF-binding protein-1 (IGFBP-1), and IGFBP-3 in 199 infants2. Our intention is to extend the measurement of leptin, IGF-I and insulin to the remainder of the ALSPAC cohort and examine additional new analytes.

This work builds on several current studies undertaken by the applicants. First is the analysis of lipids, inflammatory markers, insulin and liver function at age 9, 15 and 17 years. To date we have measured leptin, adiponectin, insulin, lipids, CRP, liver function tests, and IL-6 in non-fasting plasma samples taken at age 9 years old. Analysis of fasting lipids, glucose, insulin, CRP, liver function tests and IL-6 at age 15 years has also recently been completed. Additionally we are currently funded to analyse fasting glucose, insulin, lipids & liver function tests in the cohort at age 17 (Lawlor PI). Secondly, the proposed study will also build on the inspection and abstraction of obstetric data from the ALSPAC cohort and the follow-up studies of the mothers, with funds available and analyses currently being completed on ALSPAC mother's fasting blood for glucose, insulin, pro-insulin, lipids and inflammatory markers now 17 years after the index pregnancy (Davey Smith and Lawlor PIs). Thirdly. the proposed study will also utilise the recently completed genome-wide association study (GWAS) of the offspring (Davey Smith PI) and the recently funded GWAS of the ALSPAC mothers (Lawlor PI), to examine the independent contributions of each genotype and their interaction in the determination of cord analytes. This combination of data, together with the additional data we plan to collect in this proposal, will allow clarification of the role of maternal lifestyle and pregnancy outcome on the offspring metabolic, vascular and inflammatory phenotypes, and whether variations in these phenotypes at birth track into later childhood and early adolescence. Additionally if we can show significant correlations between cord parameters and the same measures in later life, this then provides some rationale for future birth cohort studies to have cord measures as legitimate surrogates.

We would like permission to address the following objectives in relation to cord blood analytes:

a. Describe the distribution of IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests in cord blood.

b. Determine the associations of maternal weight gain in pregnancy, blood pressure change in pregnancy, maternal smoking, alcohol and dietary behaviour in pregnancy, onset of gestational diabetes/glycosuria, mode of delivery, parity and preterm birth on cord IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests.

c. Determine whether variation in maternal vitamin D, parathyroid hormone and calcium levels in pregnancy are associated with cord IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests.

d. Determine the associations of cord IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests with offspring birthweight, placental weight and feto-placental index.

e. Determine the prospective associations of cord IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests with offspring fat mass and change in fat mass and growth trajectories from birth to age 17.

f. Determine the prospective associations of cord IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests with the previously determined repeat measures of these analytes at age 9, 15 and 17 years.

g. Determine the prospective associations of cord IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests with ultrasound assessment of liver fat deposition, liver function tests, lipids, insulin and glucose at age 17.

h. Determine the prospective associations of cord IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests with offspring educational attainment.

i Determine the association of cord IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests with maternal cardiovascular risk profile in later life (lipids, carotid intimal-medial thickness, blood pressure, height, weight, BMI, fat and lean mass, insulin and glucose).

j. With available data (10,000 mothers and 3,000 offspring or larger if additional grants funded) complete a genome wide-association study of cord IGF-I, leptin, insulin, cholesterol, triglyceride, HDL-C, CRP and liver function tests

For objectives g, h, i and j this will be performed on completion of the 17 year clinic, the BHF funded follow up study of the mothers and the GWAS of the mothers (Lawlor PI for mothers follow-up clinic and for maternal GWAS and for grant that funds taking of fasting blood samples and completion of fasting glucose, insulin, lipids, liver function and inflammatory marker tests).

In order to determine the extent to which any associations of maternal characteristics with offspring cord blood levels reflect genetic, intrauterine or background socioeconomic mechanisms we will employ and compare results from three methodological approaches - multivariable regression models; within parental comparisons and Mendelian randomization studies, using maternal genotype adjusted for offspring genotype as an instrumental variable for the causal effect of an intrauterine exposure.

Request for data access to allow generation of pilot data

To facilitate the provision of pilot data for a grant submission, we request permission to examine the previously reported cord analytes of insulin, leptin and IGF-I from the In Focus group1, 2, with subsequent anthropometric and DXA measures at age 9 and 15, and repeat measures of leptin and insulin at age 9, and insulin at age 15 if available.

Background and justification of analytes

Birthweight and fetal insulin

The epidemiologic observations that smaller size or relative thinness at birth and during infancy is associated with increased rates of coronary heart disease, stroke and type 2 diabetes mellitus, in adult life have been extensively replicated3, 4. Although epidemiology studies have largely examined the association of birthweight with adult disease outcomes, because of its ready availability in many datasets, it is increasingly recognised that birth weight is most likely acting as a proxy for other exposures, including maternal diet, smoking, alcohol and other intrauterine exposures and genetic variants.

Birthweight is determined, in part, by fetal metabolic and hormonal responses to intrauterine influences. Pedersen was the first to suggest that maternal hyperglycaemia would result in excessive transfer of glucose to the fetus and the compensatory fetal hyperinsulinaemia would drive fetal growth and deposition of fetal adipose tissue5. We and others have confirmed that in maternal type 1 diabetes maternal hyperglycaemia is associated with fetal hyperinsulinaemia and increased birthweight6. We have also identified that cord insulin is the principal positive associate of the observed increases in both birthweight and neonatal adiposity in offspring of mothers with type 1 diabetes7. More recently, these positive associations have been extended to the euglycaemic range of glucose during pregnancy, with the HAPO study clearly demonstrating that there is positive linear relationship between maternal glucose and birthweight and neonatal adiposity, and that this relationship is mediated by fetal insulin production8, 9. Furthermore, there is some evidence (most convincing from populations, such as the Pima Indians, with very high prevalence of obesity and type 2 diabetes) that higher levels of maternal glucose in pregnancy are associated with greater adiposity and abnormal glucose tolerance in offspring in later life10-12 and can influence the penetrance of genetic syndromes of diabetes13. Consistent with an insulin mediated effect, fetal hyperinsulaemia has been associated with excess adiposity and elevated plasma glucose and insulin during childhood in a Caucasian population14. Given the HAPO results, it is reasonable to wonder the extent to which these associations in later life are present across the whole range of fetal insulinaemia15. Assessment of cord insulin in the ALSPAC cohort will allow us to examine the association of fetal insulin with offspring adiposity and glucose tolerance throughout childhood and adolescence.

IGF-1

Cord IGF-I is positively associated with birthweight and placental weight2, 7 and IGF-I deletion or reduced receptor expression in humans are both associated with a reduction in birthweight and placental weight16, 17. Conversely, in animal models prolonged administration of exogenous IGF-I to growth restricted fetuses substantially increases body and placental weight18. In offspring of mothers with type 1 diabetes, we have demonstrated a relationship of IGF-I and birth weight independent of insulin7, in keeping with previous observations from the ALSPAC cohort2. Furthermore, we have recently demonstrated that cord IGF-I is the principal correlate of placental sub-structure (data submitted), and that placental and fetal growth closely correlate, even if there is fetal hyperinsulinaemia19. IGF-I is also an associate of feto-placental index in offspring of mothers with type 1 diabetes19, with experimental and epidemiological studies suggesting that mismatch of placental and fetal growth are associated with postnatal abnormalities in cardiovascular, metabolic and endocrine functions 20, 21. Therefore, although IGF-I at birth does not track during childhood22, there is significant potential for an association with later development, through its associations with placental development and matching of fetal and placental growth.

Adiposity and Leptin

Cord leptin strongly correlates with maternal and neonatal adiposity7, 23-27, and also neonatal whole body bone mineral contents and estimated volumetric bone density28. Although neonatal adiposity is largely determined by maternal glycaemia9, maternal smoking has also been associated with a reduction in cord leptin independent of influences on birthweight29. Cord leptin may therefore be influenced by maternal lifestyle behaviour - potentially through an impact on both fetal and placental leptin production. Lower cord blood leptin levels, but not variation in adiponectin levels, have recently been shown to be associated with more pronounced weight gain in the first 6 months of life and higher BMI at 3 years of age30. This is consistent with studies demonstrating that early upward crossing of growth centiles is associated with an increased risk of adiposity in later life31. However, these initial results require replication and currently the relationship between cord leptin and outcomes, including adiposity and skeletal development, in later life remain to be determined.

Inflammatory Markers

CRP and ICAM-1 are increased in adults with obesity32-36 and type 2 diabetes37, 38 and similar relationships also appear to be present in childhood39. We have previously demonstrated that offspring of mothers with type 1 diabetes (OT1DM), have increased fat mass and an associated increased circulating leptin concentration40, 41. Furthermore, we have shown that CRP and ICAM-1 are not only increased in OT1DM but are associated with cord leptin and skin fold thickness42. Notably leptin was also associated with IL-6, a major stimulus for CRP production, raising the possibility that fetal adipose tissue is not only responsible for endothelial activation but induction of a pro-inflammatory phenotype which is already evident by the time of birth. Maternal HbA1c was also associated with CRP supporting a role of maternal glycaemic control in the inflammatory phenotype at birth. This finding of an elevated CRP in OT1DM and relation with maternal glycaemia has now also been replicated43, and the possibility that this extends to lower glucose values given the continuous relationship of glucose to offspring birthweight and adiposity9 requires further investigation. Inflammatory markers are also known to track in childhood44 and to be associated with later metabolic37, 38 and vascular disease45. A single study has found raised inflammatory markers in OT1DM in childhood46. We and others have demonstrated that this inflammatory phenotype is present at birth in OT1DM and that it is particularly related to fetal leptin42, 43. Collectively this raises the possibility that there is potential in-utero effects on sub-clinical inflammatory phenotypes, which directly relate to maternal glycaemia and fetal hyperinsulinaemia and adiposity.

Lipids

With respect to lipids we have also previously determined that maternal diabetes is associated with lower fetal lipids in male offspring, in particular HDL-C (thus significantly higher cholesterol to HDL-C ratio)47. Furthermore, we identified that perturbances in IGF-1 and leptin, rather than insulin - may be the major determinants of HDL-C in-utero47. Lipid metabolism and inflammation are linked via hepatic lipid metabolism with elevations of plasma triglycerides occurring during acute adult inflammatory responses48, consequently, CRP and triglyceride are positively related in children33 and adults49. We have also demonstrated that this relationship is present at birth in OT1DM, with markers; ICAM-1, CRP and IL-6 potentially all acting as independent determinants of triglyceride. The possibility that dyslipidaemia and subclinical inflammation - major determinants of adult disease may coexist at birth, requires further study, particularly if we can identify maternal antecedents which may be modified in the future.

Liver Function Tests

Non alcoholic fatty liver disease (NAFLD) has been described in children and adolescents, with estimates of the prevalence of NAFLD based on unexplained elevated levels of alanine aminotransferase (ALT) or ultrasound range from 2-3% in general paediatric populations50, 51 to between 6-30% in obese children or adolescents52-54. Prevalence estimates in general paediatric populations may underestimate the prevalence in adolescents. A recent post-mortem study provides the most robust evidence of the potential importance of NAFLD in adolescents55. In that study, of 742 US individuals (aged 2-19 years) who had died from external causes (mostly road traffic accidents) the age, gender and ethnicity standardised prevalence of NAFLD (defined as greater than 5% steatosis on histology of the post-mortem liver) was 9.6%, with 3% of the population having non alcoholic steatohepatitis. The prevalence increased with increasing age from 0.7% in those ages 2-4 to 17.3% in those aged 15-1955. A non-human primate model has recently demonstrated that a high fat maternal diet can influence offspring liver function with an increase in fetal liver fat deposition and hepatic oxidative stress - with persistence of this phenotype at 180 days postnatal56. Furthermore, it was suggested that this related to fetal lipid rather than glucose loads. Notably cord leptin and triglyceride are associated in male OT1DM47, raising the possibility that fetal liver fat deposition may occur in conjunction with fetal adiposity. Given the potential long-term importance of NAFLD, identification of whether maternal obesity is associated with derangement of LFTs at birth and if this persists through childhood is important.

Maternal cardiovascular disease

Lastly, offspring birthweight has also been inversely associated with parental mortality, with a stronger relationship seen in mothers and a dominance of cardiovascular disease57. Given the importance of fetal metabolic and hormonal responses in determining birthweight, we hypothesise that these cord analytes will be associated with maternal cardiovascular disease risk profile in later life independent of birthweight.

Methods

All biochemical analyses will be performed at Glasgow Royal Infirmary, which adheres to UK external quality control for all parameters and is Clinical Pathology Accreditation (CPA) accredited. Plasma total cholesterol, triglyceride and HDL-C will be performed by modification of the standard Lipid Research Clinics Protocol unsing enzymatic reagents for lipid determinations. LDL-C will be calculated from total cholesterol and triglyceride using the Friedwald equation. A high degree of correlation between Friedwald calculated LDL-C and directly measured LDL-C has previously been described for cord blood58. Leptin will be measured by a highly sensitive in-house ELISA with better sensitivity at lower levels than commercial assays59. Insulin will be measured by an ELISA (Mercodia), which does not cross-react with proinsulin and has a lower sensitivity of 6pmol/l, well below the (median 22.4pmol/l, IQR15.0 -38.0) we noted in control offspring in a recent study6. CRP will be measured using a high-sensitivity, 2-site enzyme-linked immunoassay42, 60. IGF-1 will be assayed using a chemiluminescence immunoassay (Nichols Institute Diagnostics, San Juan Capistrano, CA 92675, USA) using standards referenced to WHO 1st International Reference Reagent 1988 (Insulin-Like Growth Factor-1 87/518). The limit of detection is 1*0 nmol/l. Intra- and interassay coefficients of variation (CVs) were 5*5-6.8% and 5*4-7*0%. Liver enzymes will be measured by automated analyser with enzymatic methods (all CVs less than 3%). A total of 500micro-l will be required to be shipped to Glasgow for the assays due to dead space, however it is likely that only ~300micro-l will be used.

We will request in the research grant the cost of a post-doctoral statistician to be employed at the University of Bristol under the supervision of Professors Lawlor and Davey Smith for data management and completion of the statistical analysis.

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60 Packard, C. J., O'Reilly, D. S. J., Caslake, M. J., McMahon, A. D., Ford, I., Cooney, J., Macphee, C. H., Suckling, K. E., Krishna, M., Wilkinson, F. E., Rumley, A., Lowe, G. D. O., Docherty, G., Burczak, J. D. and The West of Scotland Coronary Prevention Study, G., Lipoprotein-Associated Phospholipase A2 as an Independent Predictor of Coronary Heart Disease, N Engl J Med, 2000, 343: 1148-1155.