We wish to investigate the associations between birth outcomes, (primarily gestational age and birth weight) and SNPs with proven roles in biochemical phenotypes. Key pathways include the inflammatory response and vascular function, which has important implications for utero-placental function. We have recently performed a genome wide assocaition study of mulitple biochemistry phenotypes in the InCHIANTI study (Melzer et al PloS Genetics, May 08), that has identified, or taken to GWAS significance, many variants altering biochemical traits. This study and other recent genome-wide and large-scale association studies have provided robust evidence (P values less than 5 x 10-8) that common genetic variants, marked by key SNPs, influence the levels of various inflammatory cytokines, clotting factors, and susceptibility to aneurysm. This makes them excellent candidates for altering fetal growth and or gestational age and greatly increases the prior odds that they will influence genetic susceptibility to preterm birth through these pathways. In addition, SNPs have been identified which alter the circulating serum levels of various nutrients, hormones and growth factors, including Vitamin A, each of which is also a potential candidate for influencing susceptibility to preterm birth. In the case of Vitamin A , a known teratogen, the SNP that alters Vitamin A may alter risk of miscarriages or fetal abnormalities.

In this study we propose to test the hypothesis that each selected "functional" SNP is associated with fetal growth and or gestational age.

We wish to genotype the selected SNPs in all ALSPAC mothers and children in order to assess associations between fetal growth gestational age and both maternal and fetal genotypes.

ALSPAC is the largest and best UK study for investigating the genetics of fetal growth and perinatal outcomes as it is well-powered to detect small genetic effects, it has the availability of both maternal and fetal DNA and has detailed clinical data on birth size and pregnancy outcomes as well as other relevant maternal phenotypes. RAINE in collaboration with Lyle Palmer, and NFBC with Mark McCarthy, Leena Peltonen and Marjo-Riita Jarvelin are other potential sources of replication/meta-analysis for fetal effects.

The collaboration between Cognitive Drug Research (CDR) Ltd and the Avon Longitudinal Study of Parents And Children (ALSPAC) involved the measurement of attention in ALSPAC participants using the laptop-based CDR computerised assessment system. This collaboration was to be based around our generic collaboration agreement, a copy of which is attached, but owing to oversights, was not signed for this particular data gathering exercise.

The central dataset we use at CDR will include the CDR variables collected, and require demographic information from the ALSPAC study (gender and age required; handedness and race if available).

In keeping with the spirit of the standard CDR Collaborative Research Programme agreement (example attached), it is proposed that both parties have shared access to the data by holding identical copies.

Intended use

CDR hold a large in-house normative database of cognitive data. We propose to add the ALSPAC data to this database. Data will be anonymised, and reference to the source of the data in relation to our norms will not be made. The CDR normative database is used internally for reference and not published or sold externally.

Publication

If publication or presentation of the work occurs, both CDR and the Collaborating Group must agree the content of the publication or presentation. The 'Cognitive Drug Research computerised assessment system' must be mentioned in any publication or presentation. Representatives from both CDR and the Collaborating Group must be on the author list of any publication or presentation.

The publication policy of the ALSPAC study will be adhered to.

Generally, with any publications or conference presentations, the ALSPAC will take senior authorship, and one or more members of the CDR team who has been involved in the research will receive junior authorship.

If there are any questions relating to this proposal, Paul Snell would be happy to address them in the first instance.

05.07.2010

E-mail from Rachel - 5/7/10

I'm doing this project for my PhD - have done nearly three years, another 12 - 18 months to go I think. I've had one paper rejected, and am about to send the exec a new version. Another paper is also nearly ready for the exec.

Study Plan

In this 3-year study, the named applicant (SS) will spend 2 years working on life course models to develop and test causal pathways for myopia using the 15-year data. At the same time, data on myopic progression will be collected from cohort members who attend the 17+ clinic (estimated as 4,500?). In the third and final year, the most robust models derived from years 1 and 2 will be used to analyze the newly acquired myopia progression data (i.e. change in axial length). Outputs will include papers on both the methodologies employed, with recommendations for use in other contexts and on the predictive power of eye growth over the lifecourse analyses to predict myopia.

BACKGROUND

Steroidogenic factor-1 (SF1, NR5A1) is a nuclear receptor that plays a central role in many aspects of adrenal, reproductive and metabolic function. Deletion of Sf1 (FtzF1) in mice causes adrenal and gonadal agenesis, and several loss of function mutations in SF1 have now been reported in humans with adrenal dysfunction and/or disorders of sex development. In addition to these well-established effects, Sf1 is also emerging as an important regulator of ventromedial hypothalamic development and programming, and post-natal obesity has been reported in Sf1 knock-out mice rescued by adrenal transplantation (1, 2). Although incidental reports of weight changes in patients with SF1 mutations exist, corticosteroid administration may have influenced growth in some cases, so the true role of SF1 in humans is still poorly understood.

Recent work has shown that a non-synonymous polymorphism exists in the coding sequence of SF1 (p.G146A, rs1110061) (3, 4). Studies in our laboratory have shown that this change does not affect nuclear localization or cellular dynamics, but does result in a mild but potentially significant effect on SF1 function in the transcriptional regulation of a range of target genes. In additional limited studies published recently, this polymorphim has been shown to be associated with undescended testes or micropenis in two small cohort studies in Japan (5, 6), and with an increased incidence of type 2 diabetes mellitus in the Chinese (7).

We have now undertaken analysis of this p.G146A polymorphism in SF1 in a UK-based cohort of mothers and children in the UCL Fetal Growth Study (Professor Peter Hindmarsh). This polymorphism is present in heterozygous state in approximately 8% of the 460 children studied and is in Hardy-Weinberg equilibrium. Heterozygosity for p.G146A was associated with a reduction in placental weight (643g vs 678g, pless than 0.05) and preterm delivery (15.3% vs 4.3%; Chi-square, 7.4; p=0.02; OR 0.25, 95% CI 0.09-0.77). Although there was no significant difference in birth weight between groups, analysis of post-natal growth data in a subset of children showed that a heterozygous p.G146A polymorphism was associated with higher BMI at three years of age (17.8 kg/m2 vs 16.3 kg/m2, pless than 0.001) and a significant elevation in both systolic and diastolic blood pressure.

We are therefore interested in establishing whether these findings can be reproduced in an independent population based cohort of children in the UK. It would also be of great interest to see if an association with undescended testes or hypospadias could be seen in the UK population, although it is possible that this would be underpowered and a case-control approach would be better.

AIM

We would aim to collaborate with K biosciences to establish the SF1 polymorphism (p.G146A, G/C, rs1110061) genotype status in the ALSPAC cohort. We would agree that genetyping the entire cohort would have potential added value in the long term, and would support this approach depending on cost implications. Taqman SNP genotyping probes for this change have been obtained from ABI and validated by Professor Steve Humphries at UCL for the study of an adult cohort, thus I would assume that K biosciences would be able proceed with this project without unexpected difficulties.

We have recently shown in the ALSPAC cohort that differences are evident in food and nutrient intakes at 10 and 13 years in those young people classified as under-reporters compared to those with valid reports. Similarly, differences are evident in those completing 2/3 dietary diaries compared to 24 hour recall (papers in progress).

Following on from my PhD work, I would like to investigate any differences in dietary patterns obtained using Principal Components Analysis (PCA) and cluster analysis in these groups. To my knowledge this has not been investigated by any other studies and will therefore make a significant contribution to the literature on dietary patterns. This work will also feed into the analyses that we hope to carry out should our NIH application (dietary patterns and changes in body fat) be successful.

Attention Deficit Hyperactivity Disorder (ADHD) is a common, extremely disabling disorder that has major adverse sequelae in childhood and later life. Despite being such an important clinical problem, the aetiology and pathogenesis of ADHD is poorly understood. The available evidence shows that genetic factors are of major importance and that genes co-act and interact with environmental risk factors. The presence of antisocial behaviour in children with ADHD is an important marker of heterogeneity, indexing greater clinical severity, poorer outcome, persistent problems in adult life, a stronger association with neurocognitive deficits, especially affecting prefrontal cortical functioning and higher genetic loading.

Previous evidence had suggested that prefrontal cortical function is influenced by a valine/methionine variant in the catechol-O-methyltransferase (COMT) gene and indeed this was recently found in the ALSPAC sample (Barnett et al., 2007). We tested for association between this functional COMT gene variant and antisocial behaviour in ADHD in our well characterised sample of 240 clinic children with ADHD. We found evidence of association with the same COMT val/val genotype (Thapar et al, 2005). Since this publication, the finding has been replicated in two independent population-based samples (Caspi et al, 2008). There was significant evidence of association between the COMT val/val genotype and 1) childhood antisocial behaviour in those with ADHD in one sample, and 2) adult criminality in those with ADHD in the other. A pooled analysis of 4 published studies, showed significant association (Caspi et al, 2008).

The aim of this first stage proposal is to identify a potential mediating mechanism. Specifically we wish to test the hypothesis that

1). the COMT val/val genotype is associatied with antisocial behaviour in ADHD and

2). these effects are mediated through prefrontal cortical functioning.

The aim at this stage is to undertake analysis on existing data, develop research links with Bristol under the auspices of the Neurosciences Centres, publish findings and provide the basis for a subsequent grant /fellowship application.

The future application would aim to integrate the research we are undertaking on a clinical sample through Wellcome Trust programme funding on the genetics of ADHD with ALSPAC data. The key aims would be to test associated gene variants (gene discovery in other samples-notably our own clinical study and collaborative whole genome association studies) in a population-based sample a)for links with ADHD and antisocial behaviour in ADHD and b)investigate potential mediating mechanisms-specifically cognitive vs. emotion-based pathways.

Background

There are over 1 billion people with high blood pressure worldwide and simulations by the World Health Organisation suggest this will rise to 1.5 billion by 2020 [1]. It is estimated that blood pressure played an important part in 50% of the 16.7M cardiovascular deaths worldwide [1, 2]. In spite of the availability of multiple different therapeutic strategies, public health data from western economies shows hypertension remains poorly controlled.

The Wellcome Trust Case Control Consortium and replication of signals

In the first phase of WTCCC 2000 hypertensives from the BRIGHT Study were subjected to a genomewide scan using the Affymetrix 500 chip and compared with 3000 common controls. We found comparable numbers and distribution of suggestive association signals in the range P less than 10-4 to 10-7 compared with the 6 other diseases investigated [3]. We are actively pursuing the most promising signals from WTCCC1 and parrallel candidate gene studies in extended replication resources and have waited to approach ALSPAC until we had evidence that some of these signals had survived initial replication with blood pressure and hypertension with decreasing P values. We have also found association of SLC2A9 SNPS with urate and a borderline association with blood pressure. In a second candidate gene we have found a developmental gene that influences nephron number to relate to blood pressure. ALSPAC represents and ideal resource for evaluation of these putative associations.

Reasons for approaching ALSPAC now.

Our reasons for approaching ALSPAC childrens DNA bank now is to investigate association of these variants with early life blood pressure ( at all time points you have these measures) and flow mediated dilatation as a measure of endothelial function (at all time points). In at least 2 cases the genes are implicated in nephron development which makes investigation of early life association of blood pressure valuable. One of these genes might also be involved in control of birthweight ( testing association with measures of birth weight and anthropometry might be interesting). If there was access to renal function measures such as eGFR or urea and creatinine we would also be interested in these but we think from what we can tell these may not be available. We would be happy to receive advice on any other phenotypes you think we should test.

Genotyping at KBioscience and funding.

We provide below a list of SNPs for genotyping on fast-turn around at K Bioscience and will fund this work from our MRC Programme.

Background

Non-alcoholic fatty liver disease (NAFLD) is defined as fatty infiltration of the liver in the absence of alcohol misuse or other causes of liver damage and is probably the most common liver disorder in all ages in the developed world today. NAFLD includes a wide spectrum of liver damage, ranging from simple steatosis to advanced fibrosis and cirrhosis (this advanced form referred to as non-alcoholic steatohepetitis (NASH)). The primary cause of NAFLD is obesity and with the global epidemic of obesity the prevalence of NAFLD and its complications are increasing. NAFLD has recently been described as "a disease of our generation", as clinicians have noted a large increase in liver pathology in all ages driven by the obesity epidemic, in addition to increasing rates of alcohol consumption.

The whole spectrum of NAFLD has been described in children and adolescents. Presentation with, and progression to, cirrhosis in adolescence has also been described. Estimates of the prevalence of NAFLD, based on unexplained elevated levels of alanine aminotransferase (ALT) or USS, range from 2-3% in general paediatric populations, to between 10-30% in obese children or adolescents. These studies suggest that NAFLD is rare before adolescence, findings confirmed in a recent post-mortem study that provides the most robust evidence of the potential importance of NAFLD in adolescents. 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 NASH. The prevalence increased with increasing age from 0.7% in those ages 2-4 to 17.3% in those aged 15-19; it was very rare before the age of 10. Of importance, as duration of NAFLD in adults is associated with increased likelihood of progression to severe advanced disease, this means that it is likely that individuals with NAFLD in adolescence are at a very high risk of severe liver pathology in adulthood if fatty infiltration of their liver is not reversed (since by definition having the condition from adolescence into adulthood is a longer duration than fatty infiltration first appearing in adulthood). In one small (N=9) case study of clinically obese children who were referred because of persistently elevated ALT levels, those who complied with a hypocaloric diet were reported as having reductions in ALT levels that paralleled their weight loss. However, stronger epidemiological evidence is required to determine whether evidence of NAFLD in early life is reversible or whether changes in risk factors in early life can reverse evidence of hepatic fatty infiltration, liver damage and its associated adverse metabolic and vascular traits.

We recently found that 6% of a general (healthy) population of white American adolescents (age 12-19 years) had elevated ALT levels indicative of NAFLD (using a threshold of greater than =30 U/L to define NALFD). Furthermore, we showed marked differences in the distribution of ALT by gender (age standardised mean levels lower in girls compared with boys even after adjustment for body mass index (BMI)), which raises the question as to whether the same threshold of defining NAFLD in this age group should be used. We also found mean levels varied by ethnicity and that overtime mean ALT levels in adolescence have increased in this population. We used the publicly available US National Health and Nutrition Examination Survey (NHANES), which is an annually repeated cross-sectional survey that is used for public health surveillance and epidemiological studies in the US. Similar surveys - the Health Survey for England, the Scottish Health Survey and the Welsh Health Survey (http://www.natcen.ac.uk/natcen/pages/or_health.htm) - exist in the UK and are similarly used for public health surveillance and epidemiological studies. We sought to undertake a study of prevalence of elevated ALT and variation in ALT by gender, ethnicity and over time in adolescents in the UK but found that our national surveys did not have information on biomarkers for NAFLD. The lack of such data can be justified because, despite our and others attempts to examine the prevalence of NAFLD in adolescents using liver enzymes as biomarkers, there is currently insufficient evidence evaluating the utility of liver enzymes as biomarkers for NAFLD in adolescents in general or in the UK adolescent population in particular. Liver enzymes are relatively cheap and easy to assay. They would, therefore, be ideal for public health surveillance if they are shown to be accurate as diagnostic biomarkers in adolescents.

The diagnosis of NAFLD is suspected when there are indicative changes in liver enzymes (ALT, aspartate aminotransferase (AST), ALT:AST) and/or echogenic (fatty) liver detected by USS, together with the absence of any established causes of liver disease. A definitive diagnosis, and determination of severity, requires a liver biopsy, which is not feasible in large-scale epidemiological studies. Indeed, since liver biopsies are painful and have rare, but potentially severe, iatrogenic effects even in clinical practice liver biopsy is only recommended when there is a high level of suspicion of liver damage and clear indications that biopsy would alter clinical practice. The British Society of Gastroenterology and the British Association for the Study of the Liver guidelines for the use of liver biopsy in clinical practice state that the role of liver biopsy in the diagnosis and clinical management of NAFLD is currently not established. Similarly the American Gastroenterology Association have noted that the potential health risks, high cost and biopsy sampling errors associated with liver biopsy should be weighted against the value of the information that would be gained from performing a biopsy each patient. In clinical practice a combination of clinical history, measures of adiposity, presence of other associated morbidities, such as diabetes, elevated aminotransferases (ALT, AST and ALT:AST) and imaging modalities (USS, CT scan and MRI) are used and recommended in clinical guidelines for the diagnosis of NAFLD.

In research, elevated aminotransferases (ALT, AST, ALT:AST) have been predominantly used for case definition of NAFLD. However, there is marked variation in the thresholds used to define NAFLD across different studies, even when the same or very similar populations are used. For example, three studies have used ALT to assess NAFLD prevalence in adults from the USA NHANES survey and have used different thresholds - 19, 31 or 43 u/L in women; 40 or 43 u/L in men. Far fewer studies have examined the prevalence of NAFLD in general adolescent populations, but similar variation is found. ALT thresholds of between 30-40 u/L have been use, with identical thresholds used in females and males, despite clear evidence that in adolescence (as in adulthood) the distribution of ALT varies by gender.

There is biological plausibility for all three of ALT, AST and GGT as biomarkers for NAFLD; all are secreted by hepatocytes in response to liver damage, including NAFLD. ALT is the liver enzyme most commonly used in the assessment of liver conditions in general, including NAFLD, though its primary use has been questioned. It is a more specific marker of liver damage than AST, which is also found in the heart, skeletal muscle, kidney and other organs, and than GGT, which is expressed by most cells in humans. ALT elevation is greater than AST elevation in NAFLD and ALT is a more robust predictor of diabetes. Nonetheless elevation in both aminotransferases, ALT and AST, are recommended in clinical guidelines as biomarkers for NAFLD in adults. In NAFLD, plasma levels of both ALT and AST may be increased due to leakage from hepatocytes that have been damaged by fat accumulation. In addition, as ALT has an important role in gluconeogenesis and amino acid metabolism its greater elevation (compared to AST) may reflect increased gluconeogenesis, due to hepatic insulin resistance that is closely associated with liver fat accumulation, in addition to leakage from hepatocytes. Because of the greater ALT compared to AST elevation with NAFLD a low (below 1) AST:ALT ratio has also been suggested as a valid biomarker for NAFLD. The low AST:ALT ratio contrasts to the high (above 1) AST:ALT ratio that is typical of alcoholic liver disease.

GGT levels also increase with NAFLD, although this enzyme is not mentioned in clinical guidelines as part of the screening or diagnostic procedures for NAFLD. This is probably because it is has long been used as a marker of alcohol consumption and because it is expressed in most cell types and is therefore not a liver specific enzyme. In NAFLD, leakage from damaged hepatocytes would explain elevated levels of GGT. Thus elevated GGT levels could equally represent excess alcohol intake or NAFLD. Few adolescents are likely to have experienced the chronic alcohol abuse that is required to cause liver damage. Thus, it is biologically plausible that in adolescents GGT is also a useful biomarker for NAFLD. In this application we will use genetic variants associated with greater fat mass as proxies for fat related increase in GGT, and other enzymes (i.e. NAFLD related enzyme increase). These genetic variants are not associated with alcohol consumption and therefore we are able to use them to examine the extent to which elevated GGT, ALT and AST might be explained by NAFLD as opposed to alcohol consumption or other non-fat causes of liver damage.

Few studies have formally evaluated the utility of ALT, AST, AST:ALT and GGT as biomarkers for NAFLD in adolescents. One study in adults with the more severe form of NAFLD (non-alcoholic steatohepetitis (NASH)) examined the utility of ALT and AST as biomarkers for treatment effects in randomised controlled trials. The motivation of the investigators was their recognition of the need for relatively cheap and non-invasive surrogates even in RCTs of adults with more advanced disease (in whom biopsy would be more likely that in public health / epidemiological studies or trials of preventive interventions in health adolescents). Within a subgroup (N=102) in the trial they compared changes in ALT and AST levels to the main outcomes assessed by liver biopsy. They concluded that both aminotransferases were equally useful in RCTs of treatment for NASH in adults. Utility (assessed by receiver-operating characteristic (ROC) curves) was improved in multivariable analyses that adjusted for baseline ALT and AST levels as well as histological changes obtained from liver biopsy at baseline. In children and adolescents it has been suggested that elevated ALT levels potentially miss up to 40% of individuals with NAFLD.5;31These statements have been made largely on the basis of comparisons of elevated ALT with liver biopsy in clinical obese paediatric populations, who are unlikely to be representative of general populations. Even within clinically obese paediatric populations there are inconsistencies in the findings. For example, one study of obese Japanese children (up to age 16) found that a threshold of 30 u/L of ALT had a sensitivity of 0.92 (i.e. identified 92% of cases) for detecting NAFLD proven by USS. That study tested a particular threshold rather than using ROC curves33 to identify a threshold that maximises both sensitivity and specificity.

Thus, to date, there has been no thorough evaluation of ALT, AST, AST:ALT and GGT as biomarkers for NAFLD in general populations of adolescents. If we find that ALT, AST and GGT either alone or in combination are valid biomarkers for NAFLD they will provide a cheap and easily available marker that could be used widely for public health surveillance, epidemiological research and randomised controlled trials of preventive interventions.

In a healthy liver there should be no or very little fatty infiltration. The formal definition of NAFLD is that just 5% (or more) of hepatocytes have fatty infiltration on biopsy. On USS fat appears as a bright area. The USS indicators of fatty infiltration in the liver are bright hepatic echotexture (compared to the kidneys and/or spleen), deep attenuation and vascular blunting, and the extent of fatty infiltration can be quantified (none, mild, moderate and severe), with this quantification also shown to reflect amount of fatty infiltration on histology. In healthy individuals the liver should appear similar in echotexture to the kidneys and spleen, indicating no fatty infiltration; any indication of fatty infiltration supports a diagnosis of NAFLD if there are no other known causes. USS determined fatty liver has been shown in several studies to have high levels of sensitivity (89-90%), specificity (82-93%), positive predictive value (87-93%) and negative predictive value (87-94%), when compared with the gold standard of liver biopsy histology, for diagnosing moderate to profound levels of hepatic fatty infiltration. USS fatty infiltration has high levels of intra- and inter-rater reliability. USS cannot adequately identify liver fibrosis or cirrhosis in general asymptomatic individuals. However, neither can other radiological approaches. Indeed at present fibrosis and cirrhosis can only be diagnosed by biopsy, which as discussed above is not ethical or practical in any research study in health individuals.

Other radiological approaches including standard MRI, MR spectroscopy and CT scan are also able to accurately diagnose NAFLD. These modalities can identify different patterns of hepatic fatty infiltration, such as diffuse or nodular, but the relevance of these is currently unclear and the subject of on-going research. As yet there is no clear evidence that CT, MRI or MR spectroscopy have important benefits when compared to USS in simply identifying fatty liver infiltration.

Thus, USS provides a valid gold standard against which to evaluate ALT, AST, AST:ALT and GGT as biomarkers for NAFLD in adolescents.