(No outline received).

(No outline received).

(No outline received).

(No outline received).

Atopic diseases (including atopic dermatitis/eczema, and asthma) have increased in frequency in the last several decades (1) and now affect up to 20% of the population in the developed world. Twin and family studies have shown that predisposition to atopic disease is highly heritable (2). To date most genetic studies have focused on immunological mechanisms behind regulation of IgE and atopy, however, a primary genetic epithelial barrier defect has been anticipated (3). Recently we identified two functional genetic variants in the filaggrin gene (FLG) affecting epithelial barrier formation (Smith FJD et al., Nature Genetics, in press, preprint attached). In a second study, we have shown that these filaggrin variants show clear predisposition to this group of common conditions. Filaggrin is a key protein that facilitates terminal differentiation of the epidermis and formation of the skin barrier. Two independent variants in this gene carried by ~9% of Caucasians, result in haploinsufficiency. We have shown that these null alleles are responsible for a substantial genetic predisposition to asthma, and atopic dermatitis (Palmer C et al, under review, Nature Genetics).

In this study the two null variants were genotyped in 1008 Scottish Caucasian schoolchildren with unknown disease status (population cohort) and in 604 Scottish Children with asthma. The frequency of carriers of mutation R501X was 5.8% and the mutation 2282del4 variant was present in 3.8% of the schoolchildren, giving a combined carrier frequency of 9.6%. Both filaggrin variants were over-represented in the asthmatic cohort (mutation R501X: 9.2%, mutation 2282del4: 7.5%, combined carrier frequency: 15.7%) with carriers of either allele demonstrating a dominant risk for asthma (mutation X dominant OR = 1.6, 95%CI = 1.1-2.3, p = 0.025, mutation del4 dominant OR = 2.1 95%CI= 1.3-3.2, p = 0.002, combined genotype dominant OR = 1.8 95%CI= 1.3-2.5, p = 0.0002).

AD is co-associated with asthma (3) and since this is a major epidermal protein, null alleles should be expected to be more strongly associated with the combined AD/asthma phenotype than with asthma alone. Consistent with this, 72% of all the children in the asthma cohort carrying a null allele had AD, in contrast to only 46% of those without these filaggrin variants. A co-dominant model comparing individuals with asthma and AD to the population controls provided the best fit and the most significant association (p = 4.8x10-11).

Over the past several years, the mechanisms by which allergen exposure through the skin could initiate systemic allergy and predispose to asthma have become clearer. Epicutaneous sensitization with a protein allergen has been shown to induce a Th2-type immune response with concomitant high IgE production in mice (4). In mice sensitized with ovalbumin, cutaneous exposure of this allergen induced a T cell and eosinophil-rich cellular infiltrate along with a cytokine response that closely mirrored those seen in AD. Significantly, in addition to generating allergen-specific IgE, these mice developed airway hyper-responsiveness on intravenous challenge with methacholine after a single inhalation challenge with ovalbumin (5). These observations imply that the presence of a heritable defect in the skin barrier, in a substantial proportion of the asthmatic patients in our dataset, who would have increased susceptibility to transepidermal allergen transfer, therefore amplifying the epidermal-driven Th2 response and provoking the onset of atopic disease.

AIMS AND OBJECTIVES OF THIS PROPOSAL

This proposal seeks to further develop the initial findings detailed above and to specifically study:

1. To accurately delineate the longitudinal health risk of happloinsufficiency in this key epidermal protein with particular reference to phenotypic expression of atopic disease including atopic dermatitis, asthma, and allergic rhinitis and intermediate atopic traits such as skin prick test positivity to house dust mice and grasses and bronchial hyperesponsiveness.
2. To study the frequency of these alleles in subsets within the study population identified as having atopic dermatitis, asthma or rhinitis and all combinations of these conditions. We expect to be able to accurately quantify the population-attributable risk of these functional polymorphisms for each of these atopic phenotypes.
3. To examine possible genetic modifiers of the phenotype (gene:gene interactions) in the approximately 900 individuals we expect to identify as happolinsufficient for this key epidermal protein. Possible candidate genes for this phase of the study include genes involved in formation of the stratum corneum and in the inflammatory cascade. As this aspect of the study develops we will discuss each of these potential candidate genes individually with the committee prior to commencing any studies.
4. To explore possible gene:environment interactions in this cohort of 900 individuals. Relevant environmental parameters would include urban versus rural living, smoking by parents, keeping pets, type of accommodation. This will not form part of the preliminary study and will be discussed in detail with the committee prior to work commencing.

(No outline received).

We wish to use the ALSPAC study to investigate the effects of type 2 diabetes susceptibility variants at the KCNQ1 locus on fetal growth, growth in childhood and intermediate traits related to type 2 diabetes.

Recently, two genome-wide association (GWA) studies of East Asian subjects simultaneously reported a strong association between variants in the KCNQ1 gene and the odds of type 2 diabetes [1, 2]. The effect size estimates were large (OR 1.3-1.4) and the associations were robust, exceeding stringent criteria for statistical significance appropriate to GWA studies (Pless than 5x10-8).

This association had not been identified previously in European GWA studies due to the lower allele frequency (5% vs 40%) and consequently reduced power [3]. However associations were observed in European samples following the East Asian GWA studies, and the effect size estimates were consistent [1-3]. The index SNP, rs2237895, has also shown detectable effects on beta cell function in Europeans [4].

The associations with type 2 diabetes and beta cell function make the KCNQ1 locus an excellent candidate for influencing early growth. A variant that predisposes to reduced insulin secretion and diabetes in adulthood may also influence insulin secretion/action in utero, and thereby reduce birth weight [5]. Our preliminary data on the CDKAL1 and HHEX loci support this (PLoS Med, under review). Maternal diabetes genes may additionally influence birth weight through their effects on the intrauterine environment [6]. We have observed that the maternal risk alleles for fasting glucose and type 2 diabetes at GCK and TCF7L2, respectively, are associated with higher offspring birth weight [7, 8].

The KCNQ1 locus is of additional interest in relation to early growth because the locus is imprinted and may harbour elements that influence the imprinting of neighbouring genes [9, 10]. The region is implicated in Beckwith-Wiedemann syndrome and Silver-Russell syndrome, rare neonatal disorders of fetal overgrowth and growth restriction, respectively.

We therefore propose to analyse the polymorphisms in ALSPAC to test the following hypotheses:

1. Fetal genotype and maternal genotype are associated with fetal growth.

2. Fetal genotype and maternal genotype are associated with growth phenotypes (height, BMI, growth velocity) in childhood

3. Offspring genotype is associated with diabetes-related traits in childhood including fasting insulin, fasting glucose and insulin secretion (in the subset of offspring with OGTT data), triglycerides, HDL, LDL and total cholesterol, anthropometric measures including BMI, lean/fat body mass, WHR, waist circumference, skin folds where available.

4. Due to imprinting, association between the risk allele in the offspring and early growth is dependent upon the parent of origin (we will be able to assess this using informative mother-offspring pairs).

Whether the results are negative or positive they will help our understanding of how the KCNQ1 variants function and, if positive, provide important insights into growth and other diabetes-related phenotypes.

To do this we would like to genotype (at Kbiosciences) all ~20,000 ALSPAC samples. We will need the following phenotypes to test our hypotheses (a detailed list is in the next section):

1. Birth weight, length and head circumference

2. Growth measures in childhood (height, weight and BMI aged 7-11)

3. Covariates of birth weight to check if genotype is acting through them: gestational age, maternal age, maternal BMI, smoking , parity, twin status to exclude non-singletons, ethnicity as genotype frequency may alter with ethnic origin and confound analyses.

4. Type 2 diabetes-related intermediate traits including fasting insulin, fasting glucose and insulin secretion (in the subset of offspring with OGTT data), triglycerides, HDL, LDL and total cholesterol, anthropometric measures including BMI, lean/fat body mass, WHR, waist circumference, skin folds where available.

Plans for meta-analysis:

ALSPAC will provide the largest European-ethnicity dataset for association studies with fetal growth. However, we have access, through our own studies and extensive collaborations to samples from the Exeter family study (950 population based parent-newborn trios), the Northern Finland 1966 Birth Cohort (4600 individuals with own birth measures) and the Hyperglycemia and Adverse Pregnancy Outcomes study (4000 European mother-offspring pairs; 2000 Thai/Chinese mother-offspring pairs; 1250 African-Caribbean mother-offspring pairs; 2500 other ethnicity). We hypothesize that real genetic associations will be consistent across all of these studies - i.e. even if individually studies show only nominal significance, a meta-analysis of all studies will provide highly significant results.

Infant malnutrition is a major problem on the South Asian subcontinent. Rates of malnutrition and stunting are very high in South Asian societies. Interestingly, some evidence suggests that this phenomenon may be influenced by cultural factors. These include infant feeding practices such as a use of the supine position, feeding while sleeping, the prolonged use of the bottle, delay of protein and adult food. Other cultural factors that may influence this phenomenon have to do with the low status of women in South Asian societies. Maternal depression has been strongly linked to infant malnutrition; furthermore, female infants and children are far more likely than males to experience malnutrition and related diseases and problems, and to die before reaching the age of five. The problem of childhood malnutrition, while grave in itself, may also increase vulnerability in the South Asian population to the later development of obesity and metabolic-related problems such as diabetes.

There has been very little study of this phenomenon in communities of the South Asian diaspora, including immigrants from India, Pakistan, and Bangladesh. Such research is important. Comparative research is especially crucial in this regard, since it could permit the identification of culture-specific practices and behaviors that might be amenable to modification. Our research team has considerable experience working in the New York City South Asian community (and, mention your stuff). We would like to develop a research intervention to assist South Asian families in this regard. However, to develop and fund pilot work, we would like to present preliminary data indicating the features, correlates, and prevalence, of infant and child malnutrition in South Asians. Comparative research which shows that some practices and behaviors are especially common or uncommon among South Asian families, will be particularly helpful in this regard.

We propose to conduct a secondary analysis of ALSPAC data. The 82 families of South Asian origin will be compared to 82 families of white-European background. We will select a comparison group matched on factors such as: housing status, parity, maternal and paternal education, gestational age at birth, and birth length.

We will compare the South Asian and control groups with regard to the primary outcome variables related to growth, e.g.,: BMI, height- Z scores, and head circumference Z scores. Secondary outcomes will include the longitudinal parent-reported feeding measures of: difficulty feeding, being choosy with food, not intaking a sufficient amount of food. Co-variates will include measures of; matenral depression, sociodemogrphics, child gender, parenting scores (i.e. HOME), etc.

Note, no new data are requested. We will conduct analyses with the dataset we already have, and will not require ALSPAC assistance.

AIMS

To test this hypothesis we propose to assess the sensitivity, specificity, positive predictive value, and negative predictive value of DCDC2 allelic variations for reading performance in all 10,000 DNA samples of the ALSPAC epidemiologic collection. These children have been phenotyped with an array of age-appropriate neurocognitive and reading assessments. At a minimum we will attempt to correlate genotypes with quantitative tests of basic word reading (Wechsler Objective Reading Dimension: WORD subtests49), phoneme deletion (Auditory Analysis Test of Rosner and Simon50), and spelling (regular and irregular words). These tests are administered to the ALSPAC cohort at age 7. We will also include IQ assessments at age 8 through the Wechsler Intelligence Scale for Children III51, and other cognitive measures such as language and verbal short-term memory, and Key Stage 1 School Assessments, if available. Furthermore, our on-going lab studies of the developmental biology of DCDC2 and it's role in neuronal migration, and physiology studies through functional imaging studies at the Yale Center for the Study of Learning and Attention, will suggest correlating additional age-appropriate neurocognitive and language measures that are extant in the ALSPAC cohort.

These studies will lead to the development of a sensitive and cost-effective population screening tool that will identify children at-risk for RD before entering school and help to guide strategies for effective early intervention.Moreover, when considered in the context of our parallel studies of gene function and cognitive imaging profiles, they will lead to a comprehensive understanding of the neurodevelopmental processes subserving language, which should further guide the remediation of RD and other learning disorders.