We wish to use ALSPAC as a replication study to investigate the role of the HNF4A T130I variant (rs1800961) in fetal growth and intermediate traits related to type 2 diabetes (T2D).

The Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study is a multi-centre study of 25,000 mothers and their babies, which demonstrated a continuous relationship between maternal glucose and measures of birth size and fetal adiposity (1). A genetic association study of 1536 SNPs in ~80 candidate loci previously implicated in insulin secretion/sensitivity, has been carried out in a subset of HAPO samples to investigate associations with maternal glycemia at ~28 weeks gestation and offspring size at birth. Included in the study were 3828 mothers of European (Belfast and Manchester, UK, and Brisbane and Newcastle, Australia) and 1813 mothers of Asian (Bangkok, Thailand) ancestry and their offspring.

Among the top signals from the analysis was rs1800961, which encodes a Thrgreater than Ile amino acid change in exon 4 of HNF4A. This was strongly associated with increased fetal head circumference (0.5cm [95%CI: 0.3-0.7] per maternal risk (A) allele; P=1.2x10-7) in those of European descent. The maternal risk allele was also more weakly associated with 1-hour glucose from OGTT (4.3mg/dL [95%CI: 0.5-7.9]; P=0.03), birth length (0.7cm [95%CI: 0.2-1.1]; P=0.003), birth weight (52.6g [95%CI: -8.0-113.3]; P=0.09), and sum of skinfolds (0.3cm [95%CI: -0.1-0.6]; P=0.13). This same risk allele in the fetal genome was weakly associated with cord C-peptide (0.1ug/dL [95%CI: 0.01-0.22]; P=0.03), and head circumference (0.2cm [95%CI: -0.1-0.4]; P=0.08). The same trends were observed among the Thai (Pgreater than 0.05).

We are interested to seek replication of these results in ALSPAC for the following three reasons:

1. Statistical evidence: the maternal genotype-head circumference association survives Bonferroni correction for multiple testing, while multiple other traits show associations at Pless than 0.1 in a consistent direction

2. The SNP is has been recently associated with HDL-cholesterol levels with evidence that exceeds the generally accepted criteria for "genome-wide significance" (Pless than 5x10-8) (2). This evidence that the SNP is marking a real biological function increases the prior odds that it will be associated with fetal growth. The minor A allele, associated with increased fetal head circumference in the HAPO study, predisposes to lower HDL levels in the general population.

3. Carriers of rare diabetes-causing mutations in HNF4A experience greatly increased fetal growth (3). The associations in HAPO of the fetal allele with raised C-peptide and head circumference are consistent with this. Also, in line with the genome-wide lipid data, HDL levels are lower in individuals with HNF4A mutations (4).

Power

The SNP is relatively rare (MAF=4% in Europeans and 2% in Thai). Assuming 7000 ALSPAC mothers in the analysis, we estimate that we will have greater than 99% power to detect a change in head circumference of the magnitude seen in HAPO at Pless than 0.05. To detect a change in birth weight of 50g per allele, we will have 64% power. Even if the associations observed in HAPO represent true underlying effects on fetal growth, it is likely that the "Winner's Curse" has led to an overestimation of effect sizes. Therefore, it will be important to meta-analyse data from the individual HAPO study sites, along with ALSPAC. We will also genotype the SNP in samples from the Exeter family study (950 population based parent-newborn trios), and we will potentially be able to add further samples from the HAPO study (~1500 further Caucasian mother-offspring pairs; 1250 African-Caribbean; 800 Hispanic). We hypothesize that real genetic associations will be consistent across all of these studies - i.e. even if individually studies show only nominal significance, or even P values greater than 0.05, a meta-analysis of all studies will provide highly significant results.

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

1. Fetal genotype and maternal genotype are associated with measures of fetal growth, including head circumference, weight and length at birth.

2. 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.

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. 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.

REFERENCES

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2. Kathiresan S, Willer CJ, Peloso GM, Demissie S, Musunuru K, Schadt EE, Kaplan L, Bennett D, Li Y, Tanaka T, Voight BF, Bonnycastle LL, Jackson AU, Crawford G, Surti A, Guiducci C, Burtt NP, Parish S, Clarke R, Zelenika D, Kubalanza KA, Morken MA, Scott LJ, Stringham HM, Galan P, Swift AJ, Kuusisto J, Bergman RN, Sundvall J, Laakso M, Ferrucci L, Scheet P, Sanna S, Uda M, Yang Q, Lunetta KL, Dupuis J, de Bakker PI, O'Donnell CJ, Chambers JC, Kooner JS, Hercberg S, Meneton P, Lakatta EG, Scuteri A, Schlessinger D, Tuomilehto J, Collins FS, Groop L, Altshuler D, Collins R, Lathrop GM, Melander O, Salomaa V, Peltonen L, Orho-Melander M, Ordovas JM, Boehnke M, Abecasis GR, Mohlke KL, Cupples LA: Common variants at 30 loci contribute to polygenic dyslipidemia. Nat Genet 41:56-65, 2009

3. Pearson ER, Boj SF, Steele AM, Barrett T, Stals K, Shield JP, Ellard S, Ferrer J, Hattersley AT: Macrosomia and hyperinsulinaemic hypoglycaemia in patients with heterozygous mutations in the HNF4A gene. PLoS Med 4:e118, 2007

4. Murphy R, Ellard S, Hattersley AT: Clinical implications of a molecular genetic classification of monogenic beta-cell diabetes. Nat Clin Pract Endocrinol Metab 4:200-213, 2008.