The Epidemiology Branch is conducting a number of birth defect studies in collaboration with the Health Research Board and Trinity College, Dublin, Ireland. The main objective of these studies is to determine the relationship between folate and birth defects. The birth defects studied to date are neural tube defects (NTDs), oral clefts, congenital heart defects, Down syndrome and omphalocele. These studies focus on biochemical factors in the area of folate metabolism, and on genetic mutations in folate related genes associated with birth defects. Recent work has expanded to include the biochemical pathways related to birth defects. On going work is examining pathways in health young students of Irish genetic background (the Trinity Student Study) with several goals. These include identifying genetic factors using genome wide association study data that influence these pathways and defining how environmental influences such as smoking, alcohol and the use of supplements change the biochemical milieu. Neural tube defects (NTDs) are common birth defects (1 in 1000 pregnancies in the US and Europe) that have complex origins, including environmental and genetic factors. A low level of maternal folate is one well-established risk factor, with maternal periconceptional folic acid supplementation reducing the occurrence of NTD pregnancies by 50-70%. Gene variants in the folate metabolic pathway (e.g., MTHFR rs1801133 (677 C >T) and MTHFD1 rs2236225 (R653Q)) have been found to increase NTD risk. We hypothesized that other micronutrients and genes contribute to NTD risk. We have continued to search for environmental factors that might be related to the risk for having a child with a NTD. Animal studies have found an association between low iron status and having offspring with NTDs but human data were lacking. We tested this hypothesis in a case control study using samples from 64 pregnant women carrying an NTD affected pregnancy and 207 women with unaffected pregnancies. In the two groups the case median ferritin was 16.9 micrograms/L and the hemoglobin was 12.4g/dL vs. 15.4 micrograms/dL and 12.3 g/dL in the controls. Thus, there was no significant difference between the groups in iron status. Furthermore, there was no difference in ferritin or hemoglobin status between any subtype of NTD and the control population. Therefore, giving women iron prior to conception may improve maternal iron status but is not likely to prevent NTDs. Future investigations will explore genetic factors that have been shown to affect micronutrients of interest in our quantitative traits genome wide assocation study. For example, we have measured tryptophan pathway chemical concentrations in the over 2500 student volunteers in the Trinity Student Study and have generated genome wide data on their genetic variants. These data sets have been merged to determine the most important genetic factors that influence the tryptophan pathway taking into account the effect of gender, alcohol, smoking and oral contraceptive use. The analysis is complete and a manuscript has been prepared. These results will inform our investigation of genetic factors that may be associated with neural tube defect risk. A similar investigation is underway looking at genetic and environmental factors that affect vitamin B6 (pyridoxal phosphate and related vitamers). The data have been analyzed and a manuscript is in preparation. This study also uses the healthy student population. The genome wide association data we have generated on these students has also led to fruitful collaborations with outside investigators to study other quantitative traits. In these collaborations investigators receive samples from our study population for analysis. They assay the samples for the metabolite of interest. We then examine the genome to see what genes influence concentrations of the metabolite of interest. Our collaboration to study von Willebrand factor has recently been published in Proc Natl Acad Sci U S A . The plasma glycoprotein von Willebrand factor (VWF) exhibits fivefold antigen level variation across the normal human population determined by both genetic and environmental factors. Low levels of VWF are associated with bleeding and elevated levels with increased risk for thrombosis, myocardial infarction, and stroke. To identify additional genetic determinants of VWF antigen levels and to minimize the impact of age and illness-related environmental factors, we performed genome-wide association analysis in two young and healthy cohorts (n = 1,152 and n = 2,310) and identified signals at ABO (P <7.9E-139) and VWF (P <5.5E-16), consistent with previous reports. Additionally, linkage analysis based on sibling structure within the cohorts, identified significant signals at chromosome 2q12-2p13 (LOD score 5.3) and at the ABO locus on chromosome 9q34 (LOD score 2.9) that explained 19.2% and 24.5% of the variance in VWF levels, respectively. Given its strong effect, the linkage region on chromosome 2 could harbor a potentially important determinant of bleeding and thrombosis risk. The absence of a chromosome 2 association signal in this or previous association studies suggests a causative gene harboring many genetic variants that are individually rare, but in aggregate common. These results raise the possibility that similar loci could explain a significant portion of the """"""""missing heritability"""""""" for other complex genetic traits. We have finished a second collaboration examining plasminogen that has been accepted for publication and will be published soon. A third collaboration using normal data from the Trinity Student Study GWAS involved studying genetic risk factors for epilepsy in a large multinational investigation. The study, just published in Lancet Neurology,included 8696 cases and 26,157 controls in our analysis. Meta-analysis of the all-epilepsy cohort identified loci at 2q24.3 (p=8.71x10(-10)), implicating SCN1A, and at 4p15.1 (p=5.44x10(-9)), harbouring PCDH7, which encodes a protocadherin molecule not previously implicated in epilepsy. For the cohort of genetic generalised epilepsy, we noted a single signal at 2p16.1 (p=9.99x10(-9)), implicating VRK2 or FANCL. No single nucleotide polymorphism achieved genome-wide significance for focal epilepsy. This meta-analysis describes a new locus not previously implicated in epilepsy and provides further evidence about the genetic architecture of these disorders, with the ultimate aim of assisting in disease classification and prognosis. The data suggest that specific loci can act pleiotropically raising risk for epilepsy broadly, or can have effects limited to a specific epilepsy subtype. Future genetic analyses might benefit from both lumping (ie, grouping of epilepsy types together) or splitting (ie, analysis of specific clinical subtypes). Both Dr. Ruzong Fan and Dr. Aiyi Liu, research statisticians on our project, have used our work and data to identify important statistical research questions. Their major area is looking for more efficient and powerful methods to identify true associations in genome-wide investigations. This work has produced several important papers noted in the bibliography. These will be discussed by Dr. Fan and Dr. Liu in their reports.
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