We hypothesize that certain congenital cardiac malformations are caused by hypomethylation at the cellular level during organogenesis, leading to abnormally slow tissue growth and abnormal cell migration. Cellular methylation status strongly influenced by folic acid nutriture and the enzyme methylene tetrahydrafolate reductase (MTHFR) hypomethylation is possible if an inadequate amount of folate reaches the multiplying cells or the fetus inherits mutation in MTHFR. To test this hypothesis, we will access the University of Alabama's Congenital Heart Disease Collection, a multidisciplinary teaching and research registry of archived heart specimens with cardiovascular malformations. This registry, established and maintained by one of the co-investigators (0 F-P), contains over 550 that have been catalogued by lesion group and cross referenced by individual defect, with all known etiologies and/or diagnoses (e.g. aneuploidy, genetic syndrome, teratogen exposure, etc) noted. Approximately 100 cardiac specimens representing each of the five basic mechanisms of cardiac development will be selected for study. That is, we will include defects caused by abnormally slow tissue growth (hypoplastic left or right ventricle, aortic stenosis, bifid aortic valve, coarctation of the aorta, pulmonary atresia), abnormally cell migration (conotruncal defects), extraneous cell, (Ebstein anomaly), abnormal growth of the extracellular matrix (AV canal), and abnormal targeted growth (anomalous pulmonary venous return). We will also study a control group of similarly archived normal cardiac specimens. After employing a novel procedure to remove the formalin, DNA will be extracted from the abnormally and normal cardiac tissue samples and tested for the presence or absence of two well-characterized MTHFR mutations. We will then quantitate level of methylation in abnormal and normal heart tissue via an assay utilizing 3H]-methyl-S-adenosylmethionine an extracted genomic DNA. Finally, the specific location of any hypomethylated tissue within each cardiac structure will be determined by exposing representative sections of cardiac tissue to anti S-methylcytosine antibody. If our hypothesis is correct, cardiac defects resulting from abnormally slow tissue growth and abnormal cell migration will be the most hypomethylated. Additionally, confirmation of our hypothesis would suggest that certain types of cardiac malformations might be prevented by folic acid supplementation
