The aim of this study was to determine the association between global DNA methylation and nondisjunction of chromosome 21 during oogenesis and occurence of congenital heart defects (CHD) in Down syndrome (DS). Additionally, the imact of endogenous and exogenous factors on global DNA methylation was analyzed, including MTHFR C677T polymorphism, gender, age, body mass index, intake of folate through diet, periconceptional folic acid supplementation, smoking, alcohol drinking and medication use. Patients and methods: A total of 94 mothers of children with DS of maternal origin and with defined meiotic stage of chromosome nondisjunction have been enrolled in this study. The control group consisted of mothers of healthy children with no personal or family history (N=100). The influence of global DNA methylation on development of CHD was studied in 42 DS probands who were the children of mothers included in this study. Global DNA methylation was analyzed in peripheral blood lymphocytes by quantification of LINE-1 methylation using MethyLight method. Genotyping of MTHFR C677T polymorphism was performed by PCR-RFLP. Results: Global DNA methylation in mothers of children with DS was significantly lower than in mothers of healthy children (P=0.000), but no significant differences were found regarding the meiotic stage of nondisjunction of chromosome 21 (P=1.000) or regarding the presence of CHD in their children (P=0.951). Combination of MTHFR C677T genotype/diet significantly influenced global DNA methylation (R2=4.5%, P=0.046). The lowest values of global DNA methylation were determined in mothers with CT+TT genotype and low folate diet. The contribution of these factors was even higher among mothers of children with trisomy 21 resulted from meiotic one nondisjunction (R2=6.2%, P=0.036). The highest effect was found in mothers of children with DS-CHD+ (R2=9.9%, P=0.026), particularly among those who had children with DS and septal heart defect (R2=15.4%, P=0.018). In DS probands, no significant differences in global DNA methylation were found according to the presence of CHD (P=1.000), but values of global DNA methylation were significantly influenced by gender (R2=19.1%; P=0.025). Conclusion: Our findings revealed global DNA hypomethylation in mothers of children with DS, suggesting that this is a possible risk factor for nondisjunction of chromosomes 21 during oogenesis 21. Although, global DNA methylation was not significantly associated with development of CHD in DS, its influence can not be completely excluded, since the significant impact of MTHFR genotype and diet on global DNA methylation in mothers of DS-CHD+ was determined. Further analyses on larger sample are needed to provide an answer to this question.