Methylation profiling of paternally imprinted loci in male gametes following alcohol exposure

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in MedicineFetal Alcohol Syndrome (F AS), the most severe form of Fetal Alcohol Spectrum Disorder (F ASD), has traditionally been associated with maternal alcohol consumption during pregnancy. However, a number of animal studies have shown an association between paternal preconception alcohol consumption and developmental abnormalities in the offspring that resemble the features of F AS. Dysregulation of epigenetic factors (such as DNA methylation) in the presence of alcohol may provide a plausible mechanism by which paternal alcohol consumption could result in offspring affected with features of F AS. Imprinted genes are expressed in a parentof- origin manner due to DNA methylation at distinct differentially methylated regions (DMRs) and are essential for normal embryonic development. There are only two known paternally methylated DMRs in humans, with an additional one described in mice - associated with Rasgrfl. The first aim of this study was to determine whether the human RASGRFl gene contains a DMR and whether this DMR is paternally methylated. In order to assess the imprint status of RASGRF 1, a number of computational assessments were done to identify key features of imprinted loci. Pyrosequencing analysis was used to assess the methylation status of various CpO islands surrounding RASGRFi in peripheral blood and sperm DNA samples. The RASGRF i-associated CpO regions were not found to exhibit differential methylation in a parent-of-origin manner. The second aIm of the study was to examine the effect of paternal alcohol consumption on the methylation status of the IG-DMR locus in male gametes and to detennine whether alcohol is correlated with methylation in a dose-dependant manner. Methylation assessment was done using the quantitative pyrosequencing technology. While an overall reduction in methylation was noted in males who consumed alcohol after adjusting for confounding variables, the amount of alcohol consumed did not correlate with overall methylation. When analyzed by individual CpG sites, alcohol consumption was found to correlate preferentially with demethylation at CpG 3 while alcohol-dosage preferentially correlated with demethylation at CpG 7. Age was significantly correlated with an increase in the overall methylation at JG-DMR and at individual sites within JG-DMR. In conclusion, these findings support the hypothesis that paternal preconception alcohol consumption can lead to hypomethylation of nonnally hypennethylated DMRs of specific imprinted genes in human spenn. This in tum could have significant implications with regard to the regulation of developmentally significant genes in the zygote and fetus, resulting in developmental, behavioral and neurocognitive disorders

Appetite regulation genes are associated with body mass index in black South African adolescents: a genetic association study

PMID: 22614171 PMCID: PMC3358621BACKGROUND: Obesity is a complex trait with both environmental and genetic contributors. Genome-wide association studies have identified several variants that are robustly associated with obesity and body mass index (BMI), many of which are found within genes involved in appetite regulation. Currently, genetic association data for obesity are lacking in Africans-a single genome-wide association study and a few replication studies have been published in West Africa, but none have been performed in a South African population. OBJECTIVE: To assess the association of candidate loci with BMI in black South Africans. The authors focused on single nucleotide polymorphisms (SNPs) in the FTO, LEP, LEPR, MC4R, NPY2R and POMC genes. DESIGN: A genetic association study. PARTICIPANTS: 990 randomly selected individuals from the larger Birth to Twenty cohort (a longitudinal birth cohort study of health and development in Africans). MEASURES: The authors genotyped 44 SNPs within the six candidate genes that included known BMI-associated SNPs and tagSNPs based on linkage disequilibrium in an African population for FTO, LEP and NPY2R. To assess population substructure, the authors included 18 ancestry informative markers. Weight, height, sex, sex-specific pubertal stage and exact age collected during adolescence (13 years) were used to identify loci that predispose to obesity early in life. RESULTS: Sex, sex-specific pubertal stage and exact age together explain 14.3% of the variation in log(BMI) at age 13. After adjustment for these factors, four SNPs were individually significantly associated with BMI: FTO rs17817449 (p=0.022), LEP rs10954174 (p=0.0004), LEP rs6966536 (p=0.012) and MC4R rs17782313 (p=0.045). Together the four SNPs account for 2.1% of the variation in log(BMI). Each risk allele was associated with an estimated average increase of 2.5% in BMI. CONCLUSIONS: The study highlighted SNPs in FTO and MC4R as potential genetic markers of obesity risk in South Africans. The association with two SNPs in the 3' untranslated region of the LEP gene is novel