Contrasting the effects of intra-uterine smoking and one-carbon micronutrient exposures on offspring DNA methylation

Maternal smoking and micronutrient intake during pregnancy are two strong biological candidates for impacting the developing epigenome. The extent to which DNA methylation in offspring is modified by these intrauterine exposures has not been presented in parallel. In this review, we summarize human studies which have investigated genome-wide DNA methylation in the offspring in relation to maternal smoking and one-carbon micronutrient exposure during pregnancy. We contrast the primarily independent efforts for these two categories of exposure, and potential explanations for these differences. We emphasize methodological considerations such as power to detect methylation signals, exposure assessment, control of sources of variability, causal inference and the role of observed methylation changes in mediating downstream outcomes in the offspring

Human genetic susceptibility to mother to child transmission of HIV: a study of mother-infant pairs in Malawi

Mother-To-Child Transmission of HIV (HIV MTCT) is a worldwide public health problem and particularly burdens mothers and children in Sub-Saharan Africa, where in 2006, over 500,000 infants were newly infected with HIV 1. To more clearly understand the mechanisms of transmission, we studied genetic exposures and HIV MTCT in consenting mother-infant pairs receiving antenatal care in Blantyre, Malawi. We first examined infant genetic susceptibility to maternal infection through a genome wide association (GWA) scan of 655,000 SNPs. Top associations with HIV MTCT were found for 20 SNPs within 7 genes (p0.90) but not with other ancestry populations (r20.80, other ancestry r2<0.54). Frequencies of 4 SNPs in the lactase gene (LCT) varied greatly between the Malawi population and Maasai in Kenyawa, Kenya (Bonferroni p<1x10-33). The Malawi population was genetically homogenous but distinct from other populations. Finally, we examined the regulation of chemokine co-receptor 5 (CCR5) expression in human placenta by infant polymorphisms and maternal infection. The CCR5 promoter polymorphisms CCR5-2554T (rs2734648, β= -0.67, 95% CI= -1.23, - 0.11) and -2132T (β=-0.75, 95% CI=-0.131, -0.18) were significantly associated with reduced placental expression of CCR5. An incremental increase in CCR5 expression by expression of HS3ST3A1 (β=0.27, 95% CI=0.18, 0.35) and HS3ST3B1 (β=0.11, 95% CI=0.06, 0.18) was observed. CCR5 expression was up-regulated for higher maternal HIV viral load (β=0.76, 95% CI=0.12, 1.39; p=0.020) and malaria infection (β=0.37, 95% CI=-0.43, 1.18, p=0.362), with variable statistical significance. This cumulative body of work provides a fresh look at genetic factors involved in the risk of HIV MTCT as well as how such findings can be generalized to other populations in Africa

Longitudinal age-dependent effect on systolic blood pressure

Age-dependent genetic effects on susceptibility to hypertension have been documented. We present a novel variance-component method for the estimation of age-dependent genetic effects on longitudinal systolic blood pressure using 57,827 Affymetrix single-nucleotide polymorphisms (SNPs) on chromosomes 17-22 genotyped in 2,475 members of the Offspring Cohort of the Framingham Heart Study. We used the likelihood-ratio test statistic to test the main genetic effect, genotype-by-age interaction, and simultaneously, main genetic effect and genotype-by-age interactions (2 degrees of freedom (df) test) for each SNP. Applying Bonferroni correction, three SNPs were significantly associated with longitudinal blood pressure in the analysis of main genetic effects or in combined 2-df analyses. For the associations detected using the simultaneous 2-df test, neither main effects nor genotype-by-age interaction p-values reached genome-wide statistical significance. The value of the 2-df test for screening genetic interaction effects could not be established in this study

A whole genome association study of mother-to-child transmission of HIV in Malawi

Abstract: Background: More than 300,000 children are newly infected with HIV each year, predominantly through mother-to-child transmission (HIV MTCT). Identification of host genetic traits associated with transmission may more clearly explain the mechanisms of HIV MTCT and further the development of a vaccine to protect infants from infection. Associations between transmission and a selection of genes or single nucleotide polymorphisms (SNP)s may give an incomplete picture of HIV MTCT etiology. Thus, this study employed a genome-wide association approach to identify novel variants associated with HIV MTCT. Methods: We conducted a nested case-control study of HIV MTCT using infants of HIV(+) mothers, drawn from a cohort study of malaria and HIV in pregnancy in Blantyre, Malawi. Whole genome scans (650,000 SNPs genotyped using Illumina genotyping assays) were obtained for each infant. Logistic regression was used to evaluate the association between each SNP and HIV MTCT. Results: Genotype results were available for 100 HIV(+) infants (at birth, 6, or 12 weeks) and 126 HIV(-) infants (at birth, 6, and 12 weeks). We identified 9 SNPs within 6 genes with a P-value <5 × 10-5 associated with the risk of transmission, in either unadjusted or adjusted by maternal HIV viral load analyses. Carriers of the rs8069770 variant allele were associated with a lower risk of HIV MTCT (odds ratio = 0.27, 95% confidence interval = 0.14, 0.51), where rs8069770 is located within HS3ST3A1, a gene involved in heparan sulfate biosynthesis. Interesting associations for SNPs located within or near genes involved in pregnancy and development, innate immunological response, or HIV protein interactions were also observed. Conclusions: This study used a genome-wide approach to identify novel variants associated with the risk of HIV MTCT in order to gain new insights into HIV MTCT etiology. Replication of this work using a larger sample size will help us to differentiate true positive findings

Correction: A whole genome association study of mother-to-child transmission of HIV in Malawi

A correction to: Bonnie R Joubert, Ethan M Lange, Nora Franceschini, Victor Mwapasa, Kari E North, Steven R Meshnick andthe NIAID Center for HIV/AIDS Vaccine Immunology. A whole genome association study of mother-to-child transmission of HIV in Malawi. Genome Medicine 2010, 2:17

Longitudinal age-dependent effect on systolic blood pressure

Abstract Age-dependent genetic effects on susceptibility to hypertension have been documented. We present a novel variance-component method for the estimation of age-dependent genetic effects on longitudinal systolic blood pressure using 57,827 Affymetrix single-nucleotide polymorphisms (SNPs) on chromosomes 17-22 genotyped in 2,475 members of the Offspring Cohort of the Framingham Heart Study. We used the likelihood-ratio test statistic to test the main genetic effect, genotype-by-age interaction, and simultaneously, main genetic effect and genotype-by-age interactions (2 degrees of freedom (df) test) for each SNP. Applying Bonferroni correction, three SNPs were significantly associated with longitudinal blood pressure in the analysis of main genetic effects or in combined 2-df analyses. For the associations detected using the simultaneous 2-df test, neither main effects nor genotype-by-age interaction p-values reached genome-wide statistical significance. The value of the 2-df test for screening genetic interaction effects could not be established in this study

Regulation of CCR5 Expression in Human Placenta: Insights from a Study of Mother-to-Child Transmission of HIV in Malawi

BackgroundHuman promoter polymorphisms in the chemokine co-receptor 5 gene (CCR5) have been noted for association with mother-to-child transmission of HIV (HIV MTCT) as well as reduced receptor expression in vitro, but have not been clearly associated with CCR5 expression in vivo. Placental expression of CCR5 may be influenced by such polymorphisms as well as other in vivo regulatory factors.Methodology/Principal FindingsWe evaluated the associations between infant CCR5 polymorphisms, measures of maternal infection, and placental expression of CCR5 among mother-infant pairs in Blantyre, Malawi. RNA was extracted from placental tissue and used in multiplex real-time PCR to quantify gene expression. Through linear regression, we observed that CCR5-2554T (β = −0.67, 95% CI = −1.23, −0.11) and -2132T (β = −0.75, 95% CI = −0.131, −0.18) were significantly associated with reduced placental expression of CCR5. An incremental increase in CCR5 expression was observed for incremental increases in expression of two heparan sulfate genes involved in viral infection, HS3ST3A1 (β = 0.27, 95% CI = 0.18, 0.35) and HS3ST3B1 (β = 0.11, 95% CI = 0.06, 0.18). Among HIV infected mothers, an incremental increase in maternal HIV viral load was also associated with higher CCR5 expression (β = 0.76, 95% CI = 0.12, 1.39). Maternal HIV status had no overall effect (β = 0.072, 95% CI = −0.57, −0.72). Higher CCR5 expression was observed for mothers with malaria but was not statistically significant (β = 0.37, 95% CI = −0.43, 1.18).Conclusions/SignificanceThese results provide in vivo evidence for genetic and environmental factors involved in the regulation of CCR5 expression in the placenta. Our findings also suggest that the measurement of placental expression of CCR5 alone is not an adequate indicator of the risk of mother-to-child transmission of HIV

Comparison of genome-wide variation between Malawians and African ancestry HapMap populations

Understanding genetic variation between populations is important because it affects the portability of human genome wide analytical methods. We compared genetic variation and substructure between Malawians and other African and non-African HapMap populations. Allele frequencies and adjacent linkage disequilibrium (LD) were measured for 617,715 single nucleotide polymorphisms (SNPs) across subject genomes. Allele frequencies in the Malawian population (N = 226) were highly correlated with allele frequencies in HapMap populations of African Ancestry (AFA, N = 376), namely Yoruban in Ibadan, Nigeria (Spearman’s r2 = 0.97), Luhya in Webuye, Kenya (r2 = 0.97), African Americans in the southwest United States (r2 = 0.94), and Maasai in Kinyawa, Kenya (r2 = 0.91). This correlation was much lower between Malawians and other ancestry populations (r2 0.82, other ancestries r2 < 0.57). Principal components analyses revealed little population substructure within our Malawi sample but provided clear distinction between Malawians, AFA populations, and two European populations. Five SNPs within the lactase gene (LCT) had substantially different allele frequencies between the Malawi population and Maasai in Kenyawa, Kenya (rs3769013, rs730005, rs3769012, rs2304370; p values < 1×10−33)

25-hydroxyvitamin D in pregnancy and genome wide cord blood DNA methylation in two pregnancy cohorts (MoBa and ALSPAC)

The aim of the study was to investigate whether maternal mid-pregnancy 25-hydroxyvitamin D concentrations are associated with cord blood DNA methylation. DNA methylation was assessed using the Illumina HumanMethylation450 BeadChip, and maternal plasma 25-hydroxyvitamin D was measured in 819 mothers/newborn pairs participating in the Norwegian Mother and Child Cohort (MoBa) and 597 mothers/newborn pairs participating in the Avon Longitudinal Study of Parents and Children (ALSPAC). Across 473,731CpG DNA methylation sites in cord blood DNA, none were strongly associated with maternal 25-hydroxyvitamin D after adjusting for multiple tests (false discovery rate (FDR) > 0.5; 473,731 tests). A meta-analysis of the results from both cohorts, using the Fisher method for combining p-values, also did not strengthen findings (FDR > 0.2). Further exploration of a set of CpG sites in the proximity of four a priori defined candidate genes (CYP24A1, CYP27B1, CYP27A1 and CYP2R1) did not result in any associations with FDR < 0.05 (56 tests). In this large genome wide assessment of the potential influence of maternal vitamin D status on DNA methylation, we did not find any convincing associations in 1416 newborns. If true associations do exist, their identification might require much larger consortium studies, expanded genomic coverage, investigation of alternative cell types or measurements of 25-hydroxyvitamin D at different gestational time points

A systematic assessment of normalization approaches for the Infinium 450K methylation platform

The Illumina Infinium HumanMethylation450 BeadChip has emerged as one of the most popular platforms for genome wide profiling of DNA methylation. While the technology is wide-spread, systematic technical biases are believed to be present in the data. For example, this array incorporates two different chemical assays, i.e., Type I and Type II probes, which exhibit different technical characteristics and potentially complicate the computational and statistical analysis. Several normalization methods have been introduced recently to adjust for possible biases. However, there is considerable debate within the field on which normalization procedure should be used and indeed whether normalization is even necessary. Yet despite the importance of the question, there has been little comprehensive comparison of normalization methods. We sought to systematically compare several popular normalization approaches using the Norwegian Mother and Child Cohort Study (MoBa) methylation data set and the technical replicates analyzed with it as a case study. We assessed both the reproducibility between technical replicates following normalization and the effect of normalization on association analysis. Results indicate that the raw data are already highly reproducible, some normalization approaches can slightly improve reproducibility, but other normalization approaches may introduce more variability into the data. Results also suggest that differences in association analysis after applying different normalizations are not large when the signal is strong, but when the signal is more modest, different normalizations can yield very different numbers of findings that meet a weaker statistical significance threshold. Overall, our work provides useful, objective assessment of the effectiveness of key normalization methods