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Inter- and intra-individual variation in allele-specific DNA methylation and gene expression in children conceived using assisted reproductive technology.

Turan N, Katari S, Gerson LF, Chalian R, Foster MW, Gaughan JP, Coutifaris C, Sapienza C - PLoS Genet. (2010)

Bottom Line: We found substantial variation in allele-specific methylation at both loci in both groups.Our results show that in vitro conception is associated with aberrant methylation patterns at the IGF2/H19 locus.It is possible that this developmental difference has an effect on placental and fetal growth.

View Article: PubMed Central - PubMed

Affiliation: Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Epidemiological studies have reported a higher incidence of rare disorders involving imprinted genes among children conceived using assisted reproductive technology (ART), suggesting that ART procedures may be disruptive to imprinted gene methylation patterns. We examined intra- and inter-individual variation in DNA methylation at the differentially methylated regions (DMRs) of the IGF2/H19 and IGF2R loci in a population of children conceived in vitro or in vivo. We found substantial variation in allele-specific methylation at both loci in both groups. Aberrant methylation of the maternal IGF2/H19 DMR was more common in the in vitro group, and the overall variance was also significantly greater in the in vitro group. We estimated the number of trophoblast stem cells in each group based on approximation of the variance of the binomial distribution of IGF2/H19 methylation ratios, as well as the distribution of X chromosome inactivation scores in placenta. Both of these independent measures indicated that placentas of the in vitro group were derived from fewer stem cells than the in vivo conceived group. Both IGF2 and H19 mRNAs were significantly lower in placenta from the in vitro group. Although average birth weight was lower in the in vitro group, we found no correlation between birth weight and IGF2 or IGF2R transcript levels or the ratio of IGF2/IGF2R transcript levels. Our results show that in vitro conception is associated with aberrant methylation patterns at the IGF2/H19 locus. However, very little of the inter- or intra-individual variation in H19 or IGF2 mRNA levels can be explained by differences in maternal DMR DNA methylation, in contrast to the expectations of current transcriptional imprinting models. Extraembryonic tissues of embryos cultured in vitro appear to be derived from fewer trophoblast stem cells. It is possible that this developmental difference has an effect on placental and fetal growth.

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Pyrosequencing methylation at the IGF2/H19 DMR in placenta.(A) 31 in vivo individuals, and (B) 26 in vitro individuals. The methylation results at each CpG site for each individual are represented by a horizontal series of symbols. All but a few of the CpGs show methylation levels of 50% or higher, which is in concordance with our findings of gain of methylation on the maternal allele (M/P ≥0.15 in placenta, Figure 1).
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pgen-1001033-g002: Pyrosequencing methylation at the IGF2/H19 DMR in placenta.(A) 31 in vivo individuals, and (B) 26 in vitro individuals. The methylation results at each CpG site for each individual are represented by a horizontal series of symbols. All but a few of the CpGs show methylation levels of 50% or higher, which is in concordance with our findings of gain of methylation on the maternal allele (M/P ≥0.15 in placenta, Figure 1).

Mentions: Because the methylation-sensitive restriction endonuclease assay used to generate the data shown in Figure 1 provides a ratio of maternal alleles at which MluI sites are methylated to paternal alleles at which MluI sites are methylated rather than an absolute fraction of all alleles, we also assayed DNA methylation at the IGF2/H19 DMR by bisulfite pyrosequencing. The assay we used quantifies the methylation status of five CpGs in the IGF2/H19 DMR that are adjacent to the CpG queried in the MluI assay. If an M/P ratio greater than zero (Figure 1) represents methylation of maternal alleles in addition to methylation of DMRs on all paternal alleles, then the CpG sites in these samples/individuals should be methylated on greater than 50% of molecules assayed by bisulfite pyrosequencing (i.e. all paternal alleles plus some fraction of maternal alleles). The placenta samples with M/P ratios greater than zero show greater than 50% methylation at all five CpGs in almost all cases (Figure 2) by bisulfite pyrosequencing, indicating that both the methylation-sensitive restriction endonuclease assay and the bisulfite pyrosequencing assay are measuring gain of methylation on maternal alleles, as has also been reported in the mouse [19], [22], [23].


Inter- and intra-individual variation in allele-specific DNA methylation and gene expression in children conceived using assisted reproductive technology.

Turan N, Katari S, Gerson LF, Chalian R, Foster MW, Gaughan JP, Coutifaris C, Sapienza C - PLoS Genet. (2010)

Pyrosequencing methylation at the IGF2/H19 DMR in placenta.(A) 31 in vivo individuals, and (B) 26 in vitro individuals. The methylation results at each CpG site for each individual are represented by a horizontal series of symbols. All but a few of the CpGs show methylation levels of 50% or higher, which is in concordance with our findings of gain of methylation on the maternal allele (M/P ≥0.15 in placenta, Figure 1).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2908687&req=5

pgen-1001033-g002: Pyrosequencing methylation at the IGF2/H19 DMR in placenta.(A) 31 in vivo individuals, and (B) 26 in vitro individuals. The methylation results at each CpG site for each individual are represented by a horizontal series of symbols. All but a few of the CpGs show methylation levels of 50% or higher, which is in concordance with our findings of gain of methylation on the maternal allele (M/P ≥0.15 in placenta, Figure 1).
Mentions: Because the methylation-sensitive restriction endonuclease assay used to generate the data shown in Figure 1 provides a ratio of maternal alleles at which MluI sites are methylated to paternal alleles at which MluI sites are methylated rather than an absolute fraction of all alleles, we also assayed DNA methylation at the IGF2/H19 DMR by bisulfite pyrosequencing. The assay we used quantifies the methylation status of five CpGs in the IGF2/H19 DMR that are adjacent to the CpG queried in the MluI assay. If an M/P ratio greater than zero (Figure 1) represents methylation of maternal alleles in addition to methylation of DMRs on all paternal alleles, then the CpG sites in these samples/individuals should be methylated on greater than 50% of molecules assayed by bisulfite pyrosequencing (i.e. all paternal alleles plus some fraction of maternal alleles). The placenta samples with M/P ratios greater than zero show greater than 50% methylation at all five CpGs in almost all cases (Figure 2) by bisulfite pyrosequencing, indicating that both the methylation-sensitive restriction endonuclease assay and the bisulfite pyrosequencing assay are measuring gain of methylation on maternal alleles, as has also been reported in the mouse [19], [22], [23].

Bottom Line: We found substantial variation in allele-specific methylation at both loci in both groups.Our results show that in vitro conception is associated with aberrant methylation patterns at the IGF2/H19 locus.It is possible that this developmental difference has an effect on placental and fetal growth.

View Article: PubMed Central - PubMed

Affiliation: Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Epidemiological studies have reported a higher incidence of rare disorders involving imprinted genes among children conceived using assisted reproductive technology (ART), suggesting that ART procedures may be disruptive to imprinted gene methylation patterns. We examined intra- and inter-individual variation in DNA methylation at the differentially methylated regions (DMRs) of the IGF2/H19 and IGF2R loci in a population of children conceived in vitro or in vivo. We found substantial variation in allele-specific methylation at both loci in both groups. Aberrant methylation of the maternal IGF2/H19 DMR was more common in the in vitro group, and the overall variance was also significantly greater in the in vitro group. We estimated the number of trophoblast stem cells in each group based on approximation of the variance of the binomial distribution of IGF2/H19 methylation ratios, as well as the distribution of X chromosome inactivation scores in placenta. Both of these independent measures indicated that placentas of the in vitro group were derived from fewer stem cells than the in vivo conceived group. Both IGF2 and H19 mRNAs were significantly lower in placenta from the in vitro group. Although average birth weight was lower in the in vitro group, we found no correlation between birth weight and IGF2 or IGF2R transcript levels or the ratio of IGF2/IGF2R transcript levels. Our results show that in vitro conception is associated with aberrant methylation patterns at the IGF2/H19 locus. However, very little of the inter- or intra-individual variation in H19 or IGF2 mRNA levels can be explained by differences in maternal DMR DNA methylation, in contrast to the expectations of current transcriptional imprinting models. Extraembryonic tissues of embryos cultured in vitro appear to be derived from fewer trophoblast stem cells. It is possible that this developmental difference has an effect on placental and fetal growth.

Show MeSH
Related in: MedlinePlus