rs10732516 polymorphism at the IGF2/H19 locus associates with a genotype-specific trend in placental DNA methylation and head circumference of prenatally alcohol-exposed newborns

Abstract

Study question: Does prenatal alcohol exposure (PAE) affect regulation of the insulin-like growth factor 2 (IGF2)/H19 locus in placenta and the growth-restricted phenotype of newborns?

Summary answer: PAE results in genotype-specific trends in both placental DNA methylation at the IGF2/H19 locus and head circumference (HC) of newborns.

What is known already: PAE can disturb development of the nervous system and lead to restricted growth of the head, even microcephaly. To clarify the etiology of alcohol-induced growth restriction, we focused on the imprinted IGF2/H19 locus known to be important for normal placental and embryonic growth. The expression of IGF2 and a negative growth controller H19 are regulated by the H19 imprinting control region (H19 ICR) with seven-binding sites for the methylation-sensitive zinc-finger regulatory protein CTCF. A single nucleotide polymorphism rs10732516 G/A in the sixth-binding site has shown to associate with genotype-specific DNA methylation profiles at the H19 ICR.

Study design size duration: By grouping 39 alcohol-exposed and 100 control samples according to rs10732516 polymorphism we explored alcohol-induced, genotype-specific changes in DNA methylation at the H19 ICR and the promoter region of H19 (H19 differentially methylated region). Also, IGF2 and H19 mRNA expression level in placenta as well as the phenotypes of newborns were examined.

Participants/materials setting methods: We explored alcohol-induced, genotype-specific changes in placental DNA methylation by MassARRAY EpiTYPER and allele-specific changes by bisulphite sequencing. IGF2 and H19 expression in placenta were analyzed by quantitative PCR and the HC, birthweight and birth length of newborns were examined using national growth charts.

Main results and the role of chance: We observed a consistent trend in genotype-specific changes in DNA methylation at H19 ICR in alcohol-exposed placentas. DNA methylation level in the normally highly methylated paternal allele of rs10732516 paternal A/maternal G genotype was decreased in alcohol-exposed placentas. In addition to decreased IGF2 mRNA expression in alcohol-exposed placentas of this specific genotype (P = 0.03), we observed significantly increased expression of H19 in relation to IGF2 when comparing all alcohol-exposed placentas to unexposed controls (P = 0.006). Furthermore, phenotypic examination showed a significant genotype-specific association between the alcohol exposure and HC of newborns (P = 0.001).

Limitations reasons for caution: Owing to the exceptional character of the alcohol-exposed human samples collected in this study, the sample size is restricted. An increased sample size and functional studies are needed to confirm these data and clarify the biological significance or causality of the observed associations.

Wider implications of the findings: Our results suggest that the rs10732516 polymorphism associates with the alcohol-induced alterations in DNA methylation profiles and head growth in a parent-of-origin manner. We also introduce a novel genotype-specific approach for exploring environmental effects on the IGF2/H19 locus and ultimately on embryonic growth.

Study funding/competing interests: This work was supported by the Academy of Finland (258304), The Finnish Foundation for Alcohol Studies, Finnish Cultural Foundation, Juho Vainio Foundation, Yrjö Jahnsson Foundation and Arvo and Lea Ylppö Foundation. No competing interests are declared.

Keywords: DNA methylation; H19; epigenetics; head circumference; imprinting; insulin-like growth factor 2; placenta; pregnancy; prenatal alcohol exposure; rs10732516.

Figure 1

Schematic structure of the insulin-like growth factor 2 (IGF2)/H19 locus on chromosome 11p15.5. CTCF protein binds to the maternal (mat) unmethylated imprinting control region (H19 ICR), which blocks the interaction between downstream enhancers and the IGF2 promoter, and enables the expression of maternal H19. The methylation of paternal (pat) H19 ICR prevents the binding of CTCF protein, allowing access of downstream enhancers to the IGF2 promoter and provoking the expression of paternal IGF2.

Figure 2

Genotype-specific DNA methylation levels of CpG units at the IGF2/H19 locus in control and alcohol-exposed placentas measured by Sequenom EpiTYPER. (A) Genotype-specific DNA methylation levels in units CpG1, CpG21,22, and CpG23 at H19 ICR (CTCF6) in control placentas (P = 0.001; P < 0.0001, P < 0.0001 respectively, one-way ANOVA). (B) Genotype-specific DNA methylation levels in units CpG6 and CpG16 at the H19 DMR. Genotype-specific methylation levels were compared between control (C) and alcohol-exposed (A) placentas (units CpG6 and CpG16 in G/G genotype, a star (★) illustrates nominal P-value < 0.05, Mann–Whitney) and genotype-specific methylation levels in alcohol-exposed placentas (unit CpG6, P = 0.006, one-way ANOVA). Error bars denote the median with interquartile range. The numbers of samples are in brackets. Bonferroni post hoc test for one-way ANOVA: *P < 0.05, **P < 0.01, ***P ≤ 0.001.

Figure 3

Genotype- and allele-specific DNA methylation levels of CpG sites at H19 ICR (CTCF6) in control and alcohol-exposed placentas measured by bisulphite sequencing. Methylation levels of selected CpG sites in the (A) patG/matA genotype, (B) paternal allele of patG/matA genotype, (C) maternal allele of patG/matA genotype, (D) patA/matG genotype (E) paternal allele of patA/matG genotype and (F) maternal allele of patA/matG genotype. Error bars denote the SD. The numbers of samples are in brackets. A star (★) illustrates nominal P-value < 0.05, Mann–Whitney.

Figure 4

Expression levels of IGF2 and H19 mRNAs in control and alcohol-exposed placentas. (A) IGF2 mRNA expression in control (C) and alcohol-exposed (A) placentas. (B) H19 expression in control and alcohol-exposed placentas. (C) Increased expression ratio of H19:IGF2 in alcohol-exposed placentas compared to controls (P = 0.006, Mann–Whitney). (D) Decreased genotype-specific IGF2 expression in alcohol-exposed placentas with patA/matG genotype compared to controls (P = 0.03, Mann–Whitney). (E) Genotype-specific H19 expression in placenta. The fold change of one data point (89,62) for an alcohol-exposed placenta in genotype patG/matA is outside the axis limit. (F) Increased genotype-specific expression ratio of H19:IGF2 in genotypes with paternal rs10732516 G genotypes G/G and patG/matA compared to controls (P = 0.03, P = 0.02, respectively, Mann–Whitney). Error bars denote the median with interquartile range. The numbers of samples are in brackets.

Figure 5

Genotype-specific HCs, weights, lengths and placental weights of control and alcohol-exposed Caucasian newborns. (A) Genotype-specific variation in SDs of control (C) and alcohol-exposed (A) newborns’ HCs (P = 0.001, One-way ANOVA). The SDs of weight and length are shown in panels B and C, respectively. The genotype-specific weights of control and alcohol-exposed placentas are shown in panel D. Error bars denote the SD. The numbers of samples are in brackets. Bonferroni post hoc test for one-way ANOVA: *P < 0.05, **P < 0.01, ***P ≤ 0.001.