DNA methylation signatures in cord blood associated with maternal gestational weight gain: results from the ALSPAC cohort

Greater maternal pre- or early- pregnancy body mass index (BMI) and gestational weight gain (GWG) have been shown to be associated with health outcomes in offspring, including increased birth weight[1, 2], impaired cognitive development and behavioral problems[3, 4], increased adiposity and adverse cardiometabolic traits[5–13]. The biological mechanisms underpinning these associations remain elusive. Potential mechanisms may include genetic factors, shared postnatal environment, as well as intrauterine metabolic programming, possibly through epigenetic mechanisms[13, 14]. With respect to potential intrauterine mechanisms the main exposure is greater maternal adiposity during pregnancy, and thus one might expect a specific association of GWG in early pregnancy, when the contribution of maternal fat deposition is greater than that of fetal weight or other GWG contributions in later periods, and little or no association of GWG in mid or later pregnancy with offspring outcomes[8, 14]. In relation to offspring adiposity and cardiometabolic outcomes results from the cohort used in this study support this expectation[8].

To our knowledge only one study in humans has examined the association of maternal pregnancy adiposity with offspring DNA methylation patterns. In that study, cord blood white cell DNA methylation was compared between 25 offspring born before their mothers had undergone bypass surgery for severe obesity with 25 matched siblings born after marked weight loss by their mothers following bypass surgery, using the Illumina HumanMethylation450 BeadChip array[15, 16]. Altered methylation and gene expression profiles of genes with known glucose-metabolic and inflammatory related functions were identified between those offspring born after their mothers surgery compared to those born before[15, 16]. Recently a study conducted in African-American population has reported maternal pre-pregnancy BMI to be associated with offspring cord blood DNA methylation levels of the CpG sites in genes involved in a broad array of chronic diseases including cancer and cardiovascular diseases[17]. However, whether similar associations would be found between maternal GWG in humans is currently unknown. Nonetheless evidence from animal models strongly supports an effect of greater maternal pregnancy adiposity on offspring epigenome. For example, gene expression and DNA methylation patterns at loci involved in adipocyte commitment have been shown to be perturbed in rodent models of maternal obesity and overfeeding during pregnancy[18, 19].

We hypothesize that maternal pre-pregnancy BMI and GWG, specifically in early pregnancy, could influence risk of adverse health outcomes in offspring by inducing epigenetic changes. Thus, we aimed to examine whether maternal pre-pregnancy BMI and GWG in different periods of pregnancy were associated with epigenetic marks in newborns, in terms of gene-specific cord blood DNA methylation, and to compare the early pregnancy GWG associations with mid- and late-pregnancy GWG to test whether there was any evidence of specific early pregnancy associations. Our analyses were undertaking in a discovery sample and then in a replication sample, both taken from a large, longitudinal UK cohort of mothers and their offspring.

This study examined the association of maternal pre-pregnancy BMI and GWG in different periods of pregnancy with gene-specific DNA methylation changes in offspring cord blood. Screening analysis using the Illumina GoldenGate Panel I in a discovery sample of 88 mother-offspring pairs showed greater GWG in early pregnancy (from 0 to 18 weeks) to be associated with differential methylation at 4 CpG sites at MMP7, KCNK4, TRPM5 and NFKB1 genes after correcting for multiple statistical testing. Of these four loci we undertook validation and replication using Pyrosequencing of the top associated locus at MMP7. Results of the validation study did not support evidence for an association with early pregnancy GWG with differential methylation at this locus, but was suggestive of an association with GWG in later pregnancy and for exceeded IoM recommended GWG. However, we failed to replicate these findings for this one locus in 170 non-overlapping mother-offspring pairs of the ALSPAC cohort.

The specific association of early pregnancy GWG with differential methylation at four loci is notable, since early GWG is more likely to reflect maternal fat deposition than later GWG (which will be influenced more by fetal growth)[13], and so this specificity, together with the potential functional roles of these loci (see below), might support these findings as being important in developmental overnutrition. However, we were only able to undertake validation and replication studies for one, the most strongly associated loci (MMP7) and the specific early GWG association with differential methylation at that locus did not validate or replicate.

Our more significant finding in the discovery study was increased methylation of CpG sites at MMP7 gene in relation to greater GWG. Matrix metalloproteinases (MMPs) comprise a large family of structurally related zinc-dependent proteinases that have been classified into subgroups on the basis of their structure, substrate specificity, and cellular localization and include collagenases, gelatinases, stromelysins, and membrane-type (MT-MMPs)[28]. MMPs participate in processes such as embryonic development, angiogenesis, wound repair, reproductive cycling, and metastasis[28]. Interestingly, MMPs are essential for proper extracellular matrix remodelling, a process that takes place during obesity-mediated adipose tissue formation. Specifically, mouse models of obesity have showed that the expression of MMP7 is down regulated in obesity[29, 30]. These findings are in accordance with our results since increased methylation of MMP7 would imply lower gene expression, which could translate into a higher risk of adiposity in the offspring in response to an in utero obesogenic environment. However, these associations were not replicated in non-overlapping mother-offspring pairs; thus, present results should be interpreted cautiously and future studies are warranted to confirm the association of greater GWG with differential methylation of MMP7.

Validation and replication studies using Pyrosequencing were not undertaken for other CpG sites that were identified as being differentially methylated in relation to greater GWG and located in genes KCNK4, TRPM5 and NFKB1. However, these three identified genes may be worthy of further exploration. KCNK4 encodes for a potassium channel, subfamily K, member 4 known as Trek/TRAAK channels that has been described to be expressed in the central nervous system, modulate neuronal activity, brain metabolism and have a role in neuroprotection[31, 32]. Knockout animal models have shown a role for Trek/TRAAK channels in behaviour, learning and memory[33, 34]. Interestingly, greater maternal pre-pregnancy BMI and GWG have been shown to be associated with offspring impaired cognition and behavioural problems later in life, including in the ALSPAC study[3, 4]. In addition, TRPM5 encodes for a thermo-sensitive TRP (transient receptor potential) channel that is expressed in pancreatic β-cells and could predominantly contribute to pancreatic functions[35]. In humans, genetic variation in TRPM5 has been reported to be likely associated with pre-diabetic phenotypes and contribute to the development of type 2 diabetes mellitus[36]. Furthermore, TRPM5 knockout mice exhibit impaired glucose clearance resulting from reductions in insulin secretion[37, 38]. This evidence from animal studies is in accordance with our results showing increased methylation of TRPM5 CpG sites in relation to greater maternal GWG; this may in turn result in lower expression of TRPM5 and, thus, perturbed insulin metabolism and increased risk of diabetes in the offspring as it has been previously suggested[39]. Finally, NFKB1 encodes for nuclear factor of kappa light polypeptide gene enhancer in B-cells 1, which is responsible for activation of transcription of genes involved in immune responses, inflammation or cell proliferation[40]. Genetic variation in NKKB1 gene has been reported to be associated with a range of immune-mediated diseases such as atopy, asthma and related phenotypes[41], which are also related to maternal obesity and GWG[42, 43]. Therefore, DNA methylation changes at these three loci highlighted in the screening analysis appear to have clear functional relevance to pathways implicated in developmental programming of adverse phenotypes in offspring linked to maternal obesity and GWG and therefore deserve further investigation.

The strengths of this study are the prospective design and that potential confounding factors were taken into account in the analyses. Further, we evaluated the association of pre-pregnancy BMI and GWG in distinctive periods of pregnancy with offspring cord blood DNA methylation patterns. To our knowledge no previous human studies have examined these associations, yet these maternal exposures have been shown to be related to later offspring outcomes and it has been suggested these associations may be mediated by epigenetic mechanisms[8, 13, 14]. The ability to examine associations of different pregnancy periods of GWG is a particular strength as our expectation would be that GWG in early pregnancy would be specifically associated with outcomes and we were able to test that here.

However, the study has some limitations. Firstly, DNA methylation changes were assessed in cord blood, which limits to know how identified changes could translate to potential target tissues. However, DNA methylation patterns are largely conserved across tissues suggesting that for population based epidemiological studies methylation markers from easily accessible surrogate tissues could be used as a proxy for methylation in target tissues[44]. Because cell count information was not available we cannot rule out the potential impact of cell heterogeneity on present findings. Secondly, limited sample size of the discovery study population may have resulted in low statistical power to detect true differences in DNA methylation patterns and false positives could have occurred. In addition, lack of correlation of methylation levels in MMP7 CpG sites measured with the GoldenGate array and Pyrosequencing limits the conclusions that can be drawn from the present validation and replication studies. Thirdly, smaller effect sizes in DNA methylation were found and biological significance in terms of changes in gene expression is unknown. However, it may be a general phenomenon in complex diseases and phenotypes, where methylation at any given CpG island or specific CpG sites in affected versus unaffected individuals may vary by less than 10%, indeed they have been reported in many other human epigenetic studies. Moreover, for some genes, evidence exists that a small change in the level of DNA methylation, especially in the lower range, can dramatically alter gene expression levels[45]. We opted not to consider differentially methylated site below a threshold of 5% due to the limitations inherent in interpreting biological significance, although this cut off may have limited our findings. Fourthly, the Illumina GoldenGate Panel I was used for screening of DNA regions differently methylated in DNA cord blood. The CpG sites included in this array are based on their functional relevance to tumor development and cancer processes, which in only some aspects can be related to fetal development (such as cell proliferation). However, this array was not designed for epigenome-wide analyses, and methylation changes at other loci in genes relevant to maternal GWG may have been overlooked. The use of the 450 k Beadchip that offers greatly improved genomic coverage over the GoldenGate and 27 k platforms is warranted in future studies. Finally, we were only able to take forward the top associated loci at MMP7 for validation and replication, which limits the interpretation of results for the other three loci that were identified as being associated with GWG in early pregnancy. Given the likely functional relevance of these and the specificity of association with early GWG we feel that further study of these loci is warranted.

We found that greater GWG, specifically in early pregnancy, was associated with increased methylation at 4 CpG sites at MMP7, KCNK4, TRPM5 and NFKB1 genes in offspring cord blood DNA. These four loci were all potentially functionally relevant but we were only able to take the top associated locus at MMP7 forward for validation using Pyrosequencing and replication in non-overlapping mother-offspring pairs. The specific association with GWG in early pregnancy for that one site was not statistically significantly validated and it did not replicateple. Given this is the first study we are aware of to examine these associations and our findings might reflect limited statistical power, we advocate further exploration of identified loci in larger studies and the study of genome-wide DNA methylation data.