2) The Netherlands Famine Study: Chronic Diseases and the Underlying Epigenetic Mechanisms

The main topic of this Wiki is the paper Persistent epigenetic differences associated with prenatal exposure to famine in adults (Heijmans et al, 2008) which is discussed in the "Developmental Programming and Epigenetics" section and following sub-sections. This paper builds off of previous research which we briefly discuss in the preceding section "The Other Studies".

Introduction

The Netherlands famine was a humanitarian disaster that occurred during the winter and spring of 1944 (Schultz, 2010). The famine was not a product of crop failure or drought, but rather resulted from the proceedings of the Second World War. A railway strike was initiated by the Netherlands in order to support the allied offensive, and the Germans responded with an embargo that prevented the transportation of food (Roseboom et al, 2001). The Netherlands was forced to limit rations. People from all socioeconomic classes within this region were restricted to 400-800 calories per day at the height of the famine. After two seasons of rationed food, the War ended and the Netherlands quickly returned to its pre-war prosperity. While tragic, this period of restricted dieting offered researchers a unique opportunity to study the effects of nutrition on different dimensions of development and health.

Especially pertinent to scientific study were the pregnant women during this famine (Schultz, 2010). The children later born to these mothers formed a cohort that were the subjects of many studies examining the effect of diet on fetal development. A developing fetus has large energy demands which are met by its mother. Therefore, the Netherlands famine has allowed researchers to assess the role of maternal nutrition on fetal programming during development (Roseboom et al, 2001). Earlier studies investigate the effect of under-nutrition during fetal development on adult chronic disease. These earlier studies are discussed in The Studies section as they serve as an important precursor to the more recent studies on the molecular mechanisms that affect cell programming during fetal development exposed to famine. The underlying molecular mechanisms that affect cell programming are discussed through analysis of the study by Heijmans et al (2008) Persistent epigenetic differences associated with prenatal exposure to famine in adults. This paper uses the Netherlands famine cohort to investigate the effect of maternal nutrition during gestation on epigenetic regulation of insulin-like growth factor II.

The Other Studies

The first study on the effects of the Netherlands Famine was published in 1976 (Schultz, 2010). However, the cohort of individuals produced by the famine have become the subjects for many additional studies, several of which are discussed below. These studies address the effects of under-nutrition in fetal development, such as higher propensities for cardiovascular disease (specifically coronary heart disease or CHD), obesity, diabetes, as well as changes in cognitive function and epigenetic patterns. Some differences were found in effects between the sexes and in effects between individuals exposed to the famine during early-, mid-, and late-gestation.

The general methods of the studies were to identify individuals born during or around the time of the famine, remained living in the Netherlands throughout their lives, were still alive at the time of the study, were “singletons” (not part of a set of twins, triplets, etc.), were born after week 37 of pregnancy, and had accessible and sufficiently complete medical records. From there, the list of research subjects was further narrowed to the people who allowed the release of their address to the researchers, allowed themselves to be interviewed, and agreed to go to a clinic for additional measurements to be made and/or for samples to be taken. These individuals make up the “cohort” of the Dutch famine studies. However, often a particular subset of the cohort was used depending on the type of study. For example, the study by van Abeelen et al looked only at women, and the study by Heijmans et al looked at individuals who had a same-sex sibling that was not exposed to the famine (the sibling acted as a control). Once researches were able to establish, interview, and examine individuals of the cohort and their medical records, the bulk of the remaining work of the studies was to statistically analyze and interpret the wealth of data.

Roseboom et al (2001) took a comprehensive, overview approach to examining the health effects of the famine. They found that fewer males were born than average, and that whether birth weight of exposed individuals was higher or lower than that of unexposed individuals was dependent on when during gestation the individuals were exposed. They reported that glucose tolerance was lower than normal in people exposed during mid- to late-gestation while those exposed in early-gestation had greater risks of CHD. Additionally, people exposed during mid-gestation had increased “obstructive airways disease”. Schultz (2010) mentions that Rooij et al in their 2010 study found that there was an overall decline in cognitive abilities in exposed individuals in their late adulthood. The study by van Abeelen et al (2012) found higher instances of and increased risk of developing type 2 diabetes in exposed individuals. Heijmans et al (2008) and Tobi et al (2009) focused on examining the epigenetic effects of the famine, which will be discussed later in greater detail.

Developmental Programming and Epigenetics

While many studies have been conducted investigating the health implications that result from under-nutrition during fetal development, there are a lack of studies that investigate the underlying genetic mechanisms that cause the programming during human development that later lead to these chronic diseases. Several animal studies have concluded that environmental exposures such as famine can result in permanent epigenetic changes to the genome that can influence the phenotype of adult organisms. The results from these animal studies have been extrapolated to explain the role of epigenetics in human development. (Heijmans et al, 2008).

Epigenetics is the modification of DNA without the alteration of the nucleotide sequence. This type of regulation is accomplished primarily through histone modifications and DNA methylation. DNA methylation is the process of adding a methyl group to CpG islands. CpG islands are simply any length of DNA where a cytosine is followed by a guanine base. The addition of the methyl group prevents sequence recognition from transcription factors, thus inhibiting their ability to function and reducing transcription rates. This process is illustrated in the image on the right. As can be seen here, methyl groups physically block the CpG island sequences from being read. In some cases of hypermethylation, genes may be imprinted meaning that their expression is silenced, essentially making the trait monoallelic (Heijmans et al, 2008). Conversely, hypomethylation can result in bi-allelic gene expression. These modifications can be the result of environmental factors, such as the famine experienced by those in the Dutch famine.

The Netherlands famine has allowed researchers to translate an understanding of the role of epigenetics in development from animals to humans by investigating insulin-like growth factor II (IGF2) in the cohort of individuals gestated during the famine. IGF2 is a locus within the genome that has been found to be associated with epigenetic regulation. Its function within the body is to facilitate human growth and development. Specifically, IGF2 expression is controlled by differential methyl regulation in utero and these differential methylation patterns have been found to persist up to middle aged adults (Heijmans et al, 2008). Under normal conditions, the maternal IGF2 locus is imprinted resulting in monoallelic expression. However, under famine conditions the resulting hypomethylation of the maternal locus resulted in bi-allelic expression (Heijmans et al, 2008).

Methods

To perform this study, there were three steps that were critical for obtaining information. The first step was to find the people that were directly impacted by the Dutch Hunger Famine in Amsterdam by looking through their medical records. The records were obtained from midwifery training schools located in Amsterdam and Rotterdam, as well as the Leiden University Hospital. Medical records were retrieved to observe the birth weights in the people involved in the experiment. The individuals that were exposed to the most to the effects of the famine were compared to their same sex siblings and to unrelated individuals that were also born in the same facilities. This step included obtaining the medical records of these individuals, who were all born in 1945 and early 1946. The goal was to “achieve partial genetic matching in view of the high heritability of IGF2 DMR methylation” (Heijmans et al, 2008).

The next step called for performing several procedures. After those records were obtained, blood samples were taken from 311 exposed individuals, 311 same sex siblings, and 349 unrelated controls. Then researchers used a salting-out method for DNA extraction from cells by using a bisulfate treatment. This was to measure methylation of the IGF2 DMR protein. Once the salting-out method was complete, the DNA containing IGF2 regions were amplified using PCR.

The final step of this research included statistical analyses. Several models were created to observe the associations between IGF2 methylation and birth weight. Some of the fixed variables included CpG nucleotide associations, exposure and age. Data corresponding to exposed individuals was compared to the data that came from their sex same siblings and the unrelated individuals that served as controls.

Results and Discussion

It has been known for some time that a developing fetus can be affected by environmental factors. Perhaps the largest factor for the health of a developing fetus is proper nutrition through maternal diet. Nutritional deficiency is a continuing problem in developing countries and they can be quite detrimental to the child during gestation and after birth. In this paper, it is determined that the health problems resulting from prenatal exposure to famine is caused by epigenetic changes to the genome during development that stay with an individual throughout their lifetime (Heijmans et al, 2008).

As mentioned above, the main gene studied by Heijmans et al was the insulin-like growth factor II (IGF2) gene. The focus of the paper was the determination of the effects of varying methylation of the IGF2 gene as a result of the Netherlands famine. Researchers looked at effects both on early and late gestational periods in development. They discovered that individuals that were exposed to the famine at early stages ended up with lower rates of methylation on their IGF2 differentially methylated region (DMR) cytosine-guanine (CpG) sites. This conclusion is supported by the above data in Table 1. Table 1 compares the mean methylation fraction of cytosine residues at five CpG sites for individuals periconceptually exposed to the famine and their unexposed siblings. Out of the five sites that they investigated, they discovered that the famine subjects show lower rates of methylated in all but one as compared to the control. On the other hand, persons that were exposed to the famine at later stages of gestation showed little to no changes in their IGF2 DMR CpG sites (Heijmans et al, 2008).

There are many implications from the results of this study. The researchers compared their results to a mice experiment where the pregnant mother was given sub-par levels of nutrients in her diet and saw that similar hypomethylation patterns existed. In humans, tissue development occurs during early gestation, which led the researchers to believe that this is when epigenetic marking occurs as well. This is further strengthened by the fact that late gestation famine patients did not exhibit the same phenotypic changes as the early gestation famine patients.

Conclusions

In conclusion, Heijmans et al wanted to make clear that their work was not the final frontier in the studies of epigenetics with regard to abnormalities in fetal development. There were shortcomings with the research that they addressed, mainly the fact that they were unable to monitor the situation of the fetus in development.They were very eager to have future research similar to this continue with other diseases that affect a number of people today, such as CHD, and find a way to prevent said problems (Heijmans et al, 2008).This study by Heijmans et al serves as a linchpin connecting all of the studies conducted on the Netherlands famine cohort. The earlier studies on the Netherlands cohort examined the development of chronic diseases as a result of prenatal under-nutrition; whereas, Heijmans et al establishes the genetic mechanism that leads to these chronic diseases.

References

Heijmans, B., Tobi, E., Stein, A., Putter, H., Blauw, G., Susser, E., Slagboom, P., Lumey, L. (2008). Persistent epigenetic differences associated with prenatal exposure to famine in adults. PNAS, 105(44), 17046-17049. Retrieved February 8, 2015, from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2579375/

Ravelli, A.C.J., van der Meulen, J.H.P., Michels, R.P.J., Osmond, C., Barker, D.J.P., Hales, C.N., Bleker, O.P. (1998). Glucose tolerance in adults after prenatal exposure to famine. The Lancet, Volume 351, 173-177. Retrieved February 9, 2015 from http://www.thelancet.com/pdfs/journals/lancet/PIIS0140-6736(97)07244-9.pdf

Roseboom, T., Van der Meulen, J., Ravelli, A., Osmond, C., Barker, D., & Bleker, O. (2001). Effects of prenatal exposure to the Dutch famine on adult disease in later life: An overview. Molecular and Cellular Endocrinology, 185, 93-98. Retrieved February 8, 2015, from http://jamesclear.com/wp-content/uploads/2015/01/tessa-roseboom-effects-of-prenatal-exposure-to-the-dutch-famine-on-adult-disease-in-later-life.pdf

Schultz, L. (2010). The Dutch Hunger Winter and the developmental origins of health and disease. PNAS, 107(39). Retrieved February 7, 2015, from http://www.pnas.org/content/107/39/16757.full

Tobi, E.W., Lumey, L.H., Talens, R.P., Kremer, D., Putter, H., Stein, A.D., Slagboom, P.E., Heijmans, B.T. (2009). DNA methylation differences after exposure to prenatal famine are common and timing- and sex-specific. Hum Mol Genet. 18(21): 4046-4053. Retrieved February 9, 2015 from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2758137/

Van Abeelen, A. F. M., Elias, S. G., Bossuyt, P. M. M., Grobbee, D. E., van der Schouw, Y. T., Roseboom, T. J., & Uiterwaal, C. S. P. M. (2012). Famine Exposure in the Young and the Risk of Type 2 Diabetes in Adulthood. Diabetes, 61(9), 2255–2260. doi:10.2337/db11-1559. Retrieved February 10, 2015 from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3425424/

Image References

http://www.state.gov/img/09/34348/netherlands_map_2009worldfactbook_300_1.jpg

http://missinglink.ucsf.edu/lm/genes_and_genomes/methylation.html

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2579375/pdf/zpq17046.pdf