Investigating the multigenerational link between sperm RNA content and birth defects caused by abnormal folate metabolism

Several diseases (e.g. cardiovascular disease, diabetes) and disorders (e.g. autism, birth defects) are prevalent in the UK without a known genetic cause. It is becoming clear that the environment in which we live affects our risk for these diseases. For example, a high sugar/fat diet increases your risk for diabetes, or smoking can increase your risk of cancer.

Remarkably, scientists are beginning to unravel evidence that suggests that the 'sins' of grandparents affect the health of their children and grandchildren. This occurs even when the environmental insult is no longer present. We and other researchers are currently addressing how diseases are inherited over multiple generations when there are no genetic mutations.

This phenomenon is called transgenerational epigenetic inheritance (TEI). Epigenetics is layer of instructions that tells a cell how to control gene expression. It involves chemicals, such as methyl groups, that bind to DNA and act as 'on/off' switches, or small RNA that also regulate gene expression in an unknown manner.

It is possible that some of this epigenetic information is inherited, and when it is abnormal, there is increased disease risk in the offspring.

Much of our understanding of TEI comes from plants, fruit flies, and worms. We recently generated a mouse model of TEI that will better address how TEI occurs in humans. Mice and humans share many similarities in their DNA sequences and the epigenetic factors at work.

Furthermore, the generation time in mice is short making multigenerational research more efficient. The prevailing hypothesis for how TEI works is that an environmental stressor (e.g., vitamin deficiency, toxicant exposure) disrupts the epigenetic information in sperm or egg cells that make a new embryo. This, in turn, might affect which genes turn on/off during development of the baby and even later in that individual's lifetime.

For example, if certain genes in the offspring are affected, this might lead to birth defects (e.g. neural tube defects [NTDs]) or metabolic disease (e.g. diabetes). Metabolic disease might be an important factor in recreating abnormal epigenetic information in the new sperm or egg, thus creating a cycle of TEI. These complex questions about TEI will be explored in our proposed research.

We will study a mouse model that cannot properly metabolize folate and as a result, inheritance of birth defects over multiple generations occurs. Folate (also known as folic acid) is a vitamin that is important for producing methyl groups required for epigenetic control of gene expression. The role of folate metabolism in TEI is unclear and will be explored in this study.

In addition to abnormal folate metabolism, our mice display atypical epigenetic profiles in sperm of the grandfather including alterations in DNA methylation and small RNA content. We will determine whether small RNA content in sperm is specifically responsible for the birth defects observed in our model. To do this, we will inject specific small RNAs into 1-cell mouse embryos and assess the frequency of birth defects alongside changes in the epigenome and gene expression over multiple generations in direct comparison to our folate model.

Additionally, we will assess whether there are metabolic changes in our model outside of folate metabolism that might aid in recreating abnormal epigenetic profiles in sperm to help perpetuate TEI. Overall, our study aims to further the understanding of how TEI occurs in mammals.

In the UK, it is suggested that pregnant women take folate to reduce the risk of NTDs in the baby, though uptake is low. Our work suggests that men should also consider folate supplementation when conceiving. Furthermore, human birth defects without an apparent cause might be attributed to a folate-deficient grandparent. This research indicates the importance of folate fortification programs in the prevention of birth defects, yet the full effects may take several generations to resolve.