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Introduction

Background

Consumption of alcohol is a common aspect of today’s society; however, it is known to have detrimental effects on humans when a significant amount is consumed. Although children may not consume alcohol, they may come into contact with alcohol’s teratogenic effects during development through their parents’ habits. This may lead to fetal alcohol spectrum disorders (FASD); fetuses have decreased pre- and postnatal growth, distinct facial abnormalities, and improper development of the central nervous system, which may cause mental and cognitive disabilities (Lee et al., 2013). These defects are associated with excessive maternal alcohol consumption during pregnancy (Burd, Selfridge, Klug, & Bakko, 2003).  Prenatal alcohol exposure via the placenta allows ethanol and toxic metabolites to come into contact with the fetus (Zelner & Koren, 2013). However, studies have shown that preconception paternal consumption of alcohol have transgenerational effects on the fetus (Knezovich & Ramsay, 2012; Lee et al., 2013).

Lee et al. (2013) speculated that these effects were passed down as changes in the epigenome of the sperm. The epigenome consists of inheritable chemical groups independent of the DNA sequence that can regulate gene expression (Holliday, 2006).  These changes include methylation of cytosine bases, which is usually associated with gene silencing, and chemical groups that are added to histones. Epigenetic modifications are used to silence and activate genes in a parent-of-origin specific manner called imprinting. Imprinting control regions (ICRs) are areas of DNA which are differentially methylated when inherited maternally compared to paternally. Alcohol is shown to lower the levels of DNA methyltransferase transcript and/or activity in sperm (Bielawski, Zaher, Svinarich, & Abel, 2002). Ouko et al. (2009) found a correlation between chronic alcohol use and demethylation of normally hypermethylated imprinted regions in human sperm DNA. All these studies agree that sperm DNA could be a potential medium to transmit alcohol induced epigenetic mutations; however, there is not a study that shows a direct link.

Lee et al. (2013) treated male mice with alcohol and mated them with untreated females. They observed skull malformations in some fetuses and speculated the cause was due to changes in the methylation signatures in the sperm. Laufer et al. (2013) identified three loci that are differentially methylated when the fetus is exposed to alcohol prenatally. There is currently no link between these observations from these two studies. These cranial abnormality may be caused by a change in methylation in these three ICRs that are related to neuron development of the fetus. My specific question I would like to answer is are Stfmb2, Snrpn-Ube3a, and Dlk-Dio3 differentially methylated in the sperm DNA and fetal brain tissue DNA when CD1 male mice are treated with ethanol?

By answering this question, we would be able to finally find a direct link in how a father pass on negative transgenerational effects onto his offspring. There is already public awareness in which pregnant women should not consume alcohol as that would have a direct effect on their babies. If we can find an explanation that alcoholic fathers can still pose a risk on birth defects, then we can raise awareness for paternal alcohol related defects. Fetal alcohol spectrum disorders are easily preventable if the parents are responsible. Raising awareness and education is an excellent preventative measure for FASD.

Hypothesis

I hypothesize that Stfmb2, Snrpn-Ube3a, and Dlk-Dio3 are differentially methylated in the sperm DNA and fetal brain tissue DNA when CD1 male mice are treated with ethanol. Lee et al. (2013) observed preconception paternal consumption of ethanol resulted in skull malformation of fetuses. In another study, Knezowich and Ramsay (2012) found that preconception paternal alcohol exposure showed reduction in methylation of two ICRs (H19 and Rasgrf1) in sperm of exposed males and somatic DNA of the offspring. Although they did not observe an overall change in methylation in the sperm, they speculated that this was due to their method of identifying methylation statuses (more of this will be explained in the experimental design. Laufer et al. (2013) identified three murine ICRs that are differentially methylated after prenatal alcohol exposure: Stfbm2, Snrpn-Ube3a, and Dlk-Dio3. The Stfbm2 region contains neuron-specific transcripts expressed during development (Kagami et al., 2008). The Snrpn-Ube3a region contains a neuron-specific polycistronic transcript (de los Santos, Schweizer, Rees, & Francke, 2000). The Dlk-Dio3 region contains over 40 miRNA in two clusters that are expressed in the embryo, placenta, and in adult brains (Seitz et al., 2004). These three ICRs are relevant because they affect endophenotypes that are observed in FASD including impaired growth, craniofacial abnormalities, and behaviourial and cognitive disabilities (May & Gossage, 2001). These ICRs are associated to neuron development in fetus, which may explain the skull malformation observed by Lee et al. (2013).

Prediction

If these ICRs are found to be differentially methylated in sperm and fetal brain tissue DNA, we may be able to explain how the negative effects of alcohol could be passed on through changes in the epigenome of sperm DNA. Finding epigenetic changes in these three ICRs may be able to explain the skull malformations observed in the fetuses. There is a correlation that alcohol consumption increases the demethylation sperm DNA (Ouko et al., 2009). I predict that these changes will in methylation in sperm DNA will be passed to the offspring. This will cause an abnormal expression of some genes controlled by the three loci during development and cause skull malformations.

Experimental Design

To test the hypothesis, five CD1 mice (postnatal day 49) are treated with a 4 g/kg EtOH with 0.9% saline orally once in the morning and once in the evening, for a seven week period. Five control mice would be fed a saline solution. After the seven weeks of alcohol treatment, the mice are allowed to relax for a week then they will be mated with non-treated females. A female will be housed with a male in a cage overnight. When sperm plugs are found, that would be considered as gestation day (GD) 0. After mating, mature sperm will be harvested from all the male mice and DNA is extracted. Dams are sacrificed on GD 16.5. Brain tissues from all the fetuses will be harvested and DNA will be extracted.

A portion of each DNA sample (sperm and fetal brain tissue DNA) will be treated with potassium perruthenate (KRuO4) and sodium bisulfite for oxidative bisulfite sequencing (OxBS-seq). The other portion will be sequenced without any treatment for reference. All samples will be amplified by PCR using 3 sets of primers that each flank Sfmbt2, Snrpn-Ube3a, Dlk-Dio3 in triplicate. The samples will be then prepared and sent for sequencing. After receiving the sequences, look for 5-hydroxymethylcytosine (5-mC) by looking for cytosines in the OxBS-seq data. Using the reference, each methylated CpG will be mapped and counted for each loci. The triplicates for each sample will be averaged for the number of 5-mC for each loci. Assess for changes in methylation between the experimental and control groups for sperm DNA and fetal brain tissue DNA using Mann-Whittney U test.

Knezovich and Ramsay (2012) used a bisulfite sequencing which could not differentiate between 5-methylcytosine (5-mC) and 5-methylhydroxymethylcytosine (5-hmC) and proposed that this led to the observations of very little demethylation of the sperm DNA after preconception alcohol consumption.  Tet dioxygenases changes 5-mC to 5-hmC through hydroxylation and is implicated in active DNA demethylation (Wossidlo et al., 2011). Additionally, alcohol metabolism has been proposed to cause oxidation of 5-mC to 5-hmC (Wright, McManaman, & Repine, 1999).  Knezovich and Ramsay (2012) speculated that the undetected 5-hmC in sperm DNA would manifest itself as demethylated DNA in the offspring. In this study, OxBS-seq is used to differentiate between 5-mC and 5-hmC through selectively oxidizing 5-hmC, which will register as T when sequenced, whereas 5-mC would register as a C (Booth, Branco, Ficz, Oxley, & Krueger, 2010). This results in a better representation of the methylation status of alcohol-exposed sperm DNA. The Mann-Whittney U test is used in this study for determination of statistical significance of difference in methylation in the loci.  This test is more suitable when a particular population tends to have larger values than the other, the control is expected to have more 5-mC compared to the experimental group since alcohol induces demethylation (Bielawski et al., 2002). As a precaution, a synthetic oligonucleotide containing a known number of 5-mC and 5-mhC will be used as a control to see if OxBS-sequencing is able to distinguish between the two nucleotides.

Discussion of Possible Results

A possible outcome of this experiment is that the hypothesis is correct and alcohol does cause demethylation in the three loci in both sperm DNA and in the fetal brain tissue DNA. This will provide evidence for a link that epigenetic mutations caused by alcohol can be passed down to the fetus through sperm DNA.  Another outcome can could be that the only a change in methylation is seen in the fetus, not in sperm DNA, as observed by Knezowich and Ramsay (2012).  This means that alcohol does not necessarily affect the methylation in sperm but rather affect other mechanism such as RNA mediated effects and histone modifications.

Another possible outcome would be that these three loci are not differentially methylated in sperm DNA and/or the fetal brain tissue DNA. This means that the three loci are only affected through prenatal alcohol exposure and not by preconception paternal alcohol exposure. Perhaps there are other effect loci that are involved with toxic transgenerational effects of alcohol.

A possible technical difficult is finding primers that flank the entirety of each loci. I would need to know the sequence in order to design primers that efficiently amplify each loci. This allows for to properly map out each 5-mC that is found. There may be some difficulties assembling and mapping out the sequences. It is important to properly map out each 5-mC in order to find a significant difference in methylation.

Layperson Paragraph

Fetal Alcohol Spectrum Disorders (FASD) is a full range of disabilities, including growth deficiency, craniofacial abnormalities, and mental disabilities, caused by excessive maternal alcohol consumption during pregnancy. Alcohol and its toxic metabolites can reach the developing fetus through the placenta. However, recent studies have showed that the father may be responsible too. The researchers speculate that alcohol induces changes to the DNA in sperm (epigenetic mutations); thus, can be passed onto his offspring. Epigenetics does not change the code of DNA but changes the way how DNA is expressed through inheritable chemical groups added to DNA. There have been a few studies which showed that alcohol leads to the removal of these groups and change the expression of some genes during the development of the fetus. There is currently no experiment that proves a father can transmit transgenerational toxic effects of alcohol through changes in sperm DNA. I would like to perform an experiment that answers this question and see if alcohol induce epigenetic mutations to sperm that can be found in the offspring’s DNA in mice. If I can find evidence that these toxic effects can be passed down paternally, there will be greater awareness of the risks of birth defects caused by alcohol. FASD is an easily preventable disorder that can be countered by responsible parenting through awareness and education. 

References

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Booth, M. J., Branco, M. R., Ficz, G., Oxley, D., & Krueger, F. (2010). Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution, 336(6083), 934–937.

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Wossidlo, M., Nakamura, T., Lepikhov, K., Marques, C. J., Zakhartchenko, V., Boiani, M., … Walter, J. (2011). 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming. Nature Communications, 2, 241.

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