Discriminating Between Monozygotic Twins
Danielle Gibbes
Bio 303H
November 1, 2014
Monozygotic twins stem from a single zygote and are the result of the morula
separating during the early embryonic development (Weber-Lehmann, et al. 2013).
Monozygotic twins (MZs) are considered to be genetically identical because they
share a common genotype and have identical microsatellite profiles (WeberLehmann, et al. 2013). Because of this problem, it is difficult to distinguish genetic
differences between the twins using conventional genetic markers such as short
tandem repeats (STRs). However, monozygotic twins are not completely identical as
shown through phenotypic discordance (Li, 2013). While phenotypic discordance
can be described as the result of many things, science and technology today have
discovered methods to detect epigenetic differences between a set of identical twins
leading to the phenotypic discordance. This information has been useful for several
reasons including determining the role epigenetic differences play in the discordant
frequency and onset of diseases, how variations in epigenetic patterns can affect
phenotype, and also helping in forensics when dealing with criminal or paternity
cases involving monozygotic twins.
One method of distinguishing between a set of monozygotic twins is using
next generation sequencing to find mutations, specifically single nucleotide
polymorphisms (SNPs), that are present in one twin but not the other. A study
conducted by Jacqueline Weber-Lehmann, et al. (2013), experimented with this
technique in order to find a solution to solving “paternity and forensic cases
involving monozygotic twins as alleged fathers or originators of DNA traces.”
The study was geared toward men under the hypothesis taken from a
theoretical paper by Krawczak et al. (2012) that said more than 80% of the children
of one of the twin brothers would have at least one germline mutation that would be
detectable in their father’s sperm but not their uncle’s. To test this hypothesis,
Weber-Lehmann recruited a pair of male twins as well as the wife and child of one
twin but did not tell the laboratory team which twin the child belonged to in order
to avoid bias.
DNA was extracted from blood samples of the mother and child and from
blood, buccal mucosa, and sperm samples from the twins (Weber-Lehmann, et al.,
2013). PCR was used to confirm that the twins were monozygotic and that one was
in fact the father of the child. Using the Illumina HiSeq 2000 the DNA was sequenced
and the resulting data for the twins and the child were mapped to the human
reference genome sequence. VarScan2 software scanned for and identified somatic
mutations. This ultra deep next generation sequencing allowed the identification of
germline/somatic mutations that would have occurred after twinning during
embryonic development and therefore would only be present in the twin father but
not the twin uncle (Weber-Lehmann, et al., 2013). The SNPs were confirmed with
Sanger sequencing after amplifying regions of interest.
Twelve potential somatic SNPs were found that were present in the child and
his father but not the uncle. Seven of those SNPs had to be discarded because more
than one significant hit on the genome was found in the surrounding sequence
(Weber-Lehmann, et al., 2013).
Figures 2 and 3 show one of the five remaining SNPs that was found on
chromosome 4. As it is shown in the figures, the child has the same mutation as the
father while the uncle does not have the mutation. Four other chromosomes show
the same results. The five mutations were discovered in the sperm DNA of the twin
father but interestingly four of those five were also present in the buccal mucosa
sample and one of those four present in the blood DNA sample. Only one mutation
was found exclusively in the sperm DNA sample. These results make it possible to
determine when the mutations occurred during development. Weber-Lehmann
made the observation that “mutations that are present in one twins’ sperm and
buccal mucosa, but not in blood, will have occurred after gastrulation, but before the
separation the buccal mucosa and the precursors of the sperms” (2013). The
mutation found in each of the three samples, blood, sperm, and buccal mucosa,
would have occurred before the separation of the germ layers and therefore would
be the earliest mutation. Thus, the SNP present only in the sperm sample was the
last mutation of the five to occur. In order to distinguish twins by these SNPs, all of
the mutations must have occurred after the morula separates (Weber-Lehmann,
2013).
Studying mutations present in one twin as well as their offspring but not in
the other twin is a revolutionary advancement in solving forensic criminal or
paternity cases involving monozygotic twins. Identifying mutations that were
present in the two ectodermal tissues, buccal mucosa and sperm, allows this method
to be useful to any forensic case involving ectodermal traces found at crime scenes
(Weber-Lehmann, 2013). However, studying DNA methylation profiles between
monozygotic twins has proved to be a promising alternate way to discriminate
between the two for forensic purposes.
DNA methylation is an epigenetic modification that is one of the most
intensely studied (Kulis et al., 2010). It is associated with histone modifications and
helps ensure that gene expression and gene silencing are properly regulated. This
regulation is mediated by the covalent addition of a methyl group to cytosines that
are located within CpG dinucleotides (Kulis et al., 2010). For twins, differences in
these epigenetic modifications can be examined to distinguish them.
Chengtao Li and his colleagues (2013) conducted a study that looked at blood
samples of monozygotic twins and differences in their DNA methylation profiles.
The results of the experiment show that DNA methylation is a highly promising
epigenetic marker that can be applied to forensic genetics. Blood samples were
taken from 22 pairs of MZs, 13 female and 9 male that ranged in age. After
confirming the monozygosity of each pair, genomic DNA was modified with bisulfite.
The bisulfite converts unmethylated cytosine to uracil (Li, et al., 2013). The bisulfiteconverted genomic DNA was then analyzed at 27,578 CpG dinucleotide sites,
encompassing 14,473 well-annotated and unique human RefSeq genes (Li, et al.,
2013). The resulting data showed the methylation status for each CpG site, and a list
of significantly different methylation CpG sites was compiled.
The differences of DNA methylation profiles of each MZ pair were mapped
onto volcano plots according to the criteria listed above. Figure 3 shows the volcano
plots of three different pairs of MZs that reveals the CpG sites with differential
methylation status (Li, et al., 2013). In studying the volcano plots, 3,616 candidate
CpG sites had significant differential methylation status. The next step was to find
out which of those CpG sites were significantly different among the majority of the
22 pairs of twins. After calculating the frequencies, a pool of 92 “best” CpG sites
were chosen and every pair of twins could be distinguished using these
differentially methylated sites.
This pilot study has paved the way for using DNA methylation profiles as a
method to discriminate between monozygotic twins, which will be useful in forensic
casework. Mario Fraga et al. (2005) took DNA methylation methods a step further in
a study conducted to look at epigenetic differences that arise during the lifetime of
monozygotic twins. DNA methylation and histone acetylation was used to examine
global and locus-specific differences in order to see a correlation between the age of
twins and the amount of epigenetic differences between the twins.
The experiment used eighty twins including 30 male and 50 female
participants whose ages ranged from 3 to 74 years old. Monozygosity was
confirmed with microsatellite analysis for each pair.
A comparison of X chromosome inactivation patterns was done because X
chromosome inactivation in females is one of the most obvious places monozygotic
twins may differ (Tiberio, 1994). In Figure 1, picture B shows two examples of
differing patterns of X inactivation between two MZ pairs. Overall, only 19% of all
the female MZ twins had skewed X chromosome methylation patterns that were
different from that of their respective sibling (Fraga, et al., 2005).
Then a comparison of DNA methylation and histone acetylation content of
the twins was determined using a global approach. The 5mC (5-Methylcytosine)
genomic content levels as well as the acetylation levels of histones H3 and H4 were
determined using high performance liquid chromatography and high performance
capillary electrophoresis. Examples of three analyses are shown in the upper
portion of Figure 1C. The results show that 65% of MZ twins showed close to
identical levels of 5mC genomic content and overall acetylation levels of the H3 and
H4 histones (Fraga, et al., 2005). The other 35% of the pairs showed significantly
different results between each sibling in all three of the tested epigenetic characters.
Fraga, et al., (2005) also discovered a link between the epigenetic differences they
found and the ages of the MZ twins. A direct association showed that the younger
sets of twins were epigenetically similar while the older twins were very clearly
distinct as shown in the lower portion of figure 1C (Fraga, et al., 2005).
To examine the location of these epigenetic differences in the MZ twin
genomes, the AIMS approach, a global methylation DNA fingerprinting technique,
was used (Fraga, et al., 2005). This process “provides a methylation fingerprint
constituted by multiple anonymous bands or tags, representing DNA sequences”
that have a methylated site on either side.
The left part of figure 2A displays the mapping sequences of two sets of twins
after the samples underwent the AIMS test. Overall, there were about 600 AIMS
bands that were seen in the gels and between 0.5% and 35% of them were different
within the twin pairs (Fraga, et al., 2005). Fraga, et al., discovered that the MZ twins
with “the most differential AIMS bands were those with the greatest differences in
5mC DNA levels and acetylation levels of histones H3 and H4” as well (2005). The
results from AIMS also follow the same correlation that Fraga, et al., established
earlier that the sets of twins with the most differential bands were older (Fraga, et
al., 2005).
A pool of 53 AIMS bands that were differentially present in MZ twin pairs
were selected to be further examined for extended confirmation that they represent
clear epigenetic differences in the twins. The bands were cloned, sequenced, and run
against several sequence databases and the results showed a breakdown of
sequenced the clones matched. Of the clones, 43% matched Alu sequences and
Figure 2B shows the results of the bisulfite genomic sequencing of 12 clones that
matched the DNA sequence Alu-SP.
Fraga, et al. (2005), then repeated the experiment using epithelial mouth
cells, intraabdominal fat, and skeletal muscle biopsies. Just as in the lymphocytes,
these tissues also showed marked epigenetic differences that were present in older
twins but not in younger twins. From this information, Fraga, et al. (2005),
concluded that “distinct profiles of DNA methylation and histone acetylation
patterns among many different tissues arise during the lifetime of MZ twins that
may contribute to the explanation of some of their phenotypic discordances and
underlie their differential frequency/onset of common diseases.”
The epigenetic differences in DNA methylation and histone modification that
were discovered in the twin pairs after using whole-genome and locus-specific
approaches were dispersed throughout their genomes and play a key role in gene
expression (Fraga, et al., 2005). It was established that these markers were clearly
distinct in older MZ twins while younger twins had more identical genotypes.
Overall, science and technology has paved the way for epigenetic differences
between monozygotic twins to be examined. This information can be used to
identify the basis for phenotypic differences, predict the risk of disease, and
distinguish between individuals in forensic casework dealing with criminal and
paternity cases that involve monozygotic twins.
Works Cited
Fraga, Mario F., Ballestar E., Paz M., et al. (2005). Epigenetic Differences Arise
during the Lifetime of Monozygotic Twins. Proceedings of the National
Academy of Sciences of the United States of America 102.30: 10604-0609.
Krawczak, M., Cooper, D.N., Fändrich, F., Engel, W., and Schmidtke, J. (2012). How to
distinguish genetically between an alleged father and his monozygotic twin: a
thought experiment. Forensic Science International: Genetics. 6: 129–130
Li, Chengtao, Zhao S., Zhang N., Zhang S., Hou Y, (2013). Differences of DNA
methylation profiles between monozygotic twins’ blood samples. Molecular
Biology Reports. 40.9: 5275-5280.
Tiberio, G. (1994). Acta Genet. Med. Gemellol. 43: 207-214.
Weber-Lehmann, Jacqueline, Schilling E., Gradl G., et al. (2014). Finding the needle in
the haystack: Differentiating “identical” twins in paternity testing and
forensics by ultra-deep next generation sequencing. Forensic Science
International: Genetics. 9: 42-46.