# How Close We Are to a Complete List of Imprinted Genes

*This post summarizes some of the research I have been doing for
Bootstrap Bio AKA [kman](https://www.lesswrong.com/users/kman) and
[Genesmith](https://www.lesswrong.com/users/genesmith)*

# TL;DR

-   **We currently don’t have a list of all imprinted genes that are
    important in human development, but further long-read sequencing in
    adults and in the human placenta is going to get us close to that
    goal.**
-   **Measuring DNA methylation in a low input DNA context is really
    annoying, because usually we can't preserve methylation marks when amplifying DNA. (Edit 2025-06-16: This method is probably not as good as the [improved bisulfite sequencing method](https://www.nature.com/articles/s41587-023-02034-w) I mention below) ~~I explore
    the only way that appears tractable to me to amplify methylated DNA by combining Phi29 and DNMT1.~~ This could be useful for building an atlas of early embryo
    development and for epigenetic preimplantation embryo screening.**
-   **Correct imprinting seems particularly achievable in [Hulk
    sperm](https://www.lesswrong.com/posts/2w6hjptanQ3cDyDw7/methods-for-strong-human-germline-engineering#Hulk_sperm)**

# Epigenetics and imprinting

This post assumes basic familiarity with epigenetics. The next paragraph
is a 1-paragraph summary, but I recommend reading
[these](https://denovo.substack.com/p/what-is-epigenetics)
[two](https://denovo.substack.com/p/epigenetics-of-the-mammalian-germline)
excellent posts on epigenetics. If you know what DNA methylation, DNMT1
and imprinting are, you can safely skip those posts. You might also just
do fine by using the AI-generated glossary.

The short explanation is that DNA methylation consists of small chemical
marks on DNA that change which proteins can bind well to it. Together
with other chemical marks on
[histones](https://en.wikipedia.org/wiki/Histone), which are “spools”
that DNA is rolled around, these marks are how a cell knows that it is a
skin cell, rather than a neuron. Imprinting is a phenomenon where the
two sets of chromosomes in early mammal embryos behave differently
depending on if they were inherited from the father or the mother side.
Imprinting appears to be mostly due to differences in DNA methylation
and histone marks. DNA methylation and histone marks are maintained
across cell replications through an array of specific enzymes that both
add, maintain (DNMT enzymes in the case of methylation) and remove marks
(Tet enzymes in the case of DNA methylation). Approximate maintenance of
methylation is achieved by a balance between the actions of these
enzymes in ways that aren’t fully understood yet.

# Why we need to know all imprinting regions and genes

All [strong methods](https://www.lesswrong.com/posts/2w6hjptanQ3cDyDw7/methods-for-strong-human-germline-engineering) of germline engineering require turning cultured
cells that are not fully totipotent, back into totipotent or naive
pluripotent stem cells[^1] that an embryo can grow from. We might get to
such a state through [different
mechanisms](https://www.lesswrong.com/posts/2w6hjptanQ3cDyDw7/methods-for-strong-human-germline-engineering),
including in vitro oogenesis, spermatogenesis or maybe we are able to
find a cocktail that turns cells naive directly. While the Yamanaka
factors make it possible to turn cells into a more stem-cell like state,
they are not fully totipotent. One distinction that is often made here
is between naive and primed iPSCs. Naive iPSCs share some
characteristics, but the most important one is that they can specialize
into cells from all three germ layers and are similar to cells in the
blastocyst. Currently all methods to create naive human pluripotent stem
cells are prone to loss of imprinting. If we properly imitate oogenesis
and spermatogenesis in vitro, imprints are also erased. So in both cases
it is important that we know of and understand all imprints in order to
preserve or reestablish them.

Loss of imprinting tends to be really bad for embryos and have
non-negligible effects. For example loss of paternal ZDBF2 imprinting is associated with a
20% lower weight in 2 week old mice and this effect persists in
weaker form throughout development![^2] Confusingly though, ZDBF2 is
found on chromosome 2 and presumably if ZDBF2 has such large effects it
would have been seen as [a pattern in UPD 2
cases](https://cs-tl.de/DB/CA/UPD/2-UPDm.html)? But perhaps UPD2 is too
rare for the same doctor to have seen multiple patients to connect the
dots? I think it’s also plausible that imprinting generally has larger
effects in mice. In mice there seems to be stronger competition between
paternal and maternal line due to being less monogamous than humans (One
sign of this is that IGF2R is always imprinted in mice, but only
polymorphically imprinted in humans).

Thus if we want to make healthy embryos, we need to make sure that ALL
important imprints are correct in early embryos.[^3] For this goal, it
would be great if we had a list of all existing imprinted genes and
their imprinting control regions, so we can make sure currently
developed techniques to maintain imprinting don’t overlook any crucial
ones. In the rest of this post, I will do a deep dive through existing
papers, to explain how far we are in documenting all existing imprinted
genes and what methods we might use to better screen for them.

# How imprinted genes work and how we measure them

So there are about ~200 imprinted genes. At least in mice the pattern
seems to be that there is a bimodal distribution between genes that are
heavily biased in expression (close to 0 expression from 1 parent) and
the regular genes which are expressed in roughly equal ratios. There are
some genes that are only a little biased (think 52/48) and these tend to
occur close to existing imprinting regions (Edwards et al., 2023). I
think it is fair for the purposes of germline engineering to treat
little biased genes as not imprinted.

In humans, it appears that all imprinted genes are ultimately regulated
through some differentially methylated region (DMR). A DMR in this
context is a region that is methylated in the chromosome inherited from
one parent, but not the other. When there are differences in methylation
between different cell types, those are also often referred to as DMRs,
but those are not interesting to us in this post.

As you can see in the image below from wikipedia, methylation tends to
be low in early embryonic development and then increase
post-implantation. It is not shown in this image, but methylation tends
to increase more in the inner cell mass compared to trophoblasts which
stay more unmethylated. For this reason there are some differentially
methylated regions that are only transient in the embryo (tDMRs), but
that stick around in the placenta.
![image](https://upload.wikimedia.org/wikipedia/commons/thumb/9/98/DNA_methylation_reprogramming.png/1200px-DNA_methylation_reprogramming.png)

The majority of methylated regions only become differentially methylated
after fertilization (somatic DMRs or sDMRs) caused by some nearby DMR
that was inherited from the oocyte or sperm (germline DMRs or gDMRs). In
mice, some sDMRs are established through the histone mark H3K27me3 set
in the oocyte which then become methylated in preimplantation embryos.
That differential methylation is then lost again in the transition to postimplantation embryos, because the DMR becomes methylated on the paternal chromosome (but often stays differentially methylated in the placenta). Since these types of DMRs are not following
the “canonical” pattern of an imprint established through a methylated
gDMR, they are called non-canonical imprints. It is possible that there
are imprints in humans that are established through histones, as in the
non-canonical imprints in mice. But if that’s the case it is probably a
rare phenomenon, otherwise Daskeviciute et al. (2025) would have found
one of these DMRs when they went explicitly looking for them.

We have identified some of the proteins that are involved in maintaining
imprints. For example ZFP57 and ZNF445 appear necessary to maintain
maternally methylated DMRs. CTCF and the histone mark H3K4me3 can help
maintain DMRs unmethylated.

# Have we identified all imprinted genes?

One natural question to ask if we are interested in germline engineering
is if we have already identified all imprinted genes or if we at least
know all imprinted genes that are crucial for development.

One of the first ways how imprinted genes were discovered is through
patients with [uniparental
disomy](https://en.wikipedia.org/wiki/Uniparental_disomy), a rare
genetic disorder where for a specific chromosome the patient has
inherited two copies of a chromosome from the same parent, but none from
the other (often as the result of a “rescued” trisomy). For more info,
[see my post on
UPD](https://www.lesswrong.com/posts/49Y2WDxStNBWMCBku/draft-uniparental-disomy-as-an-upper-bound-on-the-effect-of).

Unfortunately, there isn’t one well maintained list for all known
imprints. But combining the list from
[geneimprint.com](https://www.geneimprint.com) with the list from Tucci
et al. (2019) is pretty close to such a list. For learning about how we
know specific genes are imprinted I would also recommend the [Catalogue
of Parent of Origin Effects](https://corpapp.otago.ac.nz/gene-catalogue)
over [geneimprint.com](https://www.geneimprint.com), because it tends to
cite more sources and includes more rationale for why a gene was included
or not. The genes found in these lists have been identified either
through UPD disorders or methylation and RNA sequencing in both mice and
humans.

Here’s how to find imprinted genes through RNA sequencing: Find RNA
transcripts that are more often expressed from one chromosome rather
than another. It is possible to identify if RNA fragments are from
different chromosomes through SNPs that are different between maternal
and paternal chromosome. If we have DNA data from the parents we can
even identify if they are maternally or paternally imprinted as was done
by Jadhav et al. (2019).

Some imprinted genes might be hard to track through RNA expression in
humans, specifically for genes that are only imprinted in some specific
kind of neuron or similar.

If the gene is also not imprinted in mice, one way how we could still
identify such an imprint is through screening the entire genome for
differentially methylated regions with high coverage epigenetic
sequencing. If we do such screening for a lot of diverse cell types, I’d
be relatively confident that we did not miss any imprinted genes.

Well known DMRs tend to be around one thousand base pairs long and contain
about 50 CpG sites and are about ~0-10% methylated on one chromosome and
~90% on the other (Monk et al., 2018). These are easy to identify
through bisulfite sequencing by searching for long stretches that are
about 50% methylated on average. Obviously there could be the problem
that the important DMRs that we haven’t discovered are shorter or
perhaps they are on average 70% methylated, because one chromosome isn’t
fully unmethylated. There is really no way we can be sure how many such
DMRs there are without drowning in false positives as long as we do not
phase bisulfite reads by chromosome (like Zink et al. (2018) does).

Fortunately, long-read sequencing has recently become more economical
and long-read sequencing allows us to both read methylation while also
allowing us to phase DNA reads. We also have long-read DNA data that has
higher coverage than any bisulfite sequencing that has ever been done to
date. Using phased nanopore reads Akbari et al. (2022) was able to
identify about 42 novel large differentially methylated regions. All of
these novel regions seemed to be polymorphic and not differentially
methylated in all individuals. Akbari et al. (2022) also identify 17 of
these imprints to be conserved in mice and monkeys. So I don’t think it
is off the table that these imprinted regions are essential early in
development, in the brain or the placenta, but appear “polymorphic” in
blood and other tissues. What is also suspicious is that Akbari et al.
(2022) identify some imprints (like ZNF714) as somatic DMRs because they
show less than 50% methylation in blastocysts. My best guess is that
ZNF714 actually has a gDMR on its promoter that just appears to be a
little less methylated in the blastocyst stage, but still managed to
stay maintained.

Kindlova et al. (2023) uses phased parent-of-origin-assigned nanopore
reads to investigate imprinting in 8 human placentas. Kindlova et al.
(2023) find two DMRs on genes not previously known to be imprinted, that
they describe in more detail in their paper, but supplementary table 4
lists at least another ~90 such DMRs that weren’t identified in previous
studies that show affinity for ZFP57. It’s possible that a lot of these
are false positives, but some of them are probably real. Additional
placenta samples will hopefully show which of these are genuine in the
future.

![image](https://www.biorxiv.org/content/biorxiv/early/2023/02/13/2023.02.13.528289/F5.large.jpg)

*An image of the two novel DMRs discovered by Kindlova et al. (2023). The upper image shows the DMR on an exon of ILDR2 and the lower image shows the atypical-looking DMR around the RASA1 promoter.*

# Fixing paternal imprinting might be easier than fixing maternal imprinting

Most imprints are maternally methylated and paternally unmethylated.
This means creating [Hulk
sperm](https://www.lesswrong.com/posts/2w6hjptanQ3cDyDw7/methods-for-strong-human-germline-engineering#Hulk_sperm)
with correct imprinting might be comparatively easy, because we just
erase all imprints by demethylating the cell on the way of turning it
into a spermatogonial stem cell like cell and before transferring them
into someone’s testicles, we only need to make sure that all imprints
are properly demethylated and then perhaps using some more targeted
strategy, like [epigenetic
Crispr](https://www.nature.com/articles/s41556-020-00620-7) to deal with
the few remaining paternally methylated imprints.[^4]

# PCR for methylated DNA

Both for better understanding early embryos and for preimplantation
screening of embryos, better methods of measuring methylation would be
useful. Working with embryos means we are working with little DNA. 1
cell has about 6 picograms of DNA. Illumina sequencing methods need
~1000x more DNA than that to get adequate coverage and long-read
sequencing methods have [even higher input
requirements](https://www.sequencing.uio.no/pacbio-services/dna-requirements/).

The reason for this difficulty is that methylated DNA loses its
methylation when amplified through common PCR methods. So traditionally
the only way to read methylation is to chemically convert unmethylated
cytosine into uracil (which both DNA polymerases and DNA sequencing
methods read as thymidine) before amplifying DNA for downstream
sequencing. The way this conversion has been done for the last 30 years
is by treating DNA with sodium bisulfite. Not only does this degrade DNA
(Tanaka & Okamoto, 2007), but it also reduces the complexity of the DNA
sequence, making it harder to align reads. I have no lab experience and
rudimentary chemistry knowledge, but a number often cited for how much
DNA is lost through bisulfite treatment range from 70%-95% and there are
some papers claiming to reduce the loss down to 25-35% under ideal
conditions (Rajput et al., 2012; Yi et al., 2017). Recently, methods to
convert the DNA via enzymes have also sprung up. Chatterton et al.
(2023) try this in a single cell sequencing setting, but they end up
losing more DNA through enzymatic conversion than through regular
bisulfite sequencing. Overall both methods do not solve the problem that
any procedure that we perform before amplifying the DNA is going to lead
to unrecoverable losses in breadth of coverage.

So for this reason, if we want breadth of coverage with ultra-low input
DNA, we need a method to amplify our methylated DNA. (EDIT 2025-06-16: The method I mention here has been abandoned by that lab for a better method where they [improve standard bisulfite sequencing by using ammonium instead of sodium in their salt](https://www.nature.com/articles/s41587-023-02034-w). The method has been commercialized by [Ellis Bio](https://ellisbio.com/). Someone with more chemistry knowledge than me could probably look into if there is further tractable improvements possible here.) ~~I see one tractable
way to do this that hasn’t been extensively tried before: 
Use the same molecules human cells use, the enzyme DNMT1 and the reagent SAM to maintain methylation during PCR. For both
of these there don’t exist alternative natural molecules that can
withstand the high temperature during regular PCR, since methylated
cytosine aminates too often at high temperatures, so bacteria that can
withstand high temperatures use different chemical modifications to DNA.
So we can only use polymerase that work well under low temperature
(&lt;40°C), which narrows down the DNA polymerase we can use to
Phi29.[^5] Liu et al. (2020) tried and patented[^6] this idea. From their
Figure 3a and S10 I infer that DNMT1 seems to do de-novo methylation at a
rate of 1-2% per replication and DNMT1 seems to fail to maintain
methylation a similar fraction of the time.[^7] That is a high false
positive and false negative rate, but not fatally so if we get more
coverage in return.[^8] Overall their results are confusing in some
areas, so I remain unsure if this method is any good. For example, Liu
et al. (2020) get lower breadth coverage out of their amplified 10pg
samples, compared to their unamplified controls.[^9]~~

If it turns out the idea above isn’t really tractable or Liu et al.
(2020) aren’t willing to license their idea at a reasonable price, my
best guess is that methylation sequencing isn’t going to be high
coverage enough to be of good use for preimplantation embryo screening
and (long or short-read) RNA sequencing might be more useful
¯\\*(ツ)*/¯. Possibly the enzyme conversion method
[TAPS](https://watchmakergenomics.com/news-and-events/taps-milestone/)
could still be worth it once commercially available, since it only
converts methylated Cs to Uracil and thus keeps most of the sequence
complexity intact. [This
blogpost](http://omicsomics.blogspot.com/2025/03/mission-impossible-methylomics.html)
also goes through some more intractable methylation sequencing
strategies.

# Thoughts on the role of RNA in imprinting

-   **Long non-coding RNA**
    -   The textbook example for the role of RNA in epigenetics is the
        *Xist* gene, which silences one of the X chromosomes in humans
        and other mammals.
    -   Such long non-coding RNAs are sometimes used in imprinting
        regions to suppress multiple adjacent genes in cis. Prototypical
        examples are *KCNQ1OT1* and *Airn* in mice (and presumably
        *KCNQ1OT1* functions similarly in humans). In fact, Xist appears
        to be imprinted in mice, leading to mostly the paternal
        X-chromosome being inactivated in the placenta[^10].
    -   I don’t know of any cases where the initial methylation
        vanishes, but the expression is still long-term imprinted (the maternally methylated germline DMR  for ZDBF2 is transient in the embryo, [but it establishes a new paternally methylated DMR](https://www.nature.com/articles/ng.3718) before the maternal DMR becomes methylated on the paternal chromosome as well).
        Seeing an example like that would be really interesting, but so
        far I haven’t seen one and it seems long-term imprinting is
        always maintained through methylation.
    -   People have been measuring these non-coding RNAs and there are
        not THAT many of them around. If one of them was acting like
        memory in some imprint that doesn’t have methylation involved
        similar to Xist, we would know that long non-coding RNA.
-   **microRNA**
    -   There are examples of direct *trans* effects of an imprint on
        the other allele, where the paternal allele produces an
        antisense microRNA that is breaking down the maternal mRNA for
        that imprinted gene (Haig & Mainieri, 2020). There are
        presumably more examples like this. We should not expect
        imprints to only have cis effects.

# Some useful datasets and resources if someone else wants to look further into imprinting and epigenetics in the future

~~I learned the hard way that epigenetics is too new a field to get a good
understanding by reading [the
textbook](https://cshlpress.com/default.tpl?action=full&--eqskudatarq=987),
because being 13 years out of date is in fact making a big difference.~~
(Edit 2025-06-16: [There is a free e-textbook on epigenetics from 2021 that on a quick skim seemed pretty decent](https://www.amazon.com/Introduction-Epigenetics-Learning-Materials-Biosciences-ebook/dp/B08ZXS3965))
For example for learning about histones and chromatin states, I would
now recommend [this YouTube
video](https://www.youtube.com/watch?v=0dOFztY3VJY&list=PLi807-a75VPpjir5w_pIBtW9iv-kQMbMC)
and after that just [the wikipedia
article](https://en.wikipedia.org/wiki/Histone). 

When checking if a gene is a somatic or a germline differentially
methylated region, it is important to have data about CpG methylation
for both sperm and oocytes. Unfortunately oocytes are really expensive
and even if studies end up using a handful of oocytes for bisulfite seq,
the coverage per Oocyte is abysmal (1-10% of the genome are covered, for
42 oocytes, 12 oocytes or 9 oocytes (Hernandez Mora et al., 2023; Li et
al., 2018; Zhu et al., 2018)). One exception is Okae et al. (2014),
which used about 200 oocytes, but they didn’t provide me their processed
data when I emailed them and their raw reads are in the Japan
Genotype-Phenotype Archive, which is impossible to access if you don’t
have an ethics board that has approved of whatever you want to do with
that data. Fortunately, Akbari et al. (2022) used Okae et al.’s (2014)
data to identify gDMRs in their paper and provided all the aggregated
methylation data of those oocytes in the [data repository associated
with that study](https://doi.org/10.17632/f4k2gytbh5.1)! That [same data
repository](https://doi.org/10.17632/f4k2gytbh5.1) also contains a lot
of other useful files for understanding imprinting, including both
histone and methylation marks phased and separated by parent of origin.
You can open all of these files in either [igv](https://igv.org/),
[ucsc](https://genome.ucsc.edu/) or the genome browser of your choice.

I also found some more phased methylation reads on the [1000 genomes
project’s](https://www.internationalgenome.org/) AWS bucket, that might
be worth downloading.[^11]

I can also recommend to read through some public peer review files,
which gives a better impression what the state of the art is for
academic outsiders like myself. The [peer
review](https://elifesciences.org/articles/77898/peer-reviews#content)
of Akbari et al. (2022)
[and](https://sciety.org/articles/activity/10.7554/elife.83364) [of
some](https://static-content.springer.com/esm/art%3A10.1038%2Fs41467-025-57433-1/MediaObjects/41467_2025_57433_MOESM7_ESM.pdf)
[other studies](https://f1000research.com/articles/6-2108) is publicly
available.

# Errata
I rephrased:
~~That methylation is then lost again in postimplantation embryos, but
persists in the placenta.~~ -> That differential methylation is then lost again in the transition to postimplantation embryos, because the DMR becomes methylated on the paternal chromosome (but often stays differentially methylated in the placenta).

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[^1]: In which case we need a donor for the placenta.

[^2]: “At 2 weeks of postnatal age, male and female paternal-knockout mice
were visibly smaller, with a 20% weight reduction (Fig. 6a,b,d,e) that
affected all organs uniformly (Supplementary Fig. 8c). The unimodal
distribution of the body weight data into 10% bin groups was consistent
with high penetrance and low variance of the phenotype (Fig. 6f), but
with a stronger effect in females (Fig. 6a–d). The undergrowth phenotype
tended to minimize with age, but it was nevertheless persistent, as
measured at 30 weeks (Fig. 6c).” (Greenberg et al., 2017)

[^3]: Unless we have really good reasons to believe that a particular
imprint is not crucial for development.

[^4]: So far I know of only 2 paternal imprints where I am confident that
they are not only established after fertilization and those are the
IGF2/H19 DMR and the IG-DMR. There are probably more paternal imprints,
that are important, but erased early on in development (possibly true of
RASGRF1 in humans).

[^5]: Phi29 is already a common choice in Preimplantation embryonic
testing and is used in commercial Kits like [Repli-G from
Qiagen](https://www.qiagen.com/us/products/discovery-and-translational-research/pcr-qpcr-dpcr/preamplification/repli-g-kits).

[^6]: He et al. (2021)

[^7]: Although one can’t strictly separate replications with Phi29, since
it is working continuously, but in the paper they claim to have
amplified their 10pg sample by 100x.

[^8]: Because DNMT1 alone has a tendency to not only methylate
semi-methylated, but also unmethylated cytosine (Goyal et al., 2006;
James, 2014). We might also want to add UHRF1, which Laird-Offringa et
al. (2016) claims significantly helps with this problem. They tried this
in E. coli which do not have histones. If it works in E. coli my guess
is that it should also be helpful during PCR.

[^9]: See table 2 in the paper’s appendix. Also for some reason their
100ng control ends up with lower genomic coverage than their 4ng
control? Even though the 100ng control has a higher mapping ratio?

[^10]: I think this is why rodents [turn off their X-chromosomes
twice](https://en.wikipedia.org/wiki/X-inactivation#Mechanism), but
honestly I still don’t quite understand why this happens.

[^11]: You can find them on AWS by running
`aws s3 ls s3://1000g-ont/ --recursive | grep ".bed" | grep -P "mat|pat"`
in the shell with AWS-cli installed. (2025-12-19: NOTE! These reads are confusingly named, and while they call them mat/pat they don't use trios to discern which haplotype is from which parent, so they'd be better named as just haplotype 1 and haplotype 2)