Cuddly Koala Genomics

Posted by Catherine Potenski | Categories:

Rebecca Johnson

The genome assembly of the koala is reported in a paper published online in Nature Genetics. This high quality genome represents the most complete genome sequence for a marsupial to date. The data give insight into the highly specialized koala diet, consisting of eucalyptus leaves, and provide information that may be useful to combatting infectious disease.

Koalas are a vulnerable species and part of the aim of the the project was to use the genomic data to inform conservation efforts. We spoke with lead author Rebecca Johnson to get some background on this work:

Koala

Rebecca Johnson

How did the koala genome project come to be?

The genome project started as a small group of Australian researchers (from the Australian Museum, University of the Sunshine Coast and University of Sydney) who were enthusiastic about koala conservation and using genomics to manage populations and diseases. We partnered up with colleagues at the Ramaciotti Centre at the University of New South Wales (UNSW) who were enthusiastic to try out their new sequencing equipment on a ‘de novo mammal sized genome’. This hadn’t been done before in Australia.

We decided to take a bit of a risk and announce to the world in 2013 that we were establishing the Koala Genome Consortium and sequencing the genome. This was a very effective way of getting our project on the scientific horizon but then the pressure was on us to deliver! Fortunately for me (and the koala) one of my biggest career risks (announcing the genome well ahead of time) has resulted in a brilliant collaboration of scientists producing a high quality genome with many exciting outcomes and applications.

 

What do you think were the most interesting or surprising findings that came out of the genome data?

So many interesting things have come out of this work, so it is difficult for me to pinpoint one in particular. However, as a conservation geneticist I’m particularly fond of the conservation genomics work, particularly the historical population reconstruction which infers what koala populations would have looked like through evolutionary time. It was a little surprising to discover that koalas underwent such a dramatic decrease in population size 30-40kya, which was around the time many of the megafauna were experiencing extinction in Australia. Another surprise was that the three koalas used for this analysis are from two quite geographically separate locations (~600 km apart) but both suggest a dramatic reduction in population size indicative of widespread pressures across the continent.

Having this ‘deep-time’ perspective on koala populations, combined with the contemporary population work we did as part of this study we have a long term understanding of koalas in the landscape (i.e. the importance of long-term regional gene flow). Conservation management efforts can now be based on this holistic knowledge rather than a single genetic snapshot taken in time.

 

What are the biggest threat to the koalas now?

The koala is now classified as ‘vulnerable’ due to habitat loss and widespread disease. Threats to koalas are multifaceted, with the biggest primarily due to loss and fragmentation of habitat, urbanization, climate change and disease. Current estimates put the number of koalas in Australia at only 329,000 animals (range 144,000-605,000), and a continuing decline is predicted unless measures are put in place to arrest this decline.

 

How do you envision that this genomic information can aid conservation efforts?

The benefit of the genome to conservation efforts is widespread. The population diversity information presented in our work provides the impetus for a conservation management strategy to maintain gene flow regionally while incorporating the genetic legacy of biogeographic barriers. We have also identified the huge contrast in genome-wide levels of diversity across the northern and southern populations of koalas which will be factored into future decision making. The importance of genetic diversity indices for koala conservation has been included in the recently released NSW koala strategy so we will be focusing on highlighting the genetically healthy koala populations and ensuring they maintain regional gene flow. If more intensive measures such as translocations are required (for example from the genetically diverse populations to the genetically depauperate populations), we now have the tools and data to inform those decisions.

The immune gene repertoire we report as part of the genome is also being used directly in efforts to understand the response of koalas to disease such as chlamydia and the koala retrovirus (KoRV). Several of our collaborators on this work are involved in very important work developing and trialing vaccines for both chlamydia and KoRV. The genome affords the ability to understand which immune genes are up or down regulated in response to disease or treatment and provides the platform for future therapies to be tailored to the genome level.

 

What is it like working with koalas? Do you have any good stories that you would like to share?

It never gets tiring working with koalas and it was not difficult at all to bring collaborators on board to work on this project!

Koalas are notoriously chilled out animals (spending most of their time sleeping or eating), although my friends and colleagues who wrangle them in the field do report how unpleasant it is to be on the receiving end of their extremely sharp claws and nippy diprotodon teeth!

As part of sequencing the genome, our efforts to extract suitable quality DNA from koala blood were unsuccessful (possibly because they have a high lipid content in their blood) the only way we could get suitable quality DNA was to wait for an animal to be euthanized so we could access tissues suitable for genome and transcriptome work. Our two females were euthanized because they had advanced untreatable chlamydia. It is an extremely sobering experience to be involved in these necropsies because you can see the ravages of the disease on the body. While these moments are very tough they also inspire you to work harder to ensure we are producing the best possible science to conserve this amazing species.

 

For more video information, please see:

 

https://www.youtube.com/watch?v=tcMCni28nNo&t=4s

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Sea lamprey genomics

Posted by Catherine Potenski | Categories: , ,

sea lamprey

Jeramiah Smith

The sea lamprey (Petromyzon marinus) is an important model in evolutionary biology. It was discovered in 2009 (http://www.pnas.org/content/106/27/11212.long) that the genome of the sea lamprey undergoes extensive programmed genome rearrangement during development, where ~0.5 Gb (around 20%) of DNA is eliminated from the genome. The somatic tissues contain smaller genomes and only the germ cells retain the full complement of genetic material. The genome of the sea lamprey had been sequenced previously from the blood and liver, so only the somatic genome has been thoroughly characterized (https://www.nature.com/articles/ng.2568).

Smith et al., Nature Genetics, 2018

Smith et al., Nature Genetics, 2018

In a paper published this week in Nature Genetics, Jeramiah Smith and colleagues report the germline genome sequence of the sea lamprey.  Using a combination of shot-gun and long-read sequencing integrated with scaffolding data and a meiotic map, the authors assembled a high-quality genome with near-chromosome level of contiguity. This allowed them to identify hundreds of genes that were systematically eliminated from the genome during development. Comparative analysis showed that mouse homologues of these genes are often marked by repressive complexes, indicating parallel strategies for programmed development.

We spoke with lead author Jeramiah Smith from the University of Kentucky to get some background on this research:

  • What inspired you to sequence the germline of the sea lamprey?

I have worked with lamprey for years. I originally got involved with lamprey because it holds a special place in the vertebrate tree of life that shed light on the common ancestor of all vertebrates. That was the motivation for the first lamprey genome project, which sequenced DNA from blood and liver cells.  Once we started working with lamprey we found out that the genome was much more complex than we ever anticipated. This included the fact that the genome changes its sequence content in a reproducible manner over the course of its normal development: something we call programmed genome rearrangement. The amount of DNA that is eliminated from sea lamprey is more than is present in some entire fish genomes, roughly half a billion bases. For me, this finding was the major inspiration behind sequencing the germline genome.

 

  • What do you think were the most surprising or interesting findings to come out of the sequencing?

There were quite a few, but the strong overlap between programmed genome rearrangement and Polycomb-mediated silencing was near the top. The other was the rather strong evidence suggesting the some chromosomes, including chromosomes carrying the HOX genes, appear to have duplicated rather recently and seemingly independently from the rest of the genome. It’s a really strange genome.

 

  • Can you comment on programmed genetic elimination as a developmental strategy versus Polycomb-mediated silencing? 

Polycomb-mediated silencing arose deep in our evolutionary history, and is even present in unicellular organisms. We know that lamprey possesses human homologs of all Polycomb genes, but also uses programmed elimination. The difference between programmed elimination and other mechanisms of gene silencing is that programmed elimination is essentially irreversible, given that the DNA is physically removed. This means that the genes can never be expressed after an embryonic cell lineage has undergone elimination. Other silencing mechanisms are generally reversible, meaning that gene expression can be reactivated. In some cases reactivation is important. For example, in the context of development and regeneration. But in other cases activation of genes in the wrong tissue can case diseases, such as cancer. Lamprey seems to know which genes should never be reactivated outside of the germline.

 

  • What is the most challenging part about working with sea lamprey?

The Genome! Aside from undergoing complex changes during development it also contains a large amount of repetitive DNA and a lot of sequence polymorphism. These features present substantial challenges for assembly and downstream analyses, but we’ve found that they can also be useful tools. We’ve used the abundance of sequence polymorphisms as a tool for mapping genes in lamprey and we now think that some classes of repeats are going to be critical for our future work aimed at figuring out how eliminated DNA is identified and packaged in the early embryos. Lampreys also only breed once a year and take from 5 to maybe 20 years to mature, this makes some experiments impossible, but lamprey researchers are very creative and the community has figured out how to get a lot done in this system.

  • What organisms would you like to see sequenced in the future to help resolve the evolutionary relationships of vertebrates?

There are so many! Hagfish are going to be critical. They are another deep lineage that provides important perspective on vertebrate evolution and also happen to undergo programmed DNA elimination. There are also two other deep lamprey lineages that I also think will be important. Those species live in the southern hemisphere and diverged from sea lamprey around 300 million years ago, as opposed to the roughly 600 million year divergence between lampreys and other vertebrates. A lot of evolution can happen over 600 million years and these species should help bridge that gap. Salamanders and other amphibians are also going to fill important gaps and teach us a lot about the way vertebrate genomes evolve and function. It also seems certain that new sequencing technologies are also going to give us better genomes for other important species that have already been sequenced (e.g. amphioxus, sharks and shark relatives, and even sea lamprey). Finally, I think the zebrafinch germline genome will also be really interesting. They seem to have recently evolved something similar to lamprey’s programmed eliminations, and have a chromosome that’s unique to their germline. I’d really like to know what’s on that chromosome.

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From the archives (2004): Large-scale structural variation in the human genome

Posted by Brooke LaFlamme | Categories: ,

Scherer_Lee

Iafrate et al. Nature Genetics 2004

During the past 25 years, Nature Genetics has been lucky to publish many exciting papers, more than a few of which can be described as “landmark” papers—publications that have had a dramatic and long-lasting impact on a field. In 2004, the Journal published such a study by Stephen Scherer, Charles Lee and colleagues (Iafrate et al.) in which they reported 255 loci across the human genome containing large structural variants.

In 2017, the idea that there exist large numbers of structural variants in the genome (such as rearrangements, deletions and insertions) that differ from person to person is an established fact. But in 2004, this was not the prevailing wisdom. Prof. Scherer has already written an excellent essay at The Winnower about the study and its importance to the field, so I won’t recap it in detail here—I will simply encourage you to the read the piece.

Charles Lee wrote us about the study by email. “I saw a talk by Dr. Dan Pinkel at the 2002 ASHG meeting where he presented his latest array CGH findings,” he remembers. “In his talk, one of the slides showed the array CGH results of a trisomy 18 patient and Dan remarked how cleanly his array platform performed, especially for the other chromosomes. But in fact, I (and others, I’m sure) could see that there were actually occasional clones that deviated from the expected log2 ratio of 0. During the question period, I sheepishly asked him about these clones. I really didn’t mean to criticize his platform, but I think that he took it that way. Those “blips” bothered me and when I returned to Boston, John Iafrate (who was a postdoc with me at the time) began our own array CGH experiments. Ironically, there were several other groups that were way ahead of us with respect to technical expertise and experience with array CGH, but it could be that they considered these “blips” as technical artifacts – without biological implications.”

Prof. Lee added, “In late 2003, I gave a talk at the University of Toronto and met Stephen Scherer in person for the first time. In a casual conversation, we realized that we were both using the same 1 MB chromosome microarray platform from Spectral Genomics and that we were both seeing these recurrent ‘blips’ in our data.”

Stephen Scherer also corresponded with us by email about the study and the mutual decision to collaborate with the Lee lab. “We were both were fresh enough to look beyond what others were calling ‘noise’ to realize these aberrations represented intermediate and gene-level copy number variation.”

“Many of us suspected it was there,” he said of the large-scale variation they uncovered, “based on the fact there were lots of smaller indels and that 0.6% of the population carried cytogenetic alterations. We kind of predicted it in our chromosome 7 mapping and sequence paper, but only at the chromosomal level.”

ng1416-F1

Circles to the right of each chromosome ideogram show the number of individuals with copy gains (blue) and losses (red) for each clone among 39 unrelated, healthy control individuals. Green circles to the left indicate known genome sequence gaps within 100 kb of the clone, or segmental duplications known to overlap the clone, as compared to the Human Recent Segmental Duplication Browser. Cytogenetic band positions are shown to the left.

Fig. 1 from Iafrate et al. 2004

The study by Iafrate et al. was published on August 1, 2004. Exactly one week prior, a very similar study by Michael Wigler and colleagues (Sebat et al.) was published in Science. The methods used by the two groups were different, but the findings and implications were consistent with each other. “Charles and I were happy to see the Wigler paper,” said Prof. Scherer, “because nobody believed our results.” Prof. Lee added, “This was one of the most difficult papers for me to publish. The reviewers were very skeptical. We had to keep providing more and more validation data, and one of the reviewers even commented that s/he did not believe that the paper was worthy of being an article and we had to shortened the paper into a Brief Communication. At the end, Reviewer #2, who was persistently negative wrote: ‘… I still feel hesitant about publication of this work in Nature Genetics… and I still doubt the importance and novelty of their work.” Prof. Scherer remembers similar levels of skepticism in the community. “Prior to publication I was showing the data at talks, including one at Michigan where they were trying to recruit me, and I remember getting trashed. People in my own department were mostly the same.”

[I looked up the referee reports and internal notes from the review process and Prof. Lee is correct that at least one of the reviewers was very skeptical about the impact of the study. However, I do want to note the very unusual fact, at least by today’s standards, that the study was published a little more than 2 months after initial submission, according to our records. I wish this was more common!]

After publication, however, the importance of the studies was immediately clear, at least to those working most closely in the field. Nigel Carter contributed a News and Views article in Nature Genetics about the studies. He wrote, “This unexpected level of LCV [large-scale copy-number variation] forces us to re-evaluate our view of the structure of the normal human genome.”

However, Prof. Lee remembers some ongoing skepticism about the work. “For more than 18 months after the paper was published, I had trouble getting grant funding for continuing my work in human copy number variation. Some comments that I received included, ‘If this was real, the Human Genome Project would have found it.’ I am embarrassed to say that I was forced to write for smaller grants on other topics and when funded, did everything I could to complete the projects using less money and use the ‘extra’ funds for my human copy number variation interests. It was very, very frustrating.”

In 2007, Science announced Human Genetic Variation as the Breakthrough of the Year.  “When I saw this article in Science,” Prof. Lee said, “I felt like there was finally some widespread acceptance of our findings in the general scientific community.”

“However, this came with different issues.” For example, he often received the response from the GWAS community that structural variation is interesting, but it is too difficult to incorporate into GWAS. “So, most association studies continued to focus on SNPs, which is a problem that persists to this very day.”

The findings in Iafrate et al. were based on, by today’s standards, a fairly small sample of 55 individuals profiled by array comparative hybridization array comprising ~12% of the genome (the study in Science reported results from 20 individuals using representational oligonucleotide microarray analysis). However, the impact on the field was anything but small. Part of the legacy of the studies was the establishment of the Database of Genomic Variants (originally the Genome Variation Database) that has now collected over 550,000 CNVs. The discovery that so many structural variants are present in our genomes, even in healthy individuals, opened up an entire field of study to understand the function of these variants, and much is still to be discovered (see for example a recent study on the impact of structural variation on human gene expression).

Prof. Scherer summed up the impact of the studies this way: “If you remember the fights between the public Human Genome Project and Celera Genomics, and them finger-pointing to the errors in each other’s assemblies, in many cases these were due to CNV and other structural variations. They had no idea these CNV variants existed. It was really the 2004 Nature Genetics and Science papers, coincident, pure discovery, that opened the eyes of the community and it took some longer than others to believe it.”

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From the archives (1995): Guidelines for interpreting and reporting linkage results

Posted by Brooke LaFlamme | Categories: ,

NG1995In 1995, Nature Genetics published a report by Eric Lander and Leonid Kruglyak, recommending clear statistical guidelines for reporting linkage results for complex traits. The paper had an immediate impact, setting the bar for what could or could not be called “significant” in the literature. Although originally focused on human genetic linkage studies, the guidelines set forth by Lander & Kruglyak influenced fields from model organism genetics to plant genetics, and eventually genome-wide association studies (GWAS).

The mid-1990’s was a very exciting time in genetics. The human genome project had recently been announced and advances like microsatellite linkage maps of the human genome and multiplex sequencing technology were now available. Mapping genes underlying complex phenotypes was now a real possibility, and human geneticists were busy prospecting for genetic gold. However, as Lander & Kruglyak cautioned in their paper, the lack of clear guidelines could foster a spate a false positive reports that would, if left unchecked, discredit a the nascent field (for example, see this 1993 paper in Nature Genetics finding no evidence for a previously-reported linkage region for manic depressive illness).

On the other hand, setting too high a bar for reporting significance would mean missing many true signals where they exist, an equally dangerous proposition for a new field. As explained in the paper, “striking the right balance requires both a mathematical understanding of how positive results will occur just by chance and a value judgment about the relative costs of false positives and false negatives.” The paper then outlines the mathematical and statistical arguments in favor of the standards we now all know and love.

Capture

Lander & Kruglyak, Nature Genetics 1995

I spoke with Leonid Kruglyak, co-author of this landmark paper, to get a sense of the context in which this paper came about, and the impact it had on the field at the time of publication. He first explained that it was finally possible to conduct genome-wide linkage studies with hundreds of individuals, allowing linkage mapping methods to be applied to complex traits (for example, this genome-wide screen for schizophrenia susceptibility genes published in the same issue). However, unlike Mendelian genes, there was no clue as to “how many signals there should be, or what their expected sizes were.” Thus, the need for a statistical framework.

This need was recognized as well by the Journal. As Prof Kruglyak recalls, Kevin Davies (founding editor of Nature Genetics) originally commissioned this work as a News & Views article, but it then evolved into a more extensive piece as its implications became clear. However, as he remembers, there was still a very strict deadline for the paper as it had to make the next issue (and these were still the days of hard-copy submissions). At the time, Prof Kruglyak was a young postdoc, so it fell to him to rush to the main FedEx office in downtown Boston before closing time, to make sure the manuscript got to the printer on time.

Prior to submitting the final text, Lander & Kruglyak produced some of the “original preprints”, sending a copy of the paper by snail mail or email to “everyone we knew in statistical genetics”, for comments and suggestions. After all, these guidelines would affect quite a lot of people and “signals that people would like to be results might not be real results anymore”.

Presentation1

Curtis, Nature Genetics 1996

Following publication, “the reactions came in essentially two flavors,” Prof Kruglyak recalls. There were those who thanked the authors, saying that someone really needed to do this. Others were less enthused. “They said, ‘you’re standing in the way of progress and making it harder to publish.’” In fact, Nature Genetics published two letters to the editor arguing that the proposed genome-wide significance threshold was too strict, or that at the very least additional discussion was warranted before these guidelines were adopted (see the letters here and here, and the authors’ reply here). Personally, I agree with the overall sentiment of Lander & Kruglyak as summed up in this portion of their reply: “The correspondents (all trained statisticians) argue that there is no need for guidelines because everyone should be able to interpret the genomewide significance of pointwise P values on their own. In our view, this is naïve. Most geneticists are not statisticians, and rules of thumb can be extremely helpful in promoting sensible discussion.”

The legacy of this paper is clear to anyone familiar with GWAS. “The GWAS community learned a lot from that whole experience [of false positive linkage reports],” says Prof Kruglyak. “There were many serious statistical geneticists involved [in the GWAS field] from the beginning, with a lot of carryover from the linkage era to the GWAS era.”

“Guidelines are not just ‘external gatekeepers’”, he noted.  They are not just there to tell you what you can and can’t publish. “You know what they say, the easiest person to fool is yourself.” These guidelines were developed to help researchers understand their own findings better and decide which are worth following up. “You can often make up a plausible story, but how strong is the evidence?”

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Mutation rates of Mycobacterium tuberculosis: From the archives (2013)

Posted by Catherine Potenski | Categories: ,

Mycobacterium tuberculosis- credit: NIH-NIAID (CC-BY)

Mycobacterium tuberculosis- credit: NIH-NIAID (CC-BY)

Continuing with our month-long celebration of Nature Genetics 25th anniversary, I have chosen to highlight a study by Sarah Fortune and colleagues estimating mutation rate differences between different lineages of Mycobacterium tuberculosis published in June 2013.

Multidrug resistance in M. tuberculosis is a global problem, and understanding the origins and dynamics of the emergence of resistance is an important scientific and public health endeavor.

Building on their previous work that used whole genome sequencing to estimate mutation rates of M. tuberculosis during latent infection, the authors then went on to study the rate at which different strains acquire drug resistance mutations. Using classical fluctuation tests and measuring rifampicin resistance in both clinical and laboratory isolates, they determined the mutation rates for strains from lineage 2 and lineage 4, observing an order of magnitude difference between them, with lineage 2 having the higher rate. These lineage 2 strains also acquired resistance to other antibiotics (ethambutol, isoniazid) at a higher rate than lineage 4 strains.

The authors then sought to relate the in vitro data to the in vivo infection environment. They analyzed whole-genome sequences from a lineage 4 outbreak and determined the base substitution rate; the in vivo data were in concordance with the in vitro per-day mutation rate.

Finally, the authors took these data and developed a simulation model of the evolution of drug resistance during infection in a human host. They simulate the emergence of multidrug resistance and show that in the model, individuals infected with lineage 2 strains had a substantially higher risk of acquiring multidrug resistance mutations.

Using a combination of in vitro, clinical and simulated data, Ford et al. contributed to our understanding of the emergence of multidrug resistance, highlighting the differences between strains and underscoring the importance of timely and sufficient treatment.

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Woolly mammoth hemoglobin brought to life: From the archives (2010)

Posted by Brooke LaFlamme | Categories: ,

Combarelles-mammouth

Cave painting: Mammouth gravé de la grotte des Combarelles (Dordogne, France)

As part of the ongoing celebration of the last 25 years of Nature Genetics, the editors are each choosing a few papers from our archives that we want to highlight. My first pick a paper from Kevin Campbell, Alan Cooper and colleagues on their structure-function analysis of woolly mammoth hemoglobin, published in May 2010.

I’ve picked this one to highlight because, well, who doesn’t love woolly mammoths?

The authors compared the gene sequences of the adult-expressed α- and β-like globin genes from extant elephant species (African and Asian elephants) and from a 43,000 year-old Siberian mammoth specimen reported first in Science. They found that the mammoth β-like genes (designated HBB/HBD by the authors) had 3 amino acid-altering substitutions compared to the extant species.

To test the effects of these protein-coding differences, the authors then “resurrected” the mammoth hemoglobin protein by expressing the mammoth sequence in E. coli and testing its O2 affinity at different ambient temperatures. They found that the O2 affinity of the recreated mammoth hemoglobin is less affected by temperature than that of modern-day elephants. The detailed structure-function analysis reported by Campbell et al. offered us a rare glimpse into the evolutionary process that shaped an extinct organism.

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25 years of Nature Genetics

Posted by Brooke LaFlamme | Categories: ,

 

AprilThis April marks the 25th anniversary of the first issue of Nature Genetics, and I think it’s safe to say that the field of genetics has come quite a long way. In 1992, we were still nearly a decade away from the draft human genome sequence, “omics” was not yet a word in common usage, and CRISPR/Cas9 gene editing wasn’t even a pipe dream.

Most of the content in our current issue would have possibly seemed like far-fetched science fiction to geneticists in 1992. Take for instance the new-and-improved domestic goat genome assembly reported on page 643 of this issue, for which multiple, relatively new technologies were employed to create one of the most complete and contiguous genome assemblies to date. However, as the News & Views by Kim Worley exemplifies, science marches on. While the geneticists of the past might have marveled at the possibility of a whole-genome shotgun assembly (indeed, a major advance reported in that first issue was a new technology allowing for automated sequencing of 106kb), Worley refers to the scientists of the present who are “frustrated with the highly fragmented genome sequences available for most species.”

Still, many things have remained the same.

Taking a look back at the very first editorial published in the journal, much of the journal’s mission in 1992 is still applicable to 2017. Take this passage:

“Researchers should not be dismayed that developments like this are widely reported in the general press. That is merely a measure of the widespread compassionate interest in inheritable disease. Who can be but flattered by such public testimony to the importance of a field of research?

“The research community’s interest, rather, is that there should also be a wide general understanding that the identification of an aberrant gene does not imply that there is a cure at hand for the condition for which it is responsible. […] The elucidation of the mechanisms by which genes determine the behaviour of the cells that carry them will be a general preoccupation in the years ahead. Nature Genetics intends to play its part in the publication of this important research, and also of course, in classical genetics that throws light on the human genome.”

NG1992

doi:10.1038/ng0492-1

While there is no denying that important medical advances have been enabled by the identification of disease genes, it is still painfully true that simply finding the gene does not directly lead to a cure on its own. Thus, both the identification of new disease-causing genetic alterations and studies that bring new mechanistic understanding of how a given mutation gives rise to disease are still core to the journal’s scope and aims.

The focus of the journal, as can be seen from this first editorial, was very much on human genetics at the beginning. Model organisms were considered just that, models for human biology. One of the major changes in the journal since that time has been our expansion to genetics (and genomics) more broadly, as represented by the many reference genomes and population genetics studies published for other organisms.

Too many landmarks to count

The editorial published in this month’s issue highlights a few selected articles from our among our more than 5,000 research publications over the years. These are obviously a restricted set of examples, and they are by no means the “best” papers, as such a ranking system would be ill-advised and ultimately useless. But the papers selected cover a wide range (though not all) of the sub-fields represented by the journal. This list includes landmark papers in human genome mapping (Kong et al. 2002) and cataloging of genetic variation (Iafrate et al. 2004); statistical methods that helped drive an entire field of research (Price et al. 2006); Mendelian disease gene discoveries that shed new light on biological mechanisms (Amir et al. 1999); key advances in the field of epigenetics (Heintzman et al. 2007); and advances in crop plant improvement (Ren et al. 2005).

We invite you to take a trip down memory lane and revisit these and other landmark papers from our archives. As a part of the celebration of 25 years of Nature Genetics, the editors will be blogging throughout April to highlight some of our past content.

A brief history of Nature Genetics

Nature Genetics was launched as the first of the Nature Research journals (if we ignore the very brief existence of Nature New Biology and Nature Physical Science in the early 1970s and the earlier version of Nature Biotechnology, Bio/Technology, published first in 1983).

While the history of genetics as field is by far more interesting than the history of a single journal, the occasion of our 25th anniversary has us thinking about our roots. For our 15th anniversary, founding editor Kevin Davies contributed a guest editorial telling the story of how Nature Genetics came about. I highly recommend that you check it out, if you haven’t seen it before.

Another feature of our 15th birthday celebration was the Question of the year. What would you do if the $1,000 genome were a reality today? To read the nearly 50 replies we received from leaders in the field, see the Question of the Year special here: http://go.nature.com/2mTMKBf.

The next 25 years

Just as researchers in 1992 would have been very unlikely able to predict the many breakthroughs that have occurred in genetics over the past 25 years, we have no idea where the next 25 years will take us. The goals will remain the same: to elucidate the mechanisms by which the genetic material produces the many phenotypic variations we see in nature and to identify the causes (and, more hopefully, cures) for human genetic disease.

That said, let’s take a stab at looking toward the future. What do you think will be the next major breakthrough in genetics? What will the field of genetics look like in another 25 years? Tell us below in the comments.

25 years from now, I hope to still be watching as geneticists make some of the greatest discoveries in biology. And I am confident that Nature Genetics will be there, playing its small role in announcing those discoveries to the world.

 

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A CRISPR screen for HIV targets

Posted by Catherine Potenski | Categories: ,

A new study published online this week in Nature Genetics reports the discovery of novel host targets of HIV infection identified from a high-throughput CRISPR/Cas9-based screen. This screen was performed in CD4 + T-cells and was designed to find candidate genes required for successful HIV infection, but whose inactivation did not affect cell viability. In this way, potential drug targets for anti-HIV therapy could be discovered.

Park et al., Nature Genetics 2016

Park et al., Nature Genetics 2016

Park et al., Nature Genetics 2016

Park et al., Nature Genetics 2016

 

The authors found two known (CCR5 and CD4) and three novel (ALCAM, SLC35B2 and TPST2) cellular factors that, upon abrogation, prevented HIV infection but did not have any negative effects on the cell itself. These new genes are involved in sulfation and cell aggregation pathways and represent candidate targets for interventional HIV therapy.

We spoke with first author Ryan Park to get some background on this research:

 Previous screens for host factors affecting HIV pathogenesis found a high number of hits, with low reproducibility across screens.  With your CRISPR/Cas9 approach, were you expecting similar results? Did the low number of hits in your screen surprise you?

We designed our screen stringently, as the existing literature has not been clear on what genes would potentially serve as good targets for host-directed anti-HIV therapies. Our goal was thus to identify these host factors with high confidence while maintaining an unbiased approach. The very low number of hits was certainly surprising, though, as you note, the limited overlap among the previous screens raised the suspicion of a high false positive rate and/or low reproducibility.

You find three novel genes that are dispensable for cell viability but that are needed for successful HIV infection.  Do you think that there could be natural polymorphisms in these genes in human populations that might mitigate susceptibility to HIV entry and transmission?

In the Exome Aggregation Consortium (ExAC) dataset recently published in Nature, there are individuals with truncations and/or homozygous mis-sense mutations in each of the three genes, as well as ITGAL (the loss of which we find is protective against HIV infection in primary CD4+ T cells). More work remains to be done to determine whether these individuals are relatively less susceptible to HIV infection.

Due to the high mutation rate of HIV and the emergence of resistance to drug therapies, potential targeting of host factors can be a useful strategy.  Do you envision these findings being utilized to develop novel anti-HIV therapies?

Host-targeted HIV therapies are of great interest for multiple reasons. Firstly, as you note, the emergence of drug-resistant HIV strains remains a major issue, particularly in settings where adherence to a daily antiretroviral regimen is challenging. Drug-resistant strains are less likely to emerge in the face of incomplete adherence to host-targeted therapies. Secondly, the identification of host factors may also serve as a basis for gene therapies (in which gene editing is used to produce a population of HIV-resistant target cells) that could result in a permanent HIV cure. As noted above, more work remains to be done to determine whether inactivation of these genes protects against HIV infection at the organismal level without causing detrimental effects.

How might this screen be adapted to find host factors important at other stages of the HIV life cycle and do you have future plans to explore such work?

Our screen captured all but the latest stages of the HIV life cycle (particularly virion assembly, budding, and maturation); this is because HIV Tat, which drives the GFP reporter in our cell line model, is expressed prior to these steps. Development of an alternative reporter system that is activated by virion budding or maturation would allow identification of host factors involved only at these late stages. Because completion of the HIV life cycle is not required for host cell killing by HIV, cells lacking these late-acting host factors may still not be captured in a screen; more importantly, these late-acting host factors may therefore not be attractive therapeutic targets.

Can this screening method be employed to find host factors important for infection by other viruses?  Do you speculate that there would be viruses for which a large number of non-essential host factors would be identified as important for infection?

The key elements of our approach, which include identification of a physiologically relevant cell line and the use of a high-complexity genome-wide sgRNA library, can be readily generalized to identify host factors that are critical to the propagation of any viral pathogen yet dispensable for cell viability. Our findings suggest that the number of non-essential host factors that are critical for HIV infection is quite limited, and that many candidate host factors identified by other screens or targeted studies may not be required for HIV infection or may compromise cell viability. Whether this is the case for other viruses is hard to know, but we have demonstrated that our approach can be quite powerful and specific in identifying the range of potential host targets with high confidence.

 

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Ubiquitin, keratin and skin fragility

Posted by Catherine Potenski | Categories:

Lin et al. Nature Genetics, 2016

Lin et al. Nature Genetics, 2016

Protein degradation is a highly coordinated process with multiple levels of regulation, including both targeted and autodegradation.  This sophisticated cascade of protein turnover must be precisely balanced to maintain proper physiological function. A recent article published in Nature Genetics reports the discovery of gene with protein-truncating mutations that lead to the skin condition epidermolysis bullosa, which is characterized by tendency to blister, itching and other abnormalities. The authors found 5 patients all with start codon mutations in the KLHL24 gene, which encodes Kelch-like protein 24, a substrate receptor of the cullin 3 (CUL3)–RBX1–KLHL24 ubiquitin ligase complex.

Lin et al., Nature Genetics, 2016

Lin et al., Nature Genetics, 2016

The mutant proteins from these patients were found to be stabilized, with increased levels in patient samples, leading the authors to hypothesize that KLHL24 may target a substrate that is important for the structural integrity of the skin.  Indeed, through mass spectrometry and biochemical analysis, they identify keratin 14 (KRT14) as a KLHL24 substrate, and find that KRT14 levels are decreased in patient samples. Keratin 14 is an intermediate filament component important for maintaining keratinocyte integrity and mutations in the gene are found in some epidermolysis bullosa patients. The authors further show that KLHL24 is autoubiquitinated and that the truncated mutant has reduced levels of autoubiquitination, stabilizing the protein. This increased KLHL24 stability leads to increased KRT14 degradation, resulting in the skin fragility phenotype observed in the patients.

Lin et al., Nature Genetics, 2016

Lin et al., Nature Genetics, 2016

 

 

 

 

 

 

 

 

 

 

 

Although dynamic regulation of keratins by the ubiquitin–proteasome system had been proposed, no targeting E3 ligases had been identified. This work established KLHL24 as a keratin-targeting E3 ligase.

 

We spoke with authors Dr. Xu Tan and Dr. Yong Yang to get some background on their research.

Can you briefly describe how you found the KLHL24 mutations in these different patients?

The first three epidermolysis bullosa (EB) patients were first screened for the 18 previously known causative genes but no mutations were found. Then we performed whole exome sequencing and pinned down only one common variant gene among all three patients, namely KLHL24. We then acquired samples from two additional patients without mutations in the 18 known causative genes and used Sanger sequencing to show that both of them also have the mutations in the same KLHL24 gene, confirming that this is a new causative gene of EB.

All the patients you studied had start codon mutations leading to truncations in the protein. This must have been intriguing. What where your initial thoughts about this finding?

We were shocked. The first thought was that these must be gain-of-function mutations, unlike all the other EB mutations, which are loss-of-function mutations that can occur all over the places.

 

You very nicely demonstrate a model whereby Keratin 14 is an ubiquitination substrate of KLHL24, and that the truncated mutant is stabilized, thus leading to greater Keratin 14 degradation and the skin fragility phenotype. Can you walk us through how you teased apart this model? What do you consider the key piece of evidence that supports this model?

We used an unbiased “pull down + mass spectrometry” method to look for the binding proteins to the substrate binding domain of KLHL24 and Keratin 14 was the only one we found that specifically binds KLHL24 but not a carefully designed mutant that is predicted structurally to lose the substrate binding capacity. We immediately verified the binding and also showed that knocking down/overexpressing KLHL24 can increase/decrease Keratin 14 levels. A key piece of evidence is that transfection of KLHL24 in cell lines can boost Keratin 14 ubiquitination. Afterwards, we obtained two important pieces of in vivo evidence to show the anti-correlation of KLHL24 level and Keratin 14 level (in human skin samples and a knock-in mouse model), nicely confirming that Keratin 14 is a ubiquitination substrate of KLHL24.

 

You make a knock-in mouse, which recapitulates the decreased Keratin 14 levels similar to what is seen in patients, but not the skin fragility phenotype. Can you comment on why this might be so?

Many differences exist between human and mouse skin, the most obvious is the presence of fur in the mouse skin, which might afford better mechanic support of the epidermis than that in the human skin. In addition, there is actually a small but significant difference between the degrees of Keratin 14 decrease in patients and the mouse model (~70% decrease in patients vs. ~50% decrease in mice). Previously mouse models having ~50% decrease of Keratin 14 (the Krt14+/- mouse model) also did not show skin fragility. We don’t yet know the reason for the differential decrease of levels in human and mouse skin but are working on finding out the answers.

 

Do your findings have any potential implications for novel therapies for epidermolysis bullosa?

Absolutely, as I mentioned these are the first gain-of-function mutations found for EB, which should be easier to target therapeutically than loss-of-function mutations. Inhibiting KLHL24 in patients that we identified with these types of mutations should be able to effectively treat the conditions. We are now actively working on finding a specific KLHL24 inhibitor. In addition, because KLHL24 is a negative regulator of Keratin 14, other EB patients with partial loss-of-function mutations of Keratin 14 could also be helped by treatment with a KLHL24 inhibitor. In general, drug development targeting the ubiquitin-proteasome pathway has been given high hopes but it is not very obvious how to target the pathway specifically. Our studies provide a good example showing the importance of autoubiquitination of an E3 ligase, which might suggest previously over-looked strategies to target E3s.

 

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October issue cover: What’s going on here?

Posted by Brooke LaFlamme | Categories: , ,

Oct

Convergent cabbages by Keyong Chang

For all of October, we at Nature Genetics have been admiring the lovely cabbages on our cover. The image, created by photographer Keyong Chang, was contributed by the authors of the study on page 1218 of the issue.

But what is the story behind these pretty green cabbages?

Xiaowu Wang, corresponding author of the study, gave us a behind-the-scenes look at the process that led to the picture on our cover.

The image conveys the main idea of the study, namely that Brassica oleracea (cabbage, left) and Brassica rapa (Chinese cabbage, right) have taken similar evolutionary paths to arrive at their similar, but distinct, appearances. During domestication, farmers selected for cabbages of both species to have the large, leafy heads for which they are known. As shown in the study, the farmers were unknowingly selecting for orthologous genes in these two species. Read more

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