A DNA integrity network in the yeast Saccharomyces cerevisiae

A DNA integrity network in the yeast Saccharomyces cerevisiae.

Cell, Vol. 124, No. 5. (10 March 2006), pp. 1069-1081, doi:10.1016/j.cell.2005.12.036

by Xuewen Pan, Ping Ye, Daniel S. Yuan, Xiaoling Wang, Joel S. Bader, Jef D. Boeke

I use this paper in my graduate genetics course.  It describes a global screen for synthetic defects involving DNA integrity, which reveals a network of 16 functional modules.  The paper illustrates screens based on genetic interactions (in this case, synthetic lethality or fitness defects) and the systems biology used to evaluate the results of such a screen.  It also illustrates the use of Saccharomyces cerevisiae as a model system.

A whole-genome RNAi Screen for C. elegans miRNA pathway genes

A whole-genome RNAi Screen for C. elegans miRNA pathway genes.

Current biology : CB, Vol. 17, No. 23. (4 December 2007), pp. 2013-2022, doi:10.1016/j.cub.2007.10.058

by Devin H. Parry, Jinling Xu, Gary Ruvkun

I use this paper in my graduate genetics course. It describes a whole-genome screen of C. elegans using RNA interference, provides an example of whole-genome parallel reverse genetic screens, and illustrates both the microRNA biosynthesis pathway and a bit of C. elegans biology.

All 4096 hexamers evaluated as exonic splicing elements

Exon sequences have a large effect on splicing efficiency. Specific sequences can act as ESEs (exonic splicing enhancers) to promote splicing, or as ESSs (exonic splicing suppressors) to reduce splicing. In the August 2011 issue of Genome Research, Ke et al. describe a comprehensive quantitative measure of the splicing impact of all 4,096 6-mer sequences using an Illumina Genome Analyzer to compare spliced transcripts with an input library. They tested five positions within two different internal exons in a minigene system and sequenced millions of successfully spliced transcripts after transfection of human cells. Specific hexamers had different effects in different positions, but these were correlated, and the effect on splicing of each 6-mer could be quantified. Many complications (secondary structure, synergy, effect on chromatin) are addressed by this study, which provides a huge data set. However, this is just the beginning. This paper examines only a single cell type, and it concerns only a single type of alternative splicing - exon inclusion. This high throughput approach, which captures the power of high throughput sequencing, will certainly be extended to other contexts in alternative splicing, and may prove useful for defining other nucleic acid regulatory motifs.

Assembling haplotypes in diploid sequencing projects

Shotgun sequencing has been the dominant mode of genome sequencing since the beginning of genomics. However, assembly of a complete genome can be complicated when the two haploid genomes present within the individual being sequenced are quite different, in which case the coverage is reduced by half and the two haploid genomes must be assembled separately. Problems also arise when two haploid genomes diverge over only some of their length. For example, Barrière et al. (2009) find that, despite inbreeding designed to generate a fully homozygous sample, "approximately 10% and 30% of the Caenorhabditis remanei and C. brenneri genomes, respectively, are represented by two alleles in the assemblies."

A similar problem arises when attempting to resolve the haplotypes within an individual. In the January issue of Nature Biotechnology, Kitzman et al. describe the "Haplotype-resolved genome sequencing of a Gujarati Indian individual." Sequencing pools of large-insert clones provides information about individual haplotypes across most of the genome. The power of combining "the throughput of massively parallel sequencing with the contiguity information provided by large-insert cloning" allows parallel assembly of distinct sequence from large-insert clones to provide information about genome structure that might otherwise be very difficult to tease out of a mixed assembly.

What interests me about the method of Kitzman et al. is that it can be applied directly to cases of widespread structural polymorphism, and I expect to see it used for a variety of problems in the coming years. With this approach, or similar approaches, intractably complex genomes (e.g. Drosophila subobscura - see Sánchez-Gracia and Rozas 2011), asexual species and even metagenomic samples will yield their secrets.

Missing heritability found?

The problem of missing heritability in genetic studies (primarily genome-wide association studies, or GWAS) has been a major focus of interest in genetics journals during the past year. Many recent articles cite Manolio et al. (Nature, 8 Oct. 2009) for stating the problem:

Genome-wide association studies have identified hundreds of genetic variants associated with complex human diseases and traits, and have provided valuable insights into their genetic architecture. Most variants identified so far confer relatively small increments in risk, and explain only a small proportion of familial clustering, leading many to question how the remaining, ‘missing’ heritability can be explained. Here we examine potential sources of missing heritability.

In April, I cited an excellent article by McClellan and King, who argued that "many rare alleles account for common diseases". Now, Johansen et al. (Nature Genetics Aug. 2010), in "An excess of rare variants in genes identified by genome-wide association study of hypertriglyceridemia" provide evidence that many such rare variants can be found by sequencing a small number of candidate genes in affected individuals. Although the variants described in this study increase the proportion of genetic variation explained only incrementally, it is likely that they have only skimmed the surface (since sequencing was limited to coding regions). This article follows similar results looking at candidate genes (Romeo et al. 2009). "Pooled association [statistical] tests for rare variants in exon-resequencing studies" have already been developed (Price et al. 2010), and it is reasonable to believe that these methods can be extended to complete genome sequencing data. Thus, it appears that the analysis of rare variants will be increasingly common, and will explain much of the missing heritability.

However, progress has also been made in the more straightforward approach of looking at ever larger samples. In this week's Nature, Teslovich et al. describe a study of over 100,000 individuals that yielded an amazing 95 loci affecting blood lipids at p values of less than 5 x 10^-8 ("Biological, clinical and population relevance of 95 loci for blood lipids"). The authors take this position:

It has recently been suggested that conducting genetic studies with increasingly larger cohorts will be relatively uninformative for the biology of complex human disease, particularly if initial studies have failed to explain a sizable fraction of the heritability of the disease in question (Goldstein 2009). As the reasoning goes, analysis of a few thousand individuals will uncover the common variants with the strongest effect on phenotype. Larger studies will suffer from a plateau phenomenon in which either no additional common variants will be found or any common variants that are identified will have too small an effect to be of biological interest.

Our study provides strong empirical evidence against this assertion. We extended a GWAS for plasma lipids from ~20,000 to ~100,000 individuals and identified 95 loci (of which 59 are novel) that, in aggregate, explain 10–12% of the total variance (representing ~25–30% of the genetic variance). ... We expect that future investigations of the new loci (for example, resequencing efforts to identify low-frequency and rare variants, or functional experiments in cells and animal models, as demonstrated for SORT1 in a separate study reported in the accompanying paper [Musunuru et al.]) will uncover additional important new genes.

This recent work provides optimism the heritability underlying complex human genetic disease will be found, in the form of both more genes and more variants per gene.

The GAO, Congress and the public on genetic testing

The Subcommittee on Oversight and Investigations of the House of Representatives Committee on Energy and Commerce held a hearing on "Direct-To-Consumer Genetic Testing and the Consequences to the Public Health" on Thursday. I haven't seen any news after the fact, but it was covered by the 23andMe blog "The Spittoon," where I got the link to the committee's own website (which has copies of the testimony). The hearing included a report from the GAO which made a strong case for regulation of direct-to-consumer testing, including an extremely disturbing "video" which appeared on YouTube ("video" in quotes because it's actually just recordings of telephone calls with the words printed on screen). I think that it is almost certain that new regulations will emerge soon.

23andMe customers have responded, and there is a petition calling for continued access to genetic information. I signed it myself, making this statement:

I certainly recognize the need to insure that test results are valid. However, I'm not sure that goes beyond CLIA certification. I also recognize the need to protect consumers from misinformation and bad advice from the unqualified. However, I'm not sure that is within the FDA's purview. My main point is that secure and private access to reliable personal genetic information is a valuable thing that does not put the consumer at undo risk.

That said, regulations that protect consumers from bad advice may be appropriate. However, it's going to be tricky, because we're talking about regulation of speech and education. I hope that the regulations are written in a way that encourages the broad dissemination of genetic knowledge from the many reliable sources currently available.

FDA's public hearing on genetic tests

The FDA had a public hearing on our campus this week (Monday and Tuesday) about oversight of “laboratory developed tests” (including direct-to-consumer genetic tests). I dropped by for the last two sessions (on direct-to-consumer tests and education and outreach). Each included presentations from interested parties (the “public” in this case being representatives from companies selling direct-to-consumer tests, professional and educational organizations), a panel discussion and comments. FDA officials sat on the podium but did not speak. There were a few hundred people there, and it was a sophisticated group (at least the speakers felt no need to define BRCA, GWAS, CLIA, QSR, DTC, LDT, IVDMIA, NIH, CDC, FTC, NIST, NSGC or many other acronyms). I spent a lot of the time Googling jargon with my phone.

It was very interesting. I got the feeling that genotyping companies such as 23andMe will not be able to continue to operate as they have for a lot longer. I heard several people call for physicians being involved in "ordering the test" and "interpreting the test." Already, New York and Maryland prohibit people from obtaining their own genetic information from direct-to-consumer companies.

Of course one problem is that the typical family care physician probably knows even less about genetics and how to interpret the results of these tests than the typical 23andMe customer, and that was not lost on many of the people there. Everyone recognized the need for educating both consumers and physicians. One particularly original idea was that consumers should have to pass a test analogous to a driver’s test (as is currenly done at the Personal Genome Project).

My reaction was to come home and download my personal data from 23andMe so that I can be assured of continued access to it. I am concerned to see what the FDA decides to do.

The FDA and me, and you, and our genetic information

The FDA is holding a public meeting on "Oversight of Laboratory Developed Tests (LDTs)" on my campus (University of Maryland) today and tomorrow. Registration is closed, but it's possible to view the proceedings via webcast (here). I am doing that now (I'm watching Judith Wilber right now). I stopped by briefly this morning and hope to go back tomorrow afternoon. The topic has been in the news a bit lately (see my links under right to know on delicious), and there are legitimate issues to address.

I have expressed my opinion in favor of our right to know our own genetic makeup on my blog and will only briefly restate those arguments further here. My preference would be that oversight and approval of genotype "tests" (including whole-genome sequencing) be limited to technical standards (Are the genotype results are accurate?), but that rules and oversight are appropriate for interpretation and guidance. A genotype is information, and it may be best to separate genotyping tests from the recommendations based on them to the extent possible. Asking a genotyping service to imagine the clinical and medical utility of their tests is a bit like asking the manufacturer of bathroom scales to prove that knowing one's weight is medically useful. Clinical utility should not be a criterion for genotype tests that provide accurate and reliable information. I'm not saying that the FDA should not protect consumers from bad advice, but that in the case of genotype information, this can and should be separated from the regulation of tests per se.

Many rare alleles account for common genetic diseases

The last three years have witnessed an explosion of genome-wide association studies. A catalog of results at the National Human Genome Research Institute (www.genome.gov/gwastudies) currently lists 545 publications associating 2,664 single nucleotide polymorphisms with human traits, and top journals in the field (such as Nature Genetics) have devoted themselves almost entirely to the publication of GWAS results. However, as summarized in an excellent review in the current issue of Cell (McClellan and King, "Genetic Heterogeneity in Human Disease"), it appears that "common risk variants fail to explain the vast majority of genetic heritability for any human disease, either individually or collectively (Manolio et al., 2009)." Instead, while "most human variation is ancient and shared," most alleles, including those that cause disease, are recent and rare. "Rare large-effect mutations are now recognized as the causes of many different common medical conditions." This makes sense in that deleterious alleles should be eliminated relatively quickly by selection, but leaves us unsure of how to interpret the available GWAS data. The good news is that genome sequencing will soon be used to discover rare variants in many people. At least for the researchers, "it will be fun to sort out."

Conservation of expression without conservation of regulatory sequences

Conservation is a reliable indicator of what sequence features have a function. Very often, in comparative genomics, conservation is the only clue available. However, there are many examples of highly conserved sequence for which no function can be identified, and many of these appear to be non-essential. A recent review by Weirauch and Hughes ("Conserved expression without conserved regulatory sequence: the more things change, the more they stay the same." Trends in Genetics 26:64, PMID 20083321) considers the opposite case: when expression is conserved but regulatory sequences are not. They list 17 examples of genes whose expression pattern is conserved despite divergent cis-regulatory sequences. In many of these cases the regulatory mechanisms are well known, and the review also includes a discussion of mechanisms that allow expression patterns to persist without conservation of the cis-regulatory signals.

Water fleas have new introns

The study of intron gain and loss can be frustrating, because such events are very rare. Documented cases of intron gain have been particularly elusive. That makes the recent study by Li et al. especially exciting ("Extensive, Recent Intron Gains in Daphnia populations," in Science 2009, from Michael Lynch's group at Indiana). They have found that intron gain is remarkably common in Daphnia, and that the new introns lack features expected from most hypothesized mechanisms of intron gain. The independent gain of introns in parallel at the same site in different lineages is also observed, and also unexpected. These authors hypothesize that intron gain may arise fortuitously as a consequence of DNA damage, but this remains to be established. Whatever the mechanism, the observation that new introns can arise at reasonable rates in at least one species provides both an important clue to the origins of introns and a system for further investigation.

A consideration of the allele-frequency spectrum suggests that these new introns in Daphnia (also known as the water flea) are indeed deleterious, bringing to mind a famous poem by Jonathan Swift ("On Poetry: A Rhapsody", pub. 1733):

...
"So nat'ralists observe, a flea
Hath smaller fleas that on him prey,
And these have smaller fleas that bite 'em,
And so proceed ad infinitum."
...

The 2009 Nobel in Medicine, Telomeres, Joe Gall, women and RNA

This year's Nobel Prize in Physiology or Medicine was awarded to Jack Szostak, Elizabeth Blackburn and Carol Greider for the discovery of telomeres and telomerase. If you want a nice summary of the science behind the prize, I would recommend nobelprize.org. What struck my attention was Carol Greider's mention of Joe Gall in the New York Times:

The study of telomeres is notable as a field of research in which female scientists are particularly prominent. Dr. Greider said she ascribed this to a “founder effect,” the founder being Joseph Gall of Yale University. Dr. Gall trained Dr. Blackburn and other women, and they recruited others to the field “because there is a slight tendency for women to work with other women,” Dr. Greider said. She herself trained with Dr. Blackburn.

One of those "other women" was my own thesis advisor, Joan Steitz. Another woman who is just offstage in this story is Barbara McClintock, who won the Nobel Prize in 1983 for the discovery of mobile genetic elements, but whose work on the instability of broken chromosome ends (1941 in Genetics: "The stability of broken ends of chromosomes in Zea Mays") was an important part of the background that prepared people for the discovery of telomeres (if broken ends were unstable, then normal ends had to be somehow different).

Another motif to this story is RNA. Telomerase turned out to be an RNA enzyme. Jack Szostak went on to actively investigate the origins of catalytically active RNA. And Joe Gall has been working on RNA all along.

Nested Association Mapping Recominant Inbred Lines in maize

The August 7 issue of Science includes a report on quantitative trait loci (QTLs) affecting flowering time in maize. It is important because it presents the first results with an important new technique known as nested association mapping (NAM), which utilizes a large set of recombinant inbred lines derived from diverse founder lines. The article is accompanied by an excellent piece by Trudy Mackay that puts this method into perspective. "Linkage mapping can readily detect chromosomal regions containing one or more QTLs that affect a trait ... but it is difficult to precisely localize the QTLs. This approach usually relies on crosses between two strains, thus capturing only a tiny fraction of genetic diversity in the population. By contrast, association mapping widely samples genetic diversity and requires fewer individuals, but has less power to detect QTLs when alleles are not common." The new method combines advantages of these earlier approaches. It also provides surprising results, different from what has been found in other systems. In particular, the authors find numerous genes of small effect with few genetic or environmental interactions, so that "a simple additive model accurately predicts flowering time for maize." The authors argue that their data supports "common genes with uncommon variants." Moving forward, I look forward to seeing this system applied to other traits, and to the discovery of specific genes involved in this and other traits.

Relevant links:

McMullen et al.. 2009 "Genetic Properties of the Maize Nested Association Mapping Population" Science 325: 737.
This is the main paper presented here. It describes QTLs for flowering time.

Mackay. 2009 "A-maize-ing Diversity" Science 325: 688.
A very nice summary of how NAM compares to other methods for finding QTLs and how these results compare with those from other systems.

Mackay. 2001. "The genetic architecture of quantitative traits." Annu. Rev. Genet. 35: 303.

Yu et al. "Genetic Design and Statistical Power of Nested Association Mapping in Maize" Genetics 178: 539.
An earlier paper from some of the same authors describing the NAM method.

45 microexon-rich genes in the schistosomiasis parasite genome

I have been fascinated by microexons for a long time. Many exons are relatively small (less than 100 bp.), but still large enough for recognition of the two splice sites by the splicing machinery simultaneously. In fact, the boundaries of such exons are often recognized coordinately in a process known as exon definition. Introns that are too small for that, so that the two splice sites cannot be recognized simultaneously, are termed microexons. Many are less than 10 nucleotides (see Volfovsky et al. 2003). Sometimes the downstream intron must be removed first (e.g. potato invertase). The inability of the splicing machinery to recognize both splice sites simultaneously due to physical occlusion probably comes into play with exons less than about 30 nucleotides. Although the exact length at which this occurs is difficult to know for sure (especially in a species like S. mansoni, for which we have little experimental data, it is nevertheless what I consider to be the defining characteristic of a true microexon. Microexons are characterized by alternative splicing and annotation errors.

Now, the genome of the blood fluke Schistosoma mansoni reveals "at least 45 genes with an unusual microexon structure," such that microexons make up the majority of the coding sequence in those genes. As is often true with microexons, these genes are alternatively spliced, suggesting that a "'pick and mix' strategy is used to create protein variation." These MEGs, or microexon genes, have the hallmarks of secreted proteins and are expressed in the intramammalian stages of the life cycle. It will be interesting to see what role microexon splicing or these genes turns out to play.

The mystery of ultraconserved sequences

A Science News Focus piece by Don Monroe ("Genomic Clues to DNA Treasure Sometimes Lead Nowhere") presents the concerns that "not all conserved sequences are important and, worse, that not all important sequences are conserved." While I think that formulation is a bit misleading, it does point to some very interesting and timely questions in genomics. Eddie Rubin and colleagues have shown that "deletion of ultraconserved sequences yields viable mice" (PLOS Biology 2007). While this is not the same as showing that the sequences are not important, it does point to an important specific question ("What are these noncoding ultraconserved sequences in vertebrate genomes doing?") and an important general question ("Why is the correlation between gene importance and gene evolutionary rate so weak?" Wang and Zhang 2009). The article got me thinking about those questions.

However, the conservation of nonessential sequences is not new, and there are several well-established means by which the loss of sequences important enough to be maintained by purifying selection can fail to produce a phenotype. First, the specific sequences tested can be redundant. Second, the process under selection can be important without being essential. Examples of widely conserved processes that are not essential in all species include telomerase and nonsense-mediated decay. Third, the selection can be imposed by something (such as a rare pathogen) that does not arise in the experimental system. However unlikely these cases may seem, I know of no means other than purifying selection by which a sequence can be maintained unchanged for millions of years.

More on paternal age

Today's New York Times has an article, 'It Seems the Fertility Clock Ticks for Men, Too,' by Roni Rabin, that covers both declining male fertility and genetic risks tied to a father's age. The latter is something that I have written about here before, in response to a study linking autism to paternal age. That posting generated some interesting discussion and an email that pointed me to a very interesting article by Dolores Malaspina in the schizophrenia research forum: 'schizophrenia research and the male germ line.' The observations of increased risk are dramatic. I can't add much to that excellent commentary by an expert in the field, except for the small point that double-strand breaks are missing from Malaspina's list of possible genetic mechanisms. There is evidence, from Drosophila and people that double strand breaks increase with paternal age.

As discussed in comments to my earlier post, I am especially interested in knowing what genes these surprisingly specific effects act through. A 1997 PNAS article describes a few genes that are somehow quite special. In a 2003 comment in Science, Crow describes them as "hot-spots occurring almost exclusively in males and rising steeply with age. Three genes--fibroblast growth factor receptor 3 (FGFR3, mutated in achondroplasia), FGFR2 (mutated in Apert's syndrome), and RET (mutated in multiple endocrine neoplasia)--are examples of the hot-spot class. In this class, genes carry mutations that are clustered at just one or two nucleotide sites." He then discusses at length the hypothesis that these specific mutations are selected in the male germ line, a hypothesis that Goriely et al. put right in the title of their article: "Evidence for Selective Advantage of Pathogenic FGFR2 Mutations in the Male Germ Line." The title of Crow's commentary was "There's Something Curious About Paternal-Age Effects." The effects on complex behavioral diseases like autism and schizophrenia make this story even more curious.

How far is H5N1 bird flu from 1918 all over again?

Reconstruction of the deadly 1918 influenza virus has been controversial because of the risk that the reconstructed virus might escape from the laboratory or fall into the wrong hands. On the other hand, research with this virus has indeed led to some important insights (reviewed by Garcia-Sastre and Whitley). One of the first of these was that the 1918 virus was probably avian in origin. Since that review the severity of symptoms has been attributed to an overactive innate immune response (Kobasa et al. 2007, summarized by Loo and Gale, 2007), and the infectivity to mammals has been attributed to two amino acid changes in the hemagglutinin gene (Tumpey et al., 2007, summarized by Enserink, 2007). Does this mean that the H5N1 virus now infecting birds in much of the world is only two point mutations away from causing a human pandemic? This is not clear. Certainly, there are those who think that we need to be more concerned about the birds (e.g. Juan Lobroth), and Yamada et al., 2006 present evidence that changes in hemagglutinin can be observed (already) in some H5N1 viruses isolated from humans even though human-to-human transmission remains extremely rare. Even so, we have learned what to look for.

2006 - a year for small genomes and cells

Earlier this fall, Nakabachi et al. described a 160-Kilobase genome from the bacterial endosymbiont Carsonella in Science (PubMed). Now, Baker et al. describe acidophilic Archaea that may be the smallest cells yet observed (Science, PubMed). The authors themselves express this claim cautiously ("It has not escaped our attention that if the average cell size estimated from TEM observations is accurate, the ARMAN cells have volumes of <0.006>3, making them smaller than any other known cellular life form."), but it seems to me that even if this is some sort of artifact (perhaps these cells are larger, but extremely reticulate) it still bears on the controversy regarding microfossils in the martial meteorite ALH84001, which have been thought too small to be life.

National Geographic's Genographic project

Today's New York Times reports (on the front page, in "DNA Gatherers Hit Snag: Tribes Don't Trust Them" by Amy Harmon) that the Genographic Project of National Geographic has been slowed by resistance from skeptical indigenous people, and a specific case involving "the Alaska review board" is highlighted. However, the same article also states that they have collected over 18,000 samples from remote places and 150,ooo samples from nonindigenous Americans interested in their roots. The picture is a screenshot from the National Geographic site.

Resveratrol improves health, survival, mitochondrial function and insulin resistance in mice.

Two recent articles confirm that resveratrol can benefit health. Baur et al. report in Nature that mice on a high-calorie diet not only live longer if fed resveratrol, but also show increased insulin sensitivity, increased PGC-1 activity, increased mitochondrial number and improved motor function. Lagouge et al. report in Cell that resveratrol not only greatly increases mitochondrial function and endurance but that downstream effects on gene expression depend on SIRT1-dependent deacetylation of PGC-1. These results are all consistent with resveratrol acting through the same pathway by which caloric restriction extends lifespan. In just a few weeks, these results have been widely reported and are already well known. I recommend reading the original articles and the commentary by Kaeberlein and Rabinovitch in Nature, and following the reporting by Nicolas Wade in the New York Times. I expect this story to show considerable endurance.