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Soon after SARS-CoV-2 was detected in China, scientists began analyzing viral sample and posting the genetic codes online. Mutations allowed researchers to track the spread by linking closely related viruses to understand how SARS-CoV-2 infects humans. They recognized that SARS-CoV-2 encode their genome in RNA and tends to pick up mutations quickly as they are copied inside their hosts. Yet, sequencing data suggest that coronaviruses change more slowly than most RNA viruses, probably because of a proofreading enzyme that corrects fatal copying mutations. In spite of the virus slow mutation rate, scientists have been able to classified more than 12,000 mutations in SARS-CoV-2 genomes.
Many scientists such as David Montefiori, a virologist who spent much of his career studying how chance mutations in HIV helps it evade the immune system thought that COVID-19 might cause the same thing. His laboratory in collaboration with Dr. Bette Korber investigated several thousands of coronavirus sequences for mutations that might have changed virus properties around the world.
Compared to HIV, SARS-CoV-2 seems to be changing slower than it spreads, but one mutation is obvious. That mutation includes a gene encoding the spike protein, which helps the virus particles penetrate cells. According to Korber, the 614th amino acid position of the spike protein, the amino acid aspartate was replaced by glycine, because of a mutation, D614G that altered a single nucleotide in the virus’s 29,903-letter RNA code.
To observe whether D614G mutation made the virus more transmissible, Montefiori evaluated its effects under laboratory conditions but he couldn’t study the natural SARS-CoV-2 virus in his lab, because of the biosafety containment required. So, he studied a genetically modified form of HIV that used the SARS-CoV-2 spike protein to infect cells. Such ‘pseudo virus’ particles are a workhorse of virology labs: they enable the safe study of deadly pathogens such as the Ebola virus, and they make it simpler to test the effects of mutations.
The strongest sign that D614G has a consequence on the spread of SARS-CoV-2 in humans comes from an ambitious UK effort called the COVID-19 Genomics UK Consortium, which has analyzed genomes of around 25,000 viral samples. From these data, researchers have identified more than 1,300 instances in which a virus entered the United Kingdom and spread, including examples of D- and G-type viruses.
What is clearly known is that D614G is an adaptation that helps the virus infect cells or compete with viruses that don’t carry the change, while at the same time altering a bit of information about how SARS-CoV-2 spreads between people and through a population. Some scientists believe that D614G mutation should explain how SARS-CoV-2 fuses with cells and can use that process to develop a more efficient vaccine.
At the present time, the evidence suggests that D614G doesn’t stop the immune system’s neutralizing antibodies from recognizing SARS-CoV-2, partly because the mutation is not in the spike protein’s receptor-binding domain.
Recent Progress in Gene Editing Error Reduction, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair
Recent Progress in Gene Editing Error Reduction
Larry H. Bernstein, MD, FCAP, Curator
LPBI
Advances in Genome Editing
Researchers develop a CRISPR-based technique that efficiently corrects point mutations without cleaving DNA.
Illustration of DNA ligase, one of the cell proteins involved in repairing double-strand breaks in DNA WIKIMEDIA; WASHINGTON UNIVERSITY SCHOOL OF MEDICINE IN ST. LOUIS, TOM ELLENBERGER
Most genetic diseases in humans are caused by point mutations—single base errors in the DNA sequence. However, current genome-editing methods cannot efficiently correct these mutations in cells, and often cause random nucleotide insertions or deletions (indels) as a byproduct. Now, researchers at Harvard University have modified CRISPR/Cas9 technology to get around these problems, creating a new “base editor,” described today (April 20) in Nature, which permanently and efficiently converts cytosine (C) to uracil (U) bases with low error in human and mouse cell lines.
“There are a lot of genetic diseases where you would want, in essence, to swap bases in and out,” said Jacob Corn, scientific director of the Innovative Genomics Initiative at the University of California, Berkeley, who was not involved in the research. “Trying to get this to work is one of the big challenges in the field, and I think this is a really exciting approach.”
To date, CRISPR/Cas9 genome-editing approaches have relied on a cellular mechanism called homology-directed repair, which is triggered by double-strand breaks in DNA. Researchers supply cells with a template containing the desired sequence, make a targeted double-strand break with the Cas9 enzyme, and then wait to see whether homology-directed repair incorporates the template to reconnect the strands. Unfortunately, this method is inefficient (incorporation is rare) and often introduces new errors in the form of random indels around the break, making it impractical for therapeutic correction of point mutations.
So researchers at Harvard, led by chemist and chemical biologist David Liu, tried a different approach. First, they inactivated part of Cas9 so that it couldn’t make the double-strand break. They then tethered Cas9 to an enzyme called cytidine deaminase that directly catalyzes conversion of C to U (essentially an equivalent of thymine, T), without DNA cleavage. Sending this machinery into cells creates a mismatched pair at the target, comprising the newly introduced U, and an original guanine base (G) on the opposite strand. “This [mismatch] distorts the DNA,” Liu explained. “It creates a funny little bulge that doesn’t look normal.”
The bulge alerts a different cellular repair mechanism, mismatch repair, which removes one of the mismatched bases and replaces it with the complement to the remaining one. Without any information about which base is incorrect, mismatch repair produces the desired G to A conversion about 50 percent of the time; the rest of the time it converts the U back into a C.
But mismatch repair does incorporate further information when available: it detects tiny breaks in the DNA backbone called nicks. “Cells have evolved mismatch repair machinery to prioritize old DNA over newly synthesized DNA,” said Liu. “Newly synthesized DNA tends to have some nicks in it. So we reasoned that we could manipulate mismatch repair to favor correcting the DNA strand that we don’t want, namely the strand containing the G.”
The team again modified Cas9, this time so that it would create a nick in the nonedited, G-containing strand, while leaving the edited, U-containing strand intact. “Now the cell says, ‘Aha, there’s a mismatch here, and the base at fault must be the G, because that must be a newly synthesized strand because it has a nick in it,’” said Liu. “It will preferentially correct that G, using the other strand as a template.”
Using the technique at six loci in human cells, the team reported a targeted base correction rate of up to 37 percent, with only around 1 percent of the sequences showing indels. By contrast, a normal Cas9 editing technique tested on three of those loci showed less than one percent efficiency, and more than four percent formation of indels. The researchers also demonstrated the technique’s potential to correct disease-associated mutations by converting a variant of APOE, a gene linked with Alzheimer’s, into a lower risk version in mouse cells.
“By engineering this Cas9, they’ve figured out a really nice way to trick the cell into preferring pathways that it would normally not prefer,” said Corn. However, because the method is currently only able to convert C-G to U-A (i.e., T-A) base pairs, and in some cases edits other C bases in the immediate vicinity of the target, “it’s certainly not a panacea,” he cautioned. “It doesn’t mean that you can now cure every genetic disease out there. But there are probably going to be quite a few that fit into this category.”
The University of Oxford’s Tudor Fulga called the technique “an extremely ingenious idea” to get around inefficient homology-directed repair, and to reduce unwanted indel formation. “I think this will set up a paradigm shift in the field,” he told The Scientist. “It is very likely that the impact of Cas9-mediated base editing is going to be massive—both in terms of answering basic research questions and in genome engineering–based therapeutic applications.”
Also appearing in Nature today are two studies addressing a potential alternative to Cas9: the Cpf1 enzyme. CRISPR/Cpf1 creates “sticky ends”—overhangs in cleaved DNA that leave unpaired bases either side of the break—rather than the blunt ends made by Cas9’s double-strand DNA cleavage.
Emmanuelle Charpentier and colleagues at the Max Planck Institute for Infection Biology in Germany have shown that, unlike Cas9, Cpf1 processes RNA in addition to cleaving DNA. Meanwhile, Zhiwei Huang of the Harbin Institute of Technology, China, and colleagues have described the crystal structure of CRISPR/Cpf1.
“Sticky ends are more efficient [than blunt ends] for DNA repair in cells,” Huang told The Scientist. “We believe that [understanding] the structure of Cpf1 will help us not only to know the working mechanism of Cpf1 but also to design more specific and more efficient genome-editing tools.”
D. Dong et al., “The crystal structure of Cpf1 in complex with CRISPR RNA,” Nature, doi:10.1038/nature17944, 2016.
I. Fonfara et al., “The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA,” Nature, doi:10.1038/nature17945, 2016.
A.C. Komor et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage,” Nature, doi:10.1038/nature17946, 2016.
Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage
Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction1, 2. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks1, 2. Here we report the development of ‘base editing’, a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting ‘base editors’ convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15–75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.
The CRISPR/Cas9 technique is revolutionizing genetic research: scientists have already used it to engineer crops, livestock and even human embryos, and it may one day yield new ways to treat disease.
But now one of the technique’s pioneers thinks that he has found a way to make CRISPR even simpler and more precise. In a paper published in Cell on 25 September, a team led by synthetic biologist Feng Zhang of the Broad Institute in Cambridge, Massachusetts, reports the discovery of a protein1 called Cpf1 that may overcome one of CRISPR/Cas9’s few limitations; although the system works well for disabling genes, it is often difficult to truly edit them by replacing one DNA sequence with another.
The CRISPR/Cas9 system evolved as a way for bacteria and archaea to defend themselves against invading viruses. It is found in a wide range of these organisms, and uses an enzyme called Cas9 to cut DNA at a site specified by ‘guide’ strands of RNA. Researchers have turned CRISPR/Cas9 into a molecular-biology powerhouse that can be used in other organisms. The cuts made by the enzyme are repaired by the cell’s natural DNA-repair processes.
Good, better, best?
CRISPR is much simpler than previous gene-editing methods, but Zhang thought there was still room for improvement.
So he and his colleagues searched the bacterial kingdom to find an alternative to the Cas9 enzyme commonly used in laboratories. In April, they reported that they had discovered a smaller version of Cas9 in the bacterium Staphylococcus aureus2. The small size makes the enzyme easier to shuttle into mature cells — a crucial destination for some potential therapies.
The team was also intrigued by Cpf1, a protein that looks very different from Cas9, but is present in some bacteria with CRISPR. The scientists evaluated Cpf1 enzymes from 16 different bacteria, eventually finding two that could cut human DNA.
They also uncovered some curious differences between how Cpf1 and Cas9 work. Cas9 requires two RNA molecules to cut DNA; Cpf1 needs only one. The proteins also cut DNA at different places, offering researchers more options when selecting a site to edit. “This opens up a lot of possibilities for all the things we could not target before,” says epigeneticist Luca Magnani of Imperial College London.
Cpf1 also cuts DNA in a different way. Cas9 cuts both strands in a DNA molecule at the same position, leaving behind what molecular biologists call ‘blunt’ ends. But Cpf1 leaves one strand longer than the other, creating a ‘sticky’ end. Blunt ends are not as easy to work with: a DNA sequence could be inserted in either end, for example, whereas a sticky end will only pair with a complementary sticky end.
“The sticky ends carry information that can direct the insertion of the DNA,” says Zhang. “It makes the insertion much more controllable.”
Zhang’s team is now working to use these sticky ends to improve the frequency with which researchers can replace a natural DNA sequence. Cuts left by Cas9 tend to be repaired by sticking the two ends back together, in a relatively sloppy repair process that can leave errors. Although it is possible that the cell will instead insert a designated, new sequence at that site, that kind of repair occurs at a much lower frequency. Zhang hopes that the unique properties of how Cpf1 cuts may be harnessed to make such insertions more frequent.
For Bing Yang, a plant biologist at the Iowa State University in Ames, this is the most exciting aspect of Cpf1. “Boosting the efficiency would be a big step for plant science,” he says. “Right now, it is a major challenge.”
Will the new enzyme surpass Cas9 in popularity? “It’s too early to tell,” says Zhang. “It certainly has some distinct advantages.” The CRISPR/Cas9 system is so popular — and potentially lucrative — that it has sparked a fierce patent fight between the University of California, Berkeley, and the Broad Institute and its ally, the Massachusetts Institute of Technology in Cambridge. Zhang says that his lab will make the CRISPR/Cpf1 components available to academic researchers, as it has done with its CRISPR/Cas9 tools.
For now, the results stand as a testament that researchers still have more to learn from the genome-editing systems that bacteria have evolved. “This study powerfully demonstrates that the natural evolutionary diversity of CRISPR systems is rich with potential solutions to the challenges facing the use of genome-editing agents,” says David Liu, a chemical biologist at Harvard University in Cambridge. (Zhang and Liu are both scientific advisers to Editas Medicine, a company in Cambridge that aims to develop CRISPR-based therapies.)
Microbiologist John van der Oost of Wageningen University in the Netherlands, who collaborated on the latest study with Zhang, plans to keep searching for new methods. “You never know whether one of these systems will be suitable for genome editing,” he says. “There are still surprises ahead of us.”
•Cpf1 is a CRISPR-associated two-component RNA-programmable DNA nuclease
•Targeted DNA is cleaved as a 5-nt staggered cut distal to a 5′ T-rich PAM
•Two Cpf1 orthologs exhibit robust nuclease activity in human cells
The microbial adaptive immune system CRISPR mediates defense against foreign genetic elements through two classes of RNA-guided nuclease effectors. Class 1 effectors utilize multi-protein complexes, whereas class 2 effectors rely on single-component effector proteins such as the well-characterized Cas9. Here, we report characterization of Cpf1, a putative class 2 CRISPR effector. We demonstrate that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif. Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, we identified two candidate enzymes from Acidaminococcus and Lachnospiraceae, with efficient genome-editing activity in human cells. Identifying this mechanism of interference broadens our understanding of CRISPR-Cas systems and advances their genome editing applications.
CRISPR–Cas systems that provide defence against mobile genetic elements in bacteria and archaea have evolved a variety of mechanisms to target and cleave RNA or DNA1. The well-studied types I, II and III utilize a set of distinct CRISPR-associated (Cas) proteins for production of mature CRISPR RNAs (crRNAs) and interference with invading nucleic acids. In types I and III, Cas6 or Cas5d cleaves precursor crRNA (pre-crRNA)2, 3, 4, 5 and the mature crRNAs then guide a complex of Cas proteins (Cascade-Cas3, type I; Csm or Cmr, type III) to target and cleave invading DNA or RNA6, 7, 8, 9, 10, 11, 12. In type II systems, RNase III cleaves pre-crRNA base-paired withtrans-activating crRNA (tracrRNA) in the presence of Cas9 (refs 13, 14). The mature tracrRNA–crRNA duplex then guides Cas9 to cleave target DNA15. Here, we demonstrate a novel mechanism in CRISPR–Cas immunity. We show that type V-A Cpf1 from Francisella novicida is a dual-nuclease that is specific to crRNA biogenesis and target DNA interference. Cpf1 cleaves pre-crRNA upstream of a hairpin structure formed within the CRISPR repeats and thereby generates intermediate crRNAs that are processed further, leading to mature crRNAs. After recognition of a 5′-YTN-3′ protospacer adjacent motif on the non-target DNA strand and subsequent probing for an eight-nucleotide seed sequence, Cpf1, guided by the single mature repeat-spacer crRNA, introduces double-stranded breaks in the target DNA to generate a 5′ overhang16. The RNase and DNase activities of Cpf1 require sequence- and structure-specific binding to the hairpin of crRNA repeats. Cpf1 uses distinct active domains for both nuclease reactions and cleaves nucleic acids in the presence of magnesium or calcium. This study uncovers a new family of enzymes with specific dual endoribonuclease and endonuclease activities, and demonstrates that type V-A constitutes the most minimalistic of the CRISPR–Cas systems so far described.
The crystal structure of Cpf1 in complex with CRISPR RNA
The CRISPR–Cas systems, as exemplified by CRISPR–Cas9, are RNA-guided adaptive immune systems used by bacteria and archaea to defend against viral infection1, 2, 3, 4, 5, 6, 7. The CRISPR–Cpf1 system, a new class 2 CRISPR–Cas system, mediates robust DNA interference in human cells1, 8, 9, 10. Although functionally conserved, Cpf1 and Cas9 differ in many aspects including their guide RNAs and substrate specificity. Here we report the 2.38 Å crystal structure of the CRISPR RNA (crRNA)-bound Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1). LbCpf1 has a triangle-shaped architecture with a large positively charged channel at the centre. Recognized by the oligonucleotide-binding domain of LbCpf1, the crRNA adopts a highly distorted conformation stabilized by extensive intramolecular interactions and the (Mg(H2O)6)2+ ion. The oligonucleotide-binding domain also harbours a looped-out helical domain that is important for LbCpf1 substrate binding. Binding of crRNA or crRNA lacking the guide sequence induces marked conformational changes but no oligomerization of LbCpf1. Our study reveals the crRNA recognition mechanism and provides insight into crRNA-guided substrate binding of LbCpf1, establishing a framework for engineering LbCpf1 to improve its efficiency and specificity for genome editing
The ability to sequence a person’s entire genome has led many researchers to hunt for the genetic causes of certain diseases. But without a larger set of genomes to compare mutations against, putting these variations into context is difficult. An international group of researchers has banked the full exomes of 60,706 individuals in a database called the Exome Aggregation Consortium (ExAC). The team’s analaysis, posted last month (October 30) on the preprint server bioRxiv, was presented at the Genome Science 2015 conference in Birmingham, U.K. (September 7).
Led by Daniel MacArthur from the Broad Institute of MIT and Harvard, the research team collected exomes from labs around the world for its dataset. “The resulting catalogue of human genetic diversity has unprecedented resolution,” the authors wrote in their preprint. Many of the variants observed in the dataset occurred only once.
“This is one of the most useful resources ever created for medical testing for genetic disorders,” Heidi Rehm, a clinical lab director at Harvard Medical School, told Science News.
Among other things, the team found 3,230 genes that are highly conserved across exomes, indicating likely involvement in critical cellular functions. Of these, 2,557 are not associated with diseases. The authors hypothesized that these genes, if mutated, either lead to embryonic death—before a problem can be diagnosed—or cause rare diseases that have not yet been genetically characterized.
“We should soon be able to say, with high precision: If you have a mutation at this site, it will kill you. And we’ll be able to say that without ever seeing a person with that mutation,” MacArthur said during his Genome Science talk, according to The Atlantic.
This is not the complete set of essential genes in the human body, David Goldstein, a geneticist at Columbia University in New York City, pointed out to Nature. Only by studying more exomes will researchers be able to refine that number, he noted.
Analysis of protein-coding genetic variation in 60,706 humans
Exome Aggregation Consortium, MonkolLek, KonradKarczewski, EricMinikel, KaitlinSamocha, et al.
Large-scale reference data sets of human genetic variation are critical for the medical and functional interpretation of DNA sequence changes. Here we describe the aggregation and analysis of high-quality exome (protein-coding region) sequence data for 60,706 individuals of diverse ethnicities. The resulting catalogue of human genetic diversity has unprecedented resolution, with an average of one variant every eight bases of coding sequence and the presence of widespread mutational recurrence. The deep catalogue of variation provided by the Exome Aggregation Consortium (ExAC) can be used to calculate objective metrics of pathogenicity for sequence variants, and to identify genes subject to strong selection against various classes of mutation; we identify 3,230 genes with near-complete depletion of truncating variants, 79% of which have no currently established human disease phenotype. Finally, we show that these data can be used for the efficient filtering of candidate disease-causing variants, and for the discovery of human knockout variants in protein-coding genes.
Over the last five years, the widespread availability of high-throughput DNA sequencing technologies has permitted the sequencing of the whole genomes or exomes (the 18 protein-coding regions of genomes) of over half a million humans. In theory, these data represent a powerful source of information about the global patterns of human genetic variation, but in practice, are difficult to access for practical, logistical, and ethical reasons; in addition, the inconsistent processing complicates variant-calling pipelines used by different groups. Current publicly available datasets of human DNA sequence variation contain only a small fraction of all sequenced samples: the Exome Variant Server, created as part of the NHLBI Exome Sequencing Project (ESP)1, contains frequency information spanning 6,503 exomes; and the 1000 Genomes (1000G) Project, which includes individual-level genotype data from whole-genome and exome sequence data for 2,504 individuals2.
Databases of genetic variation are important for our understanding of human population history and biology1–5, but also provide critical resources for the clinical interpretation of variants observed in patients suffering from rare Mendelian diseases6,7. The filtering of candidate variants by frequency in unselected individuals is a key step in any pipeline for the discovery of causal variants in Mendelian disease patients, and the efficacy of such filtering depends on both the size and the ancestral diversity of the available reference data.
Here, we describe the joint variant calling and analysis of high-quality variant calls across 60,706 human exomes, assembled by the Exome Aggregation Consortium (ExAC; exac.broadinstitute.org). This call set exceeds previously available exome-wide variant databases by nearly an order of magnitude, providing unprecedented resolution for the analysis of very low-frequency genetic variants. We demonstrate the application of this data set to the analysis of patterns of genetic variation including the discovery of widespread mutational recurrence, the inference of gene-level constraint against 10 truncating variation, the clinical interpretation of variation in Mendelian disease genes, and the discovery of human “knockout” variants in protein-coding genes.
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Deleterious variants are expected to have lower allele frequencies than neutral ones, due to negative selection. This theoretical property has been demonstrated previously in human population sequencing data18,19 and here (Figure 1d, Figure 1e). This allows inference of the degree of natural selection against specific functional classes of variation: however, mutational recurrence as described above indicates that allele frequencies observed in ExAC-scale samples are also skewed by mutation rate, with 10 more mutable sites less likely to be singletons (Figure 2c and Extended Data Figure 4d). Mutation rate is in turn non-uniformly distributed across functional classes – for instance, stop lost mutations can never occur at CpG dinucleotides (Extended Data Figure 4e). We corrected for mutation rates (Supplementary Information) by creating a mutability-adjusted proportion singleton (MAPS) metric. This metric reflects (as expected) strong selection against predicted PTVs, as well as missense variants predicted by conservation-based methods to be deleterious (Figure 2e).
The deep ascertainment of rare variation in ExAC also allows us to infer the extent of 19 selection against variant categories on a per-gene basis by examining the proportion of 20 variation that is missing compared to expectations under random mutation. Conceptually similar approaches have been applied to smaller exome datasets13,20 but have been underpowered, particularly for the analysis of depletion of PTVs. We compared the observed number of rare (MAF <0.1%) variants per gene to an expected number derived from a selection neutral, sequence-context based mutational model13. The model performs extremely well in predicting the number of synonymous variants, which should be under minimal purifying selection, per gene (r = 0.98; Extended Data Figure 5).
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Critically, we note that LoF-intolerant genes include virtually all known severe haploinsufficient human disease genes (Figure 3b), but that 79% of LoF-intolerant genes have not yet been assigned a human disease phenotype despite the clear evidence for extreme selective constraint (Supplementary Information 4.11). These likely represent either undiscovered severe dominant disease genes, or genes in which loss of a single copy results in embryonic lethality.
The most highly constrained missense (top 25% missense Z scores) and PTV (pLI ≥0.9) genes show higher expression levels and broader tissue expression than the least constrained genes24 (Figure 3c). These most highly constrained genes are also depleted for eQTLs (p < 10-9 for missense and PTV; Figure 3d), yet are enriched within genome-wide significant trait-associated loci (χ2 p < 10-14, Figure 3e). Intuitively, genes intolerant of PTV variation are dosage sensitive: natural selection does not tolerate a 50% deficit in expression due to the loss of single allele. It is therefore unsurprising that these genes are also depleted of common genetic variants that have a large enough effect on expression to be detected as eQTLs with current limited sample sizes. However, smaller changes in the expression of these genes, through weaker eQTLs or functional variants, are more likely to contribute to medically relevant phenotypes. Therefore, highly constrained genes are dosage-sensitive, expressed more broadly across tissues (as expected for core cellular processes), and are enriched for medically relevant variation.
Finally, we investigated how these constraint metrics would stratify mutational classes according to their frequency spectrum, corrected for mutability as in the previous section (Figure 3f). The effect was most dramatic when considering stop-gained variants in the LoF-intolerant set of genes. For missense variants, the missense Z score offers information additional to Polyphen2 and CADD classifications, indicating that gene-level measures of constraint offer additional information to variant-level metrics in assessing potential pathogenicity.
We assessed the value of ExAC as a reference dataset for clinical sequencing approaches, which typically prioritize or filter potentially deleterious variants based on functional consequence and allele frequency6. To simulate a Mendelian variant analysis, we filtered variants in 100 ExAC exomes per continental population against ESP (the previous default reference data set for clinical analysis) or the remainder of ExAC, removing variants present at ≥0.1% allele frequency, a filter recommended for dominant 16 disease variant discovery6. Filtering on ExAC reduced the number of candidate protein-altering variants by 7-fold compared to ESP, and was most powerful when the highest 18 allele frequency in any one population (“popmax”) was used rather than average (“global”) allele frequency (Figure 4a). ESP is not well-powered to filter at 0.1% AF without removing many genuinely rare variants, as AF estimates based on low allele counts are both upward-biased and imprecise (Figure 4b). We thus expect that ExAC will provide a very substantial boost in the power and accuracy of variant filtering in Mendelian disease projects.
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The above curation efforts confirm the importance of allele frequency filtering in analysis of candidate disease variants. However, literature and database errors are prevalent even at lower allele frequencies: the average ExAC exome contains 0.89 reportedly Mendelian variants in well-characterized dominant disease genes at <1% popmax AF and 0.20 at <0.1% popmax AF. This inflation likely results from a combination of false reports of pathogenicity and incomplete penetrance, as we show for PRNP in the accompanying work [Minikel et al, submitted]. The abundance of rare functional variation in many disease genes in ExAC is a reminder that such variants should not be assumed to be causal or highly penetrant without careful segregation or case-control analysis28,7.
We investigated the distribution of PTVs, variants predicted to disrupt protein-coding genes through the introduction of a stop codon or frameshift or the disruption of an essential splice site; such variants are expected to be enriched for complete loss-of-function of the impacted genes. Naturally-occurring PTVs in humans provide a model for the functional impact of gene inactivation, and have been used to identify many genes in 6 which LoF causes severe disease31, as well as rare cases where LoF is protective against disease32.
Among the 7,404,909 HQ variants in ExAC, we found 179,774 high-confidence PTVs (as 10 defined in Supplementary Information Section 6), 121,309 of which are singletons. This 11 corresponds to an average of 85 heterozygous and 35 homozygous PTVs per individual (Figure 5a). The diverse nature of the cohort enables the discovery of substantial numbers of novel PTVs: out of 58,435 PTVs with an allele count greater than one, 33,625 occur in only one population. However, while PTVs as a category are extremely rare, the majority of the PTVs found in any one person are common, and each individual 16 has only ~2 singleton PTVs, of which 0.14 are found in PTV-constrained genes (pLI 17 >0.9). The site frequency spectrum of these variants across the populations represented in ExAC recapitulates known aspects of demographic models, including an increase in intermediate-frequency (1%-5%) PTVs in Finland33 and relatively common (>0.1%) PTVs in Africans (Figure 5b).
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Discussion Here we describe the generation and analysis of the most comprehensive catalogue of 29 human protein-coding genetic variation to date, incorporating high-quality exome sequencing data from 60,706 individuals of diverse geographic ancestry. The resulting call set provides unprecedented resolution for the analysis of very low-frequency protein-coding variants in human populations, as well as a powerful resource for the clinical interpretation of genetic variants observed in disease patients. The complete frequency CC-BY-ND 4.0 International license for this preprint is the author/funder. It is made available under a bioRxiv preprint first posted online October 30, 2015; http://dx.doi.org/10.1101/030338 ; The copyright holder and annotation data from this call-set has been made freely available through a public website [exac.broadinstitute.org]
The ExAC resource provides the largest database to date for the estimation of allele frequency for protein-coding genetic variants, providing a powerful filter for analysis of candidate pathogenic variants in severe Mendelian diseases. Frequency data from ESP1 have been widely used for this purpose, but those data are limited by population diversity and by resolution at allele frequencies ≤0.1%. ExAC therefore provides 21 substantially improved power for Mendelian analyses, although it is still limited in power at lower allele frequencies, emphasizing the need for more sophisticated pathogenic variant filtering strategies alongside on-going data aggregation efforts. ExAC also highlights an unexpected tolerance of many disease genes to functional variation, and reveals that the literature and public databases contain an inflated number of reportedly pathogenic variants across the frequency spectrum, indicating a need for stringent criteria for assertions of pathogenicity.
Finally, we show that different populations confer different advantages in the discovery of gene-disrupting PTVs, providing guidance for projects seeking to identify human “knockouts” to understand gene function. Individuals of African ancestry have more PTVs (140 on average), with this enrichment most pronounced at allele frequencies above 1% (Figure 5b). Finnish individuals, as a result of a population bottleneck, are depleted at the lowest (<0.1%) allele frequencies but have a peak in frequency at 1-5% (Figure 5b). However, these differences are diminished when considering only LoF-constrained (pLI > 0.9) genes (Extended Data Figure 10). Sampling multiple populations would likely be a fruitful strategy for a researcher investigating common PTV variation. However, discovery of homozygous PTVs is markedly enhanced in the South Asia samples, which come primarily from a Pakistani cohort with 38.3% of individuals self- reporting as having closely related parents, emphasizing the extreme value of consanguineous cohorts for “human knockout” discovery (Figure 5d) [Saleheen et al., to 8 be co-submitted].
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While the ExAC dataset dramatically exceeds the scale of previously available frequency reference datasets, much remains to be gained by further increases in sample size. Indeed, the fact that even the rarest transversions have mutational rates13 on the order of 1 x 10-9 implies that almost all possible non-lethal SNVs likely exist in some person on Earth. ExAC already includes >70% of all possible protein-coding CpG transitions at well-covered sites; order of magnitude increases in sample size will eventually lead to saturation of other classes of variation.
FDA approvesEGFR mutation detection test for NSCLC drug, Tarceva
Author/Reporter: Ritu Saxena, Ph.D.
The cobas EGFR Mutation Test, Roche Molecular Diagnostics, identifies mutations in epidermal growth factor receptor (EGFR) exons 18, 19, 20 and 21 of patients. The FDA has approved the companion diagnostic for the cancer drug Tarceva (erlotinib). It would select non-small cell lung cancer (NSCLC) patients for treatment with EGFR inhibitors. This is the first FDA-approved companion diagnostic that detects EGFR gene mutations, which are present in approximately 10-30% of non-small cell lung cancers (NSCLC). The test is being approved with an expanded use for Tarceva as a first-line treatment for patients with NSCLC that has metastasized and who have certain mutations in the EGFR gene.
Lung cancer, the leading cause of cancer death among both men and women leads to death of more people than colon, breast, and prostate cancers combined. The American Cancer Society’s most recent estimates for lung cancer in the United States for 2012 reveal that about 226,160 new cases of lung cancer will be diagnosed (116,470 in men and 109,690 in women), and there will be an estimated 160,340 deaths from lung cancer (87,750 in men and 72,590 among women), accounting for about 28% of all cancer deaths. NSCLC is the most common type of lung cancer and usually grows and spreads more slowly than small cell lung cancer. Activating EGFR mutations occur in 10–30% NSCLC cases, and lead to hyperdependence of tumors on EGFR signaling and increased sensitivity of EGFR to inhibition by erlotinib. Genentech/OSI Pharmaceuticals/Roche/Chugai Pharmaceutical’s erlotinib (Tarceva) is a small molecule quinazoline and directly and reversibly inhibits the EGFR tyrosine kinase.
Tarceva has been indicated for first-line treatment of cancer with EGFR mutations including NSCLC. The approval is Tarceva’s fourth indication and the third use for lung cancer. The FDA approved Tarceva on April 16, 2010, for maintenance treatment of patients with locally advanced or metastatic NSCLC whose disease has not progressed after four cycles of platinum-based first-line chemotherapy. Tarceva was originally approved in November 2004 for the treatment of patients with locally advanced or metastatic NSCLC after failure of at least one prior chemotherapy regimen.
In a recent multicenter, open label, randomized, phase III clinical trial (EURTAC trial; NCT0044625; http://clinicaltrials.gov/ct2/show/NCT00446225 ), Tarceva was investigated in patients with advanced NSCLC with mutations in the tyrosine kinase (TK) domain of the EGFR. The EURTAC trial was initiated in February 2007 and completed in December 2012 and enrolled around 174 patients. Patients were divided into two experimental arms. Patients in arm 1 were administered Tarceva (150 mg/day) while patients in arm 2 underwent chemotherapy as platinum-based doublets. The chemotherapeutic drugs were administered as Cisplatin (75 mg/m2) / Docetaxel (75 mg/m2); Cisplatin (75 mg/m2) / Gemcitabine (1250 mg/m2; day 1 and 8); Docetaxel (75 mg/m2) /carboplatin (AUC=6); Gemcitabine (1000 mg/m2; day 1 and 8) / Carboplatin (AUC=5). Results revealed that Erlotinib is better tolerated in Chinese population (grade 3-4 toxicities 17%) then in European patients (grade 3-4 toxicities 45%). Erlotinib scored significantly better than chemotherapy in terms of progression-free survival (PFS) with 9.7 versus 5.2 months, respectively (HR 0.37, 95% CI 0.25-0.54). Thus, the results of the trial strengthen the rationale for routine baseline tissue-based assessment of EGFR mutations in patients with NSCLC and for treatment of mutation-positive patients with EGFR tyrosine-kinase inhibitors. (Gridelli C and Rossi A, J Thorac Dis. 2012 Apr 1;4(2):219-20; http://www.ncbi.nlm.nih.gov/pubmed/22833832 )
In conclusion, FDA approval of cobas EGFR Mutation Test is a recent example of how genotyping patients in clinical trials could lead to crucial information regarding personalizing the diagnostic and therapeutic approaches.
Melosky B. EURTAC first line therapy for non small cell lung carcinoma in epidermal growth factor receptor mutation positive patients: A choice between two TKIs. J Thorac Dis. 2012 Apr 1;4(2):221-2; http://www.ncbi.nlm.nih.gov/pubmed/22833833
Gridelli C and Rossi AJ. EURTAC first-line phase III randomized study in advanced non-small cell lung cancer: Erlotinib works also in European population. Thorac Dis. 2012 Apr 1;4(2):219-20; http://www.ncbi.nlm.nih.gov/pubmed/22833832
Related reading
Nguyen KS and Neal JW. First-line treatment of EGFR-mutant non-small-cell lung cancer: the role of erlotinib and other tyrosine kinase inhibitors. Biologics. 2012;6:337-45; http://www.ncbi.nlm.nih.gov/pubmed/23055691
Rewriting the Mathematics of Tumor Growth[1]; Teams Use Math Models to Sort Drivers from Passengers[2]: Two JNCI Reviews by Mike Martin Regarding Genomics, Cancer, and Mutation
Curator: Stephen J. Williams, Ph.D.
Word Cloud By Danielle Smolyar
Recently, there has been extensive interest in the cancer research and oncology community on detecting those mutations responsible for the initiation and propagation of a neoplastic cell (driver mutations) versus those mutations that are randomly (or by selective pressures) acquired due to the genetic instability of the transformed cell. The impact of either type of mutation has been a topic for debate, with a recent article showing that some passenger mutations may actually be responsible for tumor survival. In addition many articles, highlighted on this site (and referenced below) in recent years have described the importance of classifying driver and passenger mutations for the purposes of more effective personalized medicine strategies directed against tumors. Two review articles by Mike Martin in the Journal of the National Cancer Institute (JCNI) shed light on the current efforts and successes to discriminate between these passenger and driver mutations and determine impact of each type of mutation to tumor growth. However, as described in the associated article, the picture is not as clear cut as previously thought and highlights some revolutionary findings. In Rewriting the Mathematics of Tumor Growth, researchers discovered that driver mutations may confer such a small growth advantage that, multiple mutations, including the so called passenger mutations are necessary in order to sustain tumor growth. In fact, much experimental evidence has suggested at least six defined genetic events may be necessary for the in-vitro transformation of human cells. The following table shows some of the genetic events required for in-vitro transformation in cell culture systems.
3 for anchorage independence (cyclin D1, dnp53, EGFR),Cyclin D1+dnp53 for immortalization
HOSE
6
CDK4, cyclin D, hTERT plus combination of either P53DD, myrAkt, and H-ras or P53DD, H-ras, c-myc Bcl2
(f)Sasaki(Kiyono)
5
HOSE
3
hTERTSV40 earlyH-ras orK-ras
(g)Liu(Bast)
2hTERT+ SV40 early
HOSE
3
Large ThTERTH-ras orc-erB-2
(h)Kusakari(Fujii)
2hTERT+large T
Rat
Fibroblasts
2
Large TH-ras
(i)Hirakawa
Did not analyze
Fibroblasts
2
Large TH-ras
(d)Rangarajan(Weinberg)
Large T
Mouse
MOSEIn p53-/- background
3
c-mycK-rasAkt
(j)Orsulic
Pig
Fibroblasts
6
p53DDhTERT
CDK4H-ras c-myc
cyclin D1
(k)Adam(Counter)
5 need all butp53DD
Note: priming means events required to immortalize but not fully transform. * Note that both ability to form colonies in soft agarose and subsequently tested for tumor formation in immunocompromised mice.
a. Hahn, W. C., Counter, C. M., Lundberg, A. S., Beijersbergen, R. L., Brooks, M. W., and Weinberg, R. A. (1999) Creation of human tumour cells with defined genetic elements, Nature400, 464-468.
b. Kendall, S. D., Linardic, C. M., Adam, S. J., and Counter, C. M. (2005) A network of genetic events sufficient to convert normal human cells to a tumorigenic state, Cancer Res65, 9824-9828.
c. Sun, B., Chen, M., Hawks, C. L., Pereira-Smith, O. M., and Hornsby, P. J. (2005) The minimal set of genetic alterations required for conversion of primary human fibroblasts to cancer cells in the subrenal capsule assay, Neoplasia7, 585-593.
d. Rangarajan, A., Hong, S. J., Gifford, A., and Weinberg, R. A. (2004) Species- and cell type-specific requirements for cellular transformation, Cancer Cell6, 171-183.
e. Goessel, G., Quante, M., Hahn, W. C., Harada, H., Heeg, S., Suliman, Y., Doebele, M., von Werder, A., Fulda, C., Nakagawa, H., Rustgi, A. K., Blum, H. E., and Opitz, O. G. (2005) Creating oral squamous cancer cells: a cellular model of oral-esophageal carcinogenesis, Proc Natl Acad Sci U S A102, 15599-15604.
f. Sasaki, R., Narisawa-Saito, M., Yugawa, T., Fujita, M., Tashiro, H., Katabuchi, H., and Kiyono, T. (2009) Oncogenic transformation of human ovarian surface epithelial cells with defined cellular oncogenes,Carcinogenesis30, 423-431.
g. Liu, J., Yang, G., Thompson-Lanza, J. A., Glassman, A., Hayes, K., Patterson, A., Marquez, R. T., Auersperg, N., Yu, Y., Hahn, W. C., Mills, G. B., and Bast, R. C., Jr. (2004) A genetically defined model for human ovarian cancer, Cancer Res64, 1655-1663.
h. Kusakari, T., Kariya, M., Mandai, M., Tsuruta, Y., Hamid, A. A., Fukuhara, K., Nanbu, K., Takakura, K., and Fujii, S. (2003) C-erbB-2 or mutant Ha-ras induced malignant transformation of immortalized human ovarian surface epithelial cells in vitro, Br J Cancer89, 2293-2298.
i. Hirakawa, T., and Ruley, H. E. (1988) Rescue of cells from ras oncogene-induced growth arrest by a second, complementing, oncogene, Proc Natl Acad Sci U S A85, 1519-1523.
j. Orsulic, S., Li, Y., Soslow, R. A., Vitale-Cross, L. A., Gutkind, J. S., and Varmus, H. E. (2002) Induction of ovarian cancer by defined multiple genetic changes in a mouse model system, Cancer Cell1, 53-62.
k. Adam, S. J., Rund, L. A., Kuzmuk, K. N., Zachary, J. F., Schook, L. B., and Counter, C. M. (2007) Genetic induction of tumorigenesis in swine, Oncogene26, 1038-1045.
However it may be argued that the aforementioned experimental examples were produced in cell lines with a more stable genome than that which is seen in most tumors and had used traditional assays of transformation, such as growth in soft agarose and tumorigenicity in immunocompromised mice, as endpoints of transformation, and not representative of the tumor growth seen in the clinical setting.
Therefore Bert Vogelstein, M.D., along with collaborators around the world developed a model they termed the “sequential driver mutation theory”, in which they describe that driver mutations multiply over time with each mutation “slightly increasing the tumor growth rate through a process that depends on three factors”:
Driver mutation rate
The 0.4% selective growth advantage
Cell division time
This model was based on a combination of experimental data and computer simulations of gliobastoma multiforme and pancreatic adenocarcinoma. Most tumor models follow a Gompertz kinetics, which show how tumor growth is exponential but eventually levels off over time.
This new theory shows though that a tumor cell with only one driver mutation can only grow so much, until a second driver mutation is required. Using data for the COSMIC database (Catalog of Somatic Mutations in Cancer) together with analysis software CHASM (Cancer-specific High-throughput Annotation of Somatic Mutations) the researchers analyzed 713 mutations sequenced from 14 glioma patients and 562 mutations in nine pancreatic adenocarcinomas, revealing at least 100 tumor suppressor genes and 100 oncogenes altered. Therefore, the authors suggested these may be possible driver mutations, or at least mutations required for the sustained growth of these tumors. Applying this new model to data obtained from Dr. Giardiello’s publication concerning familial adenopolypsis in New England Journal of medicine in 19993 and 2000, the sequential driver mutation model predicted age distribution of FAP patients, number and size of polyps, and polyp growth rate than previous models. This surprising number of required driver mutations for full transformation was also verified in a study led by University of Texas Southwestern Medical Center biologist Jerry Shay, Ph.D., who noted “this team’s surprise nearly 45% of all colorectal candidate oncogenes (65 mutations) drove malignant proliferation”[3].
However, some investigators do not believe the model is complex enough to account for other factors involved in oncogenesis, such as epigenetic factors like methylation and acetylation. In addition the review also discusses host and tissue factors which may complicate the models, such as location where a tumor develops. However, most of the investigators interviewed for this review agreed that focusing on this long-term progression of the disease may give us clues to other potential druggable targets.
Teams Use Math Models to Sort Drivers From Passengers
A related review from Mike Martin in JNCI [2] describes a statistical method, published in 2009 Cancer Informatics[4], which distinguishes chromosomal abnormalities that can drive oncogenesis from passenger abnormalities. Chromosomal abnormalities, such as deletions, additions, and translocations are common in cancer. For instance, the well-known Philadelphia chromosome, a translocation between chromosome 9 and 22 which results in the BCR-ABL tyrosine kinase fusion protein is the molecular basis of chronic myelogenous leukemia.
In the report, Eytan Domany, Ph.D., from Weizmann Institute and several colleagues from University of Lausanne, University of Haifa and the Broad Institute were analyzing chromosomal aberrations in a subset of medulloblastoma, which had more gain and losses in chromosomes than had been attributed to the disease. Using a statistical method they termed a “volumetric sieve”, the investigators were able to identify driver versus passenger aberrations based on three filters:
Fraction of patients with the abnormality
Length of DNA involved in the aberrant chromosome
Abnormality’s copy number
Another method to sort the most “important” chromosomal aberrations from less relevant alterations is termed GISTIC[5], as the website describes is: a tool to identify genes targeted by somatic copy-number alterations (SCNAs) that drive cancer growth (at the Broad Institute website http://www.broadinstitute.org/software/cprg/?q=node/31). The method allows for comparison across multiple tumors so noise is eliminated and improves consistency of analysis. This method had been successfully used to determine driver aberrations is mesotheliomas, leukemias, and identify new oncogenes in adenocarcinomas of the lung and squamous cell carcinoma of the esophagus.
Main references for the two Mike Martin articles are as follows:
3. Eskiocak U, Kim SB, Ly P, Roig AI, Biglione S, Komurov K, Cornelius C, Wright WE, White MA, Shay JW: Functional parsing of driver mutations in the colorectal cancer genome reveals numerous suppressors of anchorage-independent growth. Cancer research 2011, 71(13):4359-4365.
4. Shay T, Lambiv WL, Reiner-Benaim A, Hegi ME, Domany E: Combining chromosomal arm status and significantly aberrant genomic locations reveals new cancer subtypes. Cancer informatics 2009, 7:91-104.
A typical cancer cell has thousands of mutations scattered throughout its genome and hundreds of mutated genes. However, only a handful of those genes, known as drivers, are responsible for cancerous traits such as uncontrolled growth. Cancer biologists have largely ignored the other mutations, believing they had little or no impact on cancer progression.
But a new study from MIT, Harvard University, the Broad Institute and Brigham and Women’s Hospital reveals, for the first time, that these so-called passenger mutations are not just along for the ride. When enough of them accumulate, they can slow or even halt tumor growth.
The findings, reported in this week’sProceedings of the National Academy of Sciences, suggest that cancer should be viewed as an evolutionary process whose course is determined by a delicate balance between driver-propelled growth and the gradual buildup of passenger mutations that are damaging to cancer, says Leonid Mirny, an associate professor of physics and health sciences and technology at MIT and senior author of the paper.
Furthermore, drugs that tip the balance in favor of the passenger mutations could offer a new way to treat cancer, the researchers say, beating it with its own weapon — mutations. Although the influence of a single passenger mutation is minuscule, “collectively they can have a profound effect,” Mirny says. “If a drug can make them a little bit more deleterious, it’s still a tiny effect for each passenger, but collectively this can build up.”
Lead author of the paper is Christopher McFarland, a graduate student at Harvard. Other authors are Kirill Korolev, a Pappalardo postdoctoral fellow at MIT, Gregory Kryukov, a senior computational biologist at the Broad Institute, and Shamil Sunyaev, an associate professor at Brigham and Women’s.
Power struggle
Cancer can take years or even decades to develop, as cells gradually accumulate the necessary driver mutations. Those mutations usually stimulate oncogenes such as Ras, which promotes cell growth, or turn off tumor-suppressing genes such as p53, which normally restrains growth.
Passenger mutations that arise randomly alongside drivers were believed to be fairly benign: In natural populations, selection weeds out deleterious mutations. However, Mirny and his colleagues suspected that the evolutionary process in cancer can proceed differently, allowing mutations with only a slightly harmful effect to accumulate.
To test this theory, the researchers created a computer model that simulates cancer growth as an evolutionary process during which a cell acquires random mutations. These simulations followed millions of cells: every cell division, mutation and cell death.
They found that during the long periods between acquisition of driver mutations, many passenger mutations arose. When one of the cancerous cells gains a new driver mutation, that cell and its progeny take over the entire population, bringing along all of the original cell’s baggage of passenger mutations. “Those mutations otherwise would never spread in the population,” Mirny says. “They essentially hitchhike on the driver.”
This process repeats five to 10 times during cancer development; each time, a new wave of damaging passengers is accumulated. If enough deleterious passengers are present, their cumulative effects can slow tumor growth, the simulations found. Tumors may become dormant, or even regress, but growth can start up again if new driver mutations are acquired. This matches the cancer growth patterns often seen in human patients.
“Cancer may not be a sequence of inevitable accumulation of driver events, but may be actually a delicate balance between drivers and passengers,” Mirny says. “Spontaneous remissions or remissions triggered by drugs may actually be mediated by the load of deleterious passenger mutations.”
When they analyzed passenger mutations found in genomic data taken from cancer patients, the researchers found the same pattern predicted by their model — accumulation of large quantities of slightly deleterious mutations.
Tipping the balance
In computer simulations, the researchers tested the possibility of treating tumors by boosting the impact of deleterious mutations. In their original simulation, each deleterious passenger mutation reduced the cell’s fitness by about 0.1 percent. When that was increased to 0.3 percent, tumors shrank under the load of their own mutations.
The same effect could be achieved in real tumors with drugs that interfere with proteins known as chaperones, Mirny suggests. After proteins are synthesized, they need to be folded into the correct shape, and chaperones help with that process. In cancerous cells, chaperones help proteins fold into the correct shape even when they are mutated, helping to suppress the effects of deleterious mutations.
Several potential drugs that inhibit chaperone proteins are now in clinical trials to treat cancer, although researchers had believed that they acted by suppressing the effects of driver mutations, not by enhancing the effects of passengers.
In current studies, the researchers are comparing cancer cell lines that have identical driver mutations but a different load of passenger mutations, to see which grow faster. They are also injecting the cancer cell lines into mice to see which are likeliest to metastasize.
Current post talks about a new technique that has been introduced by the authors as a ‘Comprehensive 1-Step Molecular Analyses of Mitochondrial Genome by Massively Parallel Sequencing’. The technique was recently published in the Clinical Chemistry journal (2012) by Zhang et al.
One mitochondria may have multiple copies of mtDNA and an interesting feature observed in mitochondria is the heteroplasmy, a phenomenon where mutant and wild-type mtDNA can co-exist. During cell division, the mutant and wild-type copies are distributed randomly in daughter cells. The impact is in the heterogeneity with respect to penetrance and expressivity along that has diverse manifestations in terms of organs being affected, age of onset and the rate of progression. With such variability, the diagnosis becomes even more challenging. Therefore, mutational analysis along with accurate heteroplasmy detection in the mtDNA is an important part of the diagnosis. Thus, there is need for accurate and faster mutation detection methods for patients that are suspected to carry a mitochondrial disease.
The current molecular diagnostic methods for the detection of mtDNA mutations involves several different and complimentary methods. The detection of mutations is approached by first screening for a panel of point mutations that have been commonly associated with the mitochondrial diseases, followed by the quantification of the mutant load. In case none of the point mutations show up in the screening, the whole genome sequencing of mtDNA is performed to identify rare or novel mutations that might be associated with the disease. Also, in order to analyze large deletions within the genome, a an additional step of Southern blotting needs to be performed. Zhang et al, however, developed a novel approach to analyze the mtDNA in “single” step.
The method employed for the 1-step technique is to first enrich the entire mtDNA using amplification by PCR followed by massively parallel sequencing to detect point mutations as well as large heteroplasmic deletions simultaneously. A total of 45 samples were analyzed for the evaluation of analytic sensitivity and specificity. As stated by the authors “Our analysis demonstrated 100% diagnostic sensitivity and specificity of base calls compared to the results from Sanger sequencing” and added ” the method also detected large deletions with the breakpoints mapped”. Apart from the fact that the 1-step technique is less complex, the detection of point mutations has been found to be more accurate compared to Sanger sequencing that doesn’t provide any quantitative information and falls short of detecting heteroplasmy lying below 15%.
Thus, the 1-step technique developed by Zhang et al has been demonstrated to be better than the combination of methods currently utilized for the detection of mtDNA mutations in terms of simplicity, cost effectiveness and accuracy.
Consultants: Aviva Lev-Ari, PhD, RN and Pnina G. Abir-Am, PhD
CONTENT:
Section I : Mitochondrial diseases and molecular understanding
Section II : Diagnosis and therapy of mitochondrial diseases
Section III: Mitochondria, metabolic syndrome and research
I. MITOCHONDRIAL DISEASES and MOLECULAR UNDERSTANDING
Mitochondrial cytopathy in adults – current understanding:
Mitochondrial cytopathies are a diverse group of inherited and acquired disorders that result in inadequate energy production leading to illnesses. Several syndromes have been linked to mutations in mitochondrial DNA. Some key features common to mitochondrial diseases are listed as follows:
Diverse manifestations of mitochondrial diseases: Although all mitochondrial diseases have the same characteristic of inadequate energy production as compared to the demand, they seem to show diverse manifestations in the form of organs being affected, age of onset and the rate of progression. Reason lies in the unique genetic makeup of mitochondria. The percentage of mtDNA carrying defects varies when the ovum divides and one daughter cells receiving more defective mtDNA and the other receiving less. Hence, successive divisions may lead to accumulation of defects in one of the developing organs or tissues. Since the process in which defective mtDNA becomes concentrated in an organ is random, this may account for the differing manifestations among patients with the same genetic defect. Also, somatic mutations and mutations occurring as a result of exposure to environmental toxins may cause mitochondrial diseases.
As stated by Robert K. Naviaux, founder and co-director of the Mitochondrial and Metabolic Disease Center (MMDC) at the University of California, San Diego;
“It is a hallmark of mitochondrial diseases that identical mtDNA mutations may not produce identical diseases…the converse is also true, different mutations can lead to the same diseases.”
Postmitotic tissues are more vulnerable to mitochondrial diseases: Postmitotic tissues such as those in the brain, muscles, nerves, retinas, and kidneys, are vulnerable for several reasons. Apart from the fact that these tissues have high-energy demands, healthier neighboring cells unlike that observed in skin cannot replace the diseased cells. Thus, mutations in mtDNA accumulate over a period of time resulting in progressive dysfunction of individual cells and hence the organ itself.
High rate of mtDNA mutation: MtDNA mutates at rate that is six-seven times higher than the rate of mutation of nuclear DNA. First reason is the absence of histones on mtDNA and second is the exposure of mtDNA to free radicals due to their close proximity to electron transport chain. Additionally, lack of DNA repair enzymes results in mutant tRNA, rRNA and protein transcripts
Spectrum of mitochondrial diseases:
Following is the list of mitochondrial diseases occurring as a result of either mtDNA mutations, alteration in mitochondrial function or those diseases that sometimes might be associated with mitochondrial dysfunction.
II. DIAGNOSIS AND THERAPY OF MITOCHONDRIAL DISEASES
Diagnosis:
Owing to the diversity of symptoms, there is no accepted criterion for diagnosis. Also, due to overlapping symptoms of several diseases with those of mitochondrial dysfunction illnesses, it is important to evaluate the patient for other conditions. A diagnosis could involve combination of molecular genetic, pathologic, or biochemical data in a patient who has clinical features consistent with the diagnosis including mutational analysis on blood lymphocytes and possibly muscle biopsy for visual and biochemical analysis.
The two main biochemical features in most mtDNA disorders are:
Respiratory chain deficiency and
Lactic acidosis.
Skeletal muscle is chosen to study the pathogenic consequence of mtDNA mutations because of the formation of ragged-red fibers (RRF) through mitochondrial proliferation and massive mitochondrial accumulation in many pathogenic situations. RRF can be detected in two ways. Mitochondrial fibers in a subset of these fibers are shown by red or purple stained area by Gomori trichrome stain; the normal or less-affected fibers stain blue or turquoise. Deep purple areas show accumulations of mitochondria as activity of succinate dehydrogenase (SDH) in the case of mitochondrial mutation.
The primary care physician should remember this relatively simple rule of thumb: “When a common disease has features that set it apart from the pack, or involves 3 or more organ systems, think mitochondria.”
Treatment:
There are no cures for mitochondrial diseases; therefore, the treatment is focused on alleviating symptoms and enabling normal functioning of the affected organs. Most patients have used cofactor and vitamins; however, there is no overwhelming evidence that they are helpful in most patients.
Coenzyme Q10 (CoQ10) is the best-known cofactor used in treating mitochondrial cytopathies with no known side effects. CoQ10, residing in the inner mitochondrial membrane, functions as the mobile electron carrier and is a powerful antioxidant with benefits such as reduction in lactic acid levels, improved muscle strength, decreased muscle fatigue and so on.
Levocarnitine (L-carnitine, carnitine), is a cofactor required for the metabolism of fatty acids. Levocarnitine therapy improves strength, reversal of cardiomyopathy, and improved gastrointestinal motility, which can be a major benefit to those with poor motility due to their disease. Intestinal cramping and pain are the major side effects.
Creatine phosphate, synthesized from creatine can accumulate in small amounts in the body, and can act as storage for a high-energy phosphate bond. Muscular creatine may be depleted in mitochondrial cytopathy, and supplemental creatine phosphate has been shown to be helpful in some patients with weakness due to their disease.
B Vitamin, are necessary for the function of several enzymes associated with energy production. The need for supplemental B vitamin therapy is not proven, aside from rare cases of thiamine (vitamin B1)-responsive pyruvate dehydrogenase deficiency.
Research – Restriction enzyme for gene therapy of Mitochondria diseases:
Mitochondrial DNA (mtDNA) is the only extrachromosomal DNA in humans and defects in this genome are now recognized as important causes of various diseases. Presently, there is no effective treatment for patients suffering from diseases that harbor mutations in mtDNA.
Tanaka et al discovered a gene therapy method to treat a mitochondrial disease associated with mtDNA heteroplasmy. Heteroplasmy is where mutant and wild-type mtDNA molecules co-exist within cells. This syndrome of neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP) is caused by mutations in mtDNA leading to amino acid replacement in the resulting protein that codes for a subunit of mitochondrial ATP synthase. Level of mutant mtDNA is crucial for the disease as above a certain threshold level of mtDNA, the disease becomes biochemically and clinically apparent. Authors hypothesized that a possible method to treat patients was by selectively destroying mutant mtDNA, thereby only allowing propagation of wild-type mtDNA. Since restriction endonucleases can recognize highly specific sequences, they were utilized for gene therapy. Tanaka et al utilized Sma1, a restriction endonuclease to destroy mutant mtDNA, leading to increase in wild-type mtDNA levels.
Thus, authors concluded, “ the present results indicate that the use of a mitochondrion-targeted restriction enzyme which specifically recognizes a mutant mtDNA provides a novel strategy for gene therapy of mitochondrial diseases.”
III. MITOCHONDRIA, METABOLIC SYNDROME & RESEARCH
Mitochondria:
Mitochondria are double-membrane organelles located in the cytoplasm and often referred to as the “powerhouse” of the cell. In simple terms, they convert energy into forms that are usable by the cell. Mitochondria are semi-autonomous in that they are only partially dependent on the cell to replicate and grow. They have their own DNA, ribosomes, and can make their own proteins. They are the sites of cellular respiration that generates fuel for the cell’s activities. Mitochondria are also involved in other cell processes such as cell division, cellular growth and cell death. Multiple essential cellular functions are mediated by thousands of mitochondrial-specific proteins, encoded by both the nuclear and mitochondrial genomes.
Interestingly, mitochondria take on many different shapes and along with serving several different metabolic functions. In fact, each mitochondrion’s shape is characteristic of the specialized cell in which it resides. The number of mitochondria too varies in difference cell types, with as high as 500-2000 in some nucleated cells and as low as zero in RBCs and 2-6 in platelets.
The standard sequence to which all human mtNDNA is compared is referred to as the “Cambridge Sequence.” It was sequenced from several different human mtDNAs by a Medical Research Council (MRC) labora- tory based at Cambridge, UK, in 1981 and as a part of this work, Fred Sanger, the received his second Nobel Prize. Several variations in the form of polymorphisms are observed from the Cambridge sequence in the mtDNA of different individuals.
Metabolic syndrome:
Metabolic syndrome is a cluster of conditions — increased blood pressure, a high blood sugar level, excess body fat around the waist or abnormal cholesterol levels — that occur together, increasing your risk of heart disease, stroke and diabetes. Metabolic syndrome is becoming more and more common in the United States. In the future, it may overtake smoking as the leading risk factor for heart disease. In general, a person who has metabolic syndrome is twice as likely to develop heart disease and five times as likely to develop diabetes as someone who doesn’t have metabolic syndrome.
The five conditions described below are metabolic risk factors. You must have at least three metabolic risk factors to be diagnosed with metabolic syndrome.
A large waistline. This also is called abdominal obesity or “having an apple shape.” Excess fat in the stomach area is a greater risk factor for heart disease than excess fat in other parts of the body, such as on the hips.
A high triglyceride level (or you’re on medicine to treat high triglycerides). Triglycerides are a type of fat found in the blood.
A low HDL cholesterol level (or you’re on medicine to treat low HDL cholesterol). HDL sometimes is called “good” cholesterol. This is because it helps remove cholesterol from your arteries. A low HDL cholesterol level raises your risk for heart disease.
High blood pressure (or you’re on medicine to treat high blood pressure). Blood pressure is the force of blood pushing against the walls of your arteries as your heart pumps blood. If this pressure rises and stays high over time, it can damage your heart and lead to plaque buildup.
High fasting blood sugar (or you’re on medicine to treat high blood sugar). Mildly high blood sugar may be an early sign of diabetes.
Role of Mitochondria in Metabolic Syndrome & Diabetes:
Impaired mitochondrial function has recently emerged as a potential causes of insulin resistance and/or diabetes progression, risk factors of metabolic syndrome.
Mitochondria plays several key functions including generation of ATP, and generating metabolites via Tricarboxylic acid cycle that function in cytosolic pathways, oxidative catabolism of amino acids, ketogenesis, urea cycle; the generation of reactive oxygen species (ROS); the control of cytoplasmic calcium; and the synthesis of all cellular Fe/S clusters, protein cofactors essential for cellular functions such as protein translation and DNA repair. These roles define the mitochondria to be involved in metabolic homeostasis and hence, a major candidate for metabolic syndrome and its associated risk factor including diabetes, obesity and insulin resistance.
Research and Therapeutic relevance:
Understanding the underlying molecular mechanism of aberrant role of mitochondria is important in developing therapeutic agents for mitochondria-associated diseases. In the recent issue of Mitonews, several papers have been published using the products of MitoSciences, which describe research pertaining to the importance of mitochondria in obesity and diabetes. Some recent research articles based on mitochondrial research (also mentioned in MitoNews) have been briefly discussed here:
Metabolic inflexibility and Metabolic syndrome: Metabolic inflexibility is defined as the failure of insulin-resistant patients to appropriately adjust mitochondrial fuel selection in response to nutritional cues. Although the phenomenon has been emphasized an important aspect of metabolic syndrome, the molecular mechanisms have not yet been fully deciphered. In a recent article by Muoio et al, published in Cell Metabolism journal, essential role of the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT) has been identified in regulating substrate switching and glucose tolerance. CrAT regulates mitochondrial and intracellular Carbon trafficking by converting acetyl-CoA to its membrane permeant acetylcarnitine ester. Using muscle muscle-specific Crat knockout mice, primary human skeletal myocytes, and human subjects undergoing L-carnitine supplementation, authors have suggested a model wherein CrAT combats nutrient stress, promotes metabolic flexibility, and enhances insulin action by permitting mitochondrial efflux of excess acetyl moieties that otherwise inhibit key regulatory enzymes such as pyruvate dehydrogenase. These findings offer therapeutically relevant insights into the molecular basis of metabolic inflexibility.
Rosiglitazone and obesity: Eepicardial adipose tissue (EAT) has been described in humans as a functioning brown adipose tissue (BAT) and has been shown in animal models to have a lower glucose oxidation rate and higher fatty acid (FA) metabolism. In obese individuals, epicardial adipose tissue (EAT) is “hypertrophied”. EAT is a source of BAT may be a source of proinflamatory cytokines. Distel et al published their studies using a rat model of obesity and insulin resistance treated with rosiglitazone. They observed that rosiglitazone, promoted a BAT phenotype in the EAT depot characterized by an increase in the expression levels of genes encoding proteins involved in mitochondrial processing and density PPARγ coactivator 1 alpha (PGC-1α), NADH dehydrogenase 1 and cytochrome oxidase (COX4) resulting in significant up-regulation of PGC1-α and COX4 protein. The authors concluded that PPAR-γ agonist could induce a rapid browning of the EAT that probably contributes to the increase in lipid turnover. Thus, important insights into the mechanism of fat metabolism and involvement of mitochondrial proteins with a therapy were presented in the article.
Mitochondrial dysfunction and diabetic neuropathy: Animal models of diabetic neuropathy show that mitochondrial dysfunction occurs in sensory neurons that may contribute to distal axonopathy. The adenosine monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) signalling axis senses the metabolic demands of cells and regulates mitochondrial function. Studies in muscle, liver and cardiac tissues have shown that the activity of AMPK and PGC-1α is decreased under hyperglycaemia. Chowdhury et al using type 1 and type 2 diabetic rat and mice models studied the hypothesis that deficits in AMPK/PGC-1 signalling in sensory neurons underlie impaired axonal plasticity, suboptimal mitochondrial function and development of neuropathy. The authors have shown there is a significant reduction in phospho-AMPK, phopho-ACC, total PGC-1α, NDUFS3and COXIV in sensory neurons of the dorsal root ganglia of 14 week old diabetic mice with marked signs of thermal hypoalgesia. These results were associated with an impaired neuronal bioenergetic profile and a decrease in the activity of mitochondrial complex I, complex IV and citrate synthase. The fact that resveratrol treatment reversed the changes observed in vitro and in vivo suggest that the development of distal axonopathy in diabetic neuropathy is linked to nutrient excess and mitochondrial dysfunction via defective signalling of the AMPK/PGC-1α pathway.
ROS and diabetes: Mitochondria generated reactive oxygen species (ROS) has been associated with kidney damage occurring in diabetes. Rosca etal, published an article investigating the source and site of ROS production by kidney cortical tubule mitochondria in streptozotocin-induced type 1 diabetes in rats. The authors observed that in diabetic mitochondria, the fatty acid oxidation enzymes were elevated with increased oxidative phosphorylation and increased ROS production. The authors observed ROS production with fatty acid oxidation remained unchanged by limiting electron flow in ETC complexes, changes in ETC substrate processing and that the ROS supported by pyruvate also remained unaltered. The authors hence concluded that mitochondrial fatty acid oxidation is the source of increased ROS production in kidney cortical tubules in early diabetes
HBV and HCV-associated Liver Cancer: Important Insights from the Genome
Author: Ritu Saxena, PhD
UPDATED on 7/21/2022
HBV drug shifts to next-gen approaches
“While we respect Assembly’s decision to discontinue clinical development of VBR, we believe that it is premature to make any conclusions about any results in this triple combination clinical trial,” Arbutus CEO William Collier said in a separate release, referring to the study that involved his company’s drug. “We intend, in collaboration with Assembly, to continue the clinical trial in order to fully and accurately assess the results.”
So as Assembly shuts the door to future trials and wraps
Study 203 — a Phase II study testing VBR plus NrtI (nucleoside analogue reverse transcriptase inhibitor) plus interferon —
Study 204 will go on, with primary endpoints being safety and tolerability.
Patients are given either
VBR, NrtI and Arbutus’ AB-729,
VBR plus NrtI, or
NrtI plus AB-729.
The RNAi drug is designed to reduce all HBV viral proteins and antigens.
For Assembly Bio, the focus now shifts to two next-generation core inhibitors that it hopes could prove potent treatments for HBV. At the same time, it’s also working on earlier-stage research programs, including
a hepatitis D virus entry inhibitor,
a liver-focused interferon-α receptor agonist and
new antivirals to be introduced later.
With CMO Luisa Stamm and CFO Michael Samar set to leave in the next few weeks, McHutchison — a former Gilead CSO — will now lead a remaining team of 70.
Meanwhile, Michele Anderson, SVP of development operations, is being promoted to chief development officer; and COO Jason Okazaki will add president to his title and finance to his slate of duties. The company now expects to have a cash runway into the first half of 2024.
(research article published in New England Journal of Medicine regarding the role of SALL4 gene in aggressive hepatocellular carcinoma)
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The incidence of HCC varies considerably with the geographic area because of differences in the major causative factors. Chronic hepatitis B and C, mostly in the cirrhotic stage, are responsible for the great majority of cases of HCC worldwide.
Hepatitis B and C viruses (HBV/HCV) can be implicated in the development of HCC in an indirect way, through induction of chronic inflammation, or directly by means of viral proteins or, in the case of HBV, by creation of mutations by integration into the genome of the hepatocyte.http://www.wjso.com/content/3/1/27
With the advent of genome sequencing methodologies, it was about time that the scientists look clues within the genome of HCC tumor cells that would provide clues for disease progression via virus integration into the liver cells.
Two studies published in the recent issue of Nature Genetics (May 2012) explored the genome of HCC cells for genetic mutations that might be related to HBV and HCV highlighting the types of genetic mutations that underlie the liver cancer hepatocellular carcinoma, including forms of the disease related to hepatitis B and hepatitis C virus infection.
In the first study, Sung et al performed an extensive whole genome analysis using a large sample size of 88 Chinese individuals with HCC http://www.ncbi.nlm.nih.gov/pubmed?term=Genome-wide%20survey%20of%20recurrent%20HBV This was in the fact first unbiased, genome-wide, HBV-integration map in HCC leading to new recurrent integration sites and molecular mechanisms.
Although integration of viral DNA sequence within HCC genome has been reported in several studies, however, fewer cases of recurring mutations within genes during these integrations have been studied. The reason might be limited sample size in these studies. Tumor and non-tumor adjacent liver cells were surveyed in 81 HBV positive and 7 HBV negative HCC tumor samples. After the survey of whole genome of the 88 patients, several viral integration sites were discovered referred to as breakpoints. The breakpoints were found to be much more common in tumor than normal samples. Although the observed breakpoints were randomly distributed across the genome, a handful or frequently occurring sites referred to as ‘hotspots’ were discovered. The frequency of integration revealed that there were five genes with recurring integrations in HBV tumors- TERT, MLL4, CCNE5, SENP1, and ROCK1.
Apart from genome analysis, expression levels of the 5 genes implicated in the study were determined. In other words, the levels of proteins formed from the genes were compared and it was observed that samples with HBV integration had significantly higher level of protein expression of TERT, MLL4 and CCNE5 than the samples harboring no HBV integration sites. Although not statistically significant, overexpression of SENP1 and ROCK1 genes was also observed in HBV integration samples. This lead to an important conclusion from the study that the five genes that harbor recurrent HBV integrations might be implicated in HCC tumor development and that overexpression of these proteins is a probable molecular mechanism of tumorigenesis.
Interestingly, analysis of the HBV analysis revealed that almost 40% of the HBV genomes were cleaved at approximately 1,800 bp and then integrated into the human genome. The cleaved HBV sites had the necessary machinery (enhancers and ORF replication sites) for protein formation.
The study also confirmed the popular belief that HBV integrations might worsen the prognosis of HCC patients revealing a significant correlation between the number of HBV integrations and the survival of patients.An interesting observation from the study that had not been reported before was that HBV integration was associated with the occurrence of HCC at a younger age.
The study presented convincing evidence that chromosomal instability of HCC genome may originate from HBV integration.
A parallel study published in the same issue of Nature Genetics explored the genome of HCC tumors to gain insights into HBV and HCV-related genomic alterations. The research team sequenced whole-exon (protein forming genomic regions) of 27 liver tumors from 25 patients and compared with the corresponding genome sequences from matched white blood cell samples.
The study involved both HBV-related and HCV-related tumors along with two samples of tumors from individuals without HBV or HCV infection. The genome wide sequencing of HCC tumor cells revealed several mutations that included deletions and mutations of genes with predicted functional consequences. “Considering the high complexity and heterogeneity of [hepatocellular carcinomas] of both etiological and genetic aspects,” they concluded, “further molecular classification is required for appropriate diagnosis and therapy in personalized medicine.” Additionally, recurrent alterations were observed in the four genes – ARID1A, RPS6KA3, NFE2L2 and IRF2 that had not been previously described in HCC. The comprehensive mutation pattern observed in the study might be indicative of specific mutagenesis mechanisms occurring in tumor cells.
Authors said “Although no common somatic mutations were identified in the multicentric tumor pairs,” further stating “their whole-genome substitution patterns were similar, suggesting that these tumors developed from independent mutations, although their shared etiological backgrounds may have strongly influenced their somatic mutation patterns.”The researchers suggested a major role of chromatin remodeling complexes and involvement of both interferon and oxidative stress pathways in hepatocellular malignant proliferation and transformation based on the genes showing recurrent mutations in the observed genes.
Thus, in both the studies new genes recurrently altered in HCC were identified along with uncovering some important clues relating to the molecular mechanism of virus-associated HCC.
Role of SALL4 in HCC
The oncofetal gene SALL4 is a marker of a subtype of HCC with progenitor-like features and is associated with a poor prognosis. Investigators at Cancer Science Institute of Singapore, National University of Singapore studied the role of oncofetal gene, SALL4 in HCC and the results were published were in a recent issue of New England Journal of Medicine ((Yong KJ, et al, Oncofetal Gene SALL4 in Aggressive Hepatocellular Carcinoma. http://www.ncbi.nlm.nih.gov/pubmed/23758232). Yong and colleagues (2013) screened specimens from patients with primary HCC for the expression of SALL4 and carried out a clinicopathological analysis. Loss-of-function studies were then performed to evaluate the role of SALL4 in hepatocarcinogenesis and its potential as a molecular target for therapy. Furthermore, in vitro functional and in vivo xenograft assays were performed to assess the therapeutic effects of a peptide that targets SALL4.
According to the results, SALL4 is an oncofetal protein that is expressed in the human fetal liver and silenced in the adult liver, but it is reexpressed in a subgroup of patients who have HCC and an unfavorable prognosis. Gene-expression analysis showed the enrichment of progenitor-like gene signatures with overexpression of proliferative and metastatic genes in SALL4-positive HCC. Loss-of-function studies confirmed the critical role of SALL4 in cell survival and tumorigenicity. The peptide targeting SALL4 blocked SALL4-corepressor interactions that released suppression of PTEN and inhibited tumor formation in xenograft assays in vivo. In conclusion, the results from the study indicate that SALL4 is a marker for a progenitor subclass of HCC with an aggressive phenotype. The absence of SALL4 expression in the healthy adult liver enhances the potential of SALL4 as a treatment target in HCC.
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