Posts Tagged ‘genetic’

Statistical Approach for Calculating Environmental Influences in Genome-Wide Association Study (GWAS) Results

The approach fills a gap in current analyses. Complex diseases like cancer usually arise from complex interactions among genetic and environmental factors. When many such combinations are studied, identifying the relevant interactions versus those that reflect chance combinations among affected individuals becomes difficult. In this study, the investigators developed a novel approach for evaluating the relevance of interactions using a Bayesian hierarchal mixture framework. The approach is applicable for the study of interactions among genes or between genetic and environmental factors.

Chris Amos, PhD, senior author of the paper said, “These findings can be used to develop models that include only those interactions that are relevant to disease causation, allowing the researcher to remove false positive findings that plague modern research when many dozens of factors and their interactions are suggested to play a role in causing complex diseases.”

The model evaluates “gene by gene” and “gene by environment” factors by looking at specific DNA sequencing variations. Complex diseases are caused by multiple factors. In some cases a genetic predisposition or abnormality may be a factor. A person’s healthy lifestyle and environment, however, may help him or her overcome a genetic vulnerability and avoid a chronic disease like cancer. In other situations, a person whose DNA does not have an abnormality may develop one when exposed to known carcinogens like tobacco smoke or sunburn.

“Understanding the combinations of genetic and environmental factors that cause complex diseases is important,” said Amos, associate director of population sciences and deputy director of Norris Cotton Cancer Center, “because understanding the genetic architecture underlying complex disease may help us to identify specific targets for prevention or therapy upon which interventions may appropriately reduce the risk of cancer development or progression.”

The study applied the model in cutaneous melanoma and lung cancer genetic sequences using previously identified abnormalities (known as single nucleotide polymorphisms or SNPs) with environmental factors introduced as independent variables. The Bayesian mixture model was compared with the traditional logistic regression model. The hierarchal model successfully controlled the probability of false positive discovery and identified significant interactions. It also showed good performance on parameter estimation and variable selection. The model cannot be applied to a complete GWAS because if its reliance on other probability models (MCMC ). It is most effective when applied to a group of SNPs.

“The method was effective for the study of melanoma and lung cancer risk because these cancers develop from a complex interaction between genetic and environmental factors but understanding how these factors interact has been difficult to achieve without the sophisticated modeling that has been developed in this study,” said Amos.

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Gene-editing technique offers new way to model cancer

Now, MIT researchers have found an alternative: They have shown that a gene-editing system called CRISPR can introduce cancer-causing mutations into the livers of adult mice, enabling scientists to screen these mutations much more quickly.

In a study appearing in the Aug. 6 issue of Nature, the researchers generated liver tumors in adult mice by disrupting the tumor suppressor genes p53 and pten. They are now working on ways to deliver the necessary CRISPR components to other organs, allowing them to investigate mutations found in other types of cancer.

“The sequencing of human tumors has revealed hundreds of oncogenes and tumor suppressor genes in different combinations. The flexibility of this technology, as delivery gets better in the future, will give you a way to pretty rapidly test those combinations,” says Institute Professor Phillip Sharp, an author of the paper.

Tyler Jacks, director of MIT’s Koch Institute for Integrative Cancer Research and the David H. Koch Professor of Biology, is the paper’s senior author. The lead authors are Koch Institute postdocs Wen Xue, Sidi Chen, and Hao Yin.

Gene disruption

CRISPR relies on cellular machinery that bacteria use to defend themselves from viral infection. Researchers have copied this bacterial system to create gene-editing complexes that include a DNA-cutting enzyme called Cas9 bound to a short RNA guide strand that is programmed to bind to a specific genome sequence, telling Cas9 where to make its cut.

In some cases, the researchers simply snip out part of a gene to disrupt its function; in others, they also introduce a DNA template strand that encodes a new sequence to replace the deleted DNA.

To investigate the potential usefulness of CRISPR for creating mouse models of cancer, the researchers first used it to knock out p53 and pten, which protect cells from becoming cancerous by regulating cell growth. Previous studies have shown that genetically engineered mice with mutations in both of those genes will develop cancer within a few months.

Studies of such genetically engineered mice have yielded many important discoveries, but the process, which requires introducing mutations into embryonic stem cells, can take more than a year and costs hundreds of thousands of dollars. “It’s a very long process, and the more genes you’re working with, the longer and more complicated it becomes,” Jacks says.

Using Cas enzymes targeted to cut snippets of p53 and pten, the researchers were able to disrupt those two genes in about 3 percent of liver cells, enough to produce liver tumors within three months.

Many models possible

The researchers also used CRISPR to create a mouse model with an oncogene called beta catenin, which makes cells more likely to become cancerous if additional mutations occur later on. To create this model, the researchers had to cut out the normal version of the gene and replace it with an overactive form, which was successful in about 0.5 percent of hepatocytes (the cells that make up most of the liver).

The ability to not only delete genes, but also to replace them with altered versions “really opens up all sorts of new possibilities when you think about the kinds of genes that you would want to mutate in the future,” Jacks says. “Both loss of function and gain of function are possible.”

Using CRISPR to generate tumors should allow scientists to more rapidly study how different genetic mutations interact to produce cancers, as well as the effects of potential drugs on tumors with a specific genetic profile.

“This is a game-changer for the production of engineered strains of human cancer,” says Ronald DePinho, director of the University of Texas MD Anderson Cancer Center, who was not part of the research team. “CRISPR/Cas9 offers the ability to totally ablate gene function in adult mice. Enhanced potential of this powerful technology will be realized with improved delivery methods, the testing of CRISPR/Cas9 efficiency in other organs and tissues, and the use of CRISPR/Cas9 in tumor-prone backgrounds.”

In this study, the researchers delivered the genes necessary for CRISPR through injections into veins in the tails of the mice. While this is an effective way to get genetic material to the liver, it would not work for other organs of interest. However, nanoparticles and other delivery methods now being developed for DNA and RNA could prove more effective in targeting other organs, Sharp says.

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Trapped: Cell-invading piece of virus captured in lab by scientists

This achievement sets the stage to use x-ray crystallography to develop complete images of HIV that include integrase, which in turn will help scientists develop new treatments for the illness.

Duane Grandgenett, Ph.D., professor at SLU’s Institute of Molecular Virology and senior author of the study, discovered integrase in 1978, little knowing the piece of virus would provide the basis for an entire class of drugs that now treats HIV.

“In 1974, we hadn’t heard of HIV yet,” Grandgenett said. “We did, however, study retroviruses, the class of viruses that includes HIV. Retroviruses spread by taking over your cell’s DNA.

“And the way the virus does this is with integrase. It’s responsible for inserting the genetic information of the virus, the DNA, into our chromosomes establishing the viral reservoir. Then, it uses our cells to replicate.

“Integrase is a key component that makes HIV pathogenic.”

When a person is infected with HIV, there is an initial burst of virus production. This is when integrase inserts the virus DNA into many human cells, including CD4 T-immune cells, brain cells and other lymph cells. HIV is particularly devastating to the immune system’s T-cells, which protect the body from infection.

“Most people do not die from virus replication but from secondary causes,” Grandgenett said. “Their immune system collapses and opportunistic infections and cancer are what really kill the person.”

Now, scientists have developed drugs that are very successful at managing HIV. Combinational drug therapy is particularly effective. The virus mutates so that it can quickly become resistant to a drug. But when three different drugs aim at three different targets, as in combination drug therapy, the probability of drug resistance is much smaller.

There is one catch, however. Patients must take the drugs every day. If they do not, the virus starts cycling again and within a few weeks the viral levels are back up.

Scientists continue to try to stay a step ahead of the virus, both to combat drug resistance and to develop better treatments.

To develop better drugs, scientists want to use a process called x-ray crystallography to develop a complete picture of how integrase inhibitors — the class of HIV drugs that target integrase– interact with the virus.

“We’re aiming to develop newer, better medicines,” Grandgenett said. “We want to better understand how the integrase inhibitor drugs interact with integrase.

“So far, everybody has failed to produce HIV integrase-DNA images via high resolution x-ray crystallography,” Grandgenett said. “No one has ever captured the mother load.”

This is Grandgenett’s goal.

“Now, we’re going after full length integrase protein with DNA,” Grandgenett said. “This is what I’ve wanted to do since 1978, even before HIV was identified.”

To do this, Grandgenett and his team, including investigators Krishan Pandey, Ph.D., and Sibes Bera, Ph.D., needed to develop an integrase-DNA complex and then kinetically stabilize the complex in the presence of the drug.

Researchers used a surrogate virus to take a shortcut. Because integrase structures are similar in all retroviruses, Grandgenett tried his approach in Rous sarcoma virus (RSV), whose integrase is more readily manipulated than HIV integrase.

All current clinical integrase inhibitors work in the same way: They block integrase which prevents HIV from replicating. Specifically, they do this by stopping viral DNA strand transfer with STIs — strand transfer inhibitors.

Those inhibitors work by binding three components together: viral DNA; viral integrase; and the drug itself. Before this study, no one had been able to produce a synaptic complex (SC) in solution, the place where these three elements meet.

The researchers developed conditions where the HIV strand transfer inhibitors (STIs) trapped the SC of the surrogate RSV integrase. Grandgenett reports that this experiment is first time anyone has ever captured an integrase-DNA-inhibitor SC in solution.

“We’ve isolated it and now we want to do x-ray crystallography on it to get a better image of HIV integrase,” Grandgenett said. “That’s the next step. Hopefully, that crystal structure will better explain how integrase drugs and DNA interact at the nanometer level.

“This will help us to design new drugs. There will be a lot of uses for this information.”

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