Boosting the Powers of Genomic Science
With two new methods, UC San Diego scientists hope to improve genome-wide association studies
As scientists probe and parse the genetic bases of what makes a human a human (or one human different from another), and vigorously push for greater use of whole genome sequencing, they find themselves increasingly threatened by the unthinkable: Too much data to make full sense of.
In a pair of papers published in the April 25, 2013 issue of PLOS Genetics, two diverse teams of scientists, both headed by researchers at the University of California, San Diego School of Medicine, describe novel statistical models that more broadly and deeply identify associations between bits of sequenced DNA called single nucleotide polymorphisms or SNPs and say lead to a more complete and accurate understanding of the genetic underpinnings of many diseases and how best to treat them.
“It’s increasingly evident that highly heritable diseases and traits are influenced by a large number of genetic variants in different parts of the genome, each with small effects,” said Anders M. Dale, PhD, a professor in the departments of Radiology, Neurosciences and Psychiatry at the UC San Diego School of Medicine. “Unfortunately, it’s also increasingly evident that existing statistical methods, like genome-wide association studies (GWAS) that look for associations between SNPs and diseases, are severely underpowered and can’t adequately incorporate all of this new, exciting and exceedingly rich data.”
Dale cited, for example, a recent study published in Nature Genetics in which researchers used traditional GWAS to raise the number of SNPs associated with primary sclerosing cholangitis from four to 16. The scientists then applied the new statistical methods to identify 33 additional SNPs, more than tripling the number of genome locations associated with the life-threatening liver disease.
Generally speaking, the new methods boost researchers’ analytical powers by incorporating a priori or prior knowledge about the function of SNPs with their pleiotrophic relationships to multiple phenotypes. Pleiotrophy occurs when one gene influences multiple sets of observed traits or phenotypes.
Dale and colleagues believe the new methods could lead to a paradigm shift in CWAS analysis, with profound implications across a broad range of complex traits and disorders.
“There is ever-greater emphasis being placed on expensive whole genome sequencing efforts,” he said, “but as the science advances, the challenges become larger. The needle in the haystack of traditional GWAS involves searching through about one million SNPs. This will increase 10- to 100-fold, to about 3 billion positions. We think these new methodologies allow us to more completely exploit our resources, to extract the most information possible, which we think has important implications for gene discovery, drug development and more accurately assessing a person’s overall genetic risk of developing a certain disease.”
Drug Treatment Corrects Autism Symptoms in Mouse Model
Autism results from abnormal cell communication. Testing a new theory, researchers at the University of California, San Diego School of Medicine have used a newly discovered function of an old drug to restore cell communications in a mouse model of autism, reversing symptoms of the devastating disorder.
The findings are published in the March 13, 2013 issue of the journal PLOS ONE.
“Our (cell danger) theory suggests that autism happens because cells get stuck in a defensive metabolic mode and fail to talk to each other normally, which can interfere with brain development and function,” said Robert Naviaux, MD, PhD, professor of medicine and co-director of the Mitochondrial and Metabolic Disease Center at UC San Diego. “We used a class of drugs that has been around for almost a century to treat other diseases to block the ‘danger’ signal in a mouse model, allowing cells to return to normal metabolism and restore cell communication.”
“Of course, correcting abnormalities in a mouse is a long way from a cure for humans,” said Naviaux, “but we are encouraged enough to test this approach in a small clinical trial of children with autism spectrum disorder in the coming year. This trial is still in the early stages of development. We think this approach – called antipurinergic therapy or APT – offers a fresh and exciting new path that could lead to development of a new class of drugs to treat autism.”
Autism spectrum disorders (ASDs) are complex disorders defined by abnormalities in the development of language, social and repetitive behaviors. Hundreds of different genetic and environment factors are known to confer risk. In this study, nearly a dozen UC San Diego scientists from different disciplines collaborated to find a unifying mechanism that explains autism. Their work is the result of one of just three international “Trailblazer” awards given by the group Autism Speaks in 2011.
Describing a completely new theory for the origin and treatment of autism using APT, Naviaux and colleagues introduce the concept that a large majority of both genetic and environmental causes for autism act by producing a sustained cell danger response – the metabolic state underlying innate immunity and inflammation.
“When cells are exposed to classical forms of dangers, such as a virus, infection or toxic environmental substance, a defense mechanism is activated,” Naviaux explained. “This results in changes to metabolism and gene expression, and reduces the communication between neighboring cells. Simply put, when cells stop talking to each other, children stop talking.”
Since mitochondria – the so-called “power plants” of the cell – play a central role in both infectious and non-infectious cellular stress, innate immunity and inflammation, Naviaux and colleagues searched for a signaling system in the body that was both linked to mitochondria and critical for innate immunity. They found it in extracellular nucleotides like adenosine triphosphate (ATP) and other mitokines – signaling molecules made by distressed mitochondria. These mitokines have separate metabolic functions outside of the cell where they bind to and regulate receptors present on every cell of the body. Fifteen types of purinergic receptors are known to be stimulated by these extracellular nucleotides, and the receptors are known to control a broad range of biological characteristics with relevance to autism.
The researchers tested suramin – a well-known inhibitor of purinergic signaling used medically for the treatment of African sleeping sickness since shortly after it was synthesized in 1916 – in mice. They found that this APT mediator corrected autism-like symptoms in the animal model, even if the treatment was started well after the onset of symptoms. The drug restored 17 types of multi-symptom abnormalities including normalizing brain synapse structure, cell-to-cell signaling, social behavior, motor coordination and normalizing mitochondrial metabolism.
“The striking effectiveness shown in this study using APT to ‘reprogram’ the cell danger response and reduce inflammation showcases an opportunity to develop a completely new class of anti-inflammatory drugs to treat autism and several other disorders,” Naviaux said.
Effects of Alcoholism: a Q & A with Marc Schuckit, Distinguished Professor of Psychiatry
The overconsumption of alcohol kills 2.5 million people each year around the globe, according to the World Health Organization. It is a major factor in many neuropsychiatric disorders and a risk factor for cardiovascular diseases, cirrhosis of the liver and various cancers.
The effects of alcohol abuse are felt far beyond the physical and psychological effects of the drinker. From drunk driving fatalities to booze-fueled violence and other behavioral disorders, alcohol abuse leaves a lasting, damaging mark on individuals and society.
Marc Schuckit, MD, is a Distinguished Professor of psychiatry at the University of California, San Diego School of Medicine and editor of the Journal of Studies on Alcohol and Drugs. He has spent his career studying the causes and effects of alcohol use and abuse. We asked for a status report.
Q: Where do you rate alcohol consumption as a social and public health problem? Is it trending up or down? Where do you see the most cause for alarm?
A: It’s close to number one. It’s a worldwide problem. Alcohol use is one of the leading risk factors in the overall burden of disease, especially among men age 15 to 60. The U.S. has an average consumption rate per person per year somewhere between eight and 10 liters of absolute alcohol. That’s somewhere in the middle among countries worldwide, but it’s still one heck of a lot of booze. Alcohol use disorders, such as dependence and the problems associated with these conditions, are much more common than, say, illicit drug disorders, as important as they are.Whether the problem is trending up or down depends upon who you’re reading. Excessive alcohol consumption has been a bad problem for decades. Not a lot has changed. It remains a bad problem.
Q: To what degree can abuse and dependence on alcohol, or alcoholism, be attributed to genetics? Does a biological predisposition outweigh all other factors?
A: If you use the general definition of alcohol abuse and dependence, this basically reflects severe repetitive problems with alcohol. The condition is roughly 60 percent genetic. Nonetheless, that still means the environment and environmental interaction with genetics is almost half the picture.The fact that researchers are now finding some genes related to the disease can help with identifying it and people at higher risk. This knowledge can help start early prevention. It’s also possible that understanding something about the genetics will help with treatment, though not always.
Q: One of your major studies concluded that sensitivity to the effects of alcohol strongly influences risk of alcohol dependence. Can you elaborate?
A: One of the characteristics through which the genes for alcohol abuse and dependence operate is the ability to consume fairly large amounts of alcohol, but have relatively little effect. This is associated with a probability of drinking a lot more per occasion than some other people and carries a higher risk for alcohol problems in the long run. Depending upon a range of factors, including their genetic make-up, other people experience a lot of effects after a few drinks, which means they have a higher sensitivity per drink, a characteristic often related to a lower alcoholism risk.We think we’ve found some of the genes that are associated with the low alcohol sensitivity (or low effect per drink) and that we may be able to use this information to identify people at greater risk and educate them. This would be very useful when working with, say, adolescents.
Q: In your opinion, what has been the most interesting or surprising discovery in alcohol research over the last decade or so?
A: For me, it’s the findings about alcohol sensitivity, which are the opposite of what we initially thought. We had hypothesized that the people at high risk for alcohol abuse and dependence were those with a higher effects, but that’s not what we found. People who can drink a lot without obvious effect tend to drink a lot and they hang out with other people who drink a lot.
Using imaging research, it looks like this low sensitivity to alcohol may be related to how the brain is processing information. People who have a low sensitivity process some kinds of information with a little bit less efficiency than others. They’re as smart as anybody else, it’s just a different intensity in the way the brain is working. That, in turn, might interfere with their ability to recognize effects of more moderate doses of alcohol.
Chronic myeloid leukemia blood cells.
Enzyme Accelerates Malignant Stem Cell Cloning in Chronic Myeloid Leukemia
An international team, headed by researchers at the University of California, San Diego School of Medicine, has identified a key enzyme in the reprogramming process that promotes malignant stem cell cloning and the growth of chronic myeloid leukemia (CML), a cancer of the blood and marrow that experts say is increasing in prevalence.
The findings are published in the Dec. 24 online early edition of the Proceedings of the National Academy of Sciences (PNAS).
Despite the emergence of new therapies, such as tyrosine kinase inhibitors, CML and other leukemias remain problematic because some cancer stem cells avoid destruction and eventually regenerate themselves, a stem cell process known as self-renewal that can result in a return and spread (metastasis) of the disease.
In the PNAS paper, principal investigator Catriona H. M. Jamieson, MD, PhD, associate professor of medicine at UC San Diego, with colleagues in the United States, Canada and Italy, report that inflammation – long associated with the development of cancer – boosts activity of an enzyme called adenosine deaminase or ADAR1.
Expressed during embryogenesis to help blood cell development, ADAR1 subsequently turns off and is triggered by viral infections where it protects normal hematopoietic stem cells from attack. In leukemia stem cells, however, overexpression of ADAR1 enhances the missplicing of RNA, which leads to greater self-renewal and therapeutic resistance of malignant stem cells.
The findings build upon previous studies by Jamieson and others that elucidate the effects of RNA missplicing and instability. “People normally think about DNA instability in cancer, but in this case, it’s how the RNA is edited by enzymes that really matters in terms of cancer stem cell generation and resistance to conventional therapy.”
The described RNA editing process, which occurs in the context of human and other primate specific sequences, also underscores the importance of addressing inflammation as “an essential driver of cancer relapse and therapeutic resistance,” Jamieson said. It also presents a new target for future therapies.
“ADAR1 is an enzyme that we may be able to specifically target with a small molecule inhibitor, an approach we have already used effectively with other inhibitors,” said Jamieson. “If we can block the capacity of leukemia stem cells to use ADAR1, if we can knock down that pathway, maybe we can put stem cells back on the right track and stop malignant cloning.”
CML is a cancer initiated by a mutant gene called BCR-ABL in blood forming stem cells that leads to an expansion of white blood cells and their precursors. It is typically slow-growing and often not diagnosed until its later stages when there can be a sudden, dramatic increase in malignant cells, known as blast crisis. Median age of diagnosis is 66 years; incidence of the disease increases with age. Despite tremendous advances in BCR-ABL tyrosine kinase inhibitor therapies, the majority of patients relapse if therapy is discontinued, in part as a result of dormant cancer stem cell resistance. This work suggests a novel mechanism for overcoming cancer stem cell resistance to therapy that may prevent relapse and progression.
The estimated prevalence of CML in the United States is 70,000 persons with the disease, projected to steadily increase to approximately 181,000 by 2050. CML is initiated by the mutant BCR-ABL gene, but scientists have not yet identified the cause of the mutation.
11 year-old Colman Chadam has been removed from his school for having the genetic mutation for cystic fibrosis but not the disease itself. (Image Source: SF Gate)
What’s in your genes? Three questions for Kevin Shaw about cystic fibrosis
When the parents of 11 year-old Colman Chadam revealed on a school medical form that their son carried the genetic mutation for cystic fibrosis, they thought they were being proactive. However, their conscientious disclosure led to school administrators having Chadam transferred to another school. Although Chadam does not have cystic fibrosis, another student at the Palo Alto school does, and administrators were worried that Chadam could put the other student at risk.
Cystic fibrosis (CF) is not contagious but it is life-threatening to those who have the disease. We’ve asked Kevin Shaw, MD, assistant clinical professor at UC San Diego School of Medicine and associate director for the Adult Cystic Fibrosis Program at UC San Diego Health System three questions about cystic fibrosis.
Question: Is it dangerous for people with cystic fibrosis to be around others with the same disease?
Answer: In theory, yes, insomuch as there is a potential for cross-contamination with highly resistant organisms. As a result, the Cystic Fibrosis Foundation recommends physical and temporal distance between cystic fibrosis patients with very few exceptions including siblings and married couples. In the past, these recommendations had not been followed. In fact, many children with cystic fibrosis attended “cystic fibrosis camp” and spent time together on a regular basis, developing bonds, making friendships, sharing in the CF experience, et cetera. It was recognized, however, that these camp experiences also contributed to the spreading of certain organisms from patient to patient, with potential for clinical decompensation as a result. In the practical world, it is almost impossible to have complete isolation between patients, but we do use standard mechanisms including wearing isolation gowns and gloves to visit them in the inpatient setting, not reusing outpatient examination rooms between cystic fibrosis patients, and keeping them from congregating together in the waiting room when they come to clinic.
Q: How can someone have the genetic mutation for CF but not have the disease? Is being a carrier common?
A: There are over 1900 recognized CFTR (the gene that can cause cystic fibrosis) mutations that have been recognized, but not all are associated with clinical manifestations of the disease. When a person is a homozygote for a disease-causing mutation, he or she typically has clinical manifestations of cystic fibrosis. These may include salty skin, sinusitis, pulmonary infections and bronchiectasis, exocrine pancreatic insufficiency, diabetes, liver disease, bowel obstructions, infertility, arthritis, or vasculitis. However, it is extremely common to be a heterozygote carrier of a CFTR mutation, meaning that a person would have one normal copy and one abnormal copy. It is present in the population anywhere between 1:25 and 1:30 people, depending on racial background and geographic location. In fact, over 10 million Americans are asymptomatic carriers of a CFTR mutation and don’t even know it! If a person has two mutations of CFTR, it is still possible that they could not have clinical manifestations of the disease, so long as one of these mutations is deemed relatively mild or does not contribute to significant dysfunction of the CFTR protein itself.
Q: Do you think that the administrators at Chadam’s school acted responsibly or is this a case of cautious behavior taken too far?
A: It is probably cautious behavior taken too far, although it is certainly difficult to truly understand everybody’s perspective on what is a challenging problem. Although Chadam does not exhibit any clinical manifestations of cystic fibrosis, the fact that he has two known CFTR mutations is enough to make school officials curious at the very least. It is difficult to expect a school administrator to function with the same level of genetic and pathophysiologic background as a physician would, especially with such a complicated scenario. I believe the administrator would argue that his primary responsibility is the safety of the other students, and I’m certain that this was the motivating factor behind the story. It is not often that you can tell somebody you have two genetic mutations for a well recognized genetic disease, yet somehow do not have that disease. It is not surprising that the administrators have trouble with this concept. However, even if he did have clinically manifested cystic fibrosis with typical pathophysiology, I still think it is overreacting to kick him out of the school or make him transfer. Patients with cystic fibrosis have been attending the same grade schools, high schools, and colleges for many years, and to my knowledge this has not led to significant problems with transfer of infections. Now that we know about infection risk and the need to avoid the same immediate physical space, it would be relatively easy to act responsibly yet still allow both children to attend the same school. Separating their physical location in a classroom or making sure that they sit in different classrooms altogether would be more than sufficient to minimize any chance of transfer of organisms.
CIRM Funds Six UC San Diego Stem Cell Researchers
The governing board of the California Institute for Regenerative Medicine (CIRM) has announced that six investigators from the University of California, San Diego Stem Cell Research program have received a total of more than $7 million in the latest round of CIRM funding. This brings UC San Diego’s total to more than $128 million in CIRM funding since the first awards in 2006.
UC San Diego scientists funded by the newly announced CIRM Basic Biology Awards IV include Maike Sander, MD, professor of Pediatrics and Cellular and Molecular Medicine; Miles Wilkinson, PhD, professor, Division of Reproductive Endocrinology; Gene Yeo, PhD, MBA, assistant professor with the Department of Cellular and Molecular Medicine and the Institute for Genomic Medicine; George L. Sen, PhD, assistant professor of cellular and molecular medicine; David Traver, PhD, associate professor with the Department of Cellular and Molecular Medicine and Ananda Goldrath, PhD, associate professor in the Division of Biological Sciences.
pictured: Human stem cells, false color.
This mold house
More than 6 million children in the United States have asthma. Genes are known to play a role. Like allergies, asthma can run in families. Exercise, the weather, some medications, even strong emotions have been linked to the disease, which is characterized by a blocking or narrowing of passageways in the lungs, resulting in difficulty breathing.
Environmental factors, however, may be the biggest factor. Specifically, childhood asthma has been strongly linked to indoor mold problems arising from unwanted moisture problems, such as leaky pipes. Molds cause breathing problems by releasing tiny spores into the air, which are inhaled, provoking allergic reactions.
A new study by researchers at the University of Cincinnati, published in the Journal of Allergy and Clinical Immunology, sheds new light on the still murky mold-asthma connection. Between 2001 and 2003, scientists analyzed dust samples from 289 households, each with at least one infant averaging 8 months in age. Thirty-six species of mold were categorized. Each home was given an Environmental Relative Moldiness Index (ERMI) score, developed by the Environmental Protection Agency to assess mold levels.
At age 7, the researchers tested the kids for allergies and asthma. They found that 69 (24 percent) of the children had developed asthma. The earlier ERMI scores proved to be good predictors of later asthma development.
Three particular species of mold were most associated with asthma: Aspergillus ochraceus, Aspergillus unguis and Penicillium variabile.
The scientists also surveyed the homes for obvious evidence of mold. Some houses with no sign of mold had high ERMI values. The researchers said their study doesn’t prove that molds cause asthma on their own, but does provide new evidence that indoor mold can contribute to asthma development.
“This stresses the urgent need for remediating water damage in homes, particularly in lower income, urban communities where this is a common issue,” said lead study author Tiina Reponen.
The schematic above depicts some of the subnetworks of interacting proteins that either promote or slow the progression of CLL.
Tumor Cells’ Inner Workings Predict Cancer Progression
Molecular markers help reveal nature of chronic lymphocytic leukemia – slow or fast
Using a new assay method to study tumor cells, researchers at the University of California, San Diego School of Medicine and UC San Diego Moores Cancer Center have found evidence of clonal evolution in chronic lymphocytic leukemia (CLL). The assay method distinguishes features of leukemia cells that indicate whether the disease will be aggressive or slow-moving, a key factor in when and how patients are treated.
The findings are published in the July 26, 2012 First Edition online issue of Blood.
The progression of CLL is highly variable, dependent upon the rate and effects of accumulating monoclonal B cells in the blood, marrow, and lymphoid tissues. Some patients are symptom-free for years and do not require treatment, which involves the use of drugs that can cause significant side effects and are not curative. In other patients, however, CLL is relatively aggressive and demands therapeutic intervention soon after diagnosis.
“Our study shows that there may not be a sharp dividing line between the more aggressive and less aggressive forms of CLL,” said Thomas J. Kipps, MD, PhD, Evelyn and Edwin Tasch Chair in Cancer Research and senior author of the study. “Instead, it seems that over time the leukemia cells of patients with indolent disease begin to use genes similar to those that are generally used by CLL cells of patients with aggressive disease. In other words, prior to requiring therapy, the patterns of genes expressed by CLL cells appear to converge, regardless of whether or not the patient had aggressive versus indolent disease at diagnosis.”
Existing markers for aggressive or indolent disease are mostly fixed and have declining predictive value the longer the patient is from his or her initial diagnosis. When the blood sample is collected, these markers cannot reliably predict whether a CLL patient will need therapy soon, particularly when the patient has had the diagnosis of CLL for many years.
Kipps and colleagues studied thousands of genes, particularly those that code for proteins, in a group of 130 CLL patients with varying risks of disease progression. They identified 38 prognostic subnetworks of interacting genes and proteins that, at the time of sample collection, indicate the relative the aggressiveness of the disease and predict when the patient will require therapy. They confirmed their work using the method on two other, smaller CLL patient cohorts in Germany and Italy.
The subnetworks offer greater predictive value because they are based not on expression levels of individual genes or proteins, but on how they dynamically interact and change over time, influencing the course of the CLL and patient symptoms.
“In a sense, we looked at families rather than individuals,” said Kipps. “If you find in an interconnected family where most genes or proteins are expressed at higher levels, it becomes more likely that these genes and proteins have functional significance.”
Genetic Test May Reduce Need for Repeat Biopsy for Prostate Cancer
Karim Kader, MD, PhD, associate clinical professor at the UC San Diego School of Medicine, together with a team of researchers from Wake Forest University School of Medicine, have developed a genetic test to predict a man’s risk for prostate cancer. Use of the test could reduce the need for repeat biopsies in men who have had a negative biopsy. Results of the multicenter study were recently published online in the journal of European Urology.
“The genetic test outperformed the PSA test in assessing cancer risk,” said Kader, co-investigator and urologic surgeon at UC San Diego Health System. “If results of this blood test were factored into prostate cancer predictors such as total free PSA, free PSA, number of core samples taken at biopsy, and family history, we would have a more accurate picture of a whether or not a man is likely to develop the sometimes fatal disease.”
Kader and researchers evaluated 1,654 men in the Reduction by Dutasteride of Prostate Cancer Events (REDUCE) clinical trial. All the men had biopsies and consented to genetic studies that looked for the presence of germline single nucleotide polymorphisms (SNPs). SNPs are genetic variations within an individual’s DNA sequence which may have a positive association with prostate cancer risk as well as other chronic diseases.
“Avoiding repeat procedures, particularly in older men, can help reduce the risk of infection and potential hospitalizations,” said Kader. “The genetic score is available at any time in a man’s lifetime and could be used as a pre-screening test thus leaving aggressive PSA screening to men at a higher genetic risk.”
Beyond Base-Pairs: Mapping the Functional Genome
Regulatory sequences of mouse genome sequenced for first time
Popularly dubbed “the book of life,” the human genome is extraordinarily difficult to read. But without full knowledge of its grammar and syntax, the genome’s 2.9 billion base-pairs of adenine and thymine, cytosine and guanine provide limited insights into humanity’s underlying genetics.
In a paper published in the July 1, 2012 issue of the journal Nature, researchers at the Ludwig Institute for Cancer Research and the University of California, San Diego School of Medicine open the book further, mapping for the first time a significant portion of the functional sequences of the mouse genome, the most widely used mammalian model organism in biomedical research.
“We’ve known the precise alphabet of the human genome for more than a decade, but not necessarily how those letters make meaningful words, paragraphs or life,” said Bing Ren, PhD, head of the Laboratory of Gene Regulation at the Ludwig Institute for Cancer Research at UC San Diego. “We know, for example, that only one to two percent of the functional genome codes for proteins, but that there are highly conserved regions in the genome outside of protein-coding that affect genes and disease development. It’s clear these regions do something or they would have changed or disappeared.”
Chief among those regions are cis-regulatory elements, key stretches of DNA that appear to regulate the transcription of genes. Misregulation of genes can result in diseases like cancer. Using high-throughput sequencing technologies, Ren and colleagues mapped nearly 300,000 mouse cis-regulatory elements in 19 different types of tissue and cell. The unprecedented work provided a functional annotation of nearly 11 percent of the mouse genome, and more than 70 percent of the conserved, non-coding sequences shared with other mammalian species, including humans.
As expected, the researchers identified different sequences that promote or start gene activity, enhance its activity and define where it occurs in the body during development. More surprising, said Ren, was that the structural organization of the cis-regulatory elements are grouped into discrete clusters corresponding to spatial domains. “It’s a case of form following function,” he said. “It makes sense.”
While the research is fundamentally revealing, Ren noted it is also just a beginning, a partial picture of the functional genome. Additional studies will be needed in other types of cells and at different stages of development.
“We’ve mapped and understand 11 percent of the genome,” said Ren. “There’s still a long way to march.”
