News in Science (7 Jul 10)

Scientists Watch as Bacteria Evolve Heat Resistance Under Stress: New Details of Evolutionary Mechanism

Scientists in Munich report evidence that high concentrations of the molecular "chaperone" proteins GroEL and GroES -- intracellular machines that can stabilize folding proteins under stress -- play a critical role in increasing the maximum temperature at which E. coli bacteria can grow. Massively and permanently elevated levels of the GroE proteins were found in bacteria adapted, step-wise over a period of years, for growth at 48.5 degrees C. This genomic change persisted for more than 600 generations, and molecular analyses ruled out other mechanisms that might account for the increase in heat resistance.

The researchers' findings, published in the June 18 edition of the Journal of Biological Chemistry, have important implications for both fundamental evolutionary studies and biotechnology applications.

In addition to being a well established resident of the human digestive tract, Escherichia coli is at home in the lab. It is a model organism as important to biological research as brewer's yeast, the fruit fly, and the mouse. Having evolved for life at our body temperature of around 37 degrees C., wild-type E. coli can be cultivated in the laboratory at temperatures up to but not beyond 44 to 46 degrees C. Pushing the upper temperature at which E. coli could grow to 48.5 degrees C. does not approach the level of heat resistance found in thermophile species, but what this experiment required was a distinct and significant redefinition of "extreme" for E. coli.

To achieve that, Dr. Jeannette Winter and colleagues at the Technische Universitaet Muenchen (TUM) founded three lines of E. coli bacteria from a common ancestor and propagated them under heat stress for hundreds of generations. The step-wise process they designed created conditions under which a combination of normal genomic instability and natural selection would be likely to produce adaptations for growth at extreme temperatures. It took around two years to reach 48.5 degrees C, but after that, adaptation to this new extreme was inherited. The researchers propagated a control population, also descended from the common ancestor, at 37 degrees C.

Exhaustive analysis of cell physiology, protein expression, and genome sequences revealed a number of significant changes. Compared with the control group, the adapted lines of E. coli showed a clearly enhanced capability for living under conditions of heat stress. This was accompanied by reduced growth rates, showing that survival came at a cost in terms of overall fitness, a typical indicator of genomic mutations. One of the most striking changes measured was a 16-fold increase in GroE levels, more than five times what a normal heat shock response in E. coli would be expected to produce. Further analyses of heat shock genes and proteins ruled out other mechanisms -- beyond the role of GroE alone -- as being critical for evolution for life at 48.5 degrees C.

GroE chaperones are known to play an active role in assisting the folding process of other proteins, especially in cases where mutations that could cause improper folding threaten the survival of the cell. This experiment shows that they likely play a uniquely important role -- by mitigating the potentially damaging effects of accumulating mutations on protein folding -- in the evolution of heat resistance in E. coli.

"The correlation between genetic changes and chaperones has been shown not only in bacteria, but also in eukaryotes such as yeast, fruit flies, and fungi," says Dr. Jeannette Winter, a researcher in the TUM Department of Chemistry and a member of the Center for Integrated Protein Science Munich. Beyond yielding insights into evolutionary history, Winter says, further research on these highly conserved mechanisms could shed light on how organisms evolve in response to climate-related stresses in the future. "Better understanding of chaperones might also open the way to targeted generation of organisms for specific purposes -- enhancing their ability, for example, to live under stressful conditions, to break down harmful pollutants, or to produce specific, biotechnologically relevant proteins."

This research was supported by the Emmy-Noether program of the German Research Foundation (DFG), the Elitenetzwerk Bayern, the Fonds der chemischen Industrie, and SFB 594.

Tibetan Adaptation to High Altitude Occurred in Less Than 3,000 Years

Tibetans have mutations in numerous genes related to how the body uses oxygen. A comparison of the genomes of 50 Tibetans and 40 Han Chinese shows that ethnic Tibetans split off from the Han less than 3,000 years ago and since then rapidly evolved a unique ability to thrive at high altitudes and low oxygen levels.

The genome-wide comparison, performed by evolutionary biologists at the University of California, Berkeley, uncovered more than 30 genes with DNA mutations that have become more prevalent in Tibetans than Han Chinese, nearly half of which are related to how the body uses oxygen. One mutation in particular spread from fewer than 10 percent of the Han Chinese to nearly 90 percent of all Tibetans.

"This is the fastest genetic change ever observed in humans," said Rasmus Nielsen, UC Berkeley professor of integrative biology, who led the statistical analysis. "For such a very strong change, a lot of people would have had to die simply due to the fact that they had the wrong version of a gene."

The widespread mutation in Tibetans is near a gene called EPAS1, a so-called "super athlete gene" identified several years ago and named because some variants of the gene are associated with improved athletic performance, Nielsen said. The gene codes for a protein involved in sensing oxygen levels and perhaps balancing aerobic and anaerobic metabolism.

The new findings could steer scientists to till-now unknown genes that play a role in how the body deals with decreased oxygen, and perhaps explain some diseases, including schizophrenia and epilepsy, associated with oxygen deprivation in the womb, he said.

Nielsen and his colleagues in China and Europe report their findings in the July 2 issue of the journal Science.

Nielsen, a computational evolutionary biologist, mines genomic information to discover genetic changes driven by natural selection as humans and animals have adapted to new environments. Changes in the frequency of DNA mutations are one clue.

"You look for rapid evolution in genes because there must be something important about that gene forcing it to change so fast," he said. "The new finding is really the first time evolutionary information alone has helped us pinpoint an important function of a gene in humans."

Adaptation to low oxygen levels has allowed many peoples, from Andeans to Tibetans, to live at high altitude. When people from lower elevations move above about 13,000 feet, where oxygen levels are about 40 percent lower than at sea level, they typically tire easily, develop headaches, produce babies with lower birth weights and have a higher infant mortality rate. Tibetans have none of these problems, despite lower oxygen saturation in the blood and lower hemoglobin levels. Hemoglobin, which gives blood its red color, binds and transports oxygen to the body's tissues.

Nielsen used genome data produced by the Beijing Genomics Institute (BGI) in Shenzhen, China's flagship genome center, to tease out the genetic changes associated with these physiological changes.

"We're looking for footprints of past selection to find something functional in our genome," Nielsen said

BGI researchers obtained DNA from 50 Tibetans living in the Tibet Autonomous Region of China and 40 Han Chinese from Beijing. The Tibetans lived in two villages located at elevations of 4,300 meters (14,100 feet) and 4,600 meters (15,100 feet). All reported at least three generations of ancestors had lived at the same site. After obtaining informed consent, the Chinese researchers took blood samples from the participants and measured oxygen saturation, red blood cell concentration and hemoglobin content in their blood.

Back in the lab, the BGI team isolated only the active genes, or exons, from each individual, then used next-generation sequencing technology to sequence these so-called exomes. This involved cutting the DNA into many short pieces, sequencing each about 18 times with state-of-the-art Illumina sequencing machines, and then using overlaps to help reassemble the complete genome of each person. That work was directed by Jun Wang of BGI and the University of Copenhagen in Denmark.

Nielsen and post-doctoral fellows John E. Pool, Emilia-Huerta Sanchez and Nicolas Vinckenbosch conducted the analysis at UC Berkeley, locating all point mutations, called single-nucleotide polymorphisms (SNPs), in the 90 genomes and then comparing Tibetan and Han separately to a control group of 100 Europeans (Danes).

The analysis revealed that the common ancestors of Tibetans and Han Chinese split into two populations about 2,750 years ago, with the larger group moving to the Tibetan plateau. That group eventually shrank, while the low-elevation Han population expanded dramatically. Today, the Han Chinese are the dominant ethnic group in mainland China. The Tibetan branch either merged with the people's already occupying the Tibetan plateau, or replaced them.

"We can't distinguish intermixing and replacement," Nielsen said. "The Han Chinese and Tibetans are as different from one another as if the Han completely replaced the Tibetans about 3,000 years ago."

The Tibetan and Han Chinese genomes are essentially identical in terms of the frequency of polymorphisms in the roughly 20,000 genes, though some 30 genes stood out because of dramatic differences between the Tibetans and the Han.

"We made a list of the genes that changed the most," Nielsen said, "and what was fascinating was that, bing!, at the top of that list was a gene that had changed very strongly, and it was related to the response to oxygen."

The SNP with the most dramatic change in frequency, from 9 percent in Han Chinese to 87 percent in Tibetans, was associated with lower red blood cell count and lower hemoglobin levels in Tibetans. That variation occurred near a gene called EPAS1, which earlier studies suggest is involved in regulating hemoglobin in the blood as a response to oxygen levels. The mutation may be in a transcription factor that regulates the activity of EPAS1.

Tibetans carrying only one allele with this mutation had about the same hemoglobin concentration as Han Chinese, but those with two mutated alleles had significantly lower hemoglobin concentration. However, they all have about the same oxygen concentration in the blood. For some reason, individuals with two copies of the mutation function well in high altitude with relatively low hemoglobin concentration in their blood. The mutation seems to provide an alternative inborn mechanism for dealing with the low oxygen levels, Nielsen said.

Other strongly selected variants were near the genes for the fetal and adult versions of the globin genes, which produce the structural proteins of hemoglobin.

Two other genes showing a dramatic shift in frequency have been linked to anemia, while several other genes have been linked to diseases, including schizophrenia and epilepsy, possibly caused by low oxygen levels in the womb.

A large team of researchers from BGI contributed to the study, some of whom are also associated with the Graduate University of the Chinese Academy of Sciences.

The research was funded by various Chinese, American and Danish organizations, including the U.S. National Institutes of Health and the National Science Foundation. The sequenced genomes were part of the international 1000 Genomes Project, which now aims to sequence 2,500 human genomes by the end of 2011.

World's Largest DNA Scan Reveals Rare Variants That Disrupt Gene Activity in Autistic Children

The world's largest DNA scan for familial autism has uncovered new genetic changes in autistic children that are often not present in their parents. Identified in less than 1 percent of the population, these rare variants occur nearly 20 percent more in autistic children.

Published in the June 9 online edition of Nature, the findings emphasize the need for larger study samples to illuminate the diverse genetic causes of the brain disorder.

UCLA researchers from the David Geffen School of Medicine and Semel Institute for Neuroscience and Human Behavior were among the lead investigators of the three-year study by the Autism Genome Project, an international consortium of scientists from more than 60 institutions in 12 countries.

"We know that 10 million gene variants consistently exist in every individual's genome," explained Rita Cantor, UCLA professor of human genetics. "We used DNA chips to collect and analyze data on 1 million of these variations to shed light on how autism develops."

Using blood samples from 996 elementary school-age children diagnosed on the autism spectrum from the United States, Canada, and Europe, the scientific teams combed the children's DNA for rare deletions and duplications. In particular, they hunted for changes in the genetic information that a child inherits from each parent. The families consisted of parents with one autistic child.

"We discovered two striking things. First, the rare variants interfered nearly 20 percent more in the genes of autistic children than in the healthy children," said Dr. Daniel Geschwind, Gordon and Virginia MacDonald Distinguished Chair in Human Genetics and UCLA professor of neurology and psychiatry. "Second, we found a number of disruptions that are new, or de novo. The autistic child is the first in their family to carry that variant. The parents do not have it.

"This suggests that tiny genetic errors may occur during formation of the parents' eggs and sperm, and these variations are copied during creation of their child's DNA," added Geschwind, who is also director of the UCLA Center for Autism Research and Treatment. "The finding parallels what takes place in chromosomal disorders like Down's syndrome."

The study confirms earlier findings in smaller samples that some children carry private genetic mutations that are unique to them, contributing to their susceptibility to autism.

"We found many more disrupted genes in the autistic children than in the control group," said Dr. Stanley Nelson, UCLA professor of human genetics and psychiatry. "But here's where it gets tricky -- every child showed a different disturbance in a different gene. When we looked at the gene's function, however, certain categories of genes emerged that were more likely to be influenced by the mutation.

"Three of the disrupted genes, for example, participate in cellular communication," Nelson explained. "They all cluster at the synapse, the site where brain cells talk to each other. One of these genes has previously been tied to autism and intellectual disabilities."

The researchers' next step will be to uncover patterns by identifying groups of disrupted genes that work together in the body to establish key functions or biological processes. The results may reveal clues to where genes go awry and increase autism risk, offering hope for common treatments.

In the meantime, families affected by autism can help advance research efforts by participating in future genetic studies.

"This study's larger sample size enabled us to pinpoint rare variations that we could not have detected in a smaller group," emphasized Nelson. "Yet these findings explain only 3.3 percent of the genetic origins of autism. In order to identify all of autism's genetic causes, we need tens of thousands of families to volunteer their DNA samples for sequencing."

Many of the study's samples were provided by families who donated blood to the Los Angeles-based Autism Genetic Resource Exchange (AGRE), a gene bank created and funded by Cure Autism Now, (since renamed Autism Speaks), and a grant from the National Institute of Mental Health to the three UCLA authors.

The consortium's research was funded by Autism Speaks (USA), the Hilibrand Foundation (USA), Health Research Board (Ireland), The Medical Research Council (UK) and the Genome Canada/Ontario Genomics Institute. Additional support for individual groups was provided by the U.S. National Institutes of Health and equivalent federal agencies in Canada, France, Italy, United Kingdom, Germany, Portugal and Sweden.

About Autism

Autism is a complex brain disorder that strikes in early childhood. The condition disrupts a child's ability to communicate and develop social relationships and is often accompanied by acute behavioral challenges. Autism spectrum disorders are diagnosed in one in 110 children in the United States, affecting four times as many boys as girls. Diagnoses have expanded tenfold in the last decade.

Polio Outbreak in Tajikistan Is Cause for Alarm

The rapidly growing polio outbreak in Tajikistan raises serious concerns that the disease could spread to other regions in the world, states an editorial in CMAJ (Canadian Medical Association Journal). It is imperative that health agencies attempt to limit further spread by ensuring high vaccination rates.

Polio is a serious disease that can cause paralysis and death in both children and adults. However, vaccines had largely eradicated the disease, until vaccination rates dipped below the minimum 90% coverage mark recommended by the WHO. There is no cure for polio -- prevention with vaccines is the only defense.

This is the first persistent outbreak of polio in a country that was previously certified to be polio-free. The outbreak represents 75% of the world's polio cases and far exceeds that of India and Nigeria, which are usually the sources of polio outbreaks.

"Too many regions and communities have ceased to worry about polio," writes Dr. Paul Hébert, Editor-in-Chief, CMAJ with Dr. Noni MacDonald, Public Health Editor. "As a consequence, vaccine uptake rates are all too often well below effective prevention levels."

Countries such as the Ukraine and Georgia are below the 90% target, and regions within Canada and some European countries have low community uptake rates. In Ontario, for example, childhood immunization rates are only in the high 70% to low 80% range, comparable to rates in Tajikistan. Concerns about vaccine safety, anti-government views and religious strictures against vaccinations have contributed to this lower uptake.

Individuals without polio symptoms started the Tajikistan outbreak with cases now appearing in Russia and Uzbekistan. Global travel can hasten the spread of the disease.

Urgent action is needed beyond the mass polio vaccine campaigns now underway in Tajikistan. The WHO must be more proactive in raising awareness of the issue beyond posting the growing numbers on its Global Polio Eradication website. The Public Health Agency of Canada must issue immediate guidance on the importance of polio vaccination, and the provinces and territories need to ramp up programs to improve vaccination uptake rates.

"The threat of polio is no longer simply theoretical," conclude the authors. "We are only one asymptomatic traveller away from an outbreak because of low vaccination rates."

Combination MMRV Vaccine Linked With Two-Fold Risk of Seizures, Study Finds

The combination vaccine for measles, mumps, rubella and chickenpox (MMRV) is associated with double the risk of febrile seizures for 1- to 2-year-old children compared with same-day administration of the separate vaccine for MMR (measles, mumps, rubella) and the varicella (V) vaccine for chicken pox, according to a Kaiser Permanente Division of Research study appearing online in the journal Pediatrics. A febrile seizure is a brief, fever-related convulsion but it does not lead to epilepsy or seizure disorders, researchers explained.

Funded by the U.S. Centers for Disease Control, the study analyzed 459,000 children 12 to 23 months old from numerous health systems across the United States receiving their first dose of measles-containing vaccine and found MMRV to be associated with a two-fold increased risk of fever and febrile seizures 7-10 days after vaccination compared with same-day administration of a separate shot for MMR and the varicella (chickenpox) vaccine. This study found that the risk for a febrile seizure after the first dose of MMRV vaccine is low, although it is higher than after MMR vaccine and varicella vaccine administered as separate injections.

The study found no evidence of an increased febrile seizure risk after any measles vaccine beyond 7-10 days post vaccination.

"Because the risk of febrile seizure is higher for the quadrivalent (combination) vaccine, providers recommending MMRV should communicate to parents that it increases the risk of fever and febrile seizure over that already associated with measles-containing vaccines," said the study's lead investigator Nicola Klein, MD, Ph.D., co-director of the Kaiser Permanente Vaccine Study Center. "But concerned parents should understand that the risk for febrile seizures after any measles-containing vaccine is low: less than 1 febrile seizure per 1,000 injections."

The CDC recently recommended that either vaccine may be used for first dose for 1-2 year olds, however families without a strong preference for MMRV should receive separate MMR +V vaccines, Klein said. The CDC reiterates that providers who consider using MMRV should discuss with families and caregivers the risk and benefits.

"While this study and the resulting CDC recommendations are very important and ones our pediatricians will follow, it is also important to emphasize that it is more common for a child to have a febrile seizure caused by a simple cold than by an immunization. And though febrile seizures are a very scary event for a family, they are not dangerous and do not lead to later epilepsy or seizure disorders," said Randy Bergen, MD, a Kaiser Permanente pediatrician and infectious disease specialist at Kaiser Permanente-Walnut Creek.

Kaiser Permanente researchers used its electronic health records and Vaccine Safety Datalink data from 2000 to 2008 to assess seizures and fever visits among children aged 12-23 months following MMRV and separate MMR +V. They compared seizure risk following MMRV to MMR +V using regression analyses and by incorporating chart-reviewed febrile seizure cases.

The Vaccine Safety Datalink project is a collaborative effort between CDC's Immunization Safety Office and eight managed care organizations: Kaiser Permanente Northern California, Kaiser Permanente Southern California, Kaiser Permanente Colorado, Kaiser Permanente Northwest, Health Partners, Group Health Cooperative, Marshfield Clinic and Harvard Pilgrim Health Care. The VSD project was established in 1990 to monitor immunization safety and address the gaps in scientific knowledge about rare and serious events following immunization. The VSD shares electronic health records from the organizations' health systems.

MMRV was licensed by the FDA in 2005. MMRV was subsequently recommended by the Advisory Committee on Immunization Practices (ACIP) in 2006. Although prelicensure studies of MMRV among 1-2 year olds noted higher rates of fever and measles-like rash one to two weeks post vaccination when compared with separate MMR + V, it was unknown at the time of MMRV's licensure whether a higher rate of fevers was similarly associated with increased risk of febrile seizures. In February 2008, Kaiser Permanente researchers alerted the ACIP to preliminary evidence of an increased risk of febrile seizures following MMRV. This study represents additional data on twice as many vaccines.

"The Vaccine Safety Datalink, which we used to conduct this study, is a premiere example of how different managed care organizations can leverage their electronic medical records to improve vaccine safety and monitoring," Klein said.

This is the latest in a series of Kaiser Permanente studies undertaken to better understand the protective effects and risks of vaccines. Recent published studies found children of parents who refuse vaccines are nine times more likely to get chickenpox and 23 times more likely to get whooping cough compared to fully immunized children. A study published last year found that herpes zoster, also known as shingles, is very rare among children who have been vaccinated against chicken pox. A recent study in the Journal of the American Medical Association found that the pneumococcal vaccination is not associated with a reduced risk of heart attacks or strokes in middle-aged men.

Additional authors on this paper include: Bruce Fireman, MS, from the Kaiser Permanete Division of Research; Katherine Yih, MPH, PhD, from Harvard Pilgrim HealthCare Institute and Harvard Medical School, Boston, MA; Edwin Lewis, MPH, from the Kaiser Permanente Vaccine Study Center; Martin Kulldorff, PhD, from Harvard Pilgrim Healthcare Institute; Paula Ray, MPH, from the Kaiser Permanente Vaccine Study Center; Roger Baxter, MD, from the Kaiser Permanente Vaccine Study Center; Simon Hambridge, MD, PhD, from Kaiser Permanente, Denver, Co; James Nordin, MD, MPA, from Health Partners Research Foundation, Minneapolis, MN; Allison Naleway, PhD, Kaiser Permanente, Portland, OR; Edward A. Belongia, MD, from Marshfield Clinic Research Foundation; Tracy Lieu, MD, MPH, from Harvard Pilgrim Health Care Institute; James Baggs, PhD, from the Immunization Safety Office, CDC; Eric Weintraub, MPH, from the Vaccine Safety Datalink. Funding for the study was provided by the Vaccine Safety Datalink contract with America's Health Insurance Plans, funded by the Centers for Disease Control and Prevention.