New genetic clues found in fragile X syndrome

Research by Vitaly Klyachko, PhD, and colleagues has shed new light on brain dysfunctions associated with fragile X syndrome. Credit: Robert Boston

Scientists have gained new insight into fragile X syndrome -- the most common cause of inherited intellectual disability -- by studying the case of a person without the disorder, but with two of its classic symptoms.

In patients with fragile X, a key gene is completely disabled, eliminating a protein that regulates electrical signals in the brain and causing a host of behavioral, neurological and physical symptoms. This patient, in contrast, had only a single error in this gene and exhibited only two classic traits of fragile X -- intellectual disability and seizures -- allowing the researchers to parse out a previously unknown role for the gene.

"This individual case has allowed us to separate two independent functions of the fragile X protein in the brain," said co-senior author Vitaly A. Klyachko, PhD, associate professor of cell biology and physiology at Washington University School of Medicine in St. Louis. "By finding the mutation, even in just one patient, and linking it to a partial set of traits, we have identified a distinct function that this gene is responsible for and that is likely impaired in all people with fragile X."

The research, appearing in the Proceedings of the National Academy of Sciences (PNAS) Online Early Edition in December and in the print issue Jan. 5, is by investigators at Washington University and Emory University School of Medicine in Atlanta.

In studying fragile X, researchers' focus long has been on the problems that occur when brain cells receive signals. Like radio transmitters and receivers, brain cells send and receive transmissions in fine tuned ways that separate the signals from the noise. Until recently, most fragile X research has focused on problems with overly sensitive receivers, those that allow in too much information. The new study suggests that fragile X likely also causes overactive transmitters that send out too much information.

"The mechanisms that researchers have long thought were the entirety of the problem with fragile X are obviously still very much in play," Klyachko said. "But this unique case has allowed us to see that something else is going on."

The finding also raises the possibility that drugs recently tested as treatments for fragile X may be ineffective, at least in part, because they only dialed down the brain's receivers, presumably leaving transmitters on overdrive.

Fragile X syndrome results from an inherited genetic error in a gene called FMR1. The error prevents the manufacture of a protein called FMRP. Loss of FMRP is known to affect how cells in the brain receive signals, dialing up the amount of information allowed in. The gene is on the X chromosome, so the syndrome affects males more often and more severely than females, who may be able to compensate for the genetic error if their second copy of FMR1 is normal.

Patients with fragile X have a range of symptoms. One of the mysteries of the syndrome is how loss of a single gene can lead to such a variety of effects in different patients. Some patients are profoundly intellectually disabled, unable to talk or communicate. Others are only mildly affected. Patients often experience seizures, anxiety and impulsive behavior. Typical physical symptoms include enlarged heads, flat feet and distinctive facial features. 

Almost one-third of patients with fragile X also show symptoms of autism spectrum disorders.

To gain insight into what else FMRP might do, the researchers plumbed genetic sequencing data from more than 900 males with intellectual disabilities but without classic fragile X syndrome. They looked for mutations in the FMR1 gene that might impair the protein but not eliminate it entirely. Even in this relatively large sample size, they only found one patient with abnormal FMRP, resulting from a change in a single letter of the gene's DNA code.

Importantly, although this individual has intellectual disability and seizures, his physical features are not typical of the syndrome, and he is not autistic.

To see what effect this mutation might have, geneticist Stephen T. Warren, PhD, and his team at Emory replicated it in mouse brain cells and tested it for the widely known functions of FMRP. To their surprise, this mutated FMRP appeared to work normally. In other words, the patient's brain cells had entirely normal receivers, which appeared to work in ways that were indistinguishable from those in healthy people.

"This single point mutation does not seem to affect the classical, well-known functions of FMRP," said Klyachko, also an associate professor of biomedical engineering. "This patient presents a case of partial fragile X syndrome associated with mutated, rather than absent, FMRP. As far as I know, this is the only known case of this. It's a unique opportunity to parse out the functions of FMRP. What does this mutation impair to cause only two symptoms of fragile X?"

To find out, Warren and his team replicated the mutation in fruit flies.

Surprisingly, according to the researchers, the fruit fly studies indicated that this single mutation increased the number of transmitters in brain cells, implicating a fundamental problem in which the brain's cells send out too many signals.

To verify this mechanism in mammals, they turned to Klyachko's lab at Washington University, which has expertise in understanding how brain cells regulate the sending of electrical signals. Indeed, in past work Klyachko showed that total loss of FMRP in mice disrupts the normal process by which brain cells send signals, causing transmitters to send out too much information. In the new study, they were able to verify the same effect from just the mutation and link it to human disease. This single mutation in FMRP has the same overactivating effect on transmissions as the total loss of the protein.

The researchers said they can't rule out the possibility that additional problems also are caused by this mutation and are present in fragile X. But this research specifies at least one additional dysfunction not previously recognized. Further studies of patients with different partial symptoms of fragile X and different mutations -- if any can be found -- might identify more.

Source: Washington University School of Medicine

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'Microlesions' in epilepsy discovered by novel technique

Clusters of differentially expressed genes predict cellular abnormalities. Credit: Jeffrey Loeb

Using an innovative technique combining genetic analysis and mathematical modeling with some basic sleuthing, researchers have identified previously undescribed microlesions in brain tissue from epileptic patients. The millimeter-sized abnormalities may explain why areas of the brain that appear normal can produce severe seizures in many children and adults with epilepsy.

The findings, by researchers at the University of Illinois at Chicago College of Medicine, Wayne State University and Montana State University, are reported in the journal Brain.

Epilepsy affects about 1 percent of people worldwide. Its hallmark is unpredictable seizures that occur when groups of neurons in the brain abnormally fire in unison. Sometimes epilepsy can be traced back to visible abnormalities in the brain where seizures start, but in many cases, there are no clear abnormalities or scaring that would account for the epileptic activity.

"Understanding what is wrong in human brain tissues that produce seizures is critical for the development of new treatments because roughly one third of patients with epilepsy don't respond to our currently available medications," said Dr. Jeffrey Loeb, professor and head of neurology and rehabilitation in the UIC College of Medicine and corresponding author on the study. "Knowing these microlesions exist is as huge step forward in our understanding of human epilepsy and present new targets for treating this disease."

Loeb and colleagues searched for cellular changes associated with epilepsy by analyzing thousands genes in tissues from 15 patients who underwent surgery to treat their epilepsy. They used a mathematical modeling technique called cluster analysis to sort through huge amounts of genetic data.

Using the model, they were able to predict and then confirm the presence of tiny regions of cellular abnormalities -- the microlesions -- in human brain tissue with high levels of epileptic electrical activity, or 'high-spiking' areas where seizures begin.

"Using cluster analysis is like using a metal detector to find a needle in a haystack," said Loeb. The model, he said, revealed 11 gene clusters that "jumped right out at us" and were either up-regulated or down-regulated in tissue with high levels of epileptic electrical activity compared to tissue with less epileptic activity from the same patient.

When they matched the genes to the types of cells they came from, the results predicted that there would be reductions of certain types of neurons and increases in blood vessels and inflammatory cells in brain tissue with high epileptic activity.

When Fabien Dachet, an expert in bioinformatics research at UIC and first author of the study, went back to the tissue samples and stained for these cells, he found that all of the prediction were correct- there was a marked increase in blood vessels, inflammatory cells, and there were focal microlesions made up of neurons that had lost most of their normal connections that allow them to communicate with one another. "We think that these newly found microlesions lead to spontaneous, abnormal electrical currents in the brain that lead to epileptic seizures," said Loeb.

Loeb and his colleagues at UIC are using the same approach to look for the clusters of differentially expressed genes associated with ALS, a neurodegenerative disease, and in brain tumors. "We now have a way to predict cellular changes by simply measuring the genetic composition, with some fairly simple calculations, between more- and less-affected epileptic human tissues," explained Loeb.

"This technique gives us the ability to discover previously unknown cellular abnormalities in almost any disease where we have access to human tissues," Loeb said. He is currently developing at UIC a national 'neurorepository' of electrically mapped and genetically analyzed brain tissue for such studies.

Source: University of Illinois at Chicago

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'Darwinian' test uncovers an antidepressant's hidden toxicity

A University of Utah test detects hidden toxic effects by subjecting mice to competition for resources. Biology professor Wayne K. Potts and biologist Shannon M. Gaukler, who recently completed a doctoral degree at the U, stand in front of the test enclosure, illuminated in red light that mice perceive as nightfall. Credit: Andy Brimhall / University of Utah

Because of undetected toxicity problems, about a third of prescription drugs approved in the U.S. are withdrawn from the market or require added warning labels limiting their use. An exceptionally sensitive toxicity test invented at the University of Utah could make it possible to uncover more of these dangerous side effects early in pharmaceutical development so that fewer patients are given unsafe drugs.

To prove the point, the U researchers ran their test on Paxil, an antidepressant that thousands of pregnant women used in the years before it was linked to an increased risk of birth defects. The U.S. Food and Drug Administration now requires a warning about use in the first trimester of pregnancy. In the U study, mice exposed during development experienced multiple problems: males weighed less, had fewer offspring, dominated fewer territories and died at a higher rate. Females took longer to produce their first litters, had fewer pups and pups that were underweight. The drug doses were relatively close to those prescribed for people. In the conventional animal safety testing reported by the drug's manufacturer, no reproductive side effects emerged until rodents took doses multiple times higher than those given to treat depression.

"We are seeing effects at a dose that is close to human levels. And we are doing it exactly the way we need to determine if it presents a risk of harm to a developing fetus," says University of Utah biologist Shannon M. Gaukler, the study's lead author who recently completed a doctoral degree at the U. The study will be published in the January-February issue of Neurotoxicology and Teratology, which has posted a preprint online.

University of Utah biology professor Wayne K. Potts, the study's senior author, says that detecting toxicity problems early in preclinical testing would not only protect patients from exposure to unsafe drugs, but also help pharmaceutical companies avoid wasting billions of dollars bringing drugs to market only to have them fail.

"If we can find these health problems early on in preclinical testing, it has the potential of saving them a lot of money," Potts says.

Survival-of-the-fittest test

The key to the test's sensitivity is the way it uses untamed house mice -- rather than docile, inbred laboratory strains -- and subjects them to a relentless, Darwinian competition for food, shelter and mates much like they would face in the wild. Mice jostle and race for a place in a roughly 300-square-foot pen divided into six territories by wire fencing that individuals must climb to invade or flee neighboring turf. Four of the territories are prime real estate with multiple hidden nesting sites and direct access to feeders. Two territories are poor, offering only open nesting sites and indirect feeder access. The test is called the organismal performance assay, or OPA.

Potts first came up with the idea as a way to explore the impact of inbreeding. Those studies revealed harmful effects of cousin-level inbreeding that had gone unnoticed for decades of research on mouse genetics. Laboratory mice that are only slightly less healthy may not appear so when given ample food and living space. But if there is a defect in any physiological system, it is likely to stand out during intense competition.

"When they really have to compete directly, males are constantly testing each other and fighting over territories," Potts says. "If they don't win a territory, females won't consider them when it comes time to choose a mate."

In a study published last year, the performance assay revealed that doses of sugar that people regularly consume -- and deemed safe by regulators -- may in fact be toxic. When mice ate a diet of 25 percent extra sugar (the mouse equivalent of drinking three cans of soda daily) females died at twice the normal rate and males were a quarter less likely to hold territory and reproduce.

Testing Paxil

In the Paxil study, the researchers gave food laced with the antidepressant to 20 breeding pairs of mice for several weeks, until all had produced up to four litters. Doses were equivalent to about 1.8 times the level typically prescribed for people. The offspring also ate Paxil-laced chow until they reached breeding age. The researchers then released the exposed offspring into the competitive arena with the offspring of a control group of mice never exposed to Paxil. Groups consisted of eight males and 14 to 16 females, creating population densities comparable to those seen in the wild. The researchers started five such populations and kept them going for six months.

Males exposed to Paxil were about half as likely to control a territory. They also lagged behind control males in body weight throughout the weeks of competition and were more likely to die. Exposed males produced 44 percent fewer offspring. Exposed females showed no significant weight or mortality differences, but they produced half as many offspring as control females at the initial assessment. Their fecundity rebounded at later time points.

Danger signals

The test can provide an early warning of possible toxic effects, but it does not identify causal pathways. In the case of Paxil, the test might have alerted the drug's developers to the risk of birth defects when taken during pregnancy at prescribed doses, not just at the high levels used in conventional safety testing. Other selective serotonin reuptake inhibitors, or SSRIs, don't appear to pose the same level of risk as Paxil, but their safety for use during pregnancy remains unclear.

"It's unknown how Paxil causes birth defects and why Paxil has a stronger correlation with birth defects than other SSRIs," Gaukler says. "We think most of our results are driven by endocrine disruption."

Previous studies have shown that Paxil can lower the level of several reproductive hormones in female rats. In male rats, doses comparable to those prescribed for people can lower testosterone and boost estradiol. Other animal studies have shown that Paxil can lower sperm count and degrade sperm quality.

Potts says the performance assay may be even more important for identifying the toxic effects of agricultural chemicals, industrial pollutants, and other manufactured chemicals released into the environment.

"We don't really have a sensitive, broad toxicity assessment system," Potts says. "That's why these things slip through the cracks and we often don't discover harmful effects until after 10 or 20 years of epidemiology studies using the public as the experimental guinea pigs."

Source:  University of Utah

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Alcohol interferes with body's ability to regulate sleep

At right: Mahesh Thakkar, PhD, associate professor and director of research in the MU School of Medicine’s Department of Neurology, and Pradeep Sahota, MD, chair of the MU School of Medicine’s Department of Neurology, have studied alcohol’s effects on sleep for more than five years. Their study found that drinking interferes with the brain’s built-in system for regulating a person’s need for sleep. Credit: Image courtesy of University of Missouri-Columbia

Researchers from the University of Missouri School of Medicine have found that drinking alcohol to fall asleep interferes with sleep homeostasis, the body's sleep-regulating mechanism.

Alcohol is known to be a powerful somnogen, or sleep inducer, and approximately 20 percent of the U.S. adult population drinks alcohol to help fall asleep. The researchers, led by Mahesh Thakkar, PhD, associate professor and director of research in the MU School of Medicine's Department of Neurology, have studied alcohol's effects on sleep for more than five years. They found that alcohol interferes with the brain's built-in system for regulating a person's need for sleep.

"The prevailing thought was that alcohol promotes sleep by changing a person's circadian rhythm -- the body's built-in 24-hour clock," Thakkar said. "However, we discovered that alcohol actually promotes sleep by affecting a person's sleep homeostasis -- the brain's built-in mechanism that regulates your sleepiness and wakefulness."

Sleep homeostasis balances the body's need for sleep in relation to how long a person has been awake. If an individual loses sleep, the body produces adenosine, a naturally occurring sleep-regulating substance that increases a person's need for sleep. When a person goes to sleep early, sleep homeostasis is shifted and he or she may wake up in the middle of the night or early morning. The researchers found that alcohol alters the sleep homeostatic mechanism and puts pressure on an individual to sleep. When this happens, the sleep period is shifted, and a person may experience disrupted sleep.

"Based on our results, it's clear that alcohol should not be used as a sleep aid," said Pradeep Sahota, MD, chair of the MU School of Medicine's Department of Neurology and an author of the study. "Alcohol disrupts sleep and the quality of sleep is diminished. Additionally, alcohol is a diuretic, which increases your need to go the bathroom and causes you to wake up earlier in the morning."

In addition to studying alcohol's impact on sleep homeostasis, the researchers explored how alcohol withdrawal affects sleep. The investigators found that after extended periods of frequent drinking, subjects would fall asleep as expected, but would wake within a few hours and would be unable to fall back asleep. When the subjects were not given alcohol, the researchers found that subjects showed symptomatic insomnia.

"During acute alcohol withdrawal, subjects displayed a significant increase in wakefulness with a reduction in rapid eye movement and non-rapid eye movement sleep," Thakkar said. "This caused insomnia-like symptoms and suggests an impaired sleep homeostasis."

The researchers hope to use these findings to explore other effects of alcohol consumption.

"Sleep is an immense area of study," Thakkar said. "Approximately one-third of our life is spent sleeping. Coupled with statistics that show 20 percent of people drink alcohol to sleep, it's vital that we understand how the two interact. If you are experiencing difficulty sleeping, don't use alcohol. Talk to your doctor or a sleep medicine physician to determine what factors are keeping you from sleeping. These factors can then be addressed with individualized treatments."

The study, "Alcohol Disrupts Sleep Homeostasis," is an invited article published in the international biomedical journal Alcohol.

Source:  University of Missouri-Columbia

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Fine particulate air pollution linked with increased autism risk

The greater the exposure a pregnant woman has to fine particulate matter, the greater the risk of their baby having autism, researchers found. Credit: © Oleg Shelomentsev / Fotolia

Women exposed to high levels of fine particulate matter specifically during pregnancy--particularly during the third trimester--may face up to twice the risk of having a child with autism than mothers living in areas with low particulate matter, according to a new study from Harvard School of Public Health (HSPH). The greater the exposure, the greater the risk, researchers found. It was the first U.S.-wide study exploring the link between airborne particulate matter and autism.

"Our data add additional important support to the hypothesis that maternal exposure to air pollution contributes to the risk of autism spectrum disorders," said Marc Weisskopf, associate professor of environmental and occupational epidemiology and senior author of the study. "The specificity of our findings for the pregnancy period, and third trimester in particular, rules out many other possible explanations for these findings."

The study appears online December 18, 2014 in Environmental Health Perspectives.

Prior studies have suggested that, in addition to genetics, exposure to airborne environmental contaminants, particularly during pregnancy and early life, may affect risk of autism. This study focused specifically on the pregnancy period.

The study population included offspring of participants living in all 50 states in Nurses' Health Study II, a cohort of more than 116,000 female U.S. nurses begun in 1989. The researchers collected data on where participants lived during their pregnancies as well as data from the U.S. Environmental Protection Agency and other sources on levels of fine particulate matter air pollution (PM2.5)--particles 2.5 microns in diameter or smaller--in locations across the U.S. The researchers identified 245 children who were diagnosed with autism spectrum disorder (ASD) and a control group of 1,522 children without ASD during the time period studied.

The researchers explored the association between autism and exposure to PM2.5 before, during, and after pregnancy. They also calculated exposure to PM2.5 during each pregnancy trimester.

Exposure to PM2.5 was significantly associated with autism during pregnancy, but not before or after, the study found. And during the pregnancy, the third trimester specifically was significantly associated with an increased risk. Little association was found between air pollution from larger-sized particles (PM10-2.5) and autism.

"The evidence base for a role for maternal exposure to air pollution increasing the risk of autism spectrum disorders is becoming quite strong," said Weisskopf. "This not only gives us important insight as we continue to pursue the origins of autism spectrum disorders, but as a modifiable exposure, opens the door to thinking about possible preventative measures."

Source: Harvard School of Public Health

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