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Archive for the ‘Genetics’ Category

Born this way: Stories of young transgender children

Our Cover Story this morning deals with children grappling with a very grown-up issue: gender identity — boys or girls believing they’re the opposite sex, saying they were born this way. Here’s Rita Braver:

She could be any 12-year-old girl, hanging out with her mom and sister, but Zoey was biologically born a boy.

“So how did you handle it when people related to you as a boy?” asked Braver.

CBS News
“Yeah, I always get upset,” she replied. “I would be like, ‘No, I’m not a boy. I’m a girl. You know, like, I like the color pink, I scream like a girl. I act like a girl. I breathe like a girl. I’m not a boy.””
When asked what she felt when she realized that her child whom she knew as a boy felt she was a girl, Zoey’s mother, Ofelia, said her first reaction was fear: “Not because of who she’s presenting to be, but of those around us. What are other people going to say? How are they going to treat her? You know, those are the scary things: What kind of life is she going to have?”

But Ofelia (at their request, we won’t be using last names of the families in this story) felt she had no choice. A single mom, she accepted Zoey’s decision two years ago to live as an openly-transgender girl.

Zoey’s family and her childhood friends in her town near Los Angeles have been supportive. But a survey of nearly 300 transgender youth found that 89 percent reported being harassed in school.

“Harsh Realities: The Experiences of Transgender Youth in Our Nation’s Schools” – Gay, Lesbian & Straight Education Network (pdf)
Zoey, too, has endured cruel treatment from her schoolmates:

“Even the kids that do seem like they’re good kids, they even make fun of me,” said Zoey. “They’ll be, like, ‘Yeah, we’re your friends, tell us more about your stuff and how you’re going through life.’ And then they’ll just turn on you and they’ll be talking about what you told them.”

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Olson is a specialist in the care of transgendered youth, at Children’s Hospital, Los Angeles. She says that her patients have a condition known as “Gender Dysphoria.”

Dr. Olson defined it as “persistent unhappiness, discomfort and distress about the incongruence between the gender that you are assigned, based on your anatomy at birth, versus the way you internally experience gender.”

Estimates on the number of Transgender Americans range as high as 0.5 percent of the adult population — about three-quarters of a million people. But more and more young people are emerging as transgender.

“I see between one and five new trans kids a week,” said Dr. Olson. “So the growth is tremendous. We’ve had something like a 330 percent increase over the year of 2013. It’s just phenomenal.”

“What do you think is happening — are there more transgender children?” asked Braver.

“It’s not so much that there are more transgendered kids; it’s that trans people are coming out earlier,” replied Dr. Olson. .

“We also know that among trans people, there are high rates of depression, anxiety, social isolation, [and] suicide attempts. All of these things we see dramatically increased in trans youth. But young people that I’ve seen, who socially transition in childhood and have support of their familes, they have a very different experience.”

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Now there is new medical treatment for young people like Zoey. Doctors have recently started administering drugs that block puberty, and keep them from developing unwanted adult characteristics, like facial hair for transgender girls, or breasts in transgender boys.

The effects of those drugs can be reversed. But the effects of hormones — which transgender youths can take later, to look more male or female — are often not reversible.

Venice says “No one really would want to be trans.”
CBS News
Dr. Olson says, by then, her patients — like Venice — are not going back:
At 13, Venice has started taking the hormone testosterone, as well as puberty blockers. He says he has always felt like a boy, though he was born, biologically, a girl.

Braver asked, “In terms of making these changes so that you can go through life as a boy, what’s the upside of that for you?”

“The upside is that I’ll actually get to have part of the body I do want.”

But he says being trans has not been easy:

“It’s been a difficult process,” said Venice, “because, well, no one really would want to be trans. No one would actually really enjoy it.”

And then there is the prospect of sex change surgery, further down the road.

“Is that something that is a little bit scary to you?” Braver asked.

“Yeah, I’m really afraid for bottom surgery, like, how like much pain it will cause,” Venice said.

There are practical problems as well. California, where Venice lives, was the first state to pass a law specifically allowing transgender schoolchildren to use bathrooms, locker room and even play on sports teams of the gender that they choose.

But opponents — like Brad Dacus of the Pacific Justice Institute, a conservative legal group — say the law puts an unfair burden on other children:

“You’re saying under this law that a 13-year-old or 14-year-old girl in a locker room has to change and dress and be naked in front of, say, a 16-year-old boy simply because a 16-year-old boy who’s a biological boy, but inside has a mental condition called gender identity dysphoria and thinks that he’s a girl,” said Dacus. “This is ludicrous, and really unreasonable.”

Dacus argues that while transgender kids should be treated with compassion, they should use separate facilities — say, in faculty or nurses’ offices.

And Venice’s response? “You can use a separate bathroom, too,” he laughed.

But Venice has had other issues to worry about. His parents are separated, and while his mother and brother have always been supportive, his father was not.

Finally, Venice’s mom, Trish, sent his father a letter: “It said pretty much, you’re on board with this or you can’t be in our lives, because a child that’s not supported doesn’t flourish. And dad looked at that and decided, ‘Well, I can either have a son or I can have nothing.'”

Today, Venice’s father, Mike, has joined Venice’s mother in a support group for parents of transgendered kids. “I was in a lot of fear and anxiety, especially when my kid came out,” Mike said.

He acknowledges that he hired a series of therapists in an effort to convince Venice to live as a girl — all to no avail.

“So I was totally in the wrong area,” he said. “Spending lots of money trying to fix a kid that, you know, wasn’t really broken.”

And parents are beginning to heed the wishes of their children at ever-younger ages. The 6-year-old happily jumping with her younger brother is Mati — originally named Mattias.

Braver said to Mati, “Your mom told me that when you were born, everybody thought you were a boy except you.”

“Yeah, right,” replied Mati.

“What did you think?”

“I was a girl.”

“You knew?”

“Yeah.”

“How’d you know?”

Mati said, “I just figured it out.”

Mati, 6.
CBS News
Mati’s parents, Cristy and Enrique, say it started when she was younger than two — going clothes shopping, for example:
“Cristy and I would go to the boys’ section, and Mati would start directly going to the girls’ section, wanting to pick clothes from there,” said Enrique.

Mati’s parents say she was miserable when treated as a boy, and by the time she entered kindergarten in San Diego last year, they felt they had to enroll her as a girl.

“You know that some people are going to see this story on television and say, ‘Oh my gosh, they should have waited. They should have insisted on waiting longer,” said Braver. “Why didn’t you wait longer?”

“She was in pain,” said Cristy. “I don’t see that there was another option. She was uncomfortable, she was unhappy. You can’t see your child suffer like that.

“Nothing’s been done that can’t be undone. So people that would say, ‘Maybe it’s too early, we should have waited’ — what did we have to lose at this point? She’s happier.”

Like all the parents we spoke to for this story, Cristy and Enrique told us they decided to go public because they want to help other families facing the same issues:

And, said Cristy, “I also want for Mati to know that it’s nothing that can’t be talked about. I also want her to know that she shouldn’t be ashamed or afraid to be who she is.”

While doctors say some children who identify themselves as the opposite of their physical gender do change their minds as they get older, there is little data on how often that occurs.

So far, Mati shows no sign of reconsidering:

“Have you ever thought maybe sometimes, ‘Gee, it might be fun to go back and dress like a boy and be more like a boy’?” asked Braver.

“I don’t want to,” Mati said. “Uh uh.”

“You know that what — how do you feel?”

“Like a girl.”

“Is there anything you’d like to say for maybe other children who people say, ‘Oh, you’re a boy,’ but they know that they’re a girl?”

“Maybe let them choose.”

http://www.cbsnews.com/news/born-this-way-stories-of-young-transgender-children/

Hormones: Chemical Messengers That Work in Parts per Trillion
From Our Stolen Future Theo Colborn, Dianne Dumanoski, John Peterson Myers (Dutton 1996)

Pushing on with her research on hormones, Theo Colborn discovered a central piece of the puzzle in the world of Frederick vom Saal, a biologist at the University of Missouri. Vom Saal’s exploration of how hormones help make us who we are is a fascinating scientific adventure in its own right. In a series of experiments with mice, he showed that small shifts in hormones before birth can matter a great deal and have consequences that last a lifetime. His work helped highlight the hazard posed by synthetic chemicals that can disrupt hormonal systems.

Vom Saal’s investigation of the wondrous world of hormones began in 1976 during his postdoctoral days at the University of Texas in Austin, inspired by the behavior of the lab mice. Like most postdoctoral biology students, vom Saal was spending the better part of his life in the lab, where his regular chores included breeding mice. As he played mouse matchmaker, arranging encounters between eager males and receptive females, he became intrigued by the interplay between the animals as he moved them from cage to cage.

In the beginning, the small, white, pink-eyed creatures had all seemed like cookie-cutter copies of each other. But as he watched the females scurrying about in the breeding cages, individuals quickly emerged from the crowd. Whenever he returned a female to a group cage holding half a dozen females, there always seemed to be one mouse who would attack the intruder. These were mice with an attitude-tough cookies who rattled their tails threateningly and lashed out at their mild-mannered companions.

Such a difference between the behavior of one female and another was striking-and puzzling. The mice were all from a single laboratory strain that had been inbred for generations. When it came to genes, they were virtually identical.

This simple observation set the course for vom Saal’s life’s work in reproductive biology. In the years that followed, he designed dozens of experiments to probe the mystery of how two mice with almost the same genetic blueprint could behave so differently.

The notion persists that genes are tantamount to destiny and that one might explain everything from cancer to homosexuality by locating the responsible genes. But in a series of scientific papers, vom Saal demonstrated that there are other powerful forces shaping individuals-females as well as males-before birth. Genes, it turned out, are not the whole story. Not by a long shot.

What vom Saal saw during those long hours observing mice in the lab contradicted everything he had read. According to the scientific literature of the period (which reflected prevailing human assumptions as much as it described animal behavior), aggression was strictly a male behavior. But if tail-rattling, chasing, and biting among the females weren’t aggression, what would one call it?

Eventually, vom Saal’s colleagues had to concede that the behavior did look like aggression, but they tended to shrug it off as unimportant. Males were the center of the action in animal societies according to the prevailing wisdom in the field of animal behavior, so what females did simply didn’t matter. They were just passive baby makers.

Vom Saal wasn’t so sure. His intuition told him what he was seeing was probably important as well as interesting. His doctoral work had centered on the role played by testosterone in development before birth, and he knew that this hormone-found at much higher levels in males-drives aggression.

From his observations, the tough females weren’t common, but they weren’t rare either. There seemed to be roughly one aggressive female for every six mice in the colony-something he noticed because the mice were housed six to a cage. If the mice were clones, something besides genes had to be shaping the aggressive females. Since birth the sisters had been raised identically, so living conditions could not explain the differences. Could the cause be something in their prenatal environment?

That set him to thinking about how mice are carried before birth. Their mother’s womb isn’t a single compartment like the human womb, but two separate compartments or “horns” that branch off to the left and the right at the top of the vagina or birth canal. The baby mice are tucked in the narrow horns like peas in a pod-as many as six on a side. This arrangement means that some of the females will develop sandwiched between two males.

Vom Saal began calculating probabilities. If there were twelve mice in the typical mouse litter and if the placement of males and females in the womb was random, how many females would end up between two males? Roughly one in six, he figured. That supported the theory taking shape in his head. Some of the females are markedly more aggressive, he suspected, because they had spent their prenatal life wedged between two males. A week before birth, the testicles in a male pup begin to secrete the male hormone testosterone, which drives his own sexual development. The female pups might be bathed in testosterone washing over from their male neighbors.

Maybe, vom Saal thought, the answer to the mystery of how genetically identical females could be so different lay in hormones — chemical messengers that travel in the bloodstream, carrying messages from one part of the body to another.

In the body’s constant conversation with itself, nerves are just one avenue of communication — the one employed for quick, discrete messages that direct a hand to move away from a hot stove. A large part of the body’s internal conversation, however, is carried on through the bloodstream, where hormones and other chemical messengers move about on the biological equivalent of the information superhighway, carrying signals that not only govern sex and reproduction but also coordinate organs and tissues that work in concert to keep the body functioning properly.

Hormones, which get their name from the Greek word meaning “to urge on,” are produced and released into the bloodstream by a variety of organs known as endocrine glands, including the testicles, the ovaries, the pancreas, the adrenal glands, the thyroid, the parathyroid, and the thymus. The thyroid, for example, produces chemical messengers that activate the body’s overall metabolism, stimulating tissues to produce more heat. In addition to eggs, a woman’s ovaries release estrogens-the female hormones that travel in the bloodstream to the uterus, where they trigger growth of the tissue lining the womb in anticipation of a possible pregnancy.

Yet another endocrine gland, the pituitary, which dangles on a stalk from the underside of the brain just behind the nose, acts as a control center, telling the ovaries or the thyroid when to send their chemical messages and how much to send. The pituitary gets its cues from a nearby portion of the brain called the hypothalamus, a teaspoon-size center on the bottom of the brain that constantly monitors the hormone levels in the blood in much the way that a thermostat monitors the air temperature in a house. If levels of a hormone get too high or too low, the hypothalamus sends a message to the pituitary, which signals the gland that produces this hormone to gear up, slow down, or shut off.

The messages travel back and forth continuously. Without this cross talk and constant feedback, the human body would be an unruly mob of some 50 trillion cells rather than an integrated organism operating from a single script.

As scientists have delved deeper into the nervous, immune, and endocrine systems-the body’s three great integrating networks they have encountered profound interconnections: between the brain and the immune system, the immune system and the endocrine system, and the endocrine system and the brain. The links sometimes seem utterly mystifying. How, for example, could a woman suffering from multiple personality disorder play with a cat for hours while she was one personality and suffer violent allergic reactions to cats when she took on another?

Nobody knows the answer to this question, but it certainly lies in this internal conversation and the constant babble of chemical messengers. Changes in one part of this complex, interconnected system can have dramatic and unexpected consequences elsewhere, often where one might least expect, because everything is linked to everything else. A brain tumor, for example, might show up as disrupted menstrual cycles and hypersensitivity of the skin rather than as headaches.

If hormones are vital to maintain proper functioning in adults, they are perhaps even more important in the elaborate process of development before birth.

But how could vom Saal test his theory?

Mouse Caesarean sections.

Just before the females were ready to give birth at the end of their nineteen-day pregnancies, vom Saal removed the tiny babies, who were approximately an inch long and about the size of an olive. He marked them based on their position relative to their neighbors in the womb. In this way, he could discover where aggressive females had spent their prenatal lives. Thus began vom Saal’s exploration of what some in the field playfully refer to as the “wombmate” effect, known formally as intrauterine position phenomenon.

Although vom Saal is now forty-nine and a professor at the University of Missouri, he still looks youthful enough to be mistaken for a graduate student. In a scientific world where many seldom venture beyond narrow specialities, vom Saal embraces the big picture, unabashedly declaring that he is interested in “womb-to-tomb biology.” He moves easily between elegant, tightly focused studies and a larger, more encompassing pursuit of fundamental questions: Why does this happen? What is the evolutionary significance?

Those first studies in Austin confirmed his theory. As the mice removed by Caesarean section matured, the aggressive females were, as predicted, the ones who had developed between brothers. Each intriguing finding raised new questions, leading to more studies and, in time, observations on thousands of mice delivered by Caesarean section. Aggression proved just the most obvious sign of profound differences between mouse sisters that could be predicted to a remarkable degree by their position in the womb.

At first blush, vom Saal’s results sound like a tale of the ugly sister and the pretty sister. Not only was the ugly sister-the mouse that had developed between males-more aggressive, but vom Saal discovered she was significantly less attractive to males than the pretty sisters who had spent their womb time between other females. Eight times out of ten, a male given a choice would chose to mate with the pretty sister.

What’s attractive to males isn’t the female’s tiny pink eyes or the curve of her tail. The social life of mice is governed by the nose, and the attractiveness of females depends on the social chemicals they give off, which are called pheromones. The pretty sisters smell “sexier” to males because they produce different chemicals than their less attractive sisters. The prenatal hormone environment leaves a permanent imprint on each sister that is recognized by males for the rest of her life.

Behavioral and reproductive differences in mice can be predicted to a remarkable degree by their position, which is related to hormone exposure, in the womb. (Adapted from vom Saal and Dhar, 1992)
Illustration by K Brown 1995

The sisters also showed dramatic differences in their reproductive cycles. Besides finding mates more readily, the pretty sister also matured faster than her ugly sister and came into heat-a period of sexual receptivity-more often. As a consequence, she had more opportunities to get pregnant and was more likely overall to produce more offspring in her lifetime than her aggressive, unattractive sister, who experienced puberty later and came into heat less frequently.

Even more amazing, studies by other researchers, including Mertice Clark, Peter Karpiuk and Bennett Galef of McMaster University, and the team of John Vandenbergh and Cynthia Huggett of North Carolina State University, have found that the wombmate effect even influences whether a female will give birth to more males or more females when she has pups of her own. This is mysterious indeed, since scientists up to now believed that the mother has no role in determining the sex of her offspring. Based on current understanding, it is the sperm contributed by the father that dictates whether the egg develops into a male or female, so how a mother influences sex ratio is still unknown. However it happens, the pretty sisters tend to have litters made up of sixty percent females, while the ugly sisters generally give birth to litters that are roughly sixty percent male. As Vandenbergh wrote of this transgenerational wombmate influence: “Brothers beget nephews.”

After hearing the tale of the two sisters, one might easily conclude that it would be wise to be a pretty sister if one had to be a mouse. They have lots of mates and babies and, judged by the evolutionary imperative of producing offspring, seem more successful than their ugly sisters.

Not so fast, vom Saal cautions. When one considers how these sisters live their lives within a mouse population that goes through boom and bust cycles, the pretty sister begins to lose her obvious edge. Typically, a mouse population builds to a very high peak and then it crashes. In ordinary times when the population isn’t too dense, the pretty sisters definitely have the advantage, but as conditions become overcrowded the pretty sisters’ ability to produce babies diminishes because the females respond to scent cues in urine that inhibit reproduction.

But these overcrowded times are precisely when the ugly sisters come into their own. Because they are relatively immune to the inhibiting cues, they are likely to be the only ones to produce offspring, and the ugly sisters are the only ones tough enough to protect their babies from attack and infanticide.

Interestingly, some studies have shown that the mother’s physical condition can also alter hormone levels in the womb and influence the offspring. Mouse mothers that experience continuous stress through the latter part of their pregnancies give birth to females who have all the physical and behavioral characteristics of females who develop between males. Maternal stress seems to override the ordinary wombmate variations and produce a litter composed solely of tough cookies.

So what’s the evolutionary lesson in this tale?

In vom Saal’s view, the real lesson is the value of variability.

The acute sensitivity of developing mammals such as mice to slight shifts in hormone levels in the womb has been shaped by evolution. This characteristic helped insure wide variation in the offspring, even wider variation than that produced by genetic shuffling alone. Variation is the way mammals have hedged their bets in the face of a rapidly shifting environment. If you don’t know what the conditions will be for your offspring, the best thing to do is produce many different kinds in the hope that at least one of them will be suited to the emerging moment.

Vom Saal’s early. investigations into the wombmate effect focused solely on females. The decision to look at males to see if female wombmates had any influence on them was almost an afterthought. Though the results would round out this line of research, vom Saal admits he frankly did not expect to find anything remarkable. It was widely assumed that male development was driven exclusively by testosterone, so being next to females should make little difference.

In fact, the results of his experiments astonished him. The wombmate effect shaped the destinies of males as well as females and in ways that no one would have ever predicted. In a major paper in the prestigious journal Science in June 1980, vom Saal and his associates laid out the case that it was exposure to the female hormone estrogen before birth that increased a male’s sexual activity in adult life.

Inside and outside the world of science, many have regarded the level of male sexual activity as an index of masculinity and a product of the male hormone testosterone. Indeed, the findings were so counterintuitive and so contrary to assumptions about the “male” hormone testosterone and the “female” hormone estrogen that one of his collaborators protested that they must have somehow mixed up the samples. Vom Saal found, however, that estrogen and testosterone each influence males-and in ways that run counter to our conventional notions of “maleness” and “femaleness.” The effect of wombmates on males proved an even more provocative vein of research than his earlier work on females.

If the females seem a story of the pretty and ugly sisters, then vom Saal’s findings on the males sound like a tale of the playboy and the good father.

As adults, the playboy males, exposed to higher levels of estrogen by their female wombmates, showed another surprising characteristic besides their higher rates of sexual activity. It would seem logical to assume that exposure to estrogen might make males more solicitous toward the young, but in fact, the opposite proved true. When placed with young mice, these males were more likely to attack and kill babies. The high-testosterone males who had had brothers for wombmates turned out to be the good daddies, who surprisingly showed almost as great an inclination to take care of pups as mouse mothers.

The playboy males were standouts in one other respect as well — the size of their prostate, the small gland that wraps around the urethra, through which urine is eliminated. The males exposed to higher levels of estrogen had prostates that were fifty percent larger than those seen in brothers who had had male wombmates. In addition, these larger prostates are more sensitive to male hormones in adulthood because they contain three times the number of testosterone receptors found in the prostates of brothers with male wombmates. More receptors generally means that the gland will grow more quickly in response to male hormones circulating in the bloodstream in adulthood.

Although human babies don’t usually have to share the womb with siblings, their development can nevertheless be affected by varying hormone levels, which occur in the womb for reasons scientists don’t completely understand. Medical problems such as high blood pressure can drive up estrogen levels, for example. Or perhaps eating tofu, alfalfa sprouts, or other foods that are high in plant estrogens during pregnancy could boost estrogen exposure. There is also the possibility that the mother’s body fat contains synthetic chemicals that disrupt hormones.

Whatever the source, a recent study on opposite-sex human twins showed that wombmate effects can be detected in people as well. The study, which focused on an obscure difference in the auditory systems of males and females that exists from birth, found that girls who had developed with a boy twin showed a male pattern, suggesting that they, like vom Saal’s female mice, had been somewhat masculinized by the hormones spilling over from a male wombmate.

In the midst of all these surprises, the male wombmate studies in mice yielded only one expected result-on male aggression. Males with male wombmates and the highest testosterone exposure before birth were indeed the most aggressive toward other adult males, and males with female wombmates were the least aggressive.

Scientists working in this field are still debating how estrogen shapes the development of males and females, particularly the development of the brain and behavior, but vom Saal believes that estrogen is helping to masculinize males by acting to enhance some effects of the male hormone testosterone. Together the two hormones influence the organization ;of the developing brain to increase the level of sexual activity the male mouse will exhibit as an adult. Vom Saal had demonstrated that, this is a prenatal effect rather than a consequence of adult hormone levels by castrating the mice shortly after birth and then in adulthood administering an identical amount of male hormone to brothers with male and female wombmates. Even with identical hormone exposure these male mice showed different levels of sexual activity-evidence that adult hormone levels are not the cause of these behavioral differences.

Those who hear about vom Saal’s work typically ask him, Which is the “normal” mouse: the-pretty sister or the ugly sister? The playboy or the good father?

“They’re all normal,” vom Saal says emphatically.

The question itself seems to stem from our dualistic notion of maleness and femaleness, which sees the two sexes as mutually exclusive categories. In fact, there are many shades of gray and overlap between behaviors thought of as typically male or female. Seen in this light, there is nothing abnormal about an aggressive female or a nurturing male. In this strain of mice, whose genetic variability has been reduced by generations of inbreeding, these individuals reflect the variability created by the natural influence of hormones before birth. What is “normal,” vom Saal says, returning to an evolutionary theme, is not one type of individual or another but the variability itself.

But variability is just one of the larger lessons emerging from vom Saal’s work. It has also opened a window on the powerful role of hormones in the development of both sexes and the extreme sensitivity of developing mammals to slight shifts in hormone levels in the womb. The wombmate studies have also underscored that hormones permanently “organize” or program cells, organs, the brain, and behavior before birth, in many ways setting the individual’s course for an entire lifetime.

It is important to remember that hormones do this without altering genes or causing mutations. They control the “expression” of genes in the genetic blueprint an individual inherits from its parents. This relationship is similar to that between the keys on a player piano and the prepunched music roll that runs through and determines the tune. Though the piano can theoretically play many tunes, it will only play the one dictated by the pattern of holes in the music roll. During development, hormones present in the womb determine which genes will be expressed, or played, for a lifetime as well as the frequency of their expression. Nothing has been changed in the individual’s genes, but if a particular note hasn’t been punched into the music roll during development, it will remain forever mute. Genes may be the keyboard, but hormones present during development compose the tune.

What is astonishing about vom Saal’s wombmate studies is how little it takes to dramatically change the tune. Hormones are exceptionally potent chemicals that operate at concentrations so low that they can be measured only by the most sensitive analytical methods. When considering hormones such as estradiol, the most potent estrogen, forget parts per million or parts per billion. The concentrations are typically parts per trillion, one thousand times lower than parts per billion. One can begin to imagine a quantity so infinitesimally small by thinking of a drop of gin in a train of tank cars full of tonic. One drop in 660 tank cars would be one part in a trillion; such a train would be six miles long.

The striking lifelong differences between a pretty sister and ugly sister stem from no more than a thirty-five parts per trillion difference in their exposure to estradiol and a one part per billion difference in testosterone. Using the gin and tonic analogy, the pretty sister’s cocktail had 135 drops of gin in one thousand tank cars of tonic and the ugly sister’s 100 drops-a difference that might not be detectable in a glass much less in a tank car flotilla.

This is a degree of sensitivity that approaches the unfathomable, a sensitivity, vom Saal says, “beyond people’s wildest imagination.” If such exquisite sensitivity provides rich opportunities for varied offspring from the same genetic stock, this same characteristic also makes the system vulnerable to serious disruption if something interferes with normal hormone levels-a frightening possibility that first dawned on vom Saal when Theo Colborn called him to talk about synthetic chemicals that could act like hormones.

To appreciate vom Saal’s concern, one must understand more about the intricate choreography of events before birth known as sexual differentiation and the key role played by hormones in this developmental ballet. In mice, elephants, whales, humans, and all other mammals as well as in birds, reptiles, amphibians, and fish, the process that creates two sexes from initially unisex embryos is guided by these chemical messengers. They are the conductors that give the cues at the right moment as tissues and organs make now-or-never choices about the direction of development. In this central drama in which boys become boys and girls become girls, hormones have the starring role.

Our understanding of what determines whether a fertilized egg becomes a male or female is very recent. Before the twentieth century, it was widely assumed that the sex of the baby was determined by environmental factors such as temperature.

It was only in 1906 that two scientists-Nettie Marie Stevens and Edmund Beecher Wilson-independently noted that each cell in women had two X chromosomes while men always had an X and a Y, an observation that led to the theory that the number of X chromosomes determined sex. In the past decade, researchers have finally established that it is a gene on the Y chromosome rather than the number of X chromosomes that determines sex.

As most of us learned in high school biology, the eggs produced by the mother all carry one X chromosome, and the sperm from the father carry either an X or a Y chromosome. The sex of the baby hangs in the balance as the sperm burst out of the starting gate and race against each other in the reproductive marathon. If this most primordial of athletic events were broadcast like the Boston Marathon, we might hear that three Ys are neck-and-neck at the entrance to the cervix, but an X is making a move on the outside in the push into the uterus. A field of 75 million sperm have been pushing hard, sweeping their tails back and forth in steady swimming motions, but in the biological equivalent of Heartbreak Hill, many are beginning to flag as they enter the fallopian tube leading from the top of the uterus. It’s a tight race right to the finish line as the competitors crowd toward the goal. At the finish line of this race, an egg awaits the victor, rather than a crown of laurel, as it crashes through. If the Y-carrying sperm gets to the egg first, the baby, who has XY chromosomes, will be a boy. If the first sperm to the egg carries an X, the XX chromosome will produce a girl.

Such stories about the race between the Xs and the Ys for the egg left many of us with the impression that the outcome was all in the genetic instructions carried by the sperm. If the sperm delivered a Y, bingo, it was a boy-what unfolded between conception and birth was all more or less automatic and dictated by that genetic blueprint. In fact, the process is much more complex. The sex-determining gene in that Y chromosome has only a quick walk-on part in the elegant and wondrous process through which boys become boys.

In animals such as birds and humans, one sex is the basic model and the other is what might be described as a custom job, since the latter requires a sequence of additional changes directed by hormones to develop properly into the opposite sex. In birds, this basic model happens to be male. In mammals, including humans, the opposite is the case, and an embryo will develop into a female unless male hormones override the program and set it off on the alternative course.

Although the sperm delivers the genetic trigger for a male when it penetrates the egg, the developing baby does not commit itself to one course or another for some time. Instead, it retains the potential to be either male or female for more than six weeks, developing a pair of unisex gonads that can become either testicles or ovaries and two separate sets of primitive plumbing-one the precursor to the male reproductive tract and the other the making of the fallopian tubes and uterus. These two duct systems, known as the Wolffian and Müllerian ducts, are the only part of the male and female reproductive systems that originate from different tissues. All the other essential equipment — which might seem dramatically different between the two sexes — develop from common tissue found in both boy and girl fetuses. Whether this tissue becomes the penis or the clitoris, the scrotal sack that carried the testicles or the folds of labial flesh around a woman’s vagina, or something in between depends on the hormonal cues received during a baby’s development.

The big moment for the Y chromosome comes around the seventh week of life, when a single gene on the chromosome directs the unisex sex glands to develop into male testicles. In doing this, the Y chromosome throws the switch initiating the very first step in male development, the development of the testes, and that is the beginning and end of its role in shaping a male. From this point on, the remainder of the process of masculinization is driven by hormone signals originating from the baby’s brand-new testicles. In adult life, the testicles produce sperm to fertilize a woman’s eggs, the male’s contribution to reproduction and posterity. But the testicles play an even more important role in a male’s life before birth. Without the right hormone cues at the right time-cues emanating from the testicles-the baby will not develop the male body and brain that go along with the testicles. It might not even develop the penis required to deliver the sperm the testicles produce.

In girls, the changes that turn the unisex glands into ovaries, the part of the female anatomy that produces eggs, begin somewhat later, in the third to fourth month of fetal life. During this same period, one set of ducts-the Wolffian ducts that provide the option for a male reproductive tract-wither and disappear without any special hormone instructions. While the development of the female body isn’t as dependent on hormone cues as the development of males, animal research suggests estrogen is essential for proper development and normal functioning of the ovaries.

The process of laying the groundwork for the reproductive tract is more complicated in males and is marked by critical stages where hormones direct now-or-never decisions. Shortly after they are formed, the testicles produce a special hormone whose function is to trigger the disappearance of the female option — the Müllerian ducts. To accomplish this milestone, the hormone message must arrive at the right time, because there is only a short period when the female ducts respond to the signal to disappear. Then the testicles have to send another message to the Wolffian ducts, because they are programmed to disappear automatically by the fourteenth week unless they receive orders to the contrary.

The messenger is the predominantly male hormone testosterone, which insures the preservation and growth of the male Wolffian ducts. Under the influence of testosterone, these ducts form the epididymis, vas deferens, and seminal vesicles-the sperm delivery system that leads from the testicles to the penis.

A potent form of testosterone guides the development of the prostate gland and external genitals, directing the genital skin to form a penis and a scrotum that holds the testicles when they finally descend from the abdomen late in a baby’s development. A naturally occurring defect dramatically illustrates what can happen if these messages do not get through.

From time to time, a young patient will show up in a gynecologist’s office because the teenager still hasn’t had her first period although all the other girls in her class have passed this milestone. Usually nothing serious is wrong.

But once in a rare while, the physician will deliver an utterly shocking diagnosis. The patient isn’t menstruating because despite all appearances, she is not female. Although such individuals have grown up as normal-looking girls, they have the XY chromosomes of males and testicles in their abdomen instead of ovaries. But because a defect makes them insensitive to testosterone, they never responded to the hormone cues that trigger masculinization. They never developed the body and brain of a male.

The pictures in medical textbooks of these unrealized males are fascinating, for there is nothing about their unclothed bodies that looks the least bit odd or unusual. As hard as one searches for a hint that a genetic male lurks inside these bodies, there is no sign of development derailed. These genetic males look like perfectly ordinary women with normally developed breasts, narrow shoulders, and broader hips.

These completely feminized males are the most extreme example of what happens when something blocks the chemical messages that guide development. If -anything interferes with the testosterone or the enzyme that amplifies its effect, the common tissue found in boy and girl fetuses will develop instead into a clitoris and other external female genitals. In less extreme cases of disruption, males may have ambiguous genitals or abnormally small penises and undescended testicles.

But sex is more than a purely physical matter. According to physicians who treat them, these feminized males not only look like women, they act and think of themselves as women. There is nothing the least bit telling in their behavior to suggest that they are really male. In most animals, the development of a properly functioning male or female involves the brain as much as the genitals, and research such as vom Saal’s shows that hormones permanently shape some aspects of behavior before birth as much as they sculpt the penis. If an individual is going to act like a male as well as look like one, the brain must receive testosterone messages from the testicles during a critical period when brain cells are making some of their now-or-never decisions.

An individual who gets the wrong hormone messages during this critical period of brain development may show abnormal behavior and fail to mate even though it has the right physical equipment. In an influential 1959 study, Charles Phoenix of the University of Kansas found that female guinea pigs exposed to high levels of testosterone in the womb acted like males. They would not show the classic female mating posture, a raised posterior, known as “lordosis,” as adults or respond normally to the female hormones that stimulate sexual behavior and reproduction.

No one debates that hormones act to give males and females different bodies and that their role in the development of animals and humans is pretty much the same. But how hormones influence the development of the human brain is hotly debated. Do they shape the brain and behavior in humans as dramatically as they do in mice or rats or guinea pigs? Are there structural differences between the brains of men and women, and is there any evidence that the differences stem from hormone influences before birth?

These questions are difficult to answer. Not only is human behavior more complex than that of vom Saal’s mice, but we aren’t free to give pregnant women various doses of hormones to see the effect on the brain development of their babies.

Those who have probed the question of whether the behavioral differences between men and women have a biological basis or are purely cultural have found evidence of some structural differences linked to hormones, but so far these sex-linked areas are fewer and less pronounced than those seen in rats. Psychologists have also reported certain general differences in the way men and women think, reporting that women have greater verbal skills as a rule and men tend to be better at solving spatial problems. Many also believe that the rough-and-tumble play and fighting seen to a much greater degree in young boys than in girls stems from biology rather than from culture or child-rearing methods.

At the same time that hormones are guiding at least some aspects of sexual development of the unborn child, these chemical messengers are also orchestrating the growth of the baby’s nervous and immune systems, and programming organs and tissues such as the liver, blood, kidneys, and muscles, which function differently in men and women. Normal brain development, for example, depends on thyroid hormones that cue and guide the development of nerves and their migration to the right area in this immensely complex organ.

For all these systems, normal development depends on getting the right hormone messages in the right amount to the right place at the right time. As this elaborate chemical ballet rushes forward at a dizzying pace, everything hinges on timing and proper cues. If something disrupts the cues during a critical period of development, it can have serious lifelong consequences for the offspring.

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Scientists discover DNA body clock

Newly discovered mechanism could help researchers understand ageing process and lead to ways of slowing it down.

by Ian Sample, science correspondent

A US scientist has discovered an internal body clock based on DNA that measures the biological age of our tissues and organs.

The clock shows that while many healthy tissues age at the same rate as the body as a whole, some of them age much faster or slower. The age of diseased organs varied hugely, with some many tens of years “older” than healthy tissue in the same person, according to the clock.

Researchers say that unravelling the mechanisms behind the clock will help them understand the ageing process and hopefully lead to drugs and other interventions that slow it down.

Therapies that counteract natural ageing are attracting huge interest from scientists because they target the single most important risk factor for scores of incurable diseases that strike in old age.

“Ultimately, it would be very exciting to develop therapy interventions to reset the clock and hopefully keep us young,” said Steve Horvath, professor of genetics and biostatistics at the University of California in Los Angeles.

Horvath looked at the DNA of nearly 8,000 samples of 51 different healthy and cancerous cells and tissues. Specifically, he looked at how methylation, a natural process that chemically modifies DNA, varied with age.

Horvath found that the methylation of 353 DNA markers varied consistently with age and could be used as a biological clock. The clock ticked fastest in the years up to around age 20, then slowed down to a steadier rate. Whether the DNA changes cause ageing or are caused by ageing is an unknown that scientists are now keen to work out.

“Does this relate to something that keeps track of age, or is a consequence of age? I really don’t know,” Horvath told the Guardian. “The development of grey hair is a marker of ageing, but nobody would say it causes ageing,” he said.

The clock has already revealed some intriguing results. Tests on healthy heart tissue showed that its biological age – how worn out it appears to be – was around nine years younger than expected. Female breast tissue aged faster than the rest of the body, on average appearing two years older.

Diseased tissues also aged at different rates, with cancers speeding up the clock by an average of 36 years. Some brain cancer tissues taken from children had a biological age of more than 80 years.

“Female breast tissue, even healthy tissue, seems to be older than other tissues of the human body. That’s interesting in the light that breast cancer is the most common cancer in women. Also, age is one of the primary risk factors of cancer, so these types of results could explain why cancer of the breast is so common,” Horvath said.

Healthy tissue surrounding a breast tumour was on average 12 years older than the rest of the woman’s body, the scientist’s tests revealed.

Writing in the journal Genome Biology, Horvath showed that the biological clock was reset to zero when cells plucked from an adult were reprogrammed back to a stem-cell-like state. The process for converting adult cells into stem cells, which can grow into any tissue in the body, won the Nobel prize in 2012 for Sir John Gurdon at Cambridge University and Shinya Yamanaka at Kyoto University.

“It provides a proof of concept that one can reset the clock,” said Horvath. The scientist now wants to run tests to see how neurodegenerative and infectious diseases affect, or are affected by, the biological clock.

“These data could prove valuable in furthering our knowledge of the biological changes that are linked to the ageing process,” said Veryan Codd, who works on the effects of biological ageing in cardiovascular disease at Leicester University. “It will be important to determine whether the accelerated ageing, as described here, is associated with other age-related diseases and if it is a causal factor in, or a consequence of, disease development.

“As more data becomes available, it will also be interesting to see whether a similar approach could identify tissue-specific ageing signatures, which could also prove important in disease mechanisms,” she added.

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“The Toxins That Affected Your Great-Grandparents Could Be In Your Genes”

Biologist Michael Skinner has enraged the chemical community and shocked his peers with his breakthrough research

By Jeneen Interlandi for Smithsonian magazine, December 2013

Michael Skinner’s biggest discovery began, as often happens in science stories like this one, with a brilliant failure. Back in 2005, when he was still a traditional developmental biologist and the accolades and attacks were still in the future, a distraught research fellow went to his office to apologize for taking an experiment one step too far. In his laboratories at Washington State University, she and Skinner had exposed pregnant rats to an endocrine disruptor—a chemical known to interfere with fetal development—in the hope of disturbing (and thereby gaining more insight into) the process by which an unborn fetus becomes either male or female. But the chemical they used, an agricultural fungicide called vinclozolin, had not affected sexual differentiation after all. The scientists did find lower sperm counts and decreased fertility when the male offspring reached adulthood, but that was no surprise. The study seemed like a bust.

By accident, though, Skinner’s colleague had bred the grandchildren of those exposed rats, creating a fourth generation, or the great-grandchildren of the original subjects. “It’s OK,” Skinner told her. “You might as well analyze them.” If nothing else, he thought, the exercise might take her mind off her mistake. So she went ahead and studied the rats’ testes under a microscope.

What they found would not only change the direction of Skinner’s research but also challenge a bedrock principle of modern biology. And Skinner would become the forerunner of a new way of thinking about the possible long-term health consequences of exposure to environmental chemicals.

His discoveries touch on the basic question of how biological instructions are transmitted from one generation to the next. For half a century it has been common knowledge that the genetic material DNA controls this process; the “letters” in the DNA strand spell out messages that are passed from parent to offspring and so on. The messages come in the form of genes, the molecular equivalent of sentences, but they are not permanent. A change in a letter, a result of a random mutation, for example, can alter a gene’s message. The altered message can then be transmitted instead.

The strange thing about Skinner’s lab rats was that three generations after the pregnant mothers were exposed to the fungicide, the animals had abnormally low sperm counts—but not because of a change in their inherited DNA sequence. Puzzled, Skinner and his team repeated the experiments—once, twice, 15 times—and found the same sperm defects. So they bred more rats, and tested more chemicals, including substances that lead to diseases in the prostate, kidney, ovaries and immune system. Again and again, these diseases also showed up in the fourth- and fifth-generation offspring of mothers exposed to a chemical.

“In essence,” Skinner explains, “what your great-grandmother was exposed to could cause disease in you and your grandchildren.”

And, startlingly, whatever disease pathway a chemical was opening in the rats’ fur-covered bodies, it did not begin or end at a mutation in the genetic code. Skinner and his team found instead that as the toxins flooded in, they altered the pattern of simple molecules called methyl groups that latch onto DNA in the fetus’ germ-line cells, which would eventually become its eggs or sperm. Like burrs stuck to a knit sweater, these methyl molecules interfered with the functioning of the DNA and rode it down through future generations, opening each new one to the same diseases. These burrs, known to be involved in development, persisted for generations. The phenomenon was so unexpected that it has given rise to a new field, with Skinner an acknowledged leader, named transgenerational epigenetics, or the study of inherited changes that can’t be explained by traditional genetics.

A study by Skinner and colleagues published last year in the journal PLOS One has upped the ante considerably. The burrs were not just haphazardly attached, Skinner found. Instead, they fastened themselves in particular arrangements. When he bathed the insides of his pregnant rats in bug spray, jet fuel and BPA, the plastics component recently banned from baby bottles, each exposure left a distinct pattern of methyl group attachments that persisted in the great-grandchildren of exposed rats.

Not only is your great-grandmother’s environment affecting your health, Skinner concluded, but the chemicals she was exposed to may have left a fingerprint that scientists can actually trace.

The findings point to potentially new medical diagnostics. In the future, you may even go to your doctor’s office to have your methylation patterns screened. Exposure of lab rats to the chemical DDT can lead to obesity in subsequent generations—a link Skinner’s team reported in October. Hypothetically, a doctor might someday look at your methylation patterns early in life to determine your risk for obesity later. What’s more, toxicologists may need to reconsider how they study chemical exposures, especially those occurring during pregnancy. The work raises implications for monitoring the environment, for determining the safety of certain chemicals, perhaps even for establishing liability in legal cases involving health risks of chemical exposure.

These possibilities have not been lost on regulators, industries, scientists and others who have a stake in such matters. “There are two forces working against me,” Skinner says. “On one side, you have moneyed interests refusing to accept data that might force stronger regulations of their most profitable chemicals. On the other side, you have genetic determinists clinging to an old paradigm.”

Michael Skinner wears a gray Stetson with a tan strap, and leans back easily in his chair in his office on the Pullman campus. His fly-fishing rod stands in the corner, and a colossal northern pike is mounted on the wall. An avid fly fisherman, Skinner, age 57, was born and raised on the Umatilla Indian Reservation in eastern Oregon. The Skinners are not of Indian descent, but his parents owned a family farm there—“a good cultural experience,” he says. His father worked in insurance, and he and his four brothers grew up just as five generations of Skinners had before them—hunting and fishing and cowboying, learning a way of life that would sustain them into adulthood.

He loved the outdoors, and his fascination with how nature worked prompted a school guidance counselor’s suggestion that a career in science might be just the thing. He was about 12, and true to form he stuck with it. In high school and then at Reed College he wrestled competitively, and today his supporters and critics alike may detect a bit of his old grappling self in how he approaches a problem—head-on. “It probably taught me how to confront, rather than avoid challenges,” he says now. The sport also led him to his future wife, Roberta McMaster, or Bobbie, who served as his high-school wrestling team’s scorekeeper. “I was fascinated that someone so young knew exactly what he wanted to do with his life,” Bobbie recalls. He proposed marriage before heading for college, and the two have been together ever since and have two grown children.

He attended Washington State University for his PhD in biochemistry, and during that time he and Bobbie often lived on game that he’d hunted. It was not unheard of to find a freshly killed deer hanging in the carport of their student housing. “They were lean years,” Bobbie says. “But they were good ones.”

After positions at Vanderbilt and the University of California, San Francisco, Skinner returned to Washington State University. “I wanted a big research college in a rural town,” he says. He spent the next decade studying how genes turn on and off in ovaries and testes, and how those organs’ cells interact. He wasn’t aiming to take on the central idea in biology for much of the 20th century: genetic determinism, the belief that DNA is the sole blueprint for traits from hair and eye color to athletic ability, personality type and disease risk.

In some sense this interpretation of genetic determinism was always oversimplified. Scientists have long understood that environments shape us in mysterious ways, that nature and nurture are not opposing forces so much as collaborators in the great art of human-making. The environment, for example, can ramp up and pull back on gene activity through methyl groups, as well as a host of other molecules that modify and mark up a person’s full complement of DNA, called the genome. But only changes in the DNA sequence itself were normally passed to offspring.

So certain was everyone of this basic principle that President Bill Clinton praised the effort to complete the first full reading of the human genome, saying in June 2000 that this achievement would “revolutionize the diagnosis, prevention and treatment of most, if not all human diseases.” When stacked against such enthusiasm, Skinner’s findings have felt like heresy. And for a while, at least, he was criticized accordingly.

***

Critics of the Skinner-led research pointed out that the doses of vinclozolin in his rat studies were way too high to be relevant to human exposure, and injecting the rats as opposed to administering the toxins through their food exaggerated the effects. “What he’s doing doesn’t have any real obvious implications for the risk assessments on the chemical,” EPA toxicologist L. Earl Gray was quoted telling Pacific Standard magazine back in 2009. Until the results are replicated, “I’m not sure they even demonstrate basic science principles.”

Skinner responds to assaults on his data by saying that risk assessment, of the type that toxicologists do, has not been his goal. Rather, he’s interested in uncovering new biological mechanisms that control growth, development and inheritance. “My approach is basically to hit it with a hammer and see what kind of response we get,” he says. He remains calm, even when called on to defend that approach. “Conflicts with individuals solve very little,” he says. “The best way to handle these things is to let the science speak for itself.”

That science has received a lot of attention (the vinclozolin study has been cited in the scientific literature more than 800 times). Recently, the journal Nature Reviews Genetics asked five leading researchers to share their views on the importance of epigenetic inheritance. A “mixture of excitement and caution,” is how the editors described the responses, with one researcher arguing that the phenomenon was “the best candidate” for explaining at least some transgenerational effects, and another noting that it might, if fully documented, have “profound implications for how we consider inheritance, for mechanisms underlying diseases and for phenotypes that are regulated by gene-environment interactions.”

Though most of Skinner’s critics have been reassured by new data from his lab and others, he says he still feels embattled. “I really try to be a scientist first and foremost,” he says. “I’m not a toxicologist, or even an environmentalist. I didn’t come to this as an advocate for or against any particular chemical or policy. I found something in the data, and I pursued it along a logical path, the way any basic researcher would.”

Read more: http://www.smithsonianmag.com/ideas-innovations/The-Toxins-That-Affected-Your-Great-Grandparents-Could-Be-In-Your-Genes-231152741.html#ixzz2mIaKLsRH
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Methylomic analysis of monozygotic twins discordant for autism spectrum disorder and related behavioural traits
Open – Molecular Psychiatry advance online publication 23 April 2013; doi: 10.1038/mp.2013.41

C C Y Wong1, E L Meaburn1,2, A Ronald1,2, T S Price1,3, A R Jeffries1, L C Schalkwyk1, R Plomin1 and J Mill1,4

Abstract

Autism spectrum disorder (ASD) defines a group of common, complex neurodevelopmental disorders. Although the aetiology of ASD has a strong genetic component, there is considerable monozygotic (MZ) twin discordance indicating a role for non-genetic factors. Because MZ twins share an identical DNA sequence, disease-discordant MZ twin pairs provide an ideal model for examining the contribution of environmentally driven epigenetic factors in disease. We performed a genome-wide analysis of DNA methylation in a sample of 50 MZ twin pairs (100 individuals) sampled from a representative population cohort that included twins discordant and concordant for ASD, ASD-associated traits and no autistic phenotype. Within-twin and between-group analyses identified numerous differentially methylated regions associated with ASD. In addition, we report significant correlations between DNA methylation and quantitatively measured autistic trait scores across our sample cohort. This study represents the first systematic epigenomic analyses of MZ twins discordant for ASD and implicates a role for altered DNA methylation in autism.

Introduction

Autism spectrum disorder (ASD) defines a collection of complex childhood neurodevelopmental disorders affecting ~1% of the population and conferring severe lifelong disability.1 ASD is characterized by a triad of impairments: (1) deficits in social interactions and understanding, (2) non-social impairments, such as repetitive behaviour and interests, and (3) impairments in language and communication development. Quantitative genetic studies indicate that ASD has a strong heritable component,2 which is supported by the recent identification of several susceptibility loci and an emerging literature implicating the relevance of de novo and inherited copy number variants (CNVs) in the disorder.3 Despite intense research effort during the past decade, however, no definitive biological or clinical markers for ASD have been identified. This can be partly explained by the highly heterogeneous nature of ASD, both clinically and aetiologically. The clinical manifestation of ASD displays considerable individual variability in the severity of impairments and quantitative genetic studies also report genetic heterogeneity between the three trait domains of ASD.4, 5, 6

Despite the high heritability estimates for ASD, there is notable discordance within monozygotic (MZ) twin pairs for diagnosed ASD, and often considerable symptom severity differences within ASD-concordant MZ twins,2 strongly implicating a role for non-genetic epigenetic factors in aetiology. Epigenetic mechanisms mediate reversible changes in gene expression independent of DNA sequence variation, principally through alterations in DNA methylation and chromatin structure.7 Epigenetic changes in the brain have been associated with a range of neurological and cognitive processes, including neurogenesis,8 brain development9 and drug addiction.10 Emerging evidence implicates epigenetic modifications in several neuropsychiatric disorders, including ASD.11, 12 In particular, epigenetic dysregulation underlies the symptoms of Rett syndrome and Fragile X syndrome, two disorders with considerable phenotypic overlap with ASD.11 Although few empirical studies have systematically examined the role of altered epigenetic processes in ASD, recent analyses provide evidence for altered DNA methylation and histone modifications in disease pathology.13, 14, 15

The use of disease-discordant MZ twins represents a powerful strategy in epigenetic epidemiology because identical twins are matched for genotype, age, sex, maternal environment, population cohort effects and exposure to many shared environmental factors.16, 17 Recent studies have uncovered considerable epigenetic variation between MZ twins,18, 19, 20 and DNA methylation differences have been associated with MZ twin discordance for several complex phenotypic traits, including psychosis21 and Type 1 diabetes.22 In ASD, Nguyen and co-workers23 recently examined lymphoblastoid cell lines derived from peripheral blood lymphocytes collected from three ASD-discordant MZ twin pairs, reporting several ASD-associated differentially methylated loci.23 Two loci (RORA and BCL2) reported as hypermethylated in ASD were found to be downregulated in RNA from post-mortem autism brains. These findings support a role for DNA methylation in ASD and highlight the successful use of peripherally derived DNA from discordant MZ twins to identify disease-associated epigenetic changes. Given the highly heterogeneous nature of ASD, however, more comprehensive genome-wide analyses across larger numbers of samples are warranted to investigate the extent to which ASD-associated epigenetic variation is individual- and symptom-specific.

Materials and methods

Samples for methylomic analysis

Participants were recruited from the Twins’ Early Development Study (TEDS), a United Kingdom-based study of twins contacted from birth records.24 For this study, a total of 50 MZ twin pairs were identified within TEDS using the Childhood Autism Symptom Test (CAST), which assesses dimensional ASD traits, at age 8 years. The CAST25 is a 31-item screening measurement for ASD, designed for parents and teachers to complete in non-clinical settings to assess behaviours characteristic of the autistic spectrum. Items within the CAST are scored additively and a score of 15 (that is, answering ‘yes’ on 15 items) is the cutoff for identifying children ‘at risk’ for ASD. On the basis of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition criteria for autism, CAST items can be divided into three subscales: impairments in social symptoms (12 items); impairments in non-social symptoms (that is, restricted repetitive behaviours and interests (RRBIs); 7 items); and communication impairments (12 items).6 The CAST has been widely used in population-based studies of singletons25 as well as in twin studies.26 Within TEDS, the CAST has been shown to have good reliability and validity.27 Supplementary Figure 1 shows the distribution of total CAST and its three subscale scores within samples selected using parent ratings. Supplementary Table 1 provides a summary of the samples included in the analyses. Whole-blood samples were collected from subjects at age 15 years by a trained phlebotomist for DNA extraction and blood cell-count analysis. Blood cell counts were assessed for all collected samples and found to be within the normal range.

Genome-wide analysis of DNA methylation

For each individual, genomic DNA (500 ng) extracted from whole blood was treated with sodium bisulphite using the EZ 96-DNA methylation kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s standard protocol. The bisulphite conversion reaction was performed in duplicate for each sample to minimize potential bias caused by variable conversion efficiency, and pooled bisulphite-treated DNA was used for subsequent array analysis. Genome-wide DNA methylation was assessed using the Illumina Infinium HumanMethylation27 BeadChip (Illumina, San Diego, CA, USA), which interrogates the DNA methylation profile of 27 578 CpG sites located in 14 495 protein-coding gene promoters and 110 microRNA gene promoters, at single-nucleotide resolution.28 Illumina GenomeStudio software (Illumina, San Diego, CA, USA) was used to extract signal intensities for each probe and perform initial quality control checks, with all data sets (except two individuals) being considered to be of high quality and included in subsequent analyses. To ensure stringent data quality, probes with a detection P-value >0.05 in any of the samples were removed across all individuals (N=1161 probes) in addition to a set of probes (N=2923) that were reported as nonspecific and potentially unreliable in a recent survey of all probes on the microarray.29

Methylation microarray data processing

All computations and statistical analyses were performed within the R statistical analysis environment (http://www.r-project.org), and all analysis scripts are available on request from the authors. A customized pipeline was used for the analysis of Illumina 27K methylation data as described in a previous study of psychosis-discordant MZ twins.21 Briefly, signal intensities for each probe were normalized using quantile normalization to reduce unwanted interarray variation. The relative methylation level of each interrogated CpG site was calculated as the ratio of the normalized signal from the methylated probe to the sum of the normalized signals of the methylated and unmethylated probes. This gave an average DNA methylation value, described as average ‘β-value’ for each CpG site, ranging from 0 (unmethylated) to 1 (fully methylated). A density plot of β-values for every sample revealed that, as expected given the known distribution of probes on the array, the data followed a bimodal distribution (Supplementary Figure 2). An empirical variance stabilizing transformation was used to adjust for the bimodal distribution of the data.21 Raw microarray data are available for download from http://epigenetics.iop.kcl.ac.uk/ASDTwins/.

Identification of ASD-associated DMRs

Two major analysis strategies were used to identify DMRs associated with ASD and related traits. First, DNA methylation differences within pairs of MZ twins were examined in MZ twin pairs discordant for ASD and ASD-related traits. Second, case–control comparisons of DNA methylation were performed between groups of individuals scoring high and low for ASD traits. With the aim of identifying real, biologically relevant within-twin and between-group DNA methylation differences, we used an analytic approach that incorporates both the significance (that is, t-test statistic) and magnitude (that is, absolute delta-β (Δβ)) of any observed differences to produce a ranked list of DMRs.21 A summary of the analysis strategy is presented in Supplementary Figure 3. This combined approach, where data are interpreted based on the combination of fold change and statistical significance, is routinely used in genome-wide gene expression studies and has been shown to produce gene lists of higher reproducibility and biological relevance.30 We recently used a similar approach successfully to identify disease-associated epigenetic changes in a psychosis-discordant MZ twin study.21 Given the known phenotypic and aetiologic heterogeneity, we also screened for large Δβ-values within each discordant MZ twin pair to examine the possibility that disease-associated epigenetic changes are potentially private and not consistent across all families. Finally, we examined whether quantitative CAST scores are correlated with DNA methylation at specific loci. The association between each of the quantitatively rated CAST subscale variables and DNA methylation at each CpG site was assessed using Pearson’s product–moment correlation.

Global DNA methylation analysis

Global levels of DNA methylation were quantified using the LUminometric Methylation Assay (LUMA).31 This method relies on DNA cleavage by methylation-sensitive and -insensitive restriction enzymes, followed by the quantification of the resulting restriction fragments using pyrosequencing.31 Positive controls, including both artificially methylated and artificially unmethylated samples, were included in all experimental steps to ensure unambiguous restriction enzyme digestions and to calibrate the experimental data, with each sample being processed in duplicate.

Fine mapping of DNA methylation using bisulphite pyrosequencing

Although the Illumina 27K array has been well validated for detecting differences in DNA methylation, we further tested specific regions nominated from the genome-wide microarray analysis using bisulphite pyrosequencing. Independent verification analyses were performed on two CpG sites (cg16474696, MGC3207; cg20507276, OR2L13) that demonstrated a large significant ASD-associated difference from the case versus control analysis. In each case, the assay spanned multiple CpG sites, including the specific CpG interrogated on the Illumina 27K array. Briefly, 500 ng DNA from each individual was independently treated with sodium bisulphite in duplicate using the EZ 96-DNA methylation kit as described above. Bisulphite-polymerase chain reaction amplification was performed in duplicate. Quantitative DNA methylation analysis was conducted using the PyroMark Q24 pyrosequencer (Qiagen, Valencia, CA, USA). The correlation between DNA methylation estimates obtained from Illumina 27K array and bisulphite pyrosequencing was assessed using Pearson’s moment–correlation coefficient. In addition, Sanger sequencing using BigDye v.3.1 terminator mix (Applied Biosystems, Foster City, CA, USA) was performed on the regions targeted by the MGC3207 pyrosequencing assay to ensure that the Illumina probe sequences and the primer binding sites for the pyrosequencing assay were free of any DNA sequence variation. The primers and assay conditions are given in Supplementary Table 2.

CNV analysis using genotyping arrays

Genomic DNA (200 ng) extracted from whole blood was genotyped using the Illumina HumanOmniExpress BeadChip (Illumina) targeting >730 000 single-nucleotide polymorphisms and Illumina GenomeStudio software was used to call genotypes based on predefined genotype cluster boundaries to denote cluster positions (HumanOmniExpress-12v1_C.egt). CNVs were identified from the genotyping data using two independent algorithms, PennCNV32 and QuantiSNP,33 with default parameters, and GC content signal preprocessing was applied. Stringent quality control steps were used to ensure that only high-confidence CNVs, that is, those >1 kb in size, covered by >5 probes and detected by both programs, were included for further analysis.

Results

ASD is not associated with systemic differences in global DNA methylation
As expected, within-twin patterns of DNA methylation were highly correlated across all MZ twin pairs (average within-twin r across all probes=0.99), indicating that ASD and related traits are not associated with systemic changes in epigenetic programming. Supplementary Figure 4a shows the correlation between genome-wide DNA methylation across all probes on the array and one example ASD-discordant MZ twin pair; data for the other ASD-discordant MZ pairs are available for download from http://epigenetics.iop.kcl.ac.uk/ASDTwins/. These data were corroborated by global DNA methylation analysis using LUMA, which identified no significant difference between affected ASD twins and their co-twins (affected ASD twins mean=65.1%, unaffected co-twins mean=65.9%; P=0.817) (Supplementary Figure 4b).

Site-specific DNA differences are widespread in MZ-discordant ASD twins

In contrast to global levels of DNA methylation, DNA methylation at individual CpG sites demonstrated considerable variability within ASD-discordant MZ twin pairs. Figure 1a shows the distribution of average absolute differences in DNA methylation (Δβ) within all MZ twins discordant for ASD and ‘control’ MZ twin pairs concordant for low autistic trait score (unaffected). The overall distribution of average within-pair DNA methylation differences showed a highly significant skew to the right in ASD-discordant twins (P<2.2e−16, Kolmogorov–Smirnov test), with a higher number of CpG sites demonstrating a larger average difference in DNA methylation. Using an analysis method designed to identify the largest and most significant differences in DNA methylation at individual CpG sites, we identified multiple CpG sites across the genome exhibiting significant ASD-associated differential DNA methylation (Table 1). Of note, variability at these sites appears to be specific to ASD-discordant twin pairs; for the 50 top-ranked ASD-associated DMRs, we observe significantly higher average within-pair differences for MZ twin pairs discordant for ASD (P<0.01; see Figure 1b). The top differentially methylated site (cg13735974) across all ASD-discordant MZ twin pairs located in the NFYC promoter was consistently hypermethylated in affected individuals compared with their unaffected co-twins (mean Δβ=0.08, range=0.04–0.10, P<0.0004). For the top 10 DMRs, Figure 2 indicates highly consistent differences across all six ASD-discordant twin pairs.

Large DNA methylation differences are observed at specific loci within individual ASD-discordant MZ twin pairs

Because ASD is a highly heterogeneous disorder,3 it is probable that many disease-associated DMRs are family-specific. We therefore screened for the largest family-specific DNA methylation differences within each discordant ASD twin pair, identifying numerous loci (average=37.4 per twin pair) showing large DNA methylation differences (Δβ0.15) within each discordant twin pair (Supplementary Figure 5 and Supplementary Table 3). Although the majority of DMRs of large magnitude are family-specific, several are common across two or more discordant twin pairs in the same direction: cg12164282, located in PXDN promoter, showed ASD hypomethylation in twin pair 2 (Δβ=−0.19) and twin pair 4 (Δβ=−0.28); cg04545708, located in exon 1 of C11orf1, showed ASD hypermethylation in both twin pair 3 (Δβ=0.23) and twin pair 6 (Δβ=0.35); cg20426860, located in exon 1 of TMEM161A, showed ASD hypermethylation in twin pair 4 (Δβ=0.21) and twin pair 6 (Δβ=0.27); and cg27009703, located in HOXA9 promoter, showed ASD hypermethylation in twin pair 1 (Δβ=0.19) and twin pair 4 (Δβ=0.21).

DNA methylation differences are observed in MZ twins discordant for ASD-related traits

We detected significant DNA methylation differences between MZ twin pairs discordant for the three ASD-associated traits: that is, social autistic traits (N=9 MZ pairs), autistic RRBIs (N=9 MZ pairs) and communication autistic traits (N=8 MZ pairs). The top-ranked DMRs for each trait are shown in Supplementary Figure 6 and Supplementary Table 4. Interestingly, these included several genes previously implicated in the aetiology of ASD, including GABRB3, AFF2, NLGN2, JMJD1C, SNRPN, SNURF, UBE3A and KCNJ10.
As ASD is composed of a triad of all three impairments, we also examined if any CpG sites are differentially methylated across all discordant twin pairs (N=32 pairs, 64 individuals), regardless of their focal impairment. The top DMRs across all discordant twin pairs are shown in Supplementary Figure 7 and Supplementary Table 5. The top-ranked DMR located in the promoter region of PIK3C3 (cg19837131) was significantly hypomethylated in affected individuals compared with their unaffected co-twins (mean Δβ=−0.04, P<0.00004). Interestingly, while the overall average difference at this locus is small, the range of within-twin methylation difference is much greater (Δβ ranges from −0.12 to 0.6) and that the direction of effect is strikingly consistent across the majority of individual twin pairs, with 25 out of 32 discordant pairs (78%) demonstrating trait-related hypomethylation (Supplementary Figure 7).

Between-group analyses identified additional ASD-associated DMRs

Our study design also permitted us to examine group-level DNA methylation differences between ASD cases and controls. Unlike the within-pair discordant MZ twin design, between-group DNA methylation differences can be attributable to both genetic and environmental factors. Given the known gender difference in DNA methylation across the X chromosome, these analyses were restricted to probes on the autosomes (N=22 678) to minimize gender-induced biases.

Numerous DNA methylation differences were observed between ASD cases and controls. Supplementary Table 6a and Supplementary Figure 8 highlight the CpG sites showing the largest absolute DNA methylation differences (mean Δβ0.15) between ASD cases and unrelated control samples. The top case–control ASD-associated DMR was located upstream of MGC3207 (cg16474696), which was significantly hypomethylated in ASD cases compared with control samples (mean Δβ=−0.24, P<0.0002). In addition to MGC3207, large significant ASD-associated differences were observed in several other loci, including CpG sites near OR2L13 (cg20507276; mean Δβ=0.18) and C14orf152 (cg20022541; mean Δβ=−0.16, data not shown). Verification experiments were conducted on MGC3207 and OR2L13 using bisulphite-pyrosequencing confirming a high correlation (r=0.91 and 0.86, for MGC3207 (total N=33) and OR2L13 (total N=35), respectively) in DNA methylation levels, detected using the Infinium microarray and pyrosequencing platforms. Although our list of DMRs was stringently filtered to exclude probes containing known polymorphic SNPs,29 several of the top-ranked case–control DMRs, including cg16474696 and cg20507276, demonstrated patterns of DNA methylation consistent with DNA sequence effects, suggesting that they may be mediated by cis effects on DNA methylation34 or potentially reflect technical artefacts caused by uncatalogued sequence variation in probe binding sequences. To exclude the latter for MGC3207, we sequenced genomic DNA across the DMR in a range of samples showing differential methylation and identified no obvious polymorphic DNA sequence variation in the immediate vicinity of the probe.

Epigenetic differences identified between sporadic and familial ASD cases

ASD is an aetiologically heterogeneous syndrome and can occur both as a sporadic and a familial disorder. Recent CNV analyses report considerably higher frequencies of de novo variation in simplex compared with multiplex ASD families,35 suggesting that they represent genetically distinct classes. To test whether these are epigenetically distinct, we compared DNA methylation between individuals with sporadic ASD (where ASD is reported in only one member of the MZ twin pair; N=6) and individuals with familial ASD (as observed in concordant ASD MZ twin pairs; N=10). The genes most proximal to the 50 top-ranked differentially methylated CpG sites from this analysis are listed in Supplementary Table 6b. The top differentially methylated CpG site (cg07665060) is located upstream of C19orf33, which was significantly hypomethylated in individuals affected by sporadic ASD compared with those affected by familial ASD (mean Δβ=−0.12, P<0.0008) (Supplementary Figure 9). Interestingly, significant DNA methylation differences were also observed near several genes that have been previously implicated in ASD, including MBD4, AUTS2 and MAP2.

There is some overlap in DMRs across analytical groups

Table 2 provides a full list of top-ranked DMRs demonstrating overlap between analytical groups and highlighting their potential relevance to different autism-associated phenotypes. Interestingly, the top-ranked locus from the ASD-discordant twin analysis, located near NFYC, was also differentially methylated in the case–control analysis (mean Δβ=0.04, P<0.003). Furthermore, we identified significant DNA methylation differences in the MBD4 promoter in both ASD-discordant twin analysis and sporadic versus familial ASD analysis, suggesting that MBD4 methylation may have functional relevance to sporadic ASD. For each of the 50 top-ranked probes in each analysis category, Supplementary Table 7 lists their corresponding rank across the other analysis groups; although there is some overlap across groups (and each ranked list is positively, although modestly, correlated; Supplementary Table 8), few CpG sites are consistently altered across multiple analytical groups.

Quantitative autistic trait scores are correlated with DNA methylation at multiple CpG sites

Supplementary Figure 1 shows the distribution of total CAST and its three trait subscale scores across our samples. Initial analyses highlighted a strong correlation between DNA methylation and CAST score at multiple CpG sites (Supplementary Table 9). Further analysis showed that many of these correlations are influenced by extreme DNA methylation levels and phenotypic scores exhibited by one male ASD-concordant MZ twin pair (Figure 3a and Supplementary Figure 10). These twins are extreme outliers for CAST score (both scored 29 out of a maximum score of 31) and DNA methylation at multiple CpG sites (Supplementary Figure 11), and both have a history of pervasive developmental problems, with severe behavioural phenotypes and early-appearing IQ deficits, with special deficits in language. Given the existing link between highly penetrant CNVs and severe ASD, we tested whether the extreme patterns of DNA methylation in these two twins were associated with the presence of genomic alterations. Interestingly, high-density SNP microarray analysis revealed significant structural genomic alterations at multiple loci, with CNVs detected in regions previously implicated in ASD (Supplementary Table 10).

DNA methylation at multiple CpG sites remained significantly correlated with CAST scores even after this extreme twin pair was excluded from analyses (Supplementary Table 11 and Figure 3b), suggesting that they do not necessarily represent epigenetic/phenotypic ‘outliers’ but have DNA methylation levels (and phenotypic scores) at the extreme end of a true quantitative spectrum. For example, there is a strong correlation between DNA methylation at cg07753644 in P2RY11 and total CAST score in both analyses (with extreme twin pair: r=0.44; P=0.000009; without extreme twin pair: r=0.35; P=0.0006). Furthermore, DNA methylation at cg16279786 in the known ASD susceptibility locus, NRXN1, is significantly correlated with social autistic trait score in both analyses with (r=−0.41; P=0.00003) and without (r=−0.28; P=0.007) the extreme twin pair.

Discussion

This study represents the first comprehensive analysis of DNA methylation differences in MZ twins discordant for ASD and autism-related traits using a genome-wide approach. We report ASD-associated DNA methylation differences at numerous CpG sites, with some DMRs consistent across all discordant twin pairs for each diagnostic category and others specific to one or two twin pairs, or one or two autism-related traits. Although sporadic cases of ASD appear to be epigenetically distinct to familial cases of ASD, some DMRs are common across both discordant MZ twin and case–control analyses. We also observed that DNA methylation at multiple CpG sites was significantly correlated with quantitatively rated autistic trait scores, with our analyses identifying one MZ twin pair, concordant for a very severe autistic phenotype, that appear to represent epigenetic outliers at multiple CpG sites across the genome. Interestingly, both individuals harbour numerous CNVs in genomic regions previously implicated in autism. Given the important role of epigenetic mechanisms in regulating gene expression, it is plausible that, like CNVs, methylomic variation could mediate disease susceptibility via altered gene dosage. Our hypothesis-free experimental design allowed us to identify disease-associated DNA methylation differences at loci not previously implicated in ASD, although we also found evidence for epigenetic changes at several genes previously implicated in autism.

Our findings have several implications for our understanding about the aetiology of ASD. First, they document the presence of numerous DNA methylation differences in MZ twins discordant for ASD and ASD-related traits, as well as between autistic individuals and control samples. This concurs with findings from a previous ASD-discordant twin study23 and further supports the association of variable DNA methylation with phenotypic differences between genetically identical individuals.21, 22 Second, the observed DNA methylation differences in MZ twins discordant for ASD and ASD-related traits, who are otherwise matched for genotype, shared environment, age, sex and other potential confounders, highlight the role of non-shared environmental and stochastic factors in the aetiology of autism. These findings concur with mounting data suggesting that environmentally mediated effects on the epigenome may be relatively common and important for disease.36 Third, our data suggest that although DNA methylation at some CpG sites is consistently altered across the entire set of discordant twins, differences at other CpG sites are specific to certain symptom groups, with considerable overall epigenetic heterogeneity between the three domains of autistic traits. These findings are in line with recent genetic research demonstrating significant genetic heterogeneity between the three core symptoms of ASD.4, 6 Fourth, the analysis of individual ASD-discordant twin pairs suggests that there is also considerable familial heterogeneity, with rare epigenetic alterations of large magnitude being potentially associated with ASD. These findings are not entirely surprising given the known heterogeneous nature of ASD revealed by molecular genetic studies,3 with an important role for highly penetrant rare genomic alterations, especially de novo mutations. Fifth, the identification of significant correlations between DNA methylation and autism symptom scores across our sample cohort suggests that there is a quantitative relationship between the severity of the autistic phenotype and epigenetic variation at certain loci. This reinforces the view of autism as the quantitative extreme of a phenotypic spectrum and highlights the potential use of epigenetic biomarkers as a predictor for severity of symptoms, although the accuracy, sensitivity and specificity of such predictors would require extended investigation. Finally, in addition to implicating a number of novel genes in the aetiology of ASD, we identified ASD-associated differential DNA methylation in the vicinity of multiple loci previously implicated in the pathogenesis of autism in genetic studies, including AFF2, AUTS2, GABRB3, NLGN3, NRXN1, SLC6A4 and UBE3A (see Supplementary Table 12 for a comprehensive list).

This study has several strengths. First, our unique sample consisted of MZ twin pairs discordant for autism and ASD-related-traits, in addition to age-matched concordant MZ twin pairs (for both ASD and low CAST score). It allowed us to perform a comprehensive analysis of the role of DNA methylation in ASD and ASD-related traits controlling for genotype, age, sex and other potential confounders. Second, by undertaking a genome-wide approach using a robust and reliable array platform, we were able to uncover phenotype-relevant differentially methylated loci in genomic regions that are both novel and have been previously associated with ASD. Third, our analysis of 32 discordant MZ twin pairs is relatively large compared with other discordant twin studies performed for other complex disease phenotypes; in this regard, for example, the only other ASD-discordant twin study assessed only three MZ twin pairs.23 Finally, we were able to complement our discordant-twin analyses by assessing group-level differences between ASD cases and controls, and also examining the relationship between DNA methylation and quantitatively rated trait scores across our entire sample cohort.

This study also has a number of limitations that should be considered when interpreting the results. First, although this is the largest and most comprehensive study of epigenetic variation in ASD performed to date, the sample size for each subgroup is small, in part because truly discordant MZ twin pairs are relatively rare. Although none of the reported differentially methylated loci reached a Bonferroni-corrected P-value cutoff (P=2.13E–05 for discordant-twin analysis and P=2.20E–05 for between-group analysis), this statistical approach is likely to be too conservative, especially given the non-independence of CpG sites37 and the small numbers of samples tested in each group. In this study, a combined analytic approach, taking into account the significance and the extent of methylation change, was used to identify differentially methylated loci that have potentially real, biological relevance to ASD. This analytic approach is widely used in genome-wide gene expression studies and is reported to produce gene lists of higher reproducibility and biological relevance compared with the convention method that relies solely on statistical significance.30 This notion is supported by the identification of differentially methylated loci near numerous genes previously implicated in ASD. Nonetheless, given the relatively small subgroup sample size, replication in larger samples is needed. Second, genome-wide DNA methylation profiling was performed on DNA extracted from whole blood, controlled for cell count, rather than the brain. Unfortunately, there is no archived collection of post-mortem brain samples from ASD-discordant MZ twins. Although there are known tissue-specific differences in DNA methylation profiles, recent studies suggest that disease-associated epimutations may be detectable across tissues,38 and our recent work suggests that some between-individual epigenetic variation is conserved across brain and blood.39 Furthermore, ASD-associated epimutations have been demonstrated to be detectable both in the brain and in peripheral tissues (that is, blood).13, 23 Moreover, our identification of DMRs in the vicinity of genes previously implicated in autism supports the notion that disease-relevant gene network and pathways can be identified from peripheral samples. Nonetheless, it would be informative for future studies to assess whether disease-associated epimutations reported from this study are also present in brain samples from ASD patients. Third, informations pertaining to the amniotic and chorionic status of our twin samples are unavailable, preventing us from further dissecting the epigenetic similarity/dissimilarity between twins sharing their placenta and/or amniotic sac. Fourth, the genome-wide platform used for this study (the Illumina 27K array), although robust and highly reliable,28 has a somewhat limited density of probe coverage, assaying only one or two CpG sites per gene. Future studies should take advantage of recent advances in genomic profiling technology and perform a more in-depth examination of methylomic differences associated with ASD. Finally, it is difficult to draw conclusions about causality for any of the ASD-associated DMRs identified in this study, in part, because we do not have corresponding RNA expression data, or DNA samples from the twins taken before they became discordant for ASD. It is thus plausible that many of the identified changes have occurred downstream of ASD, for example, resulting from exposure to medications commonly used to treat autistic symptoms. In fact, there is mounting evidence that many drugs used to treat neuropsychiatric disorders induce epigenetic changes.40 Such medication-induced changes could still be interesting; an understanding of the pathways via which these drugs work may provide information about the neurobiological processes involved in disease. The ideal study design, however, would assess DNA methylation changes in the brain longitudinally during individuals’ transition into ASD, although such a study does not appear feasible at present.

In summary, this is the first large-scale study to examine the role of genome-wide DNA methylation in ASD and ASD-related traits. Our findings show that: (1) there are numerous DNA methylation differences between MZ twins discordant for ASD and ASD-related traits, as well as between autistic individuals and control samples; (2) many of these DMRs are located in the vicinity of both novel genes and loci that have been previously implicated in ASD; (3) the nature of ASD-associated epimutations is complex with high heterogeneity between individuals; (4) there is high epigenetic heterogeneity between the triad of impairments that define ASD; and (5) there is a quantitative relationship between the severity of the autistic phenotype and DNA methylation at specific CpG sites across the genome. Overall, our findings from this study provide further support for the potential role of DNA methylation in ASD and ASD-related traits.

To review the figures you can access the full study here http://www.nature.com/mp/journal/vaop/ncurrent/full/mp201341a.html
Methylomic analysis of monozygotic twins discordant for autism spectrum disorder and related behavioural traits

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The Man Working To Reverse-Engineer Your Brain – February 29, 2012 NPR
http://www.npr.org/2012/02/29/147190092/the-man-working-to-reverse-engineer-your-brain

Our brains are filled with billions of neurons, entangled like a dense canopy of tropical forest branches. When we think of a concept or a memory — or have a perception or feeling — our brain’s neurons quickly fire and talk to each other across connections called synapses.

How these neurons interact with each other — and what the wiring is like between them — is key to understanding our identity, says Sebastian Seung, a professor of computational neuroscience at MIT.

Seung’s new book, Connectome: How the Brain’s Wiring Makes Us Who We Are, explains how mapping out our neural connections in our brains might be the key to understanding the basis of things like personality, memory, perception and ideas, as well as illnesses that happen in the brain, like autism and schizophrenia.

“These kinds of disorders have been a puzzle for a long time,” says Seung. “We can look at other brain diseases, like Alzheimer’s disease and Parkinson’s disease, and see clear evidence that there is something wrong in the brain.”

But with schizophrenia and autism, there’s no clear abnormality during autopsy dissections, says Seung.

“We believe these are brain disorders because of lots of indirect evidence, but we can’t look at the brain directly and see something is wrong,” he says. “So the hypothesis is that the neurons are healthy, but they are simply connected together or organized in an abnormal way.”

One current theory, says Seung, is that there’s a connection between the wiring that develops between neurons during early infancy and developmental disorders like schizophrenia and autism.

“In autism, the development of the brain is hypothesized to go awry sometime before age 2, maybe in the womb,” he says. “In schizophrenia, no one knows for sure when the development is going off course. We know that schizophrenia tends to emerge in early adulthood, so many people believe that something abnormal is happening during adolescence. Or it could be that something is happening much earlier and it’s not revealed until you become an adult.”

What scientists do know, he says, is that the wiring of the brain in the first three years is critical for development. Infants born with cataracts in poor countries that don’t have the resources to restore their eyesight remain blind even after surgery is performed on them later in life.

“No matter how much they practice seeing, they can never really see,” says Seung. “They recover some visual function, but they are still blind by comparison to you and me. And one hypothesis is that the brain didn’t wire up properly when they were babies, so by the time they become adults, there’s no way for the brain to learn how to see properly.”

At birth, he says, you are born with all of the neurons you will ever have in life, except for neurons that exist in two specific areas of the brain: the dentate gyrus of the hippocampus, which is thought to help new memories form, and the olfactory bulb, which is involved in your sense of smell.

“The obvious hypothesis [is] that these two areas need to be highly plastic and need to learn more than other regions, and that’s why new neurons have to be created — to give these regions more potential for learning,” says Seung. “But we don’t really have any proof of that hypothesis.”

But not everything is set in stone from birth. The complex synaptic connections that allow neurons to communicate with one another develop after babies have left the womb.

“As far as we know, this is happening throughout your life,” he says. “Part of the reason that we are lifelong learners — that no matter how old you get, you can still learn something new — may be due to the fact that synapse creation and elimination are both continuing into adulthood.”

Connectomes: Reverse-Engineering The Brain

Only one organism has had its full connectome — or neural map — mapped out by neuroscientists. It’s a tiny worm no bigger than a millimeter, but it took scientists more than a dozen years to map out its 7,000 neural connections. They started out by using the world’s most powerful knife and slicing the worm into slices a thousand times thinner than a human hair. They then put each slice in an electron microscope and created a 3-D image of the worm’s nervous system. That’s when the true labor started, says Seung.

“That’s when [neuroscientists had to] go through all these images and trace out the paths taken by all of the branches of the neurons and find the synapses, and compile all that information to create the connectome,” he says.

Each of the worm’s 300 neurons had between 20 and 30 connections. In comparison, humans have 10,000 connections of neurons — and billions of neurons. And scientists still aren’t sure what the various pathways in a worm’s nervous system mean.

“We’re still far away from understanding the worm,” says Seung. He says that scientists would like to eventually map a 1-millimeter cube of a human brain or a mouse brain, which contains 100,000 neurons and a billion connections.

“The imaging of all of those slices of brain can be automated and made much more reliable,” he says. “And now we have computers that are getting better at seeing.”

So far, though, neuroscientists have only mapped the neural connections of a piece of a mouse retina, which is very thin.

“What we know in the retina is a catalog of the types of neurons,” he says. “The next challenge is to figure out what are the rules of connection between these types of neurons. And that’s where we still don’t know a whole lot.”

Mapping more of these connections, he says, will tell us a lot about brain function and possible pathways that can be treated.

“I don’t want to promise too much, and my goal right now is simply to see what is wrong,” he says. “That’s not in itself a cure. But obviously it’s a step toward finding better treatments. The analogy I make is the study of infectious diseases before the microscope. You could see the symptoms, but you couldn’t see the microbes — the bacteria that caused disease. We’re in an analogous stage with mental disorders. We see the symptoms, but we don’t have a clear thing we can look at in the brain and say, ‘This is what’s wrong.’ ”

http://www.npr.org/2012/02/29/147190092/the-man-working-to-reverse-engineer-your-brain

Bernard Weiss
http://www2.envmed.rochester.edu/envmed/tox/faculty/weiss.html

Neurobiology and Behavior

Our brains, the ultimate product of millions of years of evolution, are what make us human. But over the past few decades, scientists have discovered that many chemicals in our environment threaten the integrity of our brains. Thousands more have never been studied for their effects. We know some of the outcomes: reduced intelligence and cognitive function, increased antisocial tendencies, impaired senory and motor function, and elevated risks of neurodegenerative disorders such as Parkinson’s disease.

Most of these chemicals are ubiquitous and persistent. We are exposed throughout our lifetimes. But some periods of life are more vulnerable than others. Early development is an especially perilous time for exposure to toxic chemicals. The brain is exquisitely sensitive during this period because of the many paths by which it expands and differentiates on the path to maturity. Cells divide and proliferate; they migrate to specific target areas; they grow connections to other cells to form massive neural networks; neurotransmitter systems take root. All these processes are candidates for interference by toxic chemicals. All are reflected in neurobehavioral outcomes that can be measured when organisms mature to a stage at which they can be tested by procedures that are sensitive to such interference. Late in life, we enter another period of enhanced vulnerability. We are not as able as during earlier periods to compensate for toxic processes and many of our organ systems operate at diminished capacity. It is also a period when these reduced capacities may begin to reflect the damage inflicted earlier in life.

My own research aims to relate behavioral measures to neurotoxicant exposure. Behavioral research occupies a special role in safety assessment because it offers the ability to trace changes in function as organisms mature and age. Among the endpoints of salient interest to regulatory agencies such as EPA and to chemical and pharmaceutical manufacturers are learning capacity, other aspects of cognitive capacity, motor and sensory performance, and differences between males and females.

My efforts have spanned a variety of agents: metals such as mercury and manganese; solvents such as toluene and methanol; air pollutants such as ozone; adventitious contaminants such as dioxin; and endocrine disruptors, which include common ingredients in consumer products such as phthalates.

For more on Bernard Weiss and his research see below.

School of Medicine & Dentistry
Molecular Toxicology & Environmental Medicine Cluster
Ph.D. Program in Toxicology

Bernard Weiss
Professor of Environmental Medicine
and Pediatrics;
Environmental Health Sciences Center,
and Center for Reproductive Epidemiology.
B.A. 1949 (New York University)
Ph.D. 1953 (University of Rochester)

http://www2.envmed.rochester.edu/envmed/tox/faculty/weiss.html

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Unnatural selection: How humans are driving evolution by Michael Le Page
New Scientist – 27 April 2011

Humans are not only causing a mass extinction – we are also the biggest force in the evolution of the species that will survive

THE Zoque people of Mexico hold a ceremony every year during which they grind up a poisonous plant and pour the mixture into a river running through a cave (pictured below). Any of the river’s molly fish that float to the surface are seen as a gift from the gods. The gods seem to be on the side of the fish, though – the fish in the poisoned parts of the river are becoming resistant to the plant’s active ingredient, rotenone.

If fish can evolve in response to a small religious ceremony, just imagine the effects of all the other changes we are making to the planet. We are turning grassland and forests into fields and cities, while polluting the air and water. We are hunting species to the brink of extinction and beyond, as well as introducing new pests and diseases to just about every part of the world. And that’s not to mention drastically altering the climate of the entire planet.

It is no secret that many – perhaps even most – species living today are likely to be wiped out over the next century or two as a result of this multiple onslaught. What is now becoming clear is that few of the species that survive will live on unchanged.

Far from being a slow process, evolution can occur extremely rapidly when the environment changes (New Scientist, 2 April, p 32). So, as we alter the planet ever faster and more drastically, we are becoming the main force driving evolution. “The intensity of the ecological effect of man is pretty obvious,” says Stephen Palumbi of Stanford University in California. “There is an amazing amount of evolutionary change as a result.”

Some of the fastest rates of evolution ever measured in the wild are in plants and animals harvested by humans. The few populations for which we have data are, on average, evolving three times as fast as populations subject only to natural pressures, for example.

Over the following pages, we look at the many ways in which plants and animals are already evolving in response to human pressures. Some of these changes, such as animals evolving to survive in highly polluted areas, can be seen as a positive thing. Others are bad from our point of view, such as animals we hunt losing the traits we value most in them, or pests becoming immune to poisons. What is clear is that whether the issue is growing enough food, conserving wild animals or keeping our beds bug-free, human-driven evolution is a factor we can no longer afford to ignore.

Unnatural selection: Hunting down elephants’ tusks

Most predators target the young or the weak. We are different, targeting the biggest and best, or those with characteristics we desire, such as large antlers. Combine this with our ability to kill in great number and the result is extremely rapid evolution of our prey.

The first clear evidence was published in 1942, and since then many examples have emerged of how hunting can transform the hunted. The targeting of large animals has resulted in a fall in the average size of caribou in some areas, for instance, while trophy hunting has led to bighorn sheep in Canada and mouflon in France evolving smaller horns.

Perhaps the most dramatic example is the shrinking of tusks in elephants, or even their complete loss. In eastern Zambia, the proportion of tuskless female elephants shot up from 10 per cent in 1969 to nearly 40 per cent in 1989 as a result of poaching (African Journal of Ecology, vol 33, p 230). Less dramatic rises in tusklessness have been reported in many other parts of Africa, with some bull elephants losing tusks too.

Humans have had an even bigger impact in Asia. Only male Asian elephants have tusks, and the proportion of tuskless bulls has soared in many areas. In Sri Lanka, where there has been a lot of poaching, under 5 per cent of males now have tusks, says Raman Sukumar of the Indian Institute of Science in Bangalore, who studies Asian elephants. Simulations by Ralph Tiedemann of the University of Potsdam in Germany and colleagues suggest that female elephants’ preference for tuskers has partly counteracted the effect of hunting. However, even if all poaching stopped, it would take a very long time for the percentage of tuskers to rise again.

It’s not just animals that are being shaped by human preferences: the harvesting of wild plants can have a similar effect to hunting and fishing. In Tibet, for example, the height of the snow lotus at flowering has nearly halved over the past century as a result of the flowers being picked for use in traditional medicine (Proceedings of the National Academy of Sciences, vol 102, p 10218).

To even the balance, some biologists are now promoting the idea of counteracting the evolutionary pressures of hunting through “compensatory culling” – killing animals with undesirable traits. This has actually long been done in some places. In Germany and Poland, for instance, there is a tradition of shooting yearling deer with poor antlers to prevent a decrease in the antler size of mature stags.

Private reserves in countries such as Zimbabwe have a similar policy. They typically charge hunters a smaller “trophy fee” for shooting tuskless elephants – $3000 versus at least $12,500 for a tusker, for example. This is partly because tuskless animals are less valuable, but it is also a deliberate attempt to eliminate the trait.

Unnatural selection: The race against climate change

In Finland, the tawny owl used to be mainly grey. But since the 1960s, the proportion of a brown subtype has risen fast. “The frequency averaged around 12 per cent in the early 60s and has increased steadily to over 40 per cent nationwide,” says Patrik Karell of the University of Helsinki, whose findings were published earlier this year (Nature Communications, DOI: 10.1038/ncomms1213).

His team found that grey owls (pictured above right) have an advantage over brown ones only when there is lots of snow. As winters have become milder, the brown subtype has thrived. It is possible that this is because brown owls are better camouflaged when there is less snow, but it could also be because brown coloration is linked to another characteristic, such as higher energy needs.

There are countless examples of how global warming is affecting life, from plants flowering earlier in spring, to species spreading to areas that were once too cold for them to survive, to birds becoming smaller. The vast majority of these changes are not genetic but due to plasticity: organisms’ built-in ability to change their bodies and behaviour in response to whatever the environment throws at them. At least a few species, however, like the owls of Finland, are already changing genetically – evolving – in response to climate change.

In North America, for instance, pitcher plant mosquitoes lay their eggs in pitcher plants and the larvae enter a state of dormancy in the winter months before resuming development in spring. Dormancy is genetically programmed, triggered not by falling temperature but by the shortening days. As the growing season has lengthened, mutant mosquitoes that keep feeding and growing for longer have thrived. Northern populations now go dormant more than a week later than in 1972, when studies began.

The earlier breeding of red squirrels in North America is also thought to be partly a result of genetic changes. Some families emerge earlier in spring, and they are doing better as the climate shifts.

Plants are changing too. When seed collected from field mustard plants (Brassica rapa) in California in 1997 and 2004 were grown in identical conditions, the 2004 strains flowered 9 days earlier on average (Proceedings of the National Academy of Sciences, vol 104, p 1278). The change was a result of drought – the plants have evolved to reproduce before they run out of water.

Rapid evolution is thus already helping some species adapt to a warming world, but it is no “Get out of jail free” card. For instance, so far pied flycatchers in the UK seem unable to shift to laying eggs earlier in spring. And according to one model that specifically takes rapid evolution into account, global warming will kill off 20 per cent of all lizard species by 2080. The problem for lizards is that as the climate warms, they have to spend more time in the shade and less time feeding.

Organisms with long generation times and slow reproductive rates are the least able to evolve, says Stephen Palumbi at Stanford University. “And they are the ones that are already threatened. It’s a double whammy.”

Even species whose evolution has kept pace with the slight warming so far will not necessarily keep up as the global temperature soars by another 4 °C or more. Rapid evolution generally depends on the existing variation within a population, rather than on new mutations. “It is limited to the kind of changes that can happen quickly,” Palumbi says.

In fact, there is a catch-22 to very rapid evolution – the faster organisms evolve, the less able they are to evolve further. This is because fast change occurs when only a small proportion of each generation manages to reproduce, resulting in a dramatic loss of genetic diversity – the fuel for further evolution. In many cases, the size of populations will also plummet, rendering them vulnerable to extinction. “You could evolve really fast but just not make it,” says Michael Kinnison of the University of Maine in Orono.

Unnatural selection: Living with pollution

Between 1947 and 1976, two factories released half a billion kilograms of chemicals called polychlorinated biphenyls (PCBs) into the Hudson river, in the north-east US. The effects on wildlife weren’t studied at the time, but today some species seem to be thriving despite levels of PCBs, many of which are toxic, remaining high.

At least one species, the Atlantic tomcod – an ordinary-looking fish about 10 centimetres long – has evolved resistance. “We could blast them with PCBs and dioxins with no effect,” says Isaac Wirgin of New York University School of Medicine.

Many of the ill effects of PCBs and dioxins are caused by them binding to a protein called the hydrocarbon receptor (Science, vol 331, p 1322). The Hudson tomcod all have a mutation in the receptor that stops PCBs binding to it, Wirgin and colleagues reported earlier this year. The mutation is present in other tomcod populations too, Wirgin says, but at low levels.

The most famous example of evolution in action was a response to pollution: as the industrial revolution got under way, cream-coloured peppered moths in northern Britain turned black to stay hidden on trees stained by soot. As the tomcod shows, though, most evolutionary changes in response to pollution are invisible.

The spoil heaps of many old mines, for instance, are covered in plants that appear normal, but are in fact growing in soil containing high levels of metals such as copper, zinc, lead and arsenic that would be toxic to most specimens of these and other species. The evolution of tolerance has occurred extremely rapidly in some cases, sometimes within just a few years of the soil being contaminated.

With very widespread pollutants, it is much harder to show that organisms are evolving in response, because all populations change at once. The comparison has been done with a common weed called plantain (Plantago major), though. Ground-level ozone, produced when sunlight strikes car exhaust fumes, greatly impairs the growth of plants. When researchers grew plantain seeds collected in 1985 and 1991 from a site in northern England where ozone pollution reached very high levels in 1989 and 1990, they found that the plants from the 1985 batch grew nearly a third more slowly when exposed to ozone, whereas the growth of those from 1991 fell by only a tenth (New Phytologist, vol 131, p 337).

Since even the remotest parts of the planet are now polluted in one way or another, it is likely that many plants and animal populations have evolved some degree of tolerance, even though few cases have been documented. “Nobody looks for resistance,” says Wirgin. “My guess is that if you look you will find a lot of it.” His own discovery was entirely accidental: the team had set out to study liver cancers, and they only noticed the tomcod’s resistance when blasting the fish with PCBs failed to produce any tumours.

However, there are obviously limits to what evolution can achieve. This is especially true for small populations that reproduce slowly and have few offspring, such as the Yangtze river dolphin. Pollution is thought to have contributed to its extinction.

What’s more, pollution resistance in one species can have unexpected consequences for others. The tomcod’s tolerance allows it to accumulate extraordinarily high levels of PCBs in its body, for instance, which are a threat to animals higher up the food chain – such as humans with a taste for these reportedly delicious fish.

Unnatural selection: Spreading sickness

Perch in Lake Windermere in the UK used to live to a ripe old age. While the average age of fish caught and released by researchers was around 5 years, a few individuals were as old as 20. Then in 1976, an unidentified disease wiped out 99 per cent of adult fish and continued to preferentially kill older fish for years afterwards. Since then, no fish older than 7 have been caught.

According to Jan Ohlberger of the University of Oslo, Norway, the perch (Perca fluviatilis) evolved very quickly in response. They now become sexually mature at an earlier age, which increases their chances of breeding before they get killed by the disease (Proceedings of the Royal Society B, vol 278, p 35).

While the disease is thought to have spread naturally in the lake, Ohlberger points out that many devastating disease outbreaks in plants and animals are a result of human activity. To mention just a few: Dutch elm disease was caused by fungi introduced from Asia; lions were hard hit by canine distemper spread by village dogs, and corals are far more susceptible to diseases when water temperatures are abnormally high, which is happening often as a result of climate change.

Anything that kills a significant proportion of a population has the potential to bring about very fast evolution. In frogs there is now some evidence of this: last year several research groups reported that some populations appear to be becoming resistant to a fungus that has decimated many amphibian species. It is also clear that human populations have sometimes evolved rapidly in response to diseases such as kuru, which attacks the nervous system.

So it seems plausible that by spreading diseases or creating the conditions in which they thrive, humans are indirectly forcing many organisms to evolve. “I think this is a common phenomenon and has not yet been described because it is simply hard to prove,” says Ohlberger. He points out that the long-running capture-and-release programme at Lake Windermere, which began in 1943 and just happened to coincide with the disease outbreak in perch, is pretty unique. In most cases we know too little about what populations were like before disease outbreaks to be able to tell if and how they have evolved in response.

Unnatural selection: The arms race against pests

Had any strange itchy bites or rashes recently? You might have fallen victim to bedbugs. The little bloodsuckers are back in a big way, thanks in part to the fact that, like head lice and human fleas, they have evolved resistance to many common pesticides.

Whatever their drawbacks, there is no doubt that pesticides have made a huge difference to our lives. They have helped eliminate diseases like malaria from some areas and made possible the switch to intensive farming. As soon as we started using them, though, resistance began to evolve.

“Insects that succumb readily to kerosene in the Atlantic states defy it absolutely in Colorado [and] washes that easily destroy the San José scale [insect] in California are ridiculously ineffective in the Atlantic states,” wrote entomologist John Smith in 1897 – the first known report of insecticide resistance.

The use of synthetic pesticides like DDT took off in the 1940s. Resistant houseflies were discovered in 1946. By 1948, resistance had been reported in 12 insect species. In 1966, James Crow of the University of Wisconsin-Madison reported that the count had exceeded 165 species. “No more convincing examples of Darwinian evolution could be found than those provided by the development of resistance in one species after another,” he noted at the time.

It’s not just bugs. Rats and mice around the world have become resistant to the poison warfarin, and in Europe some have even become resistant to warfarin’s replacement, superwarfarin (Journal of Toxicological Studies, vol 33, p 283). In Australia, meanwhile, rabbits are becoming resistant to the poison used to control their numbers, called Compound 1080.

Because of its economic importance, pesticide resistance has been studied far more closely than other kinds of ongoing evolution. In many cases we know which mutations are involved, how they make organisms resistant and sometimes even how the mutations spread through populations.

Resistance often arises due to mutations that alter the shape of proteins and thus prevent insecticides binding to their targets. For instance, DDT and pyrethroid compounds kill insects by opening sodium ion channels in nerve cells, but in the malaria-carrying mosquito Anopheles gambiae, variants of the channels that cannot be opened this way have evolved on at least four separate occasions (PLoS One, vol 2, p e1243).

The other main mechanism of resistance involves enzymes that inactivate pesticides before they can kill. Some resistant strains of A. gambiae, for instance, produce large quantities of an enzyme called CYP6Z1 that can inactivate DDT.

Pesticide resistance is becoming such a serious problem that strategies for preventing it evolving in the first place are taken increasingly seriously. One approach is to alternate the type of pesticide applied, to try to avoid applying sustained selective pressure in one direction.

At present, though, the pests seem to be evolving faster than we can develop new pesticides. In one region of Burkina Faso, A. gambiae has become resistant to all four classes of insecticides used for malaria control.

Unnatural selection: Introducing invaders

In 1935, the South American cane toad was introduced to Australia to control pests feeding on sugar cane. The cane fields were not to the toad’s liking, but the rest of the countryside was. The toad has spread rapidly at the expense of many native species.

The highly poisonous animals are having a big effect on predators. Some, such as the Australian black snake, are developing resistance to cane toad toxins. Others, such as the red-bellied black snake and green tree snake, are changing in a more surprising way – their mouths are getting smaller. The reason is simple: snakes with big mouths can eat large toads that contain enough toxin to kill them.

The toads themselves are also changing. Some are now colonising regions that were too hot for the founder population, suggesting that they are evolving tolerance to more extreme conditions. What’s more, the toads leading the invasion are becoming better colonisers: they have bigger front legs and stronger back legs than toads living in the areas already colonised. Radio tagging has confirmed that these “super-invader” toads can travel faster, as you might expect. They are probably evolving because the first toads to reach new areas benefit from more food and less competition, and thus have more offspring. The changes are likely to be transient, though – once the toads stop spreading, the “super-invader” traits will gradually be lost.

Ships and planes have turned the natural trickle of species spreading to new islands or continents into a raging torrent, and the new arrivals sometimes have a dramatic effect. In areas of the US that have been invaded by fire ants, for instance, native fence lizards have evolved longer legs. They need them: given the opportunity, a dozen fire ants can kill a lizard in minutes.

Rather than simply study the results of invasions, Michael Kinnison of the University of Maine in Orono and colleagues have been actively experimenting. In one experiment, his team moved juvenile chinook salmon from one river in New Zealand to another. The salmon were introduced to the country around a century ago, and Kinnison wanted to assess the extent to which they had adapted to conditions in individual rivers. He found drastic differences in survival, even though the fish appear identical (Canadian Journal of Fisheries and Aquatic Sciences, vol 60, p 1). “When a population was locally adapted, it performed twice as well,” he says.

Kinnison suspects that lots of small changes can add up to make a huge difference to a population’s success. “Contemporary evolution may be relatively modest on a trait-by-trait basis, but its overall contribution to the performance of populations may be immense,” he says.

Such findings help explain why there is often a lag between the introduction of new species and their rapid spread. A newly arrived species is likely to find itself in an environment that is not quite ideal, and its population may be very small, meaning there is little genetic diversity. In these circumstances, a species will spread only slowly, if at all.

As the population begins to adapt to local conditions, though – perhaps via invisible changes such as mutations in immune genes – it is likely to start to grow and spread. Because more mutations occur in larger populations, it will then evolve faster, enabling it to spread quicker and further. If this turns out to be common, it is bad news. It suggests that many introduced species that seem to be behaving themselves could yet start spreading explosively and cause serious problems.

To view the original article click on the link below.


Unnatural selection: How humans are driving evolution by Michael Le Page

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PBS Nova – Ghost in Your Genes

This is a profound & enlightening program covering the breaking field of epigenetics. It’s important to note that Dow Chemical helped fund this program but the only way you would know this is through watching it because it is mentioned at the beginning and the ending of the program. I got a DVD copy from an educator through the Howard Hughes Medical Institute. The public cannot access copies through HHMI though. This is one of the only Nova episodes that is not available to watch online. Here is why I’m guessing that Dow Chemical has not released this program for sale. The following is taken from the transcripts and has serious implications.

But in Washington state, Michael Skinner seems to have found compelling additional evidence by triggering a similar effect with commonly used pesticides. Skinner wanted to see how these chemicals would affect pregnant rats and their offspring.

MICHAEL SKINNER: And so I treated the animals, the pregnant mother, with these compounds, and then we started seeing, between six months to a year, a whole host of other diseases that we didn’t expect. And this ranged between tumors, such as breast and skin tumors, prostate disease, kidney disease and immune dysfunction.

NARRATOR: He checked that there were no genetic mutations and then proceeded to breed the rats.

MICHAEL SKINNER: The next step was for us to go to the next generation. And the same disease state occurs. So after we did several repeats, and got the third generation showing it and then a fourth generation, we sat back and realized that the phenomenon was real. We started seeing these major diseases occur in approximately 85 percent of all the animals of every single generation.

NARRATOR: His discoveries were a revelation.

MICHAEL SKINNER: We knew that if an individual was exposed to an environmental toxin that they can get a disease state, potentially. The new phenomenon is that environmental toxin no longer affects just the individual exposed but two or three generations down the line.

I knew that epigenetics existed. I knew that it was a controlling factor for DNA activity—whether genes are silenced or not—but to say that epigenetics would have a major role in disease development, so…I had no concept for that. The fact that this could have such a huge impact and could explain a whole host of things we couldn’t explain before took a while to actually sink in.

NARRATOR: Further work has revealed changed epigenetic marks in 25 segments of the affected rat’s DNA. The implications, if they apply to humans, are sobering.

The program description version of this extraordinary discovery is significantly downplayed and the name of the toxin is completely omitted from the program. It was vinclozolin (One of the most common fungicides utilized in agriculture).


Program Description

“In Washington State, a researcher finds that a toxin given to rats still affects their offspring four generations later, without producing any changes in their genes.”


Ghost in Your Genes – “Some NOVA programs are not for sale because the rights are not available.” I’m thinking that Dow Chemical owns the rights of this program. You will not find the specifics of Dr. Skinner’s research regarding pesticides and their impact on epigenetics and germ lines in the program description. (I personally don’t think that’s an accident.)

It is unbalanced in it’s emphasis on “lifestyle” choices but does cover Dr. Michael Skinner’s research on pesticide exposure and impacts on epigenetics and germ lines (transgenerational impacts). This episode also explores the research being completed on identical twins. This research is critical to understanding genetics versus environmental influence on genetic expression. This field of research will be critical to understanding the impacts from environmental toxins on human genes and the origin of disease, infertility, and birth defects. Epigenetics will show us the mechanisms through which toxins cause harm and alter us as a species.

I have discovered the transcripts!


Ghost in Your Genes – Transcripts

PBS Airdate: October 16, 2007
Go to the companion Web site

NARRATOR: Imagine sharing life with a person who seems to be you. Created from the same fertilized egg, you share exactly the same genes. So profound is their influence, everything about you appears the same: the spaces between your teeth, the way you laugh, your body language. You are, in a word, identical. Or are you?

SUSAN: As infants, they were very much alike. Their physical similarities are obvious. And all their physical milestones happened at the same time. But functioning today, for Jenna and Bridget…they’re completely different.

Jenna is enthusiastic, productive. Jenna’s going to college. She talks about it all the time now. Bridget is essentially non-verbal. She doesn’t have purposeful conversational speech. And there’s very unusual behavior. For example, she likes to spit on monitors and then rubs it in. I don’t know why, but that’s what she does.

How? How could these guys be identical and so, on such a different level, functioning-wise?

NARRATOR: So if genes don’t tell the whole story of who we are, then what does?

Scientists suspect the answer lies in a vast chemical network within our cells that controls our genes, turning them on and off.

ANDREW P. FEINBERG (Johns Hopkins University): It’s a little bit like the dark matter of the universe. I mean, we know it’s there, we know it’s terribly important, but we don’t really know all that much about how that symphony gets played out.

MARK MEHLER (Albert Einstein College of Medicine): We’re in the midst of probably the biggest revolution in biology that is going to forever transform the way we understand genetics, environment, the way the two interact, what causes disease. It’s another level of biology, which, for the first time, really, is up to the task of explaining the biological complexity of life.

NARRATOR: Ghost in Your Genes, up next on NOVA.

Major funding for NOVA is provided by the following:

For each of us, there is a moment of discovery. We understand that all of life is elemental, and as we marvel at element bonding with element, we soon realize that when you add the human element to the equation, everything changes. Suddenly all of chemistry illuminates humanity and all of humanity illuminates chemistry. The human element: nothing is more fundamental, nothing more elemental.

And by David H. Koch, and…

Discover new knowledge: HHMI.

And by the Corporation for Public Broadcasting, and by contributions to your PBS station from viewers like you. Thank you.

NARRATOR: In the early 1990s, the biggest project ever undertaken in biology captivated the world.

NEWS AUDIO: The Human Genome Project will be seen as the outstanding achievement in the history of mankind.

NARRATOR: The endeavor would reveal the chemical structure of each gene locked within our cells, the blueprint for life itself.

WOLF REIK (The Babraham Institute): The human genome is like a bible where everything was written down. The hope and the expectation was that once we had that book in front of us, and all the letters, we could just read down the pages and we would understand how the body was put together.

NARRATOR: Once the code was deciphered, scientists hoped to find the genetic cause and cure for every disease. They estimated that the human genome, the book of life, would contain around 100,000 genes.

MICHAEL SKINNER (Washington State University): And then when they started sequencing…and it popped down to 60. And then it popped down to 50. And, slowly, it went down to a much smaller number.

NARRATOR: Thirty thousand, twenty-five thousand…as the mapping drew to an end, it appeared that humans had about the same number of genes as fish and mice.

MICHAEL SKINNER: In fact, we found out that the human genome is probably not as complex and doesn’t have as many genes as plants do. So that, then, made us really question, “Well, if the genome has less genes in this species versus this species, and we’re more complex potentially, what’s going on here?”

NARRATOR: So few genes didn’t appear enough to explain human complexity. Even more startling, it turned out the same key genes that make a fruitfly, a worm or a mouse also make a human. Chimpanzees share 98.9 percent of our genome. So what accounts for the vast differences between species? Might genes not be the whole story?

Long before the genome was mapped, geneticists, like Marcus Pembrey, had caught hints of this possibility as they encountered baffling genetic conditions—Angelman syndrome, for instance.

MARCUS PEMBREY (University College London): …named after Harry Angelman, the pediatrician who first described Angelman syndrome. He referred to them as “happy puppet children,” because this described, to some extent, the features. They have a rather jerky sort of movement when they’re walking. These children have no speech; they are severely incapacitated in terms of learning but are uncharacteristically happy, and they’re smiling all the time.

NARRATION: The condition is caused by a genetic defect. A key sequence of DNA is deleted from chromosome 15.

MARCUS PEMBREY: Then we came across a paradox. At the same time, the same change, the same little deletion of chromosome 15, had been clearly associated with a quite different syndrome—much milder in terms of intellectual impairment—the Prader-Willi syndrome.

These children are characterized by being very floppy at birth, but once they started eating properly and so on, they then had an insatiable appetite and would get very, very large.

NARRATOR: Pembrey was stunned. Angelman syndrome and Prader-Willi syndrome, two completely different diseases, were caused by the same genetic abnormality.

MARCUS PEMBREY: So here we’re in a bizarre situation really. How could one propose that the same deletion could cause a different syndrome?

NARRATOR: As Pembrey looked at the inheritance pattern for the two conditions, he noticed something even stranger.

MARCUS PEMBREY: What really mattered was the origin of the chromosome 15 that had the deletion. If the deletion was on the chromosome 15 that the child had inherited from father, then you would have Prader-Willi syndrome, whereas if the deletion was inherited from the mother, you had the Angelman syndrome.

NARRATOR: It was a complete surprise that the same missing strip of DNA, depending upon its parental origin, could cause different diseases. It was as if the genes knew where they came from.

MARCUS PEMBREY: You’ve got a developing fetus manifesting this condition. How does the chromosome 15 know where it came from? There must have been a tag or an imprint placed on that chromosome, during either egg or sperm formation in the previous generation, to say, “Hi, I came from Mother.” “I came from Father, and we are functioning differently.” So that’s the key thing, that although the DNA sequence is the same, the different sets of genes were being silenced depending on whether it came from the mother or from the father.

NARRATOR: It was the first human evidence that something other than genes passed between generations. Something that could control genes directly, switch them on or off. But how exactly did these tags go about silencing a gene?

This odd strain of agouti mice provides a visual clue. Despite the difference in color and size, they’re twins, genetically identical. Both, therefore, have a particular gene, called agouti, but in the yellow mouse it’s switched on all the time.

RANDY JIRTLE (Duke University Medical Center): As a consequence, it inappropriately blocks a receptor in what’s called the satiation center of the brain, which tells mice and us when we’re full. So the yellow animals literally eat themselves into obesity, diabetes and cancer.

NARRATOR: So what switched the agouti gene off in the thin mouse? Exercise? Atkins? No, a chemical tag called a methyl molecule. Composed of carbon and hydrogen, it affixes near the agouti gene, shutting it down. Living creatures possess millions of tags like these. Some, like methyl molecules, attach to DNA directly. Other types grab the proteins called histones, around which DNA wraps, and tighten or loosen them to turn genes on or off.

JEAN-PIERRE ISSA (M.D. Anderson Cancer Center): And, in simple terms, this contact can be thought of as hugging the DNA. And if these proteins hug the DNA very tightly, then it is hidden from view for the cell. And a gene that is hidden cannot be utilized.

NARRATOR: These tags and others control gene expression through a vast network in the body called the epigenome.

RANDY JIRTLE: Epigenetics literally translates into just meaning above the genome. So if you would think, for example, of the genome as being like a computer, the hardware of a computer, the epigenome would be like the software that tells the computer when to work, how to work, and how much.

JEAN-PIERRE ISSA: Perhaps the best example of an epigenetic phenomenon…you’re actually looking at it. You see, skin and eyes and teeth and hair and organs all have exactly the same DNA. You cannot genetically tell my skin from my eyes or my teeth, yet you couldn’t really imagine that these are the same tissues.

NARRATOR: What distinguishes cells is not their genes, but how these genes are switched on or off by epigenetics.

WOLF REIK: And, as development unfolds, certain switches need to be thrown. And you can think of it as a light switch. Switch on the gene, the light is shining, the gene is active… makes the cell do a certain thing. Or the light switch is off, everything is dark. That gene is off.

And as the cells divide, the memory of whether it’s a liver cell or a brain cell, that’s brought about by these switches. And the switches are incredibly stable.

NARRATOR: But occasionally, some epigenetic switches can be flipped. To turn off the overactive agouti gene, researchers gave pregnant mothers foods rich in vitamins like B-12, or folic acid, from which they could make those methyl tags that silence genes.

The change was small, the effect huge. Fat yellow mothers gave birth to thin brown pups no longer prone to disease.

RANDY JIRTLE: This study, why it is so important is it opened the black box up and told us that this early stage of development—in the womb, basically—is linked to adult disease susceptibilities by, literally, tiny little changes in the epigenome.

NARRATOR: Agouti mice revealed the impact of an epigenetic change, one that occurred without altering a single chemical letter in the agouti gene.

It was increasingly clear that genes needed instructions for what to do, when and where. If the thousands of genes identified by the Human Genome Project symbolized the words in the book of life, it was the epigenome that determined how that book got read.

MARK MEHLER: We thought that by understanding the genetic code, we would understand life, disease, and then we’d all go home and be fine. But, in fact, the human genome project was just the beginning. What it did was it opened us up to this new world, getting us to the point where we’re understanding another level of biology which, for the first time, is up to the challenge of the biological complexity of life.

NARRATOR: If the epigenome controls the expression of our genes, could it solve the mystery of identical twins?

These rare individuals are living illustrations of the boundary point between nature and nurture. For, since their DNA is 100 percent the same, any difference should reveal the influence of the outside world.

Most identical twins appear so similar they seem the product of genes alone. Consider Javier and Carlos. Their every gesture seems the same. Or take Ana Mari and Clotilde, who show up in nearly the same red dress when, in fact, neither had a clue what the other was going to wear. They moved through life in symmetry.

CLOTILDE: When I see my sister, I see myself. If she looks good, I think, “I look pretty today.” But if she’s not wearing makeup, I say, “My god, I look horrible.”

NARRATOR: But, five years ago, symmetry appeared to break. Ana Marie was diagnosed with cancer and Clotilde was not.

CLOTILDE: I’ve been told that I am a high risk for cancer. Damocles’ sword hangs over me.

NARRATOR: In fact, it’s not unusual for one twin to get a dread disease while the other does not. But how? How can two people so alike, be so different?

Intrigued by the mystery, Spanish geneticist Manel Esteller set out, in 2005, to find the answer to that question.

MANEL ESTELLER (Spanish National Cancer Center): One of the questions of twins is, “If my twin has this disease, I will have the same disease?” And genetics tell us that there is a high risk of developing the same disease. But it’s not really sure they are going to have it, because our genes are just part of the story.

NARRATOR: Esteller suspected epigenetics was the rest. To find out, he and his team collected cells from 40 pairs of identical twins, age three to 74.

Then began the meticulous process of dissolving the cells until all that was left were the wispy strands of DNA, the master molecule that contains our genes. Next, researchers amplified fragments of the DNA, revealing both the genes and their epigenetic tags.

Those that had been turned off appear as dark pink marks on the gel. Now, notice what happens when these genes are cut out and overlapped. The epigenetic effects stand out, especially when you contrast the genes of two sets of twins who differ in age.

Here, on the left, is the overlapped DNA of six-year-old Javier and Carlos. The yellow indicates where their genes are functioning identically.

On the right, is the DNA of 66-year-old Ana Mari and Clotilde. In contrast to the younger twins’, hardly any yellow shines through. Their genome may be the same, their epigenome clearly is not.

Identical genes active in one twin maybe shut down in the other. Thus, as the years pass, epigenetic changes accumulate in twins, as in the rest of us.

MANEL ESTELLER: One of the main findings of our research is that epigenomes can change in function of what we eat, of what we smoke or what we drink. And this is one of the key differences between epigenetics and genetics.

NARRATOR: But why does the epigenome change, when the genome does not?

In Montreal, scientists Michael Meaney and Moshe Szyf believe the question contains its own answer.

MOSHE SZYF (McGill University): We have this very, very static genome, very hard to change. It could be only changed by really dramatic things like nuclear explosions or, you know, hundreds of thousands of years of evolution. On the other hand, we have the dynamic environment that changes all the time. And so what there is here is an interface between the highly dynamic world around us and the highly static genome that we have. Epigenome is an in-between creature, built in a way, to respond to changes around us.

NARRATOR: Szyf and Meaney believe that experience itself changes the epigenome. To reach this startling conclusion they studied two kinds of rats: those born to nurturing mothers who licked and groomed them intensely after birth, and those born to mothers who took a more paws-off approach.

MICHAEL MEANEY (Douglas Institute/McGill University): What we were particularly interested in is the way in which these animals might respond to stressful events. And we found the offspring of low-licking mothers, during periods of stress, show greater increases in blood pressure and greater increases in stress hormone production.

MOSHE SZYF: They will scream. They will try to bite you. Just walking into their cage, those rats will respond differently.

NARRATOR: To rule out a genetic cause, high-licking mothers were given the babies of low-licking ones and vice versa. Once again, the less-nurtured pups grew up markedly different, and not only on blood tests.

MOSHE SZYF: So the conclusion from that is, it’s not the genes that the mother brings into the game. It is the behavior of the mother that has an impact on the offspring years after the mother is already gone. And the basic question was, “How does the rat remember what kind of care it received from its mother, so that it now has better or worse health conditions?”

And we reasoned that there must be some mark in genes that marks that memory.

NARRATOR: But could such a mark, capturing memory, be found? The researchers focused on a gene which lowers the levels of stress hormones in the blood. It’s active in a part of the rat’s brain called the hippocampus. By extracting and analyzing the gene, they could compare how its activity varied between low- and high-licked rats.

The difference was striking. Less nurtured rats had multiple epigenetic marks silencing the gene.

The result? With the gene less active, stress levels in neglected rats soared. In stark contrast, nurtured rats could better handle stress because they had nothing dimming the genes’ activity.

MOSHE SZYF: The maternal behavior essentially sculpted the genome of their babies. We looked at one gene; we know hundreds of genes were changed. But for me, it was a fantastic thing that just a behavior of one subject can change the gene expression in a different subject.

NARRATOR: The most surprising phase of the experiment, however, was yet to come. Szyf and Meaney injected anxious rats with a drug known to remove epigenetic marks.

MOSHE SZYF: And as we injected the drug, the gene turned on. And when it turned on, the entire behavior of the rat changed. It became less anxious. Also, it responded to stress like a normally-reared rat. And we looked at the way that gene was marked in the brain, and we saw that we actually changed the epigenetic marking of that gene.

NARRATOR: Although the work has yet to be replicated, it appears that Szyf and Meaney have linked personality traits, albeit in a rat, to the epigenome.

Could this have implications for humans? We will not know until the completion of a 10-year study, now underway, that will look at children from both nurturing and neglected backgrounds.

But even now, says Meaney, we have clues that our own upbringings produce the same effects.

MICHAEL MEANEY: If you grow up in a family that involves abuse, neglect, harsh and inconsistent discipline, then you are statistically more likely to develop depression, anxiety, drug abuse. And I don’t think that surprises anyone. But what is interesting is that you are also more likely to develop diabetes, heart disease and obesity. And the stress hormones actively promote the development of these individual diseases.

MOSHE SZYF: So, one day, we’ll be able, perhaps, to chart the pathway from child abuse to changes in the way certain genes are epigenetically marked in the brain that unfortunately affect our health years later in life.

NARRATOR: This work is controversial. Still, many scientists now believe that epigenetic changes in gene expression may underlie human diseases.

Take a disorder like M.D.S., cancer of the blood and bone marrow. It’s not a diagnosis you would ever want to hear.

SANDRA SHELBY (Medical patient): When I went in, he started patting my hand and he was going, “Your blood work does not look very good at all,” and that I had M.D.S. leukemia, and that there was not a cure for it, and, basically, I had six months to live.

NARRATOR: With no viable treatment, Sandra entered a clinical trial experimenting with epigenetic therapy. It was the result of a radical new way of thinking about the causes of diseases like cancer.

JEAN-PIERRE ISSA: If one has a genetic basis of cancer in mind, then one is simply asking, “What causes genetic damage?” Cigarette smoking, certain types of environmental exposures and radiation causes genetic damage. But, now if I come in and say, “Well, wait a minute, epigenetic damage can also cause cancer,” then you’ve got to ask “Well, why does this come about?”

NARRATOR: The trouble begins, believes Issa, when our stem cells, the master cells that create and replace our tissues, overwork.

JEAN-PIERRE ISSA: Every time a stem cell has to repair injury, it is aging a little more. And because each time a stem cell divides there is a finite chance of some sort of epigenetic damage, what we find is that in older people there’s been an accumulation of these epigenetic events that is easily measurable in DNA.

Now where does the cancer angle come from? Well, if you count age as how many times a stem cell has divided, then cancers, which copy themselves tirelessly, are awfully old tissues.

NARRATOR: As epigenetic errors pile up, the switches that turn genes on and off can go awry, creating havoc within the cell.

ANDREW FEINBERG: There are genes that help to prevent tumors that are normally active that epigenetically become silenced. Those are called tumor suppressor genes. And there are other genes, called oncogenes, that stimulate the growth of tumors. And then the tags, such as the methylation tags, come off those genes, and those genes become activated. So both ways, turning on and turning off, is a way of getting epigenetic disease.

NARRATOR: But could misplaced tags be rearranged? In 2004, Sandra and other patients began taking a drug to remove methyl tags silencing their tumor suppressor genes.

ROY CANTWELL (Medical patient): Your number one thing is, “Okay, is it going work?” And when you know that before this there was nothing, then yeah, it makes you pretty happy that there is a chance to go forward in your life.

NARRATOR: Ironically, the drug, decitabine, was tried in conventional chemotherapy in the 1970s and deemed too toxic. Today, Issa is giving his patients a dose 20 to 30 times lower.

JEAN-PIERRE ISSA: The idea of epigenetic therapy is to stay away from killing the cell. Rather, what we are trying to do is diplomacy, trying to change the instructions of the cancer cells, reminding the cell, “Hey, you’re a human cell. You shouldn’t be behaving this way.” And we try to do that by reactivating genes.

SANDRA SHELBY: The results have been incredible. And I didn’t have, really, any horrible side effects.

ROY CANTWELL: I am in remission, and going in the plus direction is a whole lot better than the minus direction.

NARRATOR: Roy has not been cured, but he has been cancer-free for two years. And he is not alone.

JEAN-PIERRE ISSA: Spectacular results—complete disappearance of the disease—can be seen in almost half of the patients that receive this drug. And 20 years ago we wouldn’t have dreamed that a drug that affects DNA methylation could have such a profound effect on patients.

NARRATOR: As epigenetic therapy takes off, so do the expectations for this new science. Many believe that a multitude of complex diseases, from Alzheimer’s to autism, may have epigenetic triggers.

Consider autism, a mysterious disorder characterized by social withdrawal. This is Bridget. She passes her day running her fingers across her computer screen. Locked in her own world, she has spent the past 13 years drifting apart from her identical twin sister, Jenna.

SUSAN : As infants, they were very much alike. Their physical similarities are obvious. And all their physical milestones happened at the same time. And then, at their first birthday party, we had a big party at the house, lots of balloons, lots of people. And I remember watching Bridget maneuver around the house as if there were nobody there. She was fixated on a balloon, which a lot of babies would be, but something struck me that she was not in tune with everybody that was there.

NARRATOR: Bridget was eventually diagnosed with severe autism. As the girls developed, so did their differences.

SUSAN : Functioning today for Jenna and Bridget…they’re completely different. Jenna is enthusiastic, productive, you know? Jenna’s going to college, talks about it all the time now. Bridget is essentially non-verbal. She doesn’t have purposeful conversational speech, so everything she does say is very memorized because she was taught over and over again.

Do you want grilled cheese?

BRIDGET: Grilled cheese?

SUSAN: Yes or no?

BRIDGET: Yes or no? No.

SUSAN: No?

BRIDGET: Yes, grilled cheese. Yes.

SUSAN: You want grilled cheese, yes?

BRIDGET: Yes.

SUSAN: Good.

There’s no prescription that you get when your child’s diagnosed with autism.

And new things come that weren’t there before, new behaviors that are very problematic, that interfere with her ability to learn anything. So we don’t know, really, what the prognosis is.

NARRATOR: And, for a long time, doctors couldn’t really help. Despite millions of dollars and years of searching, no single definitive autism gene had been found.

But about a decade ago, scientists at the Kennedy Krieger Institute, in Baltimore, turned their high power imagers on the problem. They scanned the brains of both healthy and autistic children, searching for a biological cause of the disorder. One of the researchers involved was Walter Kaufmann.*

WALTER KAUFMANN (Kennedy Krieger Institute): For a long time, people questioned whether autism was a real entity, because the ways to diagnose autism had been behavior…behavioral abnormalities. And those sometimes are difficult to identify in a consistent and reliable way.

NARRATOR: But, in comparing the brain scans of identical twins discordant for autism, Kaufman finally saw the definitive data he was searching for: an area in the brain linked to learning, memory and emotions—called the hippocampus—was smaller in the twin with severe autism. But how could the same genes create different brain structures? Kaufmann asked Andy Feinberg at Johns Hopkins University.

ANDREW FEINBERG: And suddenly we were able to form an epigenetic hypothesis. And that hypothesis is that they have the same genome, but one of them maybe has an epigenetic change that’s leading to a difference in their brain that you don’t see in the other twin.

NARRATOR: Kaufmann and Feinberg are now searching for methyl marks in the DNA of identical twins discordant for autism. The work has just begun, but the hope is that by finding identical genes that differ in their expression, some causes of autism may emerge.

WALTER KAUFMANN: We know environmental stimulation, sensory stimulation, auditory, visual stimulation, have an impact on brain development and brain function. And this impact we know now is mediated, at least in part, by epigenetic mechanisms.

ANDREW FEINBERG: Epigenetic changes…generally they stand at the cornerstone between our genome—in other words, all of our genes, the development of the cells of our body—and the environment that we live in.

NARRATOR: So the environment molds our epigenomes. But might it do more?

At the far speculative edge of this new science, some are seeing evidence of an astonishing possibility, that genes may not be all that passes from generation to generation.

The evidence comes from this Swedish village huddled on the Arctic Circle. Overkalix stands out for one reason, its archives.

Olov Bygren, a Swedish public health expert, has been studying them from more than 20 years. What makes these records unique is their detail. They track births and deaths over centuries—and harvests. This is significant because, in years past, Overkalix’s location left it particularly vulnerable to crop failures and famines.

LARS OLOV BYGREN (The University of Umeå): In the 19th century this was a very isolated area. They could not have help from outside. As it was so poor, they really had a hard time when there was a famine, and they really had a good, good time when the harvests were good.

NARRATOR: Bygren was studying the connection between poor nutrition and health when he stumbled on something curious.

It appeared that a famine might affect people almost a century later, even if they had never experienced a famine themselves. If so, past and future generations might be linked in ways no one had imagined.

Wondering if epigenetics might explain the phenomenon, Bygren sent his research to geneticist Marcus Pembrey.

MARCUS PEMBREY: I was terribly excited to get this, completely out of the blue. And for the first time it seemed that there was some data that we could then start to explore, so that was the beginning of our collaboration.

NARRATOR: Overkalix offered Pembrey a unique opportunity to see if the events that happened in one generation could affect another decades later.

MARCUS PEMBREY: Olly first reported that the food supply of the ancestors was affecting the longevity or mortality rate of the grandchildren, so I was very excited. I responded immediately.

NARRATOR: Pembrey suspected the incidence of one disease, diabetes, might show that the environment and epigenetics were involved. So Olov trawled the records for any deaths due to diabetes and then looked back to see if there was anything unusual about the diet of their grandparents.

MARCUS PEMBREY: A few months later he emailed me to say that indeed they had shown a strong association between the food supply of the father’s father and the chance of diabetes being mentioned on the death certificate of the grandchild.

NARRATOR: In fact, a grandson was four times more likely to die from an illness related to diabetes if his grandfather had plenty of food to eat in late childhood.

This was one of the first indications that an environmental exposure in a man, one that did not cause a genetic mutation, could directly affect his male offspring.

MARCUS PEMBREY: It really did look as if there was some new mechanism transmitting environmental exposure information from one generation to the next.

NARRATOR: Because these ideas were so heretical, Pembrey knew the results could be dismissed as nothing more than a curiosity. To bolster the research, he needed to find out how a trans-generational effect impacted each sex and if it was linked to a specific period of development.

MARCUS PEMBREY: We wanted to tease out when you could trigger, in the ancestor, a trans-generational response.

NARRATOR: So he and Bygren went back to the data. The more they looked, the more patterns started to appear.

MARCUS PEMBREY: We were able to look at the food supply every year, in the grandfather and the grandmother, from the moment they were conceived right through to the age of 20. We found that there are only certain periods in the ancestors’ development when they can trigger this trans-generational response. They’re what one might call sensitive periods of development.

NARRATOR: They discovered that when a famine was able to trigger an effect was different for the grandmother than the grandfather. The grandmother appeared susceptible while she, herself, was still in the womb, while the grandfather was affected in late childhood.

MARCUS PEMBREY: And the timing of these sensitive periods was telling us that it was tied in with the formation of the eggs and the sperm.

NARRATOR: This suggested what might be happening. Perhaps environmental information was being imprinted on the egg and sperm at the time of their formation.

At last a sharper picture was beginning to emerge. The next step was to compile their findings. Bygren drew up a rough diagram and sent it to Pembrey.

MARCUS PEMBREY: Hand-drawn…this is what Olly sent me, you know he was too excited to wait for the thing to be drawn out properly. You know, he sent me the data, and, in fact, I was recovering from having something done on my heart, so he sent it saying, you know, “I hope this helps you get better quickly,” you know? Because it was so exciting.

NARRATOR: When Pembrey looked at the diagram, he was immediately struck by seemingly bizarre connections between gender, diet and health, connections that were most pronounced two generations later. Men, for example, who experienced famine at around age 10, had paternal grandsons who lived much longer than those whose grandfathers experienced plenty. Yet, women who experienced famine while in the womb, had paternal granddaughters who died on average far earlier.

MARCUS PEMBREY: Once I had plotted out the full extent of those results, it was so beautiful and such a clear pattern, I knew then, quite definitely, that we were dealing with a trans-generational response. It was so coherent—and that’s important in science, that the effect was coherent in some way—was tying in when eggs and sperm were being formed.

NARRATOR: The diagram showed a significant link between generations, between the diet in one and the life expectancy of another.

OLOV BYGREN: When you think that you have found something important for the understanding of the seasons itself, you can imagine that this is something really special.

MARCUS PEMBREY: This is going to become a famous diagram, I’m convinced about that. I get so excited every time I see it. It’s just amazing. Every time I look at it, I find it really exciting. It’s fantastic.

NARRATOR: Much about these findings puzzles researchers. Why, for instance, does this effect only appear in the paternal line of inheritance? And why should famine be both harmful and beneficial, depending on the sex and age of the grandparent who experiences it?

Nonetheless, it raises a tantalizing prospect: that the impact of famine can be captured by the genes, in the egg and sperm, and that the memory of this event could be carried forward to affect grandchildren two generations later.

MARCUS PEMBREY: We are changing the view of what inheritance is. You can’t, in life, in ordinary development and living, separate out the gene from the environmental effect. They’re so intertwined.

NARRATOR: Pembrey and Bygren’s work suggests that our grandparents’ experiences effect our health. But is the effect epigenetic? With no DNA yet analyzed, Pembrey can only speculate. But in Washington state, Michael Skinner seems to have found compelling additional evidence by triggering a similar effect with commonly used pesticides. Skinner wanted to see how these chemicals would affect pregnant rats and their offspring.

MICHAEL SKINNER: And so I treated the animals, the pregnant mother, with these compounds, and then we started seeing, between six months to a year, a whole host of other diseases that we didn’t expect. And this ranged between tumors, such as breast and skin tumors, prostate disease, kidney disease and immune dysfunction.

NARRATOR: He checked that there were no genetic mutations and then proceeded to breed the rats.

MICHAEL SKINNER: The next step was for us to go to the next generation. And the same disease state occurs. So after we did several repeats, and got the third generation showing it and then a fourth generation, we sat back and realized that the phenomenon was real. We started seeing these major diseases occur in approximately 85 percent of all the animals of every single generation.

NARRATOR: His discoveries were a revelation.

MICHAEL SKINNER: We knew that if an individual was exposed to an environmental toxin that they can get a disease state, potentially. The new phenomenon is that environmental toxin no longer affects just the individual exposed but two or three generations down the line.

I knew that epigenetics existed. I knew that it was a controlling factor for DNA activity—whether genes are silenced or not—but to say that epigenetics would have a major role in disease development, so…I had no concept for that. The fact that this could have such a huge impact and could explain a whole host of things we couldn’t explain before took a while to actually sink in.

NARRATOR: Further work has revealed changed epigenetic marks in 25 segments of the affected rat’s DNA. The implications, if they apply to humans, are sobering.

MICHAEL SKINNER: What this means, then, is what your grandmother was exposed to when she was pregnant could cause a disease in you—even though you’ve had no exposure—and you’re going to pass it on to your great-grandchildren.

NARRATOR: And if a pesticide can generate such effects, what about stress, smoking, drinking? To some, the epigenetic evidence is compelling enough already to warrant a public note of caution.

RANDY JIRTLE: We’ve got to get people thinking more about what they do. They have a responsibility for their epigenome. Their genome they inherit. But their epigenome, they potentially can alter, and particularly that of their children. And that brings in responsibility, but it also brings in hope. You’re not necessarily stuck with this. You can alter this.

NARRATOR: Might our lifestyle choices resonate down the ages, effecting people yet unborn? Such ideas remain, to say the least, controversial. But one thing many in the field can agree upon is the need to take a cue from the Human Genome Project and launch a similar effort, this time to decipher the epigenome.

ANDREW FEINBERG: Mapping the human epigenome is the most important thing that we could do right now, as a big project in science, because it will tell us some very important things about why organisms function the way they do, why cells have the behavior they do.

JEAN-PIERRE ISSA: We now know how many genes we have, but we really don’t know how they are regulated inside the cells. And mapping the epigenome will give a much better understanding of this particular process.

NARRATOR: The hurdle is that, unlike the genome, which is the same in every cell, the epigenome varies from tissue to tissue, between individuals and over time.

MARK MEHLER: If you thought sequencing the human genome took years and was difficult, you’re talking about levels of complexity that will dwarf anything we knew about the human genome. But it’s crucial, it’s essential. That’s the way that the future is going to unfold. So in a sense, the Human Genome Project was just the beginning.

NARRATOR: The end may be the realization that the code of life is more complex and interactive than we ever imagined.

MARCUS PEMBREY: I’ve thought of nothing else really for the last five years.

It is said, the first time, you know, one had a photograph of the Earth, you know this sort of delicate thing sailing through the universe, you know, it had a huge effect on the sort of “save-the-planet” type of feeling, you know? I’m sure that’s part of why the future generation think in a planetary way, because they’ve actually seen that picture, you know? And this might be the same. It may get to a point where they realize that you live your life as a sort of guardian of your genome. It seems to me you’ve got to be careful of it because it’s not just you. You can’t be selfish because you can’t say, “Well I’ll smoke,” or “I’ll do whatever it is because I’m prepared to die early.” You’re also looking after it for your children and grandchildren. It is changing the way we think about inheritance forever.

*This study was led by the Kennedy Krieger Institute’s Dr. Wendy Kates, who is now (late 2007) at the State University of New York at Upstate Medical University. NOVA would like to thank Dr. Kates for providing access to the autistic twins featured in the program.

On the Ghost in Your Genes Web site, an expert answers viewer questions about the growing field of epigenetics and its potential. Find it on PBS.org.

Educators and other educational institutions can order this or other NOVA programs for $19.95 plus shipping and handling. Call WGBH Boston Video at 1-800-255-9424.

NOVA is a production of WGBH Boston.

Major funding for NOVA is provided by the following:

For each of us, there is a moment of discovery. We understand that all of life is elemental, and as we marvel at element bonding with element, we soon realize that when you add the human element to the equation, everything changes. Suddenly all of chemistry illuminates humanity, and all of humanity illuminates chemistry. The human element: nothing is more fundamental, nothing more elemental.

And by David H. Koch, and…

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