DNA Science Blog

For Valentine’s Day, here is a fly’s eye view of my PhD research on the mutation Antennapedia. Although published in this millenium at Scientific American  blogs on April 14, 2012, I wrote “The Making of  Mutant” in 1978. Just to see if my mentor Thom Kaufman was paying attention, I stuck it into a manuscript to be submitted to the journal Genetics. As far as I know, the events and facts reported here are all accurate.

(Credit: Tommy Leung)

CHAPTER 1

She knew she was different long before her mother had told her the truth. A sensitive youngster, she could tell from the sneering glances of her neighbors that she was, somehow, not quite like them. True, her larval segments were squished, her head too small. Now, hidden in her cocoon, she pondered her plight as she awaited, with apprehension, the great eclosion.

What would it be like? She’d heard the exciting but also frightening descriptions of the event from the elders, but now that she was about to experience the change herself, it suddenly seemed more significant. Would it hurt? What new senses and skills would she have, and which would vanish? Would she still be different, or was the impending transformation a second chance?

As she felt her larval organs begin to slowly dissolve, Ann’s thoughts returned to the horror that her parents had endured. The others spoke of The Great Mutagenesis in whispers and, although no one would put it into words, all of the youngsters feared, deep down in their discs, that it could happen again.

Ann remembered when her mother had told her, limbs trembling, of how all of the males were plucked from their yeasty homes and placed in a new, sweetly reeking environment. The gullible males greedily sucked up the sticky goop at their tarsi.

Soon they became nauseated. Just as they felt about to expire, they were suddenly moved to a new home, where hundreds of sex-starved virgins waited, their still-wet-and-wrinkled wings shimmering, their eggs dropping unfertilized in anticipation.

Meanwhile, in another vial, dozens of newly-eclosed, pure females were snatched, their virgin wings still unspread, and crammed into tiny, hot capsules. Some fainted. All were terrified. They were placed in a dark chamber for several minutes, and then dumped mercilessly into dens of slavering males with only one thing on their cerebral ganglia.

Then males and females from both mass poisonings were isolated as pairs. Fortunately for Ann’s mother, her father hadn’t been too demanding. The couple produced a surprising number of stillbirths. Ann could remember, no more than a first instar herself, her parents hopefully watching their offspring try desperately to hatch, the fully-formed embryos, about to be larvae, quaking with the effort beneath their thin eggshells. Many of those who did burst free were sick. Other couples in the colony were having similar problems.

And then there was Ann. Ann Tennapedia, or “mutant,” as her insensitive bottle mates called her. As Ann grew, transitioning from one instar to the next, she noticed her parents glancing from her to the other larvae with a strange look in their ommatidia. When she reached third instar, which for some reason had taken an inordinately long time, she worried about what lay ahead, and finally went to her mother, seeking answers.

CHAPTER 2
Ann slipped slowly into a dreamless sleep as she felt her imaginal discs expand.

After four days in suspended animation, consciousness returned as Ann became aware of new muscles and sensations. She became restless – she had to move. As she rubbed her new legs, her cocoon suddenly split apart. Soon, she was wobbling about.

Antennapedia (Credit: F. R. Turner)

The world certainly seemed different with so many eye facets! Unaccustomed to this spectacular new body, Ann at first felt a little off balance. Her head ached. She tried to straighten her crinkled wings. Ann stumbled along, noticing the stares. She must be quite beautiful!

Then Ann remembered her difficult larvahood, and quickly examined herself – 6 legs, 2 wings, and quite a lovely abdomen! She was most anxious to see her beautiful new eyes. Were they ruddy red wild type, or a pretty variant?

Ann sensed a shiny drop of moisture up ahead, and danced around it to see her new eyes in the reflection. Alas, they were wild type, but sparkling and symmetrical nonetheless.

Her head was certainly sore. She lowered it slightly, as if she was under some great weight. Still looking in the mirror-drop, Ann could not, at first, believe what it reflected.

Her proboscis fell open and she could feel her abdomen distend in shock as she tried vainly to comprehend what was staring back at her. It must be an illusion. A cruel trick.

Coming out of Ann’s head, where her antennae should have been, were two enormous appendages!

CHAPTER 3

A Drosophila den is a busy place, and life must go on despite unpleasant variations.

Ann survived. Her larval handicap had accustomed her to ridicule. Since The Great Mutagenesis, quite a few oddballs like herself wandered about. Many of the elders had died off, and not enough young adults were surviving embryohood to replace them.

One day, as Ann’s colony was transferred to a fresh new home, she sensed a certain excitement. It could only mean one thing: Orgy!

No need for her to worry her heavy head over an influx of new males. Nobody wanted her. She must be the world’s only four-day-old virgin.

Ann pitifully clung to the outskirts of the colony as the frenzied mating began. Staring into a shining droplet, much like the one that had initially revealed her gross deformity to her, she realized how hideous she truly was.

She gazed at the offensive growths emanating from her head, past her opalescent ommatidia and voluptuous proboscis, down to her admittedly fine legs. But what was this? Two of the reflected legs bore massive sex combs.

She looked down in utter confusion at her own smooth legs. Slowly lifting her head, she met the gaze of Anton O. Pedia.

(Credit: Antoine Morin)

CHAPTER 4

The romance between Ann and Anton rivaled that depicted anywhere in the classical literature. Sensing in those first few awkward moments that they were indeed meant for one another, they entered a dreamland neither had thought possible. They danced together in ecstasy, oblivious to the revolted stares.

Soon, out of Ann’s hindquarters emerged dozens of shiny white fertilized eggs, glowing with health. Within a week, the colony buzzed with joyous larval feeding activity.

In her exhausted bliss, Ann didn’t notice that most of her offspring didn’t look like the others. They seemed happy as they munched through the maple-flavored goop that was their home. Anton, however, always the practical one, knew that many of their children were afflicted with the same empty-headedness that he himself had been ostracized for in his larvahood.

Finally, bulging and satiated, the offspring of Ann and Anton, one by one, abandoned their gastronomic way of life for the mysterious serenity of pupariation. As the incessant chomping of larval jaws diminished, Ann slowed her ovipositing and observed, with the satisfaction known only to mothers, her tanning youngsters. She knew many of them would be like herself, but didn’t share her fear with Anton. No need to worry his massive head.

Ann hadn’t been feeling well. Her frequent exhaustion made walking on the sticky ground difficult. Anton, growing weary too, had to help her.

And so as the days passed, the children of Ann and Anton melanized as their proud parents weakened.

On the 20th day after their meeting, Ann and Anton knew the end was near. As they lay silently in each other’s legs, they gazed in wonder as their offspring emerged from their cocoons, many bearing exquisite legs on their heads.

(Credit: FlyBase)

CHAPTER 5

The new colony prospered. The descendants of Ann and Anton were vigorous and fruitful, and it soon became quite fashionable to display one’s antennal legs at full extension. Males with especially bulky heads were most popular with the ladies, much to the envy of those not blessed with good penetrance.

The generations passed, uneventfully, for many transfers of the stock bottle. Memories of Ann and Anton, of their suffering, of those who had lived through The Great Mutagenesis, had all but vanished.

And then it happened. Without warning, couples were violently separated, the males sent to a sticky, sweet-smelling chamber where they quickly became ill. The poor females, just like their forgotten great-great-great-grandmothers, were mercilessly crammed into tiny, dark capsules and placed in a monstrous machine that pelted them with X-rays. And the unspeakable happened – the larvae were taken, too.

Most of the colony recovered. But instead of returning to their communities, they were sent into new bottles containing wild sex maniacs of all colors, bristle types, and persuasions.

Time passed. New couplings formed, eggs were laid, and life in the lab went on.

Nine days after this Second Great Mutagenesis, the new homes of Ann and Anton’s descendants were once more lined with the darkening bodies of a new generation. The survivors watched with primeval awe as their children appeared, stretching their magnificent antennal legs towards the cotton at the bottle top in anticipation of adult life.

(Credit: FlyBase)

CHAPTER 6

He knew he was different, long before his mother told him the truth. A sensitive youngster, he could tell from the sneers of his neighbors that he was somehow not quite like them. Now, hidden in his cocoon, he awaited the unknown. Would he be born again?

Casting aside his chitin covering in an orgasm of release, he tested his new body parts slowly, then stumbled forward. He looked about him at his handsome neighbors, flexing their superbly hairy antennal legs. Preening. But why were they looking at him so strangely?

Robert peered cautiously into a moisture droplet. He gazed lovingly at his well-endowed sex combs, admired and extended his phallic proboscis, and then stopped, his hemolymph turning to ice, as he scrutinized, with stunned disbelief, the perfectly-formed, wild type antennae protruding in obscene normalcy from his otherwise perfect head.

In the usual trajectory of passing on genetic information, the older tell the younger, when the time is right. Typically, a patient has a genetic test because family history, ethnic group, or some other clue suggests to an astute practitioner an increased risk of something specific.

If a test reveals a mutation that could cause a disease, then the patient and perhaps her partner discuss how, when and what to tell their children – in the best of circumstances, with the help of a genetic counselor.

Direct-to-consumer (dtc) genetic testing has flipped the normal order of things. Young adults are taking dtc tests, some “just for fun,” and through their results, their parents – demographically at the cusp of beginning to fall apart – may learn just how they may do so.

I’m watching this generation reversal in genetic testing right now – and confusion began right away, with misuse of a single word.

‘CARRIER’ HAS MULTIPLE MEANINGS

(NHGRI)

A friend of a friend I’ll call Lisa called me last week with a question: her 22-year-old son Justin had driven to nearby Vermont to mail spit to the dtc company 23andMe (it’s illegal in New York to take the tests, and also illegal to spit in public in NYC). He’d just showed her his DNA test results, with a surprise: he’s a “carrier” for one of two mutations in the BRCA1 cancer gene found mostly in people of Ashkenazi Jewish descent.

Lisa’s Ashkenazi; her husband’s not.

“But I don’t have to worry, right, because he’s only a carrier,” she said.

I hesitated. “Well, not exactly.”

“But I discussed it with three people and they all said it couldn’t affect me because I could just be a carrier, like Justin. Carriers of cystic fibrosis don’t actually get sick. So I won’t. Right?”

“In cancer it doesn’t usually work that way. Why don’t you come over and we can discuss it?”

I was worried, fearing not just high risk of cancer, but the company’s mixing up technical genetics terms with plain English. At fault: the word carrier. Forgive my foray into textbookese, but sloppy use of terminology in genetics can lead to life-altering decisions based on misunderstanding.

A carrier can be a healthy person spreading an infectious disease, like the cook Typhoid Mary, who spread typhoid fever to members of the unfortunate families she served in the New York City area in the early years of the twentieth century.

In genetics, in autosomal recessive inheritance, a carrier has one copy of a mutation and a normal (wild type) copy, and doesn’t have the associated condition. A person gets the disease by inheriting two recessive mutations from two carrier parents. Lisa was right, carriers of cystic fibrosis don’t inherit full-blown disease (although rarely they can have mild symptoms).

In autosomal dominant inheritance, in contrast, just one copy of a mutant gene causes disease. There are no carriers. If you get the mutation, you get the disease. A person too young to have recognizable symptoms of Huntington disease (HD), for example, isn’t, technically, a carrier – instead he or she is “pre-manifest” or “pre-symptomatic,” in genetics jargon. Early on, HD researchers and clinicians agreed that people under age 18 shouldn’t be tested, precisely because of the inevitability of the currently untreatable disease in “carriers.”

(NCI)

BRCA mutations are also inherited in an autosomal dominant pattern, but not as straightforward as is the case for HD. Inheriting a BRCA mutation gives a person elevated risk, not a certainty of cancer. The classic “two-hit“ hypothesis of cancer explains the situation: Justin inherited a susceptibility allele (gene variant) in all his cells, and cancer might develop if/when and where a mutation in the second copy of that gene occurs. That could be in his breast, testes, or prostate – or it might never happen.

But the inheritance of BRCA-related disease is even more complicated, because the risks of developing BRCA1 cancers vary in different population groups, due to the actions of other genes and environmental factors. And the numbers are all over the place, which is driving Lisa crazy. The chance of either breast or ovarian cancer happening by age 70 for a person with a BRCA1 mutation ranges from 40% to 86%, depending on how a particular study selected its families.

The information that Justin received from 23andMe on BRCA Cancer is accurate: “Carrier for the 5382insC BRCA1 mutation. Lifetime risk of breast cancer for women is increased from 12% to about 60% and risk of ovarian cancer is increased from less than 2% to about 40%. May significantly increase risk of prostate cancer in men. There is also an increased risk for breast cancer in men.”

MENDEL’S LAWS STILL MATTER

(Natl Library of Medicine)

When Lisa came over, I sketched a pedigree as she talked, filling in the circles and squares that represented family members with cancer. Unfortunately cancer has so many guises, and is so common, that its appearance in a pedigree can be coincidence, as it is in my own family.

In Lisa’s family, two cases of ovarian and breast cancer stood out, but the women were older than is typical for inherited cancers. And they weren’t primary relatives (parent, sib, or kid). “Incomplete penetrance” could explain the pattern of filled-in and blank symbols – Lisa and her mom silently passed the mutation on. This may look like autosomal recessive inheritance, but it isn’t.

If Justin hadn’t sent his spit to 23andMe, likely no one would have suggested Lisa be tested for BRCA genes based on her family history.

As I filled in the square symbol at the bottom of the family tree representing Justin, the implications became clear. Lisa grew teary.

“It looks like if Justin has it, I would too. Unless my husband does – but he’s not Jewish,” she said quietly.

“Right.”

“Oh. So, if I’m not a carrier like in cystic fibrosis, that means … do I have the high risk of Ashkenazi Jews of getting cancer?”

“If you have the mutation, I’m afraid so. But the risk may be a little less because the cases in your family are older, and you and your mom don’t have cancer.”

“Some other gene is protecting me?”

“Possibly.” She really got it.

Alternatively, Justin could be a “new mutation,” but that’s unlikely. His DNA would have had to have mutated by chance in the exact same way that the ancestral Ashkenazi mutation did millennia ago.

I was struck by the irony, given recent debate over how we will handle the dense, overwhelming information that is pouring out from exome and genome sequencing. Lisa’s case reveals the repercussions of a test on a single, intensely-studied gene on a family. And her distress derives from applying the work of a researcher who conducted his experiments in the 1880s – Gregor Mendel.

ACTION OR ANXIETY?

(Mitchell Schnall, MD, NCI)

“Actionable” is a word we’ll be hearing more of as genome sequencing becomes routine. Knowing that her son has a BRCA1 mutation and she probably does too, Lisa’s having a breast exam, physical, and mammogram. She’ll have a BRCA1 test from Myriad Genetics, so insurance can cover the test and follow-up, because 23andMe is not (yet) a medical testing company and licenses their test from Myriad. If Lisa has her son’s mutation, as her pedigree suggests, she’ll be followed closely. The American College of Obstetrics and Gynecology (ACOG) advises breast exams twice yearly, and mammograms and MRIs annually, to detect cancer when it’s treatable. (Even the ACOG news release quotes a physician using “carrier” inappropriately, reflecting a persistent disconnect between genetics and medicine.) And I keep telling Lisa, as did her primary care provider, that in the absence of symptoms or other evidence, a genetic test does not a diagnosis make. She can have the genotype and never develop the phenotype.

But Justin and his sister Maya don’t want to think about, let alone act on, the BRCA1 mutation (that Maya has a 50% chance of having), or anything else lurking in their genomes. After all, Justin just took the tests for fun. “Not so much fun for me,” said his mother.

Justin’s attitude is understandable. Many young adults who’ve grown up in healthy families can’t imagine that one day they might have cancer, or heart disease, or depression, or have a child with a metabolic disease.

So once again I have mixed feelings about the ease with which people can learn their DNA sequences. If common terms like “carrier” are used in ambiguous ways, even by the test providers and the physicians they quote on their websites, and risk statistics vary depending on who makes up the experimental group — for delivery of information on just one well known gene –- how is the public to fare when a genome sequence can be had for a few thousand dollars, in two days? I hear through the grapevine that this is already possible, that genomics companies are hustling to annotate “variants of uncertain significance” so that the sequences are meaningful. I can’t help but worry about the millions of DNA bases that can vary among individuals amidst the aura of genetic determinism, that our sequences say who we are, what we have.

(On a related note, I’d like to know why my husband can take hundreds of genetic tests from 23andMe, without any contact with a health care provider, yet not be allowed to learn his PSA test results without permission from his physician. It’s a crazy inconsistency.)

I’m reminded of a situation that affected a large family group who had DNA marker tests from a direct-to-consumer genetic ancestry company. Tests revealed that the 96-year-old patriarch was not actually related to any of the others. The old man must have been an “unofficial adoption” – an orphan picked up by a caring couple who never told him — the heads of the family deduced. And it had never mattered. The elders elected not to give the DNA news to the man, because his entire identity was based on his position in his family.

Yes, this is extreme paternalism. But perhaps it is prescient. Will one of my daughters one day tell me something’s in my immediate future that I might not want to know? And this isn’t just me having my head in the sand, as responses to my blog about not wanting to know my genome sequence suggested a few months ago.

I think from years of writing a human genetics textbook, I’m perhaps very aware of just how complex our genomes are. And it is common in science that we can think we know something, and then a discovery changes everything. This is why I think at least some of the information being given to people now and in the near future will not have been validated – that is, one mutation might be counteracted by another, yet to be discovered. Some mutations or lesser variants will have effects in one genetic background but not another – like BRCA1 mutations posing different cancer risks in different populations.

There’s still a lot we don’t know, and we can’t know what it is we don’t yet know. So I’m not ready to be a guinea pig. Yet. But genome sequencing is (for now) a choice.

We can, though, perhaps prevent genetic information disasters. Caveats from genomics companies, conversations among family members so each can know what the others want to know, and more genetics education at all levels can do a lot to stave off upsetting use of genetic information.

Some of my fear of rash decisions based on seeing sequences of A, C, T and G is based partly on response to my book about gene therapy – middle and high-schoolers have no problems at all getting the science, but some of their parents and grandparents struggle. They’ve never learned what genes are and do. That leaves a lot of older people vulnerable to giving too much power to genetic information, of not understanding nuances like incomplete penetrance and one gene’s expression affecting another’s or a mutation having one risk in one population, a different one in another.

Let’s hope that we can enter and embrace this age of genomics while preserving individuals’ right to determine what they do and do not want to know.

Influenza. (credit: CDC)

My immune system is still on hyperdrive from what may have been the flu 3 weeks ago. I qualify my self-diagnosis because I never had a test to tell whether viruses or bacteria had invaded my body.

I’ve long wondered why such diagnostics aren’t in routine use. Molecular biology was pioneered on the genetic details of bacteria and their viruses in the 1970s, and by now most of our pathogens have had their genomes sequenced.

A quick, easy, and early way to distinguish viral from bacterial infections would have enormous impact, by:
• getting antibiotics and antivirals to those who truly need them.
• limiting spread by diagnosing people at the first ominous throat tickle or drip – or before.
• revealing new viral variants or epidemiological trends.

In two articles in yesterday’s PLOS One, researchers describe powerful gene expression signatures that distinguish viral and bacterial infections. Unlike traditional diagnostics that take days to culture pathogens, the new tests highlight the response of the host – us. Which mRNAs our bodies make reflect which genes are active. I can tell this is important news because the list of pharmaceutical companies the researchers disclose affiliation with is almost as long as the papers.

The persisting impact of infectious diseases on us is clear from individual tragedies to emergency departments packed with flu victims. Last summer, the New York Times chronicled the rapid and unnecessary death of 12-year-old Rory Staunton at the NYU Langone Medical Center, due to miscommunication and failure to recognize a bacterial infection. And this map shows the spread of the current flu.

WAYS TO DO IT
Despite the tendency of the Today show to discuss bacteria while showing graphics of viruses, the two are quite different. Bacteria are cells and are alive; viruses are neither. So telling them apart should be straightforward, and in fact several ways to do so are already out there.

1. An antibody-based rapid test detects a carbohydrate from Streptococcus pyogenes, the bacterium behind strep throat, impetigo, and scarlet fever. It killed Muppet inventor Jim Henson, as well as young Rory Staunton. The test yields results in 15 minutes, but because it isn’t as accurate as culturing, the American Academy of Pediatrics, the American Heart Association, and the Infectious Disease Society of America recommend backup throat cultures.

2. The BACcel system from Accelerate Diagnostics uses automated microscopy and computerized image analysis to eyeball pathogens, providing diagnosis in 2 hours and antibiotic resistance analysis in 6 hours. The technology was developed for wound and trauma-related infections on the battlefield.

3. Researchers at Ben-Gurion University of the Negev developed a test that generates different bioluminescence patterns from white blood cells exposed to bacteria or viruses. It uses luminol, the stuff that Law and Order uses to highlight blood at crime scenes. Like the approaches introduced in PLOS One, this assay follows response of the host, rather than evidence from the pathogen.

4. The BC-GP test from Nanosphere Inc. highlights genes that encode antibiotic resistance in a dozen species of strep, staph, enterococcus, and listeria, which account for 65% of bloodstream infections. It’s FDA approved and can check blood cultures in 2.5 hours. Antibiotic resistance genes flit from bacterium to bacterium, like starships wandering among the planets of a solar system.

5. Last July, FDA approved Becton, Dickinson and Company’s BD MAX MRSA assay, which collects, amplifies, and fluoresces MRSA (methicillin-resistant Staphylococcus aureus) in nasal swabs. It’s intended to prevent or control spread in healthcare settings, rather than for diagnosis.

6. Speaking of snot, the Nasal Microbiome Project is looking for microbial DNA clues that flu will progress to secondary bacterial pneumonia. The project is from the Genomic Sequencing Center for Infectious Diseases at the J. Craig Venter Institute, which is looking at DNA from everywhere in the universe, including, now, stuffed nasal passages.

TRACKING THE HOST, NOT THE PATHOGEN
The new approaches combine the specificity of a genetic approach with a computational tool to sort through the responses of thousands of genes. The tool, called a Bayesian sparse factor model, zeroes in on genes whose expression varies a lot in exposed/infected vs nonexposed people. The math also aggregates genes that partake of the same biochemical pathways, which can compensate for the fact that gene expression level does not necessarily predict the importance of the corresponding protein.

The researchers, Geoffrey Ginsburg, MD, PhD, Director, Genomic Medicine, Duke Institute for Genome Sciences & Policy, Christopher Woods, MD, MPH, also at Duke, and colleagues carried out two sets of experiments, one on bacteria, the other on viruses.

Staphylococcus aureus. (credit: CDC)

In the bacterial paper, the researchers derived a “molecular classifier” to detect S. aureus in mice, and used it to guide development of a similar tool for people. “Factor analysis” boiled down data from 9,109 expressed genes to 79 factors, which proved sufficient to distinguish S. aureus infection from E. coli infection from no infection. It also could distinguish MRSA from methicillin-sensitive S. aureus (MSSA).

The viral study looked at gene expression in 24 healthy young people who volunteered to have H1N1 influenza A squirted up their noses and 17 who got H3N2. This happened at a “purpose-built quarantine clinic” in London affiliated with Retroscreen Virology Limited Retroscreen Virology Limited, which recently announced infecting its 1,000th volunteer in their quest to develop antivirals.

The volunteers gave blood thrice daily for a week, from which the researchers teased out the telltale mRNAs reflecting genes expressed in response to the viral challenge. The usual suspects emerged, such as downstream targets of interferon. Because snorting flu virus in a lab is not exactly a normal situation, the researchers validated the test on blood samples taken from emergency department patients with H1N1 in 2009, along with blinded controls.

The researchers learned, observing the volunteers, that flu symptoms appear one to three days after infection. That’s a lot of time for a person to feel okay but be spewing viruses with every exhalation. The gene expression signature that viruses have taken up residence appears a little over a day after infection. So someone sneezed on at work can have the test and find out that flu looms, soon enough to stay home the next day and halt the spread. That’s been needed for years.

“A test that could identify individuals exposed to the flu before the onset of symptoms would be an important and useful tool for guiding treatment decisions, especially with limited antiviral medications,” said Dr. Woods.

When the flu hits, nothing much helps. (credit: Cliff Lewis)

Added Dr. Ginsburg, “These studies demonstrate that analysis of genomic factors show promise for early detection and accurately diagnosing the flu and staph.” The team is working now on how best to take the tests to the average person – like me, three weeks ago.